PDF hosted at the Radboud Repository of the Radboud University

Nijmegen

The following full text is a publisher's version.

For additional information about this publication click this link.

http://hdl.handle.net/2066/114106

Please be advised that this information was generated on 2022-10-01 and may be subject to

change.

30-33

corticosterone regulation

of the immune response

to malaria during pregnancy

a. a. ). с van zon

CORTICOSTERONE REGULATION OF THE IMMUNE

RESPONSE TO MALARIA DURING PREGNANCY

1

Promotor Prof Dr С Jerusalem

Co-referent Dr W M С Elmg

The studies presented in this thesis were performed at the Department of Cytology and Histology, Faculty of Medicine, University of Nijmegen They were supported by the Wor ld Health Organization, Geneva, Switzerland, and by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO)

2

CORTICOSTERONE REGULATION OF THE IMMUNE RESPONSE TO MALARIA DURING PREGNANCY

PROEFSCHRIFT

ter verkrijging van de graad van doctor m de geneeskunde aan de Katholieke Universiteit te Nijmegen,

op gezag van de Rector Magnificus Prof Dr J H G I Giesbers

volgens besluit van het College van Dekanen in het openbaar te verdedigen op donderdag 11 oktober 1984 des namiddags te 2 uur precies

door

ADRIAAN ARNOLDUS JOSEPHUS CORNELIS VAN ZON

geboren te Breda

1984

Drukkerij Brakkenstem, Nijmegen

3

4

CONTENTS

Abbrevations

Ten Geleide

Chapter I General Introduction 11

Chapter II Depressed malarial immunity in pregnant mice 39

Chapter III Pregnancy associated recrudescence in murine malaria (p. berghei) 43

Chapter IV Pregnancy induced improvement of malaria immunity 51

Chapter V Enhanced virulence of malaria infection in pregnant mice 65

Chapter VI Corticosterone regulation of the effector function of malarial immunity during pregnancy 71

Chapter VII Malarial immunity in pregnant mice, in relation to total and unbound corticosterone 81

Chapter Vili ACTH dependent modulation of malaria immunity in mice 93

Chapter IX The effect of progesterone and DES on malaria immunity in mice 109

Chapter X Corticosterone and reduced spleen cell proli­feration in vitro in relation to malaria immunity during pregnancy 119

Summary and final considerations

Samenvatting

137

143

5

Abbreviations

ACTH adrenocorticotropic hormone

ATS rabbit-anti-mouse T-cell serum

CBG corticosteroid binding globulin

Con A concanavalin A

CTL cytolytic T-lymphocyte

DES diethylstilbestrol

dpm désintégration per minute

DTH delayed-type hypersensitivity

PCS foetal calf serum

HCG human chorionic gonadotropin

Ig immunoglobulin

i.p. intraperitoneal

i.v. intravenous

ID international unit

LPS lipopolysaccharide

MEM(Eagle) Minimum essential Medium (Eagle)

MLR mixed lymphocyte reaction

NMS normal mouse serum

NRS normal rabbit serum

P. berghei Plasmodium berghei

PE parasitized erythrocytes

PEG polyethylene glycol 400

PHA phytohaemagglutinin

PRET parasitized reticulocytes

PrMS pregnant mouse serum

RES reticuloendothelial system

RIA radioimmunoassay

RPMI 1640 Roswell Park Memorial Institute medium number 1640

SEM standard error of the mean

6

Ten geleide

Malaria is een ziekte die veroorzaakt wordt door Plasmodium, een

eencellige parasiet die voor een deel van zijn levenscyclus leeft in

de rode bloedcellen en die wordt overgebracht door muggen. De ziekte

komt zeer veel voor, vooral in ontwikkelingslanden in tropen en

subtropen. Grote problemen bij de bestrijding zijn de uitgebreid

voorkomende resistentie zowel van de parasiet tegen diverse genees­

middelen als van de muggen tegen veel gebruikte insecticiden. Ook

de socio-economische struktuur van vele malaria gebieden staat een

efficiënte aanpak van de bestrijding in de weg.

Mede ten gevolge van malaria is er in endemische gebieden (ge­

bieden waar malaria voorkomt) een hoge kindersterfte. De volwassenen

hebben een zekere graad van immuniteit opgebouwd. Zij zijn vaak wel

drager van de malaria parasiet, worden ook regelmatig geinfekteerd

als ze door een mug gestoken worden, maar het afweersysteem in het

lichaam voorkomt dat de parasiet zich sterk kan vermenigvuldigen

en ziekteverschijnselen veroorzaakt. Deze vorm van immuniteit wordt

premuniteit genoemd. Voor het in stand houden van zo'n premuniteit

is het belangrijk regelmatig geinfekteerd te worden, zodat de para­

siet door zijn aanwezigheid de afweerreakties in het lichaam steeds

weer aktiveert. Het komt voor dat inwoners van endemische gebieden,

die een immuniteit tegen malaria hadden opgebouwd, na een lang­

durig verblijf in malaria-vrije landen bij terugkeer in hun eigen

land weer problemen krijgen met malaria omdat hun immuniteit ge­

heel of gedeeltelijk verdwenen is.

Malaria veroorzaakt in endemische gebieden vaak komplikaties

tijdens zwangerschap, met name tijdens de eerste zwangerschap.

Vrouwen in verwachting hebben meer last van malaria, terwijl ze

vooraf toch immuun waren. Het lijkt erop dat tijdens zwangerschap

de afweerreaktie van het lichaam tegen malaria min of meer onder­

drukt wordt zodat de parasiet zich weer kan vermenigvuldigen en

de zwangere vrouw ziek maakt. Overigens, ook bij zwangere vrouwen

7

uit malaria-vrije gebieden verloopt de ziekte ernstiger dan bij met-

zwangeren, als zij malaria gebieden bezoeken. Komplikaties tijdens

zwangerschap zijn o.a. sterke bloedarmoede, intra-uterine dood van

de foetus of abortus, voortijdige geboorte en een te laag geboorte

gewicht. In het algemeen wordt weinig melding gemaakt van over­

dracht van malaria van moeder op kind (zgn. congenitale malaria).

Ook van een aantal andere ziekten is bekend dat zij ernstiger

kunnen verlopen tijdens zwangerschap. Dit wijst erop dat het af­

weersysteem van de vrouw verctndert als zij in verwachting is. Waar­

om dit gebeurt is m e t met zekerheid bekend, en ook m e t welke af-

weerreakties onderdrukt worden en welke faktoren in het lichaam

daarvoor verantwoordelijk zijn. In het algemeen wordt gedacht dat

een afname in de immunologi sene reaktiviteit van de moeder ervoor

zorgt dat de foetus m e t als een vreemd transplantaat wordt afge­

stoten. De foetus heeft immers kenmerken van de vader, die voor de

moeder als lichaai is vreemd gelden.

Veel malaria onderzoek in het laboratorium wordt gedaan in

proefdiermodellen, vooral apen, ratten en muizen. In ons labora­

torium wordt onderzoek gedaan aan het malaria model: Ріавтоагші

berght- in muizen. Deze knaagdier parasiet komt van nature in boom-

ratten чтг o.a. in Zaïre en is van daaruit geadapteerd aan muizen.

Een ¡nfektie in de door ons gebruikte muizenstammen is altijd dode­

lijk, tenzij geneesmiddelen worden toegediend. Geholpen door genees­

middelen zijn de muizen in staat een immuniteit tegen de parasiet

te ontwikkelen, waardoor ze achteraf ook zelfstandig een nieuwe in-

fektie kunnen weerstaan zonder ziek te worden. Net als de mens blijft

de muis ook drager van de parasiet, dus hier is ook sprake van pre-

mumteit.

In dit mui zen-mal ari a model wordt ook gevonden dat tijdens

zwangerschap de immumteit afneemt, zodat veel drachtige muizen,

die vooraf immuun waren, ziek worden en zelfs doodgaan ten gevolge

van de weer opkomende malaria.

8

Het onderzoek, beschreven in dit proefschrift, gaat over dit

verlies van malaria immuniteit, hoe dit tot stand komt en welke

faktoren erbij betrokken zijn. De resultaten zijn achter in dit

proefschrift in een samenvatting bijeen gezet.

9

10

Chapter I

General Introduction

and

Aim of this Study

General introduction

In endemie areas malaria is a serious complication of pregnancy.

Clinical manifestations of malaria become more important during preg­

nancy among members of communities where immunity has been established.

Studies in several countries show that malaria infection in general,

and particularly Plasmodium falciparum infection is more severe during

pregnancy (27, 83, 96, 98, 147). Parasite rate and parasite density

are higher in pregnant women as compared to the same individuals be­

fore pregnancy or to an age-matched, non-pregnant group (23, 71).

Pregnant women, especially Primigravidae show enlargement of the spleen

which is not otherwise common in adults in holoendemic areas (93, 96).

Cerebral malaria with a high mortality rate is more common in pregnant

women (109), though Lawson (96) believes that it is uncommon, but

repeatedly diagnosed in stead of ecciampsia. The highest infection rate

of both peripheral blood and placenta is observed in Primigravidae (22,

23, 106, 130).

Neither the mechanism of loss of malaria immunity during pregnancy,

nor the mechanism of the parity dependent decrease of the infection

rate are known. A proposed age dependent increase in maternal immuno­

logical maturity (93) is less likely in view of the usually short time

interval between first and second pregnancy (107). It is more likely

that infection in Primigravidae enhances immune reactivity or induces

additional immune reactions, that are less sensitive to depression

during subsequent pregnancy (22; see chapter 4 of this thesis).

Malaria prevalence is not constant throughout pregnancy (151).

Loss of immunity and febril attacks are reported to be more common as

pregnancy advances (83, 96, 109), though a decline of malaria pre­

valence and parasite density from the middle of the second trimester

towards term has been reported as well (23, 122). This apparent con­

tradiction has been explained by differences in acquired states of

immunity. In women living in holoendemic areas the recovery rate ex­

ceeds the infection rate later in pregnancy. This is in contrast to

12

women living in areas with lower endemicity, or to non-immune women

visiting endemic areas (22).

Pathological examinations suggest a direct correlation between

malaria infection and second trimester abortion (98), but no clear

association is seen between stillbirth and placental infection (107,

130). Malaria infection of the placenta is suggested to be one of

the causes of low birthweight and prematurity in malaria endemic

areas (26, 31, 107, 143) and maternal chemoprophylaxis during preg­

nancy leads to higher infant birthweight (22). Placental infection

and low birthweight are more frequently observed in Primigravidae

(22, 107). Placental parasitaemia and the cellular reactions against

it may affect the function of the placenta (109), and cause lower

birthweight and early delivery of the infant (107), but placenta

weight itself is not influenced by malaria.

Frequent observation of a heavy infection in the placenta asso­

ciated with a much lower parasitaemia in the peripheral blood may be

related to a local immunosuppression in the uterus during pregnancy,

which is described by Gottesman (74), or to local escape from inmune

destruction in that the newly developing vasculature of the placenta

forms a privileged site, especially for P. falaipavum with its pre­

dilection for capillaries in deep tissues (107). Results of histolo­

gical studies are contradictory. Osunkoya et al. (117) describe pre­

ferential binding of P. falatparum infected cells to the trophoblastic

xeils uf Lhe chorionic v1Ui, but this sequësTFaiiön i s "not found in

light- and electronmicroscopical analysis by others (24).

Malaria not only has a direct, but also an indirect effect on

pregnancy. Infection is associated with a more or less severe de­

pression of the immune system of the host, both in human as well as

in animal malaria (75, 167), resulting in an increased susceptibility

to other infections (129).

Though a pregnancy dependent loss of malaria immunity was noted

over 60 years (compare 22), hardly any progress is made regarding ana­

lysis of the underlying mechanism. An important factor may have been

that a suitable experimental model system was not available. Most

studies on experimental malaria and pregnancy are carried out with

13

rodents, and deal primarily with transfer of immunity from mother

to offspring (5, 28, 53, 137, 153, 173). Oduola et al. (115) observe

a more virulent malaria infection during pregnancy with enhanced

anaemia, and a severely compromised maternal-fetal relationship

leading to abortion, resorption of fetuses, or low birthweights of

offspring when infection was started late in pregnancy.

PREGNANCY AND DISEASE

Enhanced prevalence and morbidity during pregnancy is not re­

stricted to malaria, but is observed for an increasing number of

viral, bacterial and parasitic infections. In addition enhanced tumor

growth and increased mortality rates during pregnancy are described

for Burkitt's lymphoma (12) and breast cancer (125). A list of diseases

being enhanced during pregnancy is given in Table I. In these studies

suggestions on possible factors in the underlying mechanism are des­

cribed.

Naturally occurring changes in serum corticoid (amoebic colitis,

malaria, encephalomyocarditis), oestrogen (hepatitis, encephalomyo-

carditis) and progesterone (herpes infection) are considered to affect

host resistance. Enhanced susceptibility to infection or an enhanced

virulence depends on the day of pregnancy when primary infections are

started.

A local reduction of resistance is described for malaria (pla­

cental parasitaemia higher and more frequent than infections of peri­

pheral blood) and herpes infection (enhancement of infection started

by the intravaginal, not by the intraperitoneal, intravenous or intra­

nasal route). Reduction of specific cell mediated immune responsive­

ness is observed against rubella and cytomegalovirus, whereas the

humoral response against cytomegalovirus remains intact (94), and

is enhanced against Escherichia coli. Loss of a previously existing

immunity during pregnancy is described for leprosy, tuberculosis,

amoebic colitis, toxoplasmosis, malaria, and trypanosomiasis.

In sunmary several observations suggest that a pregnancy asso­

ciated reduction of part of the immune reactivity caused enhanced

prevalence and virulence of infection during pregnancy.

14

Table I. Pregnancy and disease

DISEASE HOST REFERENCE

Viral infections:

encephalomyocardi

hepatitis

herpes

poliomyelitis

polyoma

rubella

¡tis

Bacterial infections:

mouse

man

mouse

mouse

man

man

64

116

11, 92

46

158

171

Escherichia coli infection

leprosy

listeriosis

tuberculosis

mouse

man

mouse

91

57

101

man 119

Parasitic infections:

amoebic colitis

babesiosis

malaria

toxoplasmosis II

trypanosomiasis

man

man

mouse

man

mouse

man

mouse

1

127

115, 163, 164

22, 23, 27, 71, 83, 93,

98, 106, 107, 109, 122,

151

101

162

165

96,

130,

Tumors:

breast cancer

Burkitt's lymphoma man

man

125

12

15

IMMUNE REACTIVITY DURING PREGNANCY

Suppression of the maternal immune system during pregnancy is

thought to be functional in protection of the fetal graft, bearing

paternal antigens, from rejection by the mother (15, 78, 141). De­

pression especially concerns the cell mediated immune response (125),

the humoral immune response is not decreased, or even increased

during pregnancy (8, 34, 141). At the same time when during preg­

nancy an inhibition of T-cell reactivity is measured, no impairment

of development of antibody-secreting lymphocytes, of maturation of

helper T-cells, or of the B-cell dependent LPS response is found

(40, 74).

Histological changes in lymphoid organs are observed during

pregnancy. Thymus weight decreases in pregnant mice (118, 121), the

involution occurs in late pregnancy and reaches a maximum at partu­

rition (86, 108). Involution is associated with reduction of steroid-

sensitive cortical cells, and can be considered as a non-specific

phenomenon, independent of immunization against paternal histocom­

patibility antigens (105, 121). Decrease of weight of the superficial

lymph nodes is also reported, but is less marked compared to the

thymus (118). Lymph nodes draining the uterine area exhibit weight

increase, especially in allogeneic pregnancy (6, 15, 105), but no

concomittant transplantation immunity to the fetus is elicited. En­

largement may be connected to stimulation of humoral immunity (pro­

duction of antibodies) (15). Depression of at least part of cellular

immune responsiveness during pregnancy is demonstrated both in vivo and in vitro. Cardiac allografts expressing paternal antigens sur­

vive longer in pregnant than in virgin mice (10). Skin grafts with

paternal antigens showed prolonged survival time when transplanted

into the uterus of pregnant rats, but not when transplanted to the

trunks, indicating a local immunosuppression (15). A decrease of

the tuberculin reaction in pregnant women (66), and of the contact

allergic reaction to picryl chloride in pregnant mice (62) are

other indications of impaired cell mediated immunity in vivo.

16

Several reports mention a significant reduction in the lympho­

cyte response to PHA in pregnant individuals (66, 97, 125, 158).

Gottesman (74) describes a decrease in T-cell proliferative capacity

in the area of the uterus but not in the rest of the body. A rapid

increase in PHA stimulation activity early in pregnancy (73) followed

by a decrease afterwards until delivery (94) indicates that this

immune reactivity is not constant throughout pregnancy and therefore

apparently is not a vital factor to prevent fetal graft rejection

(73).

Other in vitro T-cell assays also indicate an inhibition of T-

cell activity during pregnancy. The mixed lymphocyte reaction (MLR)

of maternal lymphocytes (158), and in particular the MLR response

from mother to child (20, 159) is depressed. Harrison (81), however,

finds no alteration of the MLR in lymphocytes from primigravid rats,

unless serum from pregnant rats was added to the culture. Others des­

cribe a normal capability to develop in vitro a MLR and CTL to allo­

geneic cells, but specific immunosuppression towards paternal anti­

gens is apparent from the absence of cytotoxic T-lymphocytes specific

for paternal antigens in allopregnant primi- or multigravidae (37,

160). Several authors note a locally suppressed capacity to generate

a CTL (41, 141), especially in cells from the lymph nodes draining

the uterus during pregnancy (40, 41).

The suppression of T-cell activity during pregnancy is thought

4o-be-exerted by treïls (141)~r~SFrteen~céTTs~öF TTlopregnänt muTti-

parous mice were able to suppress in vitro MLR response and CTL ge­

neration against paternal cells (36, 37). With monoclonal antibodies

a subpopulation of splenic T-cells with suppressive activity can be

identified (36, 160). Others suggest that suppressive activity has

to be ascribed to B-cells or B-cell-like lymphoid cells (84), cells

of the trophoblast (40) or to suppressor lymphocytes in the neonatal

blood (159). The cellular suppression function is also clear from

the observation that the enhancement effect on allograft survival

during pregnancy could be transferred by para-aortic lymph node

cells, but not by maternal serum (10).

17

Immunosuppression during pregnancy is not only dependent on

(suppressor) cell activity, but is also related to the presence of

serum factors. These suppressive factors may act directly on the tar­

get cell, or indirectly by inducing a suppressor cell population (20,

41). Serum factors with immunosuppressive properties are: - an early

pregnancy factor that appears in the serum within a few hours after

fertilization (110, 111), - alpha-fetoprotein (51, 122), though this

is not supported by observations from Wajner (166), and - alpha2-

macroglobulins (67, 144, 145, 154). The significantly decreased serum

levels of alpha2-macroglobulins in pregnant women with spontaneous

abortion and fetal death (67, 156) underline the immunosuppressive

action of this substance.

Changes in hormone levels during pregnancy are also related to

immunosuppression. HCG has been reported to suppress the prolifera­

tive T- and B-cell response induced by different mitogens, to inhibit

MLR development, and to induce generation of suppressor T-cells (4,

47, 70, 79, 80). Others show, however, that HCG, when highly purified,

no longer suppresses T-cell activity (14, 30, 104)

Oestrogen, administered in pharmacological doses, suppresses in­

flammation, delays allograft rejection and decreases in vitro blasto-

genic responses (95, 102), but oestriol has no blocking effect on

lymphocyte cytotoxicity to embryonic fibroblast cells, not even at

supraphysiological concentrations (149). Mice treated with increasing

amounts of diethylstilbestrol (DES) exhibit a progressive loss of

cortical thymic lymphocytes and T-cells in the splenic white pulp and

a stimulated RES-activity (21, 99, 102). Other reports mention re­

duced resistance to bacterial or parasitic infections, and suppressed

immunoreactivity to tumor cells and tumor associated antigens after

DES treatment in mice (52), and men (2). It is doubted, however,

whether this occurs at physiologic concentrations of free circulating

oestrogens (95).

Progesterone inhibits T-cell reactivity in vitro also (42, 146),

but Clark (41) doubts whether a reduced capacity to generate CTL ,

activity in cells of pregnant mice is a direct effect of progesterone,

since maximum suppression was measured during the first half of

18

pregnancy and the blood progesterone level is maximal in the second

half. Serum levels of progesterone in the second and third trimester

of pregnancy can suppress lymphocyte reactivity in vitro (11, 149),

but not the serum concentration alone, also a pregnancy dependent

change in the binding capacity of the lymphocytes for progesterone

may be an important factor in the development of the immunosuppressive

effect (150).

Since progesterone is produced by the placenta, the local con­

centration may be high enough to suppress an immunological rejection

of the "allograft" fetus. On the other hand progesterone production

by the human placenta does not start until 6 to 7 weeks of pregnancy,

and placentae of rabbits and goats do not produce progesterone yet

no fetal rejection occurs (146).

Progesterone affinity for corticosteroid-binding globuline (CBG)

is almost equal to that of Cortisol and corticosterone (146, 169).

The high concentration of free progesterone produced in the placenta

can locally release glucocorticoid from CBG thereby increasing at

least locally the free concentration glucocorticoid; the immunosup­

pressive action of glucocorticoids is well established. In this way

the immunosuppressive action of progesterone is indirect.

During pregnancy the total and free concentration of glucocorti­

coid increases in man (25, 114, 142) and mice (13). Pregnancy levels

of Cortisol inhibit the PHA mitogenic response, but the concentration

of unbound Cortisol in these sera is not high enough (149). In addi­

tion, the inhibition of MLR by added plasma from pregnant women is

correlated to the period of gestation, but not to the concentration

Cortisol in the plasma (90). The day-night variation of unbound Cor­

tisol, however, is much diminished in late pregnancy (29, 35, 114).

Thus, maternal lymphoid tissue is exposed to an average daily concen­

tration of Cortisol two and a half times normal (29), and apart from

an increased concentration a continuous exposure to increased levels

may have an immunoregulatory effect during pregnancy as well.

19

IMMUNITY AND GLUCOCORTICOIDS

Glucocorticoids have a wide range of effects on the immune and

inflammatory response in both animals and man (9), and are used in

the treatment of many diseases with autoimmune or inflammatory

features. Patients treated with large doses of corticosteroids develop

an impaired immunological status, and are more susceptible to a wide

variety of infections (17). Regarding parasitic diseases increased sus­

ceptibility and exacerbation of latent infections during corticoid

treatment are reported for Giardia muris and Strongyloides ratti in

mice (76, 113), and Entamoeba histolytica in man, the latter leading

to acute amoebic dysentery and fulminating hepatic amoebiasis (148).

The immunosuppression produced by corticosteroids rapidly alters the

stable host-parasite relationship, but it is unknown how the steroids

produce this effect. At pharmacological doses, glucocorticoids have a

lympholytic effect (9). Sensitivity of lymphocytes to lysis is species

dependent, e.g. mouse and rat lymphocytes are more sensitive than those

of guinea pigs or man. But also within corticoid-sensitive species,

differences in sensitivity are observed between subpopulations of

lymphocytes and between young and mature cells.

In mice and rats, corticoid treatment induces depletion of blood

lymphocytes and atrophy of most lymphoid organs at variable degree,

most dramatically of the thymus (9, 56). In man, lymphopenia following

corticosteroid administration is attributed to a redistribution of

the recirculating lymphocytes, preferentially to the bone marrow (50,

72, 82). Certain lymphocyte subsets, however, may be sensitive to

the lytic effect of corticosteroids, and this may be another impor­

tant aspect of corticosteroid-mediated immunoregulation in man (50).

At physiological levels glucocorticoids are also functional in immuno­

regulation. There is an inverse relationship between lymphocyte levels

in the peripheral blood and plasma Cortisol concentrations in humans.

Patients with adrenal deficiency, and subsequently low plasma corti-

sol levels do not exhibit circadian changes in blood lymphocyte num­

bers (157). Moreover, variations in PHA induced blastogenesis and

antibody-dependent cellular cytotoxicity are related to endogenous

20

serum Cortisol (3, 152). A regulatory circuit is suggested between

the magnitude of the immune response, ACTH release by the adenohypo-

physis, glucocorticoid production of the adrenals and its modulating

effect on lymphocyte activity, acting as a centrally mediated immuno-

regulatory circuit (19).

A major effect of glucocorticoid treatment is the inhibition of

T-lymphocyte proliferation and function. Glucocorticoids especially

inhibit the functional activity of the suppressor/cytotoxic T-cell

subsets (38), subsets with a high number of glucocorticoid receptors

(54). The dose-dependent inhibition of T-cell function by corticoid

parallels the inhibition of interleukin 2 production in vitro, and

exogenous interleukin 2 restores normal function (38, 50). Interfer­

ence with production of other soluble mediators from lymphocytes such

as lymphotoxin and a monocytechemotactic factor have been reported

also (50). Thus, modulation of the release of soluble mediators

may be an important potential mechanism of corticosteroid-mediated

immunoregulation both in man and animal.

Not only lymphocytes, but also eosinophils, monocytes and macro-

hages are sensitive to glucocorticoids (50, 168). Corticoid treat­

ment suppresses the colloid clearance function of the reticulo-endo-

thelial system in mice (18), and in man it reduces the number of circu­

lating monocytes, and causes a marked impairment of the bactericidal

and fungicidal activity (131, 155). A decrease of the plasma cortico­

sterone level in mice leads to an enhanced cellular response, paral­

leled by a specific increase of the number of blood monocytes (161).

Studies in vitro suggest that glucocorticoids reduce the population

of inflammatory macrophages by suppressing lymphokine production

(e.g. macrophage mitogenic factor) (58). Glucocorticoids also in­

hibit secretion of monokines, e.g. interleukin 1, a lymphocyte-

activating factor (50). Since monocytes serve important accessory

cell functions in T- and B-lymphocyte responses, corticosteroid-

induced effects on monocytes may therefore indirectly alter the

functional activity of other cell subsets.

The humoral immune response is also sensitive to glucocorti­

coids. Administration of corticoids inhibits the production of IgG,

21

IgA and to a lesser extent of IgM (50). In general, the secondary

antibody response is less susceptible to corticoid-mediated suppression

than the primary response (9, 16).

In conclusion, the glucocorticoids have profound and complex effects

on the immune response and at physiological levels they already seem to

exert a regulatory effect on immunological processes.

MALARIA IMMUNITY AND GLUCOCORTICOIDS

The life cycle of the malaria parasite in the vertebrate host

comprises different stages, the sporozoite, the exoerythrocytic stages

in the liver, and the erythrocytic stages including the gametocytes.

The erythrocytic stages are directly harmful to the host, and our stu­

dies and discussions only consider these stages.

In experimental malaria, the severity of an infection depends on

the host-parasite combination (87). Some forms of malaria evoke a self-

limiting infection, spontaneously resulting in protection of the host,

but other forms are lethal. A number of experimental immunization pro­

cedures are described and in most instances, immunity to malaria, de­

veloped under "natural" conditions, but also in experimental models,

is of the premunition type, with small numbers of parasites persisting

in the immune host (87).

The immune response to malaria is complex, with both activation

of cellular immune mechanisms and production of antibodies (123). The

cell-mediated immune response is evident from the ability to transfer

immunity by cells, and from the positive DTH to parasitized erythro­

cytes in mice immune to P. yoelii. The intensity of the DTH response

closely correlates with the immune status of the host (48, 89).

Playfair (124) observes an accumulation of lymphoid cells in the spleen,

and especially in the liver of P. yoelii infected mice, and there is

a close correlation between the timing of cell accumulation and re­

covery from infection. This pattern of accelerated cell accumulation

can be transferred·to normal mice by lymphoid cells, but not by serum

of protected mice.

Development of protective immunity is T-cell dependent (89).

22

T-cell deficient "nude" mice are unable to develop protection to the

malar-ia parasite (39, 132), and injection with anti-T-cell serum

given during the immunization procedure decreases the percentage of

mice developing immunity (60).

Monocytes and macrophages are involved in the response to

malaria. Parasitized erythrocytes are more rapidly removed from the

circulation than normal red cells (55, 126). Mice, infected with P.

yoelii, showed a massive increase in the number of blood monocytes

(88) and accelerated carbon clearance (133). Both these processes

were not observed in infected athymic mice. Macrophages from P. berghei infected mice were more phagocytic for parasitized cells in vitro (138).

Whether phagocytosis of parasitized erythrocytes plays an important

role in vivo, however, is questionable since recovery from blood-

stage infections is not correlated with enhanced phagocytosis (33,

100). Moreover, attempts at in vivo depletion of macrophages do not

have a significant effect on the course of the infection (124).

The role of antibodies in malaria is controversial. Immunoglo­

bulins, possibly autoantibodies, have been demonstrated on the sur­

face of red cells in murine (85, 103) and human malaria (63, 135).

Anaemia in malaria is partly due to loss of uninfected red cells by

autoantibodies (172).

Specific antibodies in immune serum can passively protect against

blood-stage malaria infection (44, 59, 120). Results from in vitro ex­

periments show that immune serum acts mainly by blocking the invasion

of red cells by merozoites(45). Recent studies in mice with monoclonal

antibodies support the idea that the specific anti-merozoite action

is a major feature of protection in vivo (43, 68, 69).

There is other evidence that antibodies may be important in

controlling the early phase of a primary infection, but that they

are not essential for maintenance of immunity (89). B-cell deficient

mice cannot overcome the primary infection, but if cured by chemo­

therapy they are resistant to reinfection (132, 134). Moreover, Grun

(77) shows that B-cell deficient mice are able to terminate acute

P. chabaudi infections, and become resistant to homologous challenge.

23

In summary, the malaria parasite triggers many immune responses

in the host. Which of these are protective is unclear, and may vary

from one host-parasite combination to another, though it is evident

that the induction of immunity is T-cell dependent. Mostly, the de­

velopment of protective immunity implies achievement of a new level

of equilibrium in the host-parasite interplay, allowing a small

number of parasites to persist, yet the host is resistant to challenge

(premunition). Premunition is a dynamic state, this is emphasized by

the fact that small numbers oí persisting parasites provide the sig­

nal for maintenance of immunity and that reduction of the immunocom-

petence of the host, e.g. during malnutrition or pregnancy, can dis-

rupi the equilibrium res.ñtino in loss of immunity (87).

Results of studies on the effect of glucocorticoids on malaria

infection are controversial. Findlay (65) describes that corticoster­

one injection enhances P. berghei infection in mice with a faster in­

crease of parasitaemia and shorter mean survival time. The smaller

spleens of corticosterone-treated mice suggest inhibition of the

cellular reaction. Singer (139) and Cantre!! (32), on the other hand,

observe a reduction of parasitaemia and an increased survival time

in P. oargkei infected mice, treated with cortisone. These results

are related to a decrease in reticulocyte count in cortisone treated

animals, since P. berihe-L shows a definite predilection for these

cells. In mice, infected with P. vinakei (predilection for adult ery­

throcytes), betamethasone has no significant effect on the normal

course of infection (49). Betamethasone prevents development o f ac­

quired immunity in infected mice cured by chloroquine treatment.

Once immunity is established, however, no breakthrough is provoked

by betamethasone treatment (49). El ing (61) describes an increased

survival time in some P. berghei - mouse strains treated with dexa-methasone, but not in others. In this study increased survival

is explained by a dexamethasone-mediated suppression of immunopatho-

logical reactions.

The results in other parasite-host combinations are equally di­

vergent. A primary infection of P. aynomolgi in monkeys is not altered

24

by cortisone injections, but in monkeys with a chronic low grade in­

fection, the number and the intensity of relapses is increased by

cortisone treatment (136).

In avian malaria, the primary infection of P. reliction in

pigeons is enhanced, but no relapse can be elicited in birds with

a chronic, latent infection (128). Latent chronic infections of

P. relicbwr in sparrows, however, tend to relapse after treatment

with corticosterone (7).

For malaria in man, it is reported that in patients with a

relapse of p. νίυαχ malaria, cortisone injections do not influence

appreciably the clinical expression of the relapse (170).

In conclusion, the effect of glucocorticoid treatment on a

malaria infection is not clearly defined, and probably depends on

the parasite-host combination. Glucocorticoids seem to influence es­

pecially the generation of acquired immunity, but the effect on an

established immunity is small. Differences in the type of gluco­

corticoids used, and in the way of administration may be important

factors for the effect of the hormone in ю.

AIM OF THIS STUDY

This thesis comprises studies on malaria immunity in the Plas­

modium Ъегдкеі - mouse model in relation to pregnancy and plasma

corticosterone levels. Since in this node! an unprotected primary

infection was always lethal, immunity was induced by a drug con­

trolled infection, followed by a challenge. Acquired immunity was

of the premunition type with low numbers of surviving parasites in

the host. Repeated challenges throughout life prevented fading of

immunity, and cases of spontaneous recrudescences were very rare.

When immune mice became pregnant, a considerable percentage

lost their immunity, and developed a frequently fatal recrudescent

infection. The main topic of this thesis was the study of this preg­

nancy associated loss of malaria immunity in mice and the factors

involved. The features of the pregnancy associated loss of malaria

25

immunity were studied in several P. ЪетдНег - mouse strain combi­nations to define common and strain specific aspects (chapter 2 and

3). Since in human malaria the problem is particularly important

in Primigravidae, particular attention has been paid to the effect

of parity and the role of the parasite in the establishment of this

effect (chapter 3 and 4). Parallelism with human malaria was also

looked for with regard to the severity of a primary infection during

pregnancy (chapter 5).

In view of changes in blood corticosterone levels during preg­

nancy, and the well known immunosuppressive properties of corticoid,

a possible relation with loss of immunity during pregnancy was ana­

lysed (chapter 6 and 7). Establishment of a relation between plasma

corticosterone and loss of immunity prompted analysis of an immuno-

regulatory role of corticosterone in non-pregnant mice. The influ­

ence of ACTH treatment on endogenous corticosterone levels was corre­

lated to loss of immunity (chapter 8).

Since not only corticosterone but also other hormones are

thought to exert immunosuppression during pregnancy, experiments were

started to evaluate the effect of progesterone and oestrogen on ma­

laria immunity (chapter 9).

Immune reactivity of spleen cells in vitro was investigated to

define immune reactions involved in the pregnancy associated immune

suppression. Furthermore, the study of the effect of serum and cor­

ticosterone in serum on this in vitro response was carried out to

correlate the in vitro and in vivo situation (chapter 10).

26

LITERATURE CITED

1. Abioye, A.A. 1973. Fatal amoebic colitis in pregnancy and puerperi-uni.· a new clinico-pathological entity. J. Trop. Med. Hyg. 76: 97-100.

2. Ablin, R.J., R.A. Bhatti, P.D. Guiñan and W. Khin. 1979. Modulatory effects of oestrogen on immunological responsiveness. II. Supp­ression of tumour-associated immunity in patients with prostatic cancer. Clin. Exp. Immunol. 38: 83-91.

3. Abo, T., T. Kawate, К. Itoh and K. Kumagai. 1981. Studies on the bioperiodicity of the immune response. I. Circadian rhythms of human T, B, and К cell traffic in the peripheral blood. J. Immunol. 126: 1360-1363.

4. Adcock, E.W., E. Teasdale and C.S. August. 1973. Human chorionic gonadotropin; its possible role in maternal lymphocyte suppression. Science 181: 845-847.

5. Adler, S. and A. Fonez. 1965. Transfer of antibodies to Plasmodium vinakei through milk of immune mice. Isr. J. Med. Sci 1: 988-993.

6. Ansell, J.D., C M . McDougall, G. Speedy and C.J. Inchley. 1978. Changes in lymphocyte accumulation and proliferation in the lymph nodes draining the pregnant uterus. Clin. Exp. Immunol. 31: 397-407.

7. Applegate, J.E. 1970. Population changes in latent avian malaria infections associated with season and corticosterone treatment. J. Parasitol. 56: 439-443.

8. Baboom'an, Chr. and P. Griffiths. 1983. Is pregnancy immuno­suppressive? Humoral immunity against viruses. Br. J. Obstet. Gynaecol. 90: 1168-1175.

9. Bach, J.F. 1975. Corticosteroids, pp. 21-91. In: J.F. Bach (ed.): The mode of action of immunosuppressive agents. North Holland Publishing Co., Amsterdam.

10. Baines, M.G., K.G. Millar and H.F. Pross. 1980. Allograft enhance­ment during normal murine pregnancy. J. Reprod. Immunol. 2: 141-149.

11. Baker, D.A., Ch. A. Phillips, K. Roessner, R.J. Albertim' and L.I. Mann. 1980. Suppression by progesterone of nonspecific in vitro lymphocyte stimulation in mice as a mechanism for the enhance­ment of herpes simplex virus type II vaginal infection. Am. J. Obstet. Gynecol. 136: 440-445.

12. Bannerman, R.H.O. 1966. Burkitt's tumour in pregnancy. Br. Med. J. ii: 1136-1137.

13. Barlow, S.M., P.J. Morrison and F.M. Sullivan. 1974. Plasma corti­costerone levels during pregnancy in the mouse: the relative con­tributions of the adrenal glands and foeto-placental units. J. Endocrinol. 60: 473-483.

14. Bean, M.A., J.S. Salser, M. Newman, K. Stahl and M.E. Balis. 1977. Human chorionic gonadotropin: doubtful role as an inhibitor of cell-mediated immunity. Cancer Immunol. Immunother. 2: 85-90.

15. Beer, A.E. and R.E. Billingham. 1972. Immunobiology of mammalian reproduction. Adv. Immunol. 14: 1-84.

27

16. Benner, R., J.J.M, van Dongen and A. van Oudenaren. 1978. Corti­costeroids and the humoral immune response of mice. I. Differ­ential effect of corticosteroids upon antibody formation to sheep red blood cells in spleen and bone marrow. Cell. Immunol. 41: 52-61.

17. Berger, R. and S. Kraman. 1981. Acute military blastomycosis after "short-course" corticosteroid treatment. Arch. Intern. Med. 141: 1223-1225.

18. Besedovsky, H. and E. Sorkin. 1977. Network of immune-neuroendo-crine interactions. Clin. Exp. Immunol. 27: 1-12.

19. Besedovsky, H.O., A.E. del Rey and E. Sorkin. 1983. What do the immune system and the brain know about each other? Immunol. Today 4: 342-346.

20. Birkeland, S.A. and K. Kristoffersen. 1980. The fetus as an allo­graft: a longitudinal study of normal pregnancies with mixed lymphocyte cultures between mother-father and mother-child. Scand. J. Immunol. 11: 311-319.

21. Boorman, G.A., M.I. Luster, J.H. Dean and R.E. Wilson. 1980. The effect of adult exposure to diethylstilbestrol in the mouse on macrophage function and numbers. J. Reticuloendothel. Soc. 28: 547-560.

22. Brabin, B.J. 1983. An analysis of malaria in pregnancy in Africa. Bull. WHO 61: 1005-1016.

23. Bray, R.S. and M.J. Anderson. 1979. Раіоърагыт malaria and preg­nancy. Trans. R. Soc. Trop. Med. Hyg. 73: 427-431.

24. Bray, R.S. and R.E. Sinden. 1979. The sequestration of Plasmodium falciparum infected erythrocytes in the placenta. Trans. R. Soc. Trop. Med. Hyg. 73: 716-721.

25. Bro-Rasmussen, F., 0. Buus, F. Lundwall and D. Trolle. 1962. Variations in plasma Cortisol during pregnancy, delivery and the Puerperium. Acta Endocrinol. 40: 571-578.

26. Bruce-Chwatt, L.J. 1952. Malaria in African infants and children in Southern Nigeria. Ann. Trop. Med. Parasitol. 46: 173-200.

27. Bruce-Chwatt, L.J. 1983. Malaria and pregnancy. Br. Med. J. 286: 1457-1458.

28. Bruce-Chwatt, L.J. and F.D. Gibson. 1955. Transplacental passage of Plasmodium berghei and passive transfer of immunity in rats and mice. Trans. R. Soc. Trop. Med. Hyg. 50: 47-53.

29. Burke, C.W. and F. Roulet. 1970. Increased exposure of tissues to Cortisol in late pregnancy. Br. Med. J. 1: 657-659.

30. Caldwell, J.L., D.P. Stites and H.H. Fudenberg. 1975. Human chorionic gonadotropin effects of crude and purified prepa­rations on lymphocyte responses to phytohemagglutinin and allogeneic stimulation. J. Immunol. 115: 1249-1253.

31. Cannon, D.S.H. 1958. Malaria and prematurity in the western region of Nigeria. Br. Med. J. 2: 877-878.

28

32. Cantrell, W. and L.P. Kendrick. 1963. Cortisone and antimalarial drug activity against Plasmodium berghei. J. Infect. Dis. 113: 144-150.

33. Cantrell, W. and E.E. Elko. 1970. Plasmodium berghei: phago­cytic hyperactivity of infected rats. Exp. Parasitol. 28: 291-297.

34. Carter, J. and D.W. Dresser. 1983. Pregnancy induces an increase in the number of immuno-globulin-secreting cells. Immunology 49: 481-490.

35. Challis, J.R.G. and J.E. Patrick. 1983. Changes in the diurnal rhythms of plasma Cortisol in women during the third trimester of pregnancy. Gynecol. Obstet. Invest. 16: 27-32.

36. Chaouat, G. and G.A. Voisin. 1979. Regulatory T-cell subpopu­lations in pregnancy. I. Evidence for suppressive activity of the early phase of MLR. J. Immunol. 122: 1383-1388.

37. Chaouat, G., P. Monnot, M. Hoffmann and G.A. Voisin. 1982. Regu­latory T-cells in pregnancy. VI. Evidence for T-cell mediated suppression of CTL generation towards paternal alloantigens. Cell. Immunol. 68: 322-331.

38. Claësson, M.H. and С. Röpke. 1983. Colony formation by subpopu­lations of Τ lymphocytes. IV. Inhibitory effect of hydrocorti­sone on human and murine Τ cell subsets. Clin. Exp. Immunol. 54: 554-560.

39. Clark, I.A. and A.C. Allison. 1974. Babesia microti and Plasmodium berghei yoelii infections in nude mice. Nature 252: 328-329.

40. Clark, D.A., M.R. McDermott and M.R. Szewczuk. 1980. Impairment of host-versus-graft reaction in pregnant mice. II. Selective suppression of cytotoxic T-cell generation correlates with soluble suppressor activity and with successful allogeneic pregnancy. Cell. Immunol. 52: 106-118.

41. Clark, D.A. and M.R. McDermott. 1981. Active suppression of host-

vs-graft reaction in pregnant mice. III. Developmental kinetics, properties and mechanism of induction of suppressor cells during first pregnancy. J. Immunol. 127: 1267-1273.

42. Clemens, L.E., P.K. Si i ten' and D.P. Stites. 1979. Mechanism of immunosuppression of progesterone on maternal lymphocyte acti­vation during pregnancy. 0. Immunol. 122: 1978-1985.

43. Cohen, S. 1982. Progress in malaria vaccine development. Br. Med. Bull. 38: 161-165.

44. Cohen, S., I.A. McGregor and S. Carrington. 1961. Gamma-globulin and acquired immunity to human malaria. Nature 192: 733-737.

45. Cohen, S., G.A. Butcher and R.B. Crandall. 1969. Action of malarial antibody in vitro. Nature 223: 368-371.

46. Coleman, D.V., S.D. Gardner, С Mulholland, V. Fridiksdottir, A.A. Porter, R. Lilford and H. Valdimarsson. 1983. Human poly-oma-virus in pregnancy. A model for the study of defence mecha­nisms to virus reactivation. Clin. Exp. Immunol. 53: 289-296.

29

47. Contractor, S.F. and H. Davies. 1973. Effect of human chorionic somatomamma-trophin and HCG on phytohemagglutinin induced lymphocyte transformation. Nature (New Biology) 243: 284-285.

48. Cottrell, B.J., J.H.L. Playfair and B.J. de Souza. 1978. Cell-mediated immunity in mice vaccinated against malaria. Clin. Exp. Immunol. 34: 147-158.

49. Cox, F.E.G. 1968. The effect of betamethasone on acquired immuni­ty to Plasmodium vinokei in mice. Ann. Trop. Med. Parasitol. 62: 295-300.

50. Cupps, T.R. and A.S. Fauci. 1982. Corticosteroid-mediated immu-no-regulation in man. Immunol. Rev. 65: 133-155.

51. Czokalo, M. and L. Wisniewski. 1979. Inhibition of blastic trans­formation and immunoglobulin release in lymphocytes cultured in the presence of alpha-fetoprotein (AFP). Exp. Pathol. 17: 176-180.

52. Dean, J.H., M.I. Luster, G.A. Boorman, R.W. Luebke and L.D. Lauer. 1980. The effect of adult exposure to diethylstilbestrol in the mouse: alterations in tumor susceptibility and host resistance parameters. J. Reticuloendothel. Soc. 28: 571-583.

53. Desowitz, R.S. 1973. Some factors influencing the induction, main­tenance and degree of maternally transmitted protective immunity to malaria (Plasmodium berghei). Trans. R. Soc. Trop. Med. Hyg. 67: 238-244.

54. Distelhorst, C.W. and B.M. Benutto. 1981. Glucocorticoid recep­tor content of T-lymphocytes: evidence for heterogeneity. J. Immunol. 126: 1630-1634.

55. Dockrell, H.M., J.B. de Souza and J.H.L. Playfair. 1980. The role of the liver in immunity to blood-stage murine malaria. Immuno­logy 41: 421-430.

56. Dracott, B.N. and C.E.T. Smith. 1979. Hydrocortisone and the anti­body response in mice. I. Correlations between serum Cortisol levels and cell numbers in thymus, spleen, marrow and lymph nodes. Immunology 38: 429-435.

57. Duncan, M.E., J.M.H. Pearson, D.S. Ridley, R. Melsom and G. Bjune. 1982. Pregnancy and leprosy: the consequences of alterations of cell-mediated and humoral immunity during pregnancy and lactation. Int. J. Lepr. 50: 425-435.

58. Duncan, M.R., J.R. Sadlik and J.W. Hadden. 1982. Glucocorticoid modulation of lymphokine-induced macrophage proliferation. Cell. Immunol. 67: 23-36.

59. Edozien, J.C., H.M. Gilles, and I.O.K. gdeozo. 1962. Adult and cord-blood gamma-globulin and immunity to malaria in Nigerians. Lancet i : 951-955.

60. Eling, W.M.C. 1979. Plasmodium berghei: effect of antithymocyte serum on induction of immunity in the mouse. Exp. Parasitol. 47: 403-409.

30

61. El ing, W.M.C.. 1982. Immunopathological aspects in parasitic infections, pp. 141-155. In: Fortschritte der Zoologie, Band 27. Zbl. Bakt. Suppl. 12: Immune Reactions to Parasites. Gustav Fischer Verlag, Stuttgart, New York.

62. Fabris, N.,.L. Piantanelli and M. Muzzioli. 1977. Differential effect of pregnancy or gestagens on humoral and cell-mediated immunity. Clin. Exp. Immunol. 28: 306-314.

63. Facer, CA., R.S. Bray and J. Brown. 1979. Direct coombs anti­globulin reactions in Cambian children with Plasmodium falci­parum malaria. I. Incidence and class specificity. Clin. Exp. Immunol. 35: 119-127.

64. Färber, P.A. and L.A. Glasgow. 1968. Factors modifying host resis­tance to virus infection. II. Enhanced susceptibility of mice to encephalocarditis virus infection during pregnancy. Am. J. Pathol. 53: 463-481.

65. Findlay, G.M. and E.M. Howard. 1952. Cortisone and Plasmodium berghei infection in mice. Nature 169:547.

66. Finn, R., C.A.S. Hill, A.J. Govan, I.G. Ralfs, F.J. Gurney and V. Denye. 1972. Immunological responses in pregnancy and sur­vival of fetal homograft. Br. Med. J. 3: 150-152.

67. Fizet, D., J. Bousquet, Y. Piquet and F. Cabantous. 1983. Iden­tification of a factor blocking a cellular cytotoxicity re­action in pregnant serum. Clin. Exp. Immunol. 52: 648-654.

68. Freeman, R.R., A.J. Trejdosiewicz and G.A.M. Cross. 1980. Pro­tective monoclonal antibodies recognizing stage-specific mero-zoite antigens of a rodent malaria parasite. Nature 284: 366-368.

69. Freeman, R.R. and A.A. Holder. 1983. Characteristics of the pro­tective response of Balb/c mice immunized with a purified Plasmodium yoelii schizont antigen. Clin. Exp. Immunol. 54: 609-616.

70. Fuchs, T., L. Hammarström, d . E . Smith and J.Brundin. 1980. In vitro induction of murine suppressor T-cells by human chorionic gonadotropin. Acta Obstet. Gynaecol. Scand. 59: 355-359.

71. Gilles, H.M., J.B. Lawson, M. Sibelas, Α. Voller and N. Allan. 1969. Malaria, anaemia and pregnancy. Ann. Trop. Med. Parasitol. 63: 245-263.

72. Glasser, L., M.J. Hicks, R.E. Lindberg and J.F. Jones. 1981. The effect of in vivo dexamethasone on lymphocyte subpopu­lations : differential response of EA rosette-forming cells. Clin. Immunol. Immunopathol. 18: 22-31.

73. Goodfellow, C F . 1983. Maternal lymphocyte responses during normal and abnormal pregnancies, measured in vitro using composite trophoblast antigens and phytohaemagglutinin. Immunol. Rev. 75: 61-85.

31

74. Gottesman, S.R.S. and 0. Stutman. 1981. Cellular immunity during pregnancy. II. Response to Τ and В cell mitogens. Am. J. Reprod. Immunol. 1: 78-82.

75. Greenwood, B.M., A.J. Oduloju and D. Stratton. 1977. Lymphocyte changes in acute malaria. Trans. R. Soc. Trop. Med. Hyg. 71: 408-410.

76. Grove, D.I. and H.J.S. Dwakins. 1981. Effects of prednisolone on murine strongyloidiasis. Parasitology 83: 401-409.

77. Grun, J.L. and W.P. Weidanz. 1981. Immunity to Plasmodium ahabaudi adami in the B-cel1-deficient mouse. Nature (London) 290: 143-145.

78. Gusdon, J.P.Jr. 1983. The immunology of reproduction. Am. J. Re-prod. Immunol. 3: 5-6.

79. Hammarström, L., T. Fuchs and C.I.E. Smith. 1979. The immunode-pressive effect of human glucoproteins and their possible role in the non-rejection process during pregnancy. Acta Obstet. Gynecol. Scand. 58: 417-422.

80. Han, T. 1975. Human chorionic gonadotropin. Its inhibitory effect on cell-mediated immunity in vivo and in vitro. Immunology 29: 509-515.

81. Harrison, M.R. 1976. Maternal immunocompetence. II. Proliferative responses of maternal lymphocytes in vitro and inhibition by serum from pregnant rats. Scand. J. Immunol. 5: 881-885.

82. Haynes, B.F. and A.S. Fauci. 1978. The differential effect of in vivo hydrocortisone on the kinetics of subpopulations of human peripheral blood thymus-derived lymphocytes. J. Clin. Invest. 61: 703-707.

83. Herd, N. and T. Jordan. 1981. An investigation of malaria during pregnancy in Zimbabwe. Centr. Afr. J. Med. 27: 62-68.

84. Hoskin, D., D.C. Hooper and R.A. Murgita. 1983. Naturally occur­ring non-T suppressor cells in pregnant and neonatal mice: some functional and phenotypic characteristics. Am. J. Reprod. Immunol. 3: 72-77.

85. Hunter, K.W., F.D. Finkelman, G.T. Strickland, P.C. Sayles and I. Scher. 1980. Murine malaria analysis of erythrocyte sur­face-bound immunoglobulin by flow microfluorimetry. J. Immunol. 125: 169-174.

86. Ito, T. and T. Hoshino. 1962. Studies of the influences of preg­nancy and lactation on the thymus in the mouse. Z. Zellforsch. Mikros. Anat. 57: 667-678.

87. Jayawardena, A.N. 1981. Immune responses in malaria, pp. 85-136. In: J. Mansfield (ed.), Parasitic Diseases. Vol. 1, The Immu­nology. M. Dekker Inc., New York.

88. Jayawardena, A.N., G.A.T. Targett, R.L. Carter, E. Leuchars and A.J.S. Davies. 1977. The immunological response of CBA mice to Plasmodium yoelii. I. General characteristics, the effects of T-cell deprivation and reconstitution with thymus grafts. Immunol. 32: 849-859.

32

89. Jayawardena, A.N., D.B. Murphy, Ch.A. Janeway and R.K. Gershon. 1982. T-cell-mediated immunity in malaria. I. The Ly phenotype of T-cell mediating resistance to Plasmodium yoelii. J. Immunol. 129: 377-381.

90. Kasakura, S. 1973. Is Cortisol responsible for inhibition of MLC reactions by pregnancy plasma? Nature 246: 496-497.

91. Kenny, J.F. and M. Diamond. 1977. Immunological responsiveness to Escherichia coli during pregnancy. Infect. Immun. 16: 174-180.

92. Knox, A.W. 1950. Influence of pregnancy in mice on the course of infection with murine poliomyelitis virus. Proc. Soc. Exp. Biol. Med. 73: 520-528.

93. Kortmann, H.F.C.M. 1972. Malaria and pregnancy. M.D. Thesis., Amsterdam.

94. Kumar, Α., D.L. Madden and G.A. Nankervis. 1984. Humoral and cell-mediated immune responses to herpes virus antigens during pregnancy. A longitudinal study. J. Clin. Immunol. 4: 12-17.

95. Lawrence, R., J.A. Church, W. Richards and M. Borzy. 1980. Immunological mechanisms in the maintenance of pregnancy. Ann. Allergy 44: 166-173.

96. Lawson, J.B. 1967. Malaria and pregnancy, pp. 59-72. In: J.B. Lawson and D.B. Stewart (ed.), Obstetrics and Gynaecology in the Tropics. E. Arnold, English Language Book Society, London.

97. Leikin, S. 1972. Depressed maternal lymphocyte response to phyto-haemagglutinin in pregnancy. Lancet i: 43.

98. Lewis, R., N.H. Lauersen and S. Birnbaum. 1973. Malaria associ­ated with pregnancy. Obstet. Gynaecol. 42: 696-700.

99. Loose, L.D. and N.R. DiLuzio. 1976. Dose-related reticuloendo­thelial system stimulation by diethylsti1bestrol. J. Reticulo-endothel. Soc. 20: 457-460.

100. Lucia, H.L. and R.S. Nussenzweig. 1969. Plasmodium chabaudi and Plasmodium vinakei: phagocytic activity of mouse reticuloendo­thelial system. Exp. Parasitol. 25: 319-323.

101. Luft, B.J. and J.S. Remington. 1982. Effect of pregnancy on re­sistance to Listeria monocytogenesis and Toxoplasma gondii in­fections in mice. Infect. Immun. 38: 1164-1171.

102. Luster, M.I., G.A. Boorman, J.H. Dean, R.W. Luebke and L.D. Lawson. 1980. The effect of adult exposure to diethylstilbestrol in the mouse: alterations in immunological functions. J. Reticuloendo-thel. Soc. 28: 561-569.

103. Lustig, H.J., V. Nussenzweig and R.S. Nussenzweig. 1977. Erythro­cyte membrane-associated immunoglobulins during malaria in­fection of mice. J. Immunol. 119: 210-216.

104. Maes, R.F. and N. Claverie. 1977. The effect of preparations of human chorionic gonadotropin on lymphocyte stimulation and immune response. Immunology 33: 351-360.

33

105. Maroni, E.S. and M.A.B, de Sousa. 1973. The lymphoid organs du­ring pregnancy in the mouse. A comparison between a syngeneic and an allogeneic mating. Clin. Exp. Immunol. 13: 107-124.

106. McGregor, I.A. and D.A. Smith. 1952. A health, nutrition and parasitological survey in a rural village (Keneba) in West Kiang, Gambia. Trans. R. Soc. Trop. Med. Hyg. 46: 403-427.

107. McGregor, I.A., M.E. Wilson and W.Z. Billewicz. 1983. Malaria infection of the placenta in the Gambia, West Africa; its incidence and relationship to stillbirth, birthweight and placental weight. Trans. R. Soc. Trop. Med. Hyg. 77: 232-244.

108. McLean, J.M., J.G. Mosley and A.C.C. Gibbs. 1974. Changes in the thymus, spleen and lymph nodes during pregnancy and lac­tation in the rat. J. Anat. 118: 223-229.

109. Menon, R. 1972. Pregnancy and malaria. Med. J. Malays. 27: 115-119.

110. Morton, H., V. Hegh and G.J.A, Clunie. 1974. Immunosuppression detected in pregnant mice by rosette inhibition test. Nature (London) 249: 459-460.

111. Morton, H., V. Hegh and G.J.A. Clunie. 1976. Studies of the ro­sette inhibition test in pregnant mice: evidence of immuno­suppression? Proc. R. Soc. London Ser. В. 193: 413-419.

112. Murgita, R.A., E.A. Goidl, S. Kontiainen and H. Wigzell. 1977. Alpha-fetoprotein induces suppressor Τ cells in vitro. Nature (London) 267: 257-159.

113. Nair, K.V., J. Gillon and A. Ferguson. 1981. Corticosteroid treatment increases parasite numbers in murine giardiasis. Gut 22: 475-480.

114. Nolten, W.E. and P.A. Rueckert. 1981. Elevated free Cortisol index in pregnancy; possible regulatory mechanisms. Am. J. Obstet. Gynecol. 139: 492-498.

115. Oduola, A.M.J., T.W. Holbrook, R.M. Galbraith, H. Bank and S.S. Spicer. 1982. Effects of malaria {Plasmodium berghei) on the maternal-fetal relationship in mice. J. Protozool. 29: 77-81.

116. Ojo, O.A., T.I. Francis and A. Onifade. 1971. Massive liver necrosis in pregnancy. W. Afr. Med. J. 20: 339-344.

117. Osunkoya, B.O. and A.I.O. Williams. 1980. Microscopic obser­vations on the human placenta in malaria infection. Niger. Med. J. 10: 45-53.

118. Pepper, F.J. 1961. The effect of age, pregnancy and lactation on the thymus gland and lymph nodes of the mouse. J. Endo­crinol. 22: 335-348.

119. Petrini, В., J. Gentz, B. Winbladh, B. Sköldenberg and B. Westin. 1983. Perinatal transmission of tuberculosis: mening­itis in mother, disseminated disease in child. Scand. J. Infect. Dis. 15: 403-405.

34

120. Phillips, R.S. 1970. Plasmodium berghei: passive transfer of immunity by antisera and cells. Exp. Parasitol. 27: 479-495.

121. Phuc, L.H., M. Papiernik, S. Berrih and D. Duval. 1981. Thymic involution in pregnant mice. I. Characterization of the re­maining thymocyte subpopulations. Clin. Exp. Immunol. 44: 247-252.

122. Pingoud, E. 1968. Malariaplasmodien im Blute von Schwangeren und Nichtschwangeren in Abeokuta (West Nigeria). Z. Tropen-med. Parasitol. 20: 279-287.

123. Playfair, J.H.L. 1982. Immunity to malaria. Br. Med. Bull. 38: 153-159.

124. Playfair, J.H.L., J.B. de Souza, H.M. Dockrell, P.U. Agomo and J. Taverne. 1979. Cell-mediated immunity in the liver of mice vaccinated against malaria. Nature (London) 282: 731-734.

125. Purtilo, D.T., H.M. Hallgren and E.J. Yunis. 1972. Depressed maternal lymphocyte response to phytohaemagglutinin in human pregnancy. Lancet i: 769-771.

126. Quinn, T.C. and D.J. Wyler. 1979. Intravascular clearance of parasitized erythrocytes in rodent malaria. J. Clin. Invest. 63: 1187-1194.

127. Raucher, H.S., H. Jaffin and J.L. Glass. 1984. Babesiosis in pregnancy. Obstet. Gynecol. 63: 7s-9s.

128. Redmond, W.B. 1952. Influence of cortisone on natural course of malaria in the pigeon. Proc. Soc. Exp. Biol. Med. 79: 258-261.

129. Reinhardt, M.C. 1980. Effects of parasitic infections in preg­nant women, pp. 149-170. In: Perinatal Infections. Ciba Foun­dation Symposium 77. Excerpta Medica, Amsterdam.

130. Reinhardt, М . С , P. Ambroi se-Thomas, R. Cavallo-Serra, С. Meylan and R. Gautier. 1978. Malaria at delivery in Abidjan. Helv. Paediatr. Acta 33, suppl. 41: 65-84.

131. Rinehart, J.J., A.L. Sagone, S.P. Balcerzak, G.A. Ackerman and A.F. Lobuglio. 1975. Effects of corticosteroid therapy on human monocyte function. N. Engl. J. Med. 292: 236-241.

132. Roberts, D.W., R.G. Rank, W.P. Weidanz and J.F. Finerty. 1977. Prevention of recrudescent malaria in nude mice by thymic grafting or by treatment with hyperimmune serum. Infect. Immun. 16: 821-826.

133. Roberts, D.W. and W.P. Weidanz. 1978. Splenomegaly, enhanced phagocytosis and anemia are thymus dependent responses to malaria. Infect. Immun. 20: 728-731.

134. Roberts, D.W. and W.P. Weidanz. 1979. T-cell immunity to ma­laria in the B-cell deficient mouse. Am. J. Trop. Med. Hyg. 28: 1-3.

135. Rosenberg, E.B., G.T. Strickland, S.L. Yang and G.E. Wahlen. 1973. IgM antibodies to red cells and autoimmune anemia in patients with malaria. Am. J. Trop. Med. Hyg. 22: 146-152.

136. Schmidt, L.H. and W.L. Squires. 1951. The influence of corti­

sone on primate malaria. J. Exp. Med. 94: 501-520.

35

137. Serguiev, P.G. and N.A. Demina. 1957. Studies of immunity to Plasmodium berghei infection. I. The transmission of acquired immunity against P. berghei from female rats to their off­spring. Indian J. Malario!. 11: 129-138.

138. Shear, H.L.S., R.S. Nussenzweig and С Bianco. 1979. Immune phagocytosis in murine malaria. J. Exp. Med. 149: 1288-1298.

139. Singer, I. 1954. The effect of cortisone on infections with Plasmodium berghei in the white mouse. J. Infect. Dis. 94: 164.

140. Skinnider, L.F. and V. Laxdal. 1981. The effect of progesterone, oestrogens and hydrocortisone on the mitogenic response of lymphocytes to phytohaemagglutim'n in pregnant and non-preg­nant women. Br. J. Obstet. Gynaecol. 88: 1110-1114.

141. Slapsys, R. and D.A. Clark. 1983. Active suppression of host-versus-graft reaction in pregnant mice. V. Kinetics, specifi­city, and in vivo activity of non-T suppressor cells loca­lized to the genital tract of mice during first pregnancy. Am. J. Reprod. Immunol. 3: 65-71.

142. Smith, J.В., G. Nolan and W. Jubiz. 1980. The relationship be­tween unbound and total Cortisol: its usefulness in detecting CBG abnormalities. Clin. Chim. Acta 108: 435-445.

143. Spitz, A.J.W. 1959. Malaria infection of the placenta and its influence on the incidence of prematurity in eastern Nigeria. Bull. W.H.O. 21: 242-244.

144. Stimson, W.H. 1972. Transplantation - nature's success. Lancet i : 684.

145. Stimson, W.H. 1976. Studies on the immunosuppressive properties of a pregnancy-associated alpha-macroglobulin. Clin. Exp. Immunol. 25: 199-206.

146. Stites, D.P. and P.K. Siiteri. 1983. Steroids as immunosuppress­ants in pregnancy. Immunol. Rev. 75: 117-138.

147. Strang, Α., E. Lachman, S.B. Pitsoe, A. Marszalek and R.H. Philpott. 1984. Malaria in pregnancy with fatal complications. Case report. Br. J. Obstet.Gynaecol. 91: 399-403.

148. Stuiver, P.C. and Th. J.L.M. Goud. 1978. Corticosteroids and liver amoebiasis. Br. Med. J. ii: 394-395.

149. Szekeres, J., V. Csernus, S. Pejtsik, L. Emody and A.S. Pacsa. 1981. Progesterone as an immunologic blocking factor in hu­man pregnancy serum. J. Reprod. Immunol. 3: 333-339.

150. Szekeres-Bartho, J., V. Csernus, J.Hadraagy and A.S. Pacsa. 1983. Immunosuppressive effect of serum progesterone during preg­nancy depends on the progesterone binding capacity of the lymphocytes. J. Reprod. Immunol. 5: 81-88.

151. Taufa, T. 1978. Malaria and pregnancy. Papua New Guinea Med.

J. 21: 197-206. 152. Tavadia, H.B., K.A. Flemming, P.D. Hume and H.W. Simpson. 1975.

Circadian rhythmicity of human plasma Cortisol and PHA-in-duced lymphocyte transformation. Clin. Exp. Immunol. 22: 190-193.

36

153. Terry, R.J. 1956. Transmission of antimalarial immunity (Plas­modium berghei) from mother rats to their young during lac­tation. Trans. R. Soc. Trop. Med. Hyg. 50: 41-46.

154. Than, G.N., I.F. Csaba and D.G. Szabo. 1974. An antigen asso­ciated with pregnancy. Lancet ii: Б78-1579.

155. Thompson, J. and R. van Fürth. 1970. The effect of glucocorti-costeroids on the kinetics of monocytes and peritoneal macro­phages. pp. 255-264. In: R.v. Fürth (ed.). Mononuclear phago­cytes. Blackwell Scientific Publications, Oxford.

156. Thomson, A.W., J.K. Powrie and C.H.W. Home. 1979. Plasma preg­nancy -associated o^-glycoprotein concentrations in complica­tions of pregnancy and foetal abnormality. J. Reprod. Immunol. 1: 229-235.

157. Thomson, S.P., L.J. McMahon and C.A. Nugent. 1980. Endogenous Cortisol: a regulator of the number of lymphocytes in peri­pheral blood. Clin. Immunol. Immunopathol. 17: 506-514.

158. Thong, Y.H., R.W. Steele, M.M. Vincent, S.A. Henoen and J.A. Bell anti. 1973. Impaired in aitro cell-mediated immunity to rubella virus during pregnancy. N. Eng. J. Med. 289: 604-606.

159. Toi vanen, P. and Chr. Granberg. 1980. Mother/child mixed lympho­cyte reaction: is it depressed? Immunol. Today 1: 76-78.

160. Vanderbeeken, Y., M.P. Vlieghe, J. Duchateau and G. Delespesse. 1984. Suppressor T-lymphocytes in pregnancy. Am. J. Reprod. Immunol. 5: 20-24.

161. Van Dijk, H., J. Testerink and E. Noordegraaf. 1976. Stimulation of the immune response against SRBC by reduction of cortico­sterone plasma levels. Mediation by mononuclear phagocytes. Cell. Immunol. 25: 8-14.

162. Van Knapen, F. 1984. Immunodiagnosis of toxoplasmosis. M.D. Thesis, Amsterdam.

163. Van Zon, A.A.J.С. and W.M.C. Eling. 1980. Pregnancy associated recrudescence in murine malaria (Plasmodium berghei). Tropen-med. Parásito!. 31: 402-408.

164. Van Zon, A.A.J.С, W.M.C. Eling and C.C. Hermsen. 1983. Enhanced^ virulence of malaria infection in pregnant mice. IRCS. Med. Sci. 11: 1002-1003.

165. Viens, P., M. Roger and R. Dubois. 1983. Influence of pregnancy on mouse immunity to Trypanosoma musauli. Trans. R. Soc. Trop. Med. Hyg. 77: 274-275.

166. Wajner, M., S.S. Papiha and T.I. Wagstaff. 1983. Response of human peripheral blood lymphocytes in the presence of cord sera: relationship of lymphocyte transformation with number of pregnancies and levels of alpha-fetoprotein. Clin. Exp. Immunol. 52: 381-386.

167. Weidanz, W.P. 1982. Malaria and alterations in immune reactivity. Br. Med. Bull. 38: 167-172.

168. Weston, W.L., H.N. Claman and G.G. Krueger. 1973. Site of action of Cortisol in cellular immunity. J. Immunol. 110: 880-883.

37

169. Westphal, U. 1971. Steroid-Protein Interactions. Springer Verlag, Berlin, Heidelberg.

170. Wurl, O.A., J.O. Gillespie and W.R. Beisel. 1952. Treatment of malaria with cortisone. U.S. Arm. Forces Med. J. 3: 1347-1352.

171. Young, E.J. and C.I. Gomez. 1979. Enhancement of herpes virus type 2 infection in pregnant mice. Proc. Soc. Exp. Biol. Med. 160: 416-420.

172. Zuckerman, A. 1964. Autoimmunization and other types of indirect damage to host cells as factors in certain protozoan diseases. Exp. Parasitol. 15: 138-183.

173. Zuckerman, A. 1970. Dynamics of the passive transfer of pro­tection and anti pi asmodi al precipitin to their litters by mother rats hyperimmune to Plasmodium berghei. Isr. J. Med. Sci. 6: 461.

38

Chapter II

Depressed malarial inmunity in pregnant mice

Adriaan A.J,С van Zon

and

Wijnand M.C. Eling

Department of Cytology and Histology

Faculty of Medicine

University of Nijmegen

The Netherlands

Infection and Immunity (1980) 28: 630-632

39

IKFIXTION A M I IMMUNITY, May 1980, p. 630-S:i2 Vol. 28, No. 2 ΙΧ)19-9Γ)67/«)/(15.0(ί3Ο/03$02.00./0

Depressed Malarial Immunity in Pregnant Mice ADRIAAN A. J. С VAN ZON' AND WIJNAND M. С KLING

Department of Cytology and Histology, Faculty of Medicin·-*. Uniirrsity of S'ijniegen, 6.400 KH Nijmegen, The Netherlands

A proportion of mice solidly immune to the rodent malaria parasite Plasmo­dium berghci exhibited a preg:>ancv-as<wiated depressed immunity with a tran­sient or oven lethal recrudt'sceiire

Attenuated immunity and analysis of the im-munodepressive effect of pregnancy-associated substances have been the subject of many inves­tigations In human malaria a pregnancy-asso­ciated depression of immunity was inferrn/ froin an observed increase in parasite rate ant' para­site density in pregnant woinun Η a ^olocndemic area and from an increased ке іміу oí" mojaría during pregnancy {e.g.. cerebral maiuria) in areas with unstable malaria (13. 17,21;. й о т е authors reported that primigrávida? are at an especially high risk (2, 3,17, 20; H. Kurtmann, M.D. thesii, Hoyal Tropical Institute, Amsterdam, 1972), whereas a decline in tlv- prevalence and density of malaria parasites in pregnam as well as ¡n nonpregnant women with increasing age has been described (2, (). Prematurity and possibly abortion were also found to be associated with malaria during pregnancy in humans (3, 4, 17, 18, 21, 28). Comparable phenomena were ob­served in mice immunized against the rodent malaria p-.m^ite Plasmodium berghei during pregnancy. Moreover, with recrudescence as a markei for depressed immunity, this model can be used to analyze the relevance to the immune status of pregnancy-associated changes in im­mune responsiveness and levels of immunologi­cally activp. iwoteins and hormones during preg­nancy.

Approximately 6-week-old mice of several strains (Swiss outbred, СЗН/StZ, and B10LP inbred) were immunized against P. ber^hei (strain K173) by an intraperitoneal injection oí 10' parasitized erythrocytes, followed by sulfa­diazine treatment (30 mg/liter of drinking water: estimated daily consumption, 3 ml per mouse) from days 2 to 33 after infection. Two days laier mice were challenged with 10s parasitized eryth­rocytes given intraperitoneally to assess immu­nity (9). The acquired immunity was of the premunition type with low, subclinical numbers of persisting parasites. Spontaneous recrudes­cences were rare (7). A considerable proportion of such immune mice, however, suffered a recru­descence during pregnancy (Table 1). Patent infection (parasites are readily observed in a thin

blood smear) was found in approximately the same proportion of mice in all strains tested.

A parasitemia of more than 5% infected eryth­rocytes, as determined from thin blood films, was taken to indicate depressed immunity. Pat­ency was not observed before the last, i.e., third, week of pregnancy. As a prepatent period (pe­riod between infection and patency) of several days is common for P. berghei infection in mice, presumably the actual breakdown of malarial immunity occurred before the third week of pregnancy in at least a considerable proportion of the mice. In human malaria the situation is even less dear as some authors reported a prev­alence of malaria early in pregnancy (2, 26), and others reported it late in pregnancy (17, 21).

The results of Table 1 also show that, depend­ing on the strain, a varying proportion of mice recovered from a pregnancy-associated recru­descence. Recovery was never observed before parturition. These resul's seem to indicate that at least in a proportion of the mice the immu-nodepressed state is abrogated after parturition, in accordance with changes in many other im­munological phenomena observed during preg­nancy. Although the mechanism of recovery of mice suíí'ering a recrudescence during pregnancy is unki.own, a possible mechanism was discjssed in relation to a time-dependent waning of im­munity (8, 9a).

A proportion of mice did not suffer a recru­descence during pregnancy, possibly due to the clearance of parasites before the immunodepres-sive period. The clearance of parasites in an increasing proportion of mice in relation to in­creasing periods since the last challenge has been described (7). For evaluating the status of non-recrudescing mice, blood was subinoculated im­mediately after parturition. Of 28 Swiss mice without a recrudescence during pregnancy, 19 were found to harbor parasites after parturition. Such mice may either have developed better immunity or a less severe immunodepression during pregnancy.

The P. berghei mouse model also revealed malaria-associated prematurity and abortion.

40

VOL 28, 1980

The results of Table 2 indicate that 10% of the mice with a recrudescence delivered prema­turely (before day 20) Abortion was also ob­served

Though the percentage of premature deliver­ies was low (10%), 204 of the recrudescing mice died from malaria before parturition and were not included m the count

As to the prevalence of recrudescences in Pri­migravidae, the situation in the Ρ berghei mouse model is complicated Though the inci­dence of recrudescences was much lower in mul-tigravidae, a high proportion of the mice that recrudescenred during their first pregnancy died from the infection and could not be studied as multigravidae In a different approach to the problem, the possibility of a change in the im­mune status ol the mouse from the first to subsequent pregnancies was investigated A group of 24 immune Swiss mice recei\ ed curative treatment with chloroqume (0 β mg per mouse intrapentoneall) for 5 days before mating, sup­plemented with 100 mg of chloroqume per liter of drinking water for the duration of the first pregnancy) After parturition the mice were reinfected and allowed to mate again During the second pregnancy 46% (11 mice) exhibited a recrudescence, and this percentage was the same as that observed in Primigravidae (Table 1) A fundamental difference in -he immunological ca­pacity of the host between the first ι ad subse­quent pregnancies is, therefore, not indicated by these results

In older mice a decreased proportion of recru­descences was observed (Fig 1) Although this result is comparable with the situation in hu­mans (2, 4), the enhanced clearame of parasites in older mice (9a) mav have been influential

Our results show that the Ρ berghei mouae moael is an excellent experimental model for the anahsis of the mechanism of depressed malarial immunity during pregnane), exhibiting several similarities with the human rroHel Moreover, it is lbo suitable for the study of the reli i n t e of

I ABLE 1 Ftecrudetience rnd recoicry m pregnant /шее"

No of mice No of mice No of with a recru recovering

Mouse strain pregnant defence from a re mice during preg crudescence

nancy C» (<})

CdH/St7, 63 31 (49) 6(19) D10L1' 94 3« (40) 2J (61) hwiss 21J 107(46) 32(30)

" Age ol mice at mating varied from 11 to 22 weeks (te mliuediateU to 11 weeks after immunization) ApproximateK ¿5^ of Swiss mice wpre older at mating lup to 4b weeks,)

TABLE 2 Recrudescence and prematurity in Stiiss mice

% with parturition on day % Dead

No recrudescence 50 33 17 0 (n -48 )

Recrudescence (n 2 4 4 33 37 20 = 46)

mice with ret ruOescence d u n ^ pinone y —

100

50 " ·

О к- 1 1 . , . '5 20 25 30 Зь чо 45 weens

0 9 e „f г- *

Fie 1 Effect of age on the recrudescence of ma laria in Primigravidae The figure shows the results of 22 groups, each containing from J2 to 5b pregnant Suiss mice Mice aere immunized at the age of 6 weeks (9) Before the mice uere allowed to mate, immunity was (re)confirmed by the resistance to chai lenge (1& PL·) The Urne indicated in the figure reßects the age at mating

changes of immunological responses in general or of immunologically active substances to "n-munitv The potential immunodepressive activ-it\ of substances like prolactin (15) a-macro-globuhns (29 30 J l ) , a fetoproteins (6, 24r ¿5), soluble fetal antigens in the maternal circulation (1), progesteione (27), and corticoids (14, 19), which are present during pregnancy, has been established, but is not directly related to the actual immunological status of the host With recrudescence as a marker for depressed immu-mt> a direct correlation is possible The same holds true for pregnancy-associated changes in lymphocyte reactivity (5, 11, 12, 22, 23) or anti­body responses (10, 16, 24) As to changes in antibody responses during a malaria-associated pregnancy in humans, the situation is not clear The inhibition of an antiparasitic immunoglob­ulin G response has been suggested (2), but others did not observe changes in anti-parasitic antibody titers during pregnancy (13, Kortmann, M D thesis)

We lhank J Koekkoek and С Непти*п for lethmcal assillanle J Heitsman G Poelen M Faaseen and J vati Her Westeringh for biotechnual collaboration and tare of the

41

I N F E C T I M M U N

animals, and M L Weiss for reading the manuscript This work was supported by grants from World Health

Organization, Geneva, Switzerland, and the Dutch govern meni (International Cooperative Research Project for Malar­ial Immunity and Immunopathology)

LITERATURE CITED

1 Barg, M., R. С. Burton, J. A. Smith, G. Α. Lucken­bach, J Decker, and G. F. Mitchell. 197« EHectâ of placental tissue on immunological response» Clin Exp Immunol 34:44 l-44fl

2 Bray, R. S., and M. J. Anderson. 1979 Fakiparum malaria and pregnancy Trans R Soc Trop Med Hyg 73:427-4.11

3 Bruce-Chwatt, L. J. 1952 Malaria in African infants and children in Southern Nigeria Ann. Trop Med Parási­to] 46:173-200

4 Cannon, D. S. Η. 195Θ Malana and prematurity in the western region of Nigeria Br Med J 2:877-878

5 Clarke, F. M-, H. Morton, and G. J. A. Clunie. 197Я Detection and separation of two serum factors respon­sible for depression of lymphocyte activity in pregnancy Clin Ежр Immunol 32:318-32,1

6 Czokalo, M., and L. Wisniewaki. 1979 Inhibition of blast ic transformation and immunoglobulin release in lymphocytes cultured in the presence of alpha-felopro lem (AFP) Exp Pathol 17:176-180

7 Eling, W. 1978 Survival of parasites in mice immunized against Plasmodium berghet Tropenmed Parasi tol 2 :204-209

H Eling, W. 1978 Fading of malaria immunity in mice Tropenmed Parasitol 29:77-84

9 Eling, W. 1979 Plasmodium berghet effect of antithy mocyte serum on induction of immuni tv in the mouse Exp Parasitol 47:403-409

9a Eling, W. M С 1980 Plasmodium berghei premumtion. stenle immunity and loss of immunity in mice Exp Panmitol 49-89-ЭД

10 Εκοη, P. I)., and K. Duon. 1972 Immunological re sponses in pregnane) Br Med J 4:161-362

11 Fa bri β, N.. L· Piantanelli, and M. Muzzioli. 1977 Dif­ferential effect of pregnancy of gestagens on humoral and cell mediated immunity Clin Exp Immunol 28: 306-314

12 Finn, R.. С A. S. Hill, A. J. Govan, 1. G. Ralfs, F J. Gurney, and V. Denye. Immunological responses in pregnancy and survival of fetal homograft Br Med J 3:150-152

13 Gilles, H. M , J. Β Laweon, M. Sibelas, A. Voiler, and N. Allan. 1969 Malaria, anaemia and pregnancy Ann Trop Med Parasitol 83:245-263

14 Ito. T.. and T. Hoahino. 1462 Studies of the influences of pregnancy and lactation on the thvmus in the mouse

Ζ Zellforsch Mikrosk Anal 57:667-678 15 Karmalt. R. Α., I. Lauder, and D. F. Horrobin. 1974

Prolactin and the immune response Lancet 11:106-107 16 Kenny, J. F., and M. Diamond. 1977 Immunological

responsiveness to Еч( henchía coti during pregnancy Infect Immun 18:174-180

17 Lawson, J. В. 1967 Malana and pregnancy, ρ 59-72 In J В lawson and D В Stewart (ed ), Ohstetnes and gynaecology in the tropics E Arnold. English Language Book Society, I,ondon

18 Lewie, R., N. H. Laueraen, and S. Birnbaum. 1973 Malaria associated with pregnancy Obstet Gynecol 42:696-700

19 Maroni, E. S., and Μ. Α. Β. de Sousa. 1973 The lymphoid organs dunng pregnancy in the mouse A comparison between a syngeneic and an allogeneic mat­ing Clin Exp Immunol 13:107-124

20 McGregor, 1. Α., and I). A. Smith. 1952 A health, nutrition and parasitológica! survey in a rural village (Keneba) in West Kiang, Gambia Trans R Soc Trop Med Hyg 46:403-427

21 Menon, R. 1972 Pregnancy and malaria Med J Malays 27:115-119

22 Morton, H., V. Hegh, and G. J. A. Clunie. 1974 Im­munosuppression detected in pregnant mice bv rosette inhibition lest Nature (London) 249*459-^60

23 Morton, H-, V. Hegh, and G. J. A. Clume. 1976 Studies of the rosette inhibition test in pregnant mice evidence of immunosuppression9 Proc R Soc London Ser 193: 41.1-419

24 MurgîLa, R. Α. 1976 The immunosuppressive role of alpha-fetoprotein during pregnancy Scand J Immu­nol 5:1003-1014

25 Murgita, R. Α., E. A. GoidI, S. Kontialnen, and H. Wigzell. 1977 Alpha fetoprotein induces suppressor Τ cells m w/го Nature (London) 267:257-259

26 Pingoud, E. 1908 Malanaplasmodien im Blute von Schwangeren und Nu htschwangeren in Abeokuta (west Nigeria) 7, Tropenmed Parasitol 20.279-287

27 Siiten, Р. К., F. Febree, L. E. Clemens, R. J. Chang, H. GondoB, and D. Sites 1977 Progesterone and maintenance of pregnancy is progesterone nature's im-mumwupprcssant*' Ann NY Atad Sci 286.384-397

28 Spitz, A. .1. W. 1959 Malaria infection of the placenta and its influence on the mtidence of prematurity in еачіет Nigeria Bull WHO 21:242-244

29 Stimson, W H. 1976 Studies on the immunosuppressive properties of a pregnancy-associaLed alpha matroglob ulin Clin Exp Immunol 25.199-206

30 Than, G. N-, I. F. Caaba, and D. G. Szabo. 1974 An antigen assoc laied with pregnancy [.ancet и: 1578-1579

31 von Schoultz, В . T. Stigbrand, and A. Tarnvik. 1973 Inhibition of PHA-mduced lymphocyte stimulation b> ihe pregnancy zone protein FEBS Іліі 38:21-26

42

Chapter III

Pregnancy associated recrudescence

in murine malaria (Plasmodium berghei)

A.A.J,С van Zon and W.M.C. Eling

Department of Cytology and Histology

Faculty of Medioine

University of Nijmegen

The Netherlands

Tropenmedizin und Parasitologie (1980) 31: 402-408

43

Tropenmcd. Parasit 31 (1980) 402-408

Pregnancy Associated Recrudescence in Murine Malaria (Plasmodium berghei)*

A.AJ.C. van Zon, W.M.C. Eling Department of Cytology and Histology, University of Nijmegen, The Netherlands

Summary

Depending on the strain, a variable proportion of mice solidly immune to the rodent malaria parasite Plasmodium berghei developed a recrudescence dur­ing pregnancy that was either transient or lethal. Recrudescence was not observed in all mice, and the rate was lower in gravida II as compared to gravida I mice On the other hand a proportion of the mice that did not develop recrudescence exhibited a preg­nancy associated clearance of persisting parasites in immune mice (premunition-sterilc immunity), being more pronounced in gravida II than gravida I mice Development of the mechanism of enhanced clear­ance is apparently parasite dependent Enhanced clearance was manifest until day 4 of pregnancy Pregnancy associated immunodepression was observed most strongly between day 4 and 16 of pregnancy, when the highest rates of recrudescence and associ­ated mortality were found Immunodepression ap­parently disappears at the end of pregnancy or short­ly after parturition

Rekrudeszenz (Wiederaufflammen) der Màuse-Malaiia {Plasmodium berghei) wahrend der Trachtigkeit

Abhangig vom Mausestamm verliert wahrend der Trachtigkeit speri ode cine unterschiedlich große An­zahl der Tiere eine früher erworbene Immunitat gegen Plasmodium berghei Das Wiederaufflammen der Ma­laria ist entweder nur vorübergehend oder entwickelt sich zu einer todlich verlaufenden Infektion Nicht alle schwangeren Mause erleiden jedoch cine Rekru­deszenz, wobei der Prozentsatz in Zweit-Graviden-Tieren niedriger ist als m Erst-Graviden-Mausen. Andererseits fallt auf, daß ein Teil derjenigen Mause, die wahrend der Trachtigkeit keinen Ruckfall erlei­den, die sonst fur langere Zeit überlebenden Parasiten eliminieren (Premunitat wird zur sterilen Immunitat) Dieses Phänomen ist in Zweit-Graviden-Mausen auf­fälliger als in Erst-Graviden-Mausen.

Der Mechanismus, der den Wirt befähigt, überlebende Parasiten besser zu eliminieren, wird anscheinend von den Parasiten selbst induziert, und wirkt sich beson­ders wahrend der ersten 4 Tage der Trachtigkeit aus. Andererseits ist die Immunitat zwischen dem 4 und 16 Tag der Trachtigkeit am deutlichsten abgeschwächt, weil in dieser Zeit die höchsten Rekrudeszenz- und Lüthalitatsraten beobachtet werden Die Unterdruk-kung von Immunreaktionen endet vermutlich bereits gegen Ende der Schwangerschaft oder jedenfalls kurz nach der Geburl

Introduction

The increased prevalence and morbidity of human malaria during pregnancy and especial­ly in gravida I is well documented (Bruce-Chwatt 1952, Lawson 1967, Gilles et al 1969, Kortman 1972, Bray and Anderson 1979) In humans acquired malarial immunity is of the piemunition type (Sergent 1963) The period

*This investigation received financial support from the World Health Organization, Geneva, Switzerland, and the Dutch Government through the Internation­al Cooperative Research Project for Malarial Immuni­ty and Immunopathology

of persistence of parasites, however, varies greatly (Brown 1976) and may account for ob­served differences in prevalence and morbidity of malaria between the dry and wet season (Kortman 1972, Bray and Anderson 1979). Analysis of the role of the parasite as to pres­ence. absence or clearance before or during pregnancy, in the mechanism of pregnancy as­sociated malaria is difficult to assess in humans.

Recrudescence of malaria during pregnancy was recently observed in a Plasmodium berghei — mouse model (van Zon and Eling 1980). Im­munity to malaria in mice is also of the pre-

0303-4208/80 1600-0402 S 03.00 ) 19B0 Georg Thieme Verleg, Stuttgart · New York

44

Recrudescent Malaria in Pregnant Mice

munition type (Eling 1978a, 1980a) and the presence of parasites can be demonstrated by subinoculation. The experiments described in this paper analyse parasite persistence during pregnancy in relation to malaria recrudescence in mice.

Materials and Methods

Animals Four to seven week old random-bred Swiss, and inbred СЗН/StZ and Balb/c mice were obtained from the animal facilities of the University of Nijme­gen, and inbred B10LP mice from T.N.O Zeist, The Netherlands Mice were kept m plastic cages, and fed a standard diet (RMH-Hope Farms, The Netherlands) and drinking water ad libitum

Parasite Plasmodium berhei. stram K173, was pas­saged weekly m the different strams of mice by in­traperitoneal injection of 10s parasitized erythro­cytes (PE) Characteristics of the primary infection, which was always lethal in unprotected mice, have been described elsewhere (Eling et al 1977)

Immunization Swiss, СЗН/StZ, B10LP and Balb/c mice were immunized to Ρ berghei by sulfadiazine treatment (30 mg/1 of drinking water), starting 2 days after injection of 107 parasitized erythrocytes, as described previously (Eling 1979) In addition I group of Balb/c mice was immunized by chloroqume therapy (300 mg/l of drinking water) starting 14 days after infection with 10s parasitized erythro­cytes (Poels et al. 1977) Immunity was assessed by challenge infection In cases of prolonged periods between immunization and pregnancy, experimental mice were rechallenged shortly before mating Rein­fection of pregnancy mice was carried out intraven­ously.

Pregnant mice Animals (12-20 week old) were housed in a separate unit. Two to 3 females were placed with 1 male for 4 to 5 days, and examined for the presence of a vaginal plug every morning After this period non-mated females were removed, and 3 days later the mated females were separated from the males and rehoused in groups The day of finding the vaginal plug was day one of pregnancy. In another mating schedule, 2 to 3 females were housed with 1 male contmuously. Pregnancy was de­termined by repeated palpation starting 14 days af­ter mating. Newborn mice were removed after par-tuntion

ТаЫ 1 Recrudescence in Primigravidae with persistir

strain pregnant positive isodiagnosis

Swiss 137 7 7 % ( 1 0 6 / 1 3 7 l

B10LP 27 8 2 % ( 2 2 / 2 7 )

Balb/c 57 65% ( 3 7 /57)

Recrudescence A parasitemia of S % or more in a previously solidly immune animal was considered a recrudescence Parasitemia was determined in May-Gmnwald-Giemsa stained thm smears prepared 3 times a week either until 4 days after parturition or, m mice with a recrudescence, until death or recovery

Radical cure Persisting parasites in immune mice were eliminated by 5 daily injections of chloroqume (Nivaquine®, 0 8 mg/mouse ι ρ ) supplemented with 100 mg/1 chloroqume in the drmking water for 7 days.

Isodiagnosis Persistence of parasites was assayed by subinoculation (ι ρ ) of 0.05 ml tail blood in 0.2 ml salme + heparin (S U/mi) into a clean mouse, which was exammed for patency 2 weeks later In all groups m which persistence of parasites during pregnancy was determined by isodiagnosis, subinoculation was performed either during the penod of day 14-18 or immediately after delivery

Results

Stram specificities m gravida I mice

Previous results showed that in the majority

of immune Swiss mice parasites survived for a

considerable period after challenge (Eling

1978a, 1980a) Thus, reinfection wjthm 2

weeks before mating was considered satisfac­

tory to assure presence of parasites, and re­

crudescence in case of pregnancy associated

immunosuppression. However, a variable and

strain specific proportion of gravida 1 mice ex­

hibited persistence of parasites (positive iso­

diagnosis) during pregnancy, thereby having

the opportunity to recrudescence (Table 1)

In view of results from gravida II mice (see

below, Table 3) it is interesting to note that

23 % of Primigravidae Swiss mice had cleared

their parasites

The percentage of recrudescence, and even

more of associated mortality in mice with per­

sisting parasites varied strongly in different

strains (Table 1) Moreover, these results also

show that mice with persisting parasites do

not necessarily recrudescence

parasites

recrudescence lethal recrudescence

6 7 % ( 7 1 / 1 0 6 ) 80%

55% ( 1 2 /22) 17%

35% ( 1 3 /37) 23%

A.A S С. van Zon, W W С Eüng

Table 2 Recrudescence in gravida II mice

stram

B10LP1

СЗН/StZ1

Swiss1

SWISS2

Swiss3

Swiss2+3

number of mice

45 21 64 42 19 61

recrudescence

3( 7%) 0

12(19%) 13(31%)

7 (37%) 20 (33%)

lethal re­crudescence

33%

75% 95% 43% 70%

'Persistence of parasites was not considered in these groups

2Mice were challenged between 1st and 2nd preg nancy,

эМісе with positive isodiagnosis between 1st and 2nd pregnancy.

Recrudescence m gravida II mice

Quantitative analysis of recrudescence in mul-tigravidae is complicated by several factors, such as lethal recrudescence during the First pregnancy as well as pregnancy associated en­hanced clearance of parasites (see above)

Observations on recrudescence in gravida II mice were therefore differentiated with re­gard to measures taken to promote presence of parasites during pregnancy (Table 2) Since neither of the measures taken in Swiss-2 and Swiss-3 mice assured presence of parasites at the relevant, immunodepressive phase of preg­nancy, and the percentage of recrudescence in these groups was very similar, these results were also summarized.

The results of B10LP, СЗН/StZ and Swiss-1 mice suggest a low incidence of recrudescence in gravida II mice, whereas associated mortali­ty was close to that observed in Primigravidae (compare Table 1). Measures taken to pro­mote presence of parasites during the second

pregnancy resulted in a markedly increased re­crudescence rate (Swiss 2+3 mice), which was considerably lower, however, than in Primigra­vidae (compare Table 1). but with an equal associated mortality rate. The results of Swiss-3 mice indicate that gravida II mice may develop a recrudescence, which did not occur dunng the first pregnancy, despite an apparent pres­ence of parasites.

So far pnmigravid mice with a lethal recrudes­cence were lost for further observation. Wheth­er such mice would resist recrudescence during their second pregnancy when the infection dur­ing the first was cured, was investigated in the following experiment. In the first group para­sitemia in gravida I mice with a recrudescence was suppressed by treatment with sulfadiazine (30 mg/l drinking water) After parturition sulfadiazine treatment was stopped, the mice were reinfected (10 5 PE), allowed to mate, and observed for recrudescence during their second pregnancy. Additionally, subinoculation was performed on these mice during their sec­ond pregnancy to monitor the presence of parasites.

In the second group, mice were treated with sulfadiazine during the entire period of their first pregnancy After parturition sulfadiazine was replaced by normal drinking water, and subinoculation was performed for the selection of mice with persisting parasites Then the mice were again allowed to mate, and ob­served for recrudescence during their second pregnancy Only mice with persisting parasites during their first pregnancy were considered The results of this experiment are given in Table 3

A considerable proportion (17/26 = 65%) of mice from group I cleared the challenge given

Table 3 Recrudescence in gravida II mice in relation to recrudescence during first pregnancy

grou

1

2

ρ sulfa treatment

during recrudescence of first pregnancy

throughout first pregnancy

number of mice

26

8

positive isodi (gravida III

9/26

not done

agnosis recrudescence in gravida

5 ( 1 9 % )

5 ( 6 3 % )

II lethal recrudescence

5

5

46

Recrudescent Malaiia m Pregnant Mice

between the first and second pregnancy quick­ly, which can be compared to a value of 23% observed during first pregnancy (see above). Moreover, 4 out of 9 mice did not recrudes­cence despite presence of parasites Taken to­gether, only 19% of the mice developed an infection during their second pregnancy, which is in contrast to 63% of mice in the second group, which in tum is close to the percent­age observed dunng a first pregnancy (com­pare Table 1). The results also show that in the second group clearance of parasites asso­ciated with the second pregnancy occurred only about half as frequently as in the first group.

Recrudescence in relation to challenge during pregnancy

Previous section described that a proportion of mice cleared persisting parasites during pregnancy, which may prevent recrudescence by the simple process of eliminating parasites before the immunodepressive phase associated with pregnancy. Therefore, the effect of rein­fection during pregnancy was determined. It had previously been shown that mice retain their immunity for a time after radical cure (Eling 1978b). Immune mice were radically cured with chloroquine (materials and meth­ods) and allowed to mate On different days during pregnancy separate groups were rein­fected (10 s P.E.) and the percentage of mice with patent parasitemias and associated mor­tality determined (Figure 1). To relate the ef­fect of the day of reinfection to pregnancy the results were plotted for that day. The patency rate was distinctly higher when rein­fection was carried out between day 4 and 14 of pregnancy, being around 70%, as compared to approximately 45 % before and after this period. Patency after reinfection on day 16 or thereafter became manifest only after par-tunlion (for comparison It takes 4 to 5 days for a primary infection [10 s P.E.] to develop a parasitemia of S % or more).

The day of reinfection appeared also to exert considerable influence on the associated mor­tality. The low mortality rate when mice were reinfected on day 1 of pregnancy is remark-

RecrudMCffnce in gravida I Swiss mice —

parturition

nuiTiber of tniçt Э79ЮС10Ы 1Э ЭО lb 45 18 14 13 19 I

1 1 ' I ' I ' I 1 ' I ' I 1 1 ' . • ι 1 О 2 4 6 θ 10 12 14 16 18 20

Doy ot challenge infection dunng pnpgnancy

Fig. 1 Recrudescflncfi m Primigravidae Swiss mice in relation to the day of reinfection Immune mice were radically cured with chloroquine and allowed to mate At different days during pregnancy sepa rate groups were reinfected (10 s Ρ E.)r and the per­centage of mice with recrudescence and associated mortality of each group were plotted for the day of reinfection The number m the figure refer to the number of mica in that group о percentage of pregnant mice with recrudescence • mortality of recrudescent mice

able The mortality rate is higher in groups re­infected on day 2 and 4, but still lower than that observed in groups reinfected shortly be­fore pregnancy (Table 1) The mortality rate varies greatly when mice were reinfected in the period from day 5 till immediately after parturition

Discussion

Pregnancy associated recrudescence of malaria has been observed in humans (Lawson 1967, Gilles et al. 1969, Kortman 1972, Bray and Anderson 1979) as well as in mice (van Zon and Eling 1980, and Table 1).

In previous experiments (van Zon and Eling 1980) the observed long term persistence of parasites in immune mice (Eling 1978a, 1980a) led to the assumption of presence of parasites dunng pregnancy when a challenge was given within 2 weeks before maling. Subinoculation experiments, however, revealed a total clear­ance of parasites in a considerable proportion of mice (Table 1). Therefore, m these expen-

47

A A J С van Zon, W W С bling

ments (Table 1) the rate of recrudescence in mice with a proven persistence ol parasites (positive isodiagnosis) was higher than pre­viously reported (van Zon and Eling 1980)

The proportion of mice that cleared parasites before the end of their first pregnancy, ι e within a period of maximally 5 weeks after challenge was high compared to results in non­pregnant mice as described previously (Eling 1978a, 1980a) Since this result in immune non-pregnant mice has been observed repeated­ly, direct matched controls were not included in the experiments described in Table 1 The clearance rate was still higher in gravida II mice (Table 3, group 1) Together these re suits indicate a pregnancy associated increased clearance rate of persisting parasites in immune mice Enhanced clearance may be related to increased phagocytosis, which has been ob­served frequently in relation to pregnancy (Mitchell et al 1970, Douglas and Grogan 1970, Graham and Saba 1973) This pregnan­cy associated increased phagocytosis was, however unspecific, and possibly concerned different phases of pregnancy (compare re­sults in Figure 1)

Not all mice with persisting parasites recru deseed (Table 1) This might indicate either superior immunity, or a less severe pregnancy associated immunodepression, leavmg unex plained, however, why some of these mice de­veloped a recrudescence during their second pregnancy (Table 2)

The recrudescence associated mortality dif­fered widely in the strains tested (Table 1), which may be the result of several cooperative factors 1 immunodepression allowing recru­descence is particularly important during preg­nancy and disappears thereafter (non-lactating mothers), 2. recrudescence is not observed un­til the last week of pregnancy, 3 mice may survive a primary infection substantially lon­ger than 1 week Thus, the low recrudescence associated mortality in Balb/c mice (Table 1) may depend on survival until after parturition (mean survival time of mice with a primary mfection was 23.5 ± 5 3 days (Eling et al 1977), when the state of immunodepression disappeared, and suppression and control of the ongoing infection by a recovering immuni­

ty could take place in many of the mice The explanation for the low mortality rate in B10LP mice is even more complicated, because infected non pregnant B10LP mice did not ex hibited a comparative long survival (Eling et al 1977), whereas this was observed in immuno-depressed (athymic, splenectomi/ed) animals (bling et al 1977, Einig 1980b)

The results of Table 2, and especially of Swiss mice suggest a decreased incidence of recrudes­cence in gravida II in relation to gravida I mice, even when additional measures were taken to assure presence of parasites during the second pregnancy (Swiss groups 2 and 3) These experiments arc complicated, however, by mortality from recrudescence in Primigra­vidae, which implies that only a selection of mice was observed, and especially those able to prevent recrudescence during their first preg nancy On the other hand results of Table 3 show that clinical cure of a recrudescence in gravida I leads to a low incidence of recrudes cence in a subsequent pregnancy in such mice (group 1, Table 3) Together these results sug­gest a lower recrudescence rate m gravida II than in gravida I mice

The increased resistance to recrudescence ap­parently depends on the presence of parasites during the first pregnancy, since a normal re crudescence rate was observed in gravida II mice when parasites were absent during the first pregnancy, due to radical cure by chloro-qume treatment (van Zon and Cling 1980) Presence of parasites as such, however, is not sufficient as sulfadiazine treated mice (Table 3, group 2) from which the parasites were not eradicated during their first pregnancy ex hibited a recrudescence rate during their sec ond pregnancy comparable to that observed in Primigravidae The signal for induction of an increased resistance, therefore, may have to contain a minimal number, or metdbohcally active parasites, or particular parasitic stages A decreased recrudescence rate in pregnant mice having been cured from a recrudescence during a previous pregnancy (Table 3, group 1) strongly supports this notion

A decreased recrudescence rate in multigravidae has also been reported for human malaria

48

Recrudescent Malana in Pregnant Mice

(Bruce-Chwatt 1952, McGregor and Smith 1952, Cannon 1958, Lawson 1967, Bray and Anderson 1979) Analysis of the problem in human malaria is complicated by endemicity and transmission frequency explaining an in­creased prevalence of pregnancy associated malaria during the wet season (Bray and An­derson 1979) Cannon (1958) explamed the increased resistance in multigravidae by an age-related improvement of immunity An age-related decrease in prevalence of pregnancy associated malaria was also observed in pnmi-gravid mice (van Zon and Eling 1980) suggest­ing independence of parity An age-related ef­fect was observed in immune mice with re­gard to decreasing periods of parasite persist­ence after challenge (Eling 1980a), reflecting increased clearance capacity for parasites

An increased clearance rate, in turn, is associ ated with pregnancy, being more pronounced in multigravidae, as described above There­fore, a lower recrudescence rate m multigra vidae may depend on better immunity, being reflected by an increased clearance rate prob­ably resulting from an increased clearance capacity This improved reaction of the host is apparently induced by the parasite itself On the other hand multiple infections (boos­ters) ot immune mice did not shorten the persistence period of the parasites (Eling 1978a), suggesting that qualitative rather than quantitative changes in the immunological parasite-host confrontation are responsible for pregnancy associated and age-related increased clearance

The recrudescence rate in radically cured mice. but reinfected on day 1 to 3 of pregnancy (Figure 1) was comparable to rates observed in groups challenged before pregnancy, disre­garding persistence of parasites (van Zon and Eling 1980) A higher rate was observed in groups reinfected between day 4 and 14 being comparable to results observed in groups chai lenged before pregnancy and with persistence of parasites throughout (Table 1, Swiss mice)

If the lower recrudescence rate after reinfec­tion in the first 3 days is to be explained by enhanced clearance, the qualitative change oc­curs early after fertilization The higher rate

when reinfection was performed between day 4 and 14, may mark the manifestation of the immunodepressive phase, being in agreement with a decreased rate when reinfection was performed after 14 days of pregnancy (Figure 1) When enhanced clearance operates early during pregnancy it may constitute a protec live reaction aiming at elimination of the para­site before immunodepression evolves The results do not indicate whether immuno­depression involved blocking of enhanced clearance or that both process are independ­ent and chronological

Since it takes approximately 5 days before an infection with 105 parasites has reached a lev­el of 5% infected cells, it may well be that mice challenged late in pregnancy started de­velopment of a patent infection because of immunosuppression, but infection was con trolled and suppressed before the recrudescence level was reached because immunodepression disappeared shortly before or after delivery The abruptness of the change in the recrudes­cence rate between day 4 and 5, and day 14 and 16 (Figure 1) is remarkable

No explanation is available for the changes ob­served in pregnancy associated mortality rates (Figure 1) When return to normal immuno logical function at the end or after pregnancy could explain the decrease of the recrudescence rate when reinfection was performed on day 16 or later a reduced associated mortality rate could be expected as well, but is only observed in the group reinfected immediately after par tuntion

Acknowledgement

We thank J Smeekend Koekkoek and С Hermsen for technical assistance and J Reitsma, G Poelen, M Paassen and J ν d Westenngh for biotechnical col­laboration and care of the animals, and M L Weiss for reading the manuscript

Literature

Bray, R S, M J Anderson Falciparum malaria and pregnancy Trans R Soc Trop Med Hyg 73 (1979) 427-431

Brown, Κ N Resistence to malaria In Immunology of parasitic infections Eds S Cohen and £ H Sadun. Blackwell, Oxford (1976) 268-295

49

A.A.J.C van Zon, W.W С Elmg

Bruce-Chwatt, L.J. Malaria in African infants and children in southern Nigeria. Ann. Trop Med. Parasitol. 46 (1952) 173-200

Cannon, D.S.H.. Malaria and prematurity m the western region of Nigeria. Br. Med J. 2 (1958) 877 878

Douglas, B.H., J.B. Grogan Effect of pregnancy and hypertension on reticuloendothelial activity. Am. J. Obstet. Gynecol. 107 (1970) 44-47

Eling, W. Survival of parasites in mice immunized against Plasmodium berghei. Tropenmed. Parasit. 29 (1978a) 204 209

Eling, W. Malana immunity and premunition in a Plasmodium berghei - mouse model. Isr. J. Med. Sci. 14 (1978b) 542-553

Eling, W. Plasmodium berghei Effect of antithymo-cyte serum on induction of immunity m the mouse. Exp. Parasitol 47 (1979) 403-409

Eling, W. Phsmodium berghei' Premunition, sterile immunity, and loss of immunity in mice. Exp. Parasitol. 49 (1980a) 89-96

Elmg, M>. Strain specificities in an experimental malaria mouse model. In. Animal quality and models m biomedical research. Eds. A. Spiegel and O. Muhlbock. Gustav Fischer Verlag (1980b) in press

Eling, W., A. van Zon, С Jerusalem The course of a Plasmodium berghei infection in six different mouse strains. Z. Parasitenkd. 54 (1977) 29-45

Gilles, НЖ, J.B. Lawson, M. Sibelas, A. Voller, N. Allan Malaria, anaemia and pregnancy Ann. Trop. Med. Parasitol. 63 (1969) 245-263

A.A.J С van Zon, W.M.C. Eling, De; Nijmegen, Geert Grooteplem Ν 21,

50

Graham, C.W., T.M. Saba Opsonin levels in reticulo­endothelial regulation during and following preg­nancy. J. Reticuloendothel. Soc. 14 (1973) 121 -131

Kortmann, H.F. Malana and pregnancy. Thesis. Uni­versity of Amsterdam (1972)

Lawson, J В Malaria and Pregnancy. In. Obstetncs and Gynaecology in the Tropics. Eds. J.B. Lawson and D.B. Steward E. Arnold, London (1967) 59 72

McGregor, i.A., ОЛ. Smith. A health, nutrition and parasitológica! survey in a rural village (Keneba) in West Kiang, Gambia. Trans. R. Soc. Trop. Med. Hyg. 46 (1952) 403 427

Mitchell, GW..AA. Jacobs, V. Haddad, B.B. Paul, R.R. Strauss, A.J. Sbarra The role of the phago­cyte m host-parasite mteractions. XXV. Metabolic and bactericidal activities of leucocytes from preg­nant women. Am. J. Obstet. Gynecol. 108 (1970) 805-813

Poels, I . C , CC. van Niekerk, МЛ M. Franken Plas­modium berghei' Selective release of "protective" antigens. Exp. Parasitol. 42 (1977) 182-193

Sergent, E. Latent mfection and premunition. Some definitions of microbiology and immunology. In: Immunity to Protozoa. Eds. P.C.C. Garnham, A.E. Pierce and I. Roitt. Blackwell, Oxford (1963) 39-47

Zon, A.A.J.C. van, W.M C. Eling' Depressed malarial immunity in pregnant mice. Infection and Im­munity (1980) in press

ent of Cytology and Histology, University of IIB Nijmegen, The Netherlands

Chapter IV

Pregnancy induced improvement of malaria immunity

A.A.J.C. van Zon, W.M.C, Eling

and

CC. Hermsen

Department of Cytology and Histology

Faculty of Medicine

University of Nijmegen

The Netherlands

Submitted for publication

51

IV Pregnancy induced improvement of malaria immunity

SUMMARY

A considerable proportion of mice lose acquired immunity to Plas-

mod-Lwn Ъе дТаеъ during the first pregnancy. Immune parous mice, however,

have a better immune status than virgin mice, the risk of loss of im­

munity during a subsequent pregnancy is greatly reduced, the capacity

to clear parasites is enhanced, and the maintenance of immunity is

less dependent on certain splenic functions. The establishment of im­

proved immunity is dependent on the presence of proliferating para­

sites during the second half of pregnancy when immunosuppression re­

sults in recrudescence.

Immune reactivity is also improved after a (chemotherapeutically

controlled) recrudescent infection provoked by immunosuppressive

treatment of immune mice with corticoids or anti-T-cell serum. This

mimics the situation encountered during pregnancy. Hence, improved

immunity after pregnancy is a consequence of a reconfrontation of a

suppressed and/or convalescent immune system with proliferating para­

sites.

INTRODUCTION

Malaria presents a serious complication in pregnant women in

endemic areas. Loss of established acquired immunity has repeated­

ly been observed during pregnancy (Gilles, Lawson, Sibelas, Voller

& Allan, 1969; Taufa, 1978; Bray & Anderson, 1979; Campbell,

Martinez & Collins, 1980), and malaria infections are more severe

in pregnant women in epidemic areas as well as in those with low

endemicity (Menon, 1972). Loss of immunity as referred from an

increased incidence, higher parasite densities in peripheral blood

(Reinhardt, Ambroi se-Thomas, Cavallo-Serra, Meylan & Gautier, 1978;

Bray & Anderson, 1979) and placenta is more frequently observed

in Primigravidae as compared to multigravidae (Cannon, 1958;

52

McGregor, Wilson & Billewicz, 1983).

Recrudescent infections specifically occurring during preg­

nancy, and a higher frequency in Primigravidae as compared to multi-

gravidae was also observed in a murine Plasmodium berghei model

(Van Zon & Eling, 1980a, b). Moreover, the majority of mice with

a recrudescent infection during their first pregnancy did not ex­

hibit loss of immunity during a subsequent pregnancy (Van Zon &

Eling, 1980b). The question rises whether a lower recrudescence

rate in multigravidae is the consequence of a better immunity, how

this is accomplished, and what is the role of the parasite as anti­

genic signal for development of this better immunity. It should be

noted that loss of immunity during pregnancy cannot be prevented by

challenges given before (unpublished observations). Thus, challenges

in an immune non pregnant animal do not act as boosters that rein­

force immunity, either with regard to the pregnancy problem or

with regard to fading of immunity (Eling, 1978).

Loss of malaria immunity during human as well as animal preg­

nancy is considered to depend on a pregnancy associated depression

of part of the immune system (Bray & Anderson, 1979; Brabin, 1983).

In the P. berghei - mouse model it is related to the immunosuppres­

sive effect of a pregnancy associated increase in the plasma concen­

tration of corticosterone (Van Zon, Eling, Hermsen & Koekkoek, 1982).

This paper describes experiments in the P. berghei - mouse

model in order to show the importance of the presence of the proli­

ferating parasite during the first pregnancy for the generation of

a better immunity. Pregnancy associated immunosuppression was imi­

tated in non pregnant immune mice by temporary dexamethasone or

anti-T-cell serum treatment. In these animals, too, development of

better immunity was observed in relation to the presence of the

parasite during immunosuppression.

MATERIALS AND METHODS

Mice: Outbred Swiss mice were obtained from the animal facili-

53

ties of the University of Nijmegen, and inbred B10LP mice from TNO,

Zeist, The Netherlands. Swiss mice were used in all but one experi­

ment (table II, groups 1 and 2). Mice were kept under SPF condi­

tions in plastic cages and received water and food ad Libitum.

Parasite: Plasmodium berghei strain K173 was used in all ex­

periments. The strain was maintained by weekly intraperitoneal sub-

inoculation of infected blood into normal mice. Primary infections

are always lethal in mice. For primary infection or challenge І.Ю^

parasitized erythrocytes (PE) were injected intravenously in preg­

nant mice and intraperitoneally in non pregnant animals. Character­

istics of the primary infection have been described previously

(Eling, Van Zon & Jerusalem, 1977).

Immune mice: Mice were immunized as described previously

(Eling & Jerusalem, 1977). In short, mice were inoculated intraperi­

toneally with 1.107 parasitized erythrocytes (PE). Two days later

sulfadiazine (30 ng/L) was added to the drinking water. On day 33

after inoculation sulfadiazine treatment was stopped and the mice

were challenged 2 days later with 1.10^ PE (i.p.) to assess immuni­

ty. Only mice with established immunity were used. Immunity isof

the premunition type, which implies that low, subpatent numbers

of parasites may survive in the immune host for considerable periods

of time (Eling, 1980a). Immune mice developed neither patency after

challenge inoculations nor spontaneous recrudescences.

Recrudescence: Loss of immunity during pregnancy or during

immunosuppressive treatment was determined from parasite counts in

thin bloodsmears of tailblood stained with May-Griinwald Giemsa.

Mice with a parasitaemia of 5% or more were considered to have a

recrudescence. Recrudescent infections during pregnancy exhibit

a rapidly increasing parasitaemia which is lethal in approximately

80% of Swiss mice when left untreated (Van Zon & Eling, 1980b).

Immune pregnant mice: Two to three immune female mice were

housed with one male and checked for a vaginal plug on the next

four days. Mated females were removed from the male 3 days later.

54

The day of the vaginal plug was day 1 of pregnancy. Pregnancy was

either confirmed at autopsy or by delivery of young.

Chemotherapy: Sulfadiazine treatment was given to cure an es­

tablished (recrudescent) infection (30 mg/L drinking water), or to

prevent development of a recrudescence (2.5 or 5 mg/L) during immuno­

suppression (pregnancy dependent or corticoid induced). Sulfadia­

zine treatment is usually not radically curative.

Chloroquine treatment (5 daily intraperitoneal injections of

0.8 mg chloroquine sulphate (Nivaquine(R), Specia, Paris) supple­

mented with 100 mg chloroquine sulphate/L drinking water for 7

days) was given to achieve radical cure. After this treatment ani­

mals were free of parasites as determined by subinoculation of

blood into normal mice.

Clearance test: Immune mice were challenged with 1.10' PE

(i.p.), and 2 weeks later survival of parasites was determined by

subinoculation of tailblood into normal mice. 0.05 ml of tailblood

was diluted with medium 199 (Flow Laboratories) supplemented with

10% fetal calf serum (Flow Laboratories) and heparin (5 lU/ml).

Two control mice received 0.2 ml of this suspension, and were check­

ed for development of parasitaemia over a period of 2 weeks.

Immunosuppressive treatment: Dexamethasone (20 mg/L) was added

to the drinking water. Immune mice were challenged (І.Ю^ PE), and

received dexamethasone either until they recrudesced or for a max­

imum period of 5 weeks. Mice with a recrudescent infection were sub­

sequently cured by sulfadiazine treatment.

Treatment with rabbit-anti-mouse-T-cell serum (ATS), prepared

as described previously (Eling, 1979),consisted of two intraperi­

toneal injections of 0.3 ml of inactivated ATS 2 days apart. Devel­

opment of a recrudescence after ATS treatment was monitored during

2 weeks.

Splenectomy: Immune mice were splenectomized under ether anaes­

thesia. A lateral incision was made, the spleen exposed, the splenic

pedicles ligated, and the spleen removed. The body wall was sutured,

55

and the skin closed with wound clamps.

Statistical evaluation: Significance of differences between groups

of mice was assessed by the X^-test for the comparison of 2 proportions.

RESULTS

Improved malaria immunity in gravida II mice

Previous experiments showed that the proportion of immune mice

losing their immunity during pregnancy is considerably lower in multi-

gravid mice as compared to Primigravidae (Van Zon & Eling, 1930b).

The question is whether a pregnancy as such or the presence of the

parasite (antigen) during pregnancy is important for the reinforce­

ment of immunity. To analyse the effect of parity the recrudescence

rate in Primigravidae was compared with that of gravida II mice that

were radically cured before their first pregnancy, and reinfected be­

fore their second. In a summary of groups of immune mice that were

allowed to enter their first pregnancy untreated, a recrudescence

rate of 53% (45/85) was observed. Recrudescent infections exhibited

rapidly increasing parasitaemias and were frequently lethal indicating

a loss of preexisting immunity. In an other group the immune mice were

radically cured by chloroquine treatment before mating to prevent anti­

genic stimulation during first pregnancy. After the first pregnancy

the animals were challenged (1.105 PE/mouse; this challenge does not

provoke a patent infection, but re-establishes the state of pre-

munition) and allowed to mate for their second pregnancy. These mice

exhibited a recrudescence rate of 46% (11/24), which is close to that

observed in the group of untreated Primigravidae.

These results indicate that the recrudescence rate is not de­

pendent on physiological differences between the first and second

pregnancy. On the other hand these results should be compared to

those showing a significantly reduced recrudescence rate in gravida

II mice when they had experienced a recrudescence during their first

pregnancy (Van Zon & Eling, 1980b). Together, these results suggest

56

an important role of the parasite in generation of a better immunity.

Enhanced clearance of parasites in immune parous mice

The question is whether improved immunity after pregnancy is re­

flected by an enhanced ability to clear parasites (switch from pré­

muni ty to sterile immunity). Therefore, groups of mice were challenged

(1.10 PE/mouse) within a week after parturition, and clearance of

parasites was determined by subinoculation of tailblood into normal

controls 2 weeks after challenge. The results of this experiment are

depicted in table I.

A strongly enhanced clearance rate was found in parous mice ex­

posed to parasites during a foregoing pregnancy (group 2) as compared

to immune virgin mice (group 1), or to parous mice that were radical­

ly cured by chemotherapy before pregnancy (group 3). Thus, an enhanc­

ed clearance rate is not obtained by pregnancy alone but by the pres-

cence of parasites during pregnancy. This result is in line with the

observed effect of the parasite during pregnancy on a lower recrudes­

cence rate in gravida II mice (previous section). The combined re­

sults indicate that the presence of proliferating parasites during

pregnancy is an important factor in reinforcement of immunity.

To analyse a possible relation between improvement of immunity

and presence/proliferation of parasites during a certain period of

pregnancy, the presence of parasites in immune pregnant mice was re­

stricted to various time sections of the pregnancy period. Group 4

(table I) summarizes the data of mice in which parasites were present

either until day 5, or from day 5 to day 11 of pregnancy, after which

period chloroquine treatment radically cured the mice. Only 15% of

these mice were able to clear the challenge inoculum given after par­

turition, indicating that the presence of parasites before day 11 of

pregnancy does not act as an antigenic signal. In group 5 the effect

of the presence of parasites from day 11 of pregnancy onwards was

analysed. Immune mice were radically cured by chloroquine treatment

before mating and 1.10^ PE were inoculated intravenously on day 11 of

57

Table I. Clearance rate in relation to pregnancy and presence of

parasites

Significance compared

Clearance rate to group I

1. Immune, virgin mice 10% (3/11)

2. Parous mice; surviving

parasites during pre­

vious pregnancy 82% (14/17) Ρ < 0.005

3. Parous mice; no para­

sites during previous

pregnancy 0% (0/34) n.s.*

4. Parous mice; surviving

parasites only before

day 11 of previous

pregnancy 15% (3/20) n.s.

5. Parous mice; surviving

parasites from day 11 of

previous pregnancy on­

wards 78% (25/32) Ρ < 0.001

6. Parous mice as in group

5, but proliferation of

parasites during preg­

nancy inhibited by

sulfonamide treatment 38% (5/13) n.s.

n.s. not significant

pregnancy. Ninety percent of these mice developed a recrudescence

which was cured by sulfadiazine (30 mg/L drinking water) when para-

si taemi a exceeded 5% infected cells. After recovery mice were re­

turned to normal drinking water. When the clearance rate was deter-

58

mined in this group after pregnancy it was found that 78% of the mice

were able to clear the challenge inoculum. The results of group 4 and

5 show the importance of the presence of parasites during the second

half of pregnancy for the development of better immunity.

Finally, the importance of proliferation versus presence of

parasites during the second half of pregnancy was analysed. The mice

in group 6 as in group 5 harboured parasites from day 11 till the end

of pregnancy (tested by subinoculation), but proliferation of the

parasite during this period was largely inhibited by sulfadiazine

treatment (2.5 or 5 mg/L drinking water). The clearance rate observed

after challenge (38%) was intermediate between that of mice which

were (group 2, 5) or were not (group 3, 4) exposed to proliferating

parasites during this period of pregnancy. The results so far indi­

cate that the conditions during pregnancy for reinforcement of para­

site clearance in parous mice or for reduced recrudescence rate in

multigravidae are similar. Reinforcement of immunity in pregnant mice

is mediated by proliferating parasites during the second half of the

pregnancy period.

Enhanced clearance of parasites after temporary immunosuppression

In combination with previous work results of the present experi­

ments suggest the following hypothesis. Loss of immunity during preg­

nancy is probably caused by pregnancy dependent immunosuppression

evoked by increased serum corti coid levels during pregnancy (Van Zon

ei al., 1982), and better immunity is probably developed during the

(chemotherapeutically supported) convalescent phase of a recrudescent

infection during pregnancy (previous section). This hypothesis can be

tested further by an attempt to mimic the pregnancy situation in non

pregnant immune mice. Immunity can be depressed temporarily in immune

virgin mice by treatment with immunosuppressives. Immunosuppressive

treatment can provoke recrudescent infection, and the proliferating

parasites may then provide the signal for development of improved

immunity during the (chemotherapeutically supported) convalescent

phase.

59

In the experiments described in table II dexamethasone and anti-

T-cell serum (ATS) were used as immunosuppressives. Sulfadiazine treat­

ment was used to terminate recrudescences. Chemotherapy was not neces­

sary in ATS treated mice, since no or only low, transient recrudescent

infections were observed after treatment. After recovery the mice were

challenged, and 2 weeks later the clearance test was performed.

Table II. Cleavanae rate after temporary immunosuppression in immune

miae.

clearance rate

1. Dexamethasone treated 90% (9/10)

2. No dexamethasone treatment 27% (4/15)

3. ATS treated 80% (8/10)

4. Controls, NRS treated 40% (4/10)

Temporary suppression of malaria immunity by dexamethasone treat­

ment, and in the presence of proliferating parasites resulted in a

significantly higher clearance rate (compare group 1 and 2 of table

II; p< 0.01). Immunosuppressive treatment with ATS likewise resulted

in a high clearance rate afterwards, as compared to the mice with

normal rabbit serum (NRS), though the difference is not statistically

significant (table II, group 3 and 4).

In summary, temporary immunosuppression in combination with pro­

liferating parasites in immune non pregnant mice results in improved

immunity, as expressed by enhanced parasite clearance. This mimics

the improved immunity in multigravidae as compared to Primigravidae.

Splenectomy and immunity

The spleen plays an important role in maintenance of malaria

immunity. A high proportion of mice with an established immunity de­

veloped lethal infections after splenectomy (Eling, 1980b). This

60

raises the question whether improved immunity in parous mice was

expressed by a decreased loss of immunity after splenectomy. There­

fore, immune parous and virgin mice were splenectomized and challeng­

ed with 1.10 PE one week later. The proportion of parous mice sur­

viving challenge infection (44/53 = 83%) was significantly higher

(p < 0.001) than age matched virgin controls (10/22 = 45%).

DISCUSSION

It has repeatedly been reported that malaria is a serious problem

during human pregnancy and that it is more frequent in primi- as com­

pared to multigravidae (Reinhart et al., 1978; Bray & Anderson, 1979;

McGregor et al., 1983). This is also true for murine Plasmodium berghei malaria. Approximately 50 to 70% of the mice immune to P. berghei lose

their immunity during their first pregnancy (Van Zon & El ing, 1980a).

In contrast, the proportion of multigravid mice that lose their immu­

nity during pregnancy is significantly lower (Van Zon & El ing, 1980b).

An important question is whether this difference depends on either

better immunity in multigravidae or other than immunological differ­

ences (McGregor et al., 1983). The experiments described in this

paper clearly indicate that a previously existing malaria immunity

in mice is improved after pregnancy and that this improvement on (an)

antiparasitic immune reaction(s) developing during the convalescent

phase of an immunosuppressive period.

Thus, pregnancy is generally considered to be associated with

immunosuppression, and in particular of cell-mediated immune reac­

tivity rather than humoral immunity (Beer & Billingham, 1972; Fabris,

Piantanelli & Muzzioli, 1977; Loke, 1978). The loss of immunity to

P. berghei in pregnant mice occurred particularly during the second

half of pregnancy (Van Zon et al., 1982), and was more or less com­

plete, since in the majority of mice the recrudescent infection ran

a fatal course (Van Zon & El ing, 1980b). The fact that immune mice

that managed to survive until after parturition, despite a recru­

descence, recovered spontaneously, indicated that the state of immuno-

61

suppression has been abrogated after pregnancy.

Together with better immunity in multigravidae, parous mice also

exhibited enhanced clearance of a challenge inoculum (table I) and

the ability to develop sterile immunity instead of an otherwise long

lasting state of premunity (Eling, 1980a). The conditions for develop­

ment of enhanced clearance were the same as those necessary for develop­

ment of better immunity in multigravidae, and since the clearance test

is much less laborious, and time consuming than an analysis of repeated

pregnancies, the clearance test was preferred in the later experiments.

Since pregnancy associated immunosuppression is related to and probably

mediated by increased serum corticoid levels (Van Zon et al., 1982),

it could be hypothesized that development of better immunity is not

pregnancy specific, but depends on presentation of antigen to the

immune system in the convalescent phase of a (corticoid mediated)

immunosuppression. The enhanced clearance rate observed after tempo­

rary corticoid or anti-T-cell immunosuppression in non pregnant mice

supported this view (table II).

Improvement of immunity in multigravidae was not dependent on

parity as such, but parasites have to have been present during a

previous pregnancy. Moreover, proliferation of the parasite appeared

to be the effective signal for induction of better immunity (table I,

group 5 and table II, group 1), rather than the mere presence of

parasites (table I, group 6). It is important to note that previous

observations had shown that repeated challenges did not boost anti-

malaria immunity in this model, i.e. did not prevent fading of

immunity or convert premunity to sterile immunity (Eling, 1978), nor

prevent loss of immunity during pregnancy (unpublished observations).

This indicates that increase of the amount of parasite material per

se, as achieved by repeated challenges, was not effective in inducing

improvement of the immunity. The importance of the proliferating

parasite in the immunosuppressed host might indicate the need for a

confrontation of a threshold load of parasites, or more interesting­

ly, of immunogenic stages of the parasite (parasitized erythrocyte)

with the suppressed and convalescing immune system.

62

In the P. berghei - mouse model the spleen is important for

both development and maintenance of immunity (Wyler, 1983). The sig­

nificantly higher proportion of parous mice that maintained immunity

upon splenectomy indicates that their better immunity has become less

spleen dependent.

Consequently, better malaria immunity in parous mice might be

due to a reinforcement of existing (cell-mediated) immune reactions,

but it can also be speculated that confrontation of the parasite

(parasitized erythrocyte) with an immune system which is inhibited

in its cell-mediated reactivity leads to new, additional immune re­

actions, which are not suppressed during pregnancy and, therefore,

might be of the humoral type.

ACKNOWLEDGEMENTS

We thank J. Smeekens-Koekkoek for technical assistance, J. Reitsma and G. Poelen for biotechnical collaboration and care of the animals, and C. Jerusalem and M.L. Weiss for their help in preparation of the manuscript.

These investigations were supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO).

REFERENCES

1. Brabin, B.J. 1983. An analysis of malaria in pregnancy in Africa. Bull. WHO 61: 1005-1016.

2. Bray, R.S. and M.J. Anderson. 1979. Faloiparum malaria and preg­nancy. Trans. R. Soc. Trop. Med. Hyg. 73: 427-431.

3. Beer, A.E. and R.E. Billingham. 1972. Immunobiology of mammalian reproduction. Adv. Irmunol. 14: 1-84.

4. Campbell, C.C., J.M. Martinez and W.E. Collins. 1980. Seroepi-demiological studies of malaria in pregnant women and newborns from coastal El Salvador. Trans. R. Soc. Trop. Med. Hyg. 29: 151-157.

5. Cannon, D.S.H. 1958. Malaria and prematurity in the western region of Nigeria. Br. Med. J. 2: 877-878.

6. El ing, W.M.C. 1978. Malaria immunity and premunition in a Plasmodium berghei mouse model. Isr. J. fled. Sci. 14: 542-553.

7. El ing, W.M.C. 1979. Plasmodium berghei: effect of anti thymocyte serum on induction of immunity in the mouse. Exp. Parasitol. 47: 403-409.

0. Eling, W.M.C. 1980a. Plasmodium berghei: premunition, sterile immunity and loss of immunity in mice. Exp. Parasitol. 49: 89-96.

63

9. Eling, W.M.C. 1980b. Strain specificities in murine malaria with emphasis on immunological responsiveness. In: Animal Quality and Models in Biochemical Research (ed. A. Spiegel, S. Erichsen, H.A. Solleveld), pp. 91-95, Stuttgart, New York: Gustav Fischer Verlag.

10. Eling, W. and С Jerusalem. 1977. Active immunization against the malaria parasite Plasmodium bevghei in mice: sulfathiazole treatment of a P. berghei infection and development of immunity. Tropenmed. Paras i tol. 28: 158-174.

11. Eling, W., A. Van Zon and C. Jerusalem. 1977. The course of a Plasmodium berghei infection in six different mouse strains. Z. Parasitenkd. 54: 29-45.

12. Fabris, N., L. Piantanelli and M. Muzzioli. 1977. Differential effect of pregnancy or gestagens on humoral and cell-mediated immunity. Clin. Exp. Immunol. 28: 306-314.

13. Gilles, H.M., J.B. Lawson, M. Sibelas, Α. Voller and N. Allan. 1969. Malaria, anaemia and pregnancy. Ann. Trop. Med. Parasitol. 63: 245-263.

14. Jayawardena, A.N. 1981. Immune responses in malaria. In: Parasitic Diseases, vol. 1, The Immunology (ed. J. Manfield), pp. 85-136, New York: Marcel Dekker Inc.

15. Loke, Y.W. 1978. Immunology and immunopathology of the human foetal-maternal interaction. Amsterdam: Elsevier.

16. McGregor, I.A., M.E. Wilson and W.Z. Billewicz. 1983. Malaria in­fection of the placenta in the Gambia, West Africa; its incidence and relationship to stillbirth, birthweight and placental weight. Trans. R. Soc. Trop. Med. Hyg. 77: 232-244.

17. Menon, R. 1972. Pregnancy and malaria. Med. J. Malaysia 27: 115-119. 18. Reinhardt, M.C., P. Ambroise-Thomas, R. Cavallo-Serra, С. Meylan

and R. Gautier. 1978. Malaria at delivery in Abidjan. Helv. Paediatr. Acta 33: suppl. 41: 65-84.

19. Taufa, T. 1978. Malaria and pregnancy. Papua New Guinea Med. J. 21: 197-206.

20. Van Zon, A.A.J.С. and W.M.C. Eling. 1980a. Depressed malaria immunity in pregnant mice. Infect. Immun. 28: 630-632.

21. Van Zon, A.A.J.С. and W.M.C. Eling. 1980b. Pregnancy associated recrudescence in murine malaria {Plasmodium berghei). Tropenmed. Parasitol. 31: 402-408.

22. Van Zon, A.A.J.С, W.M.C. Eling, C.C. Hermsen and A.A.G.M. Koekkoek. 1982. Corticosterone regulation of the effector function of malarial immunity during pregnancy. Infect. Immun. 36: 484-491.

23. Wyler, D.J. 1983. The spleen in malaria. In: Malaria and the Red Cell (Ciba Foundation Symposium 94), pp. 98-116, London: Pitman.

64

Chapter V

Enhanced virulence of malaria infection

in pregnant mice

A.A.J,С van Zon, W.M.C. Eling

and C.C. Hermsen

Department of Cytology and Histology

Faculty of Medicine

University of Nijmegen

The Netherlands

IRCS Mediaal Saienae (1983) 11: 1002-1003

65

V Enhanced virulence of malaria infection in pregnant mice

An increased incidence of malaria and increased severity of the

infection have been reported in pregnant women as compared to non­

pregnant women, from endemic areas (1, 2). Such pregnant women develop

clinical infections despite an acquired immunity which protected them

before pregnancy. More virulent infections have also been described in

unprotected women during pregnancy (3, 4). A more aggravated course has

been described for other diseases during pregnancy as well (5).

During pregnancy the immune response of the mother is partly sup­

pressed, possibly to prevent rejection of the allograft (6). This sup­

pression usually concerns cell-mediated rather than humoral immune

reactivity (5). Since cell-mediated immunity is important in the de­

fence against malaria parasite (7), the pregnancy associated decrease

in protection to malaria may be a result of depressed cellular immunity.

In mice immune to the murine malaria parasite Plasmodium berghe-i,

a considerable proportion lose their immunity during pregnancy (8). In

this respect the murine model is comparable to human malaria. This paper

describes an enhanced virulence of the infection in different mouse

strains in relation to the pregnancy period and in comparison with non

pregnant controls. Changes in virulence are interpreted in the framework

of changes in immune reactivity during pregnancy and in relation to ma­

larial immunity.

MATERIALS AND METHODS

BiOLP, C57B1/Rij, СЗН/StZ and Balb/c inbred mice were obtained from

the animal facilities of the University of Nijmegen. Two to three (10 to

12 weeks old) female mice were housed with one male and checked for a

vaginal plug on the next four days. Three days later the mated females

were removed from the male. The day of the vaginal plug was day one of

pregnancy. P. berghei, strain К 173, was maintained by weekly subinocu­

lation of infected blood into mice from the corresponding strain. The

experimental animals were infected by injecting them (i.v.) with 10^ pa­

rasitized erythrocytes (PE) in diluted blood obtained from an infected

66

mouse. Animals were checked for pregnancy either by palpation of the

abdomen on day 16 to 18 of pregnancy, or by postmortem dissection.

и

s, s

со

C57BI/RIJ

RESULTS AND DISCUSSION

The virulence of a Plasmodium berghei infection in unprotected,

pregnant and non-pregnant mice was determined by the mean survival time

after the inoculation of the 10^ parasitized erythrocytes. Mean survival

time with respect to the day of pregnancy chosen to infect the mice, as

observed in B10LP, C57B1/Rij, Balb/c and СЗН/StZ mice, is depicted in

the figure. The degree of reduction in survival time was apparently de­

pendent on both mouse strains and

the day of pregnancy chosen to

infect the animals. The strongest

reduction was noted in B10LP and

C57B1/Rij mice with minimum

values of 31% and 23% respective­

ly of the control values (figures

A and B). The reduction in mean

survival time was less but still

significant (P <0.05; Wilcoxon

test) in Balb/c mice (figure C)

and became minimal in C3H/StZ

mice (not significant when infec­

ted on day 7, but significant on

day 14 of pregnancy, figure D).

. , . ., , Reduction of the survival time Survival ігте (aays) of pregnant тгое with primary malaria infection plotted was observed in mice infected

against the day of pregnancy chosen to between day 7 and 14 of pregnancy.

infect the mice. The number of each

group of mice varied from 5 to 3b mice Minimal survival times were ob-

(mean + SEM). served when the animals were in­

fected on day 10 and 12 of pregnancy. Values were comparable in the two

strains tested on these days and were close to those values found in non

pregnant СЗН/StZ mice. No changes in mean survival time were observed in

mice infected on day 1 or day 5 of pregnancy (figure A). Results obtained

when B10LP, C57B1/Rij and Balb/c mice were infected on day 14 of pregnan-

67

су suggest a return to normal values in the last part of pregnancy

(figures А, В and C). Enhanced virulence of the infection during

pregnancy was also apparent from higher parasitaemias in pregnant

as compared to non-pregnant mice (9,10).

Parasite-host interplay during a primary malaria infection is

complex and comprises several different immune reactions which either

primarily serve to protect the host, or may be regulatory and even

suppressive, serving survival of the parasite (7, 11). The virulence

of an infection depends on the presence of immunoprotective and/or

immunopathological signals on the parasitized erythrocyte and the

host's reaction to these signals. The stronger (earlier) the immuno­

protective reactions the longer the survival and the stronger (ear­

lier) the immunopathological reactions the shorter the survival.This

may explain the difference in mean survival time observed in diffe­

rent strains as well as between males and females of a given strain

(12). СЗН/StZ mice, pregnant or not, exhibit a short mean survival

time. This might depend either on poorly developed immunoprotective

or strong immunopathological reactions. When immunopathological

reactions dominate, immunosuppression will be favourable to the

host provided that immunoprotective reactions are present. C3H/StZ

mice, immunosuppressed or not, display a short mean survival time

after infection (figure D (12)), suggesting that in those mice

immunoprotective reactions are poorly triggered. As a consequence,

the pregnancy dependent reduction of the mean survival time observed

in the other strains (figure А, В and C) may be explained by a re­

duction of the host's ability to mount immunoprotective reactions.

Shortest mean survival times were observed after infection in the

second week of pregnancy, suggesting that pregnancy dependent immuno­

suppression is maximal during this period. Mice infected before this

period exhibited a normal survival pattern despite the experience of

an immunosuppressive period. This may indicate that pregnancy depen­

dent immunosuppression acts upon sensitizing reactions. Mice infected

in the first week of pregnancy, survived beyond the pregnancy period

indicating that reduced survival is not due to delivery problems in

malaria sick mice.

68

Enhanced virulence might be related to increased reticulocyto-

sis, starting from day 6 of pregnancy onwards (13) and providing a

higher number of host cells (P. berghei preferentially invades reti­

culocytes (14)). In contradiction to this is the long survival time

in mice infected on day 1 or 5 of pregnancy, unless we assume that

these mice have developed an effective protective immunity before­

hand, by which reticulocytosis at this time does not precipitate

enhanced infection.

A more severe course of malaria infection in pregnant mice

was also observed by Oduola et al. (9) who showed the harmful in­

fluence of this infection on the development of the fetuses, re­

sulting in abortion or resorption of the fetuses. A correlation

between virulence of infection and the day of pregnancy on which

infection was started was also reported for pregnant mice infected

with poliomyelitis virus (15).

The period of pregnancy with reduced survival after malaria

infection coincides with the period when previously immune mice

suffer from frequently fatal recrudescences, indicating a complete

loss of a previously existing immunity (16). In these animals preg­

nancy associated immunosuppression is supposed to block the effector

part of immunity. Since an established protective immune reaction

triggered immediately after infection is not affected by the preg­

nancy dependent immunosuppression (see above) this immune reaction

is different from the one suppressed in immune mice during pregnancy.

A different sensitivity of immune reactions to anti-T-cell treatment

during induction of immunity compared to an established immunity (17)

underlines the possibility that several different immune reactions

operate.

We thank J. Reitsma and G. Poelen for biotechnical collaboration

and care of the animals, and A. Jenks for reading the manuscript. This

work received financial support from BION-ZWO, The Netherlands.

69

1. Bray,R.S. and Anderson,M.J.(1979) Trans R.Soa.Trop.Med.Нуд., 73, 427-431

2. McGregor,I.A..Wilson,M.E.and Billewicz,W.Z.(1983):rrarcs Я.Soa.Trop. Med.Нуд., 77, 232-244

3. Menon,R.(1972) Med.J.Malays, 27, 115-119 4. Taufa,T.(1978) Papua New Guinea Med.J.,21, 197-206 5. Purtilo, D.T.,Hallgren,H.M. and Yunis,E.J.(1972) Lancet, i, 769-771 6. Gusdon,J.P.,jr.(1983) Am.J.Reprod.Immunol.,3, 5-6 7. Jayawardena.A.N.(1981) in Parasitic Diseases, Vol 1, The Immunology,

(Mansfield,J.,ed.), pp.85-136, Marcel Dekker Inc., New York 8. van Zon,A.A.J.C.and El ing,W.M.C.(1980) Inject.Immun., 28, 630-632 9. Oduola, A.M.J.,Holbrook,T.W.,Galbraith,R.M.Bank,H.and Spicer.S.S.

(1982) J.Protozoal., 29, 77-81 10. van Zon,Α.Α.J.С. ,Eling,W.M.C.,Herrasen,C.C.,van de Wiel J.J.J.M.and

Duives.M.E. Bull.Soa.Pathol. Kxot.,i η press 11. Eling.W.M.C. (1982) Fortschritte Zoöl, 27,Suppl.l2, 140-155 12. Eling.W.M.C. (1980) in Animal Quality and Models in Biochemical

Research,(Spiegel,A.,Erichsen,J.and Solleveld.H.eds), pp.91-95, Gustav Fisher Verlag,Stuttgart,New York

13. Fruhman,G.J.(1968) Blood, 31, 242-248 14. Zuckerman,A.(1957) J.Infeat.Dis., 100, 172-185 15. Knox,A.W.(1950) Proa.Soc.Exp.Biol.Med., 73, 510-528 16. van Zon,A.A.J.C..Eling.W.M.C.,Hermsen,C.C.and Koekkoek,A.A.G.M.

(1982) Infect.Immun., 36, 484-491 17. Eling.W.M.C.(1979) Exp.Parásito I., 47, 403-409

70

Chapter VI

Corticosterone regulation of the effector function

of malarial immunity during pregnancy

Adriaan A,J,С van Zon, Wijnand M.C, Eling,

C.C.Rob Hermsen and Anne A.G.M. Koekkoek

Department of Cytology and Histology

Faculty of Medicine

University of Nijmegen

The Netherlands

Infection and Immunity (1982) 36: 484-491

71

iNi-ECTiON AND IMMUNITY May 1982 ρ 484-491 0019 9567/82/050484 07S02 00/0

Vol 36 No 2

Corticosterone Regulation of the Effector Function of Malarial Immunity During Pregnancy

ADRIAAN A J С VAN ZON,* W1JNAND M С ELING С С ROB HERMSEN, AND ANNE A G M ΚΟί-ΚΚΟΕΚ

Department of Cytology and Histology Unnersity of Nijmegen, 6500 HB Nymegen, The Netherlands

Received 14 September 1981/Accepted 30 December 1981

In the experimental Plasmodium berghei mouse model, as in human malaria, reduced maternal responsiveness and even loss of immunity were observed during pregnancy Loss of immunity in the second half of pregnancy occurred during a period of elevated plasma corticoid levels Further analysis showed that plasma corticoid levels were significantly higher in immunodepressed mice than in mice that remained immune throughout pregnancy Plasma corticosterone levels dif­fered increasingly from those in mice with persistent immunity towards recrudes­cence In nonimmune infected controls, however, only a slight increase in plasma corticosterone, already present during the subpatent penod, was measured Blocking of maternal corticoid production by adrenalectomy delayed the increase of plasma corticosterone (fetoplacental origin) and reduced the number of mice that lost immunity during pregnancy considerably The role of various plasma corticoid levels in the regulation of effector function of immunity during pregnan­cy is discussed

Reduced resistance during pregnancy result­ing in more severe infections and increased mortality rates has been reported for viral, para­sitic, and fungal infections, as well as for tumors (1,4, 23, 33, 39, 42, 53) Among these is malaria with a higher incidence and seventy in pregnant women, possibly causing prematurity and low birth weight (9, 11, 13, 34, 35)

As in human malaria, loss of immunity is also observed in a considerable proportion of mice previously immune to the virulent parasite Plas­modium berghei (50, 51)

A decreased maternal immunoreactmty dur­ing pregnancy, contributing to tolerance of the fetal allograft, has been suggested frequently (6) Humoral immunity is either not affected or even slightly stimulated during pregnancy (12,22,32) The decreased mixed-lymphocyte reaction, phy-tohcmagglutinin stimulation, and lymphocyte cytotoxicity, as well as delayed rejection of cardiac allografts, during pregnancy reflect im­pairment of T-ccll function in vitro and in vivo (3, 8, 15, 16, 28, 42) Moreover, macrophage activity is reduced or increased in different peri­ods of pregnancy (25, 38) Since it is frequently assumed that T-cells and macrophages play an important role in malaria immunity (26, 29, 41, 44), a pregnancy-associated depression of their functions may lead to loss of immunity

Several factors have been implicated in preg­nancy-associated immunodepression, such as al-pha-fetoprotein (37, 46) or changes in serum hormone levels (e g , progesterone, human cho-

пошс gonadotropin, and glucocorticoids [27, 45], which either directly inhibit T-cell activity or generate suppressor T-cells (7, 24) The inhib­iting effect of glucocorticoids on the immunolog­ical functioning of Τ cells and macrophages is well known (2)

Expenments desenbed in this paper show a relation between the level of plasma corticoste­rone and loss of malaria immunity Mice that lost immunity during pregnancy exhibited higher plasma corticosterone levels than those with persisting immunity, whereas adrenalectomy be­fore pregnancy not only blocked maternal corti­costerone production, but also prevented loss of immunity, suggesting regulation of the effector function by plasma corticosterone

MATERIALS AND METHODS

Mice. Random-bred Swiss mice, obtained from the animal facilities of the University of Nijmegen or from T N O , Zeist, The Netherlands, were kept in plastic cages with food and drinking water ad libitum

Pregnant mice Two or three female mice, aged 12 to 20 weeks, were housed with one male Every morning for the next 4 days mice were examined for the presence of a vaginal plug Females not mated after 4 nights were removed from the male, mated females were removed 3 days later The day of finding the vaginal plug was day 1 of pregnancy After delivery the litter was removed from the mother All pregnan­cy-related expenments concerned pnmigravid mice

Parasite. Ρ berghei strain K173 was maintained by intraperitoneal subinoculation of infected blood (10s

72

VOL. 36,1982 PLASMA CORTICOSTERONE AND MALARIAL IMMUNITY

parasitized erythrocytes) into female Swiss mice weekly. The course of a primary infection, which is always lethal, has been described previously (21).

Immune mice. The immunization procedure (19) was as follows. Mice were infected with 107 parasitized erythrocytes and treated with sulfadiazine (30 mg/liter of drinking water) from 2 to 33 days thereafter to prevent patency. Immunity was assessed by resistance to challenge (10' parasitized erythrocytes) given 35 days after the immunizing infection. Only mice with an established immunity were used in the experiments. Immunity is of the premunition type; i.e., small, subpatent numbers of parasites persist in immune animals for different periods of lime (20). Spontaneous recrudescences were rare (18). Immune mice did not develop patency after any challenge.

Recrudescence. A pregnancy-associated recrudes­cence (parasitemia higher than 5% infected cells) was determined from thin smears prepared from tail blood and stained with May-Griinwald-Giemsa solutions. Since recrudescence can only occur when parasites are present, immune mice were given an extra chal­lenge (10fl parasitized erythrocytes) 3 days before mating.

Isodiagnosls. The presence of parasites in immune mice was determined by subinoculation of 0.02 ml of tail blood, diluted with 0.2 ml of saline conlaining heparin (5 U/ml), into a clean animal, which was subsequently checked for patency.

Adrenalectomy. Each adrenal gland was removed through a' lateral incision, separation of the muscle wall, and blunt preparation of the organ. Operation was performed under ether anesthesia. Adrenalecto-mized mice were kept at 280C for 1 week and received seven daily injections of 1 ml of saline subcutaneously, supplemented with 0.9% NaCI solution as drinking water throughout the experimental period. The time between adrenalectomy and mating was at least 2 weeks.

Plasma samples. Blood (100 to 150 μΐ) was obtained from the retro-orbital plexus of anesthetized (ether) mice, using heparinized disposable micropipettes. In this way animals could be sampled longitudinally. Blood samples were centrifuged and the plasma was frozen (-20oC). To avoid interference by diurnal changes of the corticosterone level, all samples were taken between 11:00 and 11:30 a.m.

Corticosterone. Plasma corticosterone was mea­sured by radioimmunoassay, using an antiserum raised in sheep against corticosterone-21-hemisuccinate-bo-vine senjm albumin (gift from T. Ben raad. Department of Experimental and Chemical Endocrinology, Uni­versity of Nümegen). Plasma samples were diluted (100-fold) in distilled water with 0.2% ethylene glycol, incubated overnight at 40C with I'HJcorticosterone (specific activity, 57 Ci/mmol; Amersham Internation­al Limited), and extracted with toluene. The organic phase was evaporated, and corticosterone was sepa­rated from cross-reacting substances by paper chro­matography (Whatman no. 1), using a modified Bush B5 system (1 volume of toluene, 0.5 volume of metha­nol, and 0.5 volume of water). Paper was scanned for radioactivity (Packard radiochromatogram scanner, model 7201), and the radioactive zone was eluted with ethylene-glycol-water. Recoveries after extraction and chromatography were 24 ± %% (standard deviation; η = 46). The procedure for the radioimmunoassay of

corticosterone in the eluate is the same as that de­scribed for aldosterone (17). The antiserum dilution was 225,000, and corticosterone (Stcraloids) concen­trations of 25 to 2,000 pg were used for the standard curve. The interassay coefficient of variation was 14% (л = 46).

SUtistical evaluation. Significance of differences be­tween plasma corticosterone levels in groups of mice was assessed by the Km s kal and Wallis test and the Wilcoxon test.

RESULTS

Recrudescence during pregnancy. A high per­centage of mice» immune to P. berghei, lose their immunity during pregnancy and suffer a frequently lethal infection (51). The cumulative proportion of recrudescent mice in relation to the day of pregnancy is depicted in Fig. 1. Recrudescences are almost only found in the last trimester of pregnancy. Some mice exhibited recrudescence after parturition.

Plasma corticosterone and thymus involution during pregnancy in normal and adrenalecto· mized Swiss mice. During a malaria infection there is reduced immune reactivity which is associated with thymus involution (21), and this in turn could be taken as an indirect marker for immunodepressive changes in T-cell areas. Therefore, changes in thymus weight during pregnancy were recorded and compared with the pattern of recrudescences. Moreover, since corticosterone, the physiologically important glucocorticoid of mice, is well known for its immunosuppressive properties, serum plasma levels were determined during pregnancy and compared with changes in thymus weight. The effect of plasma corticosterone on thymus invo-

cumulat ive p e r c e n t a g e of • r e c r u d e s c e n t mice

100 parturition

ІГ

J-J 10

•+-15 20

day of pregnancy FIG. 1. Cumulaiive pattern of recrudescent mice (л

= 98) in relation to the period оГ pregnancy.

73

VAN ZON ET AL I N F E C T I M M U N

lution was also determined m adrenalectomizcd mice, i.e., in the absence of maternal corticoste­rone production. The results of these experi­ments are given in Fig. 2a to d

Progressive thymus involution was observed in intact pregnant mice from day 14 onwards (Fig 2a), and this correlated with an increase in plasma corticosterone levels starting as early as day 12 of pregnancy (Fig 2b) In adrcnalecto-mized mice thymus involution was not signifi­cant until partuntion (Fig. 2c), and this was several days after an increase in plasma cortico­sterone level (Fig. 2d), which was apparently produced by the fetoplacental unit, since the increase in plasma corticosterone in adrenalcc-tomized mice was proportional to the number of

fetuses (data not shown) In intact and adrenal-ectomized mice increase in plasma corticoste­rone preceded thymus involution, but a reduc­tion in corticoid level was not closely followed by thymus regeneration

An approximately 10-fold increase of plasma corticosterone was observed during pregnancy, with peak values on day 18 and return to "non­pregnant" values after partuntion (Fig 2b and d) Individual plasma levels vaned strongly, especially during the penod of peak values

Plasma corticosterone and loss of immunity in intact pregnant mice. Increased plasma corticos­terone levels coincided with recrudescence dur­ing pregnancy This prompted analysis of plas­ma corticosterone levels in mice developing

thymus weight mg

100-

5 0

parturilion

® intact

Г""!-

parturition

i T

© adrenalectomized

plasma corticosterone /ug/100 ml 200

100-

Θ Π0Π-

pr«anant controls

2 0 Ι θ non­

pregnant controls

12 16 2 0 day ot pregnancy

FIG 2 Changes in thymus weight and plasma comcosterone in normal and adrenalectomized pregnant Swiss mice Average thymus weight (± standard deviation) was determined in groups of three to six mice, and average plasma corticosterone values (± standard deviation) were determined m groups of 4 to 11 animals Data for nonpregnant controls (open symbols) were obtained from a control group sacnfìced on different days of pregnancy together with experimental mice (closed symbols)

74

VOL 36, 1982 PLASMA CORFICOSTERONE AND MAI ARIAL IMMUNITY

recrudescence during pregnancy in comparison with mice that remained immune throughout

Female mice with an established immunity were allowed to mate Blood samples were taken on days 10, 14, 16, and 18 or 19 of pregnancy, and plasma corticosterone concen trations were determined Thin smears were prepared each time to determine recrudescence The blood of each mouse found to be smear negative in the penod between days 14 and 18 was submoculated into control mice, and all mice negative by isodiagnosis were taken out of the experiment, because without parasites no recrudescence could occur

To relate plasma corticosterone with loss of immunity, mice were divided into three catego­ries on each day of sampling (i) mice actually having a recrudescence, (и) mice developing recrudescence later in pregnancy, and (in) mice without recrudescence throughout pregnancy The combined results of two independent ex-penmenls are depicted in Fig 3 Plasma corti­costerone concentrations of mice in the three categorie;, differed significantly on days 14 and 16 of pregnancy (P < 0 05, Kruskal and Wallis test) Pairwise companson to explore this differ­ence shows significantly higher corticosterone levels in the group of mice with recrudescence compared with the levels in one of the other groups (P < 0 05, Wilcoxon test) Results on days 18 and 19 were not clear, because the number of mice involved was too small, which was due to the fact that either recrudescent mice died earlier or had to be eliminated due to prematunty or abortion (50), which caused an acute drop of the corticosterone level Never­theless, the results seem lo indicate a higher corticosterone level m recrudescent mice (day 18, Ρ = 0 05, day 19, Ρ = 0 10)

In the period of days 14 and 19 the average corticosterone concentration, as well as the magnitude of the standard deviation of mice developing recrudescence later (group 2), shift­ed from the value found in the group with no recrudescence (group 3) to that in the group actually having a recrudescence (group 1) The significantly higher corticosterone values m re­crudescent mice and the growing significance of increase of plasma corticosterone concentra­tions towards recrudescence could suggest a direct relation between plasma corticosterone levels and parasitemia Figure 4 shows plasma corticosterone in relation to primary infection A small increase in plasma corticosterone was already observed in the prepatent penod, was not related to the magnitude of the parasitemia, and was very small in companson with values observed during a pregnancy-related recrudes­cence

Plasma corticosterone and loss of immunity in

adrenalectomized pregnant mice. The close cor­relation between high levels of plasma corticos­terone and recrudescence suggests a causal rela­t ionship This implies lhat blocking and corticosterone production should prevent loss of immunity and recrudescence Maternal produc­tion of corticosterone can be blocked by adre­nalectomy

Immune mice were adrenalectomized and plasma corticosterone levels were determined 2 weeks later Then, mice were challenged with ΙΟ^ parasitized erythrocytes to assure the pres­ence of parasites, as much as possible, and allowed to male 3 days later On days 14, 16, 18 or 19, and 20 of pregnancy, as well as 1 week after partuntion, plasma corticosterone levels were determined On the same days blood smears were made to assess a possible recrudes­cence, and isodiagnosis was performed on smear-negative mice Finally, mice were sacri­ficed and examined for the presence of adrenal tissue, which was eventually verified by light microscopy Mice with remnants of adrenal tis­sues and isodiagnosis-negative mice were taken out of the experiment

As in normal adrenalectomized mice (Fig 2d), plasma corticosterone levels were only elevated from day 16 on (Fig 5) With comparatively low values until day 18, there is a considerable

corticosterone level in plasma ol I immunized mice during pregnancy

yug/IOOml 600 η « recrudescence

τ • recrudescence later о no recrudescence

5 0 0 -

4 0 0 -

300 - I ·

11 'fi 100- 14 ^ •L

*4 , , , , , ,-10 12 U 16 1Θ 20

day of pregnancy HG 3 Plasma corhcosteronc Loncentrdtions dur

ing pregnancy Of 37 mice 24 (659f ) developed recru descence Plotled corticosterone levels are mean val ues (± standard deviation) of each group of mice On day 10 of pregnancy the results were pooled since corticosterone levels of the groups were the same Symbols · miLe actually having recrudescence, + mice developing recrudescence later m pregnancy С mice without recrudescence throughout pregnancy

75

VAN ZON ET AL I N F E C T I M M U N

p l a s m a cort icosterone («yug/IOOml) _ parasi temia (о p e r c e n t a g e )

Ю-PE i

I l I. I, ' в

. 1 0 2 5 7 9 day after infection

FIG 4 Mice (л = 10) were longtludtnally sampled (sec tent) I day before (control values) and 2 5 7 and 9 days after infection Λι the same time thin smears were made to determine parasitemia Plotted data are the mean (± ^andard deviation) Nine mice survived until day 7 and three mice survived until day 9 of infection PE Parasitized erythrocytes

reduction in plasma corticosterone compared with intact immune mice (Fig 3) Of 22 animals, only 3 (14%) developed recrudescence during pregnancy, exhibiting higher plasma corticoste­rone levels than the others on day 16 of pregnan­cy

DISCUSSION

A considerable proportion (approximately 50%) of mice with an established immunity relapsed during their first pregnancy (50) In Swiss mice approximately 70% of these recru descent mice develop lethal infection (51), im plying complete loss of a previously existing immunity during pregnancy Considering that spontaneous recrudescences are rare in immune nonpregnant mice (18), these expenments re­vealed a remarkable pregnancy associated sup­pression of effector function of immunity Preg nancy-associated immunosuppression has also been observed with regard to human malaria (9, 11, 13, 34, 35), viral infections (e g , poliomyeli tis [33], influenza [42], herpes simplex virus [53], hepatitis [39], and encephalomyocarditis [23]), the protozoan infection amoebic colitis (1), and the fungal infection coccidioidomycosis (42) Also, tumor development can be enhanced dur ing pregnancy, e g , Burkitt s lymphoma (4) The Ρ berghei mouse model desenbed in this paper may be an adequate experimental model for the analysis of the mechanisms of immuno-deprcssion during pregnancy

An important result of the expenments was that mice that lost immunity during pregnancy had significantly higher plasma corticosterone levels than mice with persistent immunity (Fig 3) Analysis of changes during pregnancy re­vealed that loss of immunity as marked by

recrudescence (Fig 1 ) was observed in a period of progressive thymic involution (Fig 2a) and increasing plasma corticosterone levels (Fig 2b) Recrudescence and thymic involution chro­nologically followed elevation of plasma corti­costerone levels (Fig 1 and 2) Combined with the well-known immunosuppressive properties of glucocorticoids, this suggested a causal rela­tionship among elevation of plasma corticoid levels, thymus involution (30, 36, 40), and im­paired immune reactivity (recrudescence) In combination with the scattering of plasma corti­costerone levels particularly during peak peri­ods (Fig 2), this led to the hypothesis that mice developing plasma corticosterone values belong­ing to the upper part of the distribution during the peak period (Fig 2b) were prone to lose immunity during pregnancy Two observations supported this hypothesis first, the significantly higher corticosterone levels in mice that lost immunity during pregnancy in companson with those with persistent immunity throughout (Fig 3), and second, absence of recrudescence when early increase of plasma corticosterone was pre vented by adrenalectomy (Fig 5) Some addi tional remarks have to be made The scatlenng of corticoid levels in recrudescent mice (Fig 3) indicates that part of these mice exhibited higher corticosterone levels than nonimmune controls (Fig 2b), suggesting extra corticosterone pro­duction Moreover, the plasma corticosterone level in the group of mice developing recrudes cence some time after sampling increased pro­gressively towards the end of pregnancy in com­panson with the group with persisting immunity

Γ / j g / 1 0 0 n

4 0 0 -ι

cort costeronr level in plasma ot mmun zed , a n d a d r e n a l e c l o m z e d m ce dur ing pregnancy

• recrudescence + recrLdescence later о n o recrudescence

b»Fort pregnancy

after pregnancy

1 6 1Θ 2 0 day of p r e g n a n c y

FIG 5 Plasma corticosterone concentrations in adrenalectomizcd pregnant mice (л = 22) Plotted values are the mean (± standard deviation) of mice without recrudescence whereas values of recrudes cent mice are plotled individually Symbols · mice actually having recrudescence + mice developing recrudescence later in pregnancy О mice without recrudescence throughout pregnancy

76

V O L 36, 1982 PLASMA CORTICOSTERONE AND MALARIAL IMMUNITY

(Fig 3) It can be assumed that there is an increasing chance that recrudescence and in­creased corticosterone values occur shortly af­ter sampling towards the end of pregnancy Together, these observations suggest that devel­opment of recrudescent infection leads to in­creased corticosterone production, but, on the other hand, the plasma corticosterone level was only minimally changed during infection (Fig 4) and appeared to be independent of parasitemia This makes it unlikely that stress associated with disease was responsible for the impressive in­crease in recnidescent mice, but leaves unex­plained the apparent excessive production of plasma corticosterone in some recrudescent mice

Maximum plasma corticosterone levels in ad-renalectomized mice (Fig 2d) were not marked­ly different from those in intact controls (Fig 2b), only the rise was delayed The increased plasma corticosterone levels towards the end of pregnancy in adrenalectomiied mice is assumed to depend on production in the fetoplacental unit (5), a view that is supported by the observation that the level was related to the number of fetuses The delayed increase in plasma cortico­sterone in adrenaleclormzed mice also delayed thymic involution In view of the impressive reduction of the recrudescence rate in adrenal-ectomized mice, this may suggest that plasma corticosterone levels in adrenalectonwed mice can be high enough to suppress the effector mechanism of immunity, but that the period of sufficiently high, immunosuppressive levels starts too late and is too short to cause recrudes­cence The observation that plasma corticoste­rone values in recrudescent adrenalectomized mice were high already on day 16 of pregnancy (Fig 5) is in line with this assumption

The number of recrudescences after partun-tion was small (Fig 1), and since recrudescence was scored when parasitemia passed 5% infect­ed cells, the actual breakdown of immunity occurred even before parturition However, af­ter parturition recrudescent infections are sup­pressed spontaneously (50), suggesting that the immunosuppression phased out around partun-tion, correlating nicely with the reduction in plasma corticosterone shortly before parturition (Fig 2 and 3)

In humans, too, elevated plasma Cortisol lev­els coincide with loss of immunity during preg­nancy (9, 10) Moreover, an exogenous supply of corticoid could break malaria immunity (W Eling and A van Zon, Parasitology 77:ii, 1978) Also, immunosuppression related to elevated corticoid levels was suggested in cases of amoe­bic colitis (1, 31) These observations support the hypothesis that plasma corticoid levels regu­late the effector part of immunity against several

pathogens, although the mechanism of this regu­lation remains to be clarified T-cells and macro phages are important in malaria immunity (19, 26, 41, 44, 52), and changes in activity of these cells during pregnancy could lead to loss of immunity It is well known that elevated plasma corticoid levels decrease the number as well as the activity of circulating lymphocytes and monocytes (2, 43, 47-49) Since T-suppres-sor cells are less sensitive to corticoid (54), a relative increase of suppressor T-cells during pregnancy (29) has been taken to explain preg­nancy-associated immunodepression (7, 14) Furthermore, humoral immunity is either unaf­fected or enhanced during pregnancy (22, 27, 33), suggesting that cell mediated reactions are suppressed (4)

Biological activity of corticoids is generally related to free corticoid unbound to corlicoid-binding globulin (2), and further studies are directed to the role of free corticoid in the regulation of immunity

ACKSOWIEDGMiNTS

We are grateful to Τ Hcnraad and co workers (Depanment of Experimental and Chemical Endocrinology Lmvcrsity of NijmcBcn) for advice and assistante m development of the radioimmunoassay for corticosterone and Γοι providing the antiserum We also thank H van Druten (Department of Mathematical and Statistical Consultation Lnivcrsil> of Nij megen) for assistance with the statistical tests J Rcilsma G Poelen M baassen and J van de Westenngh for biolcchmcal collaboration and care of the animals and M L Weiss for reading the manuscnpl

These investigations were supported bv the World Health Organi7ation Geneva Switzerland and the F-oundation for f-undamental Biological Research (BIONI which is subsi dized by the Netherlands Organization for the Advancement of Pure Research (ZWO)

IlTFRVrLRECITtD

1 АЫоуе, Λ A 1973 I alai amoebic colitis in pregnancy and Puerperium a new clinico pathological entity J Trop Med Hvg 76 S17-100

2 Bach, J F 197S I orlieosleroids ρ Zl-'Jl Ini I- Bach (cd ) The mode of action of immunosuppressive agents North Holland Publishing Co Amsterdam

3 Balnes, M G К (. Millar, and H l· Pro« 1980 Allograft enhancement during normal murine pregnancy J Reprod Immunol 2 141-149

4 Bannennan, R H O 1966 Burktlt s tumour m prcgnan cy Br Med J II 1136-1117

5 Barlow, S Μ , Ρ J Morrison, and F M Sullivan 1974 Plasma corticosterone levels during pregnancy in the mouse the relative contributions of the adrenal glands and foeto placental units J Endocrinol 60 471-483

6 Beer, A E , and R E Billinghain 1972 Immunobiology of mammalian reproduction Adv Immunol 14 1-84

7 Birkeland S A , and К Krtstoffersen 1980 The fetus as an allograft a longitudinal study of normal human preg nancies studied with mixed lymphocyte cultures between mother father and mother child Scand J Immunol I I 311-319

8 Bissenden, J С , N R Ling, and Ρ Mackintosh 1980 Suppression of mixed lymphocyte reactions by pregnancy senim Clin bxp Immunol 39 19S-202

77

VAN ZON ET AL I N F E C T I M M U N

9 Bray, R. S., and M. J. Andenon. 1979 talttpanm malaria and pregnancy Trans R Soc Trop Med Hyg 73:427^11

10 Bro-Rasmussen, F., О. Buus, F. Lund wall and D Trolle. 1962 Varidlions in plasma согіічоі during prcgnancv. del ι ven· and the Puerperium Acta Cndocnnol 40:^71-S7H

It Bruce-Chwatt, L. J. 19^2 Malaria in African infants and children m Southern Nigeria Ann Trop Med Parasitol 46:171-200

12 Bui mer, R., and K. W. Hanock. 1977 Depletion of circulating Τ lymphocytes m pregnancy Clin Ьхр lm munol 2a:102-10S

13 Campbell, C. C , J. M. Maríinez. and W. E. Collins. 1980 Scrocpidcmiologiial sludies of malaria m pregnant wom­en and newborns from coastal El Salvador Am J Trop Med Hyg 29: И1-157

14 Chaoual, G., and (i. A. Voisin. 1979 Regulatory Τ cell subpopulations in pregnancy I Evidence fur suppressive activity of the early phase of Ml.R J Immunol 122:1383-1388

15 Clark, D. A.,andM. R. McDennolt. 197« Impairment of Host-versus Graft reaction in pregnant mite I Suppres­sion of суилохіс \ -cell generation in Ivmph nodes dram mg the uterus J Immunol 121:1189-1393

16 ('lark, D. A., M. R. McDermott. and M. R. Szewczuk. 1980 Impairment of Host-versus Grafi reaction in preg­nant mice II Selective suppression of cytotoxic T-cell generation correlates with soluble suppressor activity and with successful allogeneic pregnancy Cell Immunol 52:106-118

17 de Man, A. J. M , and T. J. Benraad. 1977 Aldosterone secretion rale radioimmunoassay versus double isotope dilution derivative assay Clin Chim Acta 79.489-S01

18 Lhng, W. 1978 Survival of parasites in mice immunized against Plasmodium brrghei Tropenmed Parasitol 29.204-209

19 FJinR, W. M. С 1979 Ptusnwthum ЬсгкНеі effect of antilhymoivic scrum on induction of immuni!у in the mouse Exp Parasiiol 47:401-409

20 Eling, W. M. С 1980 Pttnmodium berghei premumtion. sterile immunity, and loss of immunily in mice fcxp Parasitol 49:89-%

21 Eling, W., A. van Zon. and С Jerusalem. 1977 The course of a Plasmodium berghvi infection m six different mouse strains Ζ Parasiicnkd 54.29-45

22 l·abris, Ν., L Planlanelli, and M. МіініоН. 1977 Differen­tial elf cc ι of pregnancy or gestagens on humoral and cell mediated immunily С lin Exp Immunol 28:106-114

23 Färber, P. Α., and L, A. Glasgow. 196Я Factors modifying host resistance to virus infection II Enhanced sust,epli-bihty of mice to cncephalocarditis virus infection during pregnancy Am J Pathol 53:463-481

24 Fuchi, T., L. Hammarstr&m, С. I. F Smith, and J. Brundm. 1980 In vitro induction of munne suppressor 1 cells by human chorionic gonadotropin Ada Obstet Gynaecol Scand 59:155-349

25 Graham. С W., and Т. M. Saba. 1973 Opsonin levels in reticulo-cndothclial regulation during and following preg­nancy RES J Reticuloendothel Soc 14.121-131

26 tïrun, J. L., and W. P. Weidanz. 1981 Immunity to PUnmodium ihabaudi adonti in the B-ccH-deficient mouse Nature (London) 290.143-145

27 Gusdon, J. P. 1976 Maternal immune responses in preg­nancy, ρ 103-125 In J S Scott and W R Jones (ed ) Immunology of human reproduction Academic Press. London

28 Harrison, M. R. 1976 Maternal immunocompctence Π Proliferative responses of maternal lymphocytes in vitro and inhibition by serum from pregnant rats Scand J Immunol 5.881-885

29 Hlrahara, F., I. Goral, К. Tenuità, Y. Matsuzaki, Y. Sumiyoshl, and Y. Shlojima. 1980 Cellular immunity in pregnancy subpopulalions of T-lymphocyles bearing Fc receptors for IgG and IgM in pregnant women Clin Exp

Immunol 41:153-357 10 Ito, T., and T. Hoshino. 1962 Studies of the influences of

pregnancy and lactation on the thymus in the mouse Ζ Zellforsch 57-667-678

31 Kananl, S. R., and R. Knight. 1969 Relapsing amoebic colitis of 12 years' standing exacerbated by corticoste­roids Br Med J 2:611-614

32 Kenny, J. F., and M. Diamond. 1977 Immunological responsiveness to Ьмhenchía ioli during pregnancy ІПт feet Immun 16:174-180

33 Knox, A, W. 1950 Influence of pregnancy in mice on the course of infection with murine poliomyelitis virus Proc Soc Exp Biol Med 73:520-528

34 Lawson, J. B. 1967 Malaria pregnancy, ρ 59-72 In J B. Lawion and D В Steward (ed ), Obstetrics and gynaecology in the tropics E Arnold. English Language Book Society, London

35 Uwls, R., N. H. Lauenen, and S. Birnbaum. 1973 Malana associated with pregnancy Obstel Gynecol 42:696-700

36 McLean, j . M., J. G. Mm Icy, and А. С. C. Glbbs. 1974 Changes in the thymus, spleen and lymph nodes during pregnancy and lactation m the rat J Anat 118:221-229

37 Murglta, R. Α.. К. A. Goldl, S. Kontlanen, and H. WIRZSII. 1977 Alpha-fetoprotein induces suppressor T-cells in utro Nature Ц ondon) 267:257-259

38 Nkklin, S., and W. D. Bllllngton. 1979 Macrophage activity m mouse pregnancy J Rcprod Immunol 1.117-126

39 Ojo, O. \., T. I. Francis, and A. Onlfade. 1971 Massive liver necrosis in pregnancy W Afr Med J 20:339-144

40 Pepper, F. J. 1961 The effect of age. pregnancy and lactation on the thymus gland and lymph nodes of the mouse J Endocrinol 22.11('_348

41 Playfalr, J. II. L., J. B. de Sou ¿a, H. M. Dockrell, P. I . Agomo, and J. Taverne 1979 Cell-mediated immunity in the liver of mice vaccinated against malaria Nature (London) 282:711-734

42 Partilo, D. T.. H. M. Hellgren, and E. J Yunis. 1972 Depressed maternal lymphoevte response to phytohae-magglutinin in human pregnancy Lancet 1:769-77]

43 Rmehart, J. J., A. !.. S agone, S. P. Balcerzak, G. Λ. Ackerman, and A \. LoBugho. 1975 LffcUs of corticoste­roid therapy on human monocyte function N Engl J Med 292:236-241

44 Roberts, D W., and W. P. Weidanz. 1979 T-cell immunity to malaria in the B-cel) deficient mouse Am J Ггор Med Hyg 28:1-3

45 Sii 1er ι. P. K., F. Febres, L. E. Clemens, R. J. Chang, B. Gondos. and D. Stites. 1977 Progesterone and mainte­nance of pregnancy is progesterone nature's immunosup­pressant'' Ann Ν Y Acad Sci 2β6.184-197

46 Stlmson, W. H. 1976 Studies on the immunosuppressive properties of a pregnancy-associated alpha-macroglobu-lin Clin Exp Immunol 25:199-206

47 Thompson, J., and R. van Fürth. 1970 The effect of glucocorticostcroids on the kinetics of monocytes and peritoneal macrophages, ρ 255-264 In R V F-urth (ed ), Mononuclear phagocytes Blackwell Sdentine Publica­tions Oxford

48 Thompson, S. P., L. J. McMahon, and С A. Nugent. 1980 Endogenous Cortisol a regulator of (he number of lym­phocytes m peripheral blood Clin Immunol Immuno-pathol 17:506-514

49 Van Щк, Η., J. Testerink, and E. Noordegraaf. 1976 Stimulalion of the immune response against SRBC by reduction of corticosterone plasma levels Mediation by mononuclear phagocytes Cell Immunol 25:8-14

50 Van Zon, A. A. J. С and W. M. C. Ellng. 1980 Depressed malarial immunity in pregnant mice Infect Immun 28:630-612

51 Van Zon, A. A. J. C, and W. M. С Ellng. 1980 Pregnancy associated recrudescence in munne malaria {.Plasmodium berghei) Tropenmed Parasitol 31:402-408.

78

VOL 36,1982 PLASMA CORTICOSTERONE AND MALARIAL IMMUNITY

S2 Wyler.D. J., С N. Oster, and Т. С. Qiilnn 1979 The role of the spleen in malaria infections, ρ 181-204 /π Role oi the spleen in the immunology of parasitic diseases Tropi­cal Diseases Research Scries no 1 Schwabe & Co A G , Basel

M Young, L·. J., and C. I. Gomez. 1979 Enhancement of

herpes virus type 2 infection in pregnant mice Proc Soc Exp Biol Med 160"116-^20

54 Zan-Ban-, 1., D. Nachligal, and M Fddman. 1975 Mecha­nisms m immune tolerance U Specific immunosupprcs sion by T-lymphotytes of В memory cells in mice made tolerant to HSA Cell Immunol 17-202-214

79

80

Chapter VII

Malarial immunity in pregnant mice, in relation

to total and unbound corticosterone

A.A.J.С. van Zon, W.M.C, El ing, C.C. Hermsen

Th.J.J.ΓΙ. van de Wiel and M.E. Duives

Department of Cytology and Histology

Faculty of Medicine

University of Nijmegen

The Netherlands

Bulletin de la Société de Pathologie Exotique (1983) 76: 493-502

81

Bull Soc Path kx , 76 1983, 493-502

MALARIAL I M M U N I T Y I N P R E G N A N T MICE,

I N R E L A T I O N T O T O T A L

A N D U N B O U N D PLASMA C O R T I C O S T E R O N E

Jt\ Λ A J ( \ 111 /ON \\ 4 ( 1 1 I V . Г ( I f l ï lVsbN Th J J M Л m h \M] I \ M I Dl I\ I ь *

Si чч\п\

4 pregnancif dependent loss of malarial immunity is actompanied by an (excessive) mínase of total as »ill as free plasma iortnosterone This loss of immunity tvas largely prevented by adrenalectomy Woreoier, malarial immunity »as more sensitn-e to dexa methasone immunosuppression during pregnancy Primary infections are more virulent during pregnamy and like in reirudesient mice, (ause eutssive total and free plasma cortiíosterone levels Corticosterone may be lonsidtred an immuno regulatory arum factor during pregnancy the endoirtne regulation of »Inch is disturbed in pregnant, infected mue

Keywords MALXRIAI І Ч Ч І M T \ , Р П Ю \ А М Л ( I M M I N O S L P P R F S S I O N , COBTICOSTEHONF

HbSLMl·

HcUitions c n l n Гішшишіе paludéenne et les 1 oncentralion^ Ho cortiíosterone libre et totale ehez la souris gr<nide

t ne perte de l immunité paludéenne en rapport avei la gestation est atcompagnee par une augmtntatton importante de la Lortitosterone aussi bun Ubre que totale ( ttte pirte d*immunité est m grandi partie pres.emu par la surrenahitonne De plus Vimmunité paludéenne est plus sensible pendant la gebt ut ton a laition suppressne de la derametha-sone J <s primo invasions sont plus gra<e\ pendant la gestation et tomme tluz les souris qui présentent une recrudescente, elks entraînent un taur exiessif de cortiíosterone libre et plasmatujue La (ostuosterone peut ttre considérée tomme un fut leur stnijut d'immuno regulation pendant la gestation les mecamsmts endotnniens de sa regulation sont per­turbes chez la souris gravide

Mots-tles І ч ч і м г ь PALinihNNF, GISTVTION, IMMLNO SLPPHFSSION, ( O R T K O S T E -

HO\l·

I N T R Ü D L C T I O N

P r e g n a n t as compared to non p regnan t women exhibi t an inorcabcd inci­

dence of malar ia and higher paras i te ra tes (15 16) Heduct ion and e \ en

comple te loss of malar ial immuni ty was a b o obber \ed in a mur ine model

(*) Department of ( \ lolop\ and TIist»U>g\ Faculty of Medicine, Linvcrsilj of Nijmegen, 6500 HB Nijmegen, The Netherlands

82

BULLEII\ DE LA SOCIÉTP DE PATHOLOGIE EXOTIQUF

(Plasmodium berghei) dur ing ]>regnanc\ (24, 25I A.pproximateK 60 0 of

p regnan t mice rcmidebced (de\e loped pa t en t ιηΓριΙιοηί,Ί and the iiiajorit\ died

from the iiifcUion Vbortioii and ] i r t m a t u r i t \ were frequentlv оЬьегч ed in

reir i idescent nuée as has also heen ol)!>Lr\cd in h u m a n malaria in association

wilh pregnanes Morcoser as in h u m a n malaria reduced immuniU is more

f rcquentk obscrsed in p rmi ig ra \ idae t h a n in mii l t igrasidae 124) Reduced

malarial i m m u n i U dur ing pregnanes m a \ be a side effect of a functional

immunosuppress ion fasouring s u m s a l of a fœtal allograft (13) bnhanced sur-

s i s a l of e g cardiac allografts '•>,) and a l tera t ions m P H \ dependent U m p h o -

(Ste s t imulat ion the mixed K m p h o e s t e cul ture as well as c s l o h l i c T-lvni-

phocs t e generat ion (1 22 point to suppression of cell mediated immune reac

turns dur ing pregnanes Changes m com n i t r a t i o n of s i r u m fador s ssith

immunosuppress i se propert ies e g a l p in fa ' loprotein, glucocorticoids œs t ro

gen progesterone H( d or cor tuoid b inding globulin ^CBCii 11 >) mas be insol-

sed m th( modula t ion of the inununc response In the unirme model rediietion

of malarial і т ш и т і л ssas found to be related to mcicascd p h s n i a corticoste­

rone concentrat ions Morcosei recrudescence during ] ircginncs ssas largels

])resenLed in adrenalec lomized m u e abrogat ing coit icosterone product ion (26)

These results suggest i n s o l s e r m n l of p lasma ( o r h c o s U r o n e in the regulal ion

of malar ia n n m u n i t s during p n guani s in mice Since the free fraction of

cort icosterone (not hound to сorticold b inding globulin is considered to be

biologicalls aclis e 2 its actis its in pregnanes associated rediic lion of malaria

n n m u n i t s ssas analssed

I h c serious patliolog\ associated swlh the infection 1231 prompted a n a l s s i s

of the (ffec I of an infeclion on the plasma concentra t ion of fret c o r t u o s u r o m

in non p r e g n a n t mice as well as dur ing dilTcient pli iscs of pregnanes Гиг

thermorc it was tested sshclhcr regulal ion of i m t e m a l i i i imunils lis endoge­

nous cort icosterone resulted 111 increased susccpl i lnbls to immunosuppressum

bs c t o g e n o u s corticoid ' d e x a m c t h a s o n e

VISTHIIVL ANO SIFrilODS

Animili·, J i a n d o m bred specific pathogen free Swiss mice were obta ined

from the animal facilities of the I nisersilv of Nijmegen at the age of four

t o six sseeks The mice ssere kept in plastic cages ssith food and dr inking

ssaler ad libitum

Pregnant mm fsso or three s irgine female mice ssere housed ssith one male and checked for a saginal plug for a period of 4 d a s s I h e das t h e vaginal plug ssas found was considered to be das I of pregnan \

Parasite Plannodium berghei s t ra in К і / З ssas mainta ined bs sseekls subi-

noculat ion of infected blood into normal mice T h e infection was ei ther s t a r t e d

bs int raper i toneal (non p r e g n a n t miccj or i n t r a s e n o u s (pregnant mice) i r icc-

tion of 10 s paras i t i/ed er s throcv les PI 1 The course of a primarv infection

ss Inch is alssass lethal in iinlreateel mice has been described presiouslv (11)

83

BULLETIN DE LA SOCIÉTÉ DE PATHOLOGIE EXOTIQUE

Immune mice The immunization procedure has been described pre \iousl\ (9) In short, mice infected with ΙΟ' ΡΠ were treated with sulfa diazinc (30 mg'l drinking water) from two da\s after infection onwards Vfter 2Ç) da\s the drug was remo\ed and two da\s later a challenge infection (IO1 Vh,) was gi\en to assess immunity No or onh low transient parasitae-mia (< 5 %) was obsersed Only mice wilh established immunity were used for the experiments Iminiinitv \\as of the jjremunition type ι e mice that did not deyelop an infection after challenge may harbour parasites (detected by isodiagnosis, see below) for a long time (10) In these mice spontaneous recrudescences y\cre rare

Rlood iamphng l o r the measurement of the total and/or free corticoste­rone in the plasma a blood sample of about 150 μΐ was obtained from the retro-orbital plexus, using hepanmzed glass capillaries Aiter centrifugalion the plasma was diyided into two samples y\hich were stored at — 20° С ЛИ blood samples were collected betyycen 11 00 h and 12 00 h a m to ay old interference by the circadian yanalion of the hormone concentration

Recrudescence Mice were arbitrarily considered to haye recrudesced when parasitaemia rose to > 5 % In bwiss mice approximately 80 % of the mice dcycloping a recrudescence during pregnancy died from the infection (25), indnating a complete loss of immunity Parasitaemias were determined from a thin smear made from a drop of tail blood stained with May-Grunwald-Giemsa's solution

Isodiagnosu Io examine the presence of parasites in the blood of immune animals during pregnancy approximately ϋ 05 ml blood was subinoculatcd into normal mice which y\orc checked for parasitaemia oyer a 2 week period Immune mice with a negatiye subinoculation test were remoyed from the experiments

Cortuosterone The total concentration of plasma corticosterone yyas measu­red by a radio-immunoassay (HIVj as described prcyiously (26) The concen­tration of free eortieosLerone yvas determined bv equilibrium dialysis in a Dianorm bquilibnum Dialyser (Diachema \.G, Zurich, Switzerland) according to Hoss (20) ІЗГІСПУ plasma samples wert diluted II times in Hank's solution ЬиЯегсгі with 20 mM Ilepes and dialyzed against О 27 % Dcxlran T70 ^Phar-macia, L ppsala, Sweden) in the same buller Dextran was included to obtain equilibrium between the macromolccular osmotic pressure from diluted plasma and bulfer (15) A quantity of 3 H corticosterone (Amersham Ltd , England), approximately equal to I % of the corticosterone concentration of the sample in the plasma compartment yyas added to the dextran containing buder compartment 'Intiated corticosterone yyas purified by paper chromatography in a modified Bush B5 system (26) before use Contamination after chromato-graphy was 0 3 %, as tested by binding to an excess of the anticorticosterone antibody used in the radio-immunoassay A correction for this contamination was included in the calculation of the concentration of free corticosterone in the plasma sample The samples were dialyzcd for 5 h at 37o С in rotating dialysis cells (working volume 150 μ.1, the dialysis membrane had a molecular

84

BVLIETIN DE LA SOCIÉTÉ DE PATH010G1E EXOTIQUE

weight cut-oiî value of 5,000) Hadioaclivitv was counted after diaksis 111 a 100 μΐ aliquot of each compartment The fraction of free corticosterone (f) in the diluted plasma was derived from 2 times the radioactivil> in 100 μΐ of the buffer compailment divided bv the sum of the aclnitie» in 100 μΐ of the plasma and buffer compartments The ratio of free to bound corticosterone

(f/b) in the diluted plasma could be calculated as f/b = —, In preliminarv

experiments a direct proportionaliU was found between fib and the dilution factor of the plasma (maximal dilution tested was ^2) with a correlation of 0 999 for both normal plasma and plasma from dav 16 of pregnancv Therefore the ratio fjb in undiluted plasma could be calculated b\ dividing the latio f/b in diluted plasma li) the dilution factor ( = 22) of the dialvsis svstem From this result and the concentration of total plasma corticosterone measured in the RIA, the percentage and eoncentralion of free corticosterone in the plasma could be calculated The intra assa> coefficient of variation in the equilibrium dialjsis was 25 % (n = 4), the inter assaj variation was 15 % ( n = i 7 )

Rh SC LTS

Plasma corticosterone level during prignancy The plasma corlicostcrone concentration (bound + free) increased during pregnancv with a maximum in the third week and returned to low levels shortlv before parturition v26)

Measurement of the concentrations of free corticosterone did not reveal a comparable change in the plasma level during pregnancv (fig I A) During pregnancy sbghtlv elevated concentrations of free plasma corticosterone were found on da\ TO and 18 whereas лег low values were found immcdiatelv after parturition

Plasma corticosterone concentration and loss of immunity during pregnancy Approximatel\ half of the mice lose their malarial immunity during prcgnaiic\ (24, 25, sec also below) In these mice loss of immunitv was related to increa­sed levels of total plasma corticosterone 126) likewise, the comentration of free corticosterone was increased in the plasma of mice that had lost their immunitv during pregnancv (fig if);

The total plasma corticosterone concentration returned to low levels before parturition, independent of whether mice had lost their ininiuiutj and suffered from a patent infection or remained immune throughout pregnancj (26) The concentration of free cortuosterone, however, remained high in 3 out of 4 mice that had lost immunitv during pregnane} (fig \K\ The fourth mouse with a patent infection and л low plasma concentration of free corticosterone shortly before parturition did not exhibit patene) two dav s earlier, in contrast to the other 3 mice In mice that maintained their immunitj during pre­gnancv the plasma concentration of free corticosterone was low shortlv after parturition

The concentration of free plasma corticosterone in mice that appeared to lose immunitv at a later point during pregnane) had already increased from

85

BULLETIN DE LA SOCIÉTÉ DE PATHOLOGIE EXOTIQUE

. Free corticosterone ƒ pregnant mice

jug /lOOml

* * i ' M f ? l·

Γ" I

6 0 0 -

лоо-

2 0 0 -

к? PE

•

-щ

- Total corticosterone/pregnant mice —, with primary infection

day 7

Ρ

'À — ι — ι—'

С

day 9

,

У. I —F-i—1

о controls • int eel ed

I day 11

» э

_Free corticosterone/ immunized pregnant mice

в

• recrudescence A recrudescence later о no recrudescence

Free corticosterone/pregnant mice I with primary infection

D P P P

è iill6 l' '•

hi i jij 1&кЦ

2 0 10 15 20 10 15 2 0 10 15 20

Day of p r e g n a n c y

Γιρ I — Plasma corliroslerone cononnlratmn^ íug/loo ml — ЬК\[ , η = 3 l t ) 71

in [iregnanL jn\4f

day 14 of pregnancy onwards, when rompared to those that maintained immu­nity throughout. A comparable ohber\ation was, made earlier with regard to the total plasma corticosterone level (26).

Excessive enrticosterone levels in mice with a malaria infection during pre­gnancy: The corticosterone levels (bound + free) in mice suffering a malaria recrudescence as a result of a pregnancy associated loss of immunity were frequently higher than the range observed in normal mice (26) Total plasma corticosterone levels in non-pregnant mice with malaria infection were not or only transiently e l a t e d (table I, also 26). Excessive total plasma cortico­sterone Іел-еЬ, however, were found in non-immune mice with a primary infection during pregnancy, and the extent depended on the day of pregnancy chosen to infect the animals (fig. 1С). Infections started on da\ 9 of pregnancv resultcd in excessive total plasma corticosterone levels in comparison to the normal pregnancy range, whereas increases were smaller when mice were infec­ted on day 7 or day II of pregnancy.

Free plasma corticosterone concentrations were elevated in infected non­pregnant (table I) as well as in pregnant mice (fig. ID). In non-pregnant mice a primary infection free plasma corticosterone levels were substantially

86

BULLETIN DE LA SOCIÉTÉ DE PATHOLOGIE EXOTIQUE

T A B I Ε I

Total and free plasmi corticosterone during a malaria infection in non pregnant nuce

Da\ ι after ІПГГГІІОП

1

7 9

l'arasi Infima Co - ЬГМ . η - 5 lo io

о 7 6 - 1 7

440 ± 28 46 7 - f> 7

Plasma rorlirosteronc ΙμςΙιοο ml _ SMI η = 5 lo IO)

Toni , Froc

32 7 - 5 о io о = 8 г 2 2 8 - 7 3 47« 4 7 30 2 - 5 о

¡

о 99 — о 21 о 66 ± о 45 ο 8 ί ι о ц 3 27 о ч 3 20 а. I 25

increased from da\ 7 "f infection onwards (table I) Increased free levels were found earlier when non-immune mice were infected during pregnamv 'Ihis appears to coincide with a more virulent course of infection during pre­gnancy (see below)

Virulence of infection during pregnancy Primary infections started either on dav 7, 9 or 11 of pregnamv exhibited an increased virulence in comparison to non pregnant controls Parasitaemia developed faster in pregnant mice reaching values of I % parasitized red Mood cells on dav 2 after inoculation and 27 5 % on day 5, compared to о % and 7.6 %, respectively, in non­pregnant mue

In separate experiments it was found that the mean survival time after infection was shorter in pregnant ÍIO б ± 0 8 davs, η = 52) than in non-pre­gnant controls 2̂5 4 L 0 6 days, η = 45) The increased virulence of an infec­tion during pregnancy mav explain the earlier increase of the plasma concen­tration of free corticosterone in these mice f̂ig iDl Despite an equally increa­sed virulence of infections started either on dav 7, 9 or II of prcgnancv, the increase of plasma corticosterone was highest when the infection was started on day 9 of pregnancy (fig iC, ID)

Increased sensitivity to dexamelhasone іттипочирргечзюп during pregnancy. The relation between plasma corticosterone levels and loss of immunity during pregnancy (26) suggested the possibilitv of an increased susceptibility to gluco­corticoid immunosuppression during pregnancy

When non-pregnant, immune mice received 20 nig dexamelhasone/1 of drinking water all animals developed lethal recrudescences, indicating loss of immunity When 10 or 5 mg dexamethasone/1 were added to the drinking water 73 % (19/26) and 8 % (1/12), respectively, of the mice lost immunity When 5 mg/1 was given to immune mice during pregnancy, however, 76 % (Π/17) lost their immunity, as compared to 47 % (7/'5) of pregnant mice not additionally treated with dexamelhasone

87

HbLLETlN DE LA SOCIÉTÉ DE PATHOLOGIE EXOTIQUE

DlSCl SSION

A considerable proportion of nuce Іоье a solid malarial {Ρ berghei) imiiiu-nit\ during іігейііап(\ (24, 25) The concentration of total plasma corticoste­rone (hound + free) was increased in recrudescent mice Adrenalectoniv before pregnanc\, prc\enting production of plasma corticosterone largck reduced loss of immunitv during pregnancv Since total plasma torticoslerone concentra­tions were not markedK ele\ated in non pregnant mice during a malaria infec­tion, the mfedion per vc apparentK did not cause the increased corticoid le\els (tahle I, also 26) A. possible causal relalion between immunosupprcsbivc adion of plasma corticosterone and loss of immumts during pregnancv needed further investigation, since the biological actniU of the glucocorticoids is usuallv not ascribed to total plasma corticosleronc but to the free, non prutein-bound fraclion of the hormone

According to the results described in this paper, loss of immunitv during pregnane;, appears to be related not onh to increased concentrations of total but also of free plasma corticostcione In normal mice the increase of total plasma corticosterone during the last week of pregnancv was not accompanied bv elevated concentrations of free hormone dig I \), indicating a parallel increase of corticosterone-binding globulin In immune mice with recrudescence during pregnancv, however, the free plasma corticosterone concentration was increased (hg iB^ No indications were found that in mice losing immunity corticosterone concentrations were elevated earK 111 pregnancv Thus, on da\ IO of pregnancv the mean concentration (^t SPAT) of free plasma corti­costerone was the same in mice t i n t lost immunitv later during pregnancv as compared to those that remained immune (fig iHl

The increase of total plasma corticosterone in pregnant mice with recru­descent malaria are frequenth above the range observed during normal pre gnancv '26) This was also observed during pnmarv infection in pregnant mice (fig" iCl, but not in non-pregnant mice with a pnmarv infection (table I) Moreover the excessive plasma corticosterone concenlrations are apparcnUv related to the dav of pregnancv on which the infection was ini­tiated (lig iC) This suggests that, unlike in normal mice, in pregnant mice the plasma corticosterone regulation is deranged bv malaria infection, and that there is a phase during pregnancv (close to dav 9) when the beginning of an infection renders regulation especiallv sensitive Whether increased stress sensitivilv during gestation (4^ is responsible for these observations remains to be clarified

Increase of the concentration of free corticosterone during patent malaria infection was independent of changes in the total corticosterone concentration In pregnant mice with a well established patent infection the free concen­tration remained high shortlv before parturition (fig ]B), despite a reduction in the concentration of total plasma corticosterone at time (26) During pnmarv infection in non-pregnant mice the free concentration but not the total concentration increased with the severity of the infection (table I) These increased concentrations of free corticosterone nia\ be explained b\ a reduced

88

BUUEIIS DE IA SOCIÉTÉ Dl· PATHOLOGIE EXOTIQUE

CI3G production in Ihp li\er as a consequente of the sexere liver patholog\ observed at the end of the first week of infection ^ 1 , 23) I his ma\ also ечріаш whv the mouse a patcnev of less than 2 da\s on da\ 20 of pregnancv did noi show an increased toncentralion of free corticosterone in contrast to the other 3 on that da\ of pregnant\ (fig IB) Thus, the plasma concen tration of free «orticostcrone is jiroliahK also dependent on normal liver fune tion for CUG production

The excessive levels of total and free plasma corticosterone are (oncomitant to patent malaria vpriinar\ infeehon or recrudescence) during jiregnancv 'I his reiterated the question whether plasma (orticoslcrone levels are causallv related to loss ol immunitv The correlation between imreased plasma levels of corti­costerone and loss of immunitv, and the reduction of the recrudescence rate in adrenalertomized animals suggested a regulatorv role for corticosterone in the elTettor svstem of ininiumlv Corticoid treatment (dexamethasone) of immune mue resulted in breakdown of immunitv, and during pregnancv immune mue appeared to be more sensitive to coilicoid immunosupprcbsion than non pregnant immune mice These observations emphasize the suscepti-bihtv of the tlfeclor svstem of malaria immunitv to corticoid iinrnunosuppres sion, espeiiallv during pregnancv, and support the immunoregulatorj role of plasma corticoid

Recrudescence of occult infections after corticoid treatment vvas also shown in other parasitic diseases, e g murine giardiasis (17) strongvloidiasis (18) and amoelnasis (2Г) Moreover, a more severe course of ainoclm colitis vvas described during pregnancv {1), as well as of infections of Liberia monocyto genesis and Toioplusma gondii (14; Immunosuppression during pregnancv especiallv concerns cell mediated immunitv as indiialed bv reduced resistance to several vnal and parasitic infections m vivo (22), and reduced actiwtv in several l-cell assavs 6 71 m vitro csing pregnancv derived material This correlates with the action of glucocorticoids which are thought to exert tluir major suppressive elTcct on Τ kinphoi ν les and 1 cell dependent macrophage ailivitv, both in the initiation and the efTcctor phases of the immune res­ponse {2, S1 In contrast, humoral immunitv is not considered to be suppres­sed during jiregnancv ι e pregnancv is usuallv no contra indication for vaccination In malarial immunitv 1 -cells and macrophages ріал an important role (12Ì PregnaiKN assodateci loss of malarial immunitv is correlated with an Ulerease of the plasma concentration of both total and free corticosterone

In summarv the observations could be interpreted as follows

— changes 111 the plasma corticosterone com cntiation are functional in jiregnancv dejiendent immunorcgiilation in general,

— a nalurallv occurring high plasma corticosterone level predisposes to loss of malarial immuniH during pregnaniv,

- - jiatent infection during pregnancv causes derangement of endocrine regulation, and consequentlv excessive plasma corticosterone levels

Though these mechanisms mav equally applj to Primigravidae and multi-gravidae, loss of malarial immunitv is less frequent in mulligravidae Previous results (25) indicated that re exposure to the jiarasilc during a pregnancy

89

BL/LE77V DE LA SOCIÉTÉ DE PATHOIOGIE EXOTlQbE

dependent recrudescencp substantialK reduced the recrudesccnLC rate during

subsequent pregnancies This suggested either a re inforcement of an existing

immune response, or a shift to a tortnoid insensilne immune response л

problem which is currenth heing in\estigatcd

\cKNOWI FDCMtNTS

We are grateful to Τ BEMIAAD and Η A. Ross (Department of Experi­

mental and Chemical Kndocrinolog\, Lni\crsit\ of Nijmegen) for advices to

plasma corticosterone measurements and for providing the antiserum We also

thank J RriTSMA and G POI-LES for hioleehmcal collaboration and care of the

animals and M L \\ u s s for reading the manuscript

The investigations received financial support from BION /Λλ 0

LlTFRAICB!

1 VBIO>F 'A V — Katril amoebu colitis in pregmncv and puerpenum a new clinirn pathologic id entitv J Trop Med llyg , 1973, 76, 97 100

2 BACH Ϊ Γ — Corlicosteroids In The mode of action of immunosuppressive agents I d I I · Bach North Holland Publishing ( о V'clam 1975, ρ 21 91

3 H U M S M G , M I I L \ R К G \ P R O S I II I - Ulografl enhancement during normal murine pregnane \ J /?<prod Immunol , 1980, 2, 141 149

\ BARLOW S M , MonnisoN ι Ρ I cV bc i i ivvN Ι M — EITects of acute and chronic stress on plasnia corticosterone levels 111 the pregnant and non pregnant mouse J l·ndocr , 1975, 66 93 99

5 liriiv Π S & VNDERSON M Ï hileiparum m llana and pergnancv Trant R Чос Trop \l(d Ili/g [979, 73, 427 431

6 ( н ос т G , MONMÌT Ρ ι, ΙΙΟΓΙ-4ΑΝ% \\ λ \ O I S I N G λ Reguhtorv

Г-cclls in pregnane ν \ I bviclencc for Τ cell mediated suppression of CTI gencr ition towards paternal alloantigens ( eli Immunol 1982,68,322331

7 ( 1 ARK v l) V tV MeDiRviorr M H \ctive suppression of host ν s graft re.ic tion in pregn nit mice III Developmentil kinetics properties unci mecha nism of inclue tion of suppressor cells during first pregnane} J Immunol , 1981 127, 1267 1273

8 DLNC V> I M R , b\DLiK J R ι λ І І М ] П Е > I V\ 1 —Glucocorticoid modulation of lymphnkinc induced macrophage proliferation ( ell Immunol, 1982, 67 23 36

9 E I I N C . \\ M ( Plasmodium berphei effec t of antithvmoc vte serum on indue tion of immunity in the mouse fixp Parnsitol , 1979, 47, 403 409

10 KLINO \\ M ( 1 Plasmodium berghei premumtion, sterile immunity, and loss of immunitv in mice Ετρ Parasitai, 1980, 49, 8 9 9 6

11 biLis-G *\\ \ \ an / O N ( V λ I F R C S A I F M С - T h e course of a Plasmodium her ghei infection in six different mouse strains Ζ Parasilenk , 1977, 54, 29 45

12 IAÏ AV\ \ηοι·Ν \ ι λ N j — Immune responses 111 malaria In Parasitic Diseases \ol I The Immunology, Ld I Mansfield, \I Dekker Ine , "Sew York, 1981, ρ 85 136

13 LiwREM l· iR 1, ( ue RCH ( I V \ RICHARDS \\ ) λ B O R Í Y (Μ I —Immunologica l mechanisms in the maintenance of pregnancy 4nn of illergy, 1980, 44, 166-173

14 LtbT (B J ) λ REMINGTON ( I S — I fleet of pregnancy cm resistance to Listeria monocytogenesis and Toxoplasma gondii infections 111 mice Infect Immun, 1982, 38, I164-I171

90

BVIJ-KTIf DE LA SOCIÉTÉ DE PATHOLOGIE EXOTIQUE

15 MATSI 1 (N 1, YAMAMOTO ·\\.\ SFO H.., N'IINOMI 1M.1 & Y I T A K A Ά . ) . - Deter­mination of serum Cortisol fractions by isocolloidosmol.ir eqiiiHbrium dialysis. Changes during pregnancy. Endocrinol, .lapon., 1979, 26, 2б}-2"3·

16. MCGRFGOR ' 1 . A. 1, \\ 11 sos, \'l. R., ά. D I L I F W K Z \\. Z.Ì. - Malaria infecliim of the phuenta in the Gambia, West Africa: its incidence and relationship to stillbirth, birth«eight and placental weight. Trans. R. Soc. Trop. Med. Hi/g., 1983, 77, 232-244

17. N V I R I K . Υ , Ο Ι Ι Ι Ο Ν Л. λ. F E R R I SON Ι Λ. . — Corlicosleroid t reatment increases paiasite numbers in murine giardiasis Gut. 19ЙІ, 22, 475-480.

18. N E E H · I,. 1. , Ρ ί Μ ΐ ι ι O.', ( i v a v c i s i \ . Ρ.1 ά. H A L F H Η . . Disseminated strongyloidiasis with cerebr.il in\olvemeiil a complication of corticosteroid therapy. .Im. J. Med., 1973, 55, 832-837.

19. N u k i i s . S. 4. HiiLiNGTON \V. D.i. — Macrophage actiMty in mouse pregnancy. J. Itcprod Immunol., 1979, 1, II7-126.

20. Ross «11. \ . - S\mnielric and equilibrium dialysis for the measurement of free lodothy ronine and steroid hormones in blood. Thesis, l*ni\ersit\ of Nijmegen, the N'cthcrlands, 1980.

21. S T I V E R 11'. С.) & Gol ο ιΤ. Л. L. M.1 - Cortilosteroids and li\er amoebiasis. Br Med. ./., 1978, 2, 394-195.

22. THONG ι Y IL· , S I I H F H. \\.\ YINC 1 vr (M. M.l, H H N S F N 'S. \ : *. B E L ·

LVNTI (Л. A.l — Impaired in vitro cell-mediated immunity to rubella \irus during [iregnaney. Λ' Εημ. J. Med., 1973, 289, 604-606.

23. Лап / O N [\.ì, KiiNG Л\. (S; .II-RLSVIFM С.1. Misto- and inimiinopnthology of a malaria f Plasmodium berghei) infection in гпк e. Israel J. Sied. Sa., 1978, 14, 659-672.

24. Yaii ZON (A. A. .1. C 1 & K I I N G ΛΥ. M. C ' . — Depressed malarial immunity in pregnant mice. Infect. Immun., 1980, 28, 630-632.

25. \ a n /.i>\ (А.А.Л.С.1 λ J^iiNG Λ\. M.C.». — Pregnancy associated recrudescence in murine malaria f Plasmodium berghei). Trop.'nmed. Parasitol., 1980, 31, 402-408.

26. \ a n ¿ON А. А. Л. С 1, E L I N G \V. M. C.i, H F R M S I N С C.' 4. Koi к-

KOEK 'A. \. G. M j . — (Corticosterone regulation of the efTcelor funttion of malarial immunity (luring pregnaniy. Infect. Immun., 1982, 36, 484-'t9I.

91

92

Chapter Vili

ACTH dependent modulation of malaria immunity

in mice

Adriaan A.J.C. van Zon, Wijnand M.C, Eling

Theo P.M, Schetters and C.CRob Hermsen

Department of Cytology and Histology

Faculty of Medicine

University of Nijmegen

The Netherlands

Submitted for publication

93

Vili ACTH dependent modulation of malaria immunity in mice

ABSTRACT

Tetracosactrin, a synthetic ACTH analogue delivered from osmotic

minipumps implanted subcutaneously in mice induced a dose dependent

increase of plasma corticosterone levels. In mice with an established

immunity to Plasmodium berghei the increase of the plasma corticoste­

rone level due to tetracosactrin treatment correlated with loss of

immunity against this malaria parasite. The observed plasma corticos­

terone levels associated with loss of malaria immunity were of the

same order as those in mice that lost their immunity during pregnancy.

Adrenalectomy before administration of the ACTH analogue preven­

ted both the increase of plasma corticosterone and loss of malaria

immunity. Adrenalectorni zed mice still lost their malaria immunity

when treated with the synthetic corticoid dexamethasone. The effector

function of malaria immunity is sensitive to corticoids, and, at least

during pregnancy, the naturally occurring serum corticosterone level

appears to be an important regulator of malaria immunity.

INTRODUCTION

Glucocorticoids have a well known effect on the immune response,

though its mechanism of action is far from clear. A prominent lympho-

lytic effect is observed at supra-physiological doses (1). At physio­

logical levels a regulatory role of the immune response, possibly by

modulation of the release of soluble mediators from immunologically

active cells has been proposed (2, 7, 8, 9).

Patients treated with corticoids have an increased susceptibility

to infections. Glucocorticoid treatment may affect the host-parasite

equilibrium of a clinical immunity, and provoke recrudescence of a

subpatent infection both in humans (17, 23) and mice (14, 18). A preg­

nancy associated reduction of malaria immunity has repeatedly been des­

cribed in humans (3, 4, 5, 16, 21), as well as in mice (25, 26). In the

94

Plasmodium berghei - mouse model this pregnancy associated loss of immunity was correlated with increased plasma corticosterone levels,

and could be prevented by adrenalectomy performed before pregnancy

(27, 28). The next question was whether the regulatory function of

plasma corticosterone in malaria immunity during pregnancy could

also operate in non-pregnants.

The results reported here show that an ACTH (-analogue) induced

increase of the plasma corticosterone level correlated with loss of

malaria immunity in non-pregnant mice. Plasma corticosterone levels

in these mice were of the same order as those observed during preg­

nancy. Adrenalectomy before treatment with the ACTH analogue preven­

ted both the increase of plasma corticosterone and loss of malaria

immunity, though in these mice immunity was lost after dexamethasone

treatment.

The results show that plasma corticosterone is an effective re­

gulator of the effector function of malaria immunity, which, in turn

strongly suggests that pregnancy associated loss of immunity is caused

by increased plasma corticosterone levels.

MATERIALS AND METHODS

Mice. Random bred, specific pathogen free Swiss mice were obtained

from the animal facilities of the University of Nijmegen. They were

housed in plastic cages and received food and water ad libitum.

Parasite. Plasmodium berghei, strain K173, was maintained by

weekly subinoculation of infected blood in normal mice. The malaria

infection was always lethal in unprotected mice, the course of the in­

fection has been described earlier (11).

Immune mice. Mice were immunized to P. berghei as described by

Eling (12). In short, mice were infected with 10? parasitized erythro­

cytes (PE) and from day 2 to day 33 after infection treated with sulfa­

diazine (30 mg/L) added to the drinking water to prevent patent infec­

tion. Two days after termination of sulfadiazine treatment the mice

95

were challenged (10^ PE) to assess immunity.

Immunity was of the premunition type with low numbers of surviving

parasites in the immune host. Spontaneous recrudescences were not ob­

served. Shortly before implantation of an osmotic pump, the immune mice

were challenged again to ensure the presence of parasites, and thus the

possibility of development of a recrudescence during the time of hor­

mone treatment. Immune mice without recrudescence during hormone treat­

ment were checked for the presence of parasites by isodiagnosis after­

wards (see below).

Recrudescence. Development of a recrudescent infection was checked

in thin blood smears (May-Griinwald Giemsa staining) made from tail

blood three times a week. Mice with a parasitemia of 5% or more infec­

ted red blood cells were considered to have lost their immunity. The

5% limit was chosen, because previous experience had shown that the

majority of immune mice developing parasitemias exceding 5% finally

succumbed to the infection.

Isodiagnosis. Presence of parasites in the peripheral blood of

immune mice was examined by subinoculation of tail blood into normal

mice. These mice were checked for development of a malaria infection

over a two week period.

Adrenalectomy. Mice were anesthetized with ether, and a lateral

incision was made on each side. The muscle wall was separated and

the adrenal glands removed, using blunt forceps.

Adrenalectomized mice were injected with 1 ml saline subcutane-

ously daily for a period of 7 days. Th^received 0.9°i NaCl solution

as drinking water and were housed at 28° С for the time of the expe­

riment. The time between adrenalectomy and implantation of an osmotic

pump was at least 14 days.

Immunosuppressive treatment. Immune mice were treated with cor­

ticosterone, dexamethasone, or tetracosactrin (Synacthen, Ciba), a

synthetic polypeptide with ACTH activity. The substances were in­

jected into osmotic minipumps (Alza Coop.type 2002) which were im-

96

planted subcutaneously. According to the manufacturer the release rate

of the pumps is 0.43 μΐ/h, and completely filled pumps would be active

for a period of about 2 weeks.

For corticosterone, a solution containing 25 pg/ul, which is close

to the saturation concentration in polyethylene glycol 400 (26 yg/vil )

(29) was injected into the osmotic minipumps, resulting in a calculated

dose of 258 ug/24 h. The dexamethasone concentration was related to the

previously established immunosuppressive effect of 20 mg, 10 mg or 5

mg/L of dexamethasone when added to the drinking water of the mice (28)

Assuming a daily intake of 3 ml drinking water per mouse, the 24 h

doses of dexamethasone are 60 pg, 30 \ig and 15 yg respectively, which

is equivalent to a concentration of 5.81 yg, 2.91 yg and 1.45 ug dexa­

methasone per yl in the mi ni pump. The dexamethasone also was dissolved

in polyethylene glycol 400 (29).

For tetracosactrin the equivalent of 0.1 ID, 0.05 IU and 0.025 IU

of ACTH, released in a volume of 10.32 yl per 24 h, was injected in the

pumps. The activity of 1 mg of tetracosactrin is assumed to be equiva­

lent to 100 IU of corticotropin (Martindale, the Extra Pharmacopeia,

26th ed. 1972). Thus, tetracosactrin concentrations in the minipumps

were 0.1 yg, 0.05 yg and 0.025 yg per yl of Hanks solution buffered

with 20 mM Hepes.

Plasma corticosterone. Total plasma corticosterone was measured

by a radioimmunoassay (RIA) using a sheep-anti-corticosterone serum

(gift from T. Benraad, Dept. of Experimental and Chemical Endocrinolo­

gy, University of Nijmegen) as described previously (27). The concen­

tration of free corticosterone in the plasma was determined by equili­

brium dialysis according to the method of Ross (H.A. Ross, M.D. thesis,

University of Nijmegen, 1980), modified as described in detail else­

where (28). In brief, plasma samples, 11 times diluted in Hank's solu­

tion buffered with 20 mM Hepes, were dialysed against 0.27% dextran

T70 (Pharmacia, Sweden) in the same buffer. A quantity of 3H cortico­

sterone (Amersham Ltd., England), approximately equal to 1% of the cor-

97

ticosterone concentration in the plasma compartment, was added to the

dextran containing buffer compartment. Samples were dialysed for 5 h

at 37° С in rotating dialysis cells with a working volume of 150 μΐ

and a dialysis membrane with a molecular weight cut-off value of 5000.

After dialysis, radioactivity was counted in 100 μΐ aliquots of each

compartment. The fraction of free corticosterone (f) in the diluted

plasma was derived from 2 times the radioactivity in 100 μΐ of the

buffer compartment divided by the sum of activities in 100 yl of the

plasma and buffer compartment. The ratio of free to bound corticoste­

rone (f/b) in the diluted plasma could be calculated as: f/b = y ^ p

the ratio f/b in undiluted plasma by dividing the ratio f/b in di­

luted plasma by the dilution factor (= 22) of the dialysis system

(in preliminary experiments a direct proportionality was found between

f/b and the dilution factor). From this result and the total concen­

tration plasma corticosterone as measured in the RIA, the concentra­

tion of free hormone was calculated.

RESULTS

Effect of glucocorticoid treatment. Corticosterone in the mini-

pump (25 μς/μΐ ) resulted in a calculated release of 258 μg per 24 h.

Although this dose seems high in relation to the basal plasma level

of 0.1 μg/ml plasma (27), it did not result in a detectable increase

in the plasma corticosterone concentration during the 3 week period

following implantation of the minipumps (results not shown).

Moreover, immune mice treated with corticosterone by minipumps did

not lose their malaria immunity. Dexamethasone in the minipumps,

however, led to loss of malaria immunity. The calculated doses of

60 μg and 30 μg of dexamethasone released from the minipumps over a

24 h period caused loss of malaria immunity in all four mice tested

within 1 to 2 weeks after implantation. These regimens correspond to

20 mg and 10 mg of dexamethasone per liter drinking water, which also

led to loss of malaria immunity. A calculated dose of 15 μg dexametha-

98

soné delivered from the minipumps per 24 h did not cause loss of

immunity, which is in accordance with the lack of effect when an

equivalent dose of 5 mg/L drinking water was administered (28).

The calculated dose of 60 pg dexamethasone per 24 h delivered

from the minipumps suppressed the endogenous plasma cortiscoste-

rone level completely (result not shown).

Effect of tetracosactrin in intact immune mice

Stimulation of the adrenal steroidogenesis by continuous ACTH

administration resulted in an increase of the plasma corticosterone

concentration in rats (13). Therefore, a solution of tetracosactrin,

a polypeptide with ACTH properties, was injected in the minipumps

which were implanted subcutaneously into immune mice. In these mice,

a dose dependent increase of the plasma corticosterone concentration

was observed (Fig. 1 and 2). A dose of tetracosactrin equivalent to

0.025 ID ACTH per 24 h neither increased the total nor the free plasma

corticosterone level (Fig. 1A and 2A). Insertion of a pump secreting

an equivalent dose of 0.05 IU ACTH per 24 h resulted in no or only a

small increase of plasma corticosterone (Fig. IB and 2B), but a dose

equivalent to 0.1 IU ACTH per 24 h increased both the total and, even

more, the free plasma corticosterone levels in all mice tested (Fig.

1С and 2C). Particularly on day 2 and 4 after implantation a remar­

kable increase was observed. In some mice the plasma corticosterone

levels returned to normal on day 7 and 9 after implantation, but in

others increased levels were found for longer periods. The reason

for this relatively early return to normal plasma corticosterone le­

vels is unknown. It may be the result of a feed-back adaptation of

the mice to increased corticotropin activity or to a reduction of the

activity of the osmotic minipump.

Loss of malaria immunity was observed in all mice receiving a

dose equivalent to 0.1 IU ACTH per 24 h, and in one of the three mice

receiving a dose equivalent to 0.05 IU ACTH. In this mouse, in contrast

to the other two of this group, the plasma corticosterone level was

99

I total plasma corticosterone during tetracosactnn

(ACTH analogue) administration in immune mice

yug/100 ml o intact, no recrudescence • intact, recrudescence

4 0

2 0 -

0 n = 5 » Ax, no recrudescence » Ax, recrudescence

4 0

2 0

τ 1—ι 1 1 1 1 1 1 1 1 1 1 1 Γ-

® n=3

о ·

100-1

-8 0 -

6 0 -

-| 4 0 -

2 0 -

©

L.

ntact

• • • • • •

•

J f - r

n=7,

•

•

I

Ax n*13

:

• •

! Μ Γ I

•

• •

•

t •

I • M 10 14 16

days

Fig. 1. Total plasma oort-Laosterone during tetraaosaotrin (ACTH analogue) release from osmotic minipumps, subautaneously implanted on day 0. The aaloulated amount of tetraaosaotrin released was equivalent to: A 0.025 IU, В 0.05 IU and С 0.1 IU of ACTH per 24 h, respectively. The open sym­bols refer to mice that remained immune throughout, whereas the closed symbols refer to mice that developed lethal recrudescent infections. Circles refer to intact mice, triangles to adrenalectomized (Ax) mice. Plasma values are either depicted as mean + SEM, or they are plotted individually, since not all mice were sampled each day.

increased on day 7 after implantation of the minipump (Fig. IB). Immune

mice, receiving a dose equivalent to 0.025 IU ACTH neither lost malaria

immunity nor exhibited increased plasma corticosterone levels.

In mice that lost immunity the hormone provoked recrudescences that

became patent 7 to 9 days after implantation of the minipump, and survi-

100

(ree plasma corticosterone during tetracosactnn (ACTH analogue) administration in immune mice

/ug/ЮО ml o intact, no recrudescence • intact, recrudescence

(д) n i 5 * Ax, no recrudescence ^S » Ax, recrudescence

f I I

2 -

(В) η · 3

4-^4- τ — ι — ι — ι — Γ "

12 -

-10 -

θ -

6 -

4 -

2 -

_

(с) intact n»7 W Ax n.4

•

• • 1 •

If»*. V I I ,

• •

- т - ^ - т

•

•

1

• •

• •

•

ι ' Τ 1 1

•

ι 1 V 6 θ K) 12 M 16

days

Fig. 2. Free plasma corticosterone during tetraoosaatrin (ACTH analogue) release from osmotic minipimps. For further details see legend to Fig.l.

val periods varied from 12 to 20 days after implantation. The virulence

of the recrudescent infection suggests a more or less complete loss of

protective reactions in the previously immune mice. Mice treated with

low doses of the ACTH analogue and remaining immune troughout, did ex­

hibit a reticulocytosis in the peripheral blood, and some mice exhibi­

ted a low (2% or less) and transient parasitemia. All the immune mice

in these experiments harbored parasites in their peripheral blood

throughout the duration of the experiment, as tested by subinoculation.

Thus, all mice had the opportunity to develop recrudescences during

treatment.

101

In conclusion, treatment of immune mice with a synthetic ACTH ana­

logue induced loss of malaria immunity, when the dose administered was

sufficiently high to increase the endogenous plasma corticosterone level.

Effect of tetracosactrin on adrenalectomized immune mice

To analyse whether increased plasma corticosterone levels after ad­

ministration of the ACTH analogue to immune mice are responsible for

loss of malaria immunity, osmotic minipumps filled with tetracosactrin

solution were inserted in adrenalectomized, immune mice. A dose equiva­

lent to 0.05 IU ACTH per 24 h did not induce loss of immunity (n = 5),

and a dose equivalent to 0.1 ID ACTH resulted in loss of immunity in

only one out of 13 mice. This mouse died 10 days after implantation of

the osmotic pump, which is earlier than any of the intact mice with loss

of immunity during treatment with tetracosactrin (compare previous sec­

tion).

Adrenalectomized immune mice, treated with the ACTH analogue, did

not exhibit increased plasma corticosterone concentrations, neither of

total nor free corticosterone. Furthermore, no increase in plasma cor­

ticosterone was observed in the one adrenalectomized mouse with a fatal

recrudescence (Fig. 1С and 2C). All mice developed a reticulocytosis in

the peripheral blood during hormone treatment, and the subinoculation

test confirming presence of parasites, and thus the possibility of de­

velopment of a recrudescence, was positive in all animals.

In a group of 9 adrenalectomized mice treated with an equivalent

dose of 0.1 IU ACTH, the minipumps were removed three weeks after im­

plantation. One week later the mice received a challenge (10^ PE/mouse),

and dexamethasone (20 mg/L) was added to their 0.9% NaCl drinking water.

Within 3 weeks 5 out of these 9 mice developed a lethal malaria recru­

descence. The other 4 mice died also, but parasitemia was not observed.

In summary, treatment with an ACTH analogue did not cause loss of

immunity in adrenalectomized immune mice, though immunity of these mice

remained susceptible to glucocorticoid treatment.

102

DISCUSSION

A remarkable proportion of mice with an established immunity

against the murine malaria parasite Plasmodium berghei lose their

immunity during pergnancy (25, 26). Loss of immunity during preg­

nancy was related to increased levels of both total and free plasma

corticosterone. Moreover, when the increase in plasma corticosterone

in the mother was prevented by adrenalectomy before pregnancy, loss

of immunity during pregnancy was drastically reduced (27). These re­

sults suggested that reduced immune reactivity during pregnancy cau­

sing loss of a preexisting antimalaria immunity was causally related

to pregnancy dependent changes in corticosterone levels. They further

suggested that experimental manipulation causing an increase in plasma

corticosterone in immune, non-pregnant mice would also lead to a loss

of antimalaria immunity. The experiments described in this paper support

this hypothesis and indicate that the function of the effector system of

malaria immunity is sensitive to, and can be regulated by, normally oc­

curring plasma corticosterone levels.

A prolonged increase of the plasma corticosterone concentration by

administration of exogenous corticosterone is not easily obtained. In­

jection of corticosterone acetate neither resulted in a prolonged in­

crease of the plasma corticosterone level, nor was it very effective in

suppression of malaria immunity (W. El ing and A. van Zon, Parasitology

77: ii, 1978). Furthermore, continuous administration of a maximum at­

tainable concentration of corticosterone (29) by osmotic minipumps again

failed to increase plasma level and to break malaria immunity.

The sensitivity of the effector system of malaria immunity for cor-

ticoids, however, was demonstrated by the effect of dexamethasone treat­

ment. A dose dependent loss of malaria immunity was obtained when either

dexamethasone was added to the drinking water of the mice (28) or a

dose of dexamethasone equivalent to the daily intake through drinking

water was delivered by a subcutaneously implanted osmotic minipump. The

30-fold stronger potency of dexamethasone relative to corticosterone (1)

may explain the observed differential effect of these substances. These

103

results in turn suggested that increased plasma corticosterone levels

could be expected to suppress malaria immunity.

In view of the effect of ACTH treatment on the plasma corticoste­

rone level in rats (13) it was tried to manipulate the corticosterone

level in mice by administrating the ACTH analogue tetracosactrin by

minipump. Tetracosactrin in subcutaneously implanted minipumps provoked

a dose dependent increase of both the total and, to an even higher ex­

tent, the free corticosterone concentration in plasma. In all instances

increased plasma corticosterone levels were related to loss of malaria

immunity and vice versa. The plasma concentrations of total corticoste­

rone in tetracosactrin treated mice that had lost immunity were lower

(Fig. 1) than those in pregnant mice with loss of immunity (27). The

plasma concentration of free corticosterone, however, was higher in

tetracosactrin treated mice (Fig. 2) relative to pregnant ones (28).

All tetracosactrin treated immune mice developed a reticulocytosis,

but in spite of the preference of p. berghei for young erythrocytes (30)

no relation was found with loss of immunity. Moreover, it appeared that

the highest concentration of tetracosactrin in normal, control mice in­

duced reticulocytosis, also (result not shown).

To further reinforce the notion that corticosterone is causally

related to loss of malaria immunity, tetracosactrin was given to adre-

nalectomized mice. Corticosterone levels remained undetectable in these

mice, and loss of immunity was drastically reduced to 8% (1 out of 13)

in comparison to 100% in the corresponding intact group. Since immunity

in adrenalectomized mice remained sensitive to dexamethasone treatment,

the effects obtained with tetracosactrin are apparently directly related

to its induction of an increased adrenal output of corticosterone. That

corticosterone is not the only suppressor of malaria immunity is shown

by the one adrenalectomized mouse that developed a fatal recrudescence

upon treatment with tetracosactrin and without detectable plasma corti­

costerone.

Thus, experimentally induced changes in the serum corticosterone

levels affect the parasite-host equilibrium of immune mice. The immuno-

104

suppressive effect of corticosterone on malaria immunity had been sug­

gested earlier by the observed relationship between plasma corticoste­

rone level and loss of malaria immunity in pregnant mice (27).

The immunoregulatory role of endogenous glucocorticoids is sugges­

ted to act either by modulation of the release of soluble mediators,

such as interleukines (2, 8, 9), or by regulation of peripheral lympho­

cyte numbers (24). In this respect the leveling out or disappearance of

the diurnal changes in serum corticosterone during pregnancy (6, 19) or

during tetracosactrin treatment may play a role as well.

In jztro studies indicated a differential sensivity of subsets of

Τ cells to glucocorticoid immunosuppression, and the importance of Τ

cell growth factor inthe medium for its expression (7, 22). The immuno­

regulatory action of glucocorticoids is supposed to occur at the level

of the Τ cell-macrophageinteraction (8, 22), and both these cells play

an important role in malaria immunity (10, 15, 20). In conclusion,

changes in the plasma corticoid level have a regulatory effect on the

immune response of mice against the malaria parasite Plasnoi^m оегдкег.

ACKNOWLEDGEMENT

We are grateful to T. Benraad and H.A. Ross (Department of Experimental and Chemical Endocrinology, University of Nijmegen) for advices to plasma corticosterone measurements and for providing the antiserum. We thank J. Reitsma and G. Poelen for biotechmcal colla­boration and care of the animals and M.L. Weiss for reading the manu­script.

These investigations were supported by the Foundation for Funda­mental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO).

LITERATURE CITED

1. Bach, J.F. 1975. Corticosteroids,p. 21-91. In· J.F. Bach (ed.), The mode of action of immunosuppressive agents. North-Holland Publishing Co., Amsterdam.

2. Besedovsky, H.O., A.E. del Rey, and E. Sorkin. 1983. What do the immune system and the brain know about each other

7 Immunol.

Today 4: 342-346.

105

3. Bray, R.S., and M.J. Anderson. 1979. Faleiparun malaria and pregnancy. Trans. R. Soc. Trop. Med. Hyg. 73: 427-431.

4. Bruce-Chwatt, L.J. 1983. Malaria and pregnancy. Br. Med. J. 286: 1457-1458.

5. Campbell, C.C., J.M. Martinez, and W.E. Collins. 1980. Sero-epidemiological studies of malaria in pregnant women and newborns from coastal El Salvador. Am. J. Trop. Med. Hyg. 29: 151-157.

6. Challis, J.R.G., and J.E. Patrick. 1983. Changes in the diurnal rhythms of plasma Cortisol in women during the third trimes­ter of pregnancy. Gynecol, obstet. Invest. 16: 27-32

7. Claësson, M.H., and С Röpke. 1983. Colony formation by subpo­pulations of Τ lymphocytes. IV. Inhibitory effect of hydro­cortisone on human and murine Τ cell subsets. Clin. exp. Immunol. 54: 554-560.

8. Cupps, T.R., and A.S. Fauci. 1982. Corticosteroid-mediated immunoregulation in man. Immunol. Rev. 65: 133-155.

9. Duncan, M.R., J.R. Sadlik, and J.W. Hadden. 1982. Glucocorti­coid modulation of lymphokine-induced macrophage prolifera­tion. Cell. Immunol. 67: 23-36.

10. El ing, W.M.C. 1979. Ріазтоа мп berghei: effect of antithymocyte serum on induction of immunity in the mouse. Exp. Parasitol. 47: 403-409.

11. Eling, W., A. van Zon, and C. Jerusalem. 1977. The course of a Plasmodium berghei infection in six different mouse strains. Z. Parasitenkd. 54: 29-45.

12. Eling, W., and С Jerusalem. 1977. Active immunization against the malaria parasite Vlasmodv^m berghei in mice: Sulfathia-zole treatment of a P. berghei infection and development of immunity. Tropenmed. Parasit. 28: 158-174.

13. Freeman, R.H., J.O. Davis, and D. Fullerton. 1980. Chronic ACTH administration and the development of hypertension in rats. Proc. Soc. Exp. Biol. Med. 163: 473-477.

14. Grove, D.I., and H.J.S. Dawkins. 1981. Effects of prednisolone on murine strongyloidiasis. Parasitology 83: 401-409.

15. Jayawardena, A.N. 1981. Immune responses in malaria, p. 85-136. In: J. Mansfield (ed.), Parasitic Diseases. Vol. 1, The Immu­nology. M. Dekker Inc., New York.

16. McGregor, I.A., M.E. Wilson, and W.Z. Billewicz. 1983. Malaria infection of the placenta in The Gambia, West Africa; its incidence and relationship to stillbirth, birthweight and placental weight. Trans. R. Soc. Trop. Med. Hyg. 77: 232-244.

17. Neefe, L.I., 0. Pinilla, V.P. Garagusi, and H. Bauer. 1973. Disseminated strongyloidiasis with cerebral involvement, a complication of corticosteroid therapy. Am. J. Med. 55: 832-837.

18. Nair, K.V., J. Gillon, and A. Ferguson. 1981. Corticosteroid treatment increases parasite numbers in murine giardiasis. Gut 22: 475-480.

106

19. Molten, W.E., and P.A. Rueckert. 1981. Elevated free Cortisol index in pregnancy; possible regulatory mechanisms. Am. J. Obstet. Gynecol. 139: 492-498.

20. Playfair, J.H.L. 1982. Immunity to malaria. Br. Med. Bull. 38: 153-159.

21. Reinhardt, M.C., P. Ambroise-Thomas, R. Cavallo-Serra, С. Meylan, and R. Gautier. 1978. Malaria at delivery in Abidjan. Helv. Paediatr. Acta 33, suppl. 41: 65-84.

22. Stites, O.P., and P.K. Siiteri. 1983. Steroids as immuno­suppressants in pregnancy. Immunol. Rev. 75: 117-138.

23. Stuiver, P.C., and Th. J.L.M. Goud. 1978. Corticosteroids and liver amoebiasis. Br. Med. J. ii: 394-395.

24. Thomson, S.P., L.J. McMahon, and C.A. Nugent. 1980. Endogenous Cortisol: a regulator of the number of lymphocytes in peri­pheral blood. Clin. Immunol. Immunopathol. 17: 506-514.

25. Van Zon, A.A.J.С, and W.M.C. Eling. 1980. Depressed malarial immunity in pregnant mice. Infect. Immun. 28: 630-632.

26. Van Zon, A.A.J.С, and W.M.C. Eling. 1980. Pregnancy associated recrudescence in murine malaria [nasnodium berghei). Tropen-med. Parasit. 31: 402-408.

27. Van Zon, A.A.J.С, W.M.C. Eling, C.C. Hermsen, and A.A.G.M. Koekkoek. 1982. Corticosterone regulation of the effector function of malarial immunity during pregnancy. Infect. Immun. 36: 484-491.

28. Van Zon, A.A.J.С, W.M.C. Eling, C.C Hermsen, T.J.J.M. van de Wiel, and M.E. Duives. 1983. Malarial immunity in pregnant mice, in relation to total and unbound plasma corticosterone. Bull. Soc. Path. Ex. 76: 493-502.

29. Will, P.C., R.N. Cortright, and U. Hopfer. 1980. Polyethylene glycols as solvents in implantable osmotic pumps. J. Pharma­ceutical. Sciences 69: 747-749.

30. Zuckerman, A. 1957. Blood loss and replacement in plasmodial infections. I. Plasnodivm berghei in untreated rats of vary­ing age and in adult rats with erythropoietic mechanisms manipulated before inoculation. J. Infect. Dis. 100: 172-185.

107

108

Chapter IX

The effect of progesterone and DES

on malaria immunity in mice

A,A.J.C, van Zon, C C , Hermsen

and

W.M.C. E l ing

Department of Cytology and Histology

Faculty of Medicine

University of Nijmegen

The Netherlands

109

IX The effect of progesterone and DES on malaria immunity in mice

INTRODUCTION

Functional maternal immune reactivity is usually considered to be

partly suppressed during pregnancy (Beer and Billingham 1972, Gusdon

1983). In view of immunosuppressive properties of hormones in in oitro

assays of immune responsiveness the considerable increase in plasma

levels of these hormones during pregnancy may contribute to the preg­

nancy associated immunosuppression. In this respect glucocorticoids,

progesterone and oestrogens are of particular interest (Barlow et al.

1974, Boorman et al. 1980, Szekeres et al. 1981, Stites and Siiteri

1983).

Previous work already showed a correlation between loss of malaria

immunity during pregnancy, and an increased plasma corticosterone level

(van Zon et al. 1982, 1983). In addition, treatment of non-pregnant

immune mice with glucocorticoids or an increase of the plasma cortico­

sterone level by ACTH treatment caused loss of immunity also (van Zon

et al., manuscript submitted, is chapter 8 of this thesis). These results

suggested an immunoregulatory role for corticosterone in the effector

function of malaria immunity. Considering the immunosuppressive effects

of progesterone and oestrogen, and their increasing plasma concentra­

tions during pregnancy a similar immunoregulatory role may exist for

these hormones. This possibility was analysed by treatment of non­

pregnant immune mice with progesterone, or diethylstilbestrol (DES),

a synthetic oestrogen. Loss of malaria immunity due to treatment with

either progesterone or DES was not obtained.

MATERIALS AND METHODS

Mice: Random bred female Swiss mice were obtained from the ani­

mal facilities of the University of Nijmegen. They were housed in

plastic cages and received food and drinking water ad ІіЪгіш.

Parasite: Plasmodium berghei, strain K173, was used in all ex­

periments. Infection with this parasite was lethal in unprotected

110

mice. Mice were immunized to this parasite, as described previously

(Eling and Jerusalem 1977). Established immunity was of the pre-

munition type, i.e. small numbers of parasites survived in the

immune host. Immune mice did not develop patency after a challenge,

and spontaneous recrudescence was rare.

Recrudescence: Development of a recrudescent infection during

hormone treatment was checked by thin blood smears (May-Grünwald

Giemsa staining) made from tail blood three times a week. Mice with

a parasitaemia of 5% or more were considered to have lost immunity.

Isodiagnosis: The presence of surviving parasites in immune

mice which maintained immunity during hormone treatment was deter­

mined by subinoculation of tail blood into normal mice. These mice

were checked for development of parasitaemia for a period of two

weeks.

Hormone treatment: For oestrogen treatment diethylstilbestrol

(DES), a synthetic nonsteroidal compound possessing oestrogenic ac­

tivity, was used. Mice were injected with DES, dissolved in olive

oil, or received DES diphosphate (Honvan, Asta-Werke A.G., Germany)

in the drinking water. For the calculation of the amount of DES,

taken with the drinking water, a daily uptake of 3 ml water per

mouse was assumed.

Progesterone was administered by injection, or dissolved in the

drinking water, or through release of progesterone from subcutaneous-

ly implanted osmotic minipumps (Alza Coop, type 2002). Progesterone

administered by injection was dissolved in 1 volume benzylalcohol,

and diluted with 9 volumes of arachis oil.

Progesterone in the drinking water was offered as a saturated

solution of progesterone in tap water. A suspension of 50 mg/L was

stirred for 24 h and filtered through filter paper. The progesterone

containing drinking water was supplied in dark drinking bottles. Pro­

gesterone solution was freshly prepared every week, and the drinking

bottles were refreshed twice a week.

Osmotic minipumps were filled with saturated solutions of pro-

111

gesterone in polyethylene glycol 400 (PEG) or in ethanol. Two sus­

pensions of progesterone in PEG, 25 mg/ml and 50 mg/ml respectively,

were sonicated and kept at room temperature for 5 days. The suspen­

sions were centrifugated and the supernatant was injected into the

osmotic minipumps (maximum solubility of progesterone in PEG is 27

g/L at 37° C, according to the literature; Will et al. 1980).

A saturated solution of progesterone in ethanol was prepared

by suspending 250 mg progesterone per ml ethanol. After 5 days the

suspension was centrifugated and the supernatant was injected in the

osmotic pumps (maximum solubility of progesterone in ethanol is 125

g/L, according to the literature; Martindale, the Extra Pharmacopeia,

26th ed. 1972).

The release rate of the pumps was 0.48 μΐ/h (indicated by the

manufacturer) and completely filled pumps should be active for at

least two weeks. The calculated maximal dose administered by the os­

motic minipumps was 0.29 mg per 24 h for progesterone in PEG and 1.44

mg per 24 h for progesterone in ethanol.

Plasma progesterone: Progesterone concentration in the plasma was

measured using the radioimmunoassay described by Thomas et al. (1982).

Plasma corticosterone: Total concentration plasma corticosterone

was determined by a radioimmunoassay described previously (van Zon et

al. 1982). Free corticosterone in plasma was determined by the techni­

que of equilibrium dialysis (van Zon et al. 1983).

RESULTS AND DISCUSSION

The effect of PES and DES-diphosphate on malaria immunity

A group of 5 immune mice received daily, subcutaneous injections

of 0.2 mg DES, dissolved in 0.1 ml olive oil, for a period of 14 days.

A control group of 5 immune mice were injected with olive oil only.

Bloodsmears of the mice were checked for parasitaemia, but no recru­

descent infections were observed. A challenge with 10 parasitized

erythrocytes on day 9 of the injection period was cleared without

112

causing patent parasitaemia. It has been reported that oestrogens sti­

mulate the production of corticoid binding globulin (CBG) in the liver,

and that oestrogen levels during pregnancy are high enough to induce

the observed increase of CBG (Musa et al. 1965, Wilson et al. 1979).

Since CBG has a high affinity for Cortisol, a reduction of the circu­

lating free Cortisol by increased CBG production leads to a feedback

increase of ACTH secretion which in turn results in increased gluco­

corticoid production (Doe et al. 1969, Heap et al. 1973). In this way

oestrogens might influence the plasma glucocorticoid levels indirectly,

which in turn might affect the immune response to malaria (van Zon et al., manuscript submitted, is chapter 8 of this thesis). To check this

relation in DES treated mice, the concentration plasma corticosterone

was measured after 3 and 6 days of injections. Compared to control

values in untreated mice, the plasma corticosterone concentration was

not changed in the mice either injected with DES dissolved in oil, or

in those injected with olive oil only.

Our experiments with dexamethasone treatment had shown that in­

take through drinking water was more effective than injection (Eling

1982). Supply in the drinking water also avoids the stress of daily

injections over longer periods and local skin irritation which might

cause hormonal changes that affect malaria immunity. Therefore, in the

following experiments oestrogen treatment consisted of DES diphosphate

dissolved in the drinking water of the mice.

Since previous work of Boorman et al. (1980) had shown that DES

treatment of mice caused marked alterations in lymphoid organs, the

effect of DES-containing drinking water on thymus, spleen, and liver

weight was analysed, too. Results obtained after 7 or 8 days treat­

ment are summarized in table I. Doses of 100 mg or 150 mg per liter

drinking water resulted in a marked decrease of thymus weight, an in­

crease of liver weight, and no change in spleen weight. These results

agree with those of Boorman et al. (1980), except that they observed

an increase in spleen weight in DES-treated mice. The depletion of

the thymus is supposed to be the result of redistribution and mobili­

zation of thymic lymphocytes into the circulation, but treatment with

113

Table I. Thymus, spleen, and liver weights in miae excused to DES

(mean + SEM, η = ό to 5)

Concentration

DES diphosphate calculated thymus spleen liver

in drinking dose weight weight weight

water (mg/L) (mg/day) (mg) (mg) (mg)

О 0 96.1+8.9103.2+6.0 1005+48

100 0.30 8.5+1.6 92.9+31.9 1553 + 56

150 0.45 19.2 + 2.5 108.4+11.9 1600 + 72

high doses of DES is also reported to cause depletion of certain

subsets of lymphocytes (Seaman et al. 1978, Kal land 1980a and 1980b).

The observed changes in organ weight of mice treated with DES

in the drinking water suggested that this method might be suitable

for the analysis of the effect of oestrogenic activity on malaria

immunity in mice. For this purpose, groups of 5 immune mice received

100 mg, 150 mg, 250 mg, 500 mg or 1000 mg of DES, respectively, per

liter drinking water. Bloodsmears of these mice were checked for

parasitaemia three times a week during a period of 5 weeks. In the

group with 100 mg DES per liter drinking water, one mouse died with

high parasitaemia, and in the 500 mg group one mouse exhibited a

transient parasitaemia during 1 week. In the other mice no recrudes­

cent infections were observed.

In the groups of mice receiving 250 mg, 500 mg and 1000 mg DES

per liter drinking water, the corticosterone concentration in the

plasma was determined on several days during the first two weeks

of treatment. Plasma corticosterone concentration did not increase,

neither the total nor the free concentration, and no dose dependent

influences were observed. Thus.the CBG concentration in the plasma

was not increased by DES.

In previous experiments it had been shown that dexamethasone

added to the drinking water resulted in loss of malaria immunity in

a dose dependent way (van Zon et al. 1983). To compare the effect

114

of dexamethasone and DES both hormones were added to the drinking

water. Dexamethasone (10 mg/L drinking water) induced loss of malaria

immunity in 2 of 4 mice, dexamethasone and DES together (10 mg and

250 mg/L drinking water respectively) resulted in loss of immunity in

3 of 5 mice, and DES alone (250 mg/L drinking water) did not induce

loss of malaria immunity in 5 mice tested. The immunosuppressive

effect of dexamethasone agrees with previously observed results.

In conclusion, treatment of mice with DES to achieve oestrogenic

activity did not affect an existing malaria immunity.

Effect of progesterone on malaria immunity

It has been reported that daily injections of 2 mg progesterone

in mice resulted in an increase of progesterone concentration in the

plasma to levels observed during pregnancy (Baker and Plotkin 1978).

Progesterone at this physiological level suppressed in vitro Con A

stimulation of splenic lymphocytes and enhanced in vivo genital in­

fections with herpes simplex virus (Baker et al. 1980). To test the

effect of progesterone on malaria immunity, mice were injected sub-

cutaneously with 2 mg progesterone in a volume of 0.1 ml solvent for

12 days. A control group of immune mice was injected daily with the

solvent. Mice were checked for parasitaemia until 6 days after the

last injection. Two out of 8 mice injected with progesterone suffered

from recrudescent infection, compared with 1 of 7 control mice. These

results indicated that progesterone injections did not affect malaria

immunity.

A major disadvantage of daily injections was stress for the ani­

mals and skin irritations at the place of injection. In a subsequent

experiment progesterone was administered to mice with the drinking

water, but no loss of malaria immunity was observed. The maximal

solubility of progesterone in water may be too low to reach suffi­

ciently high doses in the mice. Therefore, progesterone was also

administered by subcutaneously implanted osmotic minipumps filled

with saturated solutions of progesterone in PEG or in ethanol. In

4 mice the plasma progesterone level was determined on the day of

115

implantation of the mi ni pump and on days 2 and 10 thereafter. Pro­

gesterone dissolved in ethanol did not influence the plasma proges­

terone level. Administration of progesterone in PEG resulted in a

small increase of plasma progesterone, but maximal serum levels ob­

served (57 ng/ml) were much lower than maximal levels reported for

pregnant mice (120 ng/ml; Murr et al. 1974). In previous experiments

it was also shown that administration of corticosterone by osmotic

minipumps did not induce an increase of the concentration cortico­

sterone in the plasma (van Zon et al. , manuscript submitted, is chapter 8 of this thesis), suggesting that the endocrine regulation

of the mice is able to compensate for the quantity of exogenously

administered hormones. The mice were checked for parasitaemia during

a period of 25 days following implantation of the minipumps. None of

the mice, treated with progesterone exhibited loss of malaria irmunity 5

or became patent after an additional challenge with 10 parasitized

erythrocytes 10 days after implantation.

In conclusion, progesterone treatment did not cause loss of

malaria immunity. The increase in serum progesterone levels in re­

lation to progesterone treatment reported by Baker et al. (1978)

could not be confirmed in our model, and observed serum levels were

not as high as those observed during pregnancy. From experiments

with corticosterone it is known that a sustained increase of plasma

hormone concentration is difficult to achieve by administration of

exogenous hormones. Further experiments are needed to clarify this

point.

There is one indirect observation that suggests that proges­

terone is not the major factor in suppression of malaria immunity

during pregnancy. Adrenalectomy largely prevents loss of malaria

immunity during pregnancy (van Zon et al. 1982), but is not known

to have an effect on progesterone production in pregnant mice.

Although the experiments tend to indicate that progesterone is

not an important factor in suppression of malaria immunity they are

not decisive, and do not rule out a possible role in the effector

function of malaria immunity.

116

ACKNOWLEDGEMENT

We are grateful to F. Walschot for his contribution to the

experiments and to C. Thomas and R. van de Berg (Dept. of Obstetrics

and Gynaecology, University of Nijmegen) for measurements of the

progesterone concentrations. We thank J. Reitsma, G. Poelen and co­

workers for biotechm'cal assistance and care of the animals, and

Dr. M.L. Weiss for reading the manuscript.

LITERATURE CITED

1. Baker, D.A. and S.A. Plotkin. 1978. Enhancement of vaginal infection in mice by herpes simplex virus type II with progesterone. Proc. Exp. Biol. Med. 158: 131-134.

2. Baker, D.A., Ch. A. Phillips, K. Roessner, R.J. Albertini and L.I. Mann. 1980. Suppression by progesterone of nonspecific in vitro lymphocyte stimulation in mice as a mechanism for the enhance­ment of herpes simplex virus type II vaginal infection. Am. J. Obstet. Gynecol. 136: 440-445.

3. Barlow, S.M., P.J. Morrison and P.M. Sullivan. 1974. Plasma corti­costerone levels during pregnancy in the mouse: the relative contributions of the adrenal glands and foeto-placental units. J. Endocrinol. 60: 473-483.

4. Beer, A.E. and R.E. Billingham. 1972. Immunobiology of mammalian reproduction. Adv. Immunol. 14: 1-84.

5. Boorman, G.A., M.I. Luster, J.H. Dean and R.E. Wilson. 1980. The effect of adult exposure to Jiethylstilbestrol in the mouse on macrophage function and numbers. J. Reticuloendothel. Soc. 28: 547-560.

6. Doe, R.P., P. Dickinson, H.H. Zinneman and U.S. Seal. 1969. Ele­vated nonprotein-bound Cortisol (NPC) in pregnancy, during estrogen administration and in carcinoma of the prostate. J. Clin. Endocr. 29: 757-766.

7. Eling, W.M.C. 1982. Immunopathological aspects in parasitic in­fections, pp. 141-155. In: Fortschritte der Zoologie, Band 27, Zbl. Bakt. Suppl. 12: Immune reactions to Parasites. Gustav Fischer Verlag, Stuttgart, New York.

8. Eling, W. and С Jerusalem. 1977. Active immunization against the malaria parasite Plasmodium berghei in mice: Sulfathiazole treatment of a P. berghei infection and development of immunity. Tropenmed. Parasit. 28: 158-174.

9. Gusdon, J.P. Jr. 1983. The immunology of reproduction. Am. J. Reprod. Immunol. 3: 5-6.

10. Heap, R.B., J.S. Perry and J.R.G. Challis. 1973. Hormonal main­tenance of pregnancy, pp. 217-260. In: R.O. Greep and E.B. Astwood (eds.). Handbook of Physiology, Section 7: Endocrino­logy, Volume II, Female reproductive System, Part 2. American Physiological Society, Washington, D.C.

117

11. Kalland, T. 1980a. Decreased and disproportionate T-cell popu­lation in adult mice after neonatal exposure to diethylstil-bestrol. Cell. Immunol. 51: 55-63.

12. Kalland, T. 1980b. Reduced natural killer activity in female mice after neonatal exposure to diethylsti1bestrol. J. Immunol. 124: 1297-1301.

13. Murr, S.M., G.H. Stabenfeldt, G.E. Bradford and I.I. Geschwind. 1974. Plasma progesterone during pregnancy in the mouse. Endo­crinology 94: 1209-1211.

14. Musa, В.U., U.S. Seal and R.P. Doe. 1965. Elevation of certain plasma proteins in man following estrogen administration: a dose response relationship. J. Clin. Endocrinol. Metab. 25: 1163-1166.

15. Seaman, W.E., M.A. Blackman, T.D. Gindhart, J.R. Roubinian, J.M. Loeb and N. Talal. 1978. 3-Estradiol reduces natural killer cells in mice. J. Immunol. 121: 2193-2198.

16. Stites, D.P. and P.K. Siiteri. 1983. Steroids as immunosuppressants in pregnancy. Immunol. Rev. 75: 117-138.

17. Szekeres, J., V. Csernus, S. Pejtsik, L. Emody and A.S. Pacsa. 1981. Progesterone as an immunologic blocking factor in human preg­nancy serum. J. Reprod. Immunol. 3: 333-339.

18. Thomas, C.M.G., L.A. Bastiaans and R. Rolland. 1982. Concentrations of unconjugated oestradiol and progesterone in blood plasma and Prostaglandine F-2a and E-2 in oviducts of hamsters during the oestrous cycle and in early pregnancy. J. Reprod. Pert. 66: 469-474.

19. Van Zon, A.A.J.C, W.M.C. Eling, C.C. Hermsen and A.A.G.M. Koekkoek. 1982. Corticosterone regulation of the effector function of ma­larial immunity during pregnancy. Infect. Immun. 36: 484-491.

20. Van Zon, A.A.J.C, W.M.C. Eling, C.C. Hermsen, T.J.J, van de Wiel and M.E. Duives. 1983. Malarial immunity in pregnant mice, in relation to total and unbound plasma corticosterone. Bull. Soc. Path. Ex. 76: 493-502.

21. Will, P.C., R.N. Cortright and U. Hopfer. 1980. Polyethylene gly­cols as solvents in implantable osmotic pumps. J. Pharmaceutical Sciences 69: 747-749.

22. Wilson, E.A., A.E. Finn, W. Rayburn and M.J. Jawad. 1979. Cortico-steroid-binding globulin and estrogens in maternal and cord blood. Am. J. Obstet. Gynecol. 135: 215-218.

118

Chapter Χ

Corticosterone and reduced spleen cell proliferation

ід vitro in relation to malaria immunity

during pregnancy

A,A,J,С van Zon, Rose-Marie Termaat,

T.P.M, Schetters and W.M.C. Eling

Department of Cytology and Histology

Faculty of Medi.oi.ne

University of Nijmegen

The Netherlands

Submitted for publication

119

X Corticosterone and reduced spleen cell proliferation in vitro in relation to malaria immunity during pregnancy

SUMMARY

Spleen cells from mice immune to Plasmodium berghei exhibited

a significantly increased ¿и vitro proliferative response to parasi­

tized reticulocytes (PRET) compared to spleen cells from normal mice.

The specific response to malaria antigen was decreased in spleen

cells from pregnant immune mice in contrast to the unspecific response

to the mitogen phytohaemagglutinin (PHA).Addition of mouse serum to

spleen cell cultures of immune mice depressed both the PHA and the

specific proliferative response, whereas serum of pregnant mice exerted

an even stronger inhibition than serum of non-pregnant mice. Charcaol

adsorption of mouse sera for the elimination of steroid hormones re­

moved the serum dependent immunosuppression from normal as well as

from pregnant serum. Corticosterone added to the spleen cell cultures

depressed also the proliferative response. These findings demonstrate

that the response to malaria antigen is decreased in immune mice during

pregnancy. The possible regulatory effect of serum corticosterone on

the depression of the immune response is discussed.

INTRODUCTION

Malaria can bring about serious complications during pregnancy,

which may vary from an increased incidence of malaria episodes in

women living in malaria endemic areas to more virulent, and even

fatal infections in areas with low endemicity or in non-immune indivi­

duals (Gilles, Lawson, Sibelas, Voller & Allan, 1969; Menon, 1972;

Bray & Anderson, 1979; Brabin, 1983). Loss of immunity during preg­

nancy is frequently associated with particularly heavy infections in

the placental vasculature (Osunkoya & Williams, 1980; McGregor, Wilson

& Billewicz, 1983) which is considered a privileged site where the

120

parasite can avoid immune reactivity of the mother (McGregor et al., 1983).

Previous work in the Plasmodium berghei - mouse model showed a more severe primary infection as well as loss of previously existing

anti - P. berghei immunity during pregnancy (Van Zon & Eling, 1980;

Oduola, Holbrook, Galbraith, Bank & Spicer, 1982; Van Zon, Eling &

Hermsen, 1983). Loss of immunity during pregnancy was associated with

increased serum levels of corticosterone and could largely be preven­

ted by eliminating maternal corticosterone production through adrenal­

ectomy before pregnancy (Van Zon, Eling, Hermsen & Koekkoek, 1982;

Van Zon, Eling, Hermsen, Van de Wiel & Duives, 1983). These results

suggested regulation of immune reactivity by serum corticoid. In gene­

ral, cell mediated immunity is considered to play an important part

in malaria immunity (Jayawardena, Targett, Leuchars, Carter, Doenhoff

& Davies, 1975; Jayawardena, 1981; Playfair, 1982) and cell mediated

rather than humoral immunity is depressed during pregnancy (Beer &

Billingham, 1972; Gusdon, 1983).

Plasmodiun infected erythrocytes can specifically stimulate

in vitro proliferation of lymphocytes in human experimental malaria

(Phillips, 1970; Wyler & Oppenheim, 1974; Weissberger, Golenser &

Spira, 1980; Troye-Blomberg, Perlmann, Patarroyo & Perlmann, 1983).

In addition, in mice the lymphocyte responsiveness to the parasite

in vitro was associated with persistence of the parasite in the pre­

mune host (Weinbaum, Evans & Tigelaar, 1976; Weinbaum, Weintraub,

Nkrumah, Evans, Tigelaar & Rosenberg, 1978).suggesting lymphocyte

responsiveness as a marker of antimalaria immunity. The experiments

described in this paper confirm the parasite dependent proliferation

of splenic lymphocytes from an immune mouse. Moreover, they show that

the in vitro lymphoproliferative response to specific antigen, and

not to the nonspecific mitogen phytohaemagglutinin, was lower in immune

pregnant mice during the period when loss of malaria was noted in vivo (Van Zon et al., 1982) compared to non-pregnant immune mice. Though

normal mouse serum added to the test system suppressed lymphocyte pro­

liferation, sera from pregnant mice were more suppressive. Suppressive

121

activity could be removed from both types of sera by preadsorption to

charcoal,a step being used to adsorb serum corticoid. Corticosterone

added to spleen cell cultures suppressed the specific as well as the

nonspecific lymphocyte proliferative response. The results suggest

that serum corticosterone can regulate immune reactivity by affecting

the number of antigen specific cells in the spleen as well as their

proliferative activity, and support the regulatory role of corticoste­

rone in malaria immunity during pregnancy.

MATERIALS AND METHODS

Mice. Inbred B10LP mice were obtained from the animal facilities

of the University of Nijmegen or from TNO, Zeist, The Netherlands. They

were housed in plastic cages and received food and water ad libitum.

Pregnant mice. Two or three female mice were housed with one male

and checked for vaginal plug on the next four days. Three days later

the mated mice were removed from the male. The day with the vaginal

plug was day one of pregnancy.

Parasite. Plasmodium berghei, strain K173, was maintained by

weekly passage of infected blood (10$ parasitized erythrocytes (PE)

i.p.) to normal animals. The primary infection is lethal in all mice

and has been described previously (Eling, Van Zon & Jerusalem, 1977).

Parasitized reticulocytes (PRET) used for spleen cell culture stimula­

tion were obtained from infected mice made anaemic by bleeding (12

drops) from the retro-orbital plexus using a glass capillary. Mice

were bled one day before and one day after infection. On day 5 after

infection sterile blood was obtained from these mice by heart puncture,

using pyrogen free heparin as anticoagulant. Leucocytes and platelets

were removed by passing the blood though a sterilized column, filled

with a 3 ml mixture of 1 part Servacel SE 23 (Serva Feinbiochemica,

Heidelberg) and 3 parts Sephadex superfine G 150 (Pharmacia) in saline.

Before use the column was washed with MEM (see below). The red blood

cell suspension was layered on a sterile 72% Percoli layer (Pharmacia),

and centrifuged at 1600 g, for 20 min. The cell layer on top of the

122

Percoli, which was enriched for parasitized reticulocytes, was removed,

washed in MEM, counted, suspended in RPMI (see below), and added to the

spleen cell cultures.

Immune mice. Mice were immunized to Plasmodium Ъетдкег as described

by Eling & Jerusalem (1977). In brief, mice were infected with IO? PE

and treated with sulfadiazine (30 mg/L drinking water) two days later.

On day 33 after infection, sulfadiazine treatment was stopped, and 2

days later the mice were challenged with 105 PE/mouse to asses immunity.

The acquired immunity was of the premumtion type, i.e. immune mice

harboured low numbers of parasites for varying periods of time. Immune

mice were radically cured with chloroquine (Nivaquine, Specia, Paris;

0.8 mg/mouse i.p. for 5 days, supplemented with 100 mg Nivaquine/L

drinking water for 7 days) 1 to 4 weeks before their spleen cells were

used in culture.

Spleen cell culture. Spleens were removed aseptically and cell

suspensionswere prepared by pressing the tissue through a plastic sieve.

Cells were suspended in MEM (Eagle) (Gibco) buffered with Tris-HCl and

supplemented with penicilline (100 IU/ml), pyrogen free heparin (5 ID/

ml) and 10% heat inactivated (56° C, 30 m m ) foetal calf serum (PCS)

(Gibco). The cell suspension was incubated on a glass-woll column at

37 С for 30 m m . The non-adherent cells were eluted with MEM and

washed. Red blood cells in the suspension were lysed in Tris-buffered

ammonium chloride (Mishell & Shngi, 1980) with 10% PCS on ice for

5 m m , washed and counted. Red blood cell contamination was less than

5%. The percentage viable cells, assayed by trypan blue (0.025%) exclu­

sion was better than 90%. RPMI-1640 (Gibco) buffered with Hepes (20 mM)

and МаНСОз (10 mM),supplemented with L-glutamine (2 mM), penicilline

(100 IU/ml), 2-iiercaptoethanol (S.IO-2 mM) and 20% PCS was used as cul­

ture medium. A number of 7.5 χ 10^ spleen cells was cultured in a total

volume of 0.15 ml in Costar microtitre plates with flat bottom wells

for 3 days in a humidified atmosphere with 5% CO2 in air at 37° С

During the last 18 - 20 h, the cultures were labeled with 1 цСі 3H-

thymidine per well (spec. act. 25 Ci/mmol, Amersham Int.). After freezing

123

and thawing, cells were harvested using a Skatron cell harvester. Filters

were dried and radioactivity was counted in a liquid scintillation coun­

ter (Packard). Spleen cells were cultured with medium only (unstimulated),

or with an optimal concentration of PHA, (Gibco; 30 μΐ/ml culture) for

nonspecific stimulation. For specific stimulations 5 χ 10^ or 5 χ 10^

parasitized reticulocytes (PRET) were added to the cultures. Each group

consisted of 4 to 8 replicate cultures. In some experiments, corticoste­

rone (Sigma), or heat inactivated syngeneic serum was added to the cul­

ture medium.

Statistical evaluation. Significance of differences between 3H-thy-

midine incorporation in spleen cell cultures was assessed by theWilcoxon

test for unpaired samples (results of Fig. 1) and the Wilcoxon signed

rank test (results of Fig. 3).

RESULTS

Stimulation of spleen cells from immune mice

Spleen cells from normal and immune mice were nonspecifically sti­

mulated with PHA (Fig. 1). Though spleen cell suspensions from 2 out of

8 immune mice exhibited a stronger PHA-stimulation, there was no statis­

tically significant difference between data from normal or immune mice

(p = 0,067).

The addition of PRET specifically stimulated spleen cells from

immune animals (Fig. 1). Stimulation was dependent on the number of PRET

added, and was found optimal when 5 χ 10^ PRET were added to each culture

No stimulation was observed with less than 1 χ 10^ PRET, whereas proli­

feration was strongly inhibited when more than 1 χ IO6 PRET were added to

the cultures. Spleen cells from control (non-immune)mice were not stimu­

lated by addition of PRET, but rather a small inhibition of Зн thymidine

incorporation was observed, as compared to the unstimulated cultures (ne­

gative netto dpm) (Fig. 1). Addition of 1 χ 10^ erythrocytes or reticu­

locytes from uninfected mice exerted no stimulating effect on spleen

cell cultures, either from normal mice or from immune mice. When more

red blood cells were added to the culture, 3H thymidine incorporation

124

3 H Thymid ine incorporat ion d p m xlO"¿

Δ n o r m a l чл_ о i m m u n e

о · i m m u n e , p r e g n a n t о

8-

o

6' Ϊ л- î

2 -

O-

10 ' IO5 IO6

P H A N u m b e r o l P R E T / c u l t u r e

Fig. 1. H-thymidine incorporation in spleen cells from normal, immune and immune pregnant mice, stimulated by PUA or PRET. The dpm plotted is the difference between the mean of stimulated cultures and that of un­stimulated cultures. Each point represents the mean of 4 to 8 replicate cultures. (SEM 4 5%). The figure swmarizes results of independent expe­riments .

was lower than in cultures with medium only, as found with comparable

number of PRET.

Stimulation of spleen cells from pregnant immune mice

To examine the immune response to malaria during pregnancy in vitro, the PRET stimulation of spleen cells from pregnant immune mice was tested

and compared with the stimulation of spleen cells from non-pregnant mice.

Spleen cell cultures were prepared from 13 to 16 days pregnant mice.This

period was chosen, since recrudescent infections in mice that were immune

before pregnancy occured in this period (Van Zon et al., 1982). To prevent

stimulation of spleen cells in vivo by a recrudescent infection, survi­

ving parasites were removed by radical cure with chloroquine before ma­

ting. PHA stimulation of spleen cells from pregnant immune mice was not

significantly different from that of normal or non-pregnant immune mice

(Fig. 1). The dose dependency of the PRET response in spleen cell cul­

tures from pregnant immune mice was similar to that observed in non­

pregnant immune mice. The magnitude of the specific stimulation, however,

3-

1 i

125

was significantly lower in spleen cell cultures from pregnant immune,

as compared to non-pregnant immune mice (p = 0..036 with 5 χ 10^ PRET,

and ρ = 0.012 with 5 χ 10^ PRET; Fig. 1). In summary the results indi­

cate a reduced specific but not nonspecific capacity in spleens of im­

mune mice during pregnancy.

Effect of corticosterone on stimulation of spleen cells

The previously established correlation between loss of malaria

immunity and the concentration of corticosterone in serum of mice

during pregnancy (Van Zon et al., 1982, 1983) prompted the analysis

of the effect of corticosterone on the stimulation of spleen cells.

3 H Thymidine incorporation d p m xlO"^

\ · unstimulated о stimulated by PRFT Δ stimulated by PHA

Ϊ I

I

\ i L

ι - ι ' ' 1 1 Г ^

0 1 2 5 5 10 50 100 Corticosterone n g / m l cullure

Fig. 2. The effect of corticosterone on H-thymidine incorporation of spleen cells from immune mice. The concentration of corticosterone in the culture is plotted in a logarithmic scale, the dpm on a linear scale. Spleen cell cultures, either unstimulated or stimulated with PHA or PRET were prepared from the same immune donor. Values plotted are the mean + SEM of 4 replicate cultures.

For this purpose corticosterone was added directly to the spleen cell

cultures. The results depicted in Fig. 2 show a dose dependent inhibi-3

tion of Η-thymidine incorporation in unstimulated as well as PHA or

PRET stimulated cultures. In PHA or PRET stimulated spleen cell cul­

lo

126

tures inhibition of lymphocyte proliferation was observed with 5 ng

corticosterone/ml or more.

Effect of mouse serum on spleen cell cultures

The inhibitory effect of corticosterone on the specific and non­

specific stimulation of spleen cells (previous section), along with the

observed correlation between serum corticoid levels and loss of malaria

immunity (Van Zon et al., 1982, 1983) prompted analysis of the effect

of serum on stimulation of spleen cells. In pregnant mice the serum cor­

ticosterone concentration increases after day 10 of pregnancy to peak

values on day 16 to 18 (Barlow, Morrison & Sullivan, 1974; Van Zon et al., 1982). Thus, an increasing amount of serum from 16 days pregnant mice

(PrMS) was added to cultures of spleen cells from immune, non-pregnant

donors. The effect was tested in unstimulated, and PHA and PRET stimu­

lated cultures compared to the effect of serum from non-pregnant mice

(NMS) under comparable circumstances. For better comparison of the ef­

fect of NMS or PrMS on spleen cell cultures the results were expressed

as the relative ^H-thymidine incorporation in the presence of mouse

serum compared to that of cultures without serum (mean dpm of cultures

with NMS or PrMS divided by the mean dpm of parallel cultures without

mouse serum multiplied by 100%). The paired observations (NMS and PrMS)

per experiment are summarized in figure 3.

Additon of mouse serum, NMS or PrMS, inhibited proliferation of

unstimulated, as well as nonspecifically (PHA) and specifically (PRET)

stimulated spleen cell cultures in a dose dependent way. PrMS inhibited

unstimulated (p < 0.05) as well as PHA stimulated (p < 0.02) and PRET

stimulated (p < 0.02) cultures significantly more strongly than NMS. To

further substantiate the relation between presence of corticosterone in

serum and inhibition of spleen cell cultures, serum samples were pre-

adsorbed with charcaol (160 mg charcaol/ml serum, 4° C, 3 h) which is

known to remove steroids from serum. Addition of preadsorbed sera to a

final concentration of 5% no longer inhibited specific or nonspecific

proliferation of spleen cells compared to cultures without added serum. 3H Thymidine incorporation was 95% + 6 (SEM) and 96% + 9 (SEM) of control

127

3 H T h v m i d i n e i n c o r p o r a t i o n i n c u l t u r e s s e r u m χ Ю 0 % 7 w i t h o u t

% 100-

8 0 -

6 0 -

i.0-

2 0 -

о N M S • P r M S

U n s t i m u l a t e d

о

0 . 0

° · m

о о

о

•

Р Н А

о

• s о

о

• •

P R E T

о

о 0

• о

0

• о

•

10 1 5 10 1 5 10 P e r c e n t a g e m o u s e s e r u m in c u l t u r e

Fig. 3. The effect of 1%, b% or 10% serum of normal (NMS) or 16 days pregnant (PrMS) mice in the culture medium of either unstimulated, or РИА, or PRET stimulated spleen cells from immune mice. The serum effect uas expressed as the percentage ¿H-thymidine incorporation in serum con­taining cultures compared to those without serum. Plotted values are the mean of Ζ io 4 replicate cultures (SEM < 10%). The results of 3 indepen­dent experiments are summarized. The results of NMS and PrMS were paired for each experiment.

values for PHA and PRET stimulated cultures respectively (n=4).

DISCUSSION

Loss of preexisting immunity during pregnancy is observed in human

and murine malaria (Gilles et al., 1969; Bray & Anderson, 1979; Van Zon

& Eling, 1930; McGregor et al., 1983; Bruce-Chwatt, 1983). Reduced im­

mune reactivity during pregnancy does not only affect immunity to malaria

parasites, but also resistance to an increasing number of pathogens (vi­

ruses, bacteria, parasites), as well as tumor immunity (Purtilo, Hallgren

& Yunis, 1972).

Reduced malaria immunity is thought to be connected to impaired ma­

ternal immunoreactivity during pregnancy (Lawson, 1967; Bray & Anderson,

1979; Brabin, 1983), though McGregor et al . (1983) considered the possi-

128

bility that the vasculature in the developing placenta represents an

immunologically privileged site for the parasite. A pregnancy depen­

dent reduction in immune reactivity and production of immunosuppres­

sive factors during pregnancy has been described repeatedly. In gene­

ral, it concerns suppression of cell mediated rather than humoral immu­

nity (Beer & Billingham, 1972; Gusdon, 1983).

Loss of malaria immunity in the murine model appears to be corre­

lated to increasing levels of corticosterone during pregnancy (Van Zon

et al., 1982, 1983). Mice that lost malaria immunity exhibited higher

serum corticosterone levels already before the infection was patent,

compared to mice that remained immune during pregnancy. Adrenalectomy

before pregnancy prevented maternal production of corticosterone during

pregnancy and also largely prevented loss of malaria immunity during

pregnancy, whereas immunity in adrenalectomized mice could still be

broken by exogenous corticoid. Finally, an increase in the serum level

of corticosterone by ACTH treatment of non-pregnant immune mice resul­

ted in loss of malaria immunity, and the serum corticosterone levels

of these mice were of the same order as those observed in pregnant mice

(Van Zon et al., manuscript submitted).

In view of the well known immunosuppressive properties of gluco­

corticoids these observations suggested that serum corticosterone might

regulate immune effector function, and prompted analysis of the effect

of corticosterone and different serum corticosterone concentrations on

immune function.

Immunity to malaria is correlated to the specific in vitro stimu­

lation of spleen cells (Weinbaum et al., 1976, 1978). In our experiments

too, spleen cells from immune but not from normal donors, could be sti­

mulated with PRET. Normal erythrocytes or reticulocytes could not re­

place the PRET.

Nonspecific stimulation of spleen cells with PHA was not signifi­

cantly different in cultures of spleen cells from either normal, or im­

mune, or immune pregnant mice, indicating a constant number and propor­

tion of Τ cells in these spleens. The observation is not in line with a

reduced PHA stimulation of peripheral blood cells from pregnant women

129

(Purtilo et al.j 1972), but supports the observation of Gottesman &

Stutman (1981) who found a change in PHA stimulation during pregnancy

in the draining lymph node of the uterus only.

The specific PRET stimulation of spleen cells from pregnant immune

mice was significantly lower than that of non-pregnant immune mice, and

indicated a reduction of specific immune reactivity to malaria antigens

during pregnancy. Whether this reduction is due to a reduction of the

number of spleen cells specifically reacting to PRET or to an increase

of suppressor cells is unknown. Changes in relative cell number in

lymphoid organs during pregnancy have been described (Smith & Powell,

1977; Nakamura, Persellin & Rüssel, 1983).

Changes in specific immune reactivity of spleen cells are impor­

tant for malaria immunity considering the possibility of transfer of

immunity with spleen cells but not with lymph node cells from immune

donors (Roberts & Tracey-Patte, 1969; Jayawardena, Targett, Leuchars

& Davies, 1978; McDonald & Phillips, 1980).

In addition to a reduced immunoreactivity in spleen cells from

immune pregnant mice the serum of normal mice and even more that of

pregnant mice, suppressed spleen cell proliferation of unstimulated

as well as stimulated cultures. The suppressive effect of serum was

unspecific, dose dependent, related to the concentration of corticos­

terone in the serum, and could be removed by charcaol adsorption which

is known to adsorb steroids. Furthermore, spleen cell proliferation

could be inhibited unspecifically, in a dose dependent way by directly

adding corticosterone to the cultures. The results described here show

that concentrations of 5 ng/ml culture medium or more increasingly in­

hibited spleen cell proliferation. This concentration is below the se­

rum concentration of total corticosterone observed during pregnancy

(approximately 1500 ng/ml around day 16 of pergnancy in mice) and in

normal mice (approximately 100 ng/ml) (Van Zon et al., 1982). Regar­

ding the amount of serum used in our spleen cell system the concentra­

tions corticosterone in the cultures are in the same range.

130

When serum containing corticosterone is added to the spleen cell

assay, it is not clear whether (in игіто)оп1у the concentration free

corticosterone exerts an immunosuppressive effect, as is claimed for

the biological activity of this hormone in vivo. The precise mechanism

of action of glucocorticoid has not been clearly defined (Bach, 1975;

Cupps & Fauci, 1982). Rosner, Ong & Kahn (1982) did not observe a dif­

ference in the suppressive effect of Cortisol or Cortisol bound to CBG

in the lymphocyte proliferation test. If this implies that for the

in vitro assay the total corticosterone concentration must be consider­

ed, then the serum suppressive effects described here can be at least

partly explained by its corticosterone content. The immunosuppressive

effect of 1% NMS (1% of approximately 100 ng/ml = 1 ng/ml ) may suggest

that other immunosuppressive factors are involved as well.

Another hormone with immunoregulatory activity and being important

during pregnancy is progesterone (Siiteri, Febres, Clemens, Chang,

Gondos & Stites, 1977; Stites & Siiteri, 1983). At pharmacological doses

progesterone blocks Τ cell activity in vitro and at physiological doses

lymphocyte cytotoxicity was depressed (Szekeres-Bartho, Csernus, Hadnagy

& Pacsa, 1983), but not PHA stimulation of spleen cells (Skinnider &

Laxdal, 1981). Our attempts to break malaria immunity by exogenous pro­

gesterone either through repeated injections or release from subcutane-

ously implanted osmotic minipumps filled with progesterone were unsuc­

cessful (unpublished observations).

The possibility needs further attention that other serum factor

with an established immunosuppressive effect in vitro, e.g. alphafoeto-

protein (Murgita, Goidl, Kontianen & Wigzell, 1977; Czokalo & Wisniewski,

1979), alpha2-macroglobulins (Stimson, 1972; Fizet, Bousquet, Piquet &

Cabantous, 1983), or pregnancy zone proteins (Von Schoulz, Stigbrand &

Tarvik, 1973) are involved.

In summary, loss of malaria immunity during pregnancy is related

to decrease in malaria specific spleen cell activity, which is related

to an increase in serum corticosterone, as reflected by the inhibitory

activity of pregnant serum and corticosterone itself on spleen cell re­

activity in vitro.

131

ACKNOWLEDGEMENT

We are grateful to A. Kiliaan and C.C. Hermsen for their substan­tial contribution to the experiments and to T. Treskon, R. Jerusalem and T. Tissen for their advices and technical assistance. We thank J. Reitsma, G. Poelen and co-workers for biotechnical assistance and care for the animals and M.L. Weiss for reading the manuscript.

These investigations were supported by the Foundation for Funda­mental Biological Research (BION), which is subsidized by the Nether­lands Organization for the Advancement of Pure Research (ZWO).

REFERENCES

1. Bach, J.F. (1975) Corticosteroid. In: The mode of action immunosup­pressive agents, (ed. by J.F. Bach) North-Holland publishing Co., Amsterdam.

2. Barlow, S.M., Morrison, P.J. & Sullivan, F.M. (1974) Plasma corti­costerone levels during pregnancy in the mouse: the relative con­tributions of the adrenal glands and foeto-placental units. J. Endocrinol. 60, 473.

3. Beer, A.E. & Billingham, R.E. (1972) Immunobiology of mammalian repro­duction. Adv. Immunol. 14, 1.

4. Brabin, B.J. (1983) An analysis of malaria in pregnancy in Africa. Bull. W.H.O. 61, 1005.

5. Bray, R.S. & Anderson, M.J. (1979) Falciparim malaria and pregnancy Trans. R. Soc. Trop. Med. Hyg. 73, 427.

6. Bruce-Chwatt, L.J. (1983). Malaria and pregnancy. Br. Med. J. 286, 1457.

7. Cupps, T.R. & Fauci, A.S. (1982) Corticosteroid-mediated immunoregu-lation in man, Immunol. Rev. 65, 133.

8. Czokalo, M. & Wisniewski, L. (1979) Inhibition of blastic transforma­tion and immunoglobulin release in lymphocytes cultured in the pre­sence of alpha-fetoprotein (AFP). Exp. Pathol. 17, 176.

9. Eling, W. & Jerusalem, С (1977) Active immunization against the mala­ria parasite Plasmodium berghei in mice: sulfathiazole treatment of a P. berghei infection and development of immunity. Tropenmed. Para-sit. 28, 158.

10. Eling, W., Van Zon, A. & Jerusalem, С (1977) The course of a Plasmodium berghei infection in six different mouse strains. Z. Parasitenkd. 54, 29.

11. Fizet, D., Bourquet, J., Piquet, Y. & Cabantous, F. (1983) Identifica­tion of a factor blocking a cellular cytotoxicity reaction in preg­nant serum. Clin. exp. Immunol. 52, 648.

12. Gilles, H.M., Lawson, J.B. Sibelas, M., Voller, A. & Allan, N. (1969) Malaria, anaemia and pregnancy. Ann. Trop. Med. Parasitol. 63, 245.

13. Gottesman, S.R.S. & Stutman, 0. (1981) Cellular immunity during preg­nancy. II Response to T- and B- cell mitogens. Am. J. Reprod. Immu­nol. 1, 78.

132

14. Gusdon, J.P. Jr. (1983) The immunology of reproduction. Am. J. Reprod. Immunol.3,5,

15. Jayawardena, A.N., Targett, G.A.T., Leuchars, E., Carter, R.L., Doenhoff, M.J. & Davies, A.J.S. (1975) T-cell activation in murine malaria. Nature 258, 149.

16. Jayawardena, A.N., Targett, G.A.T. Leuchars, E. & Davies, A.J.S. (1978) The immunological response of CBA mice to P. уоеігъ. Immunology 34, 157.

17. Jayawardena, A.N. (1981) Immune responses in malaria. In: Para­sitic Diseases. Vol. 1. The Immunology (ed. by J. Mansfield) M. Dekker Inc., New York.

18. Lawson, J.B. (1967) Malaria and pregnancy. In: Obstetrics and Gynaecology in the Tropics (ed. by J.B. Lawson & D.B. Stewart) Chap. 5. E. Arnold, London.

19. McDonald, V. & Phillips, R.S. (1980) Plasmodiim ahabaud%: Adop­tive transfer of immunity with different spleen cell populations and development of protective activity in the serum of lethally irradiated recipient mice. Exp. Parasitol. 49, 26.

20. McGregor, I.A., Wilson, M.E. & Billewics, W.Z. (1983) Malaria in­fection of the placenta in the Gambia, West Africa; its incidence and relationship to stillbirth, birthweight and placental weight. Trans. R. Soc. Trop. Med. Hyg. 77, 232.

21. Menon, R. (1972) Pregnancy and malaria. Med. J. Malays. 27, 115.

22. Mishell, B.B. & Shngi, S.M. (1980) Preparations of mouse cell suspensions. In: Selected Methods in Cellular Immunology, (ed. by B.B.Mishell & S.M.Sngi) pp. 3-27. W.H.Freeman and Company, San Francisco.

23. Murgita, R.A., Goidl, E.A. Kontianen, S. & Wigzell, H. (1977) Alpha-fetoprotein induces suppressor T-cell s гп miro. Nature 267, 257.

24. Nakamura, T., Persellin, R.H. & Russell, I.J. (1983) Induction of suppressor cell activity by human pregnancy serum. Am. J. Reprod. Immunol. 4, 133.

25. Oduola, A.M.J., Holbrook, T.W., Galbraith, R.M., Bank, H. & Spicer, S.S. (1982) Effects of malaria {РЪазтоагит berghev) on the mater­nal-fetal relationship in mice. J. Protozool. 29, 77.

26. Osunkoya, B.O. & Williams, A.I.O. (1980) Microscopic observations on the human placenta in malaria infection. Niger. Med. J. 10, 45.

27. Phillips, R.S. (1970) Ріаътоагът Ъе дкег: passive transfer of im­munity by antisera and cells. Exp. Parasitol. 27, 479.

28. Playfair, J.H.L. (1982) Immunity to malaria. Br. Med. Bull. 38, 153.

29. Purtilo, D.T., Hallgren, H.M. & Yums, E.J. (1972) Depressed mater­nal lymphocyte response to phytomaemagglutimn in human pregnancy. Lancet ι, 769.

30. Roberts, J.A. & Tracey-Patte, P. (1969) Adoptive transfer of immunity to Plasmodbim berghez. J. Protozool. 16, 728.

133

31. Rosner, W., Ong, K.S. & Khan, M.S. (1982) The effect of corticoste­roid binding globulin on the in vitro incorporation of thymidine into human lymphocytes. J. Clin. Endocrinol. Metab. 54, 201.

32. Skinnider, L.F. & Laxdal, V. (1981) The effect of progesterone, oestroqens and hydrocortisone on the mitogenic response of lymphocytes to phytohaemagglutinin in pregnant and non-pregnant women. Br. J. Obstet. Gynaecol. 88, 1110.

33. Siiteri, P.K., Febres, F., Clemens, L.E., Chang, R.J., Gondos, B. & Stites, D. (1977) Progesterone and maintenance qf pregnancy: is progesterone nature's immunosuppressant? Ann. N.Y. Acad. Sci. 286, 334.

34. Smith, R.N. & Powell, A.E. (1977) The adoptive transfer of preg­nancy-induced unresponsiveness to male skin grafts with thymus-dependent cells. J. Exp. Med. 146, 899.

35. Stimson, W.H. (1972) Transplantation-nature's success. The Lancet 684.

36. Stites, D.P. & Siiteri, P.K. (1983) Steroids as immunosuppressants in pregnancy. Immunol. Rev. 75, 117.

37. Szekeres-Bartho, J., Csernus, V., Hadnagy, J. & Pacsa, A.S. (1983) Immunosuppressive effect of serum progesterone during pregnancy depends on the progesterone binding capacity of the lymphocytes. J. Reprod. Immunol. 5, 81.

38. Troye- Blomberg,M., Perlmann, H., Patarroyo, M.E. & Perlmann, P. (1983) Regulation of the immune response in Plasmodium ¡'alaipavum malaria. II Antigen specific proliferative responses in vitro. Clin. Exp. Immunol. 53, 345.

39. Van Zon, A.A.J.С. & Eling, W.M.C. (1980) Depressed malarial immu­nity in pregnant mice. Infect. Immun. 28, 630.

40. Van Zon, A.A.J.С, Eling, W.M.C., Herrasen, C.C. & Koekkoek, A.A.G.M. (1982) Corticosterone regulation of the effector function of mala­rial immunity during pregnancy. Infect. Immun. 36, 484.

41. Van Zon, A.A.J.С, Eling, W.M.C. & Herrasen, C.C. (1983) Enhanced virulence of malaria infection in pregnant mice. IRCS Med. Sci. 11, 1002.

42. Van Zon, A.A.J.С, Eling, W.M.C., Herrasen, C.C., Van de Wiel, T.J.J.M. & Duives, Μ.E. (1983) Malarial immunity in pregnant mice, in rela­tion to total and unbound plasma corticosterone. Bull. Soc. Path. Ex. 76, 493.

43. Von Schoultz, В., Stigbrand, T. & Tarnvik, A. (1973) Inhibition of PHA-induced lymphocyte stimulation by the pregnancy zone protein. FEBS Lett. 38, 23.

44. Weinbaum, F.I., Evans, Ch.B. & Tigelaar, R.E. (1976) An in vitro assay for T-cell immunity to malaria in mice. J. Immunol. 116, 1280.

45. Weinbaum, F.I., Weintraub, J., Nkrumah, F.K., Evans, Ch.B., Tigelaar, R.E. & Rosenberg, Y.J. (1978) Immunity to Plasmodium berghei yoelii in mice.II Specific and nonspecific cellular and humoral responses during the course of infection. J. Immunol. 121, 629.

134

46. Weisberger, H., Golenser, J. & Spira, D.T. (1980) Plasmodium berghei: specific stimulation of rat lymphocytes by soluble antigens released in vitro from infected erythrocytes. Exp. Parasitol. 50, 136.

47. Wyler, D.J. & Oppenheim, J.J. (1974) Lymphocyte transformation in human Plasmodium falciparum malaria. J. Immunol. 113, 449.

135

136

Summary

and

Final Considerations

137

Summary and final considerations

The decrease in anti-malaria responses during pregnancy is well

recognized in human malaria. Reports from several endemic areas men­

tioned higher incidence and severity of malaria infection, especially

Plasmodium falaipamm, in pregnant women. In experimental malaria,

however, studies concerning pregnancy associated loss of malaria

immunity have not yet been described.

In the murine P.berghei malaria model studied in our laboratory

a considerable proportion of previously immune mice developed a re­

crudescence during pregnancy. This thesis comprises studies that ana­

lyze pregnancy associated changes in malaria immunity in the experi»

mental model, and the regulatory action of plasma corticosterone on

the anti-malaria immune responses.

Chapter 1 presents asurveyof the literature on depressed immunity

during pregnancy, its consequences with regard to development of dis­

ease, particularly with regard to malaria, and an analysis of immune

function during pregnancy. In view of changes in cell mediated immu­

nity, rather than humoral immunity during pregnancy, and of change^

in plasma glucocorticoids which have well known immunosuppressive

properties, special attention is given to their possible contri­

bution to the problem of loss of malaria immunity during pregnancy.

Chapter 2 and 3 describe the experimental Plasmodium berghei mouse model used in our studies. Some strain specificities were ob­

served with regard to loss of immunity during pregnancy. Random bred

Swiss and inbred СЗН/StZ and B10LP mice exhibited a similar percen­

tage of mice that lost malaria immunity during pregnancy, whereas

a lower percentage was found in inbred Balb/c mice. In Swiss and

СЗН/StZ mice the mortality rate of pregnancy related recrudescent

infections was higher than in B10LP and Balb/c mice. Intrauterine

death, abortion, and premature delivery were associated with recru­

descent infections during pregnancy.

Pregnancy dependent loss of immunity in relation to parity and

the role of the parasite in development of an improved immune res-

138

ponse in multigravidae is described in chapters 3 and 4. A lower re­

crudescence rate during the second pregnancy is dependent on the

presence of parasites during the second semester of the first preg­

nancy. When parasites were radically eliminated by chloroquine treat­

ment before the first pregnancy and re-introduced before the second

pregnancy, the recrudescence rate during this second pregnancy was

the same as otherwise found in Primigravidae. When parasites were

present during the second semester of the first pregnancy and recru­

descence infections were cured by chemotherapy during this period,

then a substantially lower recrudescence rate was observed during

the second pregnancy. The reconfrontation of a partly suppressed

immune system (cell mediated immunity more suppressed than humoral

immunity) with the proliferating parasite induces immunity, which

is less dependent on responses that are normally suppressed during

pregnancy.

Reduced immune reactivity during pregnancy not only caused loss

of immunity but also enhanced virulence of primary infections in non­

immune mice (chapter 5 and 7). In all mouse strains tested the para-

sitaemia developed faster. Whereas survival periods were considerably

reduced in B10LP and C57B1/Rij mice (from approximately 25 days to

7 days), in СЗН/StZ nice which normally exhibit such a short sur­

vival period no further reduction was observed during pregnancy.

These results are taken to indicate that immune reactivity that nor­

mally protects against early mortality (B10LP, C57B1/Rij) cannot be

developed during pregnancy.

A reduced survival period in non-immune mice was dependent on

infection of the animals between day 6 and 14 of pregnancy. This

indicates that pregnancy dependent immunosuppression is maximally

effective during this period. Considering that a normal survival

period was observed when animals were infected before day 6 of preg­

nancy, it further indicates that immunosuppression during pregnancy

is effective in sensitizing reactions operative early in infection.

139

Loss of an existing malaria immunity in pregnant mice occurred

in the second semester of pregnancy during a period of elevated plas­

ma corticoid levels. Further analysis showed that total as well as

free plasma corticosterone levels were significantly higher in mice

that lost immunity than in mice that remained immune throughout preg­

nancy (chapter 6 and 7). Blocking of maternal corticoid production

by adrenalectomy prevented maternal corticosterone production (but

plasma corticosterone increased later in pregnancy through foetal

production) and reduced the number of mice that lost immunity during

pregnancy considerably. These results led to the conclusion that

corticosterone can be considered as an immunoregulatory serum factor

during pregnancy.

Not only plasma corticosterone levels affect anti-parasite

immune responses, but patent infections disturb endocrine regulation

of plasma corticosterone levels also (chapter 6 and 7). In pregnant

mice with either a primary or a recrudescent infection serum corti­

costerone levels (both bound and free corticosterone) were excessive

compared to these normally found during pregnancy. In non-pregnant

mice only the free fractions of plasma corticosterone increased dra­

matically, which may be related to reduced liver function as a con­

sequence of severe liver pathology developing during infection, and

hence inhibition of CBG production.

The immunoregulatory action of plasma corticosterone in malaria

immunity was also investigated in non-pregnant immune mice. Treat­

ment of immune mice with dexamethasone, administered in the drinking

water or by subcutaneously implanted osmotic minipumps, resulted in

a dose dependent loss of immunity (chapter 7 and 8), indicating that

exogenous corticoids suppressed anti-malaria immune responses. To

examine the involvement of the endogenous plasma corticosterone

levels in immunoregulation, osmotic minipumps filled with an ACTH

analogue were inserted in immune mice (chapter 8). Plasma cortico­

sterone, both total and free concentration, increased in these mice

in a dose dependent way. Immune mice with increased plasma cortico­

sterone concentration developed a lethal, recrudescent infection.

140

In adrenalectomi2ed immune mice, neither an increase of plasma cor­

ticosterone nor loss of malaria immunity could be provoked by ACTH

treatment. These results strongly support the idea that plasma gluco­

corticoids are involved in the regulation of malarial immune res­

ponses in mice.

Two other hormones, oestrogen and progesterone, which are re­

ported to be important in the regulation of the maternal immunore-

activity during pregnancy, were also examined with regard to their

possible suppressive effect on malaria immunity in non-pregnant

mice (chapter 9). The synthetic oestrogen DES did not provoke loss

of malaria immunity, despite the use of high doses. Progesterone

was administered either by repeated injections, or by drinking

water, or by osmotic mi ni pumps. In the latter plasma progesterone

levels were measured, but only a small increase was observed. In

mice treated with progesterone, no effect on their malaria immunity

could be detected.

Decrease of malaria immunity in pregnant mice could also be

demonstrated in in vitro experiments (chapter 10). The response to

malaria antigen was diminished in spleen cells from pregnant immune

mice as compared to spleen cells from non-pregnant immune mice.

Moreover, serum of 16 days pregnant mice, which contained a high

concentration of corticosterone, inhibited anti-malaria responses

in spleen cell cultures more than serum of non-pregnant mice. Cor­

ticosterone itself also inhibited the spleen cell response to

malaria antigen.

The results of the investigations described in this thesis,

indicate that increased severity of a primary malaria infection or

loss of previously existing immunity in pregnant mice are a conse­

quence of decreased immune responsiveness during pregnancy. An in­

crease of the plasma concentration of corticosterone, either natu­

rally occurring during pregnancy or provoked by ACTH treatment,

plays an important role in the depression of the malaria immune

response. Reconfrontation with antigen during a pregnancy dependent

141

immunosuppression may induce immune reactions that are not sup­

pressed during pregnancy and may account for the improved immunity

observed in multigravidae.

The Plasmodium berghei - mouse model may therefore be a sui­table model for the analysis of features and mechanism of depressed

immune reactivity during pregnancy, whereas the naturally occurring,

selective and probably functional depression of immune reactivity

during pregnancy may be a suitable model for the analysis of the

mechanism(s) of malaria immunity. The model is not only relevant

to malaria, but to an increasing number of diseases as well as to

tumor immunity during pregnancy.

142

Samenvatting

In muizen, immuun tegen de malaria parasiet Plasmodium berghei, wordt tijdens zwangerschap vaak een recidiverende, letale malaria

infektie waargenomen, wat duidt op een verlies van immuniteit.

In dit proefschrift is het onderzoek aan dit verlies van immuni­

teit beschreven. In hoofdstuk 1 is een literatuur overzicht gegeven

over de onderdrukking van de immuunreaktiviteit tijdens zwangerschap

en de gevolgen hiervan voor een aantal ziekten, in het bijzonder

voor malaria. De celgebonden immuniteit blijkt meer onderdrukt te

zijn dan de humorale immuniteit. Bovendien wordt tijdens zwanger­

schap een sterke stijging waargenomen van glucocorticoiden con­

centratie in het plasma. Van glucocorticoiden is bekend dat zij een

sterke inmunosuppressievewerking hebben. Met name deze twee verander­

ingen zijn bekeken in het licht van hun mogelijke betrokkenheid bij

het verlies van malaria immuniteit tijdens zwangerschap.

In hoofdstuk 2 en 3 zijn de belangrijkste karakteristieken van

het onderzoek model beschreven. In drie van de vier onderzochte

muizenstammen ("random bred" Swiss en inteelt BIOLP en СЗН/StZ

muizen) was het percentage muizen met verlies van malaria immuniteit

tijdens zwangerschap ongeveer gelijk. In inteelt Balb/c muizen werd

een lager percentage gevonden. In Swiss en СЗН/StZ muizen was de

recidiverende infektie meestal letaal, terwijl in BIOLP en Balb/c

muizen de mortaliteit veel lager was. Vaak voorkomende gevolgen van

de malaria infektie tijdens zwangerschap waren intra-uterine dood

van de foeten, abortus en het voortijdig werpen van de foeten.

Het percentage muizen met verlies van malaria immuniteit was

hoger tijdens de eerste zwangerschap dan in de tweede. Dit bleek

het gevolg van een verbeterde immuniteit die de muizen ontwikkelden

tijdens de eerste zwangerschap (hoofdstuk 3 en 4). Hiervoor was de

aanwezigheid van prolifererende parasieten in de drachtige immune

muis noodzakelijk. De resultaten suggereren dat een hernieuwde kon-

frontatie van een onderdrukt en zich weer herstellend immuun systeem

4

143

van de drachtige immune muis met de prolifererende malaria parasiet

resulteert in een achteraf verbeterde immuniteit.

Ten gevolge van de afname in immuunreaktiviteit tijdens zwanger­

schap werd ook een verhoogde virulentie van de malaria infektie in

normale, niet immune drachtige muizen waargenomen. Het gevolg was

een sneller opkomende parasitemie en een kortere overlevingstijd

(hoofdstuk 5 en 7). Vooral in muizenstammen die normaal vrij lang

overleven werd tijdens zwangerschap een veel kortere overleving

na infektie waargenomen. De beschermende immuunreakties die in deze

stammen een snelle dood na infektie voorkomen, lijken tijdens zwanger­

schap onderdrukt te zijn.

De recidiverende malaria infekties in drachtige immune muizen

werden altijd waargenomen in de tweede helft van de zwangerschap, in

dezelfde tijd dat het plasma corticosteron gehalte sterk steeg. On­

derzoekingen toonden aan dat zowel de totale als de vrije concentra­

tie plasma corticosteron significant hoger waren in drachtige muizen

met verlies van malaria immuniteit dan in muizen die hun immuniteit

niet verloren (hoofdstuk 6 en 7). Het verhinderen van de cortico­

steron produktie in drachtige muizen door adrenalectomie vooraf re­

sulteerde in een sterke afname van het percentage drachtige muizen

met verlies van malaria immuniteit. Deze resultaten leidden tot de

konklusie dat het plasma corticosteron gehalte een regulerende funk-

tie heeft in de malaria immuniteit tijdens zwangerschap.

Een komplikatie in deze onderzoekingen was het feit dat de

endocriene regulatie tijdens een patente malaria infektie verstoord

bleek. Plasma corticosteron concentraties bleken toe te nemen naar­

mate de infektie ernstiger werd; in niet drachtige muizen alleen

de vrije concentratie, in drachtige dieren zowel de totale als de

vrije concentratie.

De regulerende werking van corticosteroiden op malaria immuni­

teit is ook in niet drachtige immune muizen bestudeerd (hoofdstuk 7

en 8). Behandeling met dexamethason (een synthetisch glucocorticoid)

resulteerde in een dosis afhankelijk verlies van malaria immuniteit.

144

Het verhogen van de endogene plasma corticosteron concentratie door

toediening van een synthetisch ACTH analogon veroorzaakte ook een

verlies van immuniteit. Adrenalectomie ббг de ACTH behandeling

voorkwam zowel een stijging van de corticosteron concentratie als

het verlies van malaria immuniteit.

Naast corticosteron zijn ook oestrogenen en progesteron, hormo­

nen die als belangrijk beschouwd worden in de regulatie van de im-

muunreaktiviteit tijdens zwangerschap, onderzocht naar hun invloed

op malaria immuniteit (hoofdstuk 9). Diethylstilbestrol (synthetisch

oestrogeen preparaat) en progesteron werden op diverse manieren aan

immune muizen toegediend: per injektie, opgelost in het drinkwater,

of via subcutaan geïmplanteerde osmotische pompjes. Met geen van de

gebruikte methoden werd verlies van malaria immuniteit bewerkstelligd.

De afname van malaria immuniteit tijdens zwangerschap kon ook

worden aangetoond in in vitro experimenten (hoofdstuk 10). De spe­

cifieke respons van immune miltcellen tegen geparasiteerde erythro-

cyten was verminderd in miltcellen van drachtige muizen. Bovendien

werd deze respons meer geremd door serum van 16 dagen drachtige

muizen, waarin een hoog gehalte corticosteron zat, dan door het

serum van niet-drachtige muizen. Chemisch gezuiverd corticosteron,

toegevoegd aan cel kweken, remde ook de respons tegen malaria antigeen.

De resultaten van het onderzoek zoals beschreven in dit proef­

schrift, hebben aangetoond dat de komplikaties van malaria tijdens

zwangerschap een gevolg zijn van een afname van de immuniteit tegen

malaria. De toename van het plasma corticosteron gehalte, zoals

van nature aanwezig tijdens zwangerschap dan wel geïnduceerd door

ACTH behandeling, blijkt betrokken te zijn bij de onderdrukking van

de immuunreaktiviteit. Een hernieuwde konfrontatie van de proli-

fererende malaria parasiet met het onderdrukte immuun systeem kan

immuunreakties induceren, die niet gevoelig zijn voor de zwanger-

schapsafhankelijke onderdrukking en daardoor in een volgende zwanger­

schap voor een (betere) bescherming kunnen zorgen.

Het Plasmodium berghei - muis model lijkt een goed bruikbaar

145

model om de onderdrukking van de immuunreaktiviteit tijdens zwanger­

schap te onderzoeken, terwijl analyse van de selektief onderdrukte

immuunreaktiviteit tijdens zwangerschap een beter inzicht kan ver­

schaffen in het mechanisme van malaria immuniteit. Een dergelijk

model is niet alleen bruikbaar voor malaria immuniteit, maar kan

ook van belang zijn voor het onderzoek betreffende de immuniteit

tegen een aantal andere ziekten en tegen tumoren tijdens zwanger­

schap.

146

Woorden van dank,

Hoewel op de titelpagina van dit proefschrift slechts één auteurs­

naam vermeld staat, moge het duidelijk zijn dat ook vele anderen er

vaak zeer intensief mee bezig zijn geweest. Diegenen, die"dagelijks"

meewerkten bij de experimenten en alles er omheen zijn reeds genoemd

als co-auteur of in de "acknowledgements" van de diverse hoofdstukken.

Daarnaast wil ik bedanken Coby van Run, Frans van Wingaarden en Kees

Moolhuyzen voor het verzorgen van het histologische werk en Theo

Hafmans voor zijn fotografisch werk. Mijn dank gaat ook uit naar de

medewerkers van het Centraal Dierenlaboratorium (hoofd: Dr. W.van de

Gulden), van de afdeling Medische Illustratie, van de afdeling Medische

Fotografie (hoofd: dhr.A.T.A.Reynen) en van de Medische Bibliotheek

(hoofd: dhr.E.de Graaff) voor de werkzaamheden die zij voor mij gedaan

hebben.

Zeer erkentelijk ben ik de studenten biologie en geneeskunde die

tijdens hun verblijf op onze afdeling veel hebben bijgedragen aan dit

onderzoek.

Tenslotte wil ik zeker ook mijn dank uitspreken aan Annelies Oostrom,

Mary Scholten en Jeanne van der Kamp die in de loop van de tijd het

typewerk hebben verzorgd van de manuscripten voor publicaties en uit­

eindelijk voor dit proefschrift.

147

CURRICULUM VITAE:

Adriaan (Ab) A.J.C, van Zon

geboren 16 november 1945 te Breda

gymnasium 1958-1964

biologie studie. Katholieke Universiteit, Nijmegen 1967-1974

vanaf 1975 werkzaam op de afdeling Cytologie en Histologie,

Faculteit der Geneeskunde en Tandheelkunde, Katholieke

Universiteit, Nijmegen.

148

STELLINGEN

I

Het ontwikkelen van een malaria vaccin via DNA hybridisatie of door

gebruik van monoclonalen heeft alleen zin als de eventuele antigene

varianten van de malaria parasiet bekend zijn.

- J. Maddox, Nature 310 (1984) 541

II

Het verloren gaan van een bestaande malaria immuniteit tijdens zwanger­

schap maakt het waarschijnlijk dat humorale immuunreacties op zich geen

bescherming bieden tegen malaria.

- H.F.C.M. Kortmann, Thesis Amsterdam, 1972 - J. Carter and D.W. Dresser, Irmunology 49 (1981) 481-490 - I.A. McGregor et al.. Trans. R. Soa. Trop. Med. Нуд. 77 (1983)

233-244

III

De toepasbaarheid en het nut van een toekomstig malaria vaccin in

holoendemische malaria gebieden zal mede afhangen van de mate waarin

gevaccineerden ook beschermd zijn tijdens zwangerschap.

- Dit proefschrift

IV

De beste en op dit moment enig afdoende maatregel voor westerse

vrouwen om geen malaria te krijgen tijdens zwangerschap is zich te

onthouden van exotische reizen.

- L.J. Bruce-Chtíatt, Br. Med. J. 286 (1983) 1457-1458

V

Het endocriene systeem is belangrijk bij de regulatie van immuno­

logische reacties.

-Dit proefschrift - H.O. Besedovsky et al., Imrmnol. Today 4 (1983) 342-346

VI

Endothelialisatie van vaatprcthesen met een klein lumen vindt plaats

vanuit de vaat-uiteinden.

- F. Hess et al., J. Cardioi)asc. Surg. 24 (1983) 516-524

VII

Het zgn. hepatische triglyceride lipase speelt een belangrijke rol

bij de opname van chylomicronen in de lever.

Vili

Het regelmatig voorkomen van darinbloedingen bij marathonlopers onder­

steunt het aloude gezegde "hardlopers zijn doodlopers".

- M.T. Buchnann, Ann. Intern. Med. 101 (1984) 127-128

IX

De veronderstelling dat testosteron spiegels tijdens de prenatale

ontwikkeling een belangrijke rol spelen in de relatie tussen leer­

problemen, auto-immuun ziekten en linkshandigheid is onterecht.

- N. Geschuind and P. Behan, Proa. Natl. Aoad. Soi. USA 79 (1982) 5097-S100

- D. Wofsy, Immunol. Today 5 (1984) 169-170

X

Bouwstijl en beeldhouwwerk van het "Kasteeltje" Heyendaal zijn des­

tijds gekozen om de maatschappelijke positie van de opdrachtgever te

onderstrepen. Na aankoop van de villa door de St. Radboudstichting is

deze oorspronkelijke representatieve funktie verloren gegaan.

- I. Pey. Nwnaga, 30 (1983) 11-16

XI

Snelheidsbeperkende maatregelen in woongebieden zijn alleen zinvol

als zij daadwerkelijk snelheidsbeperkend zijn.

XII

In plaats van het creëren van gratis parkeerterreinen b i j de NS

stations zou het de ANWB beter passen te streven naar gratis fietsen­

stallingen.

Nijmegen, 11 oktober 1984 A.A.J.C. van Zon

De uitgave van dit proefschrift werd financieel ondersteund door de Jan Dekkerstichtmg, de Dr. Ludgardine Bouwmanstichting en het Remmert Adriaan Laan-Fonds.