Morphological Quantitative Changes in the

Number of Lymphocytes,Macrophages and

Plasma Cells in the Uterus and Lymph Nodes of

Rats Exposed to the Systemic Administration of

BCG MEHMET KANTER,ABDULAZIZ GUL,ISMAIL MERAL,AHMET KOC,MAHMUT

ILHAN and ENDER ERDOGAN

Department of Histology and Embryology, Faculty of Veterinary Medicine,

Department of Obstetrics and Gynecology, Faculty of Medicine, Department

of Physiology, Faculty of Veterinary Medicine, Department of Oncology, and

Department of Histology and Embryology, Faculty of Medicine, Yuzuncu Yil

University, Van, Turkey

KANTER,M.,GUL,A.,MERAL,I.,KOC,A.,ILHAN,M.and ERDOGAN,E.Morphological Quantitative Changes in the Number of Lymphocytes, Macrophages

and Plasma Cells in the Uterus and Lymph Nodes of Rats Exposed to the Systemic

Administration of BCG. Tohoku J.Exp.Med.,2003,199(4),219-228――This

study was designed to investigate the effect of systemic administration of BCG on

the morphological quantitative changes in the number of lymphocytes,macro-phages and plasma cells in the uterus and lymph nodes of rats.Thirty female

virgin Wistar Albino rats,aging 6 months and weighing between 200-250 g,were

assigned to the two experimental groups;BCG treated and controls(n＝15).BCG group received 0.1 ml BCG in tail skin and control group received 0.1 ml

saline at the same place.Two weeks after injections,rats in both groups were

anesthesized with a high dose of ether and decapitated.Uterus and ileocecal

lymph nodes were processed to determineαnapthyl acid esterase(ANAE)-positive

T lymphocytes and macrophages.The plasma cells were stained with the methyl

green-pyronin method.It was found that the numbers of T lymphocytes,macro-phages and plasma cells in the uterus and the ileocecal lymph nodes of BCG

treated group significantly increased indicating the presence of an immune

response to the systemic BCG administration.It was concluded that the systemic

administration of BCG increases humoral and cellular immunity in endometrium,myometrium and regional lymph nodes.The immune deficiency system plays an

important role in the pathogenesis of endometriosis.Therefore,the endometriosis

might be prevented by using periodical administration of BCG.However,further

experimental and clinical studies associated with these issue are required.――――

Tohoku J.Exp.Med.,2003,199,219-228

Received November 21,2002;revision accepted for publication April 22,2003.Address for reprints:Dr.Mehmet Kanter,Yuzuncu Yil Universitesi,Veteriner Fakultesi,Histoloji ve

Embriyoloji Anabilim Dali,65080,Kampus,Van,Turkey.e-mail:mehmetkanter65＠hotmail.com

219

BCG;endometriosis;uterus;lymph nodes;ratⒸ2003 Tohoku University Medical Press

The use of Bacillus Calmette-Guerin (BCG)in cancer has been encouraged by animal studies

indicating that it can prevent growth of tumor

transports and eliminate established tumors(Bast et al.1974).BCG was originally isolated

by Calmette and Guerin(Guerin 1957;Inooka et

al.1962)through progressive attenuation of a

virulent strain of Mycobacterium bovis.Hun-dreds of millions of vaccinations have been

performed with negligible mortality and mor-bidity(Lemonde et al.1971;Bansal and Sjogren

1973;Schwartz 1973).BCG is currently one of

the most effective agents for the prophylaxis

and cure of superficial bladder cancer(Haaff et

al.1985;Debruijine et al.1989).Although the

mechanisms of its antitumor activity are un-known,they are generally believed to act by the

immune system,since various components of

that system are known to be activated(Mitchell

and Murahata 1979;De Jong et al.1987).By

using immunohistochemical staining,De Jong et

al.(1987)have showed that the numbers of T

cells increased in the bladder wall after intra

vesical BCG administration in guinea pigs.Widely scattered B cells and macrophages were

also present,but were much fewer in number

than T cells(De Jong et al.1987).BCG is also a potent and persistent inducer

of lymphocyte trapping,particularly in regional

lymph nodes,where it also markedly enhances

the trapping response to a subsequent antigenic

challenge(Zats 1976).It has been suggested

that BCG activates both macrophages and T

cells in vitro(Mokyr and Mitchell 1975),and

that its effects are mediated in vivo by T cell

systems(Ariyan and gershon 1973;Mackaness

et al.1973;Lagrange and Mackaness 1975).It

has been also reported that B lymphocytes

increase in lymphoid organs following the

intraperitonal administration of BCG(Meyer et

al.1979).

Although many women have reflux seeding

of menstrual debris into the peritoneal cavity

but do not all develop endometriosis,there may

be genetic or immunologic factors that influence

susceptibility(Speroff et al.1999).Dmowski

and coworkers(1981)demonstrated that mon-keys with endometriosis had decreased cellular

immunity to endometrial tissue and women

with endometriosis demonstrate a decrease in

various measurements of immune response(Oosterlynck et al.1992;Vigano et al.1992).However,the presence of abnormal immunity in

the“precursor-stage”and the inverse correla-tion between these abnormalities and the stag

favor the altered immune system preceding the

endometriosis(Dmowski et al.1989;Gleicher

and Pratt 1993;Laçin et al.1998).This study was designed to investigate the

effect of systemic administration of BCG on the

morphological quantitative changes in the num-ber of lymphocytes,macrophages and plasma

cells in the uterus and lymph nodes of rats and

whether systemic prophylaxis with BCG can be

used for prevention of endometriosis.

MATERIALS AND METHODS

In this study 30 female 6 months old virgin

Wistar Albino rats weighing between 200-250 g

were used.Rats were assigned to two groups,BCG and controls(n＝15),and fed with stan-dard feed and water ad libitum.In order to

maintain their biological rhythms stable 12

hours artificial and 12 hours dark require was

applied.All rats received humane care accord-ing to the criteria outlined in the“Guide for the

Care and Use of Laboratory Animals”prepared

by the National Academy of Sciences and publi-shed by the National Institutes of Health.BCG group received 0.1 ml BCG in tail skin

(Gul et al.2001)and control group received 0.1

M.Kanter et al.220

ml saline at the same place.Two weeks after

injections rats in both groups were anesthesized

with high dose ether and decapitated.Uterus

and ileocecal lymph nodes were processed to

determine a naphthyl acid esterase(ANAE)-positive T lymphocytes and macrophages.Briefly,the uterine and ileocecal lymph nodes

were removed and fixed in previously cooled

formalin-sucrose solution at＋4℃ for 22 hours

and then they were kept＋4℃ in Holt solution

for 22 hours(Mueller et al.1975).Tenμthin slides obtained using cryostat

(Microm,Germany)were transferred into the

formalin-gelatin covered glass.The materials

were left to dry at room temperature in order to

determine ANAE positive lymphocytes(Mueller

et al.1975)and macrophages(Zicca et al.1981).The slides were stained in an incubation solu-tion described by Mueller et al.(1975)at pH 7.2 for 5 minutes.After ANAE staining,the

slides were washed and processed for nucleus

staining for five minutes with 1% methyl green

dissolved in acetate buffer(pH 4.2).Materials’water was removed by preceding with alcohol

solutions at different concentrations and mate-rials were shinned with xylol and covered with

entellane.In order to plasma cells,same of the tissue

samples were fixed in formalin-alcohol fixation

solution for 24 hours.The routine histological

tissue processing protocol was applied to the

tissue samples.Methyl green-pyronine staining

was applied to 6μm-thick slides(Bancroft and

Cook 1984)obtained using rotary microtome

(Leica 2135,Leica Instruments GmbH,Nussloch,Germany).Materials were examined through

a research microscope (Nikon Optiphot 2,Tokyo).In order to determine the numerical distri-

bution of cell in the tissue samples,20 areas in

the endometrium-myometrium of the uterus and

the cortex-medulla of the lymph nodes were

chosen randomly and the cells were counted in

each area.The cell(100 square mm)densities

in each area were calculated and recorded as

cell number/mm.The data were expressed as

means with standard deviations(S.D.).Student’s

t-test was used to compare BCG treated vs.control rats.

RESULTS

T lymphocytes in the endometrium were

identified by ANAE enzyme staining with the

numerous specific brown granules around large

nuclei. Although the numbers of T

lymphocytes of both groups were almost the

same in the endometrium close to the epith-elium,they were higher(p＜0.01)in BCG treated

rats than in controls in the endometrium close to

the myometrium (Table 1,Figs.1 and 2).In

addition,the T lymphocyte number in the

myometrium was also increased(p＜0.01)by

BCG treatment(Table 1,Figs.1 and 2).Macrophages could not be seen in the en-

dometrium.These cells,however,were easily

identified by their diffuse brown coloration

around large nuclei in the myometrium.The

Quantitative Changes in the Uterus and Lymph Nodes of BCG-Exposed Rats

TABLE 1. The numbers (cell number/mm )of T cells, macrophages and plasma cells in the

endometrium and myometrium of control and BCG treated rats

Cells Control

Endometrium Myometrium

BCG treated

Endometrium Myometrium

T cells 17.32±1.27 10.47±0.13 64.11±2.42 28.12±1.56

Macrophages － 35.18±2.01 － 86.43±4.25

Plasma cell 10.38±1.37 1.67±0.30 45.60±2.57 1.81±0.44

p＜0.01 compared to control.The data are expressed as mean±S.D.(n＝15).

221

macrophage number in the mymoterium was

increased(p＜0.01)by BCG treatment(Table 1,Figs.3 and 4).Similarly the number of plasma cells

stained with the methyl green-pyronin in the

endometrium was higher(p＜0.01)in BCG treat-ed rats than controls (Table 1).In the

myometrium,their numbers were few and not

different between experimental groups(Table

1).

T lymphocytes in lymph nodes were recog-nized with one or two specific brown granules in

cytoplasm by ANAE enzyme staining(Figs.5,6

and 7).Macrophages,on the other hand,were

easily identifiable by their darker coloration(Figs.5,6 and 7).Plasma cells scattered widely

in the medulla of lymph nodes They are spheri-cal or ovoid cells,with an eccentrically placed

nucleus having a coarse pattern of hetero-chromatin.The cytoplasm is intensely baso-

Fig.1.The distribution of T lymphocytes in endometrium and myometrium in BCG group.E,endometrium;T,T lymphocyte;Ms,circular muscle in myometrium;Ml,longitudinal

muscle in myometrium.ANAE,original magnification×360.

Fig.2.The distribution of T lymphocytes in endometrium and myometrium in control group.E,endometrium;T,T lymphocyte;Ms,circular muscle in myometrium;Ml,longitudinal

muscle in myometrium.ANAE,original magnification×360.

M.Kanter et al.222

philic.(Figs.8,9 and 10).The lymph nodes of

rats treated with BCG had a much higher(p＜0.01)number of T lymphocytes,macrophages

and plasma cells than those of controls(Table

2).

DISCUSSION

The humoral and cellular immunity of

BCG administrated organs,which have lumen

such as gall bladder and uterus has been report-

ed in the following articles.In the mucosal

membrane of the uterine horns in Wistar white

rats,mainly small and middle lymphocytes and

rather small amount of plasmocytes and macro-phages have been observed after an intrauterine

administration of BCG.The local cellular type

immune reaction developed with maximum at

the first week before the total immune response,with its peak occurring on the 14th day of the

experiment(Yakhnista et al.1988).The im-munostimulating and antitumor activities of

Fig.4.The distribution of macrophages in myometrium in control group.M,myometrium;thick arrow,macrophage;Bv,blood vessel.ANAE,original magnifica-tion×360.

Fig.3.The distribution of macrophages in myometrium in BCG group.M,myometrium;thick arrow,macrophage;Bv,blood vessel.ANAE,original magnifica-tion×360.

223 Quantitative Changes in the Uterus and Lymph Nodes of BCG-Exposed Rats

BCG have been well established(Davies 1982).It is generally assumed that BCG operates by an

activation of the immune system.Repeated

intravesical administration of BCG generates a

granulomatous reaction in the bladder wall,as

revealed by histological studies(Leong et al.1990). Immunohistochemical and flow-cytofluorometric studies have shown that T

lymphocytes,monocytes/macrophages and

polymorphnuclear leukocytes are the main in-filtrating cells in response to intravesical BCG

treatment(De Boer et al.1991).It was found

that the numbers of T lymphocytes,macro-phages and plasma cells increased in the uterus

indicating the presence of an immune response

to the intrauterine BCG administration(Kanter

et al.2002).There is no much study exploring

the effect of systemic administration of BCG on

the endometrium and myometrium.In our

experiment it was observed that the systemic

administration of BCG increased the T

lymphocytes,plasma cells in endometrium-

Fig.5.The distribution of macrophages in lymph node in BCG group.c,cortex;m,medulla;f,follicle. macrophage.ANAE,original magnification×90.

Fig.6.Higher magnification of Fig.5.m,medulla;thick arrow,macrophage.ANAE,original magnification×520.

M.Kanter et al.224

myometrium and macrophages in myometrium.The systemic administration of BCG caused the

similar humoral and cellular immunity as intra-uterine administration of BCG in the uterus and

lymph nodes.Because,the intrauterine admin-istration of BCG is needed laparatomy or

opened cervix in estrous cycle,systemic admin-istration of BCG might be preferred.The administration of BCG evokes a T

cell-mediated immune response in mouse

lymphoid organs(Ten Dam et al.1976).The

BCG induced activation of macrophages has

been adequately documented(Kelly 1976).It

has been also suggested that B lymphocytes

increases in lymphoid organs following the

intraperitoneal administration of BCG(Meyer

et al.1979).The enlargement of the lymph node T cell

areas,a systemic effect of BCG,may be due in

part to increased influx or trapping of recir-culating T cells as demonstrated by Zatz(1976)in peripheral lymph nodes after intravenous

Fig.7.The distribution of macrophages in lymph node in control group.c,cortex;m,medulla;f,follicle. macrophage.ANAE,original magnification×80.

Fig.8.The distribution of plasma cells in lymph node in BCG group.c,cortex;m,medulla;f,follicle. plasma cells.Methyl green-pyronine,original magnifica-tion×80.

225 Quantitative Changes in the Uterus and Lymph Nodes of BCG-Exposed Rats

Fig.9.The distribution of plasma cells in lymph node in control group.c,cortex;m,medulla;f,follicle;thick arrow,plasma cell.Methyl green-pyronine,original

magnification×80.

Fig.10.Higher magnification of Fig.9.m,medulla;thick arrow,plasma cell.Methyl green-pyronine,original magnification×520.

TABLE 2. The numbers (cell/mm )of T cells, macrophages and plasma cells in the cortex-medulla of lymph nodes of control and BCG treated rats

Cells Control

Cortex Medulla

BCG treated

Cortex Medulla

T cells 420.22±39.16 60.19±3.42 496.19±39.41 172.92±12.98

Macrophages 16.10±0.82 29.16±1.49 68.32±3.47 72.12±4.09

Plasma cell 13.17±0.55 51.53±3.97 17.08±0.60 536.38±46.90

p＜0.01 compared to control.The data are expressed as mean±S.D.(n＝15).

M.Kanter et al.226

injection of BCG.The increase of germinal

center areas also suggests a BCG-induced prolif-eration of B cells(Meyer et al.1979).In addi-tion,the number of T lymphocytes,macro-phages and plasma cells also increased in the

ileocecal lymph nodes of rats to the intrauterine

administration of BCG (Kanter et al.2002).Systemic administration of BCG was also found

to increase the T lymphocytes,macrophages

and plasma cells in lymphoid organs.Hence

the present data support previous investigation.There is ample evidence suggesting that the

immune system plays an important role in the

pathogenesis of endometriosis,and an altered

immune responsiveness has been proposed to

explain why some women develop en-dometriosis,whereas others do not(Braun and

Dmowski 1998).Because we did not try to

produce an endometriosis experimentally after

the systemic BCG administration,we do not

know whether systemic administration of BCG

could prevent the implantation of endometriosis.However,Gul et al.(2001)suggested that sys-temic prophylaxis with BCG prevented the im-plantation of endometriosis in rats.In our

study,since systemic BCG administration in-creased the immune responsiveness,we thought

it might also prevent the implantation of en-dometriosis.However,more studies are need-ed.In conclusion,systemic administration of

BCG increases humoral and cellular immunity

in endometrium,myometrium and regional

lymph nodes.The immune deficiency system

plays an important role in the pathogenesis of

endometriosis.Therefore,the endometriosis

might be prevented by using periodical adminis-tration of BCG.However,further experimen-tal and clinical studies associated with these

issue are required.

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