1989 73: 678-683   

 CS Johnson, DJ Keckler, MI Topper, PG Braunschweiger and P Furmanski reversal of erythroid suppression with erythropoietinerythroid progenitors, suppression of late-stage erythropoiesis, andstimulation of granulocytic, monocytic, megakaryocytic, and early In vivo hematopoietic effects of recombinant interleukin-1 alpha in mice:

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678 Blood, Vol 73. No 3 (February 15), 1989: pp 678-683

In Vivo Hematopoietic Effects of Recombinant Interleukin-la in Mice:Stimulation of Granulocytic, Monocytic, Megakaryocytic, and Early Erythroid

Progenitors, Suppression of Late-Stage Erythropoiesis, and Reversal ofErythroid Suppression With Erythropoietin

By Candace S. Johnson, Douglas J. Keckler, Maureen I. Topper, Paul G. Braunschweiger, and Philip Furmanski

Interleukin-1 a (IL-i a) is a macrophage-derived, multifunc-

tional cytokine that broadly potentiates myelopoiesis and

induces the synthesis of hematopoietic colony-stimulating

factors (CSF) in vitro and in vivo. To evaluate the possibility

for use of IL-i a in ameliorating in vivo bone marrow

suppression induced by drugs or radiation, we examined

the in vivo effects of the cytokine on erythropoietic and

other hematopoietic progenitor cells. Normal mice were

treated with a single intraperitoneal (IP) injection of recom-

binant human IL-i a at varying doses and were assayed at

various times post-treatme;lt. By six hours postinjection. a

significant suppression of mature erythroid progenitors

(CFU-E) was observed in animals treated with IL-i a (0.5

ig/mouse). with maximum suppression of CFU-E and

peripheral blood reticulocyte counts occurring at 24 hours.

H EMATOPOIESIS is regulated by colony-stimulating

factors (CSF) and other cytokines, which can exhibit

either lineage-restricted or multipotent activities.’3 Interleu-

kin-la (IL-la) is a multifunctional cytokine that appears

capable of broadly potentiating hematopoiesis. Produced

mainly by macrophages and monocytes,4’5 IL-la plays a

primary role in T- and B-cell activation,5’6 induces significant

peripheral neutrophilia,7” stimulates the synthesis of CSF in

vitro and in vivo,79 acts in concert with CSF to stimulate

primitive hematopoietic ‘ enhances myeloid

recovery following 2 and 5-fluorouracil

treatment,’ and protects mice from the lethal effects of

3

The diverse myelopoietic effects of IL-i, its known syner-

gistic or additive activity with other regulatory molecules,

and its ability to enhance recovery from severe hematopoietic

insult have led to great interest in this cytokine as a myebopo-

tentiating agent for treatment of bone marrow toxicity or

insufficiency.

Studies of the in vivo hematopoietic effects of IL-la have

focused mainly on cells of the granulocyte-macrophage lin-

cage. We have demonstrated that tumor necrosis factor

From the Laboratories of Experimental Hematology, Cell Biol-

ogy and Experimental Therapeutics, AMC Cancer Research Cen-

ter, Denver.

Submitted August 30, 1988; accepted October 17, 1988.

Supported by grants CA33188, CA33939, and CA48O77from the

National Institutes of Health and by a gift to the AMC Cancer

Research Centerfrom Gerald M. Quiat.

Address reprint requests to Dr Candace S. Johnson, Laboratory

ofExperimental Hematology, AMC Cancer Research Center, 1600

Pierce St. Denver, CO 80214.

The publication costs ofthis article were defrayed in part by page

charge payment. This article must therefore be hereby marked

“advertisement” in accordance with 18 U.S.C. section 1 734 solely to

indicate this fact.

C 1989 by Grune & Stratton, Inc.

0006-4971/89/7303-0007$3.00/0

Decreases in peripheral blood hematocrit did not occur

after a single IL-i a injection but were observed after

multiple injections of the cytokine. The suppressive effects

of IL-i a on late-stage erythropoiesis were abrogated by

simultaneous administration of erythropoietin (EPO). At 48

hours post-treatment, a marked stimulation was observed

in the numbers of spleen and marrow immature erythroid

(BFU-E), macrophage (CFU-M), granulocyte (CFU-G), gran-

ulocyte-macrophage (CFU-GM), and megakaryocyte (CFU-

meg) progenitor cells. These results demonstrate the

potential use of IL-i a as a generalized stimulator of hema-

topoiesis and show that the cytokine-induced suppression

of late-stage erythropoiesis can be prevented by EPO.

a 1989 by Grune & Stratton, Inc.

(TNF), which shares many biological activities with IL-i,’4

significantly increases numbers of macrophage, granulocyte,

megakaryocyte, and immature erythroid progenitor �

However, TNF also markedly suppresses late-stage erythro-

poiesis, leading eventually to anemia in chronically treated

animals.

We report here that, like TNF, IL-ia significantly sup-

pressed late-stage erythropoiesis in normal mice, while the

number of more immature erythroid progenitors, as well as

granubocytic, monocytic, and megakaryocytic precursors

were markedly stimulated. Suppression of erythropoiesis was

abrogated by simultaneous administration of the primary

regulator of erythropoiesis, erythropoietin (EPO). These

results demonstrate that IL-la has profound effects on

hematopoiesis that could be exploited for therapeutic pur-

poses.

MATERIALS AND METHODS

Mice. These experiments were carried out in inbred NIH/PLCR mice that were originally obtained from the VeterinaryResources Branch, NIH, and inbred in our laboratories by brother-sister mating. The colonies were regularly monitored for the absenceof adventitious viruses using mouse antibody production tests. Micewere age and sex matched for experimental use. For hematopoieticcolony assays, spleens or femurs were removed from three to five

animals per treatment group and assayed individually. All experi-ments reported here were replicated at least three times.

IL-i. Recombinant human IL-la was generously provided byDr Peter Lomedico, Hoffmann-LaRoche, Nutley, NJ. The specific

activity for the recombinant IL-ia was 2.5 x l0� U/mg protein, asdetermined using the DlO.G4.1 assay.’6 All solutions were madewith pyrogen-free reagents; the endotoxin content of the IL-la usedwas 0.05 EU/mg.

Reagents. Endotoxin (lipopolysaccharide W Escherichia coli/

0l27:B8) was obtained from Duo, Detroit. Human recombinantEPO for in vivo use was obtained from Amgen, Thousand Oaks, CAand had a specific activity of 70,000 U/mg ofprotein. Iron (Imferon,iron dextran, 50 mg/mL) was obtained from Merrell Dow, Cincin-

nati.Hematopoietic progenitor assays. The plasma clot method of

McLeod et al’7 was used for the culture of mature erythroid

For personal use only. by guest on November 24, 2013. bloodjournal.hematologylibrary.orgFrom

wC,z

0

100

50

0

-50

IN VIVO EFFECTS OF IL-la 679

progenitors (CFU-E) as previously described. A modification of themethylcellulose method of Iscove et al” was adapted to agar for theculture of immature erythroid burst-forming (BFU-E), granulocyte-macrophage (CFU-GM), granulocyte (CFU-G), and megakaryo-

cyte (CFU-meg) progenitors, as follows: 0.25 mL of spleen or bone

marrow cells (1 x i0� and 2 x 106 cells/mL, respectively) weremixed with 0.25 mL of 10% deionized bovine serum albumin (BSA;

containing 0.17% NaHCO3), 0.5 mL EPO (10 U/mL, TCepo,rEPO, Amgen, Thousand Oaks, CA), 0.4 mL heat-inactivated horseserum, 0.4 mL pokeweed mitogen-stimulated spleen cell-conditioned

medium,’9 0.5 mL heat-activated fetal calf serum (FCS), 0.2 mL

NCTC 109 medium, 0.020 mL hemin (10 mmol/L),2#{176}and 1.5 mL ofan agar mixture containing 1.0 mL of Bacto agar (1 .5%), 1 .0 mL 2 xRPM! 1640, 0.5 mL heat-inactivated FCS, and 0.050 mL 2-mercaptoethanol (102 mol/L). Abiquots of 0.5mL (3 x l0� spleenand 6 x i0� bone marrow cells/well) were plated into four wells of around-bottom well (2.2 x 1.4 cm) flexible culture plate (Linbro,

#76-354-05). After seven to nine days incubation at 37#{176}Cin an N2atmosphere containing 5% CO2 + 5% 02, the agar plugs were fixedwith 5% glutaraldehyde, adhered to glass slides, stained with di-

methoxybenzidine and hematoxylin, and scored on the basis ofmorphology. Agar cultures for macrophage (CFU-M) progenitorswere performed as described previously using L-cell-conditioned

media as a source of CSF.2’

RESULTS

Effect of IL-i on erythropoiesis. To determine the in

vivo effect of IL- i on normal erythropoiesis, groups of

IL-i-treated and untreated animals were examined for

changes in their erythroid progenitor compartments. Normal

mice were injected intraperitoneally (IP) with 0.5 �sg of

recombinant human IL- ia (1 .25 x i06 U/mouse), the dose

which in vivo maximally stimulates an increase in CFU-

GM.7 After 48 hours, spleen and bone marrow were removed

and examined for absolute numbers of CFU-E and BFU-E

(Table 1). Numbers of mature CFU-E were significantly

suppressed in the spleen and bone marrow of IL-ia-treated

animals. No significant change was observed in spleen

weight or total viable numbers of nucleated cells per spleen

or femur from animals treated with IL-la. The effect of

IL-ia treatment on late-stage erythropoiesis was also mani-

fest in peripheral blood reticulocyte counts, decreasing the

percent reticulocytes from 9.5 ± 0.3 to 2.2 ± 0.7 (P < .001).

No significant difference was observed in the hematocrits of

normal (49 ± 2) and treated animals (49 ± 1) after a single

IL-la injection. In contrast to CFU-E, the immature cry-

Table 1 . Effect of IL-la on Eryth ropoiesis in Normal Animals

ColonyType Treatment

No. colonies5

Per Spleen(x10’)

P& Femur� (x10’) %C

CFU-E

BFU-E

None

IL-la

None

IL-la

604.9 ± 92.6

248.3 ± 29.3�

15.7 ± 2.6

4O.8±9.O�

104.9 ± 16.9

-59 49.9 ± 7.2�

6.6 ± 1.5

+160 lO.4±2.8�

-52

+58

C Mean ± SD per total spleen or femur was determined in three to five

animals that were assayed individually (three cultures counted/animal) 48

hours after IP injection of 0.5 �g of IL-la.

tP&cent change from control.�Significantly different than control, no treatment (individual animals),

P < .001 (Student’s ttest).

throid progenitor, BFU-E, was significantly stimulated by in

vivo IL-l treatment (Table i).

To quantitate the response of normal erythropoiesis to

IL-ia, animals were injected IP with various doses of the

cytokine and assayed for progenitor colony formation. As

shown in Fig 1 , CFU-E suppression was observed with a

single injection of 0.125 gig/mouse or more. The BFU-E

compartment was significantly stimulated in a dose-depen-

dent manner between 0.25 zg and 1.0 pg/mouse, the highest

dose analyzed. As previously reported,22 severe side effects

(hair ruffling, diarrhea, lethargy) and mortality were

observed in animals treated with doses greater than i.0 sg

IL-i (2.5 x 106 U/mouse).

The time course for effects of IL- ia on erythropoiesis in

normal animals was determined following a single injection

of 0.5 jsg/mouse. As shown in Fig 2, numbers of spleen

CFU-E were suppressed as early as six hours after IL-ia

(3 1 % ± 9%, decrease, P < .05), reached a nadir at 24 hours,

and remained significantly suppressed for 72 hours post-

treatment. By four days postinjection, CFU-E numbers

returned to normal, and after five days a significant stimula-

tion was observed in the splenic CFU-E compartment. The

BFU-E compartment (Fig 2) was significantly stimulated 48

200

150

100

0.00 0.25 0.50 0.75 1.00

DOSE (ug/mouse)

Fig 1 . Dose response for IL-i a effects on normal CFU-E (#{149})and BFU-E (0). Animals were treated with various doses ofIL-la IPand assayed 48 hours post-treatment for numbers of CFU-E andBFU-E colony-forming cells in the spleen. Points represent mean

percent of change ± SD calculated from three to five animalsassayed individually (three cultures counted/animal) at each timepoint. Numbers of colony-forming cells at doses equal to or greaterthan 0.25 iig (BFU-E) or 0.125 sg (CFU-E) per mouse weresignificantly different from control without IL-i a (P < .001 . Stu-dent’s t test).

For personal use only. by guest on November 24, 2013. bloodjournal.hematologylibrary.orgFrom

0 24 48 72 96 120 144

HOURS POST INJECTION

680 JOHNSON ET AL

Li0

I0

150

100

50

0

-50

-100

Fig 2. Time course for in vivo effects of IL-i a on CFU-E (#{149})andBFU-E (0). Normal animals were treated with 0.5 pg/mouse ofIL-ia IP and assayed for CFU-E or BFU-E colony-forming cells in thespleen at various times post-IL-i a treatment. Points representmean percent of change ± SD calculated from groups of three tofive animals assayed individually (three cultures counted/animal)

at each time point. Numbers of colony-forming cells at 48 hours(BFU-E) or six, 12. 24. 48, 72, 120. and i44 hours (CFU-E) weresignificantly different from control with IL-i a (P < .001 , Student’sttest).

hours after a single injection of 0.5 �tg with numbers of

BFU-E returning to normal by 72 hours. The dose and time

parameters for the effects of IL-ia on bone marrow CFU-E

and BFU-E were identical to those for spleen erythroid

progenitors.

To determine the effects on late-stage erythropoiesis of

multiple IL-ia injections, normal animals were treated with

0.5 zg of IL- la every 1 2 hours for three days. Twelve hours

after the last IL- la injection, spleen CFU-E numbers were

markedly decreased in treated mice (70,340 ± 10,220 SD)

compared with controls injected with vehicle alone

(820,000 ± 54,000, P < .001). Hematocrits were also signif-

icantly decreased in animals treated with repeated injections

of IL-ia compared with controls [43 ± 2 SD and 48 ± 1,

respectively, P < .001].

To establish that the effects on erythropoiesis were not due

to contaminating endotoxin animals were injected with

either heat-treated preparations of IL-ia (100#{176}Cfor 60

minutes) or varying doses of purified endotoxin alone. Heat

treatment abrogates IL-ia activity but does not affect endo-

toxin levels. No effect on erythropoiesis was observed when

animals were injected with either heat-treated IL-la or

endotoxin at concentrations as high as 0.4 EU (0.05 ng,

i6,000 times the amount in 0.5-sg doses of recombinant

IL-ia per mouse, data not shown).

The acute phase response, which is thought to be mediated

by IL- 1 ,� is accompanied by suppression of serum iron

levels.23’24 Significantly decreased levels of iron are observed

within six hours of IL-i injection. To determine whether

IL-ia induced suppression of CFU-E might be due to

reduced serum iron, animals were injected simultaneously

with 0.5 �tg IL-ia and 5 mg/mouse of iron, a dose expected

to saturate available serum iron-binding sites.25 The supple-

mentation of iron had no effect on the suppression of CFU-E

induced by IL-ia (IL-ia plus iron, 32% suppression v IL-ia

only, 43%).

To determine whether IL-ia-induced suppression was due

to a direct effect of IL-i on CFU-E, varying amounts of the

cytokine were added directly to the in vitro CFU-E colony-

forming assay. IL-ia at doses up to 1,000 U/mL (0.4

ng/mL) had no effect on in vitro CFU-E colony formation

(data not shown). Similarly, IL-ia had no effect on CFU-E

colony formation in the presence of varying concentrations of

EPO.

Reversal ofin vivo CFU-E suppression with erythropoie-

tin (EPO). Suppression of erythropoiesis by IL-ia was

observed in the EPO-dependent CFU-E but not in the

EPO-independent BFU-E compartment. To determine

whether EPO could thus abrogate the suppressive effect on

CFU-E, animals were treated simultaneously with 0.5 sg

IL- ia and varying doses of human recombinant EPO

(70,000 U/mg). As shown in Table 2, 0.5 U/mouse of EPO

were sufficient to fully reverse the suppressive effects of

IL-ia on CFU-E; EPO alone at this dose had no significant

effect on spleen CFU-E numbers in normal animals (data

not shown).26

Effect of IL-la on CFU-M, CFU-GM, CFU-G, and

CFU-meg progenitors. In vivo treatment of normal ani-

mals with IL-ia has been shown to increase numbers of

granulocyte-macrophage progenitors.79 To extend these

observations, the effects of IL-ia on the various hemato-

poietic progenitor compartments were determined. As shown

in Table 3, a single dose of IL-ia (0.5 sg) resulted in

significant stimulation of total bone marrow and spleen

CFU-M, CFU-GM, CFU-G, and CFU-meg at 48 hours

postinjection.

In dose-response studies (Fig 3), a significant stimulation

of CFU-M and CFU-G was observed following injection of

IL-ia at 0.125 gig/mouse (increases of 62% ± 18% and

52% ± 15%, respectively, P < .001), with maximum stimu-

lation obtained with 1 .0 sg/mouse (the highest dose tested,

see above). Significant stimulation of CFU-GM and CFU-

Table 2. Reversal of CFU-E Suppression with In Vivo Treatment

with EPO

rEPO Dose

No. coloniest

Per SpleenTreatment U/Mouse (x 1O�) %Ct

None 0 568.5 ± 86.6

IL-la 0

0.1

0.5

1.0

3.0

234.0 ± 20.0�

279.4 ± 29.l�

552.1 ± 62.7

594.8 ± 45.6

712.5 ± 94.011

-59

-51

+3

+5

+25

Human rEPO, 70,000 U/mg protein, was given P simultaneously

with IL-la.

tMean ± SD per total spleen was determined in three to five animalsthat were assayed individually (three cultures counted/animal) 48 hours

after IP injection of 0.5 �g of IL-la.

�Percent change from control.

§Significantly different than control, no treatment (individual animals),

P < .001 (Student’s ttest).

II�< .01.

For personal use only. by guest on November 24, 2013. bloodjournal.hematologylibrary.orgFrom

Table 3. Effect of IL-ia on CFU-M, CFU-GM, CFU-G, and

1000

meg was observed following injection of IL-ia at 0.5 jzg/

mouse (increases of 83% ± 22% and 52% ± i5%, respec-tively, P < .001) with maximum stimulation obtained with

1.0 sg/mouse. As previously described for CFU-GM,7 peak

stimulation of CFU-M, CFU-G, and CFU-GM was seen at

48 hours after a single injection of 0.5 sg IL-ia (Fig 4) with

return to normal numbers by 72 hours. For the CFU-meg

progenitors, maximum stimulation was observed at 72 hours

post-treatment with a return to normal numbers by 96 hours.

The dose and time parameters for the effects of IL-ia on

bone marrow progenitors were identical to those for spleen

CFU-M, CFU-GM, CFU-G, and CFU-meg.

DISCUSSION

Suppression of hematopoiesis is the major toxic side effect

of most cancer chemotherapy regimens. Damage to the

hematopoietic system often restricts dosage and frequency of

drug administration, limiting potential therapeutic effect.27’2’

Chemotherapy-induced toxicity occurs in the hematopoietic

compartments of the bone marrow, spleen, lymph nodes, and

thymus.29’3#{176} The result is severe immunosuppression and

increased susceptibility to infections, hemorrhage, and ane-

mia. Agents such as the CSFs and IL-ia, which prevent or

reverse these dose-limiting hematopoietic toxicities and per-

mit the use of higher or more frequent doses of cytotoxic

350

300

250

200Iii

� 150

100

50

0

-50

LiiC,

z

0

400

300

200

100

00.00 0.25 0.50 0.75 1.00

DOSE (ug/mouse)

IN VIVO EFFECTS OF IL-la 681

C FU-meg Pr ogenitor Cell s in Normal Animals

Colony Type Treatment

No. Colonies

P� Spleen(x i0�)

P& Femur%Ct (x lOs) %C

CFU-M None 14.2 ± 4.9 25.5 ± 1.9

CFU-GM

IL-ia

None

55.2 ± 5.6t

18.6 ± 3.2

+288 43.5 ± 3.it4.8 ± 1.1

+71

CFU-G

IL-ia

None

28.8 ± 3.8�

18.4 ± 5.2

+55 7.6 ± 0.5�

15.6 ± 1.4

+58

CFU-meg

IL-la

None

IL-la

42.1 ±6.9t

10.2 ± 5.3

19.0 ± 5.8�

+128 37.2±9.7t

4.4 ± 1.3

+86 7.5 ± 2.i�

+138

+70

Mean ± SD per total spleen or femur was determined in three to five

animals that were assayed individually (three cultures counted/animal) 48

hours after IP injection of 0.5 sg of IL-la.

tPercent change from control.

�Significantly different than control, no treatment (individual animals),

P < .001 (Student’s ttest).

§P< .o�.

Fig 3. Dose response for in vivo IL-i a stimulation of normalCFU-M (0). CFU-G (s), CFU-GM (t�J. and CFU-meg (A). Normalanimals were treated with 0.5 sg/mouse of IL-ia IP and assayedfor colony-forming cells in the spleen at various times post-IL-i atreatment. Points represent mean percent of change ± SD calcu-lated from groups of three to five animals assayed individually

(three cultures counted/animal) at each time point. Numbers ofcolony-forming cells at doses of IL-i a equal to or greater than0.125 M9 (CFU-M and CFU-G), 0.25 �ig (CFU-GM). or 0.50 sg(CFU-meg) per mouse were significantly different from control(P < .OOi , Student’s t test).

0 12 24 36 48 60 72 84

HOURS POST INJEC11ON

Fig 4. Time course for stimulation of CFU-M (0). CFU-G (#{149})

CFU-GM (L�j, and CFU-meg (A). Normal animals were treated with0.5 ag/mouse of IL-ia IP and assayed for colony-forming cells inthe spleen at various times post-IL-i a treatment. Points repre-sent mean percent of change ± SD calculated from groups of three

to five animals assayed individually (three cultures counted/animal) at each time point. Numbers of colony-forming cells at 6,i2, 24, and 48 hours (CFU-M), 24 and 48 hours (CFU-G andCFU-GM), or 48 and 72 hours (CFU-meg) were significantlydifferent from control without IL-i a (P < .OOi , Student’s t test).

For personal use only. by guest on November 24, 2013. bloodjournal.hematologylibrary.orgFrom

682 JOHNSON ET AL

drugs, could thus greatly enhance the therapeutic index of

many cytotoxic drugs.

Recent advances in recombinant DNA technology have

made large quantities of highly purified human IL-i readily

available.31’32 There are at least two biochemically distinct

types of human IL-i, isofocusing at pls of 5 (IL-ia) and 7

(IL-i/3). Although a comparison of the primary structures of

IL-la and IL-i$ revealed only 26% homology,32 they share

many activities in vitro and the target cell specificities for

both appear to be the same.33 Preliminary data suggest that

IL-ia is primarily membrane bound while IL-i$ is mostly

secreted.34 Mochizuki et al’#{176}demonstrated that IL-ia but

not IL-ifl potentiates hematopoietic responses to GM-CSF

and is identical to hemopoietin-i, a previously described

growth factor for primitive hematopoietic cells. In contrast,

Morrissey et al35 have demonstrated that both IL-ia and

IL-i�3 hasten the recovery of myeloid compartments in

sublethally irradiated mice.

The effects of IL- ia reported here on various normal

hematopoietic compartments have also been observed follow-

ing in vivo treatment of normal animals with TNF: TNF

significantly suppresses CFU-E and stimulates BFU-E,

CFU-M, CFU-GM, and CFU-meg.’5 Other studies have

demonstrated that TNF can suppress in vitro CFU-E colony

formation.36’37 IL-ia stimulates macrophage production of

TNF’4’3’ and also GM-CSF79 and more IL-i.’4 TNF stimu-

bates macrophages to produce IL- 1 ,�‘ prostaglandin-E2,39

IFN,�#{176}GM-CSF,4’ and more TNF.’4 Prostaglandin-E2 and

interferons have been shown to inhibit erythropoiesis.42’43

IL-i could thus act alone, through TNF, or both cytokines

could influence hematopoiesis through other mediators.

The mechanism of IL- ia suppression of in vivo mature

erythroid progenitor numbers is unknown. Because no effect

was observed on in vitro CFU-E colony formation, IL-ia

may act either directly on erythroid progenitors at a point in

the lineage between the BFU-E and the CFU-E or indirectly,

as noted above, through T cells or macrophages or their

products.

IL-ia’s in vivo suppressive effect on CFU-E appears to

involve interactions with the primary erythropoietic regula-

tory hormone, EPO. IL-ia is only suppressive to CFU-E, the

EPO-dependent progenitor, and these effects are reversed by

simultaneous administration of EPO. Schooley et al� dem-

onstrated that IL-la and IL-ifl inhibit in vitro erythropoie-

sis, apparently by interfering with the effect of EPO on

mature erythroid progenitors, as measured in a 3H-TdR

incorporation assay. Interestingly, our preliminary studies

have demonstrated that TNF-a-induced suppression of

CFU-E is also reversed with simultaneous administration of

EPO (unpublished observations), suggesting that TNF-a

and IL-a affect late-stage erythroid progenitors through

similar mechanisms.

IL-ia and TNF have been shown to induce cachexia,22’45

which could influence erythropoiesis in several ways. Fasting

rats, for example, have extremely low levels of EPO,� and

this occurrence forms the basis for the starved rat EPO

assay.47 In our studies effects on CFU-E were observed

within six hours of injection, and mice given a single injection

of IL-ia exhibited no changes in eating habits in the course

of observation and were not noticeably cachectic at sacrifice.

Treatment with IL-la, however, could result in lowered EPO

levels prior to the manifestation of cachexia.

These studies were carried out using recombinant human

IL-ia. In vivo activities of IL-i appear to lack species

specificity.4’ Using recombinant murine and human IL-ia,

however, we have observed quantitative differences in the

effects of the cytokines on isologous v homologous cells

(manuscript in preparation).

In summary, IL-la significantly increases BFU-E, CFU-

M, CFU-G, CFU-GM, and CFU-meg numbers in the spleen

and bone marrow of normal animals. This stimulation is

accompanied by a significant suppression of late-stage eryth-

ropoiesis (CFU-E) that can lead to anemia in animals

treated with multiple doses of IL-ia. The suppression of

CFU-E was reversed when animals were simultaneously

treated with EPO. These studies form the basis for gaining

an understanding of the hematologic effects of IL-la and for

further evaluation of IL-ia as an agent to mitigate the bone

marrow toxicities that occur during treatment of human

malignancy. Recent studies in our laboratories have shown

that IL- la causes acute hemorrhagic necrosis and marked

decrease in clonogenic cellularity of solid tumors4950;

together with the hematologic effects reported here, the

results point to potentially important uses of IL-i in tumor

therapy.

ACKNOWLEDGMENT

We thank Cathrine Allen for preparation of the manuscript.

REFERENCES

1 . Metcalf D: The molecular biology and functions of the granu-bocyte-macrophage colony-stimulating factors. Blood 67:257, 1986

2. Burgess AW: Haemopoietic growth factors: Structure andreceptor interactions. Ciba Found Symp 116:148, 1985

3. Quesenberry PJ: Synergistic hematopoietic growth factors. mtJ Cell Cloning 4:3, 1986

4. Dinarello CA: Interleukin-l. Rev Infect Dis 6:51, 1984

5. Gery I, Gershon RK, Waksman BH: Potentiation of theT-lymphocyte response to mitogens. I. The responding cell. J Exp

Med 136:128, 19726. Smith KA, Lachman LB. Oppenheim JJ, Favata MF: The

functional relationship of the interleukins. J Exp Med 151:1551,

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