Macrophage-mediated suppression of immune responses in Toxoplasma-infected mice

CELLULAR

85,417-427

IMMUNOLOGY

(1984)

Macrophage-Mediated Suppression of Immune Responses in Toxoplasma-Infected Mice I. inhibition of Proliferation of Lymphocytes in Primary Antibody Responses’ YASUHIRO Department

SUZUKI AND AKIO KOBAYASHI

of Parasitology, Nishi-Shinbashi,

Received

October

Jikei University School of Medicine, Minato-Ku, Tokyo 105, Japan

11, 1983; accepted December

3-25-8

21, 1983

The suppressor cells induced by Toxoplasma infection were shown to be macrophages, since they adhered to plastic, and their suppressive activity in anti-sheep erythrocytes (SRBC) antibody responses was abrogated by treatment with silica or carrageenan, which are selectively cytotoxic for macrophages. The suppressor macrophages strongly inhibited the uptake of tritiated thymidine ([3H]TdR) by normal mouse spleen cells in the responses to SRBC and Toxoplasma antigens. Supematant fluids from the suppressor macrophages could not passively transfer the suppressive effect on anti-SRBC antibody responses. Furthermore, when the suppressor macrophages were isolated by a cell-impermeable membrane from normal mouse spleen cells, the antibody responses of normal spleen cells were not suppressed. These results indicate that suppression of antibody responses in Toxoplasma-infected mice is caused by an inhibitory effect of the suppressor macrophages upon proliferation of lymphocytes via direct contact with responder target cells. The suppressive effect of the macrophages was not counteracted by indomethacin, a potent inhibitor of prostaglandin synthesis, or catalase, a catabolic enzyme for hydrogen peroxide (H,O,).

INTRODUCTION The occurrence of suppression in antibody responses to unrelated antigens has been reported in mice with Toxoplusma infection ( l-5). The suppression is nonspecific for antigens and immunoglobulin classes (4, 5). Both primary responses (l-4) and initiation of memory cells (2, 5) are suppressed by infection. Because of the antigen nonspecificity of the suppressive effect, it is presumed that suppression may also occur on antibody responses to the parasite itself, and play a role in making suitable circumstances for parasite proliferation. Concerning the mechanisms of this immunosuppression, we have reported that irradiation-resistant and plastic-adherent suppressor cells are present in the spleens of infected mice (3). In the present study, we demonstrate that the suppressor cells induced by Toxoplasma infection are macrophages, and that they inhibit the proliferative responses of lymphocytes to antigens both related and unrelated to T. gondii. The suppressor macrophages appear to affect lymphocytes via a cell-cell contact with responder target cells rather than by a release of soluble mediators. ’ This work was supported by a grant-in-aid from the Ministry of Education, Science, and Culture, Japan. 417 0008-8749184 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any font reserved.

418

SUZUKI

MATERIALS

AND KOBAYASHI

AND

METHODS

Animal. Eight-week-old female C57BL/6 mice purchased from Shizuoka Agricultural Cooperative Association for Laboratory Animals (Hamamatsu, Japan) were used for all experiments. Toxoplasma infection. Bradyzoites of the avirulent Fukaya strain of T. gondii were used for infection. The bradyzoites were obtained from the brains of chronically infected mice by treatment with 0.25% trypsin as described previously (4). Released bradyzoites were suspended in phosphate-buffered saline at 2.5 X lo4 organisms/ml, and mice were inoculated with 0.2 ml of the suspension intraperitoneally. Antigens. SRBC were purchased from Nippon Bio-Test Laboratories, Inc. (Tokyo, Japan). Toxoplasma antigens were prepared by lysis of toxoplasmas in distilled water (6). Briefly, tachyzoites of the virulent RH strain of T. gondii from the peritoneal fluid of mice 3 days postinfection were filtered to remove host cells, and washed in phosphate-buffered saline. The organisms were destroyed by suspension in distilled water. The antigen preparation was centrifuged at 9000g for 30 min, and the supernatant was used as solubel Toxoplasma antigens. Spleen cell culture. Normal mouse spleen cells were suspended at a concentration of 5 X lo6 cells/ml in a completely supplemented Eagle minimal essential medium containing Hanks’ salts, 50 U of penicillin/ml, 50 pg of streptomycin/ml, 5 X low5 M 2-mercaptoethanol, and 10% fetal calf serum (Flow Laboratories, Inc., Stanmore, New South Wales, Australia) as described elsewhere (4). Portions of 200 ~1 ( lo6 cells) were incubated with 4 X lo5 SRBC in Microplate II culture plates (Falcon Plastics, Oxnard, Calif.) for 4 days at 37°C in 5% CO2 in air. Experimental groups were composed of three identical wells. Dzfision chamber experiments. Six million normal mouse spleen cells in 1 ml medium and 2 X lo6 SRBC were added to 24-well Linbro culture plates (Linbro Scientific, Inc., Hamden, Conn.), which were used as the outer chamber. Aliquots of 0.2 ml containing 3 X lo6 normal or infected mouse spleen cells were added to a small glass vessel sealed by a Nuclepore membrane (pore size 0.4 pg, Nucleopore Corp., Pleasanton, Calif.), and the vessel was placed in the well of the Linbro culture plate as the inner chamber. After 4 days culture, the plaque-forming cells (PFCp responses of normal mouse spleen cells in the outer chamber were measured. Assays fir PFC. At the termination of the culture, cells from each group were harvested by aspiration, pooled, and sedimented by centrifugation. The sedimented cells were suspended in an amount of Hanks’ balanced salt solution equal to the original volume. The number of PFC was determined by the methods described by Pierce et al. (7). Four hundred microliters of 0.6% agarose (Nakarai Chemicals Ltd., Kyoto, Japan) in Hanks’ balanced salt solution, 50 ~1 of an 8% suspension of SRBC, and 100 ~1 of the cultured cell suspension were mixed at 47°C and poured onto microscope slides. Duplicate slides, prepared from each group, were incubated for 1.5 hr at 37°C in a humid atmosphere, and for another 1.5 hr with guinea pig complement (Toshiba Chemicals, Tokyo, Japan) diluted l/20 in Hanks’ balanced salt solution. ’ Abbreviations used: PFC, plaque-forming cell; SRBC, sheep erythrocytes; [‘H]TdR, tritiated thymidine; H202, hydrogen peroxide.

SUPPRESSOR MACROPHAGE

IN TOXOPLASMA

MICE

419

Preparation of plastic-adherent suppressor cells. The spleen cells were obtained from mice on the seventh day of infection, when the strongest suppression was observed (4). The cells were suspended in the medium described above at a concentration of 5 X lo6 or 1 X lo7 cells/ml. Portions of 200 ~1 were incubated for 2 hr in the culture plates, and nonadherent cells were removed by gentle pipetting. The remaining cells were used as plastic-adherent suppressor cells. Treatment with silica or carrageenan. X-Carrageenan was purchased from Sigma Chemical Company (St. Louis, MO.), and Wakogel LC-5H (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was used as silica particles about 5 pm in diameter. Plastic-adherent suppressor cells from 1 X lo6 infected mouse spleen cells were incubated with I mg/ml of X-carrageenan or 200 pg/ml of silica for 15 hr. After removal of the agents by thorough washing with Hanks’ balanced salt solution, the cells were added to normal mouse spleen cells. Assaysfor lymphocyte proliferation. Plastic-adherent cells from 2 X IO6 normal or infected mouse spleen cells were added to 1 X lo6 normal mouse spleen cells. In the anti-SRBC antibody responses, the culture period, 4 days, was divided into four parts, and for each 24 hr, the cells were labeled with 1 &i [3H]TdR, sp act 21 Ci/mmol (The Radiochemical Centre, Amersham, Buckinghamshire, England). To examine the responses of lymphocytes to Toxoplasma antigens, the cells were incubated with 50 p@rnl of Toxoplasma antigens for 3 days without 2-mercaptoethanol in the culture medium. For the final 24 hr, the cells were labeled with 1 &i of [3H]TdR. The cells from each group were pooled and collected on glass-fiber filters (Whatman Ltd., Maidstone, Kent, England), suspended in a toluene-based scintillation fluid, and counted on a liquid scintillation counter. Treatment with indomethacin and catalase. Indomethacin (Sigma Chemical Co.) was dissolved in 95% ethanol at 1.2 X 10e2 M. Catalase (bovine liver, twice crystallized, 33,900 Sigma units/mg protein) was also obtained from Sigma Chemical Company and dialyzed in phosphate-buffered saline to eliminate any possible effect of thymol, which is used as a preservative in the catalase preparation. Both regents were diluted to appropriate concentrations in Hanks’ balanced salt solution, and 20 ~1 of each solution was added to the culture. RESULTS Course of infection. The spleen was significantly enlarged by Toxoplasma infection. An increase of spleen weight was already detectable 3 days after infection, and most marked between the first and second week of infection, i.e., about five times larger than the uninfected control. By the fourth week of infection, the spleen weight decreased to be only twice that of the control, the level at which it remained through at least 16 weeks after infection. Anti-SRBC antibody responses were suppressed in Toxoplasma-infected mice as previously reported (4). This suppression was observed by the third day of infection, persisted throughout 1 month, and was strongest 1 week after infection. E#ect of silica or carrageenan on the activity of plastic-adherent suppressor cells. Spleen cells were obtained from mice on the seventh day of infection, when the most potent suppression had been observed (4). Figure 1 shows that in vitro anti-SRBC

420

SUZUKI

AND KOBAYASHI

NSC ISC NSC

+

ISC

NSC

+

IA

NSC + :ErntiT b&c + :$&

,A

NSC

+ NSC

NSC

+

NA

NSC + %?r$?,? NSC + %?d

NA

0

50 %

of mtrol

100 PFC responses

FIG. 1. Effect of treatment with silica or carrageenan on activity of plastic-adherent splenic suppressor cells from Toxoplasma-infected mice in primary anti-SRBC antibody responses. Whole cells or plasticadherent cells from 1 X lo6 spleen cells of either normal or infected mice were added to 1 X lo6 normal mouse spleen cells, and cultured with SRBC for 4 days. In other cultures, plastic-adherent cells from 1 X lo6 normal or infected mouse spleen cells were incubated with 200 &ml silica or 1 mg/ml carrageenan for 15 hr and after thorough washing, the cells were added to 1 X lo6 normal mouse spleen cells and cultured with SRBC for 4 days. Each bar represents the mean + SEM. NSC, normal mouse spleen cells; ISC, infected mouse spleen cells; NA, adherent cells from NSC; IA, adherent cells from ISC.

antibody responses were strongly suppressed in infected mouse spleen cells. Furthermore, the addition of 1 X lo6 infected mouse spleen cells drastically suppressed the response of an equal number of normal mouse spleen cells. The addition of the plastic-adherent cells from 1 X lo6 infected mouse spleen cells caused the same suppression of the antibody responses by normal mouse spleen cells (Fig. 1). These results confirmed the findings of our previous report (4). In order to determine the nature of the suppressor cells more clearly, we examined whether the activity of the suppressor cells was abrogated by treatment with silica or carrageenan, selective cytotoxic agents for macrophages. Plastic-adherent cells obtained from 1 X 1O6infected mouse spleen cells were incubated with 200 pg/ml silica or 1 mg/ml carrageenan for 15 hr. After removal of the agents by thorough washing, the cells were added to 1 X lo6 normal mouse spleen cells to examine their effect on anti-SRBC antibody responses of normal mouse spleen cells. The adherent cells from 1 X lo6 normal mouse spleen cells which were treated in the same way were added as a control. Results are also shown in Fig. 1. The suppressive activity of the adherent cells from the infected mice was inhibited by treatment with either silica or carrageenan. On the other hand, treatment with these agents did not show any significant effects in the normal cell control cultures. This demonstrates that the suppressor cells in the spleens of Toxoplasma-infected mice are macrophages. Inhibition of an early step of lymphocyte stimulation in primary antibody responses by suppressor macrophages. As a first step in an analysis of the action of the suppressor macrophages, a relationship between the time of addition of the suppressor macrophages during the course of culture of normal mouse spleen cells and the suppressive effect of the added cells on antibody responses of normal spleen cells was investigated.

SUPPRESSOR MACROPHAGE

IN TOXOPLASMA

MICE

421

Infected mouse spleen cells (5 X 10’) were added to the cultures of 5 X lo5 normal mouse spleen cells at intervals of 24 hr after the start of the incubation of normal spleen cells with SRBC. As a control, 5 X lo5 normal mouse spleen cells were added instead of infected mouse spleen cells. Figure 2 shows the PFC responses of the cultured cells on Day 4 of culture. A marked suppression of the PFC responses was observed when infected mouse spleen cells were added either immediately after, or 24 hr after, the start of the culture of normal mouse spleen cells with SRBC. However, the addition of infected mouse spleen cells 48 hr after the start of culture did not affect the antibody responses of normal mouse spleen cells. These results suggest that the suppressor macrophages in infected mouse spleen cells inhibit an early step of lymphocyte stimulation in primary antibody responses. Inhibition ofproliferation of lymphocytes by suppressor macrophages from the infected mice. The effect of the suppressor macrophages on the proliferation of lymphocytes, which is an early step of lymphocyte stimulation in antibody responses, was studied by measuring the uptake of [3H]TdR. The suppressor macrophages from 2 X lo6 infected mouse spleen cells were added to 1 X lo6 normal mouse spleen cells at the start of the culture with SRBC, and the cells were cultured for 4 days. The cells were labeled with 1 &i of [3H]TdR for 24 hr, at intervals of 24 hr. As a control, macrophages from 2 X lo6 normal mouse spleen cells were added to normal mouse spleen cells. As shown in Fig. 3, in the control, the uptake of [3H]TdR increased remarkably during the course of culture. By contrast, in the experimental group, the uptake of the isotope was constantly kept at a low level throughout all of the culture periods. This indicates that the suppressor macrophages from Toxoplasma-infected mice suppress the proliferation of lymphocytes in primary antibody responses to SRBC. Eflect of addition of culture fluids of suppressor macrophages on primary antibody responses of normal mouse spleen cells. The macrophages from 1 X 1O6 and 4 X 1O6 of normal and infected mouse spleen cells were cultured for 24 or 48 hr, and supernatant fluids were obtained from them. One hundred microliters of each fluid was added to an equal volume of the suspension of normal mouse spleen cells (1 X lo6 cells),

100.

Day of addition

d

of suppessor

cells

FIG.2. Relationship between the time of addition of suppressor macrophages from Toxoplasma-infected mice and strength of the suppressive effect during the course of culture in primary anti-SRBC antibody responses. Infected mouse spleen cehs (5 X 10’) were added to 5 X lo5 normal mouse spleen cells at various intervals after the start of culture with SRBC. As a controi, 5 X IO5 normal mouse spleen cells were added instead of infected mouse spleen cells. Each bar represents the mean + SEM.

422

SUZUKI

Period

AND KOBAYASHI

of pulse

label

by3H-thymidme

FIG. 3. Suppressive effect of splenic macrophages from Toxoplasma-infected mice on the uptake of [‘H]TdR by normal mouse spleen cells in primary anti-SRBC antibody responses. Adherent cells from 2 X lo6 infected mouse spleen cells were added to 1 X lo6 normal mouse spleen cells, and cultured with SRBC for 4 days. As a control, adherent cells from 2 X lo6 normal mouse spleen cells were added to normal mouse spleen cells. The cells were labeled with 1 pCi of [3H]TdR for 24 hr, at 24-hr intervals. 0, Control; 0, experimental. Each point represents the mean +- SE of the duplicated assays.

Culture

Source

NA

NA

IA

IA

1 x106

4x106

MO6

4x106

of cells

to obtain

culture

period

fluids

FIG. 4. Effects of the addition of supematant fluids from the suppressor macrophages from Toxophmzinfected mice on primary anti-SRBC antibody responses of normal mouse spleen cells. Adherent cells from 1 X lo6 and 4 X 106 of normal and infected mouse spleen cells were incubated for 24 or 48 hr. One hundred microliters of the supematant fluid from each culture was added to 100 ~1 ( 106 cells) of the suspension of normal mouse spleen cells, and cultured with SRBC for 4 days. NA, adherent cells from normal mouse spleen cells; IA, adherent cells from infected mouse spleen cells.

SUPPRESSOR MACROPHAGE Outer c+swxder, llqrar

IN TOXOPLASMA

MICE

423

Inner

NSC

i Medium

NSC

f NSC

Nsc

;

ISC

NSC NSC+NSC NSC+ ISC

%

of control

PFC respcmes

FIG. 5. Requirement for cell contact between suppressor macrophages and responder spleen cells in order to demonstrate suppression in vitro. To 6 X lo6 normal mouse spleen cells, 3 X lo6 normal or infected mouse spleen cells were added. The added cells were placed in contact with, or isolated by a cell-impermeable membrane from, the responder cells. The responder normal mouse spleen cells were cultured with SRBC for 4 days. Each bar represents the mean f SEM. NSC, normal mouse spleen cells; ISC, infected mouse spleen cells.

and the anti-SRBC antibody responses of the cells were examined. Figure 4 shows PFC responses of the cultured cells. Anti-SRBC antibody responses were not suppressed by the addition of any of the supematant fluids; instead they were enhanced by some fluids, indicating that the supernatant fluids from the suppressor macrophages did not have suppressive activity on primary antibody responses. Efect of isolation of suppressormacrophagesfrom responder normal mouse spleen cells by a cell-impermeable membrane on their suppressiveactivity. In order to examine

whether cell contact with responder spleen cells is required for the suppressive action of the macrophages, 3 X 1O6 infected mouse spleen cells were added to 6 X lo6 normal mouse spleen cells in contact with, or isolated by a cell-impermeable membrane from, normal mouse spleen cells. PFC responses are shown in Fig. 5. Infected mouse spleen cells provoked more than 60% suppression of the antibody responses of normal mouse spleen cells in the mixture with the normal mouse spleen cells. On the other hand, when infected mouse spleen cells were isolated by a cell-impermeable membrane, the antibody responses of normal mouse spleen cells were not suppressed, but rather enhanced by the addition of infected mouse spleen cells, as observed in the experiment of the culture fluids from the suppressor macrophages. In a control, normal mouse spleen cells did not affect the antibody responses of normal mouse spleen cells either with or without contact with responder normal mouse cells. This suggests that the suppressor macrophages from the infected mice require cell-cell contact with responder target cells to demonstrate their suppressive activity. Eflect of indomethacin on the activity of the suppressormacrophages.Prostaglandins are known as soluble mediators of negative immunoregulatory influence by macrophages (g-10). We examined whether the activity of the suppressor macrophages induced by Toxoplasma infection was abrogated by indomethacin, a potent inhibitor of prostaglandin synthesis. The suppressor macrophages from 2 X lo6 infected mouse spleen cells were added to 1 X lo6 normal mouse spleen cells in the presence of 2 X 10e5 to 2 X 10d7 M indomethacin, and the cells were cultured with SRBC for 4

424

SUZUKI

AND KOBAYASHI

days. PFC responses of the cultured cells are shown in Fig. 6. The suppressive effect of the macrophages was not inhibited by the presence of various doses of indomethacin. Eflect of catalase on the activity of the suppressor macrophages. H202 is known as a short-lived mediator which is released by macrophages and mediates their suppressive effect on proliferation of lymphocytes (11, 12). In order to evaluate the role of H202 in the suppressive effect of Toxoplasma-induced macrophages, catalase (a catabolic enzyme for H202 (13)) was added to the cultures of normal mouse spleen cells and the suppressor macrophages from Toxoplasma-infected mice. Addition of 0.5 X lo4 to 2 X lo4 units/ml catalase did not alter the suppressive effect of the macrophages on anti-SRBC antibody responses of normal mouse spleen cells (Fig. 7). PFC responses of the cultures with both the suppressor macrophages and catalase were slightly lower than the responses of the culture with the suppressor only. In control cultures of normal mouse spleen cells, addition of catalase induced an increase of PFC responses (data not shown). E,tfect of the suppressor macrophages from Toxoplasma-infected mice on proliferation of lymphocytes to Toxoplasma antigens. We examined whether the suppressor macrophages induced by Toxoplasma infection suppress the responses of lymphocytes to Toxoplasma antigens themselves, as observed in the responses to SRBC, which is an unrelated antigen to T. gondii. The macrophages obtained from 1 X lo6 or 2 X lo6 of normal or infected mouse spleen cells were added to 1 X lo6 normal mouse spleen cells, and the cells were cultured with 50 pg/ml of soluble Toxoplasma antigens for 3 days. During the final 24 hr, the cells were labeled with 1 &i of [3H]TdR. As shown in Table 1, the macrophages from the infected mice strongly suppressed the uptake of [3H]TdR by normal mouse spleen cells. Normal mouse spleen cells incorporated more than 3000 cpm of [3H]TdR in the responses to Toxoplasma antigens. When the suppressor macrophages were present in the culture, the uptake of t3H]TdR was not induced by addition of Toxoplasma antigens. In a control, addition of the macrophages from 2 X lo6 normal mouse spleen cells resulted in an enhancement of uptake of [3H]TdR. These results demonstrate that the suppressor macrophages induced by Toxoplasma infection inhibit the proliferative responses of lymphocytes to Toxoplasma antigens themselves. cells

lndomethacin

NSC

0

N!X+IA

0

El-

NSC+IA

2xid7M

g-

NSC+IA

2&M

1

N§C+IA

2~40-~M

-

4

0

50 % of cmtrol

100

PFC responses

FIG. 6. Effect of indomethacin on the activity of the suppressor macrophages from Toxoplasma-infected mice in primary anti-SRBC antibody responses. Adherent cells from 2 X lo6 infected mouse spleen cells were added to 1 X lo6 normal mouse spleen cells in the presence of various doses of indomethacin, and cultured with SRBC for 4 days. Each bar represents the mean -C SEM. NSC, normal mouse spleen cells; IA, adherent cells from infected mouse spleen cells.

SUPPRESSOR MACROPHAGE

IN TOXOPLASMA

425

MICE

cells

NSC ONSC+IA

0

NSC+IA

0.5

ffiC+IA

i

NSC+IA

2

t

I I 0

50 %

Of wntroi

100

PFC responses

FIG. 7. Effect of catalase on the activity of the suppressor macrophages from Toxoplasma-infected mice in primary anti-SRBC antibody responses. Adherent cells from 2 X lo6 infected mouse spleen cells were added to 1 X lo6 normal mouse spleen cells in the presence of various doses of catalase, and cultured with SRBC for 4 days. Each bar represents the mean f SEM. NSC, normal mouse spleen cells; IA, adherent cells from infected mouse spleen cells.

DISCUSSION Using an in vitro primary anti-SRBC antibody response system, the mechanism of immunosuppression in Toxoplasma-infected mice was investigated. We previously reported that the suppression of antibody responses was caused by irradiation-resistant and plastic-adherent suppressor cells (3). The present study has established that the activity of these adherent suppressor cells is abrogated by treatment with silica or carrageenan, which are known to be selectively cytotoxic for macrophages (14-16). Identification of the suppressor cells in the infected mice as macrophages is thereby supported. It was demonstrated that the suppressor macrophages inhibited an early stage of lymphocyte stimulation in the antibody responses to SRBC, and suppressed the uptake of [3H]TdR by normal spleen cells in the responses. The addition of suppressor macrophages to normal mouse spleen cells 48 hr after the start of culture with SRBC did not affect the antibody responses of the normal cells, suggesting that suppression TABLE 1 Effect of Suppressor Macrophages from Toxoplasma-Infected Mice on Proliferative Responses of Normal Mouse Spleen Cells to Toxophna Antigens Uptake of [3H]TdR (cpm)

NSC NSC NSC NSC NSC

+ + + +

Cell”

Expt I

Expt 2

NA (1 X 106) NA (2 X 106) IA (1 X 106) IA (2 X 106)

3191 1771 N.D. b 0’ N.D.

3600 1948 4676 0 0

0 NSC, normal mouse spleen cells; NA, adherent cells from NSC, IA, adherent cells from infected mouse spleen cells. b Not done. ’ The counts in the culture with antigens were less than that without antigens.

426

SUZUKI

AND KOBAYASHI

is not caused by a cytotoxic effect of the macrophages on lymphocytes. In our previous studies (3, 5), it was shown that the functions of both hapten-specific B cells and carrier-specific helper T cells were suppressed by Toxoplasma infection in the antibody responses to hapten-conjugated protein antigens. Thus, it is conceivable that the suppressor macrophages inhibit the proliferation of both T and B cells. This is supported by the findings of Strickland et al. (17), that the blastogenic responses to both T- and B-cell mitogens are suppressed in infected mouse spleen cells. Nonspecific suppression for antigens and immunoglobulin classes in the infected mice (4, 5) could be caused by the nonspecificity of the suppressive effect of the macrophages on various subpopulations of lymphocytes. Supernatant fluids from suppressor macrophages did not reduce the primary antiSRBC antibody responses of normal mouse spleen cells. Furthermore, when isolated by a cell-impermeable membrane from normal mouse spleen cells, the macrophages could not suppress their antibody responses. Therefore, it seems likely that suppressor macrophages affect lymphocyte proliferation in the immune response via cell-cell contact. Supernatant fluids from suppressor macrophages showed the converse effect, augmentation of antibody responses by normal spleen cells. The suppressive effect of macrophages via cell-cell contact may inhibit the stimulatory effect of the soluble factor(s). Further studies are needed to determine whether the enhancing soluble factors are released by the suppressor macrophages or by other cells present in splenic adherent cell fractions. The suppressive activity of the macrophages from Toxoplasma-infected mice was not abrogated by indomethacin, a potent and irreversible inhibitor of prostaglandin synthesis. Hence their suppressive activity seems unlikely to be mediated by prostaglandins. The possibility that suppressor macrophages possess sufficient prostaglandins, already produced in vivo, which would not be affected by indomethacin, could not be completely ruled out by this experiment. However, this possibility seems unlikely because the supematant fluids from the macrophages showed no suppressive effect on antibody responses. In Ttypanosoma rhodesiense-infected mice, Wellhausen and Mansfield ( 18) reported that splenic suppressor macrophages from infected mice similarly require cell-cell contact in order to suppress the responses of normal mouse spleen cells to SRBC, and here also activity was not counteracted by indomethacin. On the other hand, it has been reported in Trypanosoma cruzi-infected mice that the suppressed responsiveness of the infected mouse spleen cells to mitogens, in which suppressor macrophages were responsible, was improved by treatment with indomethacin (19). The action of suppressor macrophages, therefore, may not be the same in all parasitic protozoan infections. It was reported recently that H202 is a short-lived mediator which is released by suppressor macrophages and inhibits proliferative responses of lymphocytes (11, 12). There is no information about the participation of H202 in the suppressive action of macrophages induced by parasitic infections. In the present study, however, it was shown that catalase did not alfect the suppressive activity of macrophages induced by Toxoplasma infection. It appears unlikely, therefore, that the activity of suppressor macrophages from infected mice could be mediated by H202. The results of these experiments with indomethacin and catalase support the possibility that the suppressor macrophages induced by Toxoplasma infection affect lymphocytes, not by a release of soluble mediators, but via cell-cell contact with responder target cells. Two different

SUPPRESSOR MACROPHAGE

IN TOXOPLASMA

MICE

427

mechanisms have been also reported in the mode of action of suppressor macrophages in mice injected with tumors and bacteria, i.e., via cell-cell contact (20-22) and by release of soluble mediators (23). Suppressor macrophages from Toxoplasma-infected mice inhibited the proliferative responses of lymphocytes to Toxoplasma antigens as well as the responses to SRBC, suggesting impairment of the antibody responses to T. gondii itself. It is known that anti-Toxoplasma antibodies are responsible for acquired resistance to Toxoplasma infection (24). It is conceivable, therefore, that the antigen-nonspecific effect of the suppressor macrophages induced by Toxoplasma infection is detrimental in the acute phase of infection with T. gondii. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

Huldt, G., Gard, S., and Olovson, S. G., Nature (London) 244, 301, 1973. Strickland, G. T., and Sayles, P. C., Infect. Immun. 15, 184, 1977. Suzuki, Y., and Kobayashi, A., Infect. Immun. 40, 1, 1983. Suzuki, Y., Watanabe, N., and Kobayashi, A., Infect. Immun. 34, 30, 1981. Suzuki, Y., Watanabe, N., and Kobayashi, A., Infect. Immun. 34, 36, 1981. Jacobs, L., and Lunde, M. N., J. Parasitol. 43, 308, 1957. Pierce, C. W., Johnson, B. M., Gershon, H. E., and Asofsky, R., J. Exp. Med. 134, 395, 1971. Goodwin, J. S., Bankhurst, A. D., and Messner, R. P., J. Exp. Med. 146, 1719, 1977. Gordon, D., Bray, M. A., and Morley, J., Nature (London) 262, 401, 1976. Mattingly, J. A., and Kemp, J. D., Cell. Immunol. 48, 195, 1979. Metzger, Z., Hoffeld, J. T., and Oppenheim, J. J., J. Immunol. 124, 983, 1980. Sestini, P., Bozelka, B. E., deShazo, R. D., and Salvaggio, J. E., Cell. Immunol. 73, 264, 1982. Schonbaum, G. R., and Chance, B., In “The Enzymes” (P. D. Boyer, Ed.), Vol. 3, pp. 363-458. Academic Press, New York, 197 1. Allison, A. C., Harington, J. S., and Birbeck, M., J. Exp. Med. 124, 141, 1966. Kessel, R. W. I., Monaco, L., and Marchisio, M. A., hit. J. Exp. Pathol. 44, 351, 1963. Catanzaro, P. J., Schwartz, H. J., and Graham, R. C., Amer. J. Pathol. 64, 387, 1971. Strickland, G. T., Ahmed, A., and Sell, K. W., Clin. Exp. Immunol. 22, 167, 1975. Wellhausen, S. R., and Mansfield, J. M., Cell. Immunol. 54, 414, 1980. Kierszenbaum, F., J. Immunol. 129, 2202, 1982. Eggers, A. E., and Wunderlich, J. R., J. Immunol. 114, 1554, 1975. Klimpel, G. R., Okada, M., and Henney, C. S., J. Immunol. 123, 350, 1979. Pope, B. L., Whitney, R. B., and Levy, J. G., J. Immunol. 120, 2033, 1978. Allison, A. C., Immunol. Rev. 40, 3, 1978. Krahenbuhl, J. L., Ruskin, J., and Remington, J. S., J. Immunol. 108, 425, 1972.