Immunoregulation in experimental schistosomiasis

CELLULAR

IMMUNOLOGY

83, 161-l 69 ( 1984)

lmmunoregulation

in Experimental

Schistosomiasis

III. Role of Macrophages in Soluble Egg Antigen-Induced Chronic Spleen Cell Augmentation of Baseline Lymphocyte Reactivity’

STEPHENG. KAYES* AND DANIEL G. COLLEY Veterans Administration Medical Center and Department of Microbiology, Vanderbilt University School of Medicine, Nashville, Tennessee37203 Received June 30, 1983; accepted August 30, 1983 Spleen cells from mice infected for 20 weeks with Schistosoma mansoni, exposed in vitro to soluble schistosomal egg antigens (SEA), treated with mitomycin C (MC), and cocultured with syngeneicresponder spleen cells increasedthe baseline proliferation of the otherwise unstimulated responder cells in cocultuns. The role of macrophages in this “spontaneous” thymidine incorporation was studied directIy by removal of macrophageson Sephadex G- 10 columns. Removal of esterase-positive,Sephadex G-IO-adherent cells (macrophages)greatly reduced the amount of SEA-induced, chronically infected spleen cell-mediated stimulation observed in cocultures. It also reduced an elevated background of spontaneous DNA synthesis seen with control cultures of spleen cells from infected animals. Depletion of T lymphocytes from chronic spleen cell populations by treatment with anti-Thy 1.2 serum and complement prior to exposure to SEA partially abrogatedthe augmentation effect.Comparison of theseresultswith mitogen (concanavalin A)-induced spleencell-mediated stimulation (which is elevated,rather than reduced,by macrophage removal) and with known alterations in splenic T- and B-lymphocyte ratios in chronic murine schistosomiasis suggeststhat antigen-stimulated, chronically infected splenic macrophagedependent baseline augmentation may depend on specific T-lymphocyte-derived lymphokine induction. These results may reflect a general mechanism whereby animals harboring a persistent, chronic infection can respond quickly to a second or challenge infection or a tlareup of the primary infection.

INTRODUCTION Suppression by cells with macrophage-like characteristics has been reported in both clinical (1) and experimental settings (2, 3), including experimental schistosomiais (4). Depending on the ratio of macrophagesto lymphocytes, adherent cells can augment or suppress (2) an immune response. Relatively little is known about the role of phagocytic cells or macrophagesin the host responsetoSchisto.soma mansoni.Coulis et al. (4) found that macrophages inhibited the generation of alloantigen-specific cytotoxic T cells in the spleens of infected mice. Removal of adherent/phagocytic cells from the peripheral blood of S. mansonipatients resulted in increased blastogenic responsesby nonadherent cells to larval and adult stage antigens but had little effect ’ Supported in part by the National Institutes of Health (Grant AI 11289)and the Veterans Administration Medical Center, Nashville, Tenn. 37203. * Present address and to whom requests for reprints should be addressed: Department of Anatomy, College of Medicine, University of South Alabama, Mobile Ala. 36688. 161 0008-8749184$3.00 Copyight Q 1984 by Academic Pms, Inc. All rights of repmduction in any form reserved.

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on egg antigen responses(5, 6). Mononuclear phagocytes have also been implicated as an effector cell in killing larval schistosomes (7, 8). We have examined the in vitro system used by Colley et al. (9) for the induction of human peripheral blood suppressor cells to study spleen cell responsesto SEA of mice infected with S. mansoni (10). The resultant data were originally interpreted as indicative of suppression (10). However, in contrast to human peripheral blood mononuclear cells, 48 hr exposure of murine splenocytes to SEA followed by mitomycin C (MC) treatment leads to the development of a population of cells that upon coculture augment the amount of [3H]thymidine (TdR) incorporation by resting, control syngeneic cells (see companion paper). This augmenting effect is antigen-specific, proportional to the number of cells added to the responder population, requires 48 hr prior exposure to SEA, and is partially T lymphocyte dependent. When concanavalin A (Con A) is substituted for SEA during the pre-Mc exposure period, the amount of augmentation is only about one-half that seen following antigen exposure. Chensue and Boros have reported that the relative number of T cells is reduced in the spleens of chronically infected mice (11). It seemedthat several cell types could be interacting in the altered splenic environment to account for the augmenting effect. We have, therefore, investigated the role of the macrophage in the mouse model system. The selective removal of macrophages resulted in a marked reduction of the SEA-augmentation effect while increasing the Con A-elicited response. MATERIALS

AND METHODS

Mice. CBA/J male mice were obtained from Jackson Laboratories (Bar Harbor, Maine). All animals usedin this study were maintained in AAALAC approved facilities. Mice were infected with 30-40 cercariae of a Puerto Rican strain of S. mansoni and used 8 or 20 weekslater providing that the livers had obvious pathological indications of infection. Induction and assay. These procedures have been described previously (10). Briefly, chronically infected spleen cells at 4 X 1O6cells/2 ml of culture medium were exposed to 2.5 &ml of Con A or 4.0 &ml of SEA for 48 hr, treated with mitomycin C (50 pg/ml), and washed extensively. MC-treated cells (3 X 105)were then cocultured for 3 to 5 days with 5.0 X 10’ spleen cells from either normal mice (NSC) or mice infected for 8 weeks. At 8 hr before harvesting, 0.5 &i [3H]TdR was added to each coculture and the cells were then collected on glass-fiber filter strips and counted in a liquid scintillation spectrometer. In some experiments, cells previously exposed to Con A for 48 hr, were treated with 0.1 M cu-methyl-Dmannoside (AMM; (1 ml/l .2 X 10’ cells) Sigma Chemical Co., St. Louis, MO.), a competitive inhibitor of Con A (12), for 30 min at 37°C followed by four washes in fresh RPM1 1640. These cells were then used in the coculture assay described above. Likewise, cells previously exposed for 48 hr to SEA were treated with trypsin (10 mg/2 ml/ 1.2 X 10’ cells; Worthington Biochemicals, Freehold, N.J.) for 45 min at 37°C (13). The reaction was stopped by the addition of excess soybean trypsin inhibitor (Sigma Chemical Co.); cells were washed three times with fresh RPM1 1640 and incorporated into the coculture assay as above. Removal of esteruse-positive cells. The method of Ly and Mishell (14) was used as described by Mishell et al. (15) with horse serum being substituted for fetal calf serum. Briefly, 35 ml of sterile Sephadex G- 10 (Sigma Chemical Co.) was packed on 10 g of cleaned, sterile Cataphote Class IVa glass beads (Ferro Manufacturing Co.,

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Greenwood, Miss.) in a 50-ml Plastipak syringe barrel (Becton-Dickenson, Oxnard, Calif.). The column was washed with 150-200 ml of warm Dulbecco phosphatebuffered saline (DBSS; Flow Laboratories, McLean, Va.) containing 10%horse serum and 1%antibiotics. Chronic spleen cells (600 X lo6 cells) in 4 ml of the supplemented DBSS were allowed to enter the column and the first 10 ml of DBSS containing passagedcells was collected and used for the induction procedure as above. Esterase staining (16) of the passagedcells using cY-napthylbutyrate as substrate showed less than 1% of the recovered cells to be positive. Starting chronic spleen cell populations were between 13 and 20% esterasepositive. After allowing the remaining cells to elute from the column, the retained cells were washed with an additional 150 ml of supplemented medium and the entire contents of the column transferred to a 75cm2 sterile tissue culture flask containing 30 mA4 lidocaine (8.75 ml 1% lidocaine (Astra Laboratories, Worcester, Mass.), 41.25 ml DBSS, and 0.7 ml NaCOx). The flask was agitated firmly for 1 min and the glass beads and gel were allowed to settle for 3-5 min. The supematant fluid containing the released gel-adherent cells was collected, centrifuged, and resuspended in RPM1 1640 (Flow Laboratories). These cells were greater than 65% esterasepositive. Anti-Thy 1.2 and complement treatment. To destroy lymphocytes bearing surface Thy 1.2 antigen spleen cells from mice chronically infected with S. mansoni were adjusted to 50 X lo6 cells/ml in AKR antiC3H antiserum diluted l/3 in RPM1 1640 and allowed to stand at room temperature for 30 min with occasional mixing. The cells were washed once and resuspended in 1.O ml of a l/ 10 dilution of guinea pig serum (GIBCO; Grand Island, N.Y.) as a source of complement (C) and incubated 30 min at 37°C. After washing, the cells were subjected to the in vitro induction procedure described above. Control cells were treated in a like manner except normal AKR mouse serum (NMS) was used in lieu of anti-Thy 1.2 serum. Cells treated with anti-Thy 1.2 and C could undergo blastogenesis to bacterial lipopolysaccharide but could not respond to Con A, PHA, or any of the commonly used schistosome-derived antigens (data not presented). Cells treated with NMS responded vigorously to all of these potential stimulants. RESULTS Effect of MC-Treated Cell Concentration in Cocultures of Spleen Cells of Mice Infected 8 Weeks with S. mansoni MC-treated control cells and MC-treated SEA-stimulated cells were titrated from a ratio of l/50 to l/ 1 with responder cells. The data from one of severalsuch experiments are shown in Fig. 1. The responder population consisted of 5 X 1O5spleen cells from mice infected for 8 weeks to which various numbers of control or SEA-stimulated cells from a chronically (20 weeks)infected mouse were added. The cells were cultured for 5 days and the amount of t3H]TdR incorporated by the responder cells was determined. The addition of unstimulated cells had no effecton [3H]TdR incorporation of responder cells at any of the cell concentrations examined. However, increasing the number of SEA-MC cells to 3 X lo5 cells per culture increased the amount of augmented labeling by the responder cells to over five times that of cocultures containing a comparable number of unstimulated cells. Based on these observations, 3 X lo5 MC-treated cells were used in all subsequent experiments.

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16

IO MITOMYCIN

so C-TREATED

100

300

so0

CELLSx IO-‘/CULTURE

FIG. 1. Baseline [3H]TdR incorporation of cocultures. Chronic spleen cells which were either unstimulated Mc-treated (control) or SEA-stimulated and Me-treated were cocultured with the responder cells (5 X lo5 spleen cells from a mouse infected with S. mansoni for 8 weeks) in the quantities indicated.

Eflect of Removing Con A or SEA From the Surface of Chronic Spleen Ceils on Augmented Thymidine Incorporation To assessthe possibility that cells induced for 48 hr with either Con A or SEA were “carrying over” mitogenic or antigenic moieties, respectively, aliquots of cells were treated with either AMM, a competitive inhibitor of Con A, or, in the case of the protein antigen, SEA, trypsin; and then tested for their ability to promote increased [3H]TdR incorporation by otherwise, unstimulated spleen cells. Treatment of Con A-stimulated, MC-treated chronic spleen cells with AMM reduced augmenting activity by these cells 53% (data not shown). In contrast, treatment of SEA-induced, Mctreated chronic spleen cells with trypsin did not alter the baseline [3H]TdR augmenting properties of these cells (Table 1). These results suggestthat none of the SEA-induced activity and half of the Con A-elicited augmenting activity was due to carry over from the induction culture to the assay culture. E#ect of Removing Esterase-Positive Ceilsfrom Chronic Spleen Cell Populations Prior to Mitogen or Antigen Exposure Esterase-positive cells (macrophages) were removed from chronic spleen cell populations by passageon columns of Sephadex G-10. The resulting nonadherent cells were exposedto Con A or SEA, treated with MC, washed,and cocultured with responder cells from mice harboring 8 week S. mansoni infections. Nonadherent chronic spleen cells exposedto Con A increasedthe amount of [‘H]TdR incorporated by the responder cells (Fig. 2). In each of six separate experiments this level of incorporation was elevated in relationship to that usually seen using whole spleen cell populations. However, the mean increase due to adherent cell removal was not statistically sig-

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TABLE 1 Effect of Trypsin Treatment on SEA-Elicited, Augmented Baseline Thymidine Incorporation by Spleen Cells from Mice with Chronic Schistosomiasis Mansoni [‘H]TdR incorporation (dpm) Culture

Expt 1

Expt 2

Responder cells only (RCO)“ RCO + Unstim., MC-CHR SC’ RCO + Unstim., MC and Tryp.-CHR SC’ RCO + SEA-stim., MC-CHR SC’ RCO + SEA-sum., MC & Tryp.-CHR SC’

9,848b 9,438 11,697 19,682 19,953

11,387 13,934 I 1,092 33,808 26,650

’ Culture of S X 10’ spleen cells from mice infected with S. mansoni for 8 weeks. ’ Mean of triplicate cultures. ’ In addition to RCO cells, 3 X lo5 spleen cells from mice infected with S. mansoni for 20 weeks, cultured in vitro in RPM1 1640 for 48 hr and MC treated, were included. ‘Same culture as previous group except MC-treated cells were trypsin treated prior to coculture. ’ In addition to RCO cells, 3 X lo5 spleen cells from mice infected with S. mansoni for 20 weeks, cultured with 4 &ml SEA for 48 hr and MC-treated, were included. ‘Same culture as previous group except MC-treated cells were trypsin treated prior to cocuhum..

nificant. In contrast, coculturing nonadherent, SEA-exposedchronic spleen cells with responder cells resulted in isotope incorporation levels which were below those generated when whole spleen cells were used (Fig. 2). If the gel-adherent cells were recovered following release from the gel by use of 30 mJ4 lidocaine solution and added back to the nonadherent cells, isotope incorporation occurred at levels consistent with antigen-stimulated whole chronic spleen cell cocultures (Fig. 3). Moreover, the

(6)

*

NS

(6)

(6) 4

8wk SC ONLY

8wk 8wk set set Con A G-IO-Con A CHR SC CHR SC

JT

8wk SC ONLY

!.I

8wk SC+ G-IO-SEA CHR SC

FIG. 2. (3H]TdR incorporation of spleen cells from mice infected for 8 &zks with S. mansoni (left bar of each panel) by cocultures of these I-week responder cells and Con A-activated, MC-treated chronic spleen cells before (middle bar left panel) and after passageon Sephadex G-10 columns (right bar left panel), or SEA-activated, MC-treatedchronic spleen cells before (middle bar right panel) and after passageon Sephadex G-10 columns (right bar right panel). (Mean +- SE.)

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RAYES AND COLLEY

: WSC- WSCCONTROL SEA

G-IO SEA

1

G-IO

G-IO

G-IO

“5” NSC

“FA G-IO M9

M+ SEA

FIG. 3. [‘H]TdR incorporation by cocultures of spleen cells from mice infected with S. mansoni for 8 weeks with unstimulated chronic whole-spleen cells (WSC-control), with SEA-activated chronic wholespleen cells (WSC-SEA), with Sephadex G-10 passaged,SEA-activated chronic spleen cells (G-IO-SEA), with Sephadex G-10 passaged,normal spleen cell reconstituted, SEA-activated chronic spleen cells (G-lOSEA + NSC), with SephadexG- 10passaged,gel-adherent macrophagesreconstituted, SEA-activated chronic spleen cells (G-IO-SEA + G-IO M&), and with Sephadex G-IO-adherent M+, SEA-activated chronic spleen cells (G-10 M&-SEA). (Mean * SE.)

role of gel-adherent cells in reconstituting the SEA-elicited augmenting effect could be fulfilled by normal spleen cells serving as the source of macrophages. Further analysis of the role of adherent, esterase-positive cells in this system was performed by determining baseline thymidine incorporation levels by whole spleen cells or nonadherent spleen cells from chronically infected mice. The removal of gel-adherent cells resulted in a 77% reduction of the background labeling of DNA of unstimulated chronic spleen cells as compared with the labeling observed in whole spleen cell populations (chronic whole spleen cells, 94 14 + 1729 dpm; chronic G- 10 nonadherent spleen cells, 2,174 f 292 dpm; mean f SE; n = 4).

Efect ofAnti-Thy 1.2 Serum and Complementon Chronic SpleenCells Subsequently Exposed to SEA Spleen cells from mice chronically infected with S. mansoni were treated with either normal mouse serum or anti-Thy 1.2 serum plus complement and then exposed to SEA prior to being treated with MC. The cells were then added to responder spleen cell populations obtained from mice infected for 8 weeks.Anti-Thy 1.2 and C reduced the level of thymidine incorporation in three of four experiments. The mean results of these three experiments are depicted in Fig. 4. DISCUSSION The results of the cell titration experiments in which the number of MC-treated cells was varied across a wide range indicate that the assay used, namely, thymidine incorporation, within certain limits, is capableof quantitating the amount of blastogenic

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167

(3)

18 -

15:

0 * 5 a 0

NS (3)

12-

97 6-

(3)

3-

8WKSC ONLY

8WK SC NMS SEA-Cl+? SC

BWK SC Anti-Thy 1.2+C SEA-CHR SC

FIG. 4. Baseline [“H]TdR incorporation of 5 X lo5 8-week-infected spleen cells (left bar) and 5 X lo5 of these cells cocultured with either 3 X lo5 chronic spleen cells exposed to NMS and C, then to SEA and MC-treated (center bar), or 3 X 10’ chronic spleen cells exposed to anti-Thy 1.2 and C, then to SEA- and MC-treated (right bar). (Mean + SE).

activity produced by the cell population under study. Since the subsequentexperiments were performed using a constant number of MC-treated cells, the changesin [3H]TdR incorporation between normal spleen cells and spleen cells from infected animals may reflect changes in the relative number of spleen cells that are driven by one of the two stimulants employed. For example, cocultures of Con A-stimulated, Mctreated NSC and responder cultures of NSC incorporated over 12,000 dpm while Con A-stimulated, MC-treated chronic spleen cells caused the incorporation of less than half this amount of isotope. This finding is consistent with the possible presence of a nonspecific suppressor cell in chronically infected spleens, similar to the system described by Pierce and colleagues (17). However, recent findings by Chensue and Boros ( 11) indicate that the relative number of T lymphocytes in 20-week postinfection murine spleens is decreased in relation to B lymphocytes. This could also explain the lesser ability of Con A-stimulated, MC-treated chronic spleen cells to promote isotope incorporation in cocultures with NSC. This hypothesis would be predicated on the supposition that Con A acts on a given portion of the T cells present in the exposed population and that there are fewer such T cells in a 300,000-cell aliquot of chronic cells than in a similar aliquot of normal spleen cells. Chensue and Boros (11) have suggestedthat the suppressedantigen and mitogen blastogenic responsesthat have been reported by others may in fact be explained by this relative reduction of T cells in the spleens of infected animals. The data obtained in the present study are at least compatible with their hypothesis. Direct evidence for the involvement of macrophages in the SEA-induced, chronic spleen cell-mediated responder cell baseline augmentation response was obtained by

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removing macrophages from the chronic cell culture prior to exposing them to SEA. The results of the experiments using Sephadex G-10 to remove the gel-adherent, esterase-positivecells indicate that SEA-stimulation leading to increased proliferation differs from that driven by Con A. Chronic spleen cells depleted of macrophages and then exposed to Con A promote more proliferation than parallel unseparated cells. This finding may suggest the presence of a regulatory macrophage, or may be attributable to the Con A-stimulation of a population enriched for T lymphocytes. This is in marked contrast to the findings when chronic spleen cells are exposed to SEA. In this case,removal of the macrophages led to a cell population that promoted less isotope incorporation than did parallel whole spleen cell populations. Such a finding has also been reported by Ljungstrom and Sunqvist (18) wherein splenic macrophages from mice infected with Trichinella spiralis stimulated “spontaneous DNA synthesis” by T lymphocytes. In their study, spontaneous DNA synthesis was reduced following removal of macrophages by ingestion of silica powder. It is likely that the antigen-induced, chronic spleencell-mediated mitogen effectis likely dependent upon a T-cell-macrophage interaction. BecauseNSC were suitable substitutes in the reconstitution of gel-nonadherent SEA responsesby chronic cells and because SEA failed to induce augmentation in whole NSC cultures ( 10) it appears that the T cell confers antigen specificity while the role of the macrophage is apparently nonspecific. In the companion paper it was demonstrated that SEA-elicited baseline augmenting activity can be effectedthrough releaseof a soluble mediator which lacks H-2 restriction and is apparently nonspecific in its action. Examination of macrophage dependency of T-cell mediator (lymphokine) production suggeststhat the production of most lymphokines by either antigen or mitogen is macrophage dependent (19). The observation that macrophages from chronically infected spleens can be recovered from Sephadex G-10 columns and elaborate or cause augmenting activity argues for this explanation, These findings offer further evidence of the complex nature of the chronic host cellular response to S. mansoni. While the in vivo significance of the SEA-specific augmenting effect can only be conjectural at this time, the mechanisms and mediators involved can now be analyzed in vitro to gain insight into the cell-cell interactions that may occur in the infected host. It is clear that splenic macrophages can play a regulatory role in antigen-stimulated lymphocyte blastogenesis.In the system described herein we have observed positive regulation. Our findings may reflect a general mechanism whereby a chronicalIy parasitized host can rapidly cope with a challenge infection or a recrudescence of the initial infection (7, 20, 21). REFERENCES 1. Stobo, J. D., J. Immunol. 119, 918, 1977. 2. Kirchner, H., Holden, H. T., and Herberman, R. B., J. Immunol. 115, 1212, 1975. 3. Unsgaard, G., and Lamvik, J., Acta Pathol. Microbial. &and. Sect. C 85, 373, 1977. 4. Coulis, P. A., Lewert, R. W., and Fitch, F. W., J. Immunol. 120, 1074, 1978. 5. Todd, C. W., Goodgame, R. W., and Colley, D. G., J. Immunol. 122, 1440, 1979. 6. Ottesen, E. A., J. Immunoi. 123, 1639, 1979. 7. Elmer, J. J., and Mahmoud, A. A. F., J. Immunol. 123, 949, 1979. 8. James, S. L., Sher, A., Lazdins, J. K., and Meltzer, M. S., J. Immunol. 128, 1535, 1982. 9. Colley, D. G., Lewis, F. A., and Goodgame, R. W., J. Immunol. 120, 1225, 1978. 10. Kayes, S. G., and Colley, D. G., J. Immunol. 122, 2340, 1979. 11. Chensue, S. W., and Bores, D. L., Amer. J. Trop. Med. Hyg. 28, 291, 1979.

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12. Naidorf, K. F., Ptak, W., and Gershon, R. K., &and. J. Immunol. 11, 109, 1980. 13. James, S. L., and Colley, D. G., Ceil. Immunol. 38, 35, 1978. 14. Ly, I. A., and Mishell, R. I., J. Immunol. Methods 5, 239, 1974. 15. Mishell, B. B., Mishell, R. I., and Shiigi, J. M., In “Selected Methods in Cellular ImmunoIogy” (B. B. Mishell and S. M. Shiigi, Eds.). Freeman, San Francisco, 1980. 16. Koski, I. R., Poplack, D. G., and Blaese, R. M., In “In Vitro Methods in Cell Mediated and Tumor Immunity” (B. R. Bloom and J. R. David, Eds.), pp. 359-362. Academic Press,New York, 1976. 17. Pierce, C. W., Tadakuma T., Kuhner, A., and David, J. R., In “Mitogens in Immunobiology” (J. J. Gppenheim and D. L. Rosenstreich, Eds.), pp. 583-595. Academic Press,New York, 1976. 18. Ljungstrom, I., and Sundqvist, K.-G., Clin. Exp. Immunol. 38, 38 1, 1979. 19. Unanue, E. R., Immunol. Rev. 40, 227, 1978. 20. Goodwin, J. S., Bankhurst, A. D., and Messner, R. P., J. Exp. Med. 146, 1719, 1977. 21. Prouse, S. J., Austral. J. Exp. Biol. 59, 695, 1981.