CELLULAR
IMMUNOLOGY
45, 377-388
(1979)
Enhancing and Suppressive Effects on T-Lymphocyte Stimulation
of Macrophages in Vitro1
S. YOUDIM~ Department
of Surgery,
University of California La Jolla, California Received
San Diego 92093
School
of Medicine,
July 24, 1978
The immunoregulatory effect of peritoneal and splenic macrophages on Con A-stimulated mouse splenic T lymphocytes was investigated in vitro using [L251]UdR incorporation as a measure of lymphocyte proliferation. [1251]UdR incorporation was enhanced by the addition of increasing numbers of splenic or low doses of peritoneal adherent cells to macrophagedepleted splenic lymphocytes. The addition of increasing numbers ofperitoneal macrophages beyond 5-lo%, however, proportionally suppressed T-cell proliferation. Activated splenic macrophages obtained from mice 6 days after infection with Listeria monocytogenes were suppressive, whereas macrophages obtained from immune donors 9- 10 days after infection were not, so that a chronological association appeared to exist between macrophage activation and immunosuppression. The addition of 2-mercaptoethanol to the cell cultures increased [‘*51]UdR incorporation without affecting the stimulatory and suppressive effects of splenic and peritoneal macrophages, respectively. Heat-killed and freeze-thawed macrophages lost their capacity to enhance or inhibit lymphocyte transformation. Macrophages treated with mitomycin C to inhibit DNA synthesis retained their regulatory functions. These studies suggest differential regulatory roles for spleen versus peritoneal macrophages on T-lymphocyte responses to Con A stimulation in vitro.
INTRODUCTION Experimental models of antibody formation and cellular immune responses indicate that macrophages critically influence these lymphocyte responses in vitro. The primary antibody response of B lymphocytes3 (l-3) and the proliferative responses of T lymphocytes to antigens (4,5), to mitogens (6,7), and to allogeneic cells in the mixed lymphocyte reaction (8,9) are all M0 dependent. These adherent cells may also exert the reverse influence and inhibit T-lymphocyte proliferation (lo- 12) and antibody production (13). Functionally and morphologically, M0 are a heterogeneous cell population and differ with respect to size (15), density of Fc receptors (16) Ia (I-region products of the MHC) antigens (17) and phagocytic 1 This work was supported by USPHS Grant CA 177299 from the National Institutes of Health. 4 To whom reprint requests should be addressed at the University of California San Diego School of Medicine, Department of Surgery-Q-058, La Jolla, California 92093. 3 Abbreviations used: SM0, spleen macrophages; PM@, peritoneal macrophages; PC, peritoneal cells; SC, spleen cells, B lymphocytes, bone marrow-derived cells; T lymphocytes, thymus-derived cells; Con A, concanavalin A; LM, Listeria monocytogens; 2ME, 2-mercaptoethanol; M-D gas, Mishell-Dulton gas mixture; MHC, major histocompatibility complex. 377
0008-8749/79/080377-12$02.00/O Copyright 0 1979by Academic Press, Inc. All rights of reproduction in any form reserved.
378
SYOUDIM
(18) and chemotactic (19) activities. The anatomical site of these cells also appears to be associated with their functional diversity. For example, we previously noted (12) that antigen (LM)-primed spleen T lymphocytes in association with their complementary adherent cells become nonspecifically cytotoxic for B-16 target tumor cells in the presence of the specific antigen, however, PM0 suppressed this cooperative endeavor. We now report that a similar collaborative suppressive macrophage lymphocyte interaction occurs during Con A-induced lymphocyte transformation in vitro. MATERIALS
AND METHODS
Animals. Inbred C57BL/6 mice 6 to 9 weeks old were obtained from Strong Research Foundation, San Diego, Calif., and used in all experiments. Bacterial cultures and immunization. Cultures and innocula of Listeria monocytogens were prepared as described previously (20). All mice were immunized with 5 x lo3 viable LM intravenously. Tissue culture materials and media. All media and sera were obtained from Grand Island Biological Company, Grand Island, N.Y., or SCOR C, University of California, San Diego. The common culture medium was complete medium (CM) consisting of RPMI-1640 containing 2 mM L-glutamine supplemented with 10% fetal calf serum (FCS), 100 pg of streptomycin, and 100 units of penicillin per milliliter. Preparation of spleen ‘cells (SC). Spleens were aseptically removed and placed in 60-mm tissue culture dishes containing 5 ml cold (4°C) complete Hanks’ balanced salt solution (HBSS). Single cell suspensions were prepared by gently teasing the spleens. Preparation ofperitoneal cells (PC). These were collected from the peritoneal cavity of mice after injecting 5 ml of HBSS containing 5 units of sodium heparin/ml. Separation of adherent and nonadherent cells on plastic surfaces. Two to 5 x 10’ SC or PC were placed in 5 ml of prewarmed (37°C) CM in a plastic tissue culture flask (Falcon No. 3024) and incubated for 45 min at 37°C in humidified air containing 5% CO,. At the end of this incubation period, the nonadherent cells and two successive washings of 2.5 ml each were transferred to a second flask. Five milliliters of warm CM was added to the original flask and both flasks were incubated as above for a further 45 to 60 min. Nonadherent cells were decanted and further treated with carbonyl iron as described below. The adherent cells were washed six times in warm CM with moderate agitation of the flasks. Two and a half milliliters of cold (4°C) CM was layered on each monolayer of adherent cells, the flasks were placed at 4°C for 10 min, and adherent cells were gently scraped with a sterile rubber policeman. The loosened cells were counted, their viability was determined, and they were kept in cold CM at 4°C until used. Carbonyl iron treatment. Carbonyl iron powder, 40 mg (General Aniline and Film Cot-p, Linden, N.J.), was washed five times in 50 ml HBSS. Spleen cells, 1.5 to 3 x lo’, or PC, 1.5 to 3 x 106, were then suspended in 15 ml CM in a 250-ml flask containing the carbonyl iron and incubated for 1 hr at 37°C with periodic agitation. The contents of the flask were pipetted into a 50-ml centrifuge tube, a strong magnet was applied to the sides of the tube for 5 min, and the suspension was decanted into a second tube while holding the magnet in place.
MACROPHAGE
REGULATION
OF LYMPHOPROLIFERATION
379
This procedure was repeated two or three times more or until all iron powder was removed. The supematant cells were finally washed twice, suspended in CM, and counted. Cell viability was determined by trypan blue exclusion. Nylon wool column. Spleen cells were passed through nylon wool columns as described previously (12). The packed, autoclaved, and dried columns were rinsed with 50 ml of prewarmed (37°C) CM, allowed to drain, covered, and incubated at 37°C for 1 hr. Each column was then flushed with 30 ml of warm CM, and 1.5 to 3 x lo7 cells in 1 ml of warm medium were washed into the nylon wool, followed by a further 2.5 ml of warm CM. The columns were incubated in an upright position for 45 min at 37°C in a humidified CO, incubator. Effluent cells were collected by slowly flushing with 30 ml of prewarmed CM. The cell suspension was then washed and counted, and its viability was determined as before. Mitomycin C treatment of cells. In certain experiments cells were incubated with mitomycin C (50 pg/ml, Sigma Chemical Co., St. Louis, MO.) for 30-45 min at 37°C. The cells were then washed three times in cold HBSS. Heat inactivation and fysis of macrophages. Cells were heated for 30 min at 56°C and washed as above. Macrophages were lysed by five cycles of freezing and thawing. Concanavalin A. This was prepared by and was a gift of Dr. Doug Redelman, University of California, San Diego. Lymphocyte proliferation assay. Different numbers of PM0 or SMO suspended in CM were added to 3 x lo5 M0depleted spleen cells in a total volume of 0.1 ml and put in individual wells of microtiter flat-bottom plates (Falcon Plastic, Oxnard, Calif.). Con A was diluted in CM and 0.6, 0.3, or 0.15 pg in 0.1 ml added to each well. Control cultures received 0.1 ml of CM. Other controls contained 3 x 1W purified spleen cells plus similar concentrations of Con A. All cultures were incubated for 48 hr in 5% CO, + 95% air or, where stated, in 7% 0, + 10% CO, + 83% Nz (Mishell-Dutton gas mixture) and incubated in a sealed incubator chamber at 37°C. Each well was then pulsed with 0.1 @Zi of [lZ51]iodo-2-deoxyuridine ([‘2”I]UdR; Radiochemical Centre, Amersham England; 851 &i/ml) and cultured for a further 18 hr. Cells were harvested on glass-fiber filters using a MASH II (multiple automatic sample harvester) harvesting machine (Microbiological Associates, Bethesda, Md.). The filters were dried, placed in tubes, and counted in a Packard Auto-Gamma scintillation spectrometer. RESULTS Effect of Macrophage
Depletion
and Reconstitution
on T-Cell Mitogenesis
Macrophages were removed from the PC or SC suspensions by allowing them to adhere to plastic surfaces. The nonadherent SC were then further treated with carbonyl iron to remove residual phagocytic cells or passed through nylon wool columns as described. SMO or PM0 were then added to the nonadherent spleen (3 x 105/well) T cells at concentrations of O-40 or O-60% (0- 1.2 x 10” to 0- 1.8 x 105) MO per well. The results of representative experiments are depicted in Figs. 1 and 2. Macrophage-depleted T cells were minimally stimulated by 0.15,0.3, or 0.6 pg Con A/well. In the absence of Con A, there was no stimulation regardless of the number of MO added. Con A-stimulated T-cell proliferation increased progressively with the addition of increasing numbers of SM0 or low
S. YOUDIM I
I
I
I
I
,4
I5 pg CON A/ WELL (SMh A---
-A
, /’
,,$
4
3~9
CONA/ WELL(SM'#'I
6pg
CONAlWELL
(sM'#'j
E----a
2
FIG. 1. Normal SM0 or PM@ (O-40%) added to a constant number of (3 x 105) splenic lymphocytes depleted of adherent and phagocytic cells by carbonyl iron and magnet technique.
doses of PM0 at all concentrations of Con A. The addition of increasing numbers of PM0 beyond 5 to lo%, however, .proportionally suppressed lymphocyte proliferation. Optimum results were obtained at concentrations of 0.15 or 0.3 pg Con A/well. Effect of Peritoneal and Spleen Macrophages from T-Cell Responses to Con A
Listeria-Znfected Mice on
Spleen and peritoneal MO were collected from donor mice 6 days (infected or’ inflammatory state) after injection of 3-5 x lo3 viable LM intravenously. The effects of these activated M0 on [1251]UdR incorporation by M0-depleted spleen lymphocytes are shown in Fig. 3. At low concentrations of up to lo%, SM0 enhanced lymphocyte incorporation of [lz51]UdR. At higher concentrations of up to 60%, [lz51]UdR incorporation remained stationary and was no longer enhanced. In a subsequent experiment, SM0 obtained from mice 6 days post-LM injection in fact slightly suppressed [Y]UdR incorporation at concentrations beyond lo-20%. These data were in direct contrast to the enhancing effect of normal SM0 at the same concentrations (compare Fig. 3 to Figs. 1 and 2). Peritoneal MO (Fig. 3) initially enhanced, but beyond a 10% concentration sharply suppressed, lymphocyte proliferation. These data were similar to the inhibiting effect of normal PM0. Macrophages obtained from mice 9 days after LM injection (immune state, Fig. 4) essentially showed similar enhancing and suppressive effects as normal MO (compare Fig. 4 to Figs. 1 and 2).
MACROPHAGE
8
I
I
REGULATION I
I
I
I
6
I
4
0
5
IO
20
30
40
381
OF LYMPHOPROLIFERATION
50
3pg
CON A/ WELLM4$1
o-----o
15pg
CON A/WELL
A----A
kM$I
60
M$
%
FIG. 2. Normal SM@ or PM0 (O-60%) added to a constant number of (3 x 105) splenic lymphocytes depleted of adherent cells and filtered through nylon wool columns.
Effect of Addition of 2-Mercuptoethanol and Incubation in Mishell-D&ton Mixture on the Reconstitutive and Suppressive Potential of Macrophages
Gas
It is reported that the reducing agent 2ME promotes the viability of lymphoid cells in culture and may substitute for MO in the in vitro antibody response by lymphoid cells to sheep red blood cells (21). 2-Mercaptoethanol alone, however, 6
I
4
-
I
I
I
I
I
I
,~----~--------~---------p
3pg
CONA/WELL(SM@
15pg
CONA/WELL
O----*
“0
s z
(SM#
A----a
2
0 0
5
IO
20
30
40
50
60
% M+
FIG. 3. Spleen MO or PM0 (O-60%) obtained from mice 6 days after LM infection added to splenic lymphocytes treated as for Fig. 1.
S. YOUDIM I
I
I
I
I
I
,.p--.--
I
--+.3pg
CON A/WELL
--+5pgCON
20
30
40
50
o----o
A/WELLMh#')
A---d
.15pg
CONA/WELL(PM#)A--4
.3pg
CON A/WELL
NO CON A (SM ) NO CONAIPM )
0 5 IO
KM#
(PM+w o----o
60
% M# FIG. 4. Spleen M@ or PM0 (O-60%) obtained from mice 9 days after LM infection added to splenic lymphocytes treated as for Fig. 1.
is not sufficient to allow M0-depleted guinea pig lymphocytes to respond to PHA (6,22) or murine lymphocytes to Con A (23). Some preliminary data showed that [1251]UdR incorporation was improved when cultures were incubated in M-D gas mixture in preference to CO,. Accordingly, experiments were performed using 2ME at a 5 x lo+ M final concentration in CM and M-D gas to determine the effect of these variables on the reconstitutive and suppressive effects of SMO and PM0, respectively. Data from representative experiments are shown in Figs. 5 and 6. Addition of 2ME and incubation in M-D gas mixture dramatically improved [Y]UdR incorporation by Con A-stimulated lymphocytes without altering the enhancing effect of SMO or the suppressive effect of PM0 (Fig. 5). In general, DNA synthesis increased 2- to 4-fold when comparing 2MEsupplemented (Figs. 5 and 6) to 2ME-deficient cultures (Fig. 1). Figure 6 is a good example of the M012 ME synergy at low concentrations (5- 10%) of PM0. DNA synthesis increased over threefold at 0.15 pg and over fourfold at 0.3 ,ug Con A/well in 2ME-supplemented versus 2ME-depleted cultures. Beyond 10% however, the inhibitory effect of PM0 on T-cell proliferation was not compromised by the addition of 2ME and incubation in M-D gas mixture. 2-Mercaptoethanol apparently partially substituted for MO, since at 0% MO, [1251]UdR incorporation by spleen T cells was increased at both 0.15 and 0.3 pg Con A/well (Figs. 5 and 6 and Table I at 0% M0 + Con A). Effects of Mitomycin, Heat-Treated, ative Responses to Con A
and Lysed Macrophages on T-Cell Prolifer-
Peritoneal and SM0 which had been lysed by freeze-thawing or heated to 56°C for 30 min did not reconstitute or inhibit T-lymphocyte proliferation. On
MACROPHAGE
REGULATION
OF LYMPHOPROLIFERATION
383
FIG. 5. Normal SM0 or PM0 (O-60%) added to a constant number of (3 x loj) spleen lymphocytes treated as for Fig. 1. Cultures contained 2ME, 5 x 10-j M and incubated in M-D gas mixture.
the other hand, SMO and PM0 treated with cytostatic concentrations of mitomycin C (50 y&ml) maintained their respective inhibitory and suppressive effects (Table 1). All cultures contained 2ME and were incubated in M-D gas mixture. DISCUSSION By removal of adherent and phagocytic cells from suspensions of mouse spleen cells, it was possible to isolate a population of lymphocytes which were severely depressed or did not synthesize DNA in response to Con A. The responding cell population was previously shown (12) to be susceptible to lysis by anti-thy-l and a preparation of rabbit anti-mouse brain antiserum plus complement. The carbonyl iron and magnet technique of T-lymphocyte enrichment also removes substantial
384
S. YOUDIM
NO CON AtFMh (-EHEI NO CON A (pM4)ItZMEl 3~4 CON A/WELL (PM41 (+ZME) -15~9 CON A/WELL (PM'& 1+2ME) l5j4CONAlWELL (PM# (-2MEI 3kq CONA/WELLIPu+j I-2ME)
5 IO
20
30
40
50
o----o A----A o-----o
60
FIG. 6. Normal PM0 (O-60%) added to a constant number of (3 x 105) spleen lymphocytes treated as for Fig. 1. Cultures were supplemented with or were deficient in 2ME (5 x 10m5M final concentration).
numbers of B lymphocytes (24). The peritoneal adherent cell population was susceptible to the lytic action of a preparation of antimacrophage serum plus complement (25) and phagocytic for LM and carbonyl iron (unpublished). Based on these observations, the interacting cell populations are considered to be mainly T lymphocytes and macrophages. To ensure that loss of response to Con A was not due to dilution of the separated splenic T-cell population, nonadherent, carbonyl iron-treated cells were cultured at concentrations of 3 x lo5 and 4.8 x lo5 cells/well, i.e., equivalent to the lowest (0% MO) and to the highest (60% M0) number of combined M0/T-cell populations tested. At 0.15 pg Con A/well, mean cpm for triplicate cultures were 466 k 67 to 811 + 132, and at 0.3 pg Con A/well, 609 f 42 and 1615 f 58; therefore, it is obvious that
MACROPHAGE
REGULATION
385
OF LYMPHOPROLIFERATION
TABLE
1
Effect of Mitomycin-Lysed and Heat-Treated Macrophages on [1Z51]UdR Incorporation of Con A-Stimulated MB-Deficient Spleen Lymphocytes Source of lymphocytes” Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal
spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen
Macrophage treatmentb
% Macrophages’
PM0 normal PM0 normal PM0 normal Heat killed Heat killed Heat killed Freeze-thawed Freeze-thawed Freeze-thawed Mito c Mito c Mito c SMO normal SMO normal SMO normal Heat killed Heat killed Heat killed Freeze-thawed Freeze-thawed Freeze-thawed Mito c Mito c Mito c -
5 30 60 5 30 60 5 30 60
-
30 60 30 60 30 60 5 30 60 5 30 60 0 0
Con A addedd
+ + + +
+ + + + + +
+ + + + -
[1251]UdR incorporation (cpm ? SEM)’ 15,489 2,577 938 8,350 8,899 9,181 8,627 7,513 9,246 9,219 7,184 500 8,590 15,195 19,373 6,214 5,549 5,538 5,544 5,821 6,419 8,613 12,755 9,819 7,132 329
-t 979 t 338 + 276 +- 822 k 274 f 433 2 1123 i- 519 2 226 k 554 ” 85 _f 124 t 606 k 471 2 141 t 369 + 288 2 107 t 390 +- 140 f 82 2 177 + 837 + 135 k 530 + 17
u Spleen nonadherent cells depleted of MO by carbonyl iron and magnet technique as described in Methods. * Peritoneal or spleen MO mitomycin c or heat treated or freeze-thawed as described in Methods. c Percentage MO of 3 x lo5 lymphocytes/culture well. d Con A, 0.3 &culture well. ’ Mean _’ SEM of triplicate cultures measured at 66 hr.
restoration of the proliferative response is M0 dependent. As observed in Figs. 1 and 2, addition of small numbers of PM0 or SMO or much larger numbers of SMO restored the Con A-induced proliferative response of M0depleted lymphocytes. These observations are consistent with the findings of others that monocyte/macrophage depletion inhibits lectin-induced proliferative responses of murine (7, 23), guinea pig (6, 26), and human (27, 28) T lymphocytes. However, it is possible that the cell separation procedures select for T-cell subpopulations that respond to Con A in the absence of macrophages. This is unlikely since the exact procedures also abrogated the release of a cytotoxic lymphokine (25) by nonproliferating T lymphocytes. A number of mechanisms are probably involved in T-cell activation by macrophages. First, macrophages appear to maintain the viability and functional integrity of lymphocytes in culture (21,22). Second, macrophages may be involved
386
S.YOUDIM
in “presenting” mitogens to lymphocytes (6, 22, 26) or in facilitating effective antigen- or mitogen-lymphocyte interactions (29). Such interactions at close proximity or in the form of M0/T-cell aggregates apparently initiate DNA synthesis (30) and promote antigen (31, 33)- and mitogen (7, 22)-induced lymphocyte proliferation in vitro. A most interesting aspect of these observations is that T cells may respond to lectins in association with macrophage MHC antigens, as appears to be the case with some antigen-induced T-cell responses (33-36). Functional expression of MO Ia antigens appears to be a requirement for T-cell activation in vitro (23, 35, 36). In mice T cells with specificity for both antigen and I-A-region products on macrophages seem to be necessary for optimal contact between MO and lymphocytes as a prelude to the release of lymphokines that mediate delayed-type hypersensitivity (33, 34). Although optimal mitogenic stimulation occurs when MO and T lymphocytes are in physical contact, nonetheless, this is not an absolute requirement, since T-lymphocyte proliferation occurs when these cells are physically separated across semipermeable membranes from accessory adherent cells (6,37). These observations have led to the postulate that yet one more function of accessory cells is to provide a second signal for T-cell mitogenesis via release of such soluble products (38) as mitogenic protein(s) (39) or nonmitogenic lymphokine (40). The mechanism(s) of macrophage-mediated immunosuppression is not well understood; it seems that whenever macrophages stimulate an immune function, excess macrophages inhibit that particular function (41, 42). These findings are consistent with our data in so far as PM0 are involved (Figs. 1 and 2). A similar observation was reported previously (12) where antigen-reactive T cells cooperated with SMO to become cytotoxic to target tumor cells ostensibly via the release of lymphokine (25); however, these interactions were progressively inhibited or suppressed by the addition of increasing numbers of PM0 (12). Therefore, a functional dichotomy, that is, collaborative versus suppressive, is exhibited by high numbers of SMO versus PM0 under in vitro culture conditions. Cowing et al. (17) recently reported that at least three subpopulations of MO may be distinguished: those that lack Ia antigens and predominate in the peritoneal exudate; cells bearing I-A-subregion antigens that are the majority of splenic macrophages and a minor population in the peritoneum; and cells bearing I-Csubregion antigens that are a minor population in both anatomical sites. Given the possibility that mitogens or antigens are presented to T cells in association with Ia, it is conceivable that excessive numbers of PM0 create a case of “nonIt is possible that under conditions of presentation” or “mispresentation.” crowding, the “nonpresenting” subset of PM0, which constitutes the majority of the peritoneal cells, simply displaces the “presentor” subset. Alternatively, it may be that differential expression of Ia molecules influences antigen or mitogen binding in a positive or negative manner or activates or suppresses different T-cell subclasses either directly by contact or via stimulatory and suppressive (39, 41) soluble products. A candidate suppressor factor may be macrophage-derived prostaglandin. In experiments involving B-lymphocyte clonal proliferation, excessive numbers of adherent PM0 suppressed colony promotion (43). A glass-adherent prostaglandinproducing suppressor cell is thought to be responsible for the hyporesponsiveness to PHA seen with Hodgkin’s disease lymphocytes (44). Both these effects were largely prevented by indomethacin, an inhibitor of prostaglandin synthesis. An
MACROPHAGE
REGULATION
OF
LYMPHOPROLIFERATION
387
alternative suppressor factor may be cold thymidine (45, 46) or iododeoxyuridine (47) released by cultured macrophages or lymphocytes which competes with or has a dilution effect on incorporation of the radioactive precursor [3H]thymidine or [1251]UdR. Some other possible mechanisms of MO-mediated suppression are prevention of T-cell differentiation (48), inhibition of blast formation (49), and selective toxicity for proliferating lymphocytes (50). These latter two points seem to be attributes of both SM0 or PM0 when these cells are in an “activated” (51, 52) state. Macrophages from BCG (48, 53), C. parvum (54), and LM (Fig. 3) (12, 55), all potent activators of the reticuloendothelial system, are suppressive for B- or T-lymphocyte activation, presumably due to one or more of the reasons discussed above; Listeria by itself is not suppressive for proliferating (unpublished observations), nonproliferating (12), or antibody-secreting (55) cells. Finally, our data (Table 1) show that viable but not dividing (mitomycin suppressor effect, thus suggesting that C-treated) M0 maintain their “helper” both functions are due to metabolically active events rather than passive attachment of mitogen to the cell surface membrane. Further studies are required before the events and mechanism(s) of MB-dependent lymphocyte activation or suppression are elucidated. ACKNOWLEDGMENTS The author wishes to acknowledge and Glen Olander.
the technical assistance of Ms. E. Benveniste, B. Rothenberg,
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. 25. 26. 27. 28.
Pierce, C. W., J. Exp. Med. 130, 345, 1%9. Mosier, D. E., Science 158, 1575, 1%7. Feldman, M., J. Exp. Med. 135, 1049, 1972. Seeger, R. C., and Oppenheim, J. J., J. Exp. Med. 132, 44, 1970. Waldron, J. A., Horn, R. G., and Rosenthal, A. S., J. Immunol. 111, 58, 1973. Rosenstreich, D. L., Farrar, J. J., and Dougherty, S., J. Immunol. 116, 131, 1976. Stoecker, C. A., Pickard, B. M., and Abel, C. A., Cell. Immunol. 35, 362, 1978. Alter, B. J., and Bach, F. H., Cell. Immunol. 1, 207, 1970. Greineder, D. K., and Rosenthal, A. S., J. Immunol. 114, 1541, 1975. Keller, R., Cell. Immunol. 17, 542, 1975. Folch, H., Yoshinaga, M., and Waksman, B. H., J. Immunol. 110, 835, 1973. Youdim, S., and Sharman, M., J. Immunol. 117, 1860, 1976. Hoffman, M., Immunology 18, 791, 1970. Walker, W. S., Immunology 26, 1025, 1974. Pope, B. L., Whitney, R. B., Levy, J. G., and Kilburn, D. G., J. Immunol. 116, 1342, 1976. Rhodes, J., J. Immunol. 114, 976, 1975. Cowing, C., Schwartz, B. D., and Dickler, H. B., J. Zmmunol. 120, 378, 1978. Rice, S. G., and Fishman, M., Cell. Immunol. 11, 130. 1974. Ward, P. A., J. Exp. Med. 128, 1201, 1%8. Youdim, S., Moser, M., and Stutman, O., J. Nat. Cancer Inst. 50, 193, 1974. Chen, C., and Hirsch, J. G., J. Exp. Med. 136, 609, 1972. Ellner, J. J., Lipsky, P. E., and Rosenthal, A. S., J. Zmmunol. 116, 876, 1976. Habu, S., and Raff, M. C., Eur. J. Immunol. 7, 451, 1977. Fernandez, L. A., and Macsween, J. M., J. Immunol. Methods 18, 193, 1977. Youdim, S., Cancer Res. 37, 572, 1977. Lipsky, P. E., Ellner, J. J., and Rosenthal, A. S., J. Immunol. 116, 866, 1976. Schmidtke, J. R., and Hatfield, S., J. Zmmunol. 116, 357, 1976. Gonanholm, E. H., and Pettersson, D., Clin. Exp. Immunol. 22, 223, 1975.
388
S. YOUDIM
29. Rosenstreich, D. L., and Wilton, J. M., In “Immune Recognition” (A. S. Rosenthal, Ed.), p. 113. Academic Press, New York, 1975. 30. Braendstrup, O., Anderson, V., and Wendelin, O., Cell. Immunol. 25, 207, 1976. 31. Lipsky, P. E., and Rosenthal, A. S., .I. Exp. Med. 138, 900, 1973. 32. Lipsky, P. E., and Rosenthal, A. S., J. Exp. Med. 141, 138, 1975. 33. Miller, .I. F. A. P., Vadas, M. A., Whitelaw, A., and Gamble, J.,Proc. Nat. Acad. Sci. 72,5095,1972. 34. Miller, J. F. A. P., Vadas, M. A., Whitelaw, A., and Gamble, J., Proc. Nat. Acad. Sci. 73, 2486, 1976. 35. Shevach, E. M.,.!. Zmmunol. 116, 1482, 1976. 36. Thomas, D. W., Yanmashita, U., and Shevach, E. M., J. Immunol. 119, 223, 1977. 37. Keller, N., Cell. Immunol. 17, 542, 1975. 38. Grey, I., Gershon, R. K., and Waksman, B. H., J. Exp. Med. 136, 128, 1972. 39. Calderon, J., and Unanue, E. R., Nature 253, 359, 1975. 40. Paetkau, V., Mills, G., Gerhart, S., and Monticone, V., J. Immunol. 117, 1320, 1976. 41. Nelson, D. S., Nature 246, 306, 1973. 42. Yoshinaga, M., Yoshinaga, A., and Waksman, B. H., J. Exp. Med. 136, 956, 1972. 43. Kurland, .I. I., Kincade, P. N., and Moor, M. A. S., J. Exp. Med. 146, 1420, 1977. 44. Goodwin, J. S., Messner, R. J., Bankhurst, A. D., Peake, G. T., Saiki, J. H., and Williams, R. C., Jr., N. Engl. .I. Med. 297, 963, 1977. 45. Opitz, H. G., Niethammer, D., Jackson, R. C., Lemke, H., Huget, R., and Flad, H. D., Cell. Immunol. 18, 70, 1975. 46. Tadashi, K., and Shioiri-Nakano, K., J. Immunol. 116, 1251, 1976. 47. Evans, R., and Booth, C. G., Cell. Immunol. 26, 120, 1976. 48. Klimpel, G. R., and Henney, C. S., J. Immunol. 120, 563, 1978. 49. Baird, L. G., and Kaplan, A. M., Cell. Immunol. 28, 36, 1977. 50. Viet, B. C., and Feldman, J. D., J. Immunol. 117, 655, 1976. 51. Unanue, E. R., Amer. J. Pathol. 83, 3%, 1976. 52. Nathan, C. F., Karnovsky, M. C., and David, J. R., J. Exp. Med. 133, 1356, 1971. 53. Florentin, I., Huchet, R., Bruley-Rosset, M., Halle-Pannenko, O., and Mathe, G., Cancerlmmunol. Immunother. 1, 31, 1976. 54. Kirchner, R., Glasser, M., and Herberman, R. B., Nature 257, 396, 1975. 55. Kongshavan, P. A. L., Ho, A., and Sebaldt, R. J., Cell. Immunol. 28, 284, 1977.
IMMUNOLOGY
45, 377-388
(1979)
Enhancing and Suppressive Effects on T-Lymphocyte Stimulation
of Macrophages in Vitro1
S. YOUDIM~ Department
of Surgery,
University of California La Jolla, California Received
San Diego 92093
School
of Medicine,
July 24, 1978
The immunoregulatory effect of peritoneal and splenic macrophages on Con A-stimulated mouse splenic T lymphocytes was investigated in vitro using [L251]UdR incorporation as a measure of lymphocyte proliferation. [1251]UdR incorporation was enhanced by the addition of increasing numbers of splenic or low doses of peritoneal adherent cells to macrophagedepleted splenic lymphocytes. The addition of increasing numbers ofperitoneal macrophages beyond 5-lo%, however, proportionally suppressed T-cell proliferation. Activated splenic macrophages obtained from mice 6 days after infection with Listeria monocytogenes were suppressive, whereas macrophages obtained from immune donors 9- 10 days after infection were not, so that a chronological association appeared to exist between macrophage activation and immunosuppression. The addition of 2-mercaptoethanol to the cell cultures increased [‘*51]UdR incorporation without affecting the stimulatory and suppressive effects of splenic and peritoneal macrophages, respectively. Heat-killed and freeze-thawed macrophages lost their capacity to enhance or inhibit lymphocyte transformation. Macrophages treated with mitomycin C to inhibit DNA synthesis retained their regulatory functions. These studies suggest differential regulatory roles for spleen versus peritoneal macrophages on T-lymphocyte responses to Con A stimulation in vitro.
INTRODUCTION Experimental models of antibody formation and cellular immune responses indicate that macrophages critically influence these lymphocyte responses in vitro. The primary antibody response of B lymphocytes3 (l-3) and the proliferative responses of T lymphocytes to antigens (4,5), to mitogens (6,7), and to allogeneic cells in the mixed lymphocyte reaction (8,9) are all M0 dependent. These adherent cells may also exert the reverse influence and inhibit T-lymphocyte proliferation (lo- 12) and antibody production (13). Functionally and morphologically, M0 are a heterogeneous cell population and differ with respect to size (15), density of Fc receptors (16) Ia (I-region products of the MHC) antigens (17) and phagocytic 1 This work was supported by USPHS Grant CA 177299 from the National Institutes of Health. 4 To whom reprint requests should be addressed at the University of California San Diego School of Medicine, Department of Surgery-Q-058, La Jolla, California 92093. 3 Abbreviations used: SM0, spleen macrophages; PM@, peritoneal macrophages; PC, peritoneal cells; SC, spleen cells, B lymphocytes, bone marrow-derived cells; T lymphocytes, thymus-derived cells; Con A, concanavalin A; LM, Listeria monocytogens; 2ME, 2-mercaptoethanol; M-D gas, Mishell-Dulton gas mixture; MHC, major histocompatibility complex. 377
0008-8749/79/080377-12$02.00/O Copyright 0 1979by Academic Press, Inc. All rights of reproduction in any form reserved.
378
SYOUDIM
(18) and chemotactic (19) activities. The anatomical site of these cells also appears to be associated with their functional diversity. For example, we previously noted (12) that antigen (LM)-primed spleen T lymphocytes in association with their complementary adherent cells become nonspecifically cytotoxic for B-16 target tumor cells in the presence of the specific antigen, however, PM0 suppressed this cooperative endeavor. We now report that a similar collaborative suppressive macrophage lymphocyte interaction occurs during Con A-induced lymphocyte transformation in vitro. MATERIALS
AND METHODS
Animals. Inbred C57BL/6 mice 6 to 9 weeks old were obtained from Strong Research Foundation, San Diego, Calif., and used in all experiments. Bacterial cultures and immunization. Cultures and innocula of Listeria monocytogens were prepared as described previously (20). All mice were immunized with 5 x lo3 viable LM intravenously. Tissue culture materials and media. All media and sera were obtained from Grand Island Biological Company, Grand Island, N.Y., or SCOR C, University of California, San Diego. The common culture medium was complete medium (CM) consisting of RPMI-1640 containing 2 mM L-glutamine supplemented with 10% fetal calf serum (FCS), 100 pg of streptomycin, and 100 units of penicillin per milliliter. Preparation of spleen ‘cells (SC). Spleens were aseptically removed and placed in 60-mm tissue culture dishes containing 5 ml cold (4°C) complete Hanks’ balanced salt solution (HBSS). Single cell suspensions were prepared by gently teasing the spleens. Preparation ofperitoneal cells (PC). These were collected from the peritoneal cavity of mice after injecting 5 ml of HBSS containing 5 units of sodium heparin/ml. Separation of adherent and nonadherent cells on plastic surfaces. Two to 5 x 10’ SC or PC were placed in 5 ml of prewarmed (37°C) CM in a plastic tissue culture flask (Falcon No. 3024) and incubated for 45 min at 37°C in humidified air containing 5% CO,. At the end of this incubation period, the nonadherent cells and two successive washings of 2.5 ml each were transferred to a second flask. Five milliliters of warm CM was added to the original flask and both flasks were incubated as above for a further 45 to 60 min. Nonadherent cells were decanted and further treated with carbonyl iron as described below. The adherent cells were washed six times in warm CM with moderate agitation of the flasks. Two and a half milliliters of cold (4°C) CM was layered on each monolayer of adherent cells, the flasks were placed at 4°C for 10 min, and adherent cells were gently scraped with a sterile rubber policeman. The loosened cells were counted, their viability was determined, and they were kept in cold CM at 4°C until used. Carbonyl iron treatment. Carbonyl iron powder, 40 mg (General Aniline and Film Cot-p, Linden, N.J.), was washed five times in 50 ml HBSS. Spleen cells, 1.5 to 3 x lo’, or PC, 1.5 to 3 x 106, were then suspended in 15 ml CM in a 250-ml flask containing the carbonyl iron and incubated for 1 hr at 37°C with periodic agitation. The contents of the flask were pipetted into a 50-ml centrifuge tube, a strong magnet was applied to the sides of the tube for 5 min, and the suspension was decanted into a second tube while holding the magnet in place.
MACROPHAGE
REGULATION
OF LYMPHOPROLIFERATION
379
This procedure was repeated two or three times more or until all iron powder was removed. The supematant cells were finally washed twice, suspended in CM, and counted. Cell viability was determined by trypan blue exclusion. Nylon wool column. Spleen cells were passed through nylon wool columns as described previously (12). The packed, autoclaved, and dried columns were rinsed with 50 ml of prewarmed (37°C) CM, allowed to drain, covered, and incubated at 37°C for 1 hr. Each column was then flushed with 30 ml of warm CM, and 1.5 to 3 x lo7 cells in 1 ml of warm medium were washed into the nylon wool, followed by a further 2.5 ml of warm CM. The columns were incubated in an upright position for 45 min at 37°C in a humidified CO, incubator. Effluent cells were collected by slowly flushing with 30 ml of prewarmed CM. The cell suspension was then washed and counted, and its viability was determined as before. Mitomycin C treatment of cells. In certain experiments cells were incubated with mitomycin C (50 pg/ml, Sigma Chemical Co., St. Louis, MO.) for 30-45 min at 37°C. The cells were then washed three times in cold HBSS. Heat inactivation and fysis of macrophages. Cells were heated for 30 min at 56°C and washed as above. Macrophages were lysed by five cycles of freezing and thawing. Concanavalin A. This was prepared by and was a gift of Dr. Doug Redelman, University of California, San Diego. Lymphocyte proliferation assay. Different numbers of PM0 or SMO suspended in CM were added to 3 x lo5 M0depleted spleen cells in a total volume of 0.1 ml and put in individual wells of microtiter flat-bottom plates (Falcon Plastic, Oxnard, Calif.). Con A was diluted in CM and 0.6, 0.3, or 0.15 pg in 0.1 ml added to each well. Control cultures received 0.1 ml of CM. Other controls contained 3 x 1W purified spleen cells plus similar concentrations of Con A. All cultures were incubated for 48 hr in 5% CO, + 95% air or, where stated, in 7% 0, + 10% CO, + 83% Nz (Mishell-Dutton gas mixture) and incubated in a sealed incubator chamber at 37°C. Each well was then pulsed with 0.1 @Zi of [lZ51]iodo-2-deoxyuridine ([‘2”I]UdR; Radiochemical Centre, Amersham England; 851 &i/ml) and cultured for a further 18 hr. Cells were harvested on glass-fiber filters using a MASH II (multiple automatic sample harvester) harvesting machine (Microbiological Associates, Bethesda, Md.). The filters were dried, placed in tubes, and counted in a Packard Auto-Gamma scintillation spectrometer. RESULTS Effect of Macrophage
Depletion
and Reconstitution
on T-Cell Mitogenesis
Macrophages were removed from the PC or SC suspensions by allowing them to adhere to plastic surfaces. The nonadherent SC were then further treated with carbonyl iron to remove residual phagocytic cells or passed through nylon wool columns as described. SMO or PM0 were then added to the nonadherent spleen (3 x 105/well) T cells at concentrations of O-40 or O-60% (0- 1.2 x 10” to 0- 1.8 x 105) MO per well. The results of representative experiments are depicted in Figs. 1 and 2. Macrophage-depleted T cells were minimally stimulated by 0.15,0.3, or 0.6 pg Con A/well. In the absence of Con A, there was no stimulation regardless of the number of MO added. Con A-stimulated T-cell proliferation increased progressively with the addition of increasing numbers of SM0 or low
S. YOUDIM I
I
I
I
I
,4
I5 pg CON A/ WELL (SMh A---
-A
, /’
,,$
4
3~9
CONA/ WELL(SM'#'I
6pg
CONAlWELL
(sM'#'j
E----a
2
FIG. 1. Normal SM0 or PM@ (O-40%) added to a constant number of (3 x 105) splenic lymphocytes depleted of adherent and phagocytic cells by carbonyl iron and magnet technique.
doses of PM0 at all concentrations of Con A. The addition of increasing numbers of PM0 beyond 5 to lo%, however, .proportionally suppressed lymphocyte proliferation. Optimum results were obtained at concentrations of 0.15 or 0.3 pg Con A/well. Effect of Peritoneal and Spleen Macrophages from T-Cell Responses to Con A
Listeria-Znfected Mice on
Spleen and peritoneal MO were collected from donor mice 6 days (infected or’ inflammatory state) after injection of 3-5 x lo3 viable LM intravenously. The effects of these activated M0 on [1251]UdR incorporation by M0-depleted spleen lymphocytes are shown in Fig. 3. At low concentrations of up to lo%, SM0 enhanced lymphocyte incorporation of [lz51]UdR. At higher concentrations of up to 60%, [lz51]UdR incorporation remained stationary and was no longer enhanced. In a subsequent experiment, SM0 obtained from mice 6 days post-LM injection in fact slightly suppressed [Y]UdR incorporation at concentrations beyond lo-20%. These data were in direct contrast to the enhancing effect of normal SM0 at the same concentrations (compare Fig. 3 to Figs. 1 and 2). Peritoneal MO (Fig. 3) initially enhanced, but beyond a 10% concentration sharply suppressed, lymphocyte proliferation. These data were similar to the inhibiting effect of normal PM0. Macrophages obtained from mice 9 days after LM injection (immune state, Fig. 4) essentially showed similar enhancing and suppressive effects as normal MO (compare Fig. 4 to Figs. 1 and 2).
MACROPHAGE
8
I
I
REGULATION I
I
I
I
6
I
4
0
5
IO
20
30
40
381
OF LYMPHOPROLIFERATION
50
3pg
CON A/ WELLM4$1
o-----o
15pg
CON A/WELL
A----A
kM$I
60
M$
%
FIG. 2. Normal SM@ or PM0 (O-60%) added to a constant number of (3 x 105) splenic lymphocytes depleted of adherent cells and filtered through nylon wool columns.
Effect of Addition of 2-Mercuptoethanol and Incubation in Mishell-D&ton Mixture on the Reconstitutive and Suppressive Potential of Macrophages
Gas
It is reported that the reducing agent 2ME promotes the viability of lymphoid cells in culture and may substitute for MO in the in vitro antibody response by lymphoid cells to sheep red blood cells (21). 2-Mercaptoethanol alone, however, 6
I
4
-
I
I
I
I
I
I
,~----~--------~---------p
3pg
CONA/WELL(SM@
15pg
CONA/WELL
O----*
“0
s z
(SM#
A----a
2
0 0
5
IO
20
30
40
50
60
% M+
FIG. 3. Spleen MO or PM0 (O-60%) obtained from mice 6 days after LM infection added to splenic lymphocytes treated as for Fig. 1.
S. YOUDIM I
I
I
I
I
I
,.p--.--
I
--+.3pg
CON A/WELL
--+5pgCON
20
30
40
50
o----o
A/WELLMh#')
A---d
.15pg
CONA/WELL(PM#)A--4
.3pg
CON A/WELL
NO CON A (SM ) NO CONAIPM )
0 5 IO
KM#
(PM+w o----o
60
% M# FIG. 4. Spleen M@ or PM0 (O-60%) obtained from mice 9 days after LM infection added to splenic lymphocytes treated as for Fig. 1.
is not sufficient to allow M0-depleted guinea pig lymphocytes to respond to PHA (6,22) or murine lymphocytes to Con A (23). Some preliminary data showed that [1251]UdR incorporation was improved when cultures were incubated in M-D gas mixture in preference to CO,. Accordingly, experiments were performed using 2ME at a 5 x lo+ M final concentration in CM and M-D gas to determine the effect of these variables on the reconstitutive and suppressive effects of SMO and PM0, respectively. Data from representative experiments are shown in Figs. 5 and 6. Addition of 2ME and incubation in M-D gas mixture dramatically improved [Y]UdR incorporation by Con A-stimulated lymphocytes without altering the enhancing effect of SMO or the suppressive effect of PM0 (Fig. 5). In general, DNA synthesis increased 2- to 4-fold when comparing 2MEsupplemented (Figs. 5 and 6) to 2ME-deficient cultures (Fig. 1). Figure 6 is a good example of the M012 ME synergy at low concentrations (5- 10%) of PM0. DNA synthesis increased over threefold at 0.15 pg and over fourfold at 0.3 ,ug Con A/well in 2ME-supplemented versus 2ME-depleted cultures. Beyond 10% however, the inhibitory effect of PM0 on T-cell proliferation was not compromised by the addition of 2ME and incubation in M-D gas mixture. 2-Mercaptoethanol apparently partially substituted for MO, since at 0% MO, [1251]UdR incorporation by spleen T cells was increased at both 0.15 and 0.3 pg Con A/well (Figs. 5 and 6 and Table I at 0% M0 + Con A). Effects of Mitomycin, Heat-Treated, ative Responses to Con A
and Lysed Macrophages on T-Cell Prolifer-
Peritoneal and SM0 which had been lysed by freeze-thawing or heated to 56°C for 30 min did not reconstitute or inhibit T-lymphocyte proliferation. On
MACROPHAGE
REGULATION
OF LYMPHOPROLIFERATION
383
FIG. 5. Normal SM0 or PM0 (O-60%) added to a constant number of (3 x loj) spleen lymphocytes treated as for Fig. 1. Cultures contained 2ME, 5 x 10-j M and incubated in M-D gas mixture.
the other hand, SMO and PM0 treated with cytostatic concentrations of mitomycin C (50 y&ml) maintained their respective inhibitory and suppressive effects (Table 1). All cultures contained 2ME and were incubated in M-D gas mixture. DISCUSSION By removal of adherent and phagocytic cells from suspensions of mouse spleen cells, it was possible to isolate a population of lymphocytes which were severely depressed or did not synthesize DNA in response to Con A. The responding cell population was previously shown (12) to be susceptible to lysis by anti-thy-l and a preparation of rabbit anti-mouse brain antiserum plus complement. The carbonyl iron and magnet technique of T-lymphocyte enrichment also removes substantial
384
S. YOUDIM
NO CON AtFMh (-EHEI NO CON A (pM4)ItZMEl 3~4 CON A/WELL (PM41 (+ZME) -15~9 CON A/WELL (PM'& 1+2ME) l5j4CONAlWELL (PM# (-2MEI 3kq CONA/WELLIPu+j I-2ME)
5 IO
20
30
40
50
o----o A----A o-----o
60
FIG. 6. Normal PM0 (O-60%) added to a constant number of (3 x 105) spleen lymphocytes treated as for Fig. 1. Cultures were supplemented with or were deficient in 2ME (5 x 10m5M final concentration).
numbers of B lymphocytes (24). The peritoneal adherent cell population was susceptible to the lytic action of a preparation of antimacrophage serum plus complement (25) and phagocytic for LM and carbonyl iron (unpublished). Based on these observations, the interacting cell populations are considered to be mainly T lymphocytes and macrophages. To ensure that loss of response to Con A was not due to dilution of the separated splenic T-cell population, nonadherent, carbonyl iron-treated cells were cultured at concentrations of 3 x lo5 and 4.8 x lo5 cells/well, i.e., equivalent to the lowest (0% MO) and to the highest (60% M0) number of combined M0/T-cell populations tested. At 0.15 pg Con A/well, mean cpm for triplicate cultures were 466 k 67 to 811 + 132, and at 0.3 pg Con A/well, 609 f 42 and 1615 f 58; therefore, it is obvious that
MACROPHAGE
REGULATION
385
OF LYMPHOPROLIFERATION
TABLE
1
Effect of Mitomycin-Lysed and Heat-Treated Macrophages on [1Z51]UdR Incorporation of Con A-Stimulated MB-Deficient Spleen Lymphocytes Source of lymphocytes” Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal
spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen spleen
Macrophage treatmentb
% Macrophages’
PM0 normal PM0 normal PM0 normal Heat killed Heat killed Heat killed Freeze-thawed Freeze-thawed Freeze-thawed Mito c Mito c Mito c SMO normal SMO normal SMO normal Heat killed Heat killed Heat killed Freeze-thawed Freeze-thawed Freeze-thawed Mito c Mito c Mito c -
5 30 60 5 30 60 5 30 60
-
30 60 30 60 30 60 5 30 60 5 30 60 0 0
Con A addedd
+ + + +
+ + + + + +
+ + + + -
[1251]UdR incorporation (cpm ? SEM)’ 15,489 2,577 938 8,350 8,899 9,181 8,627 7,513 9,246 9,219 7,184 500 8,590 15,195 19,373 6,214 5,549 5,538 5,544 5,821 6,419 8,613 12,755 9,819 7,132 329
-t 979 t 338 + 276 +- 822 k 274 f 433 2 1123 i- 519 2 226 k 554 ” 85 _f 124 t 606 k 471 2 141 t 369 + 288 2 107 t 390 +- 140 f 82 2 177 + 837 + 135 k 530 + 17
u Spleen nonadherent cells depleted of MO by carbonyl iron and magnet technique as described in Methods. * Peritoneal or spleen MO mitomycin c or heat treated or freeze-thawed as described in Methods. c Percentage MO of 3 x lo5 lymphocytes/culture well. d Con A, 0.3 &culture well. ’ Mean _’ SEM of triplicate cultures measured at 66 hr.
restoration of the proliferative response is M0 dependent. As observed in Figs. 1 and 2, addition of small numbers of PM0 or SMO or much larger numbers of SMO restored the Con A-induced proliferative response of M0depleted lymphocytes. These observations are consistent with the findings of others that monocyte/macrophage depletion inhibits lectin-induced proliferative responses of murine (7, 23), guinea pig (6, 26), and human (27, 28) T lymphocytes. However, it is possible that the cell separation procedures select for T-cell subpopulations that respond to Con A in the absence of macrophages. This is unlikely since the exact procedures also abrogated the release of a cytotoxic lymphokine (25) by nonproliferating T lymphocytes. A number of mechanisms are probably involved in T-cell activation by macrophages. First, macrophages appear to maintain the viability and functional integrity of lymphocytes in culture (21,22). Second, macrophages may be involved
386
S.YOUDIM
in “presenting” mitogens to lymphocytes (6, 22, 26) or in facilitating effective antigen- or mitogen-lymphocyte interactions (29). Such interactions at close proximity or in the form of M0/T-cell aggregates apparently initiate DNA synthesis (30) and promote antigen (31, 33)- and mitogen (7, 22)-induced lymphocyte proliferation in vitro. A most interesting aspect of these observations is that T cells may respond to lectins in association with macrophage MHC antigens, as appears to be the case with some antigen-induced T-cell responses (33-36). Functional expression of MO Ia antigens appears to be a requirement for T-cell activation in vitro (23, 35, 36). In mice T cells with specificity for both antigen and I-A-region products on macrophages seem to be necessary for optimal contact between MO and lymphocytes as a prelude to the release of lymphokines that mediate delayed-type hypersensitivity (33, 34). Although optimal mitogenic stimulation occurs when MO and T lymphocytes are in physical contact, nonetheless, this is not an absolute requirement, since T-lymphocyte proliferation occurs when these cells are physically separated across semipermeable membranes from accessory adherent cells (6,37). These observations have led to the postulate that yet one more function of accessory cells is to provide a second signal for T-cell mitogenesis via release of such soluble products (38) as mitogenic protein(s) (39) or nonmitogenic lymphokine (40). The mechanism(s) of macrophage-mediated immunosuppression is not well understood; it seems that whenever macrophages stimulate an immune function, excess macrophages inhibit that particular function (41, 42). These findings are consistent with our data in so far as PM0 are involved (Figs. 1 and 2). A similar observation was reported previously (12) where antigen-reactive T cells cooperated with SMO to become cytotoxic to target tumor cells ostensibly via the release of lymphokine (25); however, these interactions were progressively inhibited or suppressed by the addition of increasing numbers of PM0 (12). Therefore, a functional dichotomy, that is, collaborative versus suppressive, is exhibited by high numbers of SMO versus PM0 under in vitro culture conditions. Cowing et al. (17) recently reported that at least three subpopulations of MO may be distinguished: those that lack Ia antigens and predominate in the peritoneal exudate; cells bearing I-A-subregion antigens that are the majority of splenic macrophages and a minor population in the peritoneum; and cells bearing I-Csubregion antigens that are a minor population in both anatomical sites. Given the possibility that mitogens or antigens are presented to T cells in association with Ia, it is conceivable that excessive numbers of PM0 create a case of “nonIt is possible that under conditions of presentation” or “mispresentation.” crowding, the “nonpresenting” subset of PM0, which constitutes the majority of the peritoneal cells, simply displaces the “presentor” subset. Alternatively, it may be that differential expression of Ia molecules influences antigen or mitogen binding in a positive or negative manner or activates or suppresses different T-cell subclasses either directly by contact or via stimulatory and suppressive (39, 41) soluble products. A candidate suppressor factor may be macrophage-derived prostaglandin. In experiments involving B-lymphocyte clonal proliferation, excessive numbers of adherent PM0 suppressed colony promotion (43). A glass-adherent prostaglandinproducing suppressor cell is thought to be responsible for the hyporesponsiveness to PHA seen with Hodgkin’s disease lymphocytes (44). Both these effects were largely prevented by indomethacin, an inhibitor of prostaglandin synthesis. An
MACROPHAGE
REGULATION
OF
LYMPHOPROLIFERATION
387
alternative suppressor factor may be cold thymidine (45, 46) or iododeoxyuridine (47) released by cultured macrophages or lymphocytes which competes with or has a dilution effect on incorporation of the radioactive precursor [3H]thymidine or [1251]UdR. Some other possible mechanisms of MO-mediated suppression are prevention of T-cell differentiation (48), inhibition of blast formation (49), and selective toxicity for proliferating lymphocytes (50). These latter two points seem to be attributes of both SM0 or PM0 when these cells are in an “activated” (51, 52) state. Macrophages from BCG (48, 53), C. parvum (54), and LM (Fig. 3) (12, 55), all potent activators of the reticuloendothelial system, are suppressive for B- or T-lymphocyte activation, presumably due to one or more of the reasons discussed above; Listeria by itself is not suppressive for proliferating (unpublished observations), nonproliferating (12), or antibody-secreting (55) cells. Finally, our data (Table 1) show that viable but not dividing (mitomycin suppressor effect, thus suggesting that C-treated) M0 maintain their “helper” both functions are due to metabolically active events rather than passive attachment of mitogen to the cell surface membrane. Further studies are required before the events and mechanism(s) of MB-dependent lymphocyte activation or suppression are elucidated. ACKNOWLEDGMENTS The author wishes to acknowledge and Glen Olander.
the technical assistance of Ms. E. Benveniste, B. Rothenberg,
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. 25. 26. 27. 28.
Pierce, C. W., J. Exp. Med. 130, 345, 1%9. Mosier, D. E., Science 158, 1575, 1%7. Feldman, M., J. Exp. Med. 135, 1049, 1972. Seeger, R. C., and Oppenheim, J. J., J. Exp. Med. 132, 44, 1970. Waldron, J. A., Horn, R. G., and Rosenthal, A. S., J. Immunol. 111, 58, 1973. Rosenstreich, D. L., Farrar, J. J., and Dougherty, S., J. Immunol. 116, 131, 1976. Stoecker, C. A., Pickard, B. M., and Abel, C. A., Cell. Immunol. 35, 362, 1978. Alter, B. J., and Bach, F. H., Cell. Immunol. 1, 207, 1970. Greineder, D. K., and Rosenthal, A. S., J. Immunol. 114, 1541, 1975. Keller, R., Cell. Immunol. 17, 542, 1975. Folch, H., Yoshinaga, M., and Waksman, B. H., J. Immunol. 110, 835, 1973. Youdim, S., and Sharman, M., J. Immunol. 117, 1860, 1976. Hoffman, M., Immunology 18, 791, 1970. Walker, W. S., Immunology 26, 1025, 1974. Pope, B. L., Whitney, R. B., Levy, J. G., and Kilburn, D. G., J. Immunol. 116, 1342, 1976. Rhodes, J., J. Immunol. 114, 976, 1975. Cowing, C., Schwartz, B. D., and Dickler, H. B., J. Zmmunol. 120, 378, 1978. Rice, S. G., and Fishman, M., Cell. Immunol. 11, 130. 1974. Ward, P. A., J. Exp. Med. 128, 1201, 1%8. Youdim, S., Moser, M., and Stutman, O., J. Nat. Cancer Inst. 50, 193, 1974. Chen, C., and Hirsch, J. G., J. Exp. Med. 136, 609, 1972. Ellner, J. J., Lipsky, P. E., and Rosenthal, A. S., J. Zmmunol. 116, 876, 1976. Habu, S., and Raff, M. C., Eur. J. Immunol. 7, 451, 1977. Fernandez, L. A., and Macsween, J. M., J. Immunol. Methods 18, 193, 1977. Youdim, S., Cancer Res. 37, 572, 1977. Lipsky, P. E., Ellner, J. J., and Rosenthal, A. S., J. Immunol. 116, 866, 1976. Schmidtke, J. R., and Hatfield, S., J. Zmmunol. 116, 357, 1976. Gonanholm, E. H., and Pettersson, D., Clin. Exp. Immunol. 22, 223, 1975.
388
S. YOUDIM
29. Rosenstreich, D. L., and Wilton, J. M., In “Immune Recognition” (A. S. Rosenthal, Ed.), p. 113. Academic Press, New York, 1975. 30. Braendstrup, O., Anderson, V., and Wendelin, O., Cell. Immunol. 25, 207, 1976. 31. Lipsky, P. E., and Rosenthal, A. S., .I. Exp. Med. 138, 900, 1973. 32. Lipsky, P. E., and Rosenthal, A. S., J. Exp. Med. 141, 138, 1975. 33. Miller, .I. F. A. P., Vadas, M. A., Whitelaw, A., and Gamble, J.,Proc. Nat. Acad. Sci. 72,5095,1972. 34. Miller, J. F. A. P., Vadas, M. A., Whitelaw, A., and Gamble, J., Proc. Nat. Acad. Sci. 73, 2486, 1976. 35. Shevach, E. M.,.!. Zmmunol. 116, 1482, 1976. 36. Thomas, D. W., Yanmashita, U., and Shevach, E. M., J. Immunol. 119, 223, 1977. 37. Keller, N., Cell. Immunol. 17, 542, 1975. 38. Grey, I., Gershon, R. K., and Waksman, B. H., J. Exp. Med. 136, 128, 1972. 39. Calderon, J., and Unanue, E. R., Nature 253, 359, 1975. 40. Paetkau, V., Mills, G., Gerhart, S., and Monticone, V., J. Immunol. 117, 1320, 1976. 41. Nelson, D. S., Nature 246, 306, 1973. 42. Yoshinaga, M., Yoshinaga, A., and Waksman, B. H., J. Exp. Med. 136, 956, 1972. 43. Kurland, .I. I., Kincade, P. N., and Moor, M. A. S., J. Exp. Med. 146, 1420, 1977. 44. Goodwin, J. S., Messner, R. J., Bankhurst, A. D., Peake, G. T., Saiki, J. H., and Williams, R. C., Jr., N. Engl. .I. Med. 297, 963, 1977. 45. Opitz, H. G., Niethammer, D., Jackson, R. C., Lemke, H., Huget, R., and Flad, H. D., Cell. Immunol. 18, 70, 1975. 46. Tadashi, K., and Shioiri-Nakano, K., J. Immunol. 116, 1251, 1976. 47. Evans, R., and Booth, C. G., Cell. Immunol. 26, 120, 1976. 48. Klimpel, G. R., and Henney, C. S., J. Immunol. 120, 563, 1978. 49. Baird, L. G., and Kaplan, A. M., Cell. Immunol. 28, 36, 1977. 50. Viet, B. C., and Feldman, J. D., J. Immunol. 117, 655, 1976. 51. Unanue, E. R., Amer. J. Pathol. 83, 3%, 1976. 52. Nathan, C. F., Karnovsky, M. C., and David, J. R., J. Exp. Med. 133, 1356, 1971. 53. Florentin, I., Huchet, R., Bruley-Rosset, M., Halle-Pannenko, O., and Mathe, G., Cancerlmmunol. Immunother. 1, 31, 1976. 54. Kirchner, R., Glasser, M., and Herberman, R. B., Nature 257, 396, 1975. 55. Kongshavan, P. A. L., Ho, A., and Sebaldt, R. J., Cell. Immunol. 28, 284, 1977.
