Suppression of primary antibody response in genetically susceptible mice infected with Salmonella typhimurium: Restoration by catalase

Q ELSEVIER Paris 1989

Res. Immunol. 1 C~@C~

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SUPPRESSION OF PRIMARY ANTIBODY RESPONSE IN GENETICALLY SUSCEPTIBLE MICE INFECTED W I T H SALMONELLA

T Y F H I M U R I U M : RESTORATION BY CATALASE

M. Desch~nes

(1),

M. Guenounou (1) and C. Naucie! (])

(2)

(]) Laboratoire de Microbiologie, Facult~ de M~decine de Paris-Ouest,

92380 Garches (France), and (2) Unitd d'Immunochimie des protdines, Institut Pasteur, 75724 Paris Cedex 15

SUMMARY Susceptible C57BL/6 mice infez:ed with a temperature-sensitive mutant of Salmonella typhimurium exhibi~:ed a marked depression of in vivo and in vitro primary antibody response to sheep erythrocytes which persisted for several weeks. The antibody response to a T-independent antigen (TNPpolyacrylamide) was also depressed. Cell-mixing experiments indicated that spleen cells from infected animals contained adherent suppressor cells and that the functional activity of T and B cells was unaffected. The antibody ~cspo~se ol spleen ceils ~rom ~ntectea mice was restored by me addition of catalase, suggesting that hydrogen peroxide is involved in the mechanism of suppression. KEY-WORDS" Salmonella typhimurium, Immunoregulation, Macrophage, Catalase; Mouse. INTRODUCTION Salmonella typhimurium is a facultative intracellular bacterium which induces in mice a disease similar to typhoid fever. The natural resistance of mice to S. typhimurium infection is regulated by several genes [18], the main one being the !ty gene [19] which controls the growth rate of bacteria during the early phase of infection [4].

Submitted July 21, 1988, accepted November 10, 1988.

M. DESCHENES A N D COLL.

56

Susceptible (Ity s) mice are rapidly killed by virulent strains of S. typhimurium. Infection with a temperature-sensitive mutant of S. typhimurium induces a protracted infection in both susceptible and resistant mice, allowing the effect of infection on immune responses to be studied [16]. S. typhimurium infection can depress several immune responses. We have previously shown that S. typhimurium infection induces, in susceptible C57BL/6 mice, a suppression both of lymphocyte proliferative responses to T and B mitogens and of interleukin-2 production, whereas the responses of resistant (Ity r) mice are unaffected [3]. A similar suppression of the proliferative response to mitogens has been reported in infected mice of the C3H lineage [10]. Macrophages were found to be involved in this infection-induced suppression [3, 10]. S. typhimurium infection also induces in genetically susceptible mice a depression of the delayed-type hypersensitivity response to sheep red blood cells (SRBC) [15]. The present study shows that S. typhimurium infection can induce a marked suppression of in vivo and in vitro primary antibody responses in susceptible C57BL/6 mice. Suppression was mediated by adherent cells and could be restored by the addition of catalase, suggesting that hydrogen peroxide plays a role in this suppression.

MATERIALS AND METHODS Animals.

Female C57BL/6 mice between 6 and 7 weeks of age were obtained from the Pasteur Institute (Paris, France).

S. typhimurium infection. S. typhimurium strain C5TS, a temperature-sensitive mutant derived from the virulent strain C5 [5], kindly provided by Dr Hormaeche, was grown for 24 h at 30°C in tryptic soy broth. Mice were infected intravenously with 106 bacteria in 0.2 ml of saline. Antigens.

SRBC were obtained from a selected animal from the ~Centre National de Recherches Zootechniques>> (Jouy-en-Josas, France). Trinitrophenyl-polyacrylamide (TNP-PAA) was prepared as described by Inman and Dinitzis [6].

FCS HBSS PFC

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f o e t a l calf s e r u m . H a n k ' s b a l a n c e d salt s o l u t i o n . p l a q u e - f o r m i n g cell.

SIRS = SRBC = TNP-PAA =

soluble i m m u n e response supressor. s h e e p r e d b l o o d cell. trinitrophenyl-polyacrylamide.

CA T A L A S E I N R E S T O R A T I O N OF A N T I B O D Y R E S P O N S E

57

Immunization.

Infected and control mice were immunized intravenously with 0.2 ml of a 10 % SRBC suspension. After 4 days (unless otherwise stated), mice were sacrificed. Spleen cell suspensions were prepared, washed twice in Hank's balanced salt solution (HBSS) and resuspended in HBSS. The number of direct plaque-forming cells (PFC) was determined by the Jerne plaque assay [7]. In vitro antibody response.

Spleen cells from infected mice and from age-matched controls (3 mice/group) were dissociated, washed twice in HBSS and resuspended in RPMI-1640 medium (Biomerieux, Marcy-l'Etoile, France) supplemented with 2 mM glutamine, 100 U of penicillin/ml, 25 ~tg of gentamicin/ml, 5 x 10-5 M 2-mercaptoethanol, 0.5 mM sodium pyruvate and 10 % heat-inactivated foetal calf serum (FCS) (M.A. Bioproducts, Walkersville, MD). Spleen cells were cultured by the method of Mishell and Dutton [12]. Cell suspensions (10 x 106 cells/ml) were incubated in 0.5-ml volumes in 24-well microplates (Falcon 3008, Beckton-Dickinson, Grenoble, France) and stimulated with 50 I~1SRBC (3 × 106) or 50 I~l TNP-PAA (5 x 103 beads). Cells were harvested at various times and the anti-SRBC PFC assay was performed as described in the pre~,eding section. TNP-specific PFC responses were meashred using TNP-coupled SRBC as target cells. TNP-SRBC were prepared according to the Rittenberg and Pratt method [20]. Results were expressed as the mean number of PFC per 106 cells + standard deviation (SD) of triplicate cultures. Cell fraetionation.

Preliminary experiments showed that spleen cells from infected mice contained a higher proportion of adherent cells than spleen cells from normal mice. 1-o obtain a similar number of adherent cells, spleen cell suspensions were adjusted to 10 × 106 cells/ml for normal mice and to 5 x 106 cells/ml for infected mice, in RPMI 1640 medium supplemented with 10 % FCS. Cell suspensions were seeded under a volume of 1 ml in 24-well microplates and incubated for 2 h at 37°C in a 5 % CO2 atmosphere. Non-adherent cells were then poured off and the wells washed four times with HBSS. Under these conditions, approximately 2.5 x 105 adherent cells remained in each well, as shown by microscopic examination with an ocular grid. Non-adherent spleen cells were obtained after depletion of adherent cells by incubating 5 ml of spleen cell suspension (10x 106 cells/mi) for 2 h at 37°C in 25 cm2 plastic flasks (Falcon). T-cell-depleted populations were obtained by selective cytolysis of T lymphocytes with monoclonal IgM anti-Thy1.2 antibody (F7D5, Serotec, Bicester, Oxon, England) plus complement (Low-Tox rabbit complement, Cedarlane Laboratories, Hornby, Ontario, Canada). Spleen cells (10x 106/ml) were incubated with a 1/2,000 dilution of anti-Thyl.2 antibody for 30 min at room temperature. The cells were then pelleted by centrifugation, suspended in 1/10 diluted complement, incubated for an additional 45 min at 37°C, and washed 3 times in culture medium. T-cell-depleted fractions obtained from control mice by this method retained full responsiveness to lipopolysaccharide stimulation, but had lost their ability to respond to concanavalin A. B-cell-depleted populations were recovered from plastic flasks coated with sheep anti-mouse-IgG antibodies (Biosys, Compi6gne, France) according to the method of

M. DESCHENES A N D COLL.

58

Wysocki and Sato [.22]. Briefly, spleen cells (10x 106/ml) were incubated for l h at room temperature m coated flasks. Non-adherent cells were then pelleted by centrifugation and resuspended in culture medium. These cells contained less than 2 °70 Ig-positive cells. Reagents.

A stock solution of 10 mg/ml of indomethacin (Sigma Chemical Co., St. Louis, MO) in 95 o7,0ethanol was prepared. Further dilutions were made in culture medium. The final concentration of ethanol in cell cultures did not exceed 0.1 °7/0. Control experiments showed that this concentration did not modify the antibody response of spleen cells. Cycloheximide and catalase (bovine liver, purified powder, 11,000 units/mg protein, thymol-free) purchased from Sigma, were dissolved in tissue culture medium. Statistical analysis. The statistical significance (P < 0.05) of the difference between infected and normal mice was evaluated by Student's unpaired t test.

RESULTS E f f e c t o f S. typhimurium i n f e c t i o n o n a n t i - S R B C r e s p o n s e in vivo.

C 5 7 B L / 6 mice were inoculated intravenously with 106 S. typhimurium C5TS and immunized at various times with SRBC. The number o f direct P F C in their spleens was determined 4 days later and c o m p a r e d to the response o f uninfected mice. A m a r k e d depression o f the antibody response (expressed either as P F C per 10 6 cells or as P F C per spleen) was present in infected sss,,,,~, i u

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TABLE I. - - Anti-SRBC response in vivo at various times after infection in C 5 7 B L / 6 mice.

Days after infection 5 10 17 21 37

PFC/106 spleen cells +- SD Control Infected (*) 369+_ 100 570+_160 497+_ 91 439+_ 120 391 +- 165

248 +__67 (67) 107+_43(19) 25_____14(5) 19+_ 15 (4) 24 +- 8 (6)

PFC/spleen +_SD Control Infected 37,100+_11,405 54,150+_10,200 55,912+10,237 36,217 + 9,900 41,055 + 14,975

46,176+ 14,785 (124) 15,515+ 6,235 (28) 4,625 +- 2,590 (8) 2,850+__ 2,250(8) 3,480+_ 232(8)

Results are the mean +_SD o f 3 mice/group. Numbers in brackets are the per cent response of infected mice relative to controls. (*) M,_'ce were infected i.v. with 10 6 CSTS, and immunized with 10 8 SRBC at indicated times. The number of P F C was determined 4 days after immunization.

CA TALASE IN RESTORATION

OF ANTIBODY

RESPONSE

59

difference between infected and normal mice was not related to a delay in the response of infected mice, since the response of infected mice remained lower than in controls when measured at day 5 or 6 after immunization (data not shown).

Effect of S. typhimurium infection on in vitro antibody response. The ability of spleen cells from infected mice to mount an antibody response in vitro was investigated. A strong depression of in vitro antibody response was found from 2 weeks up to at least 9 weeks post-infection (data not shown). Further experiments were carried out 3 weeks after infection when the response of infected mice was approximately 10 % of the controls. The response to a T-independent antigen, T N P - P A A , was reduced by as much as the response to SRBC (table II). Varying the duration of in vitro immunization did not restore the antiSRBC response of infected mice relative to control values (fig. 1).

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FIG. 1. -- Kinetics of in vitro anti-SRBC response in normal C57BL/6 mice (0) and mice infected 3 weeks prior to assay (®). Spleen cell cultures were immunized with 3 x 106 SRBC. Each point represents the mean of triplicate cultures of pooled splenocytes from 3 mice/group.

M. D E S C H E N E S A N D COLL.

60 TABLE II. - - I n

vitro antibody response against SRBC and T N P - P A A in infected C 5 7 B L / 6 mice.

Mice

PFC/106 cells _+SD Anti-SRBC Anti-TNP-PAA

Normal Infected (*)

2,667 + 401 320_+ 180

2,42 ! + 262 152_+ 20

Pooled spleen cells from 3 mice were immunized with SRBC or T N P - P A A (see ~ Materials and Methods ~) and the number of PFC was determined after 4 days' culture with SRBC and 5 days' with TNP-PAA. (*) Mice infected 3 weeks prior to assay with 106 C5TS.

E v i d e n c e o f s u p p r e s s o r cells in i n f e c t e d m i c e .

T o determine w h e t h e r suppression mechanisms were involved, various a m o u n t s o f spleen cells f r o m infected mice were a d d e d to a c o n s t a n t n u m b e r (4 x 106) o f spleen cells f r o m controls a n d stimulated with S R B C . As s h o w n in table III, the a d d i t i o n o f 20 °70 o f cells f r o m infected mice reduced the response o f n o r m a l spleen cells to 44 070 that o f the controls. 'When 50 070 o f spleen cells from infected mice were a d d e d , the a n t i - S R B C response was approximately 15 070 t h a t o f the control. T o assess the potential role o f T cells in suppression, spleen cells f r o m infected mice were treated with a n t i - T h y l . 2 a n t i b o d y plus c o m p l e m e n t . Such

TABLE III. - - Cell-mixing experiments. Cell source Normal

infected (*)

Anti-SRBC PFC/106 spleen cells-± SD

4 × I06 5 × 106 6 x l06 8 x 106 --

---~ 4x106 5 x 106 6 x 106 8X 106 1 × 106 2 x 106 4 x 106

3,347 + 1295 2,906+ 178 2,221 + 419 1,841+ 154 159_+ 27 229_+ 70 240_+ 15 385_+ 57 1,259_+ 154 720_+ 150 252_+ 27

-4 x 106 4 x 106 4 x 106

Spleen cells were immunized with 3 x 106 SRBC. The number of PFC was determined after 4 days' culture. (*) Mice infected 3 weeks prior to assay with 106 C5TS.

CA T A L A S E I N R E S T O R A T I O N OF A N T I B O D Y RESPONSE

61

TABLE IV. - - Evidence that suppressor cells were not B cells.

Mice

Cells added to normal cells (*)

PFC/106 cells +_SD

Unfractionated Unfractionated B-cell-depleted B-cell-depleted

1,931 + 225 425 + 48 1,142 + 76 331 + 51

Normal Infected (**) Normal Infected

(*) Thirty per cent of unfractionated or B-cell-depleted spleen cells from normal or infected mice were mixed with 70 % of normal spleen cells and incubated for 4 days with SRBC. (**) Mice were infected 3 weeks previously.

treated cells were found to be as suppressive as untreated cells (data not shown). This indicates that suppressor cells are not T cells. W h e n B cells were depleted f r o m spleen cells o f infected mice, suppressive activity was not reduced, suggesting that B cells were not involved in suppression either (table IV).

S u p p r e s s i v e activity o f a d h e r e n t cells.

To determine the cell population responsible for the inability of spleen cells from infected mice to m o u n t a n o r m a l antibody response, the functional activity o f adherent and n o n - a d h e r e n t cell populations was studied. Table V

TABLE V.

-

-

Functional activity of splenic adherent and non-adherent cells from

S. typhimurium-infected C 5 7 B L / 6 mice and effect of anti-Thyl.2 treatment. Adherent

Source of spleen cells (*) Non-adherent

Normal -Infected (**) -Normal Normal Infected Infected Normal + anti-Thy1.2 + C Infected + anti-Thyl.2 + C Normal unfractionated Infected unfractionated

m Normal -Infected Normal Infected Normal Infected Normal Normal

Anti-SRBC PFC/106 cells _+SD 0 235 ___61 0 716 + 139 4,131 + 576 5,161 + 460 857 +_ 25 822 + 214 4,063 + 968 419 + 146 2,395 +_588 812 + 166

(*) Non-adherent spleen cells (5 x 106) were mixed with 5 % adherent cells and incubated for 4 days with 3 x l0 6 SRBC. (**) Infected 3 weeks prior to assay with l0 6 C5TS.

62

M. DESCHENES A N D COLL. TABLE VI. - - Effect of cycloheximide and indomethacin on adherent cell suppressive activity.

Treatment (**)

Concentration

None C~ycloheximide

-2 t~g/ml 5 ~g/ml 2 l~g/ml 5 l~g/ml

Indomethacin

Anti-SRBC PFC/106 cells +_SD of normal non-adherent cells (*) mixed with: normal adherent cells infected adherent cells 1,525 +_13 1,218 +_193 1,199+_ 147 1,463 +_23 2,323 _+51

241 _+59 605 +_65 851 +_50 153 +_50 177 +- 10

(*) Non-adherent spleen cells from normal mice (5 x 106) were mixed with 5 o70 adherent cells from normal mice or mice infected 3 weeks previously and incubated for 4 days with 3 x 106 SRBC. (**) Cycloheximide was added during cell adhesion and adherent cells were washed before the addition of non-adherent cells and SRBC. Indomethacin was added at the initiation of in vitro immunization.

shows that adherent and non-adherent cells were unable separately to mount a normal anti-SRBC response. Non-adherent spleen cells from infected mice, mixed with 5 °70 of normal adherent cells displayed a normal anti-SRBC response. The same number of adherent cells from infected mice did not enable the development of an anti-SRBC response when mixed with normal nonadherent cells. These data suggest that suppression of the anti-SRBC response in infected C57BL/6 mice is associated with adherent cells. Treatment of adherent spleen cells from infected mice with anti-Thy1.2 antibody and complement did not inhibit their suppressive activity, indicating that this activity is not mediated by adherent T cells. To analyse the mechanism by which adherent cells exert their suppressive activity, the effects of cycloheximide, indomethacin and catalase were studied. Table VI shows that pretreatment of adherent cells with cycloheximide significantly reduced their suppressive effect, indicating that protein synthesis is required for adherent cells to exert their suppressive effect. The addition of indomethacin, a potent inhibitor of prostaglandin production, did not affect the suppressive activity of adherent cells from infected mice. In contrast, the addition of catalase, which inactivates hydrogen peroxide, abolished the suppressive activity of adherent cells from infected mice (table VII). The effect of catalase was totally lost after heating at 100°C for 5 min (data not shown). Catalase also enhanced the response of cells from control mice but to a lesser extent.

DISCUSSION

The data presented here extend previous findings concerning the suppression of immune responses induced by S. typhimurium infection [3, 15]. Infected C57BL/6 mice exhibited a pronounced depression of in vivo primary

CA TALASE IN RESTORATION OF ANTIBODY RESPONSE TABLE VII. - - E f f e t o f catalase o n adherent cell suppressive

Catalase (**) 5,000 U/ml

63

activity.

Anti-SRBC PFC/106 cells_+SD of normal non-adherent cells (*) mixed with: normal adherent cells

infected adherent cells

1 525 + 13 4445 + 169

241 + 59 2043 + 558

4 0 7 0 + 219 6 659 _+223

829 _+86 4 089 + 434

Exp. 1 + Exp. 2 +

(*) Non-adherent spleen cells from normal mice (5 x 106) were m ~ e d with 5 07,0adherent cells from normal mice or mice infected 3 weeks previously and incubated for 4 days with 3 x 106 SRBC. (**) Catalase was added at the initiation of in vitro immunization.

antibody response to SRBC. In vitro primary antibody responses to SRBC and TNP-PAA, a T-independent antigen, were also depressed. The presence of suppressor cells in the spleens of infected animals was demonstrated by cell-mixing experiments. Suppression was not mediated by either T cells or B cells since treatment by anti-Thy1.2 plus complement and B cell depletion did not affect suppressive activity. Suppressor cells were present in the adherent cell fraction and were presumably macrophages. Non-adherent cells from infected mice were able to mount a normal anti-SRBC response when mixed with normal adherent cells. Taken together these results indicate that depression of antibody response was essentially mediated by adherent suppressor cells. Adherent suppressor ceils have previously been found to depress the lymphocyte responses to mitogens in S. typhimurium infection [3, 10] and in other experimental infections [8, 9, 13]. Suppression of antibody response by adherent suppressor cells has also been reported in Mycobacterium lepraemurium [2], Histoplasma capsulatum [17] and Toxoplasma gondii [21] infections. The mechanism of action of adherent suppressor cells in S. typhimuriuminfected mice was investigated. These cells needed to oe metabollicaly active, since cycloheximide treatment inhibited their suppressive activity. The addition of indomethacin failed to inhibit their activity indicating that prostaglandins were not involved in immunosuppression. A major finding of the present study was that catalase abolished the suppressive activity. This suggests that the production of hydrogen peroxide by adherent cells plays a major role in immunosuppression. Metzger et al. [11] have shown that hydrogen peroxide together with prostaglandins participate in the macrophage-mediated suppression of lymphocyte responses to mitogens. The mechanism by which hydrogen peroxide induces immunosuppression is not known. Hydrogen peroxide can activate a soluble immune response

64

!~t. D E S C H E N E S A N D COLL.

suppressor (SIRS) produced by T cells [1]. In S. typhimurium-induced suppression, SIRS does not seem to be involved, since suppressive activity was not present in either T cells or in cell culture supernatants (unpublished results). Hydrogen peroxide is an important product of activated macrophages which is involved in their microbicidal and tumouricidal activities [14]. Previous studies have shown that susceptible C57BL/6 mice infected with S. typhimurium C5TS develop resistance to reinfection with S. tyohimurium or Listeria monocytogenes [16]. The simultaneous occurence during infection of increased resistance and immunodepression has been reported by other authors and has been ascribed to macrophage activation [8, 10]. The present study suggests that the production of hydrogen peroxide by activated macrophages may be involved in this phenomenon.

RI~SUMI~ SUPPRESSION DE LA RI~PONSE PRIMAIRE ANTICORPS CHEZ DES SOURIS D'UNE LIGNITE SENSIBLE, INFECTi~ES PAR SALMONELLA TYPHIMURIUM: RESTAURATION PAR LA CATALASE

La lign6e sensible C57BL/6 infect6e par un mutant thermosensible de Salmonella typhimurium pr6sente pendant plusieurs semaines une profonde d6pression de la r6ponse anticorps contre les globules rouges de mouton, in vivo et in vitro. La r6ponse anticorps contre un antig6ne thymo-ind6pendant (TNP-polyacrylamide) est 6galement affect6e. Les exp6riences de cocultures montrent que les rates de souris infect6es contiennent des cellules suppressives adh6rentes et que l'activit6 fonctionnelle des lymphocytes Bet T n'est pas touch6¢. La r6ponse anticorps des cellules spl6niques des souris infect6es est restaur6e par l'addition de catalase, ce qui sugg6re que l'eau oxyg6n6e joue un r6!e dans la suppression. MOTS-CLFS" Salmonella typhimurium, lmmunor6gulation. Macrophage, Catalase;

ACKNOWLEDGEMENTS

M. Desch~nes was in receipt of a grant from the Minist6re de la Recherche (UA CNRS 040622).

REFERENCES

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[4] HORMAECHE,C.E., Natural resistance to Salmonella typhimurium in different inbred mouse strains. Immunology, 1979, 37, 311-318. [5] HORMAECHE,C.E., PETTIFOR, R.A. & dROCK, K.J., The fate of temperaturesensitive Salmonella mutants in vivo in naturally resistant and susceptible mice. bnmunology, 1981, 42, 569-576. [6] INMAN, J.K. & DINITZIS, H.M., The derivatization of cross-linked polyacrylamide beads: controlled introduction of functional groups for the preparation of special purpose biochemical absorbants. Biochemistry, 1969, 8, 4074-4082. [7] JERNE, N.K. & NORDIN, A.A., Plaque formation in agar by single antibodyproducing cells. Science, 1963, 110, 405. [8] JERRELLS, T.R., Immunosuppression associated with the development of chronic infections with Rickettsia tsutsugamushi: adherent suppressor cell activity and macrophage activation. Infect. Immun., 1985, 50, 175-182. [9] KIERSZENBAUM,F., Immunologic deficiency during experimental Chagas' disease (Trypanosoma cruzi infection): role of adherent, nonspecific esterase-positive splenic cells. J. ImmunoL, 1982, 129, 2202-2205. [10] LEE, J.C., GmSON, C.W. & ElSENSXEIN,T.K., Macrophage-mediated mitogenic suppression induced in mice of the C3H lineage by a vaccine strain of Salmonella typhimurium. Cell. lmmunol., 1985, 91, 75-91. [11] METZGER,Z., HOFFELD,J.T. & OPPENHEIM, J.J., Macrophage-mediated suppression, n I. Evidence for participation of both hydrogen peroxide and prostaglandins in suppression of murine lymphocyte proliferation. J. Immunol., 1980, 124, 983-988. [12] MISHELL, R.I. & DUTTON, R.W., Immunization of dissociated spleen cell cultures from normal mice. J. exp. Med., 1967, 126, 423-442. [13] MURRAY,H.W., CARRIER'9,S.M. & DONELLY,D.M., Presence of a macrophagemediated suppressor cell mechanism during cell-mediated immune response in experimental leishmaniasis. Infect. Immun., 1986, 54, 487-493. [14] NATHAN,C.F., Secretory products of macrophages. J. din. Invest., 1987, 79, 319-326. [15] NAUCIEL, C., RONCO, E. & GUENET, J.L., Genetic control of Salmonella typhimurium-induced depression of delayed-type hypersensitivity to sheep erythrocytes in mice. Infect. Immun., ! 988, 56, 310-313. [llgl lMA,,r.,t:, C , VILDE, F. ~. RONCO, E., Host response to infection with a temperature-sensitive mutant of Salmonella typhimurium in a susceptible and a resistant strain of mice. Infect. Immun., 1985, 49, 523-527. [17] NICKERSON, D.A., HAVENS, R.A. & BULLOCK,W.E., Immunoregulation in disseminated histoplasmosis: characterization of splenic suppressor cell populations. Cell. Immunol., 1981, 60, 287-297. [18] O'BRIEN, A.D., TAYLOR, B.A. & ROSENSTREICH,D.H., Genetic control of natural resistance to Salmonella typhimurium in mice during the late phase of infection. J. Immunol., 1984, 133, 3313-3318. [19] PLANT,J. cg~GLYNN,A.A., Genetics of resistance to infection with Salmonella typhimurium in mice. J. infect. Dis., 1976, 133, 72-78. [20] RITTENBERG,M.B. & PRATT, K.L., Anti-nitrophenyl (TNP) plaque assay. Primary response of BALB/c mice to soluble and particular immunogen. Proc. Soc. exp. Biol. (N.Y.), 1969, 132, 575-581. [21] SUZUKI, Y., WATANABE,N. • KOBAYASHI,A., Nonspecific suppression of primary antibody responses and presence of plastic adherent suppressor cells in Toxoplasma gondii-infected mice. Infect. Immun., 1981, 34, 30-35. [22] WYSOCKI,L.J. & SATO, V.L., ~ Panning>> for lymphocytes: a method for cell separation. Proc. nat. Acad. Sci. (Wash.), 1978, 75, 2844-2848. IL A ~ . r j

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