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Exogenous interleukin-2 abrogates differences in the proliferative responses to T cell mitogens among inbred strains of mice
CELLULAR
IMMUNOLOGY
142, 177-l 85 ( 1992)
Exogenous Interleukin-2 Abrogates Differences in the Proliferative Responses to T Cell Mitogens among inbred Strains of Mice MARIE LIPOLDOVA, ALENA ZAJ~COVA, JAROSLAV ST~DRA, AND VLADIMIR Ho~Afi Institute of‘ Molecular Genetics, Czechoslovak Academy @“Sciences, Flemingovo n&n. 2, 166 37 Prague 6, Czechoslovakia Received January 24, 1992; accepted February 25, 1992
The proliferative response of spleen cells from BALB/c mice to stimulation with a T cell mitogen, concanavalin A (Con A), was two or more times stronger than that of cells from C57BL/ 1OSnSc(BlO) mice. In contrast, the cells from BlO mice responded better to B cell mitogen bacterial lipopolysaccharide (LPS). The differences in the proliferative responseto Con A stimulation were not associatedwith the function of macrophagesnor did they depend on IL- 1. Spleen cells from BALB/c and B 10 mice synthesized comparable amounts of mRNA for IL- 1LY,and the production of biologically active IL-l was even higher in the BlO strain. Indomethacin, an inhibitor of prostaglandin synthesis, had no effect on the differences in reactivity between the cells from BALB/c and BlO mice. In addition, no differences in the synthesis of mRNA for the inducible 55-kDa interleukin-2 (IL-2) receptors were found between the spleen ceils from BALB/c and BlO mice. However, Con A-stimulated spleen cells from BlO mice produced a significantly lower amount of biologically active IL-2 than similarly stimulated cells from BALB/c mice. In the presence of exogenous IL-2, these low responder spleen cells from the BlO mice responded by proliferation to Con A stimulation to the same extent as cells from the BALB/c mice. These results thus show that a low proliferative response to Con A stimulation in BlO mice was a consequenceof a lower production of IL-2 and possibly abrogated the proliferative hyporeactivity produced by exogenous IL-2. We suggestthat the differencesin the ability to produce IL-2 could be a reason for the discrepanciesobserved in the immunological responsivenessbetween BALB/ c and BlO mice. 0 1992 Academic PESS. I~C.
INTRODUCTION Considerable differences exist among individual lines and inbred strains of mice in their ability to respond upon antigenic challenge ( l-6). The strains responding weakly by antibody production to one antigen can, however, respond quite well to other antigens or can resist dosesof pathogens that are lethal to high responder strains (79). The reasons for these differences are not known in most cases,and many factors have been considered responsible for them. Thus far, differences in T and B cell activity, different handling of antigens by macrophages, and/or various degrees of suppressorcell activation have all been considered as possible causesof these discrepancies( lo- 14). Immunogenetic studies using recombinant and congeneicmouse strains revealed that more individual loci, both within the major histocompatibility complex (MHC) and outside it, influence the high or low responder phenotype of particular strains (15, 16). 177 0008-8749/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form resewed.
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We analyzed the differences in the proliferative responsivenessin cells from BALB/ and C57BL/lOSnSc (BlO) mice in vitro. The proliferative response of spleen cells from BALB/c mice to stimulation with the T cell mitogen concanavalin A (Con A) was two or more times stronger than that of similarly stimulated cells from B10 mice, whereas the cells from BlO mice responded better to B cell mitogen bacterial lipopolysaccharide (LPS). No significant role for macrophagesor IL- 1 in the determination of a high or low responder phenotype was found in this model. However, the low response of the spleen cells from B 10 mice to T cell mitogen was associated with a lower production of IL-2 by mitogen-stimulated BlO cells. Indeed, exogenous IL-2 increased the proliferative response of Con A-stimulated BIO cells to the level of the cells from BALB/c mice. The results thus suggestthat the lower production of IL-2 by BlO cells might be a reason for the poor response of the BlO cells to Con A and for the generally weak reactivity of BlO mice in T-cell-dependent immune responses (2, 3). c
MATERIALS AND METHODS Animals. Mice of the inbred strains BALB/c and BlO of both sexesat 7-10 weeks of age were obtained from the breeding colony of our Institute. Reagents. Con A and indomethacin were obtained from Sigma Chemical Co. (St. Louis, MO). LPS isolated from Escherichia coli 055:B5 was purchased from Difco Laboratories (Detroit, MI). Recombinant human IL-la (rIL-101, sp act, 1 X lo8 U/ mg) expressedin E. coli (17) was obtained from Dr. P. T. Lomedico, Hoffman-LaRoche Inc. (Nutley, NJ). Human recombinant IL2 (sp act, 2 X lo6 U/ml) was kindly provided by Dr. E. Gren, Institute of Organic Synthesis (Riga, Latvia). Proliferative assays.Spleencells at a concentration 2 X lo5 cells/well were stimulated in a volume of 0.2 ml RPM1 1640 medium supplemented with antibiotics (penicillin, streptomycin), glutamine, 10 mM Hepes buffer, 5 X 10m5M 2-mercaptoethanol, and 10% fetal calf serum (complete RPM1 1640 medium) in 96-well flat-bottom tissue culture plates (Sterilin Ltd., Feltham, UK). Con A (5 pg/ml), LPS (40 pg/ml) or indomethacin ( lop5 M), and/or interleukins (at the concentrations given below) were added to the cultures. The cultures were incubated at 37°C in a humidified atmosphere of 5% CO2 for 72 hr, and [3H]thymidine (sp act, 22.8 Ci/mmol) at a concentration of 0.5 pCi/well was added to the cultures for the last 6 hr of the incubation period to determine cell proliferation ( 18). Production of IL-l and assay systemfor IL-1 activity. Peritoneal exudate cells (PEC), obtained by washing the peritoneal cavity of individual BALB/c and B 10 mice, were left to adhere to plastic dishes for 2 hr. The nonadherent cells were removed and the adherent cells were collected, adjusted to a concentration of 1 X lo6 cells/ml, and incubated in complete RPM1 1640 medium with 10 pg/ml of LPS and lop5 M indomethacin. After 24 hr of incubation the supernatants obtained were tested for IL1 activity in the classical thymocyte costimulatory assay (19) as we have described elsewhere (20). Production ofIL-2 and assay systemfor IL-2 activity. Spleen cells at a concentration of 5 X lo6 cells/ml were incubated in 1 ml of complete RPM1 1640 medium in 24well tissue culture plates (Linbro, Flow Laboratories Inc., McLean, VA) in the presence of 5 pg/ml Con A. After a 24-hr incubation, the supematants were harvested and assessedfor the ability to support the growth of the IL-2-dependent CTLL-2 cell line (21).
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Separation of cell populations. Macrophage-rich cell populations were obtained by washing the peritoneal cavity of the mice and by using adherence of the cells to the plastic petri dishes. After each of two 2-hr adherence procedures the nonadherent cells were removed and the adherent cells were collected. T-cell-enriched cell populations were obtained by passaging of spleen cells through a nylon-wool column according to Julius et al. (22). The nylon-wool nonadherent cells were cultured on petri dishes coated with swine anti-mouse immunoglobulin antibodies to remove the remaining immunoglobulin-positive cells using the panning technique of Wysocki and Sato (23). Northern blot analysis. Total RNA was isolated using the guanidine isothiocyanate method (24) from 2 X 10’ spleen cells which were either unstimulated or stimulated for 16 hr with 5 pg/ml Con A or 20 pg/ml LPS. The RNA was subjected to electrophoresis, blotted onto Hybond-N nitrocellulose membranes (Amersham International, Amersham, UK), and hybridized with a 32P-labeled IL-2 receptor (IL-2R) probe (25) or an IL-la probe (17) as we have described elsewhere (26). The blots were then stripped and rehybridized with a cDNA probe for actin to demonstrate comparable quantities of RNAs in individual lanes. RESULTS
Reactivity of Spleen Cells,fiom BALB/c and BIO Mice to Mitogens The proliferative reactivity of spleen cells from BALB/c mice to stimulation with Con A was two or more times stronger than that of spleen cells from BlO mice (Fig. 1). In contrast, spleen cells from BlO mice responded better to LPS stimulation than
- ConA BALBk
LPS cells
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ConA LPS B 10 cells
FIG. I. Proliferative responses of spleen cells from BALB/c and B IO mice to stimulation with Con A or LPS. Spleen cells (1 X lo6 cells/ml) from BALB/c and BlO mice were stimulated with 5 Kg/ml Con A or 40 pg/ml LPS, and cell proliferation was determined by the addition of [3H]thymidine into the cell cultures for the last 6 hr of the 72-hr incubation period.
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cells from the BALB/c donors (Fig. 1). These patterns of reactivity were not dependent on mitogen concentrations, concentrations of cells per milliliter, or incubation intervals (data not shown). Discrepanciesin the Proliferative Activity Do Not Depend on the Activity ofkfacrophages or IL-l Since the proliferative responsesof T cells to stimulation with Con A depend on the presence of IL-l and macrophages (or other antigen-presenting cells), we first compared the production of IL- 1 by cells from BALB/c and B 10 mice. As shown in Fig. 2, unstimulated or LPS-stimulated cells from BALB/c mice synthesized comparable or even higher levels of mRNA for IL- 1CY than the cells from B 10 mice. However, LPS-stimulated macrophages from the B 10 mice produced slightly more biologically active IL-l than similarly stimulated macrophages from the BALB/c mice (Fig. 3). When purified T cells from BALB/c and B 10 mice were stimulated with Con A in the presence of purified BALB/c or BlO macrophages, both types of macrophages stimulated proliferative responsesin T cells to the same degree(Fig. 4). However, the proliferative responsesof the T cells from BALB/c mice were invariably higher than the responsesof the T cells from BlO mice, regardless of which type of macrophage was used (Fig. 4). Spleen Cellsfrom BALB/c and BlO Mice Synthesize Comparable Quantities of mRNA for IL-2R Spleen cells from BALB/c and BlO mice were stimulated with Con A, and total RNA was isolated. The RNA was hybridized with an IL-2R probe. As shown in Fig. 5a, Con A-stimulated cells from both strains of mice synthesizedcomparable quantities of IL-2R mRNA, which was hybridized as five bands corresponding to different types of RNA coding for the 55kDa chain of IL-2R (27). After hybridization with the IL2R probe, the blots were stripped and rehybridized with an actin probe to demonstrate that there were comparable amounts of RNA in individual lanes (Fig. 5b).
a) IL- la
b) Actin
FIG. 2. Synthesis of mRNA for IL- 101in spleen cells from BALB/c and B 10 mice. Spleen cells from B IO (lanes 1 and 3) or BALB/c (lanes 2 and 4) mice were cultured for 16 hr unstimulated (lanes I and 2) or stimulated with 20 &ml LPS (lanes 3 and 4). Total RNA was isolated and hybridized (a) with an IL-la probe or (b) after stripping with an actin probe.
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FIG. 3. Production of biologically active IL-1 by PEC from BALB/c and BlO mice. PEC from two individual BALB/c (0) and BIO (0) mice were incubated for 24 hr with LPS (20 &ml) and indomethacin (lo-’ M) and the supernatants obtained were tested in a thymocyte costimulatory assay for their ability to support the proliferation of Con A-stimulated thymocytes.
Exogenous IL-2 Abrogates Discrepancies in Con A-Induced Cell Proliferation Spleen cells from B 10 mice responded by a significantly weaker proliferative reaction to Con A stimulation than spleen cells from BALB/c mice. This lower reactivity of B 10 cells was not enhanced by exogenous IL- 1 or by indomethacin, an inhibitor of prostaglandin synthesis (Fig. 6). However, exogenous IL-2, added to the cultures of Con A-stimulated spleen cells from BALB/c and B 10 mice, enhanced the proliferation of BlO cells in such a way that the cells from both strains of mice reacted to Con A with comparable proliferative responses (Fig. 6).
BALB/c Spleen Cells Produced More Biologically Active IL-2 than BlO Cells The results in Fig. 6 suggest that the shortage of IL-2 in BlO cell cultures might be a reason for the lower reactivity of B 10 spleen cells to Con A stimulation. To test this possibility, spleen cells from BALB/c and BlO mice were stimulated with Con A for 24 hr, and the supernatants obtained were tested for their ability to support the growth of IL-2-dependent CTLL-2 cells. It is shown in Fig. 7 that Con A-stimulated sp!een cells from BALB/c mice produced considerably more biologically active IL-2 than similarly stimulated cells from B 10 mice. DISCUSSION Differences among individual inbred strains or lines of mice in the ability to mount antibody responses after antigenic challenge in vivo ( l-6) or to resist parasite infections (9,28-30) have been well documented. In the present study we have concentrated on
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BALB/c 1 cells
FIG. 4. Effectsof purified BALB/c and B 10 macrophageson the proliferative responseof Con A-stimulated T cells. Purified T cells from BALB/c and BIO mice were cultured unstimulated (Cl), stimulated with 5 pg/ ml Con A alone @I), or with Con A in the presence of BALB/c (H) or BlO (H) macrophages (the T cell: macrophage ratio was 10:I).
another aspectof immunological responsiveness,namely proliferative responseto mitogenic stimulation in vitro. We have found that the response of spleen cells from BALB/c mice to stimulation with a T cell mitogen, Con A, was two or more times stronger than that of similarly stimulated spleen cells from B 10 mice. In contrast, spleen cells from B 10 mice responded comparably or even better than cells from BALB/c mice to stimulation with LPS. The observed higher proliferative response of spleen cells from BALB/c mice to T cell mitogen stimulation in vitro correlates with the stronger responsiveness of BALB/c mice than of B 10 mice to T cell-dependent antigens in vivo (3 1). We have focused on elucidating cellular and molecular mechanisms responsible for the observed differences. It has been demonstrated in the model of antibody production in high and low responder strains of mice (lo- 12) and rats (32) that the differences in antigen handling and presentation were the causesof discrepancies in immune responsiveness. We therefore first elucidated the functions of macrophages in BALB/c and B 10 mice. It was found that spleen cells, both unstimulated and LPS-stimulated, from BALB/c mice synthesized a comparable or even higher level of mRNA for IL-la than did spleen cells from BlO mice. However, the production of biologically active IL-1 was higher in BlO mice. Similar discrepancies between the levels of specific mRNA and the particular interleukin production have been described as a result of different regulations at the translation level (33, 34). Also, our other experiments, in which we utilized combinations of purified populations of T cells and macrophages, suggested that the macrophageswere not the critical cell population responsible for the differences in proliferative responsesto Con A stimulation observed between cells from BALB/c and B 10 mice.
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4
a)lL-2R
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FIG. 5. Synthesis of mRNA for the inducible 55-kDa IL-2R in spleen cells from BALB/c and BIO mice. Spleen cells from BALB/c (lanes 1 and 3) or BlO (lanes 2 and 4) mice were cultured for 16 hr unstimulated (lanes I and 2) or stimulated with 5 pg/ml Con A (lanes 3 and 4). Total RNA was isolated and hybridized (a) with an IL-2R probe and (b) after stripping with an actin probe.
In the model of the Biozzi mice, Nakano et al. (13) showed that nonspecific suppressor cells were more numerous and active in low responder mice, and it was considered that these suppressor cells might play a role in the induction of low antibody response.We also tested the activity of nonspecific Con A-induced suppressorcells in our BALB/c and BlO mice, but their levels of activity were comparable (data not shown).
BALBlc
cells
B 10 cells
FIG. 6. Exogenous IL-2 abolished the discrepancies in Con A-induced proliferative responses between BALB/c and BlO cells. Spleen cells from BALB/c and BlO mice were cultured tutstimulated (I), stimulated with 5 pg/ml Con A alone (m) or with Con A in the presence of 100 U/ml rIL-la (IWI), 100 U/ml rIL-2 (a), or lo-‘M indomethacin (0).
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1:16 DiMon
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1:64
1:256
1:1024
of supernatants
FIG.7. Production of IL-2 by Con A-stimulated spleen cells from BALB/c and B 10 mice. Spleen cells (5 X lo6 cells/ml) from two individual BALB/c (0) or BlO (0) mice were stimulated for 24 hr with Con A (5 rg/ml) and the supernatants obtained were tested for their ability to support the growth of IL-2-dependent CTLL-2 cells.
Next we compared the activity of T cells. After stimulation with Con A, spleen cells from BALB/c and B 10 mice synthesized comparable quantities of mRNA for the inducible 55kDa IL-2R. However, we found that Con A-stimulated spleen cells from BALB/c mice produced significantly more biologically active IL-2 than similarly stimulated spleen cells from B 10 mice. To test the possibility that the lower IL-2 production by the B 10 cells could be a reason for their weaker proliferative responses,spleen cells from BALB/c and B 10 mice were stimulated with Con A in the presenceof exogenous IL-2. We found that IL-2 completely abrogated the discrepancies in proliferative responsesbetween cells from these two strains of mice. Con A-stimulated spleen cells from B 10 mice proliferated in the presence of exogenous IL-2 as did the cells from BALB/c mice. These observations suggestthat the shortage of IL-2 in the cultures of Con A-stimulated BlO cells is a reason for their lower proliferation. We prefer the idea that the shortage of IL-2 in the BlO cell cultures was caused by a lower IL-2 production by Con A-stimulated BlO cells rather than the possibility that it was due to the presence of some immunosuppressive molecules in the BlO supernatants. If the latter is the case, such inhibitory molecules should also inhibit the activity of exogenous IL-2. Experiments are now under way to determine the genetic background for the differences in IL-2 production between the BALB/c and B 10 strains of mice. In conclusion, the results showed lower proliferative responses of cells from B10 mice to Con A stimulation as a consequence of a lower production of IL-2. Since exogenous IL-2 can increase the low proliferative responsesin Con A-stimulated B 10 cells to level of that in BALB/c cells, we believe that the low production of IL-2 could be a reason for the weaker proliferative responsesof BlO cells to the T cell mitogen, Con A. It is suggested that the low production of IL-2 might also be a cause of
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the low antibody production to the T-cell-dependent antigens observed in BlO mice (2, 3). ACKNOWLEDGMENTS The authors thank Dr. P. T. Lomedico, Hoffmann-LaRoche Inc. (Nutley, NJ), for providing rlL-la and IL-1 (Yprobe, Dr. E. Gren, Institute of Organic Synthesis, Riga, Latvia, for the kind gift of rlL-2 and Dr. T. Honjo, Kyoto University, Japan, for providing the IL-2R probe. We are also grateful to Ms. E. FoitovL for expert technical work and Ms. L. Keleher for editing assistance.
REFERENCES 1. Biozzi, G., Asovsky, R., Lieberman, R., Stieffel, R., Mouton, D. and Benacerraf, B., J. Exp. Med. 132, 152, 1970. 2. Silver, D. M., McKenzie, 1. F. C., and Winn, H. J., J. Exp. Med. 136, 1036, 1972. 3. I?ihovP, B., and diha, I., Am. J. Reprod. Immunol. 1, 168, 1981. 4. Unanue, E. R., Biozzi, G., and Benacerraf, B., Proc. Sot. Exp. Biol. Med. 145, 1243, 1974. 5. Howard, J. G., Christie, G. H., Courtenay, B. M., and Biozzi, G., Eur. J. Irnmunol. 2, 269, 1972. 6. Liacopoulos-Briot, M., Bouthillier, Y., Mouton, D., Lambert, F., Decreusefond, C., Stiffel, C., and Biozzi, G., Transplantation 14, 590, 1972. 7. Howard, J. G., Courtenay, B. M., and Desaymard, C., Eur. J. Immunol. 4, 453, 1987. 8. Wiener, E., and Bandieri, A., Eur. J. Immunol. 4, 457, 1974. 9. Wu-Hsien, H., Infect. Immun. 57, 3788, 1989. 10. Doria, G., Agarossi, G., and Biozzi, G., Immunology 34, 999, 1978. 11. RihovL, B., Vttvicka, V., I&ha, I., and HolPH, V., Thymus 8, 16 1, 1986. 12. Adorini, L., and Doria, G., Eur. J. Immunol. 11,984, 1981. 13. Nakano, K., Cinader, B., and Sabiston, B. H., Eur. J. Immunol. 9, 1014, 1979. 14. Malek, T. R., Shevah, E. M., and Danis, K. M., J. Immunol. 143, 439, 1989. 15. Silver, D. M., and Winn, H. J., Cell. Immunol. 7, 237, 1973. 16. Fiengold, N., Fiengold, J., Mouton, D., Bouthillier, Y., Stiffel, C., and Biozzi, G., Eur. J. Immunol. 6, 43, 1976. 17. Lomedico, P. T., Gubler, U., Hellman, C. P., Dukovich, M., Giri, J. G., Pan, Y. C. E., Collier, K., Semionov, R., Chua, A. O., and Mizel, S. B., Nature 312, 458, 1984. 18. LipoldovB, M., Zajicovi, A., and HolBii, V., Immunology 71,497, 1990. 19. Mizel, S. B., Dukovitch, M., and Rothstein, J., J. Immunol. 131, 1834, 1983. 20. HolBfi, V.. and LipoldovB, M., Br. J. Cancer 61, 667, 1990. 21. Gillis, S., and Smith, K. A., Nature 268, 154, 1977. 22. Julius, M. H., Simpson, E., and Herzenberg, L. A., Eur. J. Immunol. 3, 645, 1973. 23. Wysocki, L. J., and Sato, V. L., Proc. Natl. Acad. Sci. USA 75, 2844, 1978. 24. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J., Biochemistry 18, 5294, 1979. 25. Shimizu, A., Kondo, S., Takeda, S.-l., Yodai, J., lshida, N., Sabe, H., Osawa, H., Diamantstein, T., Nikaido, T., and Honjo, T., Nucleic Acids Res. 13, 1505, 1985. 26. LipoldovL, M., Londei, M., Gruberk-Loebenstein, B., Feldman, M., and Owen, M. J.. J. Autoimmun. 2, 1, 1989. 27. Miller, J., Malek, T. R., Leonard, W. J., Greene, W. C., Shevach, E. M., and Germain, R. N., J. Immunol. 134,4212, 1985. 28. Cannat, A., Bousquet, C., and Serre, A., Ann. Immunol. (Paris) 129, 669, 1978. 29. Skamene, E., James, C. L., Meltzer, M. S., and Nesbitt, M. N., J. Leucocyte Biol. 35, 65, 1984. 30. Hale, C., and Howard, J. G., Parasite Immunol. 3, 45, 1981. 31. I%ihovl, B., VdtviEka, V., and Kincade, P. W., Immunopharmacology 15, 169, 1988. 32. Tada, N., ltakura, K., and Aizawa, M., J. Immunogenet. 3, 49, 1976. 33. Arend, W. P., Gordon, D. F., Janson, R. W., Joslin, F. G., and Jameel, S., J. Immunol. 143, 118, 1989. 34. Turner, M., Chantry, D., Buchan, G., Barett, K., and Feldman, M., J. Immunol. 143, 3556, 1989.
IMMUNOLOGY
142, 177-l 85 ( 1992)
Exogenous Interleukin-2 Abrogates Differences in the Proliferative Responses to T Cell Mitogens among inbred Strains of Mice MARIE LIPOLDOVA, ALENA ZAJ~COVA, JAROSLAV ST~DRA, AND VLADIMIR Ho~Afi Institute of‘ Molecular Genetics, Czechoslovak Academy @“Sciences, Flemingovo n&n. 2, 166 37 Prague 6, Czechoslovakia Received January 24, 1992; accepted February 25, 1992
The proliferative response of spleen cells from BALB/c mice to stimulation with a T cell mitogen, concanavalin A (Con A), was two or more times stronger than that of cells from C57BL/ 1OSnSc(BlO) mice. In contrast, the cells from BlO mice responded better to B cell mitogen bacterial lipopolysaccharide (LPS). The differences in the proliferative responseto Con A stimulation were not associatedwith the function of macrophagesnor did they depend on IL- 1. Spleen cells from BALB/c and B 10 mice synthesized comparable amounts of mRNA for IL- 1LY,and the production of biologically active IL-l was even higher in the BlO strain. Indomethacin, an inhibitor of prostaglandin synthesis, had no effect on the differences in reactivity between the cells from BALB/c and BlO mice. In addition, no differences in the synthesis of mRNA for the inducible 55-kDa interleukin-2 (IL-2) receptors were found between the spleen ceils from BALB/c and BlO mice. However, Con A-stimulated spleen cells from BlO mice produced a significantly lower amount of biologically active IL-2 than similarly stimulated cells from BALB/c mice. In the presence of exogenous IL-2, these low responder spleen cells from the BlO mice responded by proliferation to Con A stimulation to the same extent as cells from the BALB/c mice. These results thus show that a low proliferative response to Con A stimulation in BlO mice was a consequenceof a lower production of IL-2 and possibly abrogated the proliferative hyporeactivity produced by exogenous IL-2. We suggestthat the differencesin the ability to produce IL-2 could be a reason for the discrepanciesobserved in the immunological responsivenessbetween BALB/ c and BlO mice. 0 1992 Academic PESS. I~C.
INTRODUCTION Considerable differences exist among individual lines and inbred strains of mice in their ability to respond upon antigenic challenge ( l-6). The strains responding weakly by antibody production to one antigen can, however, respond quite well to other antigens or can resist dosesof pathogens that are lethal to high responder strains (79). The reasons for these differences are not known in most cases,and many factors have been considered responsible for them. Thus far, differences in T and B cell activity, different handling of antigens by macrophages, and/or various degrees of suppressorcell activation have all been considered as possible causesof these discrepancies( lo- 14). Immunogenetic studies using recombinant and congeneicmouse strains revealed that more individual loci, both within the major histocompatibility complex (MHC) and outside it, influence the high or low responder phenotype of particular strains (15, 16). 177 0008-8749/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form resewed.
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We analyzed the differences in the proliferative responsivenessin cells from BALB/ and C57BL/lOSnSc (BlO) mice in vitro. The proliferative response of spleen cells from BALB/c mice to stimulation with the T cell mitogen concanavalin A (Con A) was two or more times stronger than that of similarly stimulated cells from B10 mice, whereas the cells from BlO mice responded better to B cell mitogen bacterial lipopolysaccharide (LPS). No significant role for macrophagesor IL- 1 in the determination of a high or low responder phenotype was found in this model. However, the low response of the spleen cells from B 10 mice to T cell mitogen was associated with a lower production of IL-2 by mitogen-stimulated BlO cells. Indeed, exogenous IL-2 increased the proliferative response of Con A-stimulated BIO cells to the level of the cells from BALB/c mice. The results thus suggestthat the lower production of IL-2 by BlO cells might be a reason for the poor response of the BlO cells to Con A and for the generally weak reactivity of BlO mice in T-cell-dependent immune responses (2, 3). c
MATERIALS AND METHODS Animals. Mice of the inbred strains BALB/c and BlO of both sexesat 7-10 weeks of age were obtained from the breeding colony of our Institute. Reagents. Con A and indomethacin were obtained from Sigma Chemical Co. (St. Louis, MO). LPS isolated from Escherichia coli 055:B5 was purchased from Difco Laboratories (Detroit, MI). Recombinant human IL-la (rIL-101, sp act, 1 X lo8 U/ mg) expressedin E. coli (17) was obtained from Dr. P. T. Lomedico, Hoffman-LaRoche Inc. (Nutley, NJ). Human recombinant IL2 (sp act, 2 X lo6 U/ml) was kindly provided by Dr. E. Gren, Institute of Organic Synthesis (Riga, Latvia). Proliferative assays.Spleencells at a concentration 2 X lo5 cells/well were stimulated in a volume of 0.2 ml RPM1 1640 medium supplemented with antibiotics (penicillin, streptomycin), glutamine, 10 mM Hepes buffer, 5 X 10m5M 2-mercaptoethanol, and 10% fetal calf serum (complete RPM1 1640 medium) in 96-well flat-bottom tissue culture plates (Sterilin Ltd., Feltham, UK). Con A (5 pg/ml), LPS (40 pg/ml) or indomethacin ( lop5 M), and/or interleukins (at the concentrations given below) were added to the cultures. The cultures were incubated at 37°C in a humidified atmosphere of 5% CO2 for 72 hr, and [3H]thymidine (sp act, 22.8 Ci/mmol) at a concentration of 0.5 pCi/well was added to the cultures for the last 6 hr of the incubation period to determine cell proliferation ( 18). Production of IL-l and assay systemfor IL-1 activity. Peritoneal exudate cells (PEC), obtained by washing the peritoneal cavity of individual BALB/c and B 10 mice, were left to adhere to plastic dishes for 2 hr. The nonadherent cells were removed and the adherent cells were collected, adjusted to a concentration of 1 X lo6 cells/ml, and incubated in complete RPM1 1640 medium with 10 pg/ml of LPS and lop5 M indomethacin. After 24 hr of incubation the supernatants obtained were tested for IL1 activity in the classical thymocyte costimulatory assay (19) as we have described elsewhere (20). Production ofIL-2 and assay systemfor IL-2 activity. Spleen cells at a concentration of 5 X lo6 cells/ml were incubated in 1 ml of complete RPM1 1640 medium in 24well tissue culture plates (Linbro, Flow Laboratories Inc., McLean, VA) in the presence of 5 pg/ml Con A. After a 24-hr incubation, the supematants were harvested and assessedfor the ability to support the growth of the IL-2-dependent CTLL-2 cell line (21).
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Separation of cell populations. Macrophage-rich cell populations were obtained by washing the peritoneal cavity of the mice and by using adherence of the cells to the plastic petri dishes. After each of two 2-hr adherence procedures the nonadherent cells were removed and the adherent cells were collected. T-cell-enriched cell populations were obtained by passaging of spleen cells through a nylon-wool column according to Julius et al. (22). The nylon-wool nonadherent cells were cultured on petri dishes coated with swine anti-mouse immunoglobulin antibodies to remove the remaining immunoglobulin-positive cells using the panning technique of Wysocki and Sato (23). Northern blot analysis. Total RNA was isolated using the guanidine isothiocyanate method (24) from 2 X 10’ spleen cells which were either unstimulated or stimulated for 16 hr with 5 pg/ml Con A or 20 pg/ml LPS. The RNA was subjected to electrophoresis, blotted onto Hybond-N nitrocellulose membranes (Amersham International, Amersham, UK), and hybridized with a 32P-labeled IL-2 receptor (IL-2R) probe (25) or an IL-la probe (17) as we have described elsewhere (26). The blots were then stripped and rehybridized with a cDNA probe for actin to demonstrate comparable quantities of RNAs in individual lanes. RESULTS
Reactivity of Spleen Cells,fiom BALB/c and BIO Mice to Mitogens The proliferative reactivity of spleen cells from BALB/c mice to stimulation with Con A was two or more times stronger than that of spleen cells from BlO mice (Fig. 1). In contrast, spleen cells from BlO mice responded better to LPS stimulation than
- ConA BALBk
LPS cells
-
ConA LPS B 10 cells
FIG. I. Proliferative responses of spleen cells from BALB/c and B IO mice to stimulation with Con A or LPS. Spleen cells (1 X lo6 cells/ml) from BALB/c and BlO mice were stimulated with 5 Kg/ml Con A or 40 pg/ml LPS, and cell proliferation was determined by the addition of [3H]thymidine into the cell cultures for the last 6 hr of the 72-hr incubation period.
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cells from the BALB/c donors (Fig. 1). These patterns of reactivity were not dependent on mitogen concentrations, concentrations of cells per milliliter, or incubation intervals (data not shown). Discrepanciesin the Proliferative Activity Do Not Depend on the Activity ofkfacrophages or IL-l Since the proliferative responsesof T cells to stimulation with Con A depend on the presence of IL-l and macrophages (or other antigen-presenting cells), we first compared the production of IL- 1 by cells from BALB/c and B 10 mice. As shown in Fig. 2, unstimulated or LPS-stimulated cells from BALB/c mice synthesized comparable or even higher levels of mRNA for IL- 1CY than the cells from B 10 mice. However, LPS-stimulated macrophages from the B 10 mice produced slightly more biologically active IL-l than similarly stimulated macrophages from the BALB/c mice (Fig. 3). When purified T cells from BALB/c and B 10 mice were stimulated with Con A in the presence of purified BALB/c or BlO macrophages, both types of macrophages stimulated proliferative responsesin T cells to the same degree(Fig. 4). However, the proliferative responsesof the T cells from BALB/c mice were invariably higher than the responsesof the T cells from BlO mice, regardless of which type of macrophage was used (Fig. 4). Spleen Cellsfrom BALB/c and BlO Mice Synthesize Comparable Quantities of mRNA for IL-2R Spleen cells from BALB/c and BlO mice were stimulated with Con A, and total RNA was isolated. The RNA was hybridized with an IL-2R probe. As shown in Fig. 5a, Con A-stimulated cells from both strains of mice synthesizedcomparable quantities of IL-2R mRNA, which was hybridized as five bands corresponding to different types of RNA coding for the 55kDa chain of IL-2R (27). After hybridization with the IL2R probe, the blots were stripped and rehybridized with an actin probe to demonstrate that there were comparable amounts of RNA in individual lanes (Fig. 5b).
a) IL- la
b) Actin
FIG. 2. Synthesis of mRNA for IL- 101in spleen cells from BALB/c and B 10 mice. Spleen cells from B IO (lanes 1 and 3) or BALB/c (lanes 2 and 4) mice were cultured for 16 hr unstimulated (lanes I and 2) or stimulated with 20 &ml LPS (lanes 3 and 4). Total RNA was isolated and hybridized (a) with an IL-la probe or (b) after stripping with an actin probe.
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O-0
CA0 0’ g-a,, --@\a\
\ \
0 0 \
1’
I
/
I
I:4
1:8 Dilution
I:16
l,:\i\-
.
6
1:32
1:64
I
1:128
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FIG. 3. Production of biologically active IL-1 by PEC from BALB/c and BlO mice. PEC from two individual BALB/c (0) and BIO (0) mice were incubated for 24 hr with LPS (20 &ml) and indomethacin (lo-’ M) and the supernatants obtained were tested in a thymocyte costimulatory assay for their ability to support the proliferation of Con A-stimulated thymocytes.
Exogenous IL-2 Abrogates Discrepancies in Con A-Induced Cell Proliferation Spleen cells from B 10 mice responded by a significantly weaker proliferative reaction to Con A stimulation than spleen cells from BALB/c mice. This lower reactivity of B 10 cells was not enhanced by exogenous IL- 1 or by indomethacin, an inhibitor of prostaglandin synthesis (Fig. 6). However, exogenous IL-2, added to the cultures of Con A-stimulated spleen cells from BALB/c and B 10 mice, enhanced the proliferation of BlO cells in such a way that the cells from both strains of mice reacted to Con A with comparable proliferative responses (Fig. 6).
BALB/c Spleen Cells Produced More Biologically Active IL-2 than BlO Cells The results in Fig. 6 suggest that the shortage of IL-2 in BlO cell cultures might be a reason for the lower reactivity of B 10 spleen cells to Con A stimulation. To test this possibility, spleen cells from BALB/c and BlO mice were stimulated with Con A for 24 hr, and the supernatants obtained were tested for their ability to support the growth of IL-2-dependent CTLL-2 cells. It is shown in Fig. 7 that Con A-stimulated sp!een cells from BALB/c mice produced considerably more biologically active IL-2 than similarly stimulated cells from B 10 mice. DISCUSSION Differences among individual inbred strains or lines of mice in the ability to mount antibody responses after antigenic challenge in vivo ( l-6) or to resist parasite infections (9,28-30) have been well documented. In the present study we have concentrated on
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BALB/c 1 cells
FIG. 4. Effectsof purified BALB/c and B 10 macrophageson the proliferative responseof Con A-stimulated T cells. Purified T cells from BALB/c and BIO mice were cultured unstimulated (Cl), stimulated with 5 pg/ ml Con A alone @I), or with Con A in the presence of BALB/c (H) or BlO (H) macrophages (the T cell: macrophage ratio was 10:I).
another aspectof immunological responsiveness,namely proliferative responseto mitogenic stimulation in vitro. We have found that the response of spleen cells from BALB/c mice to stimulation with a T cell mitogen, Con A, was two or more times stronger than that of similarly stimulated spleen cells from B 10 mice. In contrast, spleen cells from B 10 mice responded comparably or even better than cells from BALB/c mice to stimulation with LPS. The observed higher proliferative response of spleen cells from BALB/c mice to T cell mitogen stimulation in vitro correlates with the stronger responsiveness of BALB/c mice than of B 10 mice to T cell-dependent antigens in vivo (3 1). We have focused on elucidating cellular and molecular mechanisms responsible for the observed differences. It has been demonstrated in the model of antibody production in high and low responder strains of mice (lo- 12) and rats (32) that the differences in antigen handling and presentation were the causesof discrepancies in immune responsiveness. We therefore first elucidated the functions of macrophages in BALB/c and B 10 mice. It was found that spleen cells, both unstimulated and LPS-stimulated, from BALB/c mice synthesized a comparable or even higher level of mRNA for IL-la than did spleen cells from BlO mice. However, the production of biologically active IL-1 was higher in BlO mice. Similar discrepancies between the levels of specific mRNA and the particular interleukin production have been described as a result of different regulations at the translation level (33, 34). Also, our other experiments, in which we utilized combinations of purified populations of T cells and macrophages, suggested that the macrophageswere not the critical cell population responsible for the differences in proliferative responsesto Con A stimulation observed between cells from BALB/c and B 10 mice.
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4
a)lL-2R
b) Actin
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FIG. 5. Synthesis of mRNA for the inducible 55-kDa IL-2R in spleen cells from BALB/c and BIO mice. Spleen cells from BALB/c (lanes 1 and 3) or BlO (lanes 2 and 4) mice were cultured for 16 hr unstimulated (lanes I and 2) or stimulated with 5 pg/ml Con A (lanes 3 and 4). Total RNA was isolated and hybridized (a) with an IL-2R probe and (b) after stripping with an actin probe.
In the model of the Biozzi mice, Nakano et al. (13) showed that nonspecific suppressor cells were more numerous and active in low responder mice, and it was considered that these suppressor cells might play a role in the induction of low antibody response.We also tested the activity of nonspecific Con A-induced suppressorcells in our BALB/c and BlO mice, but their levels of activity were comparable (data not shown).
BALBlc
cells
B 10 cells
FIG. 6. Exogenous IL-2 abolished the discrepancies in Con A-induced proliferative responses between BALB/c and BlO cells. Spleen cells from BALB/c and BlO mice were cultured tutstimulated (I), stimulated with 5 pg/ml Con A alone (m) or with Con A in the presence of 100 U/ml rIL-la (IWI), 100 U/ml rIL-2 (a), or lo-‘M indomethacin (0).
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1:4
1:16 DiMon
ET AL.
1:64
1:256
1:1024
of supernatants
FIG.7. Production of IL-2 by Con A-stimulated spleen cells from BALB/c and B 10 mice. Spleen cells (5 X lo6 cells/ml) from two individual BALB/c (0) or BlO (0) mice were stimulated for 24 hr with Con A (5 rg/ml) and the supernatants obtained were tested for their ability to support the growth of IL-2-dependent CTLL-2 cells.
Next we compared the activity of T cells. After stimulation with Con A, spleen cells from BALB/c and B 10 mice synthesized comparable quantities of mRNA for the inducible 55kDa IL-2R. However, we found that Con A-stimulated spleen cells from BALB/c mice produced significantly more biologically active IL-2 than similarly stimulated spleen cells from B 10 mice. To test the possibility that the lower IL-2 production by the B 10 cells could be a reason for their weaker proliferative responses,spleen cells from BALB/c and B 10 mice were stimulated with Con A in the presenceof exogenous IL-2. We found that IL-2 completely abrogated the discrepancies in proliferative responsesbetween cells from these two strains of mice. Con A-stimulated spleen cells from B 10 mice proliferated in the presence of exogenous IL-2 as did the cells from BALB/c mice. These observations suggestthat the shortage of IL-2 in the cultures of Con A-stimulated BlO cells is a reason for their lower proliferation. We prefer the idea that the shortage of IL-2 in the BlO cell cultures was caused by a lower IL-2 production by Con A-stimulated BlO cells rather than the possibility that it was due to the presence of some immunosuppressive molecules in the BlO supernatants. If the latter is the case, such inhibitory molecules should also inhibit the activity of exogenous IL-2. Experiments are now under way to determine the genetic background for the differences in IL-2 production between the BALB/c and B 10 strains of mice. In conclusion, the results showed lower proliferative responses of cells from B10 mice to Con A stimulation as a consequence of a lower production of IL-2. Since exogenous IL-2 can increase the low proliferative responsesin Con A-stimulated B 10 cells to level of that in BALB/c cells, we believe that the low production of IL-2 could be a reason for the weaker proliferative responsesof BlO cells to the T cell mitogen, Con A. It is suggested that the low production of IL-2 might also be a cause of
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the low antibody production to the T-cell-dependent antigens observed in BlO mice (2, 3). ACKNOWLEDGMENTS The authors thank Dr. P. T. Lomedico, Hoffmann-LaRoche Inc. (Nutley, NJ), for providing rlL-la and IL-1 (Yprobe, Dr. E. Gren, Institute of Organic Synthesis, Riga, Latvia, for the kind gift of rlL-2 and Dr. T. Honjo, Kyoto University, Japan, for providing the IL-2R probe. We are also grateful to Ms. E. FoitovL for expert technical work and Ms. L. Keleher for editing assistance.
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