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Suppressor cells in levamisole-treated mice: A possible role of T-cell-mediated feedback suppression in the drug-induced suppression of the humoral immune response
Suppressor Cells in Levamisole-treated Mice: A Possible Role of T-Cell-mediated Feedback Suppression in the Drug-induced Suppression of the Humoral Immune Response Shinichi Kurakata and Kouichi Kitamura Abstract: The suppressive effect of leuamisole (LMS) on the primary IgM plaque-forming cell (PFC) response to sheep erythrocytes in mice was studied. The suppressive effect of LMS on day 4 PFC response was found to be modified by dose, the timing of drug administration, and the amount of antigen injected. The experiments, using an inhibitory dose of 10mg/kg LMS, showed that: 1) T cells play an essential role in LMS-induced suppression; 2) antigen-specific suppressor cells are induced in the spleen of I_MS-treated mice; 3) the spleen cells from LMS-treated donors either suppress or enhance the PFC response in the recipients, depending on the amount of antigen injected and the number of spleen cells transferred; and 4) a dose of 10 mg/kg LMS, which is inhibitory when assessed on day 4 PFC response, actually enhances the response three and a half days after the immunization. These results suggest that LMS induces precursors of both helper and suppressor cells, and their differentiation and/or maturation to LMS-primed antigen-specific suppressor T cells are modulated, at least in part, by T-cell-mediated feedback suppression.
Key Words: Levamisole; Plaque-forming cells; Sheep erythrocytes; Cell transfer; Suppressor T cells; Feedback suppression; T cell regulation.
INTRODUCTION
Levamisole (LMS), a synthetic antihelminthic drug, has been studied extensively since Renoux and Renoux (1971) first described its immunologic properties, and the drug has been used in such diseases as rhematoid arthritis, systemic lupus erythematosus, and some types of malignancy (Symoens and Rosenthal, 1977). However, the precise mechanism of action of LMS in these diseases is still not clear. The majority of the experiments have shown that LMS modifies cellmediated immune reactions in vivo and in vitro (Symoens et al., 1979). These findings demonstrated to some extent how LMS functions in relation to B cell differentiation, but the role of LMS in immunoglobulin production was not clarified. In fact, conflicting results have been obtained in the
Received December 15, 1982; revised and accepted July 12, 1983. From the BiologicalResearch Laboratories, Sankyo Co., Ltd., 2-58, Hiromachi 1-chome, Shinagawa-ku, Tokyo 140, Japan. Address requests for reprints to: Shinichi Kurakata, BiologicalResearch Laboratories, Sankyo Company, Ltd., 2-58, Hiromachi l-chome, Shinagawa-ku, Tokyo 140, Japan. © ElsevierSdence PublishingCo., Inc., 1983 52 Vanderbi]tAve., NewYork, N.Y. Immunopharmacology6, 279-287 ( 1 9 8 3 )
279 0162-3109/83/$03.00
280
S. Kurakata and K. Kitamura
studies of the effect of LMS on the primary IgM antibody response to SRBC. For instance, Spreafico et al. (1975) reported LMS-mediated enhancement of direct PFC response in aged female CDF 1mice with drug posttreatments, while Bruley-Rosset et al. (1978) failed to confirm the potentiating effects on anti-SRBC responses in similarly aged female BDF i mice. Renoux and Renoux (1974, 1979) reported that LMS either enhanced or suppressed the PFC response to SRBC, depending on experimental factors such as sex, age, strain of mice, timing of drug administration, and dose of the drug. These factors may at least in part explain the conflicting results reported. In order to further explain the diverse effects of LMS, a number of studies have been reported on the mode of action of LMS both in vivo and in vitro. In particular, on the mechanism of immunosuppression by LMS, Sampson and others have presented evidence that LMS was capable of inducing nonspecific suppressor T cells in vitro in mitogen-induced lymphocyte responses (Sampson and Lui, 1976; Moriya et al., 1979; Pasquali et al., 1981). However, no confirmative mechanism which could explain the suppressive effect on primary antibody response in vivo has yet been reported. To further clarify the mode of action of LMS in vivo, we studied the LMS-induced suppression in the primary IgM antibody response in mice by an adoptive transfer system, which indicated a possible role of T-cell-mediated feedback suppression in the LMS-induced suppression in vivo.
MATERIALS AND METHODS
Animals Female mice ( 8 - 1 0 wk old) of BALB/c CrSlc and ICR/jcl strains were used. The mice were purchased from Shizuoka Agricultural Cooperative Association for Laboratory Animals (Slc), Hamamatsu, and Clea-Japan Inc. (jcl), Tokyo. They were raised under specific-pathogen-free conditions. The animals were kept in roomy cages and fed commercial mouse diet pellets and water ad libitum.
Levamisole Crystalline levamisole (LMS) was obtained from Aldrich Chemical Co. (Milwaukee, WI). The LMS was dissolved in sterile saline just before use, and injected into the mice intravenously (i.v.) at a dose of 10 mg/kg.
Antigen and Immunization S h e e p erythrocytes (SRBC) were obtained from Nippon Bio-Sup Center (Tokyo). The erythrocytes were washed three times with saline before use, and an appropriate number of the cells in saline (0.1 ml) was injected to the mice intraperitoneally (i.p.).
Detection of Plaque-forming Cells Splenic plaque-forming cells (PFC) were counted four days after immunization by the hemolytic plaque technique reported by Cunningham and Szenberg (1968). Single cell suspensions of spleen cells were prepared in Eagle's minimum essential medium by pressing spleens through
Abbreviations. LMS: levamisole; SRBC: sheep erythrocytes; PFC: plaque-forming cells; IgM: immunoglobulin M; i.p.: intraperitoneally; i.v.: intravenously.
Suppressor Cells in Levamisole-treated Mice
281
stainless-steel mesh. The concentrations of the suspensions were adjusted so as to result in expected yields of 5 0 - 1 0 0 plaques per culture.
Cell
Transfer
Donor mice were given LMS (I0 mg/kg) i.v. and, after an appropriate time interval, their spleens were removed for the preparation of single cell suspensions. Spleen cells (2 × 107) of these mice were suspended in Hanks' balanced salt solution and transferred i.v. into normal recipient mice. Immediately after cell transfer, the recipients were immunized i.p. with SRBC.
Statistics The statistical significance of the data were determined by Student's t-test where a p value <0.05 was considered significant.
RESULTS Suppression of the Primary lgM Antibody Responseby LMS As shown in Table 1, the numbers of PFC per 106 spleen cells in BALB/c and ICR/jcl mice decreased on treatment with 10 mg/kg LMS, depending on the timing of drug administration. The strongest suppression was exhibited in the mice to which LMS had been given six days or four days prior to immunization, while no significant inhibition in PFC was observed in the mice to which LMS had been given concomitantly with antigen or eight days prior to the injection of antigen. A similar depression could also seen when the results were calculated on PFC per spleen (data not presented). Among the mice treated with various doses of LMS four days before the immunization, the suppression in the PFC response was seen only in the high dose (10 mg/kg) treated group. At a lower dose of 2.5 mg/kg, the antibody response was rather enhanced, and neither suppression nor enhancement was observed below 2.5 mg/kg (data not shown). Figure 1 shows the dose-response effect of SRBC on the PFC response in BALB/c mice injected with or without LMS. The optimal antigen dose for PFC production in both control and LMS-treated mice was shown to be 2 × 10s SRBC. At this optimal antigen dose, LMS decreased the number of PFC by 50%. On the other hand, with a suboptimal antigen dose of 2 × 107 SRBC, the same dose significantlyincreased the number of PFC by up to 2 0 0 ~ . The results were similar in the ICR]jcl strain (data not presented).
Table I
Effect of timing of LMS administration on the IgM PFC response a lgM PFC/IO 6 spleen cellsb
Day after LMS administration 0 2 4
BALB /c 570 528 221 121
-+ 69 + 58 +_55 c _ 31 c
6
I01 - 44c
8
485 _+ 73
ICR /)cl 1160 905 701 588
_ 89 +_ 128 _ 68e + 46 c
6 8 4 +_ 109 c
1334 _+ 139
QMice were immunized with 2 x 10a SRBC 0, 2, 4, 6, and 8 days after LMS administraUon. bThe data represent the mean _ SEM of five mice. cSignificantly different from control, p < 0.01.
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2 X 108
2 X l09
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Figure I Dose-response effects of SRBC on the IgM PFC formation in mice injected with or without L.MS. BALB /c mice were injected with the indicated numbers of SRBC and each IgM PFC was evaluated four days later. Animals received I 0 mg/kg LMS (0) or saline (0)four clays before antigen administration. Each value represents an average of five mice and the vertical bars represent + SEM. *Significantly different from control, p <0.01. Thus, it was confirmed in these experiments that LMS-induced suppression of the PFC response was dependent on dose, the timing of drug administration, and the amount of antigen injected. These observations are consistent with those reported by Renoux and Renoux (1974), except for the amount of antigen injected.
Kinetics of Primary lgM Antibody Response in Mice Treated with LMS The influence of LMS on the induction rate of the primary IgM antibody response in ICR/jcl mice was studied. Mice were given 2 × 10~ SRBC four days after administration of LMS, and each IgM PFC was counted. As shown in Figure 2, the peak response in the LMS-treated mice was 12 hr earlier than that in the control group, without reducing the peak numbers of PFC. There was no difference between control and LMS-treated mice in hemagglutination titers for circulating antibody four days after immunization (data not shown). This indicated that the LMS-induced suppression of the PFC response was not due to an inhibition of overall PFC production, but due to an early shift in the formation of the peak level of reactivity.
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Figure 2 Time course of lgM PFC formation in mice treated with or without LMS. ICR /jcl mice received 10 mg/kg LMS (Q) or saline (0), and four days laterthey were immunized with 2 × 10s SRBC. IgM PFC production was determined on the indicated day. Each value represents an average of 5 to 10 mice and vertical bars represent + SEM. *Significantly different from control, p <0.01.
Induction of Suppressor Cells by LMS In order to examine whether suppression induced by LMS could be transferred to normal recipients, 2 × 107spleen cells from BALB/c and ICR/jcl mice previously injected with LMS were transferred into normal syngeneic and/or isogeneic recipients at various time intervals thereafter. Immediately after the cell transfer, the recipients were immunized with 2 × 108 SRBC, and the PFC was enumerated four days later. As shown in Table 2, the spleen cells from donors that had received LMS two to six days prior to the cell transfer decreased the number of PFC in the recipients. The formation of LMS-induced suppressor cells was coincident with the suppression of the PFC response observed in vivo (Table 1). The suppressive activity of spleen cells from LMS-treated donors was abolished by the treatment of spleen cells with anti-Thy 1.2 antiserum plus complement, but not with normal mouse serum plus complement (data not shown). This finding suggests an essential role of T cells in the suppression by LMS. LMS-induced suppressor cells were presumed to be antigen-specific, since, in the system of the
284
S. Kurakata and K. Kitamura
Table 2
Induction of suppressor cells by LMS a
Spleen cells transferred Nil Normal LMS-treated LMS-treated LMS-treated LMS-treated
Day after LMS administration
2 4 6 8
IgM PFC/106 spleen cellsb BALB /c ICR /jcl 772 714 432 425 380 695
± ± ± ± ± ±
93 54 48 c 55 c 42 a 83
1543 1499 857 658 571 1620
± ± ± ± ± ±
108 93 77 a 101 a 123 J 111
°Spleen cells (2 × 107) from LMS-treated donor mice were transferred to recipient mice 2, 4, 6, and 8 days after the drug administration. bValues are mean PFC _+ SEM of five mice per group. CSignificantlydifferent from control, p ~ 0.05. aSignificanfly different from control, p < 0.01.
pretreatment of donors with both LMS and the antigen (SRBC or horse RBC), the suppression in the recipients was observed only when the donors received the same antigen as that of the recipients (data not presented). The above results suggest that LMS induces antigen-specific suppressor T cells.
Effects of Number of Spleen Cells and SRBC on the PFC Response in the Recipients Figure 3 shows a dose-response profile of spleen cells from LMS-treated donors on the PFC response in normal recipients. Spleen cell doses greater than 2 × 106 suppressed the PFC response (about 50% suppression), whereas 2 × 105 spleen cells enhanced the response (about 50% enhancement). Neither suppression nor enhancement of the PFC response was observed in recipients of less than 2 x 104 spleen cells. As shown in Table 3, however, the same number of spleen cells (2 × 107) from the same LMStreated donors either suppressed or enhanced the PFC response in normal recipients, depending on the amount of antigen injected in the recipients. With 2 × 107 spleen cells from the same LMStreated donors, a suppression of the PFC response was observed in the recipients at the optimal antigen dose of 2 × 10SSRBC, while an enhancement was observed with the suboptimal antigen dose of 2 × 107 SRBC. This finding may reflect the dose-response effect of SRBC on the PFC response in LMS-treated mice (Figure 1), The results shown in Table 3 and Figure 3 demonstrate that the spleen cells from LMS-treated donors could exert either suppressor or helper cell activity on the PFC response in recipients, depending on the amount of antigen injected as well as the number of spleen cells transferred to the recipients.
DISCUSSION With the data presented in this paper, we have attempted to clarify the suppression mechanism(s) of a high dose (10 mg/kg) of LMS on primary IgM antibody response in normal mice. It should be noted that the enhancement of the response was usually seen at lower doses (Renoux, 1978). Using this high dose of LMS, we have demonstrated that the spleen cells from LMS-treated mice either suppressed or enhanced the PFC response in the recipient mice, depending on the amount of antigen injected and the number of spleen cells transferred (Table 3 and Figure 3). These results suggest that 10 mg/kg LMS induces precursors of both helper and suppressor cells in the spleen, and that the differentiation and/or maturation of the spleen cells from LMS-treated donors to either helper or suppressor cells in the recipients is influenced by a number of factors (mostly antigen
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Table 3
Effects of SRBC dose on the IgM PFC response in mice transferred with spleen cells from LMS-treated donor mice a
Spleen cells
IgM PFC/IO 7spleen cellsb
transferred
SRBC dose
BALB /c
ICR /)cl
Nil Normal LMS-treated
2 x I0 s 2 x 108 2 x 108
6000 _+270 6170 + 360 3150 _+ 150 c
20700 _+ 1450 19600 __ 780 9900 _+ 1300 c
Nil Normal LMS-treated
2 x 107 2 x 107 2 x 107
263_+ 30 249 ___ 41 673 _+ 33c
569_+ 42 605 _+ 37 1010 _+ 105 c
aSpleen cells (2 x 107) from LMS-treated donor mice were transferred to recipient mice four days after the drag administration.Immediatelyafter the celltransfer,the recipientswere immunizedwith either 2 × 108 or 2 x 107 SRBC. bValues are mean PFC __ SEM of five mice per group. cSignificantlydifferentfrom control, p < 0.01. dose). We found that the PFC response in the recipients was enhanced either when the recipients were transferred a smaller number (2 x 10s) of the spleen cells and immunized with an optimal antigen dose (2 x 10s) of SRBC, or when the recipients were transferred a larger number (2 × 107) of the spleen cells and immunized with a suboptimal dose (2 × 107) of the antigen. On the other hand, the PFC response was suppressed only when the recipients were transferred the larger number of the spleen cells and immunized with the optimal antigen dose of SRBC. Thus, it seems likely that the supressor cell activity appears only when the degree of total stimulation in the transferred spleen cells by the antigen reaches a certain threshold. The essential role of T cells in the LMS-induced suppression was also suggested by the finding that the treatment of spleen cells from LMS-treated donors with anti-Thy 1 antiserum plus complement relieved the suppression in the PFC response in the recipients. Since B cells are resistant to the above treatment, suppressor B cells, which were assumed to be important in the feedback suppression of humoral immune response in a variety of systems (Stockinger et al., 1979; Shimamura et al., 1982), are not responsible for the suppression by LMS. Macrophage-like suppressor cells, which were reported to be nonspecific suppressor cells by Schrier et al. (1980), are also unlikely to be involved in this suppression process because only antigen-specific suppressor cells were induced by LMS. Eardley and associates (1977, 1978, 1980) have recently postulated an immunomodulatory circuit of T-cell interactions as basic elements in feedback suppression. In brief, Ly I helper T cells that are stimulated by antigen (SRBC) induce a second nonimmune set of T cells (Ly, 1,2,3 cells), which in turn either differentiate into Ly 2,3 suppressor T cells or activate Ly 2,3 suppressor T cells. These suppressor T cells regulate the activity of B cells to secrete antibody through modulation of Ly I helper T cell activity. Since, a) T cells may play an essential role in LMS-induced suppression; b) LMS may induce precursors of both helper and suppressor cells in the spleen; c) the differentiation and/or maturation of LMS-primed spleen cells to either helper or suppressor cells in the recipients is influenced by the amount of antigen injected and the number of spleen cells transferred (Table 3 and Flgure 3); and d) a dose of 10 mg/kg LMS actually shifts the peak response in PFC earlier than the control (Figure 2), the above described T-cell-mediated feedback regulation may be involved in LMS-induced suppression. Thus, the following interpretation could be compatible with our findings. LMS primarily induces precursors of both helper and suppressor T cells. Subsequent to antigen (SRBC) injection, the helper precursors are preferentially stimufated by the antigen to form mature helper cells. These mature cells may eventually induce antigen-specific suppressor T cells.
Suppressor Cells in Levamisole-treated Mice
287
The authors acknowledgewiththanks MissMikikoTomatsufor technicalassistanceand Drs. IsaoKanekoand Yoshihiko Baba for advice and encouragement.
REFERENCES Bruley-Rosset M, Florentin I, Kiger N, Davigny M, Mathe G (1978) Effects of Bacillus CalmetteGu~rin and levamisole on immune responses in young adult and age-immunodepressed mice. Cancer Treat Rep 62:1641. Cantor H, Boyse EA (1977) Lymphocytes as models for the study of mammalian cellular differentiation. Immunological Rev 33:105. Cantor H, McVay-Boudreau L, Hugenberger J, Naidorf K, Shen FW, Gershon RK (1978) Immunomodulatory circuits among T-cell sets. II. Physiological role of feedback inhibition in vivo: absence in NZB mice. J Exp Meal 147:1116. Cunningham AJ, Szenberg A (1968) Further improvements in the plaque technique for detecting single antibody-forming cells. Immunology 14: 599. Eardley DD, Hugenberger J, McVay-Boudreau L, Shen FW, Gershon RK, Cantor H (1978) Immunomodulatory circuits among T-cell sets. I. T-helper cells induce other T-cell sets to exert feedback inhibition. J Exp Med 147:1106. Eardley DD (1980) Feedback suppression: an immunoregulatory circuit. Federation Proc 39:3114. Moriya N, Miyawaki T, Seki H, Kubo M, Nagaoki T, Okuda N, Taniguch N (1979) Induction of suppressor activity on B-cell differentiation in human T cell subset without Fc(IgG) receptors by levamisole administration. Scand J Immunol 10:535. Pasquali J-L, Tsoukas CD, Fong S, Carson DA, Vaughan JH (1981) Effect of levamisole on pokeweed mitogen stimulation of immunoglobulin production in vitro. Immunopharmacology 3:289. Renoux G, Renoux M ( 1971) Effect immunostimulant d' un imidothiazole dans r immunisation des souris contre l'infection par Brucella abortus. CR Acad Sci (Paris) 272:349. Renoux G, Renoux M (1974) Modulation of immune reactivity by phenylimidothiazole salts in mice by sheep red blood cells. J Immunol 113: 779. Renoux G, Renoux M, Guillaumin J-M (1979) Genetic and epigenetic control of levamisoleinduced immunostimulation. Int J Immunopharmacol 1:43. Sampson D, Lui A (1976) The effect of levamisole on cell mediated immunity and suppressor cell function. Cancer Res 36:952. Schrier DJ, Allen EM, Moore VL (1980) BCG-induced macrophage suppression in mice: specific and nonspecific antibody-mediated and cellular immunologic responses. Cell Immunol 56:347. Shimamura T, Hashimoto K, Sasaki S (1982) Feedback suppression of the immune response in vivo. 1. Immune B cells induce antigen-specific suppressor T cells. Cell Immunol 68:104. Spreafico F, Vecchi A, Mantovani A, Poggi A, Franchi G, Anaclerio A, Garattini S (1975) Characterization of the immunostimulants levamisole and tetramisole. Eur J Cancer 11:555. Stockinger B, Botzenhardt U, Lemmel EM (1979) On the feedback regulation of humoral immune response. I. Evidence for 'B suppressor cells'. Immunology 36:87. Symoens J, Rosenthal M (1977) Levamisole in the modulation of the immune response: the current experimental and clinical state. J Reticu Ioendothel Soc 21:175. Symoens J, Rosenthal M, Brabander MD, Goldstein G (1979) Immunoregulation with levamisole. Springer Semin Immunopathol 2:49.
Key Words: Levamisole; Plaque-forming cells; Sheep erythrocytes; Cell transfer; Suppressor T cells; Feedback suppression; T cell regulation.
INTRODUCTION
Levamisole (LMS), a synthetic antihelminthic drug, has been studied extensively since Renoux and Renoux (1971) first described its immunologic properties, and the drug has been used in such diseases as rhematoid arthritis, systemic lupus erythematosus, and some types of malignancy (Symoens and Rosenthal, 1977). However, the precise mechanism of action of LMS in these diseases is still not clear. The majority of the experiments have shown that LMS modifies cellmediated immune reactions in vivo and in vitro (Symoens et al., 1979). These findings demonstrated to some extent how LMS functions in relation to B cell differentiation, but the role of LMS in immunoglobulin production was not clarified. In fact, conflicting results have been obtained in the
Received December 15, 1982; revised and accepted July 12, 1983. From the BiologicalResearch Laboratories, Sankyo Co., Ltd., 2-58, Hiromachi 1-chome, Shinagawa-ku, Tokyo 140, Japan. Address requests for reprints to: Shinichi Kurakata, BiologicalResearch Laboratories, Sankyo Company, Ltd., 2-58, Hiromachi l-chome, Shinagawa-ku, Tokyo 140, Japan. © ElsevierSdence PublishingCo., Inc., 1983 52 Vanderbi]tAve., NewYork, N.Y. Immunopharmacology6, 279-287 ( 1 9 8 3 )
279 0162-3109/83/$03.00
280
S. Kurakata and K. Kitamura
studies of the effect of LMS on the primary IgM antibody response to SRBC. For instance, Spreafico et al. (1975) reported LMS-mediated enhancement of direct PFC response in aged female CDF 1mice with drug posttreatments, while Bruley-Rosset et al. (1978) failed to confirm the potentiating effects on anti-SRBC responses in similarly aged female BDF i mice. Renoux and Renoux (1974, 1979) reported that LMS either enhanced or suppressed the PFC response to SRBC, depending on experimental factors such as sex, age, strain of mice, timing of drug administration, and dose of the drug. These factors may at least in part explain the conflicting results reported. In order to further explain the diverse effects of LMS, a number of studies have been reported on the mode of action of LMS both in vivo and in vitro. In particular, on the mechanism of immunosuppression by LMS, Sampson and others have presented evidence that LMS was capable of inducing nonspecific suppressor T cells in vitro in mitogen-induced lymphocyte responses (Sampson and Lui, 1976; Moriya et al., 1979; Pasquali et al., 1981). However, no confirmative mechanism which could explain the suppressive effect on primary antibody response in vivo has yet been reported. To further clarify the mode of action of LMS in vivo, we studied the LMS-induced suppression in the primary IgM antibody response in mice by an adoptive transfer system, which indicated a possible role of T-cell-mediated feedback suppression in the LMS-induced suppression in vivo.
MATERIALS AND METHODS
Animals Female mice ( 8 - 1 0 wk old) of BALB/c CrSlc and ICR/jcl strains were used. The mice were purchased from Shizuoka Agricultural Cooperative Association for Laboratory Animals (Slc), Hamamatsu, and Clea-Japan Inc. (jcl), Tokyo. They were raised under specific-pathogen-free conditions. The animals were kept in roomy cages and fed commercial mouse diet pellets and water ad libitum.
Levamisole Crystalline levamisole (LMS) was obtained from Aldrich Chemical Co. (Milwaukee, WI). The LMS was dissolved in sterile saline just before use, and injected into the mice intravenously (i.v.) at a dose of 10 mg/kg.
Antigen and Immunization S h e e p erythrocytes (SRBC) were obtained from Nippon Bio-Sup Center (Tokyo). The erythrocytes were washed three times with saline before use, and an appropriate number of the cells in saline (0.1 ml) was injected to the mice intraperitoneally (i.p.).
Detection of Plaque-forming Cells Splenic plaque-forming cells (PFC) were counted four days after immunization by the hemolytic plaque technique reported by Cunningham and Szenberg (1968). Single cell suspensions of spleen cells were prepared in Eagle's minimum essential medium by pressing spleens through
Abbreviations. LMS: levamisole; SRBC: sheep erythrocytes; PFC: plaque-forming cells; IgM: immunoglobulin M; i.p.: intraperitoneally; i.v.: intravenously.
Suppressor Cells in Levamisole-treated Mice
281
stainless-steel mesh. The concentrations of the suspensions were adjusted so as to result in expected yields of 5 0 - 1 0 0 plaques per culture.
Cell
Transfer
Donor mice were given LMS (I0 mg/kg) i.v. and, after an appropriate time interval, their spleens were removed for the preparation of single cell suspensions. Spleen cells (2 × 107) of these mice were suspended in Hanks' balanced salt solution and transferred i.v. into normal recipient mice. Immediately after cell transfer, the recipients were immunized i.p. with SRBC.
Statistics The statistical significance of the data were determined by Student's t-test where a p value <0.05 was considered significant.
RESULTS Suppression of the Primary lgM Antibody Responseby LMS As shown in Table 1, the numbers of PFC per 106 spleen cells in BALB/c and ICR/jcl mice decreased on treatment with 10 mg/kg LMS, depending on the timing of drug administration. The strongest suppression was exhibited in the mice to which LMS had been given six days or four days prior to immunization, while no significant inhibition in PFC was observed in the mice to which LMS had been given concomitantly with antigen or eight days prior to the injection of antigen. A similar depression could also seen when the results were calculated on PFC per spleen (data not presented). Among the mice treated with various doses of LMS four days before the immunization, the suppression in the PFC response was seen only in the high dose (10 mg/kg) treated group. At a lower dose of 2.5 mg/kg, the antibody response was rather enhanced, and neither suppression nor enhancement was observed below 2.5 mg/kg (data not shown). Figure 1 shows the dose-response effect of SRBC on the PFC response in BALB/c mice injected with or without LMS. The optimal antigen dose for PFC production in both control and LMS-treated mice was shown to be 2 × 10s SRBC. At this optimal antigen dose, LMS decreased the number of PFC by 50%. On the other hand, with a suboptimal antigen dose of 2 × 107 SRBC, the same dose significantlyincreased the number of PFC by up to 2 0 0 ~ . The results were similar in the ICR]jcl strain (data not presented).
Table I
Effect of timing of LMS administration on the IgM PFC response a lgM PFC/IO 6 spleen cellsb
Day after LMS administration 0 2 4
BALB /c 570 528 221 121
-+ 69 + 58 +_55 c _ 31 c
6
I01 - 44c
8
485 _+ 73
ICR /)cl 1160 905 701 588
_ 89 +_ 128 _ 68e + 46 c
6 8 4 +_ 109 c
1334 _+ 139
QMice were immunized with 2 x 10a SRBC 0, 2, 4, 6, and 8 days after LMS administraUon. bThe data represent the mean _ SEM of five mice. cSignificantly different from control, p < 0.01.
S. Kurakata and K. Kitamura
282
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I
2 X 107 SRBC
2 X 108
2 X l09
DOSE
Figure I Dose-response effects of SRBC on the IgM PFC formation in mice injected with or without L.MS. BALB /c mice were injected with the indicated numbers of SRBC and each IgM PFC was evaluated four days later. Animals received I 0 mg/kg LMS (0) or saline (0)four clays before antigen administration. Each value represents an average of five mice and the vertical bars represent + SEM. *Significantly different from control, p <0.01. Thus, it was confirmed in these experiments that LMS-induced suppression of the PFC response was dependent on dose, the timing of drug administration, and the amount of antigen injected. These observations are consistent with those reported by Renoux and Renoux (1974), except for the amount of antigen injected.
Kinetics of Primary lgM Antibody Response in Mice Treated with LMS The influence of LMS on the induction rate of the primary IgM antibody response in ICR/jcl mice was studied. Mice were given 2 × 10~ SRBC four days after administration of LMS, and each IgM PFC was counted. As shown in Figure 2, the peak response in the LMS-treated mice was 12 hr earlier than that in the control group, without reducing the peak numbers of PFC. There was no difference between control and LMS-treated mice in hemagglutination titers for circulating antibody four days after immunization (data not shown). This indicated that the LMS-induced suppression of the PFC response was not due to an inhibition of overall PFC production, but due to an early shift in the formation of the peak level of reactivity.
Suppressor Cells in Levamisole-treated Mice
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Figure 2 Time course of lgM PFC formation in mice treated with or without LMS. ICR /jcl mice received 10 mg/kg LMS (Q) or saline (0), and four days laterthey were immunized with 2 × 10s SRBC. IgM PFC production was determined on the indicated day. Each value represents an average of 5 to 10 mice and vertical bars represent + SEM. *Significantly different from control, p <0.01.
Induction of Suppressor Cells by LMS In order to examine whether suppression induced by LMS could be transferred to normal recipients, 2 × 107spleen cells from BALB/c and ICR/jcl mice previously injected with LMS were transferred into normal syngeneic and/or isogeneic recipients at various time intervals thereafter. Immediately after the cell transfer, the recipients were immunized with 2 × 108 SRBC, and the PFC was enumerated four days later. As shown in Table 2, the spleen cells from donors that had received LMS two to six days prior to the cell transfer decreased the number of PFC in the recipients. The formation of LMS-induced suppressor cells was coincident with the suppression of the PFC response observed in vivo (Table 1). The suppressive activity of spleen cells from LMS-treated donors was abolished by the treatment of spleen cells with anti-Thy 1.2 antiserum plus complement, but not with normal mouse serum plus complement (data not shown). This finding suggests an essential role of T cells in the suppression by LMS. LMS-induced suppressor cells were presumed to be antigen-specific, since, in the system of the
284
S. Kurakata and K. Kitamura
Table 2
Induction of suppressor cells by LMS a
Spleen cells transferred Nil Normal LMS-treated LMS-treated LMS-treated LMS-treated
Day after LMS administration
2 4 6 8
IgM PFC/106 spleen cellsb BALB /c ICR /jcl 772 714 432 425 380 695
± ± ± ± ± ±
93 54 48 c 55 c 42 a 83
1543 1499 857 658 571 1620
± ± ± ± ± ±
108 93 77 a 101 a 123 J 111
°Spleen cells (2 × 107) from LMS-treated donor mice were transferred to recipient mice 2, 4, 6, and 8 days after the drug administration. bValues are mean PFC _+ SEM of five mice per group. CSignificantlydifferent from control, p ~ 0.05. aSignificanfly different from control, p < 0.01.
pretreatment of donors with both LMS and the antigen (SRBC or horse RBC), the suppression in the recipients was observed only when the donors received the same antigen as that of the recipients (data not presented). The above results suggest that LMS induces antigen-specific suppressor T cells.
Effects of Number of Spleen Cells and SRBC on the PFC Response in the Recipients Figure 3 shows a dose-response profile of spleen cells from LMS-treated donors on the PFC response in normal recipients. Spleen cell doses greater than 2 × 106 suppressed the PFC response (about 50% suppression), whereas 2 × 105 spleen cells enhanced the response (about 50% enhancement). Neither suppression nor enhancement of the PFC response was observed in recipients of less than 2 x 104 spleen cells. As shown in Table 3, however, the same number of spleen cells (2 × 107) from the same LMStreated donors either suppressed or enhanced the PFC response in normal recipients, depending on the amount of antigen injected in the recipients. With 2 × 107 spleen cells from the same LMStreated donors, a suppression of the PFC response was observed in the recipients at the optimal antigen dose of 2 × 10SSRBC, while an enhancement was observed with the suboptimal antigen dose of 2 × 107 SRBC. This finding may reflect the dose-response effect of SRBC on the PFC response in LMS-treated mice (Figure 1), The results shown in Table 3 and Figure 3 demonstrate that the spleen cells from LMS-treated donors could exert either suppressor or helper cell activity on the PFC response in recipients, depending on the amount of antigen injected as well as the number of spleen cells transferred to the recipients.
DISCUSSION With the data presented in this paper, we have attempted to clarify the suppression mechanism(s) of a high dose (10 mg/kg) of LMS on primary IgM antibody response in normal mice. It should be noted that the enhancement of the response was usually seen at lower doses (Renoux, 1978). Using this high dose of LMS, we have demonstrated that the spleen cells from LMS-treated mice either suppressed or enhanced the PFC response in the recipient mice, depending on the amount of antigen injected and the number of spleen cells transferred (Table 3 and Figure 3). These results suggest that 10 mg/kg LMS induces precursors of both helper and suppressor cells in the spleen, and that the differentiation and/or maturation of the spleen cells from LMS-treated donors to either helper or suppressor cells in the recipients is influenced by a number of factors (mostly antigen
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Table 3
Effects of SRBC dose on the IgM PFC response in mice transferred with spleen cells from LMS-treated donor mice a
Spleen cells
IgM PFC/IO 7spleen cellsb
transferred
SRBC dose
BALB /c
ICR /)cl
Nil Normal LMS-treated
2 x I0 s 2 x 108 2 x 108
6000 _+270 6170 + 360 3150 _+ 150 c
20700 _+ 1450 19600 __ 780 9900 _+ 1300 c
Nil Normal LMS-treated
2 x 107 2 x 107 2 x 107
263_+ 30 249 ___ 41 673 _+ 33c
569_+ 42 605 _+ 37 1010 _+ 105 c
aSpleen cells (2 x 107) from LMS-treated donor mice were transferred to recipient mice four days after the drag administration.Immediatelyafter the celltransfer,the recipientswere immunizedwith either 2 × 108 or 2 x 107 SRBC. bValues are mean PFC __ SEM of five mice per group. cSignificantlydifferentfrom control, p < 0.01. dose). We found that the PFC response in the recipients was enhanced either when the recipients were transferred a smaller number (2 x 10s) of the spleen cells and immunized with an optimal antigen dose (2 x 10s) of SRBC, or when the recipients were transferred a larger number (2 × 107) of the spleen cells and immunized with a suboptimal dose (2 × 107) of the antigen. On the other hand, the PFC response was suppressed only when the recipients were transferred the larger number of the spleen cells and immunized with the optimal antigen dose of SRBC. Thus, it seems likely that the supressor cell activity appears only when the degree of total stimulation in the transferred spleen cells by the antigen reaches a certain threshold. The essential role of T cells in the LMS-induced suppression was also suggested by the finding that the treatment of spleen cells from LMS-treated donors with anti-Thy 1 antiserum plus complement relieved the suppression in the PFC response in the recipients. Since B cells are resistant to the above treatment, suppressor B cells, which were assumed to be important in the feedback suppression of humoral immune response in a variety of systems (Stockinger et al., 1979; Shimamura et al., 1982), are not responsible for the suppression by LMS. Macrophage-like suppressor cells, which were reported to be nonspecific suppressor cells by Schrier et al. (1980), are also unlikely to be involved in this suppression process because only antigen-specific suppressor cells were induced by LMS. Eardley and associates (1977, 1978, 1980) have recently postulated an immunomodulatory circuit of T-cell interactions as basic elements in feedback suppression. In brief, Ly I helper T cells that are stimulated by antigen (SRBC) induce a second nonimmune set of T cells (Ly, 1,2,3 cells), which in turn either differentiate into Ly 2,3 suppressor T cells or activate Ly 2,3 suppressor T cells. These suppressor T cells regulate the activity of B cells to secrete antibody through modulation of Ly I helper T cell activity. Since, a) T cells may play an essential role in LMS-induced suppression; b) LMS may induce precursors of both helper and suppressor cells in the spleen; c) the differentiation and/or maturation of LMS-primed spleen cells to either helper or suppressor cells in the recipients is influenced by the amount of antigen injected and the number of spleen cells transferred (Table 3 and Flgure 3); and d) a dose of 10 mg/kg LMS actually shifts the peak response in PFC earlier than the control (Figure 2), the above described T-cell-mediated feedback regulation may be involved in LMS-induced suppression. Thus, the following interpretation could be compatible with our findings. LMS primarily induces precursors of both helper and suppressor T cells. Subsequent to antigen (SRBC) injection, the helper precursors are preferentially stimufated by the antigen to form mature helper cells. These mature cells may eventually induce antigen-specific suppressor T cells.
Suppressor Cells in Levamisole-treated Mice
287
The authors acknowledgewiththanks MissMikikoTomatsufor technicalassistanceand Drs. IsaoKanekoand Yoshihiko Baba for advice and encouragement.
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