Clin. exp. Immunol. (1977) 30, 317-322.
Some immunological effects of penicillamine D.-M. CHEN, G. DI SABATO, L. FIELD,* A. A. GALLO* & S. HARSHMANt Department of Molecular Biology, and *Department ofChemistry, and tDepartment ofMicrobiology (School ofMedicine), Vanderbilt University, Nashville, Tennessee, U.S.A. (Received 4 April 1977)
SUMMARY
Immunological effects of D- and D,L-penicillamine (PA) were studied in efforts to develop assays for syntheticD or D,L analogs and to contribute to the understanding of the mechanism(s) of action of 1-PA in rheumatoid arthritis. At the highest doses tolerated by mice, D,L-PA did not significantly inhibit the development of haemagglutinating antibodies in vivo. In studies in vitro with T lymphocytes, D-PA at 1 mm concentration inhibited both concanavalin A- and phytohaemagglutinin-induced transformation as assayed by [3H]thymidine incorporation, but D-PA concentrations of 5 mm were required to inhibit concanavalin A-induced amino acid uptake. No effect of D-PA was observed either on the induction of cytotoxic T cells or on the attack of specifically sensitized T cells on target cells. It is of interest that D-PA at 1 mm concentration did inhibit lipopolysaccharide-induced transformation, which predominately stimulates B lymphocytes. The effects of PA on the induced transformation of T and B cells deserve further attention for studies with analogs of PA. INTRODUCTION
The D enantiomer of penicillamine [PA, 13,f3-dimethylcysteine (CH3)2C(SH)CH(NH2)(COOH)] affords effective treatment for active rheumatoid arthritis, although adverse reactions may require withdrawal with some patients (Multicentre Trial Group, 1973). Various aspects of the chemistry and pharmacology of PA have been extensively reviewed (Lyle, 1973) and various explanations have been offered for the action of this compound on rheumatoid arthritis (for examples and references see Lyle, 1973; Jaffe, 1965, 1970, 1975; Jaffe, Merryman & Ehrenfield, 1974; Sweetman et al., 1971). Nevertheless, no satisfying explanation is yet at hand for the effect of PA in this disease, nor does any consensus seem to be developing. Since 'there is ample evidence that immunologic mechanisms play an important role in the induction and maintenance of the rheumatoid inflammatory process' (see Jasin et al., 1973, for leading references), the possibility that the action of PA in rheumatoid arthritis involves immunological mechanisms requires careful scrutiny and that a search for superior agents might well best be based on immunological parameters. In studies of patients with rheumatoid arthritis, D-PA has been reported to lower significantly serum immunoglobulin and C3 levels (Bluestone & Goldberg, 1973; Huskisson & Berry, 1974; Jaffe, 1975). However, none of these studies demonstrated suppression of the humoral immune response towards a number of test antigens. Similarly, clinical studies of D-PA effects on cellular immunity failed to show significant suppression (Crouzet et al., 1972), although it was reported that a subnormal number of T lymphocytes was observed (Brandt & Svensson, 1975). Recent studies by Roath & Wills (1974) demonstrated that D,L-PA inhibited in vitro the phytohaemagglutinin (PHA) induced transformation of human lymphocytes. The significance of T and B cells in rheumatoid arthritis has been reviewed recently by Messner (1974) and it is clear that, if we are to understand the effectiveness of D-PA in this disease, a Correspondence: Dr G. Di Sabato, Department of Molecular Biology, Box 1820, Station B, Vanderbilt University, Nashville, Tennessee 37235, U.S.A. K
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better understanding of the effects of D-PA on T and B cells is crucial. Whether D and/or D,L-PA cause discernible immunological effects in animals seems uncertain (Altman & Tobin, 1965; Hubner & Gengozian, 1965; Liyanage & Currey, 1972). For the purpose of evaluating less toxic and/or more active counterparts of PA that we wish to synthesize (Sweetman et al., 1971; Field et al., 1973), it is essential that a suitable test system be devised. Since organic synthesis frequently produces D,L mixtures of enantiomers from which resolution of one enantiomer may not be easy, we sought a suitable assay system for evaluating the response to D,L as well as to D-PA. The purpose of our work was to assess possible effects of D,L-PA on the level of circulating antibodies* and to examine the effect of D-PA on T and B lymphocytes. While our work was in progress, Schumacher, Maerker-Alzer & Schaaf (1975b) reported that although D-PA had no effect on the formation of haemagglutinating antibodies at levels of 200 mg/kg, it inhibited the formation of rosettes and, to a lesser extent, of plaque-forming IgM antibodies. They also reported that D-PA inhibited the responsiveness of human lymphocytes to PHA, concanavalin A (Con A) or tuberculinpurified protein derivative (1975a). The results reported here confirm and extend a number of their findings.
MATERIALS AND METHODS Effects of D,L-PA on levels ofcirculating antibodies. * Animals. Swiss Webster random-bred mice (Harlan Farms, Indiana) of both sexes, averaging 20-25 g were used. The mice were given Purina Animal Chow and water ad libitum. Preparation of PA solution. D,L-Penicillamine (Aldrich, 99% Gold Label) was dissolved in phosphate-buffered saline (PBS), pH 7-3, to give a solution 0-14 M in D,L-penicillamine. The solution was sterilized by filtration, stored in an air-tight container and administered by intraperitoneal (i.p.) injection 15 min after preparation. Preparation of sheep red blood cells (SRBC). SRBC in Alsevers' solution (Microbiological Associates, Maryland) were stored at 4VC and used within 7 days of delivery. SRBC were washed three times with PBS, and a 10% suspension of SRBC in PBS was used as the antigen. Treatment of animals and separation of serum. A collection of sixty mice was randomly divided into two groups. One group received 108 mg/kg/day of D,L-PA for 6 days. This dose was lethal within 5 days for 20-25% of the animals. On the seventh day (day 0) each mouse (both D,L-PA-treated and untreated controls) received 0-1 ml of SRBC, i.p. The treated group continued to receive i.p. injections of D,L-PA until the day of blood sampling. The control animals received 0-1 ml of PBS i.p. instead of D,L-PA. Blood was taken by cardiac puncture after anaesthesia on days 4, 7, and 12 after antigen injection (groups (a), (b), and (c) respectively). The samples were centrifuged in sterile blood tubes (1000 rev/min), and the serum was separated and kept at - 200C until titrated. Titration of serum. Samples of serum from each animal were titrated individually. Dilutions were made such that 0 25 ml of serum and 0-75 ml of PBS were placed in tube 1. Thereafter, serial two-fold dilutions of 0 5 ml were made in PBS for twelve tubes. 0 5 ml of 0.5% SRBC in PBS were added to each tube, mixed, and left at room temperature for 4 hr. The titres were read as the reciprocal of the highest dilution giving visible agglutination and expressed as log2. Effects of D-PA on Con A-, PHA- and LPS-induced transformation of lymphocytes. Animals. Male mice of strains CBA/J about 8 weeks of age were obtained from Jackson Laboratory, Bar Harbor, Maine. Materials. RPMI 1640 medium containing 10,000 units of penicillin and 10,000 ug of streptomycin per 100 ml was used throughout for preparing solutions of n-PA (Aldrich n-penicillamine, puriss.) and suspensions of all other materials. Pooled human serum (PHS) was obtained from the American Red Cross and decomplemented at 560C for 30 min. PHA was purchased from Burroughs-Wellcome, lipopolysaccharide B (LPS; Escherichia coli 0111 :B4) from Difco Laboratories, Con A from Sigma Chemical Company and [3H]thymidine (sp. act. 1 mCi/0 005 mg) from New England Nuclear Company. Methods. Experiments to stimulate lymphocytes with PHA, Con A and LPS were carried out as previously described (Di Sabato, Chen & Erickson, 1975). Briefly, single cell suspensions from spleens of CBA mice were incubated in microwells of Falcon disposable plates with RPMI 1640, 10% PHS and, when required, appropriate concentrations of mitogen and n-PA. After 48 hr ofincubation at 370C in a humidified atmosphere of 95% air and 5% C02, the microcultures were labelled with 1 JUCi of [3H]thymidine. Cells were harvested 24 hr later (Di Sabato et al., 1975). The incorporation of [3H]thymidine was taken as a measure of lymphocyte transformation. Optimal amounts of mitogen for the stimulation of murine spleen lymphocytes were established in previous work (Di Sabato et al., 1975). LCM studies with D-PA. In phase 1 of these experiments (kindly carried out by P. C. Doherty and M. Dunlop), a daily dose of 100 mg/kg of D-PA was injected i.p. into each of five adult mice for 3 days. Lymphocytic choriomeningitis virus * Note that D,L-PA rather than D-PA was used only in the circulating antibody studies. The reason for this exception is given in the two sentences preceding the mention of such use in the Introduction. The D,L and D forms, of course, are not interchangeable.
Immunological effects of penicillamine
319
(LCM) was injected intracerebrally, after which D-PA was continued daily for 7 more days. Specific 51Cr release from LCMinfected fibroblasts overlaid with LCM-immune spleen cells from the mice treated with rPA was then determined (Zinkernagel & Doherty, 1974). In phase 2 of the assay, target cells (fibroblast and mastocytoma cells) were cultured together with LCM-immune spleen cells at a ratio of 6: 1 and 30: 1 -PA (1 mM solution) was present when appropriate.
RESULTS Effect of D,L-PA on levels of circulating antibodies Each of the groups (a), (b) and (c) (see Methods) initially comprised twenty mice, ten being treated with DL-PA and ten serving as controls. Group (d) was an untreated control group. There was a 30% mortality rate within each group treated with D,L-PA, which may be attributed to the use of the racemic form since Kuchinskas & du Vigneaud (1957) have shown that the L-isomer is relatively more toxic to rats and inhibits growth. Clearly there is no significant effect of D,L-PA on the titres of haemagglutinating antibodies (Table 1). Calculated F-test ratios were less than the significance limit at the 95% confidence level (P
Some immunological effects of penicillamine D.-M. CHEN, G. DI SABATO, L. FIELD,* A. A. GALLO* & S. HARSHMANt Department of Molecular Biology, and *Department ofChemistry, and tDepartment ofMicrobiology (School ofMedicine), Vanderbilt University, Nashville, Tennessee, U.S.A. (Received 4 April 1977)
SUMMARY
Immunological effects of D- and D,L-penicillamine (PA) were studied in efforts to develop assays for syntheticD or D,L analogs and to contribute to the understanding of the mechanism(s) of action of 1-PA in rheumatoid arthritis. At the highest doses tolerated by mice, D,L-PA did not significantly inhibit the development of haemagglutinating antibodies in vivo. In studies in vitro with T lymphocytes, D-PA at 1 mm concentration inhibited both concanavalin A- and phytohaemagglutinin-induced transformation as assayed by [3H]thymidine incorporation, but D-PA concentrations of 5 mm were required to inhibit concanavalin A-induced amino acid uptake. No effect of D-PA was observed either on the induction of cytotoxic T cells or on the attack of specifically sensitized T cells on target cells. It is of interest that D-PA at 1 mm concentration did inhibit lipopolysaccharide-induced transformation, which predominately stimulates B lymphocytes. The effects of PA on the induced transformation of T and B cells deserve further attention for studies with analogs of PA. INTRODUCTION
The D enantiomer of penicillamine [PA, 13,f3-dimethylcysteine (CH3)2C(SH)CH(NH2)(COOH)] affords effective treatment for active rheumatoid arthritis, although adverse reactions may require withdrawal with some patients (Multicentre Trial Group, 1973). Various aspects of the chemistry and pharmacology of PA have been extensively reviewed (Lyle, 1973) and various explanations have been offered for the action of this compound on rheumatoid arthritis (for examples and references see Lyle, 1973; Jaffe, 1965, 1970, 1975; Jaffe, Merryman & Ehrenfield, 1974; Sweetman et al., 1971). Nevertheless, no satisfying explanation is yet at hand for the effect of PA in this disease, nor does any consensus seem to be developing. Since 'there is ample evidence that immunologic mechanisms play an important role in the induction and maintenance of the rheumatoid inflammatory process' (see Jasin et al., 1973, for leading references), the possibility that the action of PA in rheumatoid arthritis involves immunological mechanisms requires careful scrutiny and that a search for superior agents might well best be based on immunological parameters. In studies of patients with rheumatoid arthritis, D-PA has been reported to lower significantly serum immunoglobulin and C3 levels (Bluestone & Goldberg, 1973; Huskisson & Berry, 1974; Jaffe, 1975). However, none of these studies demonstrated suppression of the humoral immune response towards a number of test antigens. Similarly, clinical studies of D-PA effects on cellular immunity failed to show significant suppression (Crouzet et al., 1972), although it was reported that a subnormal number of T lymphocytes was observed (Brandt & Svensson, 1975). Recent studies by Roath & Wills (1974) demonstrated that D,L-PA inhibited in vitro the phytohaemagglutinin (PHA) induced transformation of human lymphocytes. The significance of T and B cells in rheumatoid arthritis has been reviewed recently by Messner (1974) and it is clear that, if we are to understand the effectiveness of D-PA in this disease, a Correspondence: Dr G. Di Sabato, Department of Molecular Biology, Box 1820, Station B, Vanderbilt University, Nashville, Tennessee 37235, U.S.A. K
317
318
D.-M. Chen et al.
better understanding of the effects of D-PA on T and B cells is crucial. Whether D and/or D,L-PA cause discernible immunological effects in animals seems uncertain (Altman & Tobin, 1965; Hubner & Gengozian, 1965; Liyanage & Currey, 1972). For the purpose of evaluating less toxic and/or more active counterparts of PA that we wish to synthesize (Sweetman et al., 1971; Field et al., 1973), it is essential that a suitable test system be devised. Since organic synthesis frequently produces D,L mixtures of enantiomers from which resolution of one enantiomer may not be easy, we sought a suitable assay system for evaluating the response to D,L as well as to D-PA. The purpose of our work was to assess possible effects of D,L-PA on the level of circulating antibodies* and to examine the effect of D-PA on T and B lymphocytes. While our work was in progress, Schumacher, Maerker-Alzer & Schaaf (1975b) reported that although D-PA had no effect on the formation of haemagglutinating antibodies at levels of 200 mg/kg, it inhibited the formation of rosettes and, to a lesser extent, of plaque-forming IgM antibodies. They also reported that D-PA inhibited the responsiveness of human lymphocytes to PHA, concanavalin A (Con A) or tuberculinpurified protein derivative (1975a). The results reported here confirm and extend a number of their findings.
MATERIALS AND METHODS Effects of D,L-PA on levels ofcirculating antibodies. * Animals. Swiss Webster random-bred mice (Harlan Farms, Indiana) of both sexes, averaging 20-25 g were used. The mice were given Purina Animal Chow and water ad libitum. Preparation of PA solution. D,L-Penicillamine (Aldrich, 99% Gold Label) was dissolved in phosphate-buffered saline (PBS), pH 7-3, to give a solution 0-14 M in D,L-penicillamine. The solution was sterilized by filtration, stored in an air-tight container and administered by intraperitoneal (i.p.) injection 15 min after preparation. Preparation of sheep red blood cells (SRBC). SRBC in Alsevers' solution (Microbiological Associates, Maryland) were stored at 4VC and used within 7 days of delivery. SRBC were washed three times with PBS, and a 10% suspension of SRBC in PBS was used as the antigen. Treatment of animals and separation of serum. A collection of sixty mice was randomly divided into two groups. One group received 108 mg/kg/day of D,L-PA for 6 days. This dose was lethal within 5 days for 20-25% of the animals. On the seventh day (day 0) each mouse (both D,L-PA-treated and untreated controls) received 0-1 ml of SRBC, i.p. The treated group continued to receive i.p. injections of D,L-PA until the day of blood sampling. The control animals received 0-1 ml of PBS i.p. instead of D,L-PA. Blood was taken by cardiac puncture after anaesthesia on days 4, 7, and 12 after antigen injection (groups (a), (b), and (c) respectively). The samples were centrifuged in sterile blood tubes (1000 rev/min), and the serum was separated and kept at - 200C until titrated. Titration of serum. Samples of serum from each animal were titrated individually. Dilutions were made such that 0 25 ml of serum and 0-75 ml of PBS were placed in tube 1. Thereafter, serial two-fold dilutions of 0 5 ml were made in PBS for twelve tubes. 0 5 ml of 0.5% SRBC in PBS were added to each tube, mixed, and left at room temperature for 4 hr. The titres were read as the reciprocal of the highest dilution giving visible agglutination and expressed as log2. Effects of D-PA on Con A-, PHA- and LPS-induced transformation of lymphocytes. Animals. Male mice of strains CBA/J about 8 weeks of age were obtained from Jackson Laboratory, Bar Harbor, Maine. Materials. RPMI 1640 medium containing 10,000 units of penicillin and 10,000 ug of streptomycin per 100 ml was used throughout for preparing solutions of n-PA (Aldrich n-penicillamine, puriss.) and suspensions of all other materials. Pooled human serum (PHS) was obtained from the American Red Cross and decomplemented at 560C for 30 min. PHA was purchased from Burroughs-Wellcome, lipopolysaccharide B (LPS; Escherichia coli 0111 :B4) from Difco Laboratories, Con A from Sigma Chemical Company and [3H]thymidine (sp. act. 1 mCi/0 005 mg) from New England Nuclear Company. Methods. Experiments to stimulate lymphocytes with PHA, Con A and LPS were carried out as previously described (Di Sabato, Chen & Erickson, 1975). Briefly, single cell suspensions from spleens of CBA mice were incubated in microwells of Falcon disposable plates with RPMI 1640, 10% PHS and, when required, appropriate concentrations of mitogen and n-PA. After 48 hr ofincubation at 370C in a humidified atmosphere of 95% air and 5% C02, the microcultures were labelled with 1 JUCi of [3H]thymidine. Cells were harvested 24 hr later (Di Sabato et al., 1975). The incorporation of [3H]thymidine was taken as a measure of lymphocyte transformation. Optimal amounts of mitogen for the stimulation of murine spleen lymphocytes were established in previous work (Di Sabato et al., 1975). LCM studies with D-PA. In phase 1 of these experiments (kindly carried out by P. C. Doherty and M. Dunlop), a daily dose of 100 mg/kg of D-PA was injected i.p. into each of five adult mice for 3 days. Lymphocytic choriomeningitis virus * Note that D,L-PA rather than D-PA was used only in the circulating antibody studies. The reason for this exception is given in the two sentences preceding the mention of such use in the Introduction. The D,L and D forms, of course, are not interchangeable.
Immunological effects of penicillamine
319
(LCM) was injected intracerebrally, after which D-PA was continued daily for 7 more days. Specific 51Cr release from LCMinfected fibroblasts overlaid with LCM-immune spleen cells from the mice treated with rPA was then determined (Zinkernagel & Doherty, 1974). In phase 2 of the assay, target cells (fibroblast and mastocytoma cells) were cultured together with LCM-immune spleen cells at a ratio of 6: 1 and 30: 1 -PA (1 mM solution) was present when appropriate.
RESULTS Effect of D,L-PA on levels of circulating antibodies Each of the groups (a), (b) and (c) (see Methods) initially comprised twenty mice, ten being treated with DL-PA and ten serving as controls. Group (d) was an untreated control group. There was a 30% mortality rate within each group treated with D,L-PA, which may be attributed to the use of the racemic form since Kuchinskas & du Vigneaud (1957) have shown that the L-isomer is relatively more toxic to rats and inhibits growth. Clearly there is no significant effect of D,L-PA on the titres of haemagglutinating antibodies (Table 1). Calculated F-test ratios were less than the significance limit at the 95% confidence level (P
