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Nonspecific activation of murine lymphocytes
CELLULAR
IMMUNOLOGY
65, 337-351 (1981)
Nonspecific
Activation
of Murine Lymphocytes
VIII. Effects of o-Penicillamine’ MICHAEL G. GOODMAN* AND WILLIAM Department
of Immunopathology, La Jolla, Received
July
0. WEIGLE
Scripps Clinic and Research California 92037
21, 1981; accepted
October
Foundation,
25. 1981
The effects of D-penicillamine on proliferation and polyclonal activation of lymphocytes were studied in cultures of spleen cells from a variety of murine strains. Inclusion in serumfree or serum-containing medium of optimal concentrations of D-penicillamine resulted in the uptake of tritiated thymidine in a dose-dependent fashion, with both lower and higher doses causing less marked effects. The kinetic peak of these responses was found to occur at Day 2 of culture. Experiments examining the responsiveness of B-cell-enriched and T-cell-enriched populations demonstrated that D-penicillamine, like 2-mercaptoethanol, stimulates both types of cell. The magnitude of the response remained unchanged in populations depleted of adherent cells. In correlation with previous results seen for 2-ME, tqenicillamine was mitogenically active both in reduced and oxidized forms. The oxidized form failed to enhance the response to LPS, in contrast to the marked effect of the reduced form. Additionally, D-penicillamine failed to evoke a mitogenic response from B cells of CBA/N mice, a strain characterized by a deficit in the function of a particular set of mature B cells. Young mice from autoimmune strains responded to D-penicillamine as well as normal mice did. No relationship could be observed between responsiveness to tr-penicillamine and the H-2 phenotype of the cell donor, and in A/J mice. hyporesponsiveness was shown to be a function of their background rather than H-2. D-Penicillamine was found to function as a polyclonal B-cell activator, and to significantly enhance the primary humoral immune response to sheep erythrocytes in vitro. These immunomodulatory effects of rqenicillamine are discussed in relation to possible pathogenetic mechanisms of its spectrum of autoimmune side effects.
INTRODUCTION The use of D-penicillamine in the treatment of rheumatoid arthritis has become increasingly accepted since its use was extended to this disease by Dresner and Trombly (1) and by Griffin et al. (2). It currently constitutes one of the small number of effective therapeutic modalities for the treatment of rheumatoid arthritis ’ This is publication No. 2305 from the Department of Immunopathology, Scripps Clinic and Research Foundation, La Jolla, Calif. This work was supported in part by United States Public Health Service Grant AI07007, American Cancer Society Grant IM-421, and Biomedical Research Support Grant RRO-55 14. * Recipient of United States Public Health Service Grant AI15284 and United States Public Health Service Research Career Development Award A100374. 337 OOOS-8749/81/180337-15$02.00/O Copyright Q 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.
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(RA).3 Its efficacy has been established by a number of clinical trials (3-6) and its use in the treatment of RA has been sanctioned by the FDA. The mechanism of action by which D-penicillamine ameliorates this disease is unclear. One suggestion well supported by experimental evidence is that this agent acts in an immunosuppressive capacity to diminish the immune responsiveness of patients with rheumatoid arthritis. Thus, penicillamine has been noted to depress levels of circulating rheumatoid factor and frequently also of immunoglobulin as well as immune complexes (7, 8). Several studies have demonstrated that D-penicillamine depresses the primary humoral immune response of experimental animals in vivo (9, 10) and diminishes mitogenic activation of human peripheral blood lymphocytes in vitro (11). Recently, Lipsky and Ziff (12) have demonstrated in vitro suppression of the primary humoral immune response of human PBL mediated by a selective suppression of the helper T-cell arm of this response. Clinical use of D-penicillamine, however, is limited by its high rate of complication. Among these are the group of so-called “autoimmune” side effects which include myasthenia gravis, thyroiditis, polymyositis and dermatomyositis, autoimmune hemolytic anemia, thrombotic thrombocytopenic purpura, and a drug-induced lupus-like syndrome, among others. Case reports of undesirable immunemediated side effects of penicillamine therapy suggest that the drug, rather than being solely immunosuppressive, may possess a variety of immunoregulatory properties, i.e., it may have immunopotentiating or activating effects as well. The report of Tobin and Altman (13) concerning the enhancement of the humoral immune response in vivo by D-penicillamine in rabbits supports such a concept. In previous studies we have explored the immunopotentiating and de novo-activating properties of 2-mercaptoethanol, a low MW hydroxy-thiol compound. Our observations that several other functional groups are active when present on thiol compounds led us to probe the effects of D-penicillamine (/3-mercaptovaline) with respect to lymphocyte activation. Results of the current communication indicate that in normal mice, mitogenicity, polyclonal B-cell activation, and adjuvanticity are properties of this compound. MATERIALS
AND
METHODS
Mice. C3H/St, B6D2FI, BlO, BlO.A, and Balb/c mice, 8-12 weeks of age, were obtained from the mouse breeding facility at Scripps Clinic and Research Foundation, La Jolla, California. CBA/CaJ, A/J, C57B l/65 and SJL mice, 8- 12 weeks of age, were purchased from the Jackson Laboratory, Bar Harbor, Maine. A breeding nucleus of CBA/N mice was provided by the animal production section, National Institutes of Health, Bethesda, Maryland. NZW, NZB, and BxWFl female mice, 8-12 weeks of age, were obtained from the Jackson Laboratory. All mice were maintained on Wayne Lab-Blox F6 pellets (Allied Mills, Inc., Chicago, Ill.) and chlorinated water acidified with HCl to a pH of 3.0. Culture reagents. Constituents of the serum-free culture medium employed in these studies have been described elsewhere (14). For serum-containing medium, supportive FCS was substituted for 5% of the volume of RPM1 1640, and Hepes ’ Abbreviations used: olTG, athioglycerol; ME, 2-mercaptoethanol; RA, rheumatoid phenyl.
Ig, immunoglobulin; LPS, bacterial lipopolysaccharide; arthritis, SRBC, sheep red blood cells; TNP, 2,4,6-trinitro-
2-
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buffer was omitted. Escherichia coli 055:B5 LPS, prepared by the Boivin procedure, was purchased from the Difco Laboratories, Detroit, Michigan. D-Penicillamine was purchased from the Sigma Chemical Company, St. Louis, Missouri, and from the Aldrich Chemical Company, Milwaukee, Wisconsin. D-Penicillamine disulfide was purchased from the Aldrich Chemical Company. LPS and D-penicillamine were diluted in phosphate-buffered saline and sterilized by filtration. Further dilution in complete medium was used for microcultures. D-Penicillamine was made up fresh from powder for each experiment. Cell preparation. Spleen cell suspensions were prepared as described previously ( 14). Spleen cells enriched for T lymphocytes were prepared by passage through nylon wool (NW) columns, as described previously (15). B-Cell-enriched populations were prepared by treating lo8 spleen cells with a 1: 1000 dilution of monoclonal anti-Thy 1.2 (New England Nuclear, Boston, Mass.) for 30 min at 4°C. Treated cells were then centrifuged at 280g for 10 min, the antibodies removed, and the cells resuspended in a I:6 dilution of C3H RBC-absorbed guinea pig complement at 37°C for 45 min. Cells were then washed and cultured as above. Adherent cells were depleted by passage over columns of Sephadex G-10 as described by Ly and Mishell(l6). This procedure reduced the percentage of esterasepositive cells from 7% in unseparated spleen cells to approximately 0.1% in nonadherent populations. Lymphocyte cultures. Murine spleen cells were cultured in microculture plates (No. 3042, Falcon Plastics, Oxnard, Calif.) at a cell density of 5 X IO6 viable cells/ ml in a volume of 0.1 ml ( 17). Microcultures were incubated at 37°C in a humidified atmosphere of 10% CO2 in air. Cultures were fed daily with 8 ~1 of nutritional cocktail ( 18). Measurement of DNA synthesis. During the final 24 hr of culture, cells were radiolabeled with 1.O &i of [ 3H]TdR/culture (5 Ci/mM, Amersham Radiochemicals, Amersham, England). The microcultures were harvested with a Brandel cell harvester, Model M24V (Biological Research and Development Laboratories, Rockville, Md.) onto glass fiber filter strips (Reeve Angel, Clifton, N.J.). Filter disks were transferred to plastic scintillation vials (Kimball Products, Owens-Illinois, Toledo, Ohio) covered with liquid scintillation cocktail (Scintiverse, Fisher Scientific Co., Fairlawn, N.J.) and counted in a Beckman LS-230 liquid scintillation counter. Assay of plaque-forming cells (PFC). For polyclonal activation, 10 X 1O6 spleen cells/ml were incubated in a l.O-ml volume of 5% FCS-containing medium. PFC to TNP were assayed after 2 days of culture ( 19) using a modification of the hemolytic plaque assay of Jerne and Nordin (20). For the primary humoral response to SRBC, 10 X lo6 murine spleen cells were cultured in 1 ml of 5% FCS containing medium for 4 days in the presence of 2 X lo6 SRBC/culture. RESULTS Dose dependency of the DNA-synthetic response to D-penicillamine. The ability of D-penicillamine to activate DNA synthesis was investigated in cultures of spleen cells from C3H/St mice. Spleen cells were incubated in serum-free medium in the presence of variable concentrations of D-penicillamine. Resultant activation was measured by determination of [3H]TdR uptake during the final 24 hr of d 2-day
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culture period. The results shown in Fig. 1 indicate that this thiol compound does indeed promote the uptake of tritiated thymidine in a dose-dependent manner. Lower concentrations were suboptimal while higher concentrations appeared to be inhibitory. Peak responsiveness appeared to be between 1O-3 and lo-’ it4 D-penicillamine. Peak responsiveness varied somewhat from experiment to experiment but appeared to be highly consistent within individual experiments. Kineticprofile of the response to o-penicillamine. The time at which the maximal D-penicillamine mitogenic effect occurred was assessed in C3H/St mice. Spleen cells were cultured either in the presence or absence of lop3 M D-penicillamine under serum-free culture conditions. Subsequently, one set of cultures was harvested daily after a 24-hr pulse of [3H]TdR. Maximal [3H]TdR uptake was noted to occur at Day 2 and declined thereafter (Fig. 2). Response of separately cultured B-enriched penicillamine. C3H/St spleen cell populations
and T-enriched
lymphocytes
to
D-
enriched for B cells by treatment with monoclonal anti-Thy 1.2 plus guinea pig complement, or for T cells by passage over NW columns as described, were cultured under conditions utilizing medium containing 5% FCS. Serum was used in these experiments because of the inability of separated T cells to proliferate in the absence of serum (28). The results of such experiments (Fig. 3) demonstrate that T lymphocytes and B lymphocytes can proliferate in response to D-penicillamine. In some of the experiments, the response by B cells was greater than that exhibited by T cells, and in no case was the reverse observed. B-Cell-enriched cultures failed to respond to the T-cell mitogen concanavalin A, and T-cell cultures gave only 8% of the unseparated cell response to LPS. Ability of o-penicillamine to activate nonadherent spleen cells. In order to gain
Concentration of D.Penlcillamine IMI
FIG. 1. Dose-response profile for D-penicillamine in serum-free culture. 5 X lo5 viable C3H/St spleen cells were cultured for 2 days in 0.1 ml of serum-free medium in the presence of various concentrations of D-penicillamine. Cells were pulsed with 1.0 pCi of [‘H]TdR for the final 24 hr of culture. Results are expressed as the arithmetic mean of five replicate cultures k the SE.
LYMPHOCYTE
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1 t I
I 1
2
3
Day al Harvest FIG. 2. Kinetic profile of the response to D-penicillamine. 5 X lo5 viable C3H/St spleen cells were cultured in 0.1 ml of serum-free medium in the presence or absence of lo-’ M D-penicillamine. On each day, one set of parallel cultures was harvested after a 24-hr pulse of 1.0 pCi of [3H]TdR. Results are expressed as the arithmetic mean of five replicate cultures containing D-penicillamine minus that of five replicate control cultures + the SE. Control values were 6630 + 510, 2310 + 190, and 1540 f 180 on Days 1, 2, and 3, respectively.
insight into the role played by adherent cells in the response to D-penicillamine, experiments were designed to determine whether the proliferative response to this thiol compound is mediated through such cells or is independent of them. C57Bl/ 65 spleen cells were passed over Sephadex G-10 columns and the effluent cells used as the source of nonadherent cells. Content of esterase-positive cells was greatly diminished in this population (see Materials and Methods). The results of these experiments demonstrate that nonadherent cells respond to D-penicillamine to the same degree as do unseparated cells (Fig. 4). Effects of reduced and oxidized o-penicillamine on murine spleen cells. Previous work has indicated that 2-ME, in addition to having mitogenic effects, is able to enhance the in vitro response of murine spleen cells to mitogens ( 15, 21, 22). In preliminary experiments, it was learned that D-penicillamine could similarly enhance the mitogenic response to bacterial LPS. Therefore, experiments were designed to investigate the requirement for free sulfhydryl groups both in D-penicillamine-mediated mitogenesis and in its LPS-enhancing effects. C3H/St spleen cells were incubated in the presence of incremental concentrations of either reduced or oxidized o-penicillamine under serum-free culture conditions (Fig. 5). Under these circumstances, both compounds were found to have mitogenic effects. The magnitude of the responses was generally equivalent, although lower doses of the disulfide were more effective than the equivalent doses of the thiol, resulting in a slight shift of the dose-response curve to the left for the disulfide. When these same
342
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WEIGLE
Concentration of Wenlcillamine
IMI
FIG. 3. Response of B-enriched and T-enriched lymphocyte cultures to D-penicillamine. 5 X 10’ viable C3H/St B-enriched, T-enriched, or unseparated splenic lymphocytes were cultured in 0.1 ml of 5% FCS-containing medium for 2 days in the presence of various concentrations of o-penicillamine. Cells were pulsed with 1.0 pCi of [‘H]TdR for the final 24 hr of culture. Results are expressed as the arithmetic mean of five replicate cultures containing D-penicillamine minus control cultures + the SE. Control values were 1920 + 220, 1530 + 250, and 880 f 120 for unseparated, B, and T cells, respectively.
two compounds were evaluated for their ability to enhance the mitogenic response to LPS, it was found that only reduced D-penicillamine was capable of enhancing this response (Fig. 6). This effect occurred at the same dosage range as the optimal concentration for mitogenesis by D-penicillamine. Nonetheless, it is unlikely that this effect can be accounted for by a simple additivity of mitogenic responses, since the magnitude of the increased LPS response was considerably greater than that of the mitogenic response within the same experiment and since the disulfide was ineffective. Effect of o-penicillamine in mice with an X-linked B-cell defect. Characterization of the immune deficit of the CBA/N mouse has been carried out by a number of investigators. Amsbaugh et al. (23) first described an X-linked defect in the CBA/N response to thymus-independent antigens. These animals have subsequently been reported to contain a diminished number of Ig-bearing cells in their spleens (24), to mount subnormal responses to thymus-dependent antigens (25), and to possess cell surface Ig and microdensities and organ distributions which suggest a deficit of mature B lymphocyte (26). Moreover, work characterizing the nature of the B-cell subset responsive to 2-ME demonstrated that the subpopulation of B cells responding to 2-ME was a mature set which was functionally absent in the CBA/N mouse (15). Therefore, it was of interest to assess the reactivity of cultures of CBA/N B cells to D-penicillamine. B cells were prepared as described under Materials and Methods. A direct comparison of the responsiveness of B lymphocytes derived from this strain with those from the histocompatible CBA/ CaJ strain (Fig. 7) indicates that in contrast to the CBA/CaJ response (generally
LYMPHOCYTE
ACTIVATION
BY D-PENICILLAMINE
343
I 0
3110’
10’
Concentration
3r10
10’
of LLPenicillamine [MI
FIG. 4. Ability of D-penicillamine to activate unseparated and G-10 passed spleen cells. 5 X 10’ viable C57BL/6J spleen cells, either unseparated (-) or passed over column of Sephadex G-10 (- - -) were cultured for 2 days in 0.1 ml of serum-free medium in the presence of various concentrations of Dpenicillamine. Cultures were pulsed with 1.O &i of [‘H]TdR for the final 24 hr of culture. Results are expressed as the arithmetic mean of five replicate cultures + the SE.
Concentratlan IMI
FIG. 5. Effects of reduced and oxidized D-penicillamine on murine spleen cells. 5 X lo5 viable C3H/ St spleen cells were cultured for 2 days in 0.1 ml of serum-free medium in the presence of various concentrations of either the thiol or the disulfide form of D-penicillamine. Cultures were pulsed with 1.0 pCi of [‘H]TdR for the final 24 hr of culture. Results are expressed as the arithmetic mean of five replicate cultures containing D-penicillamine minus control cultures & the SE. Control uptake was 1310 + 230 cpm.
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GOODMAN.AND
10.’ Concentration
WEIGLE
10-1 10.’ of O-Penicillamine IMI
FIG. 6. Ability of reduced and oxidized tqenicillamine to enhance the mitogenic response to LPS. 5 X lo5 viable C3H/St spleen cells were cultured for 2 days in 0.1 ml of serum-free medium together with 100 &g/ml LPS in the presence of various concentrations of either the thiol or disultide form of D-penicillamine. Cells were pulsed with 1.0 @i of [‘H]TdR for the final 24 hr of culture. Results are expressed as the arithmetic mean of five replicate cultures k the SE.
of similar magnitude to that of the C3H/St), unresponsive to D-penicillamine.
B cells from the CBA/N
mouse were
Ability of o-penicillamine to induce polyclonal B-cell activation. Since mitogenicity and polyclonal B-cell activation are frequently very closely linked, the ability of D-penicillamine to promote polyclonal immunoglobulin secretion was evaluated. Adherent cell-depleted spleen cells ( 107) were cultured in 5% FCScontaining medium in the presence of incremental concentrations of D-penicillamine. This agent was found to be a weak polyclonal B-cell activator, significant responses being detectable only on SRBC conjugated with TNP rather than on SRBC alone (Fig. 8). Peak responsiveness again was within the range seen for peak mitogenicity. Ability of o-penicillamine to activate spleen cells from normal and autoimmune mouse strains. In order to examine the effect of autoimmune predisposition on
responsiveness to D-penicillamine, spleen cells from female C3H/St, CBA/CaJ, NZW, NZB, and NZB X NZWF, mice (8-12 weeks of age) were cultured in the presence or absence of 10e3 M D-penicillamine. The results of these experiments (Fig. 9) indicate that there is no major quantitative difference in the stimulated response to this thiol compound although background thymidine uptake varied considerably. Thus, a predisposition to develop autoimmune disease did not appear to correlate with increased susceptibility of spleen cells to activation by D-penicillamine. Ability of o-penicillamine to activate spleen cells from mice of different H-2 phenotypes. Studies were undertaken to screen for a relationship between H-2
phenotype and degree of responsiveness to D-penicillamine. Spleen cells from a variety of murine strains were cultured with a range of concentrations of this thiol compound. Peak responsiveness occurred in the range of 3-6 X lop3 M. Although H-2k and H-2d mice responded to D-penicillamine, A/J mice (a natural k/d re-
LYMPHOCYTE
ACTIVATION
0-Penicillamme
BY D-PENICILLAMINE
Concentration
345
[MI
FIG. 7. The effect of o-penicillamine in mice with an X-linked B-cell defect. 5 X 10’ viable CBA/CaJ or CBA/N splenic B lymphocytes were cultured for 2 days in 0.1 ml of serum-free medium in the presence of various concentrations of o-penicillamine. Cultures were pulsed with 1.0 &i of [‘H]TdR for the final 24 hr of culture. Results are expressed as the arithmetic mean of five replicate cultures 2 the SE.
combinant) responded poorly or not at all (Fig. 10). No other obvious relationship could be observed. In other experiments, the inability of A/J mice to respond to D-penicillamine was dissected using B 10 and B 10.A mice (Fig. 11). The data indicate that hyporesponsiveness is a property of the A/J genetic background and is not exclusively linked to its H-2 phenotype. Effect of o-penicillamine on the primary humoral response to SRBC in vitro. Work by Click et al. (27) established that the primary humoral immune response to SRBC was augmented when cultures were supplemented with 2-ME. The effect of D-penicillamine on the primary humoral response to SRBC was therefore evaluated in order to determine whether this thiol compound similarly could enhance the response to specific antigen. The data presented in Fig. 12 indicate that addition of D-penicillamine to culture results in an enhanced response to SRBC. This effect
%k lb
l@’ I@’ Concentration ot O.Penicitlamine IMI
10.’
FIG. 8. Ability of D-penicillamine to induce polyclonal B-cell activation. 10 X lo6 viable C3H/St nonadherent spleen cells were cultured for 2 days in 1.0 ml of 5% FCS-containing medium in the presence of incremental concentrations of D-pe&ihniIIe. Results are expressed as the arithmetic mean of trip licate cultures f the SE.
346
GOODMAN
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WEIGLE
:,9
*.Jr
FIG. 9. Ability of o-penicillamine to activate spleen cells from normal and autoimmune mouse strains. 5 X 10’ viable spleen cells from female C3H/St, CBA/CaJ, NZW, NZB, or NZB X WF, mice were cultured for 2 days in 0.1 ml of serum-free medium in the presence of lo-’ M o-penicillamine. Cells were pulsed with 1.0 &i of [‘H]TdR for the final 24 hr of culture. Results are expressed as the arithmetic mean of five replicate cultures k the SE. Hatched bars represent unstimulated cultures, open bars represent stimulated cultures.
was dose-dependent but occurred at lower concentrations mitogenicity or for polyclonal activation.
than were required for
DISCUSSION The effectiveness of D-penicillamine for treatment of rheumatoid arthritis has been validated by a number of clinical trials and is responsible for its increasing clinical usage. This thiol compound has been thought to function primarily as an immunosuppressor, based on in vivo (7- 10) and in vitro ( 11, 12) studies. The data presented in the current communication, however, are more compatible with the thesis that D-penicillamine is a modulator of the immune response, capable of both enhancing and initiating immune activation as well as suppressing it. Previous studies exploring the effects of 2-mercaptoethanol(2-ME) in the murine system in vitro demonstrated that certain sulfhydryl compounds induce a state of activation in resting splenic lymphocytes. Such activation involves T as well as B lymphocytes (28, 29) with the latter population of cells being induced to secrete polyclonal immunoglobulin. Igarashi et al. (30) further showed that 2-ME is responsible for the induction of polyclonal cytotoxicity in T cells. In addition, 2-ME has been shown to have immunopotentiating activity in a diversity of systems (3 l34). Therefore, the possibility that similar effects might be mediated by D-penicillamine was examined experimentally. Addition of D-penicillamine to cultures of murine spleen cells was found to result in a consistent increase in uptake of [‘H]TdR. This phenomenon occurred in a dose-related fashion, such that suboptimal doses elicited lesser responses, and supraoptimal doses caused suppression below background, similar to that seen by
LYMPHOCYTE
ACTIVATION
CBAlCaJ Ikl
Balblc (dl
AIJ la1
BY D-PENICILLAMINE
C57BWJ Ibl
SJL ISI
341
Ibldl
FIG. 10. Effect of D-penicillamine on mice of different H-2 phenotypes. 5 X 10’ viable spleen cells from CBA/CaJ, Balb/c, A/J C57BL/6J, SJL, or B6D2FI mice were cultured for 2 days in 0.1 ml of serum-free medium in the presence of medium only (solid bars), 3 X 1O-3 M D-penicillamine (hatched bars), or 6 X 1O-3 M D-pcnicillamine (open bars). Cells were pulsed with 1.0 pCi of [‘H]TdR for the final 24 hr of culture. Results are expressed as the arithmetic mean of five replicate cultures f the SE.
other investigators. The observation of increased [3H]TdR uptake by spleen cells incubated with D-penicillamine contrasts with observations by some other investigators (35), but this discrepancy is in all likelihood explained by differences in the culture system and dose range employed. When cells were cultured in serumcontaining medium, induction of significant stimulation by D-penicillamine was found to require a minimum of twofold higher concentrations of the drug, probably as a consequence of mixed disulfide formation. The data presented indicate that both B- and T-lymphocyte populations repond to D-penicillamine. A similar capacity to activate both B and T lymphocytes has been reported for 2-ME and L~TG (22, 28), although in those cases B cells have been found to be more responsive than have T cells. Macrophages do not appear to play a critical role in the activation of lymphocytes by D-penicillamine. Depletion of macrophages from spleen cells does not result in a concomitant decrease in the uptake of [3H]TdR; on the contrary, stimulation of the G-10 nonadherent cell population is nearly equivalent to that seen in unseparated cells. Both the oxidized and reduced forms of D-penicillamine induce mitogenesis in spleen cell populations to approximately the same degree, while only the reduced form enhances the mitogenic response to LPS, in parallel with data obtained for reduced and oxidized 2-ME (37). Supraoptimal D-penicillamine (as well as 2-ME) inhibits the underlying LPS response. These data suggest that the free sulfhydryl group is critical to the mitogen-enhancing effect of D-penicillamine, since the disulfide fails to demonstrate this effect. Similarly, Kendall and Hutchins (35) found that D-penicillamine enhanced the responses to concanavalin A or allogeneic cells,
348
GOODMAN
AND WEIGLE
I 0
10.’ Concentration
10” of O-Penicillamine IMI
lo”
FIG. 11. Effect of D-penicillamine in A/J, BIO, and Bl0.A mice. 5 X 10’ viable spleen cells from A/ J (0 - - - 0). BlO (A. . . A) or BIO.A (x-x) mice were cultured for 2 days in 0.1 ml of serum-free medium in the presence of various concentrations of D-penicillamine. Cultures were pulsed with 1.0 &i of [‘H]TdR for the final 24 hr of culture. Results are expressed as the arithmetic mean of five replicate cultures + the SE.
and that higher doses of the drug were suppressive. In contrast, a free sulfhydryl group does not appear to be mandatory for mitogenicity. Thus, one might speculate that the mitogenic effects of D-penicillamine and its disulfide occur at an intracellular site where the redox equilibrium favors the reduced form; the LPS-enhancing effect may be mediated via a membrane site where such reduction would not occur (otherwise one would expect enhancement from the disulfide as well). The effectiveness of the disulfide at concentrations approximately 1 log lower than the sulfhydryl may be due to the fact that the disulfide can pass through the membrane without its sulfur atoms reacting as thiol groups (i.e., they are protected), and that each disulfide molecule can be reduced to liberate two thiol molecules. Previous work investigating the ability of CBA/N mice to respond to 2-ME demonstrated that the mature B-cell subset activated by 2-ME in normal strains is functionally silent in CBA/N mice (14). Similarly, the inability of B cells from this strain to respond to D-penicillamine implicates the function of a mature subpopulation of B cells in the generation of this response. Polyclonal B-cell activation induced by D-penicillamine was found to be associated with cellular proliferation. The magnitude of the polyclonal response, however, was consistently lower than that of LPS, suggesting that if this immunostimulatory effect of D-penicillamine is responsible for the spectrum of autoimmune side effects observed in clinical situations, it must occur as a result of a very favorable microenvironment (perhaps genetically determined) or of alteration of D-penicillamine to a more highly stimulatory form (perhaps also genetically determined).
LYMPHOCYTE
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349
FIG. 12. Effect of tqenicillamine on the primary humoral response to SRBC in vitro. 10 X lo6 viable C3H/St spleen cells were cultured for 4 days in 1.0 ml of 5% FCS-containing medium together with 2 X lo6 SRBC in the presence of various concentrations of o-penicillamine. Anti-SRBC PFC/culture are presented as the arithmetic mean of triplicate cultures f the SE.
The troublesome autoimmune side effects observed in patients treated with Dpenicillamine are not limited in their occurrence to patients being treated for rheumatoid arthritis. Therefore, it was of interest to assess the responsiveness of lymphoid cells from both the normal and autoimmune murine models to D-penicillamine. The results indicate that cells from both normal and autoimmune strains (NZBxW, NZB) were responsive to this thiol compound, but that rather than being exaggerated in autoimmune strains, responses were generally of the same order of magnitude. When a variety of murine strains was examined with respect to their capacity to respond mitogenically to D-penicillamine, no hyperresponder group was identified. A/J mice (H-2a) were consistently hyporesponsive or unresponsive to this thiol compound. This strain is a natural k/d recombinant; however, spleen cells from C3H and CBA mice ( H-2k), Balb/c mice ( H-2d), and BIO.A mice (H-2”) responded well to D-penicillamine. Thus, any genetic proclivity toward hypo- or hyperresponsiveness does not appear to be a function simply of H-2 phenotype. The observed effects of D-penicillamine are not likely to be attributable to endotoxin comtamination since (i) oxidized and reduced preparations gave very different results, (ii) T cells are responsive to D-penicillamine, and (iii) the endotoxinresponsive strains CBA/N and A/J both failed to respond to D-penicillamine. Enhancement and suppression of the response of murine spleen cells to concanavalin A or in mixed lymphocyte cultures by D-penicillamine has been described by Kendall and Hutchins (35). Binderup et al. have described similar modulation of the Con A response by rat macrophages preincubated with this thiol (36). In the current report, augmentation of the primary humoral immune response to SRBC occurred at a dose of D-penicillamine lower than the other effects observed,
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perhaps indicative of a separate mechanism. Again, supraoptimal doses suppressed basal response to SRBC. The observation of immunopotentiating effects attributable to D-penicillamine raises this as a possible alternate mechanism whereby this compound induces the clinical spectrum of autoimmunity observed. Whether de nova activation or immunopotentiation is the relevant mechanism is at present unresolved. While it is possible that the high concentrations of D-penicillamine used in vitro cannot be achieved in vim, it is also possible that high local concentrations occur in special, localized anatomical sites. These could vary from the gut-associated lymphoid tissue to lymph nodes or to the cell surface of lymphocytes or other cells in their immediate microenvironment; the compound could then be presented in the form of a mixed disulfide at the surface of a presenting cell. Genetic control of such factors is not an unlikely possibility. An understanding of the immunomodulatory effects of D-penicillamine, together with the molecular requisites for stimulation (37) may ultimately lead to the formulation of an agent which is therapeutically effective and lacks immunostimulatory activity. ACKNOWLEDGMENTS The authors wish to express their appreciation to Mrs. Terry technical assistance, and to Ms. Janet Kuhns and Mrs. Barbara in the preparation of the manuscript.
Else and Miss Anne Zumbrun for superb Marchand for excellent secretarial work
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
Dresner, E., and Trombly, P., Clin. Res. 8, 16, 1960. Griffin, S. W., Ulloa, A., Henry, M., Johnston, M. L., and Holley, H. L., Clin. Res. 8, 87, 1960. Multicentre Trial Group, Lancet 1, 275, 1973. Dixon, A. St. J., Davies, J., Dormandy, T. L., Hamilton, E. B. D., Holt, P. J. L., Mason, R. M., Thompson, M., Weber, J. C. P., and Zutshi, D. W., Ann. Rheum. Dis. 34, 416, 1975. Tsang, I., Patterson, C. A., Stein, H. B., Robinson, H. S., and Ford, D. K., Arthritis Rheum. 20, 666, 1977. Shiokawa, Y., Horiuchi, Y., Honma, M., Kagiyama, T., Okada, T., and Ozuma T., Arthritis Rheum. 20, 1464, 1977. Goldberg, L. S., and Barnett, E. V., Arch. Int. Med., 125, 145, 1970. Jaffe, I. A., Ann. N.Y. Acad. Sci. 256, 330, 1975. Hubner, K. F., and Gingozian, N., Proc. Sot. Exp. Biol. Med. 118, 561, 1965. Hunneyball, I. M., Stewart, G. A., and Stanworth, D. R., Immunology 34, 1053, 1978. Lipsky, P. E., and Ziff, M., J. Immunof. 120, 1006, 1978. Lipsky, P. E., and Ziff, M., J. Chin Invest. 65, 1069, 1980. Tobin, M. S., and Altman, K., Proc. Sot. Exp. Biol. Med. 115, 225, 1964. Goodman, M. G., Fidler, J. M., and Weigle, W. O., J. Immunol. 121, 1905, 1978. Julius, M., Simpson, E., and Herzenberg, L. A., Eur. J. Immunol. 3, 645, 1973. Ly, I. A., and Mishell, R. I., J. Immunol. Methods 5, 239, 1974. Goodman, M. G., and Weigle, W. O., J. Immunol. 122, 1433, 1979. Mishell, R. I., and Dutton, R. W., J. Exp. Med. 126, 423, 1967. Coutinho, A., Gronowicz, E., Bullock, W. W., and Moller, G., J. Exp. Med. 139, 74, 1974. Jerne, N. K., and Nordin, A. A., Science 140, 405, 1963. Broome, J. D., and Jeng, M. W., J. Exp. Med. 138, 574, 1973. Noelle, R. J., and Lawrence, D. A., Cell. Immunol. 50, 416, 1980. Amsbaugh, D. F., Hansen, C. T., Prescott, B., Stashak, P. W., Asofsky, R., and Baker, P. J., J. Exp. Med. 139, 1499, 1974.
LYMPHOCYTE 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.
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BY D-PENICILLAMINE
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Scher, I., Sharrow, S. O., and Paul, W. E., J. Exp. Med. 144, 507, 1976. Janeway, C. A., Jr., and Barthold, D. R., J. Immunol. 115, 898, 1975. Finkelman, F. D., Smith, A. H., Scher, I., and Paul, W. E., J. Exp. Med. 142, 1316, 1975. Click, R. E., Benck, L., and Alter, B. J., Cell. Immunol. 3, 156, 1972. Goodman, M. G., and Weigle, W. O., J. Exp. Med. 145, 473, 1977. Lemke, H., and Opitz, H. G., J. Immunol. 117, 388, 1976. Igarashi, T., Kada, M. O., Kishimoto, T., and Yamamura, Y., J. Immunol. 118, 1697, 1977. Heber-Katz, E., and Click, R. E., Cell. Immunol. 5, 410, 1972. Bevan, M. J., Epstein, R., and Cohn, M. J. Exp. Med. 139, 1025, 1974. Engers, H. D., MacDonald, H. R., Cerotiini, J.-C., and Brunner, K. T., Eur. J. Immunol. 5, 223, 1975. Metcalf, D., Nossal, G. J. V., Warner, N. L., Miller, J. F. A. P., Mendel, T. E., Layton, J. E., and Gutman, G. G., J. Exp. Med. 142, 1534, 1975. Kendall, P. A., and Hutchins, D., Immunology 35, 189, 1978. Binderup, L., Bramm, E., and Arrigoni-Martelli, E., Scund. J. Immunol. 7, 259, 1978. Goodman, M. G., and Weigle, W. 0.. J. Immunol. 126, 20, 1981.
IMMUNOLOGY
65, 337-351 (1981)
Nonspecific
Activation
of Murine Lymphocytes
VIII. Effects of o-Penicillamine’ MICHAEL G. GOODMAN* AND WILLIAM Department
of Immunopathology, La Jolla, Received
July
0. WEIGLE
Scripps Clinic and Research California 92037
21, 1981; accepted
October
Foundation,
25. 1981
The effects of D-penicillamine on proliferation and polyclonal activation of lymphocytes were studied in cultures of spleen cells from a variety of murine strains. Inclusion in serumfree or serum-containing medium of optimal concentrations of D-penicillamine resulted in the uptake of tritiated thymidine in a dose-dependent fashion, with both lower and higher doses causing less marked effects. The kinetic peak of these responses was found to occur at Day 2 of culture. Experiments examining the responsiveness of B-cell-enriched and T-cell-enriched populations demonstrated that D-penicillamine, like 2-mercaptoethanol, stimulates both types of cell. The magnitude of the response remained unchanged in populations depleted of adherent cells. In correlation with previous results seen for 2-ME, tqenicillamine was mitogenically active both in reduced and oxidized forms. The oxidized form failed to enhance the response to LPS, in contrast to the marked effect of the reduced form. Additionally, D-penicillamine failed to evoke a mitogenic response from B cells of CBA/N mice, a strain characterized by a deficit in the function of a particular set of mature B cells. Young mice from autoimmune strains responded to D-penicillamine as well as normal mice did. No relationship could be observed between responsiveness to tr-penicillamine and the H-2 phenotype of the cell donor, and in A/J mice. hyporesponsiveness was shown to be a function of their background rather than H-2. D-Penicillamine was found to function as a polyclonal B-cell activator, and to significantly enhance the primary humoral immune response to sheep erythrocytes in vitro. These immunomodulatory effects of rqenicillamine are discussed in relation to possible pathogenetic mechanisms of its spectrum of autoimmune side effects.
INTRODUCTION The use of D-penicillamine in the treatment of rheumatoid arthritis has become increasingly accepted since its use was extended to this disease by Dresner and Trombly (1) and by Griffin et al. (2). It currently constitutes one of the small number of effective therapeutic modalities for the treatment of rheumatoid arthritis ’ This is publication No. 2305 from the Department of Immunopathology, Scripps Clinic and Research Foundation, La Jolla, Calif. This work was supported in part by United States Public Health Service Grant AI07007, American Cancer Society Grant IM-421, and Biomedical Research Support Grant RRO-55 14. * Recipient of United States Public Health Service Grant AI15284 and United States Public Health Service Research Career Development Award A100374. 337 OOOS-8749/81/180337-15$02.00/O Copyright Q 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.
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(RA).3 Its efficacy has been established by a number of clinical trials (3-6) and its use in the treatment of RA has been sanctioned by the FDA. The mechanism of action by which D-penicillamine ameliorates this disease is unclear. One suggestion well supported by experimental evidence is that this agent acts in an immunosuppressive capacity to diminish the immune responsiveness of patients with rheumatoid arthritis. Thus, penicillamine has been noted to depress levels of circulating rheumatoid factor and frequently also of immunoglobulin as well as immune complexes (7, 8). Several studies have demonstrated that D-penicillamine depresses the primary humoral immune response of experimental animals in vivo (9, 10) and diminishes mitogenic activation of human peripheral blood lymphocytes in vitro (11). Recently, Lipsky and Ziff (12) have demonstrated in vitro suppression of the primary humoral immune response of human PBL mediated by a selective suppression of the helper T-cell arm of this response. Clinical use of D-penicillamine, however, is limited by its high rate of complication. Among these are the group of so-called “autoimmune” side effects which include myasthenia gravis, thyroiditis, polymyositis and dermatomyositis, autoimmune hemolytic anemia, thrombotic thrombocytopenic purpura, and a drug-induced lupus-like syndrome, among others. Case reports of undesirable immunemediated side effects of penicillamine therapy suggest that the drug, rather than being solely immunosuppressive, may possess a variety of immunoregulatory properties, i.e., it may have immunopotentiating or activating effects as well. The report of Tobin and Altman (13) concerning the enhancement of the humoral immune response in vivo by D-penicillamine in rabbits supports such a concept. In previous studies we have explored the immunopotentiating and de novo-activating properties of 2-mercaptoethanol, a low MW hydroxy-thiol compound. Our observations that several other functional groups are active when present on thiol compounds led us to probe the effects of D-penicillamine (/3-mercaptovaline) with respect to lymphocyte activation. Results of the current communication indicate that in normal mice, mitogenicity, polyclonal B-cell activation, and adjuvanticity are properties of this compound. MATERIALS
AND
METHODS
Mice. C3H/St, B6D2FI, BlO, BlO.A, and Balb/c mice, 8-12 weeks of age, were obtained from the mouse breeding facility at Scripps Clinic and Research Foundation, La Jolla, California. CBA/CaJ, A/J, C57B l/65 and SJL mice, 8- 12 weeks of age, were purchased from the Jackson Laboratory, Bar Harbor, Maine. A breeding nucleus of CBA/N mice was provided by the animal production section, National Institutes of Health, Bethesda, Maryland. NZW, NZB, and BxWFl female mice, 8-12 weeks of age, were obtained from the Jackson Laboratory. All mice were maintained on Wayne Lab-Blox F6 pellets (Allied Mills, Inc., Chicago, Ill.) and chlorinated water acidified with HCl to a pH of 3.0. Culture reagents. Constituents of the serum-free culture medium employed in these studies have been described elsewhere (14). For serum-containing medium, supportive FCS was substituted for 5% of the volume of RPM1 1640, and Hepes ’ Abbreviations used: olTG, athioglycerol; ME, 2-mercaptoethanol; RA, rheumatoid phenyl.
Ig, immunoglobulin; LPS, bacterial lipopolysaccharide; arthritis, SRBC, sheep red blood cells; TNP, 2,4,6-trinitro-
2-
LYMPHOCYTE
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buffer was omitted. Escherichia coli 055:B5 LPS, prepared by the Boivin procedure, was purchased from the Difco Laboratories, Detroit, Michigan. D-Penicillamine was purchased from the Sigma Chemical Company, St. Louis, Missouri, and from the Aldrich Chemical Company, Milwaukee, Wisconsin. D-Penicillamine disulfide was purchased from the Aldrich Chemical Company. LPS and D-penicillamine were diluted in phosphate-buffered saline and sterilized by filtration. Further dilution in complete medium was used for microcultures. D-Penicillamine was made up fresh from powder for each experiment. Cell preparation. Spleen cell suspensions were prepared as described previously ( 14). Spleen cells enriched for T lymphocytes were prepared by passage through nylon wool (NW) columns, as described previously (15). B-Cell-enriched populations were prepared by treating lo8 spleen cells with a 1: 1000 dilution of monoclonal anti-Thy 1.2 (New England Nuclear, Boston, Mass.) for 30 min at 4°C. Treated cells were then centrifuged at 280g for 10 min, the antibodies removed, and the cells resuspended in a I:6 dilution of C3H RBC-absorbed guinea pig complement at 37°C for 45 min. Cells were then washed and cultured as above. Adherent cells were depleted by passage over columns of Sephadex G-10 as described by Ly and Mishell(l6). This procedure reduced the percentage of esterasepositive cells from 7% in unseparated spleen cells to approximately 0.1% in nonadherent populations. Lymphocyte cultures. Murine spleen cells were cultured in microculture plates (No. 3042, Falcon Plastics, Oxnard, Calif.) at a cell density of 5 X IO6 viable cells/ ml in a volume of 0.1 ml ( 17). Microcultures were incubated at 37°C in a humidified atmosphere of 10% CO2 in air. Cultures were fed daily with 8 ~1 of nutritional cocktail ( 18). Measurement of DNA synthesis. During the final 24 hr of culture, cells were radiolabeled with 1.O &i of [ 3H]TdR/culture (5 Ci/mM, Amersham Radiochemicals, Amersham, England). The microcultures were harvested with a Brandel cell harvester, Model M24V (Biological Research and Development Laboratories, Rockville, Md.) onto glass fiber filter strips (Reeve Angel, Clifton, N.J.). Filter disks were transferred to plastic scintillation vials (Kimball Products, Owens-Illinois, Toledo, Ohio) covered with liquid scintillation cocktail (Scintiverse, Fisher Scientific Co., Fairlawn, N.J.) and counted in a Beckman LS-230 liquid scintillation counter. Assay of plaque-forming cells (PFC). For polyclonal activation, 10 X 1O6 spleen cells/ml were incubated in a l.O-ml volume of 5% FCS-containing medium. PFC to TNP were assayed after 2 days of culture ( 19) using a modification of the hemolytic plaque assay of Jerne and Nordin (20). For the primary humoral response to SRBC, 10 X lo6 murine spleen cells were cultured in 1 ml of 5% FCS containing medium for 4 days in the presence of 2 X lo6 SRBC/culture. RESULTS Dose dependency of the DNA-synthetic response to D-penicillamine. The ability of D-penicillamine to activate DNA synthesis was investigated in cultures of spleen cells from C3H/St mice. Spleen cells were incubated in serum-free medium in the presence of variable concentrations of D-penicillamine. Resultant activation was measured by determination of [3H]TdR uptake during the final 24 hr of d 2-day
340
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culture period. The results shown in Fig. 1 indicate that this thiol compound does indeed promote the uptake of tritiated thymidine in a dose-dependent manner. Lower concentrations were suboptimal while higher concentrations appeared to be inhibitory. Peak responsiveness appeared to be between 1O-3 and lo-’ it4 D-penicillamine. Peak responsiveness varied somewhat from experiment to experiment but appeared to be highly consistent within individual experiments. Kineticprofile of the response to o-penicillamine. The time at which the maximal D-penicillamine mitogenic effect occurred was assessed in C3H/St mice. Spleen cells were cultured either in the presence or absence of lop3 M D-penicillamine under serum-free culture conditions. Subsequently, one set of cultures was harvested daily after a 24-hr pulse of [3H]TdR. Maximal [3H]TdR uptake was noted to occur at Day 2 and declined thereafter (Fig. 2). Response of separately cultured B-enriched penicillamine. C3H/St spleen cell populations
and T-enriched
lymphocytes
to
D-
enriched for B cells by treatment with monoclonal anti-Thy 1.2 plus guinea pig complement, or for T cells by passage over NW columns as described, were cultured under conditions utilizing medium containing 5% FCS. Serum was used in these experiments because of the inability of separated T cells to proliferate in the absence of serum (28). The results of such experiments (Fig. 3) demonstrate that T lymphocytes and B lymphocytes can proliferate in response to D-penicillamine. In some of the experiments, the response by B cells was greater than that exhibited by T cells, and in no case was the reverse observed. B-Cell-enriched cultures failed to respond to the T-cell mitogen concanavalin A, and T-cell cultures gave only 8% of the unseparated cell response to LPS. Ability of o-penicillamine to activate nonadherent spleen cells. In order to gain
Concentration of D.Penlcillamine IMI
FIG. 1. Dose-response profile for D-penicillamine in serum-free culture. 5 X lo5 viable C3H/St spleen cells were cultured for 2 days in 0.1 ml of serum-free medium in the presence of various concentrations of D-penicillamine. Cells were pulsed with 1.0 pCi of [‘H]TdR for the final 24 hr of culture. Results are expressed as the arithmetic mean of five replicate cultures k the SE.
LYMPHOCYTE
ACTIVATION
BY D-PENICILLAMINE
341
1 t I
I 1
2
3
Day al Harvest FIG. 2. Kinetic profile of the response to D-penicillamine. 5 X lo5 viable C3H/St spleen cells were cultured in 0.1 ml of serum-free medium in the presence or absence of lo-’ M D-penicillamine. On each day, one set of parallel cultures was harvested after a 24-hr pulse of 1.0 pCi of [3H]TdR. Results are expressed as the arithmetic mean of five replicate cultures containing D-penicillamine minus that of five replicate control cultures + the SE. Control values were 6630 + 510, 2310 + 190, and 1540 f 180 on Days 1, 2, and 3, respectively.
insight into the role played by adherent cells in the response to D-penicillamine, experiments were designed to determine whether the proliferative response to this thiol compound is mediated through such cells or is independent of them. C57Bl/ 65 spleen cells were passed over Sephadex G-10 columns and the effluent cells used as the source of nonadherent cells. Content of esterase-positive cells was greatly diminished in this population (see Materials and Methods). The results of these experiments demonstrate that nonadherent cells respond to D-penicillamine to the same degree as do unseparated cells (Fig. 4). Effects of reduced and oxidized o-penicillamine on murine spleen cells. Previous work has indicated that 2-ME, in addition to having mitogenic effects, is able to enhance the in vitro response of murine spleen cells to mitogens ( 15, 21, 22). In preliminary experiments, it was learned that D-penicillamine could similarly enhance the mitogenic response to bacterial LPS. Therefore, experiments were designed to investigate the requirement for free sulfhydryl groups both in D-penicillamine-mediated mitogenesis and in its LPS-enhancing effects. C3H/St spleen cells were incubated in the presence of incremental concentrations of either reduced or oxidized o-penicillamine under serum-free culture conditions (Fig. 5). Under these circumstances, both compounds were found to have mitogenic effects. The magnitude of the responses was generally equivalent, although lower doses of the disulfide were more effective than the equivalent doses of the thiol, resulting in a slight shift of the dose-response curve to the left for the disulfide. When these same
342
GOODMAN
AND
WEIGLE
Concentration of Wenlcillamine
IMI
FIG. 3. Response of B-enriched and T-enriched lymphocyte cultures to D-penicillamine. 5 X 10’ viable C3H/St B-enriched, T-enriched, or unseparated splenic lymphocytes were cultured in 0.1 ml of 5% FCS-containing medium for 2 days in the presence of various concentrations of o-penicillamine. Cells were pulsed with 1.0 pCi of [‘H]TdR for the final 24 hr of culture. Results are expressed as the arithmetic mean of five replicate cultures containing D-penicillamine minus control cultures + the SE. Control values were 1920 + 220, 1530 + 250, and 880 f 120 for unseparated, B, and T cells, respectively.
two compounds were evaluated for their ability to enhance the mitogenic response to LPS, it was found that only reduced D-penicillamine was capable of enhancing this response (Fig. 6). This effect occurred at the same dosage range as the optimal concentration for mitogenesis by D-penicillamine. Nonetheless, it is unlikely that this effect can be accounted for by a simple additivity of mitogenic responses, since the magnitude of the increased LPS response was considerably greater than that of the mitogenic response within the same experiment and since the disulfide was ineffective. Effect of o-penicillamine in mice with an X-linked B-cell defect. Characterization of the immune deficit of the CBA/N mouse has been carried out by a number of investigators. Amsbaugh et al. (23) first described an X-linked defect in the CBA/N response to thymus-independent antigens. These animals have subsequently been reported to contain a diminished number of Ig-bearing cells in their spleens (24), to mount subnormal responses to thymus-dependent antigens (25), and to possess cell surface Ig and microdensities and organ distributions which suggest a deficit of mature B lymphocyte (26). Moreover, work characterizing the nature of the B-cell subset responsive to 2-ME demonstrated that the subpopulation of B cells responding to 2-ME was a mature set which was functionally absent in the CBA/N mouse (15). Therefore, it was of interest to assess the reactivity of cultures of CBA/N B cells to D-penicillamine. B cells were prepared as described under Materials and Methods. A direct comparison of the responsiveness of B lymphocytes derived from this strain with those from the histocompatible CBA/ CaJ strain (Fig. 7) indicates that in contrast to the CBA/CaJ response (generally
LYMPHOCYTE
ACTIVATION
BY D-PENICILLAMINE
343
I 0
3110’
10’
Concentration
3r10
10’
of LLPenicillamine [MI
FIG. 4. Ability of D-penicillamine to activate unseparated and G-10 passed spleen cells. 5 X 10’ viable C57BL/6J spleen cells, either unseparated (-) or passed over column of Sephadex G-10 (- - -) were cultured for 2 days in 0.1 ml of serum-free medium in the presence of various concentrations of Dpenicillamine. Cultures were pulsed with 1.O &i of [‘H]TdR for the final 24 hr of culture. Results are expressed as the arithmetic mean of five replicate cultures + the SE.
Concentratlan IMI
FIG. 5. Effects of reduced and oxidized D-penicillamine on murine spleen cells. 5 X lo5 viable C3H/ St spleen cells were cultured for 2 days in 0.1 ml of serum-free medium in the presence of various concentrations of either the thiol or the disulfide form of D-penicillamine. Cultures were pulsed with 1.0 pCi of [‘H]TdR for the final 24 hr of culture. Results are expressed as the arithmetic mean of five replicate cultures containing D-penicillamine minus control cultures & the SE. Control uptake was 1310 + 230 cpm.
344
GOODMAN.AND
10.’ Concentration
WEIGLE
10-1 10.’ of O-Penicillamine IMI
FIG. 6. Ability of reduced and oxidized tqenicillamine to enhance the mitogenic response to LPS. 5 X lo5 viable C3H/St spleen cells were cultured for 2 days in 0.1 ml of serum-free medium together with 100 &g/ml LPS in the presence of various concentrations of either the thiol or disultide form of D-penicillamine. Cells were pulsed with 1.0 @i of [‘H]TdR for the final 24 hr of culture. Results are expressed as the arithmetic mean of five replicate cultures k the SE.
of similar magnitude to that of the C3H/St), unresponsive to D-penicillamine.
B cells from the CBA/N
mouse were
Ability of o-penicillamine to induce polyclonal B-cell activation. Since mitogenicity and polyclonal B-cell activation are frequently very closely linked, the ability of D-penicillamine to promote polyclonal immunoglobulin secretion was evaluated. Adherent cell-depleted spleen cells ( 107) were cultured in 5% FCScontaining medium in the presence of incremental concentrations of D-penicillamine. This agent was found to be a weak polyclonal B-cell activator, significant responses being detectable only on SRBC conjugated with TNP rather than on SRBC alone (Fig. 8). Peak responsiveness again was within the range seen for peak mitogenicity. Ability of o-penicillamine to activate spleen cells from normal and autoimmune mouse strains. In order to examine the effect of autoimmune predisposition on
responsiveness to D-penicillamine, spleen cells from female C3H/St, CBA/CaJ, NZW, NZB, and NZB X NZWF, mice (8-12 weeks of age) were cultured in the presence or absence of 10e3 M D-penicillamine. The results of these experiments (Fig. 9) indicate that there is no major quantitative difference in the stimulated response to this thiol compound although background thymidine uptake varied considerably. Thus, a predisposition to develop autoimmune disease did not appear to correlate with increased susceptibility of spleen cells to activation by D-penicillamine. Ability of o-penicillamine to activate spleen cells from mice of different H-2 phenotypes. Studies were undertaken to screen for a relationship between H-2
phenotype and degree of responsiveness to D-penicillamine. Spleen cells from a variety of murine strains were cultured with a range of concentrations of this thiol compound. Peak responsiveness occurred in the range of 3-6 X lop3 M. Although H-2k and H-2d mice responded to D-penicillamine, A/J mice (a natural k/d re-
LYMPHOCYTE
ACTIVATION
0-Penicillamme
BY D-PENICILLAMINE
Concentration
345
[MI
FIG. 7. The effect of o-penicillamine in mice with an X-linked B-cell defect. 5 X 10’ viable CBA/CaJ or CBA/N splenic B lymphocytes were cultured for 2 days in 0.1 ml of serum-free medium in the presence of various concentrations of o-penicillamine. Cultures were pulsed with 1.0 &i of [‘H]TdR for the final 24 hr of culture. Results are expressed as the arithmetic mean of five replicate cultures 2 the SE.
combinant) responded poorly or not at all (Fig. 10). No other obvious relationship could be observed. In other experiments, the inability of A/J mice to respond to D-penicillamine was dissected using B 10 and B 10.A mice (Fig. 11). The data indicate that hyporesponsiveness is a property of the A/J genetic background and is not exclusively linked to its H-2 phenotype. Effect of o-penicillamine on the primary humoral response to SRBC in vitro. Work by Click et al. (27) established that the primary humoral immune response to SRBC was augmented when cultures were supplemented with 2-ME. The effect of D-penicillamine on the primary humoral response to SRBC was therefore evaluated in order to determine whether this thiol compound similarly could enhance the response to specific antigen. The data presented in Fig. 12 indicate that addition of D-penicillamine to culture results in an enhanced response to SRBC. This effect
%k lb
l@’ I@’ Concentration ot O.Penicitlamine IMI
10.’
FIG. 8. Ability of D-penicillamine to induce polyclonal B-cell activation. 10 X lo6 viable C3H/St nonadherent spleen cells were cultured for 2 days in 1.0 ml of 5% FCS-containing medium in the presence of incremental concentrations of D-pe&ihniIIe. Results are expressed as the arithmetic mean of trip licate cultures f the SE.
346
GOODMAN
AND
WEIGLE
:,9
*.Jr
FIG. 9. Ability of o-penicillamine to activate spleen cells from normal and autoimmune mouse strains. 5 X 10’ viable spleen cells from female C3H/St, CBA/CaJ, NZW, NZB, or NZB X WF, mice were cultured for 2 days in 0.1 ml of serum-free medium in the presence of lo-’ M o-penicillamine. Cells were pulsed with 1.0 &i of [‘H]TdR for the final 24 hr of culture. Results are expressed as the arithmetic mean of five replicate cultures k the SE. Hatched bars represent unstimulated cultures, open bars represent stimulated cultures.
was dose-dependent but occurred at lower concentrations mitogenicity or for polyclonal activation.
than were required for
DISCUSSION The effectiveness of D-penicillamine for treatment of rheumatoid arthritis has been validated by a number of clinical trials and is responsible for its increasing clinical usage. This thiol compound has been thought to function primarily as an immunosuppressor, based on in vivo (7- 10) and in vitro ( 11, 12) studies. The data presented in the current communication, however, are more compatible with the thesis that D-penicillamine is a modulator of the immune response, capable of both enhancing and initiating immune activation as well as suppressing it. Previous studies exploring the effects of 2-mercaptoethanol(2-ME) in the murine system in vitro demonstrated that certain sulfhydryl compounds induce a state of activation in resting splenic lymphocytes. Such activation involves T as well as B lymphocytes (28, 29) with the latter population of cells being induced to secrete polyclonal immunoglobulin. Igarashi et al. (30) further showed that 2-ME is responsible for the induction of polyclonal cytotoxicity in T cells. In addition, 2-ME has been shown to have immunopotentiating activity in a diversity of systems (3 l34). Therefore, the possibility that similar effects might be mediated by D-penicillamine was examined experimentally. Addition of D-penicillamine to cultures of murine spleen cells was found to result in a consistent increase in uptake of [‘H]TdR. This phenomenon occurred in a dose-related fashion, such that suboptimal doses elicited lesser responses, and supraoptimal doses caused suppression below background, similar to that seen by
LYMPHOCYTE
ACTIVATION
CBAlCaJ Ikl
Balblc (dl
AIJ la1
BY D-PENICILLAMINE
C57BWJ Ibl
SJL ISI
341
Ibldl
FIG. 10. Effect of D-penicillamine on mice of different H-2 phenotypes. 5 X 10’ viable spleen cells from CBA/CaJ, Balb/c, A/J C57BL/6J, SJL, or B6D2FI mice were cultured for 2 days in 0.1 ml of serum-free medium in the presence of medium only (solid bars), 3 X 1O-3 M D-penicillamine (hatched bars), or 6 X 1O-3 M D-pcnicillamine (open bars). Cells were pulsed with 1.0 pCi of [‘H]TdR for the final 24 hr of culture. Results are expressed as the arithmetic mean of five replicate cultures f the SE.
other investigators. The observation of increased [3H]TdR uptake by spleen cells incubated with D-penicillamine contrasts with observations by some other investigators (35), but this discrepancy is in all likelihood explained by differences in the culture system and dose range employed. When cells were cultured in serumcontaining medium, induction of significant stimulation by D-penicillamine was found to require a minimum of twofold higher concentrations of the drug, probably as a consequence of mixed disulfide formation. The data presented indicate that both B- and T-lymphocyte populations repond to D-penicillamine. A similar capacity to activate both B and T lymphocytes has been reported for 2-ME and L~TG (22, 28), although in those cases B cells have been found to be more responsive than have T cells. Macrophages do not appear to play a critical role in the activation of lymphocytes by D-penicillamine. Depletion of macrophages from spleen cells does not result in a concomitant decrease in the uptake of [3H]TdR; on the contrary, stimulation of the G-10 nonadherent cell population is nearly equivalent to that seen in unseparated cells. Both the oxidized and reduced forms of D-penicillamine induce mitogenesis in spleen cell populations to approximately the same degree, while only the reduced form enhances the mitogenic response to LPS, in parallel with data obtained for reduced and oxidized 2-ME (37). Supraoptimal D-penicillamine (as well as 2-ME) inhibits the underlying LPS response. These data suggest that the free sulfhydryl group is critical to the mitogen-enhancing effect of D-penicillamine, since the disulfide fails to demonstrate this effect. Similarly, Kendall and Hutchins (35) found that D-penicillamine enhanced the responses to concanavalin A or allogeneic cells,
348
GOODMAN
AND WEIGLE
I 0
10.’ Concentration
10” of O-Penicillamine IMI
lo”
FIG. 11. Effect of D-penicillamine in A/J, BIO, and Bl0.A mice. 5 X 10’ viable spleen cells from A/ J (0 - - - 0). BlO (A. . . A) or BIO.A (x-x) mice were cultured for 2 days in 0.1 ml of serum-free medium in the presence of various concentrations of D-penicillamine. Cultures were pulsed with 1.0 &i of [‘H]TdR for the final 24 hr of culture. Results are expressed as the arithmetic mean of five replicate cultures + the SE.
and that higher doses of the drug were suppressive. In contrast, a free sulfhydryl group does not appear to be mandatory for mitogenicity. Thus, one might speculate that the mitogenic effects of D-penicillamine and its disulfide occur at an intracellular site where the redox equilibrium favors the reduced form; the LPS-enhancing effect may be mediated via a membrane site where such reduction would not occur (otherwise one would expect enhancement from the disulfide as well). The effectiveness of the disulfide at concentrations approximately 1 log lower than the sulfhydryl may be due to the fact that the disulfide can pass through the membrane without its sulfur atoms reacting as thiol groups (i.e., they are protected), and that each disulfide molecule can be reduced to liberate two thiol molecules. Previous work investigating the ability of CBA/N mice to respond to 2-ME demonstrated that the mature B-cell subset activated by 2-ME in normal strains is functionally silent in CBA/N mice (14). Similarly, the inability of B cells from this strain to respond to D-penicillamine implicates the function of a mature subpopulation of B cells in the generation of this response. Polyclonal B-cell activation induced by D-penicillamine was found to be associated with cellular proliferation. The magnitude of the polyclonal response, however, was consistently lower than that of LPS, suggesting that if this immunostimulatory effect of D-penicillamine is responsible for the spectrum of autoimmune side effects observed in clinical situations, it must occur as a result of a very favorable microenvironment (perhaps genetically determined) or of alteration of D-penicillamine to a more highly stimulatory form (perhaps also genetically determined).
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FIG. 12. Effect of tqenicillamine on the primary humoral response to SRBC in vitro. 10 X lo6 viable C3H/St spleen cells were cultured for 4 days in 1.0 ml of 5% FCS-containing medium together with 2 X lo6 SRBC in the presence of various concentrations of o-penicillamine. Anti-SRBC PFC/culture are presented as the arithmetic mean of triplicate cultures f the SE.
The troublesome autoimmune side effects observed in patients treated with Dpenicillamine are not limited in their occurrence to patients being treated for rheumatoid arthritis. Therefore, it was of interest to assess the responsiveness of lymphoid cells from both the normal and autoimmune murine models to D-penicillamine. The results indicate that cells from both normal and autoimmune strains (NZBxW, NZB) were responsive to this thiol compound, but that rather than being exaggerated in autoimmune strains, responses were generally of the same order of magnitude. When a variety of murine strains was examined with respect to their capacity to respond mitogenically to D-penicillamine, no hyperresponder group was identified. A/J mice (H-2a) were consistently hyporesponsive or unresponsive to this thiol compound. This strain is a natural k/d recombinant; however, spleen cells from C3H and CBA mice ( H-2k), Balb/c mice ( H-2d), and BIO.A mice (H-2”) responded well to D-penicillamine. Thus, any genetic proclivity toward hypo- or hyperresponsiveness does not appear to be a function simply of H-2 phenotype. The observed effects of D-penicillamine are not likely to be attributable to endotoxin comtamination since (i) oxidized and reduced preparations gave very different results, (ii) T cells are responsive to D-penicillamine, and (iii) the endotoxinresponsive strains CBA/N and A/J both failed to respond to D-penicillamine. Enhancement and suppression of the response of murine spleen cells to concanavalin A or in mixed lymphocyte cultures by D-penicillamine has been described by Kendall and Hutchins (35). Binderup et al. have described similar modulation of the Con A response by rat macrophages preincubated with this thiol (36). In the current report, augmentation of the primary humoral immune response to SRBC occurred at a dose of D-penicillamine lower than the other effects observed,
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perhaps indicative of a separate mechanism. Again, supraoptimal doses suppressed basal response to SRBC. The observation of immunopotentiating effects attributable to D-penicillamine raises this as a possible alternate mechanism whereby this compound induces the clinical spectrum of autoimmunity observed. Whether de nova activation or immunopotentiation is the relevant mechanism is at present unresolved. While it is possible that the high concentrations of D-penicillamine used in vitro cannot be achieved in vim, it is also possible that high local concentrations occur in special, localized anatomical sites. These could vary from the gut-associated lymphoid tissue to lymph nodes or to the cell surface of lymphocytes or other cells in their immediate microenvironment; the compound could then be presented in the form of a mixed disulfide at the surface of a presenting cell. Genetic control of such factors is not an unlikely possibility. An understanding of the immunomodulatory effects of D-penicillamine, together with the molecular requisites for stimulation (37) may ultimately lead to the formulation of an agent which is therapeutically effective and lacks immunostimulatory activity. ACKNOWLEDGMENTS The authors wish to express their appreciation to Mrs. Terry technical assistance, and to Ms. Janet Kuhns and Mrs. Barbara in the preparation of the manuscript.
Else and Miss Anne Zumbrun for superb Marchand for excellent secretarial work
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
Dresner, E., and Trombly, P., Clin. Res. 8, 16, 1960. Griffin, S. W., Ulloa, A., Henry, M., Johnston, M. L., and Holley, H. L., Clin. Res. 8, 87, 1960. Multicentre Trial Group, Lancet 1, 275, 1973. Dixon, A. St. J., Davies, J., Dormandy, T. L., Hamilton, E. B. D., Holt, P. J. L., Mason, R. M., Thompson, M., Weber, J. C. P., and Zutshi, D. W., Ann. Rheum. Dis. 34, 416, 1975. Tsang, I., Patterson, C. A., Stein, H. B., Robinson, H. S., and Ford, D. K., Arthritis Rheum. 20, 666, 1977. Shiokawa, Y., Horiuchi, Y., Honma, M., Kagiyama, T., Okada, T., and Ozuma T., Arthritis Rheum. 20, 1464, 1977. Goldberg, L. S., and Barnett, E. V., Arch. Int. Med., 125, 145, 1970. Jaffe, I. A., Ann. N.Y. Acad. Sci. 256, 330, 1975. Hubner, K. F., and Gingozian, N., Proc. Sot. Exp. Biol. Med. 118, 561, 1965. Hunneyball, I. M., Stewart, G. A., and Stanworth, D. R., Immunology 34, 1053, 1978. Lipsky, P. E., and Ziff, M., J. Immunof. 120, 1006, 1978. Lipsky, P. E., and Ziff, M., J. Chin Invest. 65, 1069, 1980. Tobin, M. S., and Altman, K., Proc. Sot. Exp. Biol. Med. 115, 225, 1964. Goodman, M. G., Fidler, J. M., and Weigle, W. O., J. Immunol. 121, 1905, 1978. Julius, M., Simpson, E., and Herzenberg, L. A., Eur. J. Immunol. 3, 645, 1973. Ly, I. A., and Mishell, R. I., J. Immunol. Methods 5, 239, 1974. Goodman, M. G., and Weigle, W. O., J. Immunol. 122, 1433, 1979. Mishell, R. I., and Dutton, R. W., J. Exp. Med. 126, 423, 1967. Coutinho, A., Gronowicz, E., Bullock, W. W., and Moller, G., J. Exp. Med. 139, 74, 1974. Jerne, N. K., and Nordin, A. A., Science 140, 405, 1963. Broome, J. D., and Jeng, M. W., J. Exp. Med. 138, 574, 1973. Noelle, R. J., and Lawrence, D. A., Cell. Immunol. 50, 416, 1980. Amsbaugh, D. F., Hansen, C. T., Prescott, B., Stashak, P. W., Asofsky, R., and Baker, P. J., J. Exp. Med. 139, 1499, 1974.
LYMPHOCYTE 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.
ACTIVATION
BY D-PENICILLAMINE
351
Scher, I., Sharrow, S. O., and Paul, W. E., J. Exp. Med. 144, 507, 1976. Janeway, C. A., Jr., and Barthold, D. R., J. Immunol. 115, 898, 1975. Finkelman, F. D., Smith, A. H., Scher, I., and Paul, W. E., J. Exp. Med. 142, 1316, 1975. Click, R. E., Benck, L., and Alter, B. J., Cell. Immunol. 3, 156, 1972. Goodman, M. G., and Weigle, W. O., J. Exp. Med. 145, 473, 1977. Lemke, H., and Opitz, H. G., J. Immunol. 117, 388, 1976. Igarashi, T., Kada, M. O., Kishimoto, T., and Yamamura, Y., J. Immunol. 118, 1697, 1977. Heber-Katz, E., and Click, R. E., Cell. Immunol. 5, 410, 1972. Bevan, M. J., Epstein, R., and Cohn, M. J. Exp. Med. 139, 1025, 1974. Engers, H. D., MacDonald, H. R., Cerotiini, J.-C., and Brunner, K. T., Eur. J. Immunol. 5, 223, 1975. Metcalf, D., Nossal, G. J. V., Warner, N. L., Miller, J. F. A. P., Mendel, T. E., Layton, J. E., and Gutman, G. G., J. Exp. Med. 142, 1534, 1975. Kendall, P. A., and Hutchins, D., Immunology 35, 189, 1978. Binderup, L., Bramm, E., and Arrigoni-Martelli, E., Scund. J. Immunol. 7, 259, 1978. Goodman, M. G., and Weigle, W. 0.. J. Immunol. 126, 20, 1981.