Inhibition of Human B Cell Activation by Gold Compounds

CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY

Vol. 81, No. 2, November, pp. 175–181, 1996 Article No. 0174

Inhibition of Human B Cell Activation by Gold Compounds SHUNSEI HIROHATA Second Department of Internal Medicine, Teikyo University School of Medicine, Tokyo 173, Japan

The mechanism of action of gold compounds, which are effective in the treatment of rheumatoid arthritis (RA), has not been clearly identified. Although one of the characteristic features of RA is chronic stimulation of B cells, the effects of gold compounds on B cells have not been precisely assessed. We therefore examined the effects of gold sodium thiomalate (GST) on human B cells. IgM production was induced from highly purified B cells obtained from healthy donors by stimulation with Staphylococcus aureus Cowan 1 (SA) plus IL-2. T cell proliferation and IFN-g production were induced from highly purified T cells by stimulation with immobilized mAb to CD3. As little as 0.1 mg/ml (0.25 mM) GST almost completely suppressed B cell IgM production, whereas it did not suppress T cell proliferation or IFN-g production. The inhibition of IgM production by GST is not due to its thiomalate, but is most likely due to its gold components, since thiomalate alone did not inhibit IgM production. GST was required at the initiation of cultures to exert optimal suppressive effects on IgM production. Moreover, GST suppressed the expression of IL-2R (CD25) and transferrin receptor (CD71) on SA-stimulated B cells. These results indicate that GST preferentially inhibits the function of B cells at concentrations much lower than those which inhibit the function of T cells by interfering with the initial activation of B cells. The direct inhibitory effects of GST on human B cell activation described here may contribute at least in part to its therapeutic effect in RA. q 1996 Academic Press, Inc.

INTRODUCTION

Gold salts have been used in the treatment of rheumatoid arthritis (RA) for more than 50 years. A characteristic feature of treatment with gold compounds is a delayed onset of clinical effects and, possibly, an ability to slow or even prevent the progress of the disease (1, 2). Although a number of mechanisms have been proposed to explain the mechanisms of action of gold compounds in RA (3), none has been generally accepted. One of the characteristic features of RA is chronic stimulation of B cells, evidenced clinically by the production of rheumatoid factors (RF) (4, 5), which play an important role in the pathogenesis of the disease

(6, 7). In fact, accumulating clinical observation has disclosed that serum immunoglobulin (Ig) levels and RF titers often decrease in RA patients treated with gold compounds (8, 9), suggesting the possibility that gold compounds may suppress the functions of B cells. Although a number of studies have examined the effects of gold compounds on the function of T cells (10), monocytes (11), and endothelial cells (12, 13), the effects on B cells have not been precisely assessed. The current studies were therefore undertaken to explore the capacity of gold sodium thiomalate (GST) to modulate the function of human B cells. Particular attention was paid to the differential effects of a variety of concentrations of GST on B cells and T cells. The results indicate that GST directly suppresses the IgM production of B cells at concentrations much lower than those which inhibit the functions of T cells. The data therefore suggest that the primary target of GST in the treatment of RA may be B cells rather than T cells. METHODS

Reagents Several mAbs were used in the current studies, including 64.1, a murine IgG2a mAb directed to the CD3 complex on mature T cells (a generous gift of Dr. Peter E. Lipsky, University of Texas Southwestern Medical Center, Dallas, TX), fluorescein isothiocyanate (FITC)conjugated anti-interleukin-2 (IL-2) receptor (Becton– Dickinson, Mountain View, CA), a murine IgG1 mAb directed to the a-chain of the human IL-2 receptor (CD25), FITC-conjugated anti-transferrin receptor (CD71) (mouse IgG1, Becton–Dickinson), and FITCconjugated mouse IgG1 control mAb (Becton–Dickinson). Formalinized Cowan I strain Staphylococcus aureus (SA) was purchased from Calbiochem-Behring (San Diego, CA) and was used at a concentration of 1/ 240,000 (v/v). Recombinant human IL-2 (TGP-3) was a gift of Takeda Chemical Industries (Osaka, Japan); unit activity was determined by the providers (4.2 1 104 units/mg protein). GST was a gift of Shionogi Pharmaceutical Co. (Osaka, Japan), and thiomalic acid (TMA) was purchased from Kanto Chemical Co. (Tokyo, Japan). GST and TMA were dissolved in culture medium and added to cultures.

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0090-1229/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Culture Medium RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with penicillin G (100 units/ml), streptomycin (10 mg/ml), L-glutamine (0.3 mg/ml), and 10% fetal bovine serum (Gibco) was used for all cultures. Cell Preparation Peripheral blood mononuclear cells were obtained from healthy adult volunteers by centrifugation of heparinized venous blood over sodium diatrizoate–Ficoll gradients (Histopaque; Sigma, St. Louis, MO) and were depleted of monocytes and NK cells by incubation with 5 mM L-leucine methyl ester HCl (Sigma) in serumfree RPMI 1640, as described elsewhere (14). The treated cell population was washed twice and then incubated with neuraminidase-treated sheep red blood cells (N-SRBC). The rosetting and nonrosetting populations were then separated by centrifugation on sodium diatrizoate–Ficoll gradients. The nonrosetting cells obtained from the interface were again rosetted with NSRBC and centrifuged on sodium diatrizoate–Ficoll gradients to remove residual T cells. The resultant population of B cells contained õ1% CD14/ monocytes and õ1% CD2/ CD3/ T cells, as determined by analysis with flow cytometry. The cells were additionally characterized as containing ú90% CD20/ B cells and no CD16/ NK cells. The sedimented N-SRBC rosetteforming cells from the first centrifugation were treated with isotonic NH4Cl to lyse the N-SRBC and were then passed over a nylon–wool column. The resultant population of T cells contained õ0.1% esterase-positive monocytes and õ0.5% CD20/ B cells. Cell Culture Techniques for Induction of Immunoglobulin Production B cells (5 1 104/well) were cultured alone in wells of 96-well U-bottom microtiter plates (No. 3799, Costar, Cambridge, MA) with SA / IL-2 (0.5 unit/ml). Cultures were carried out in duplicate in a total volume of 200 ml. The cells were incubated for 11–12 days at 377C in a humidified atmosphere of 5% CO2 and 95% air.

Culture Techniques for Induction of T Cell Proliferation and IFN-g Production MAb 64.1 was diluted in RPMI 1640 (2 mg/ml), and 50 ml was placed in each well of 96-well U-bottom microtiter plates (No. 3799, Costar) and incubated at room temperature for 1 hr. The wells were then washed once with culture medium to remove nonadherent mAb before the cells were added. Approximately 14–20% of the added mAb adhered to the wells (16). Cultures were carried out in triplicate in a total volume of 200 ml. T cells (1 1 105/well) were cultured in wells with immobilized anti-CD3 for 5 days. After the incubation, the supernatants were harvested for the assay of IFN-g contents, and the proliferation of T cells was assessed by colorimetric assay using an MTT cell growth assay kit (Chemicon, El Segundo, CA) (17). Measurement of IFN-g IFN-g contents in the supernatants were assessed using a solid-phase enzyme immunoassay (18). Briefly, wells of a 96-well microtiter plate (Cooke) coated with rabbit anti-human IFN-g (Hayashibara, Okayama, Japan) were incubated with cell-free culture supernatants or various concentrations of recombinant IFN-g (Chemicon) in PBS containing 1% bovine serum albumin. Bound IFN-g was detected with peroxidase-conjugated rabbit anti-human IFN-g. The detection limit of the assay was approximately 5.0 units/ml of IFN-g. The assay was specific for natural and recombinant human IFN-g. Immunofluorescence and Flow Cytometry B cells stimulated with SA / IL-2 for 72 hr were stained with saturating concentrations of FITC-conjugated anti-CD25, anti-CD71, or control mAb and incubated at 47C for 30 min. The cells were then washed in cold PBS containing 2% normal human serum and analyzed by flow cytometry using an EPICS XL flow cytometer (Coulter, Hialeah, FL). RESULTS

Measurement of IgM Microtiter plates (Cooke; Dynatech, Alexandria, VA) coated with F(ab*)2 fragments of goat anti-human IgM (Cappel) were incubated with cell-free culture supernatants or IgM standards in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (Miles, Elkhart, IN). Bound IgM was detected with peroxidaseconjugated F(ab*)2 fragments of goat anti-human IgM (Cappel) as previously described (15). Preliminary experiments demonstrated that as much as 1 mg/ml of GST did not interfere with this assay system for IgM.

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Differential Suppressive Effects of GST on the Functions of T Cells and B Cells Initial experiments were carried out to test the influences of various concentrations of GST on the production of IgM by B cells and on the proliferation and IFN-g production of T cells. IgM production was induced from highly purified B cells by stimulation with SA / IL-2. T cell proliferation and IFN-g production were induced by stimulation with immobilized antiCD3 in the complete absence of monocytes (16). As can

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whereas the suppressive effects on T cell responses were not observed at lower concentrations, as is consistent with previous reports (10). It should be noted that the inhibition of T cell proliferation by 2.5 mM GST, although significant, was very modest. By contrast, GST strongly suppressed the production of IgM of SAstimulated B cells at concentrations as low as 0.25 mM. Thus, GST as low as 0.25 mM almost completely suppressed B cell responses, whereas it did not significantly suppress T cell proliferation or IFN-g production. These results indicate that GST at low concentrations preferentially suppresses the function of B cells, but not that of T cells. Effect of TMA on B Cell Responses Recent studies have disclosed that the thiomalate component of GST inhibits the expression of vascular cell adhesion molecule-1 and E-selectin on cytokinestimulated endothelial cells (13). On the other hand, it has been previously shown that TMA does not inhibit the function of human T cells (10). It was possible that the selective suppression of B cell responses by GST is mediated by the TMA component of GST. However, as shown in Fig. 2, as much as 2.5 mM TMA did not significantly influence the production of IgM by SAstimulated B cells. The data therefore indicate that the selective suppression of B cell responses by low concentrations of GST is not due to its TMA component alone. GST Inhibits the Initial Stages of B Cell Activation To explore the stages of B cell activation in which GST exerts its suppressive effects, GST was added at different time points after the initiation of culture. As

FIG. 1. Comparison of the effects of GST on T cell proliferation and IFN-g production and on B cell IgM production. T cells (1 1 105/ well) were cultured in wells with immobilized anti-CD3 (64.1, 100 ng/well) for 5 days. B cells (5 1 104/well) were cultured with SA / IL-2 (0.5 unit/ml) for 10 days. Various concentrations of GST were added as indicated. After incubation, T cell proliferation was assessed by the colorimetric assay as described under Methods. The supernatants in T cell or in B cell cultures were harvested and assayed for IFN-g or IgM content, respectively, by ELISA. The significance of the effects of GST in seven different individuals was evaluated by Wilcoxon’s signed rank test. *P õ 0.05.

be seen in Fig. 1, GST significantly inhibited the proliferative responses of highly purified T cells induced by immobilized anti-CD3 at a concentration of 2.5 mM,

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FIG. 2. TMA does not inhibit B cell IgM production. B cells (5 1 104/well) were cultured with SA / IL-2 (0.5 unit/ml) in the presence of various concentrations of GST or TMA. After 10 days of incubation, the supernatants were harvested and assayed for IgM content by ELISA. Data shown are representative of six independent experiments.

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characteristic of the G1 phase in the cell cycle (19, 20). B cells were stimulated with SA / IL-2 for 48 or 72 hr in the presence or the absence of GST, and the expression of CD25 and CD71 was assessed by flow cytometry. As shown in Fig. 4, unstimulated B cells did not express CD25 after 48 hr of culture, whereas stimulation of B cells with SA / IL-2 induced the expression of CD25 and CD71. GST at 0.25 mM almost completely inhibited the expression of CD25 and CD71 on SA-stimulated B cells. These results confirm that GST blocks the early events in B cell activation, thus inhibiting the progression from the G0 to the G1 phase of B cells. DISCUSSION

FIG. 3. GST is required at the initiation of culture to exert optimal suppressive effects on the differentiation of B cells. B cells (5 1 104/well) were cultured with SA / IL-2 (0.5 unit/ml). GST at 0.25 mM or culture medium was added at various times after the initiation of culture. After 10 days of total incubation, the supernatants were harvested and assayed for IgM content by ELISA. Error bars denote SEM.

shown in Fig. 3, the suppressive effects of GST on B cell responses were most marked when it was added at the initiation of culture. Moreover, the suppressive effects of GST were significantly decreased when it was added 72 hr after the initiation of culture and were totally absent when it was added after 120 hr of culture. These results indicate that GST could not suppress the proliferation or differentiation of previously activated B cells. The data suggest instead that GST may inhibit the initial stages of B cell activation. The addition of GST 72 hr after the initiation of incubation resulted in complete (Experiment 1) or partial (Experiment 2) abrogation of its suppressive influences, which might be due to variability in the responses of B cells to SA / IL-2 in healthy individuals. To further specify the stages of B cell activation in which GST exerts its suppressive effects, we next examined the influences of GST on the expression of the a-chain of IL-2R (CD25) and transferrin receptor (CD71) on SA-stimulated B cells, the cellular events

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The results in the current studies have clearly demonstrated that GST at concentrations less than 0.25 mM selectively suppresses the function of B cells, but not that of T cells. Previous studies have shown that GST at concentrations greater than 10 mM (3.9 mg/ml) significantly inhibited the proliferative responses of T cells (10) and endothelial cells (12) and the cytokineinduced expression of adhesion molecules on endothelial cells (13). In previous studies the mean whole blood gold concentration in GST-treated patients was shown to be 2.39 mg/ml (21). It is therefore possible that T cells or endothelial cells are not primary targets of GST in vivo. Of course, it should be pointed out that previous studies did not show a significant correlation between gold concentration and clinical response in RA patients treated with GST. Nevertheless, the data in the current studies, demonstrating that GST suppresses B cell responses at concentrations much lower than those which inhibit the functions of T cells and endothelial cells, support the conclusion that B cells are also one of the targets of gold compounds in vivo. It has been shown that the TMA component of GST contributes to an inhibition of the expression of adhesion molecules on endothelial cells (13). By contrast, TMA has been found to be unable to suppress the function of human T cells (10). The results in the present studies showed that TMA did not inhibit the production of IgM by SA-stimulated B cells. On the other hand, low concentrations (0.01 mg/ml) of auranofin (AUR), which does not contain a TMA component, also selectively inhibited the function of B cells, but not that of T cells (data not shown). The data therefore suggest that the gold component of GST and AUR contributes to the selective suppression of B cell responses. GST suppressed the expression of CD25 and CD71 on SA-stimulated B cells. Thus, GST is considered to inhibit the initial stages of B cell activation. In this regard, the suppressive effects of GST are different from those of other disease-modifying anti-rheumatic drugs, including bucillamine and mizoribine, which do not inhibit the initial activation of B cells (22, 23). Rather, bucillamine and mizoribine suppress matura-

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FIG. 4. GST suppresses the expression of IL-2R (CD25) and transferrin receptor (CD71) on SA-activated B cells. B cells were cultured with or without SA / IL-2 (0.5 unit/ml) in the presence or the absence of GST (0.25 mM). After 48 (A) or 72 hr (B) of culture, the cells were stained with FITC-conjugated anti-CD25 mAb (IgG1), anti-CD71 mAb (IgG1), or control IgG1 mAb and then analyzed by flow cytometry. The percentages of cells positive for CD25 and CD71 are shown. MFI, mean fluorescence intensity.

tion of previously activated B cells, presumably by interfering with the progression of the cell cycle after the G1 phase (22, 23). The data in the current studies therefore show the efficacy of a combination of gold compounds with compounds that inhibit the maturation of previously activated B cells, including bucillamine and mizoribine. It has been shown that in vivo Au dissociates from

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its carrier molecule, such as thiomalate, and binds to the thiol groups of proteins (24, 25). On the other hand, it has been demonstrated that modification of thiol groups in a number of cellular kinases that may play a role in regulating immune responses, including PKC and PKA, results in inactivation of these enzymes (26, 27). In fact, previous studies have demonstrated that GST inhibits the activity of T cell PKC (10). It is there-

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fore likely that GST and AUR also interact with thiol groups of enzymes that participate in the activation of human B cells. However, why low concentrations of GST selectively inhibit the function of B cells is currently unknown. One possible explanation is a differential capacity of B cells and T cells to take up gold compounds. It should be noted that B cells are able to take up exogenous antigens and present them to T cells effectively (28). Thus, recent studies have revealed that low concentrations of GST (0.3–2 mM) suppress the presentation of peptides containing two or more cysteine residues by murine spleen cells or B tumor cells to antigen-specific T cell hybridomas (29). It is presumed that in B cells or B lineage cells treated with low concentrations of GST, gold components dissociate from the carrier molecule and bind to cysteine residues of exogenous antigens (29). It is therefore likely that the dissociated gold components also bind to the cysteine residues of a variety of cellular kinases in human B cells, thus resulting in the inhibition of their activities. In summary, the current studies have demonstrated that GST selectively inhibits the function of B cells at concentrations much lower than those which suppress the function of T cells. It is therefore likely that the selective immunosuppressive effects on B cells of low concentrations of GST reported here contribute to the beneficial effect of gold compounds in the treatment of RA. One of the characteristic features of RA is chronic stimulation of B cells, evidenced clinically by the production of rheumatoid factors, which play an important role in the pathogenesis of the disease (6, 7). It has been suggested that in many patients with RA, the production of RF appears to be T cell independent. Thus, the production of RF has not been decreased by thoracic duct drainage (30), total lymphoid irradiation (31), or anti-T cell mAb treatment (32). Therefore, the data in the present study indicating that low concentrations of gold compounds directly suppress B cell functions by interfering with the initial activation of B cells may well explain the clinical observation that serum Ig levels and RF titers often decrease in RA patients treated with gold compounds (8, 9). Moreover, the observations in the present study also suggest the possible efficacy of the combination of gold compounds with antirheumatic drugs that inhibit the maturation of previously activated B cells, including mizoribine and bucillamine, in the treatment of RA. ACKNOWLEDGMENTS The authors thank Tamiko Yanagida and Haremi Watanabe for technical assistance and Chise Kawashima for preparing the manuscript. This work was supported by a 1995 grant from the Rheumatoid Arthritis Research Committee, the Ministry of Health and Welfare of the Japanese Government, and a grant from Manabe Medical Foundation, Tokyo, Japan.

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Received June 3, 1996; accepted with revision August 13, 1996

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