D-penicillamine cooperates with copper sulfate to enhance the surface expression of functional Fas antigen in rheumatoid synovial fibroblasts via the generation of hydrogen peroxide S. Harada, E. Sugiyama, H. Taki, K. Shinoda, T. Fujita, M. Maruyama, M. Kobayashi First Department of Internal Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan. Abstract Objective D-penicillamine (DP) has been shown to cooperate with copper ion to inhibit cell growth in a variety of cell types. To determine whether this inhibitory action is involved in Fas-mediated apoptosis, we examined the effect of DP and copper sulfate on the expression and function of Fas antigen in rheumatoid synovial fibroblasts (RSFs). Methods The expression of Fas antigen on the cell surface was determined by flow cytometric analysis. Western blot analysis was performed to examine the protein expressions of Fas and Fas-ligand. In addition, the amounts of apoptotic cells were determined by 4’, 6-diamidino-2’-phenylindol dihydrochloride (DAPI) and propidium iodide (PI) staining. Results Although DP and copper sulfate alone did not affect the surface expression of Fas antigen on RSFs, both in combination augmented the Fas expression in dose- and time-dependent manners. The enhanced expression of Fas antigen on their surface was also observed in interleukin-1 (IL-1 ) and/or tumor necrosis factor (TNF ) stimulated RSFs. On the other hand, the combination of DP and copper sulfate did not increase the amounts of cellular Fas protein, as determined by Western blot analysis. To determine whether the induced Fas antigen is functional, we examined the effect of DP and copper sulfate on Fas-mediated apoptosis, using an agonistic anti- Fas antibody. The treatment of this antibody induced the apoptosis in untreated RSFs, as determined by DAPI staining. The combination of DP and copper sulfate further enhanced the Fas-mediated apoptosis. The enhanced apoptosis and cell surface expression of Fas was completely prevented by catalase, indicating that hydrogen peroxide may be involved in these effects of DP and copper sulfate. The protein expression of Fas-ligand, a natural ligand for Fas antigen, in RSFs. was expressed in untreated RSFs. However, the protein levels were not modulated by DP and copper sulfate. Conclusions Our data demonstrated that DP cooperated with copper sulfate to enhance the cells surface expression of func- tional Fas antigen in RSFs. In addition, Fas-ligand was expressed in the RSFs. These findings suggested that DP might regress rheumatoid synovial hyperplasia via Fas-mediated apoptosis. Key words Rheumatoid arthritis, synovial fibroblast, D-penicillamine, Fas-mediated apoptosis, copper sulfate. Clinical and Experimental Rheumatology 2002; 20: 469-476. Fas expression by D-penicillamine in RA / S. Harada et al. Shuji Harada, MD; Eiji Sugiyama, MD, Introduction tion was also seen in RA patients tak- PhD; Hirofumi Taki, MD, PhD; Kouichiro Rheumatoid arthritis (RA) is an inflam- ing DMARDs including DP for longer Shinoda, MD; Tadashi Fujita, MD, PhD; matory joint disease in which perpetua- pe r iods (12). The re fo r e, aggres s i ve ther- Muneharu Maruyama, MD, PhD; Masashi tion of the chronic synovitis leads to apy with standard DMARDs according Kobayashi, MD, PhD. bone and cartilage degradation (1). Ini- to “sawtooth” strategy (13) or combi- Please address correspondence and tial histological features of RA are cha- n ation therapy ap p e a rs to still have reprint requests to: Dr. Eiji Sugiyama, First Department of Internal Medicine, racterized by synovial lining hyperpla- some impact on the RA treatment. Toyama Medical and Pharmaceutical sia, and the synovial cells play critical Although a number of pharmacological University, Sugitani 2630 Toyama 930- roles in the pathogenesis of RA. These actions of DP were reported, the mech- 0194, Japan. rheumatoid synovial cells have trans- anisms underlying its beneficial effects E-mail: [email protected] formed phenotypes, which express the h ave not been adequat e ly ex a m i n e d. Received on July 17, 2001; accepted protooncogenes ras, myc, fos, or jun Previous studies reported that DP syn- in revised form on March 1, 2002. (2). These protooncogenes up-regulate thesized hydrogen peroxide in the pres- © Copyright CLINICAL AND the proliferation of synovial cells and ence of copper ion, and that the gener- EXPERIMENTAL RHEUMATOLOGY 2002. induce the production of cysteine pro- ated hy d rogen peroxide inhibited the teinase, collagenase, and stromelysin, pro l i fe r ation of T lym p h o c ytes (14, 15 ) , which are involved in cartilage destruc- endothelial cells (16) and fi b ro bl a s t s tion (3). In addition, the synovial cells (17). However, the mechanism of these also produce large amounts of inflam- inhibitory actions still remains unclear. matory cytokines such as interleukin-1 The oxidative stress has been shown to (I L - 1 ) , tumor necrosis factor (T N F ), be critical in triggering cell death by and interleukin-6 (IL-6), which perpet- various stimuli (18), and that the reac- uate rheumatoid inflammation (4, 5). tive oxygen intermediate such as super- Th e re fo re, these synovial cells are oxide (19) or hydrogen peroxide (20) thought to be main targets for the treat- may be involved in apoptosis. These ment of RA. findings prompted us to examine the Dysregulation of apoptosis (program- effect of DP on Fas-mediated apoptosis med cell death) may be one of the rea- in rheumatoid synovial cells. In this sons for synovial hyperplasia in RA. study, we evaluated the effect of DP in The apoptosis of cells is generally initi- the presence of copper ion on the ex- ated by molecular and cellular interac- pression and function of Fas antigen tions such as the binding of Fas to Fas- and Fas-ligand in rheumatoid synovial ligand or TNF to its TNF receptor. fibroblasts. Recent studies have shown that the Fas/Fas-ligand pathway may be dimin- Patients and methods ished by pro i n fl a m m at o ry cy t o k i n e s , Cell preparation s o l u ble Fa s , or soluble Fa s - l i gand in C u l t u red synovial cells used in this RA (6-8), and the decreased signaling s t u dy we re derived from 15 pat i e n t s resulted in imbalance between prolifer- with RA who we re underwent total ation and cell death. Th e re fo re, t h e knee replacement or synovectomy. In- induction of Fas-mediated apoptosis of fo rmed consent was obtained fro m rheumatoid synovial cells is believed to e a ch patient. All patients we re diag- be useful for the treatment of RA (9). nosed as having RA according to the The treatment of patients with RA has 1987 criteria of American College of ch a n ged dra m at i c a l ly in the past 3 Rheumatology (21). The synovial tis- years, and the new drugs targeting in- sues were cut into fragments of 1-3 mm fl a m m at o ry cytokines such as T N F in diameter, and incubated in 0.5-1 and IL-1 were introduced for the treat- mg/ml of collagenase and 5-10 g/ml ment. However, standard disease modi- of deoxyribonuclease I for 2-3 hours. fying anti-rheumatic drugs (DMARDs) After digestion, the resultant single cell such as gold or D-penicillamine (DP) suspension was wa s h e d, fi l t rat e d seems to be beneficial for patients with through sterile gauze and nylon mesh, mild RA in regard to their cost effec- and finally resuspended in Dulbecco’s tiveness and side effects. DP has been modified Eagle’s medium supplement- reported to improve the symptoms of ed with 10% heat inactivated fetal calf RA and to bring to clinical remission serum (FCS), penicillin 100 U/ml, gen- (10, 11). In addition, improved func- tamicin 60 mg/ml, N - 2 - hy d rox ye t h l- 470 Fas expression by D-penicillamine in RA / S. Harada et al. p i p e razine-N-2-ethanesulphonic acid fl u o ride). The cell ly s ates we re cen- To determine the amounts of the apop- 12.5 mM, and L-glutamine 2 mM trifuged at 12,000 g for 10 min at 4°C, totic cells, we measured the amounts of (DMEM medium). After overnight in- and the supernatants were used for the hypodiploid nuclear DNA contents by c u b at i o n , n o n a d h e rent cells we re re- subsequent experiments. Protein con- flow cytometric analysis of PI staining. m ove d. When adherent cells became centrations were measured by Bradford After incubation with various reagents, confluent, the cells were harvested by methods (Bio-Rad protein assay; Bio- the cells wer e harves t e d , and immediate- trypsinisation, washed, and one third of rad, Richmond, CA). Thirty g of the ly immobilized by 70% cold ethanol them were resuspended in culture dish- protein preparations were separated by overnight. After washing with PBS, the es. The synovial cells at three or more electrophoresis on a 7.5% sodium do- pellet was resuspended in 40 mM cit- p a s s ages became morp h o l ogi c a l ly fi- decyl sulfate-polyacrylamide gel, and rate buffer for 30 min, incubated with broblast-like, and all negative for CD14 transferred onto an Immobilon-P mem- 100 g/ml RNase in PBS for 30 min at antigen and HLA-DR antigen on their brane (Millipore, Bedford, MA).