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.
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-1alpha (IL-1alpha) and/or tumor necrosis factor a (TNFalpha) 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 functional 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.
Harada
S
S
First Department of Internal Medicine, Toyama Medical and Pharmaceutical University, Japan.
Sugiyama
E
E
Taki
H
H
Shinoda
K
K
Fujita
T
T
Maruyama
M
M
Kobayashi
M
M
D018501:Antirheumatic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001172:Arthritis, Rheumatoid / Q000473:pathology*; D015153:Blotting, Western; D002374:Catalase / Q000494:pharmacology; D002478:Cells, Cultured; D019327:Copper Sulfate / Q000494:pharmacology*; D004247:DNA / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D004357:Drug Synergism; D053222:Fas Ligand Protein; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007375:Interleukin-1 / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000096:biosynthesis; D008856:Microscopy, Fluorescence; D010396:Penicillamine / Q000494:pharmacology*; D013583:Synovial Membrane / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D019014:fas Receptor / Q000096:biosynthesis*
D018501:Antirheumatic Agents; D004338:Drug Combinations; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D007375:Interleukin-1; D008562:Membrane Glycoproteins; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor; D004247:DNA; D006861:Hydrogen Peroxide; D002374:Catalase; D010396:Penicillamine; D019327:Copper Sulfate