Background Hyperplasia of synovial membrane, a characteristic feature of rheumatoid arthritis (RA), is proposed to result from either the enhanced proliferation of fibroblast-like synoviocytes (FLS) or the resistance of these cells to apoptotic death. Therapeutic approaches to inhibit an excessive growth of RA FLS are not satisfactory. We have previously shown that taurine chloramine (Tau-Cl), a chlorinated derivative of a dominant free amino acid taurine (Tau), formed by neutrophils during respiratory burst, inhibits FGF-triggered proliferation of RA FLS (Arthritis Rheum 1999;42:2552).
Objectives The aim of present study was to investigate the mechanism of Tau-Cl inhibition of RA FLS growth.
Methods FLS, isolated from the synovial membrane of RA patients, were stimulated in vitro for 18–72 h with 10 ng/ml of either rhPDGF or rhTNF-α. Tau or Tau-Cl were added (at 100–500 μM concentration) together with the stimuli. Cell proliferation was determined by incorporation of 3H-TdR. To evaluate the cytotoxicity of tested compounds, and to estimate the recovery of the cells from the cultures, the lactate dehydrogenase (LDH) activity was measured in culture supernatants and in cell lysates, respectively. The expression of proteins regulating the cell-cycle progression or apoptosis, was estimated by Western bloting.
Results The rate of spontaneous proliferation of FLS ranged from 176 to 4639 cpm. Both, PDGF and TNF-α raised it significantly by 6 and 2 times, respectively. Tau was neither cytotoxic nor affected the recovery/proliferation of RA FLS. In contrast, Tau-Cl inhibited both the proliferation and the cell recovery in a dose-dependent manner. Independent of the type of stimulus, both responses were reduced by 70% in the presence of 250 μM, and were almost completely blocked (by 90%) in the presence of 500 μM concentration of Tau-Cl. At the same concentrations (250 and 500 μM) Tau-Cl inhibited PDGF- and TNF-α -triggered expression of PCNA (proliferating cell nuclear antigen) but has no effect on the expression of either the Cdk-4 (the cyclin-dependent kinase-4), Cdk-inhibitors (p16, p21, p27) or the anti-apoptotic, Bcl-2, protein. At the late time points (48–72 hrs) only the higher (500 μM) concentration of Tau-Cl exerted cytotoxic (50%) effect.
Conclusion We report that Tau-Cl, a physiologic factor originated from activated neutrophils, blocks proliferation of RA FLS by affecting the common event critical for the cell cycle progression: the expression of PCNA protein, known to act as a cofactor for DNA polimerase δ. Thus, Tau-Cl might be promising therapeutic agent for RA. Whether Tau-Cl triggers apoptosis of RA FLS is under investigation.
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