Development of anti-cytokine drugs for treatment of endometriosis

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Peritoneal fluid (PF) in women with endometriosis contains an increased number of activated macrophages that secrete a variety of cytokines. We have previously shown that the PF levels of tumor necrosis factor alpha (TNFa) and interleukin-8 (IL-8) were significantly higher in patients with endometriosis. There was a significant correlation between PF levels of TNFa and IL-8. The purpose of the study is to investigate the role of TNFa in proliferation of endometriotic stromal cells. We also examined the activation of nulear factor-kB (NF-kB) during the induction of IL-8 by TNFa.Chocolate cyst linings of the ovaries in patients with endometriosis (n=39) were a source of endometriotic tissue. The receptors for IL-8 and TNFa were examined by RT-PCR. Proliferation of endometriotic cells was studied using MTT assay. The expression of IL-8 gene and protein was analyzed by Northern blotting and ELISA, respectively. Western blot analysis and electrophoretic mobility shift assay (EMSA) were used … More to detect NF-kB activation by TNFa. Effects of inhibitor for NF-kB, TPCK, on TNFa action were also examined.Transcripts of IL-8 receptor type A, TNF receptor type I and II were detected in endometriotic stromal cells. TNFa (10 and 100 pg/mL) induced the gene and protein expression of IL-8 in endometriotic stromal cells in a dose-dependent fashion. Addition of IL-8 (25-100 pg/mL) or TNFa (5-100 pg/mL) to the culture medium stimulated the proliferation of stromal cells. The stimulatory effects of TNFa were abolished by adding anti-IL-8 antibody. These results suggest that the action of IL-8 mediates the stimulatory effects of TNFa on stromal cell proliferation. The activation of NF-kB is usually associated with phosphorylation of IkB, followed by its degradation by the proteasome and NF-kB nuclear translocation. In order to detect the activation of NF-kB in stromal cells during inducible expression of IL-8 by TNFa, Western blotting was performed using an antibody for phosphorylated IkB (p-IkB). Addition of TNFa (100 pg/mL) rapidly induced p-IkB in stromal cells. EMSA revealed that adding TNFa promoted translocation of activated NF-kB.Preincubation with TPCK reduced TNFa inducible expression of IL-8 gene and protein. TNFa action mediated by IL-8 may contribute to progression of endometriosis by promoting the growth of endometriotic cells. We demonstrated here that NF-kB activation was involved in the induction of IL-8 by TNFa in endometriotic tissues. Thus, TNFa and its signal transduction molecule, NF-kB, play important roles in the pathophysiology of endometriosis. Less