TNF-Binding Protein Complex Inhibits TNF-α-Mediated Effects in Human Endometriotic Cell Line.

Endometriosis is characterized by the presence of elevated proinflammatory cytokines like TNFα in the peritoneal cavity. Inhibition of TNFα by TNF binding protein-1 (TBP-1), the soluble form of TNFα receptor-1, was effective in animal models of endometriosis. Recently at EMD Serono, a novel high-affinity inhibitor of TNFα was created by fusing TBP-1 to both the α and β subunits of hCG. This dimeric protein called TNF-soluble high affinity receptor complex (TNF-SHARC) was described to have prolonged pharmacokinetic profile compared to TBP-1 and effective in inhibiting collagen-induced arthritis in mice. In the current study we examined the efficacy of TNF-SHARC in a cellular model of endometriosis. Immortalized human endometriotic cells (12Z cells) respond to TNF by increased secretion of cytokines. In addition these cells have undergone epithelial-mesenchymal transition (EMT) as evidenced by increase in the expression of N-cadherin but still retain cytokeratin, a marker for epithelial cells. EMT is believed to increase the invasive phenotype of cells, thus reversal of EMT could lead to ameliorating the disease state. We examined the effect of TBP and TNF-SHARC on TNFα induced secretion of cytokines by multiplex measurement as well as monitored the expression of N-cadherin and the associated signaling molecule β-catenin by immunocytochemistry. TNFα increased the secretion of IL8, IL6, GMSCF, and MCP-1. TBP-1 and TNF-SHARC dose-dependently inhibited the secretion of all four cytokines induced by TNFα (10 ng/ml). The IC50 values (nM) for TBP-1 vs TNF-SHARC to inhibit- GMCSF is 65 vs 0.71; IL6 is 16 vs 0.61; IL8 is 66 vs 0.69; and MCP-1 is 1814 vs 1.37. Immunostaining of 12Z cells was negative for E-cadherin while positive for N-cadherin. Untreated 12Z cells display a peripheral staining of N-cadherin while treatment with TNFα caused dispersal of the N-cadherin with irregular cytoplasmic staining. TBP or TNF-SHARC treatment reduced the peripheral staining. In contrast, there was no difference in panCytokeratin staining with TNFα or TBP. Staining for β-catenin, the downstream signaling molecule of N-cadherin, was expressed in cytosol in untreated cells. Treatment of cells with TNFα (10ng/ml) localized the staining to nucleus, which was altered by TNF antagonists. To summarize, the present study demonstrate TNF-SHARC to be more effective than TBP-1 to inhibit TNFα-induced cytokines in12Z cells, while both antagonists were capable of reducing the expression of N-cadherin. Thus we provide evidence for supporting the development of TNF-SHARC as a therapeutic candidate for treating endometriosis in human.