TNF-alpha/TNFRI in primary and immortalized first trimester cytotrophoblasts

During the first trimester of pregnancy endogenous expression of tumour nec rosis factor (TNF)-alpha has been detected in villous, as well as in prolif erating and invading extravillous, trophoblasts suggesting that the protein could be involved in trophoblast differentiation. To gain insights into th e putative role of the TNF-alpha signalling pathway, we investigated expres sion of its receptors, TNFR I and II, in first trimester placentae and earl y trophoblasts, and studied the influence of the cytokine on cell prolifera tion and apoptosis. ELISA and RT-PCR revealed secretion/expression of TNFRI protein/mRNA in immortalized ED27 cells and purified first trimester cytot rophoblasts, while soluble TNFRII was undetectable in cell culture supernat ants. In agreement, immunohistochemical analyses of first trimester placent ae showed that TNFRI is localized to the villous cyto- and syncytiotrophobl ast, to the proliferating cytotrophoblasts of the cell islands and cell col umns, as well as to extravillous cells invading decidual tissue. TNFRII, ho wever, was absent in early trophoblast populations. Interleukin (IL)-1 and phorbol 12-myristate 13-acetate (PMA) induced shedding of TNFRI from ED27 a nd primary cells suggesting that under inflammatory conditions the soluble receptor protein may protect from cytotoxic effects of TNF-alpha. Upon incu bation with increasing amounts of TNF-alpha no significant changes in DNA-c ontent or cell numbers were found, suggesting that the cytokine does not au gment proliferation of primary cytotrophoblasts. High doses of TNF-alpha, h owever, provoked growth arrest in ED27 cells as evaluated by cell counting, but did not induce necrosis/apoptosis as was assessed by TUNEL assay. In f irst trimester cells addition of elevated amounts of TNF-alpha resulted in the appearance of TUNEL-positive cells and an increase in caspase-3 enzyme activity suggesting that the TNF-alpha-dependent apoptotic cascade is execu ted in a portion of the early cytotrophoblasts. (C) 2000 Harcourt Publisher s Ltd.