DIFFERENTIAL EFFECT OF TUMOR-NECROSIS-FACTOR-ALPHA ON THROMBOMODULIN GENE-EXPRESSION BY HUMAN MONOCYTOID (THP-1) CELLS VERSUS ENDOTHELIAL-CELLS

Human mononuclear phagocytes (M empty set) express a functional form o f thrombomodulin (TM), the anticoagulant molecule typically considered purely in the context of regulation of conversion of protein C (PC) t o activated PC (aPC) by thrombin-bound TM al the endothelial cell surf ace. We have been interested in the anti-inflammatory actions of aPC, including its ability to suppress M empty set production of multiple p ro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-al pha) and interleukin-1 (IL-1), leading us to consider whether M empty set surface expression of TM and resultant local aPC generation, might contribute to autoregulation of M empty set activation at sites of in flammation involving thrombin and fibrin formation. Since TNF-alpha an d IL-1 are known to downregulate endothelial expression of TM, this st udy investigated the effects of TNF-alpha on production of TM by the m onocytic leukemic cell line, THP-1. THP-1 cells display many monocyte- like properties, providing a convenient source for biochemical and mol ecular studies. Western blotting of lysates of THP-1 cells versus cult ured human umbilical vein endothelial cells showed that after 24 h of stimulation, TNF-alpha decreased TM protein expression in endothelial but not THP-1 cells and comparable responses were noted by flow cytome try. Subsequent Northern blot analysis showed that at 24 h, TNF-alpha diminished TM steady stale mRNA in endothelial but not THP-1 cells, al though Northern analysis of the kinetics of TM steady state mRNA did s how a rapid and transient modulation by TNF-alpha at 2 h of stimulatio n, which was confirmed by nuclear run-on analysis of the effect of TNF -alpha on TM gene transcription rates in THP-1 cells, analysis of prot ein expression by flow cytometry and Western blotting showed similar e ffects. In contrast to the divergent effects of TNF-alpha on THP-1 vs endothelial cells, agonists such as cyclic adenosine monophosphate (c- AMP) and phorbol ester (PMA) had comparable effects on THP-1 and endot helial cells, resulting in parallel increases or decreases in TM mRNA and protein expression, respectively. Hence, there is a 'split' in the nature of endothelial vs THP-1 cellular responses to TNF-alpha as com pared to non-inflammatory stimuli, suggesting cell-specific difference s in regulation of the TM promoter. We conclude that in contrast to it s effects on TM expression by endothelial cells, exposure of THP-1 cel ls to TNF-alpha causes a rapid and transient decrease in TM mRNA produ ction which is followed by sustained and high level expression, suppor ting the concept that M empty set expression of TM may contribute to r egulation of M empty set activation and cytokine production at inflamm atory sites. (C) 1998 Elsevier Science Ireland Ltd. All rights reserve d.