RELEASE OF THROMBOMODULIN FROM ENDOTHELIAL-CELLS BY CONCERTED ACTION OF TNF-ALPHA AND NEUTROPHILS - IN-VIVO AND IN-VITRO STUDIES

Inflammatory cytokines decrease the expression of thrombomodulin (TM) on the endothelial cell surface by suppression of TM transcription and translation or internalization with subsequent degradation. Neverthel ess, elevated serum TM levels are found in diseases associated with sy stemical or locally increased levels of inflammatory cytokines. To stu dy directly the in vivo effects of tumour necrosis factor-alpha (TNF-a lpha) we determined the course of serum TM after systemic recombinant human (rh)TNF-alpha therapy. The TM levels were determined by enzyme-l inked immunosorbent assay (ELISA). Systemic rhTNF-alpha therapy result ed in a marked and significant increase of serum TM. Using a mouse mod el we studied whether increased serum TM is associated with a decrease d expression of TM on the endothelial surface in vivo. The immunohisto chemical staining of the vasculature of meth-A sarcoma transplanted in mice showed a loss of TM immunoreactivity 4 hr after intravenous TNF- alpha application. To study the mechanism of TNF-alpha mediated releas e of TM, cultured endothelial cells were incubated with neutrophils an d TNF-alpha. Incubation with TNF-alpha alone did not lead to an increa se of TM in vitro. However TM was released into the culture supernatan t when endothelial cells pretreated with TNF-alpha were exposed to neu trophils. This was associated with morphological evidence of endotheli al cell damage. Therefore, the concerted action of cytokine-stimulated endothelial cells and neutrophils results in release of TM from cultu red endothelial cells after rhTNF-alpha therapy. This might explain th e increased serum TM levels observed in diseases associated with incre ased systemic or local levels of inflammatory cytokines despite the in duced internalization and the direct inhibitory effects of TNF-alpha o n TM transcription and translation.