INDUCTION OF INTERLEUKIN-6 (IL-6) BY HYPOXIA IN VASCULAR CELLS - CENTRAL ROLE OF THE BINDING-SITE FOR NUCLEAR FACTOR-IL-6

The pathologic picture in ischemic tissue injury shares features with the inflammatory response, including production of proinflammatory cytokines. Hypoxia-mediated induction of interleukin-6 (IL-6), a cytokine with anti-inflammatory properties, could set in motion mechanisms limiting inflammation in ischemia, Exposure of cultured endothelial cells (ECs) to H (pO(2) approximate to 12-16 torr) increased transcription of IL-6, elevated levels of IL-6 mRNA, and induced elaboration of IL-6 antigen in a time-dependent manner, Exposure of mice to hypoxia increased IL-6 transcripts in the lung, and immuno-staining revealed a striking increase in IL-6 antigen in pulmonary vasculature. Transfection of ECs with deletion chimeric IL-6 promoter-chloramphenicol acetyltransferase (CAT) constructs showed hypoxia-mediated 9-11-fold induction with -1200/+13, -596/+13, and -225/+13 but no induction with -111/+13. Electrophoretic mobility shift assays (EMSAs) using -225/-111 as the labeled probe demonstrated enhanced binding activity in nuclear extracts of hypoxic ECs and lung; the appearance of the gel shift band was prevented by excess unlabeled probe (-225/-111), and hypoxia-mediated enhancement of the band was blocked by a probe corresponding to the nuclear factor (NF)-IL-6 site (-158/-145). The hypoxia-enhanced band on EMSA displayed a supershift with antibody to CCAAT-enhancer-binding protein beta (C/EBP-beta), but antibody to C/EBP-alpha or -delta was without effect. Transfection of ECs with a construct comprising thymidine kinase promoter, -225/-111 in either the 5' to 3' or 3' to 5' orientation, and the reporter CAT showed this region to be an enhancer (approximate to 8-fold) under hypoxia, EMSA with the NF-IL-6 probe revealed a prominent induction of binding activity with nuclear extracts from hypoxic ECs and whole lung, Constructs with -158/-145 and the CAT reporter gene showed induction when transfected into hypoxic ECs, whereas a similar construct with the NF-IL-6 motif mutationally inactivated failed to display hypoxia-induced expression. Furthermore, the tumor necrosis factor (TNF) gene, whose product contributes to ischemic pathology and contains a putative regulatory NF-IL-6 site, demonstrated enhanced binding activity for its NF-IL-B motif and induction of TNF mRNA based on analysis of hypoxic lung, These data indicate that hypoxia induces expression of IL-6, most likely a result of hypoxic activation at the NF-IL-B site, and suggest that other genes with regulatory NF-IL-6 sites may also be induced by a similar mechanism.