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 cyt okines. Hypoxia-mediated induction of interleukin-6 (IL-6), a cytokine with anti-inflammatory properties, could set in motion mechanisms lim iting 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 incre ased IL-6 transcripts in the lung, and immuno-staining revealed a stri king increase in IL-6 antigen in pulmonary vasculature. Transfection o f ECs with deletion chimeric IL-6 promoter-chloramphenicol acetyltrans ferase (CAT) constructs showed hypoxia-mediated 9-11-fold induction wi th -1200/+13, -596/+13, and -225/+13 but no induction with -111/+13. E lectrophoretic mobility shift assays (EMSAs) using -225/-111 as the la beled probe demonstrated enhanced binding activity in nuclear extracts of hypoxic ECs and lung; the appearance of the gel shift band was pre vented by excess unlabeled probe (-225/-111), and hypoxia-mediated enh ancement of the band was blocked by a probe corresponding to the nucle ar factor (NF)-IL-6 site (-158/-145). The hypoxia-enhanced band on EMS A displayed a supershift with antibody to CCAAT-enhancer-binding prote in beta (C/EBP-beta), but antibody to C/EBP-alpha or -delta was withou t effect. Transfection of ECs with a construct comprising thymidine ki nase promoter, -225/-111 in either the 5' to 3' or 3' to 5' orientatio n, and the reporter CAT showed this region to be an enhancer (approxim ate to 8-fold) under hypoxia, EMSA with the NF-IL-6 probe revealed a p rominent induction of binding activity with nuclear extracts from hypo xic ECs and whole lung, Constructs with -158/-145 and the CAT reporter gene showed induction when transfected into hypoxic ECs, whereas a si milar construct with the NF-IL-6 motif mutationally inactivated failed to display hypoxia-induced expression. Furthermore, the tumor necrosi s factor (TNF) gene, whose product contributes to ischemic pathology a nd 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 si tes may also be induced by a similar mechanism.