THE DOMINANT ROLE OF EXOGENOUS OR ENDOGENOUS INTERLEUKIN-1-BETA ON EXPRESSION AND ACTIVITY OF INDUCIBLE NITRIC-OXIDE SYNTHASE IN RAT MICROVASCULAR BRAIN ENDOTHELIAL-CELLS

In the brain large amounts of nitric oxide are produced in response to various pathological stimuli such as infectious agents, ischemia and trauma. Although it is known that endothelial cells can express the in ducible isoform of nitric oxide synthase (iNOS) upon activation, the i mpact of different cytokines on iNOS expression in rat microvascular e ndothelial cells remains unclear. We now investigated iNOS mRNA expres sion and enzyme activity in primary cell cultures of rat microvascular brain endothelial cells after treatment with the proinflammatory cyto kines interleukin-1 beta (IL-1 beta), Tumor necrosis factor-alpha (TNF -alpha), and interferon-gamma (IFN-gamma) alone or in combination. Cel ls were characterized by immunocytochemistry staining for von-Willebra nd-factor and the rat brain endothelial antigen recognized by monoclon al antibody Ox2. iNOS-enzyme activity was determined by measurement of nitrite in the supernatants of cell culture using the Griess-reaction . In addition mRNA expression was analysed by RT-PCR with iNOS and IL- 1 beta specific primers. All cells in the endothelial cell culture wer e found to express the antigenic phenotype vWF(+)/Ox2(+)/Ox43(-), thus identifying the cells as rat brain endothelial cells of microvascular origin. IL-1 beta was the only cytokine that as a single stimulus ind uced iNOS mRNA expression and iNOS-enzyme activity in these endothelia l cells. All combinations of two cytokines, including that of TNF-alph a and IFN-gamma or the triple combination led to expression of iNOS-mR NA and active protein. Cell activation by the combination of TNF-alpha + IFN-gamma led to an early expression of IL-1 beta by the endothelia l cells suggesting iNOS induction as a consequence of endogenous IL-1 beta production under this challenge. The experiments prove that rat b rain microvascular endothelial cells express iNOS and produce large am ounts of NO under inflammatory conditions. Furthermore, our results in dicate a decisive role of IL-1 beta in iNOS expression and NO generati on. (C) 1997 Elsevier Science Ireland Ltd.