CYTOKINE-INDUCED NEUTROPHIL-MEDIATED INJURY OF HUMAN ENDOTHELIAL-CELLS

To understand the pathogenesis of vasculitides, we analyzed how cytoki ne stimulation of HUVEC in vitro activates the cytotoxic capacity of p olymorphonuclear (PMN) granulocytes. IL-1 beta, IFN-gamma, or TNF-alph a caused highly significant dose and time-dependent HUVEC injury, TNF- alpha-treated HUVEC activated the PMN by means of phospholipase C-rela ted event, since coincubations conferred PMN to react with a rise of c ytosolic calcium concentrations, [Ca2+](i), Ab blockade of ICAM-1 on H UVEC inhibited 50 to 70% of the injury induced by these cytokines, whe reas a mAb to E-selectin reduced 45 to 65% of IL-1 beta- and TNF-alpha -, but not IFN-gamma-induced cytotoxicity, The role of nitric oxide (N O) was of significance since injury induced by each cytokine was reduc ed by 60 to 87% by specific NO-synthase inhibitors, as well as by scav enging extracellular NO by oxyhemoglobin. In contrast, injury induced by TNF-alpha was inhibited by neither superoxide dismutase or catalase , alpha(1)-antitrypsin, alpha(2)-macroglobulin, nor the platelet-activ ating factor receptor antagonist WEB-2086, Moreover, PMN from a patien t with chronic granulomatous disease were fully capable of mediating c ytotoxicity, The possibility that IL-8, produced by HUVEC in response to TNF-alpha, mediated activation of PMN was not corroborated since ad dition of an IL-8-blocking mAb did not modify HUVEC injury, Nonetheles s, the IL-8 mAb (but not WEB-2086) blocked the rise of [Ca2+](i), Thus , in this in vitro model of vasculitis, the effect of IL-1 beta, IFN-g amma, and TNF-alpha as promoters of cytekine-mediated neutrophil-depen dent injury to HUVEC is a process dependent on expression of adhesion molecules and probably associated with NO produced in the system.