Tumor necrosis factor-alpha generates reactive oxygen species via a cytosolic phospholipase A(2)-linked cascade

Reactive oxygen species (ROS) are important regulatory molecules implicated in the signaling cascade triggered by tumor necrosis factor (TNF)-alpha, a lthough the events through which TNF-alpha induces ROS generation are not y et well characterized. We therefore investigated selected candidates likely to mediate TNF-alpha-induced ROS generation. Consistent with the role of R ac in that process, stable expression of Rac(Asn-17), a dominant negative R ad mutant, completely blocked TNF-alpha-induced ROS generation. To understa nd better the mediators downstream of Rac, we investigated the involvement of cytosolic phospholipase A(2) (cPLA(2)) activation and metabolism of the resultant arachidonic acid (AA) by B-lipoxygenase (5-LO). TNF-alpha-induced ROS generation was blocked by inhibition of cPLA(2) or 5-LO, but not cyclo oxygenase, suggesting that TNF-alpha-induced ROS generation is dependent on synthesis of AA and its subsequent metabolism to leukotrienes. Consistent with that hypothesis, TNF-alpha Rac-dependently stimulated endogenous produ ction of leukotriene B-4 (LTB4), while exogenous application of LTB4 increa sed levels of ROS. In contrast, application of leukotrienes C-4, D-4, and E -4 or prostaglandin E-2 had little effect. Our findings suggest that LTB4 p roduction by 5-LO is situated downstream of the Rac-cPLA(2) cascade, and we conclude that Rac, cPLA(2), and LTB4 play pivotal roles in the ROS-generat ing cascade triggered by TNF-alpha.