Tumor necrosis factor-α generates reactive oxygen species via a cytosolic phospholipase A2-linked cascade

Reactive oxygen species (ROS) are important regulatory molecules implicated in the signaling cascade triggered by tumor necrosis factor (TNF)-alpha, although the events through which TNF-alpha induces ROS generation are not yet well characterized. We therefore investigated selected candidates likely to mediate TNF-alpha-induced ROS generation. Consistent with the role of Rac in that process, stable expression of Rac(Asn-17), a dominant negative Rad mutant, completely blocked TNF-alpha-induced ROS generation. To understand 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 cyclooxygenase, 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 production of leukotriene B-4 (LTB4), while exogenous application of LTB4 increased 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 production 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-generating cascade triggered by TNF-alpha.