The mechanism of release of proinflammatory cytokines by peripheral blood monocytes is unknown. Peroxynitrite (ONOO(-)) formed by the reaction of nitric oxide (NO) and superoxide is released predominantly by inflammatory cells at the site of injury in several inflammatory pathologies. Here we show that human monocytes treated with ONOO(-) at micromolar concentrations induce a dose-dependent release of proinflammatory cytokines. These effects were not antagonized by up to 100 microm epigallocatechin gallate, an inhibitor of protein nitration. However, the proteasome inhibitor Z-Ile-Glu(OtBu)-Ala-Leu-CHO and 5,10,15,20-tetrakis(4-sulfonatophenyl)prophyrinato iron (III) chloride, a cell-permeable scavenger of ONOO(-), almost completely inhibited the release of cytokines and the nuclear translocation of the nuclear factor (NF)-kappaB transcription factor. SDS-PAGE electrophoresis separation with Western blotting of cell extracts also indicated that phosphorylation and nitration of tyrosine residues in IkappaB-alpha molecules correlated with NF-kappaB translocation and cytokine release. In addition, the DNA binding activity of the NF-kappaB from the nuclear extracts also correlated with its nuclear translocation. These findings indicate ONOO(-) plays an essential role in the mechanism of proinflammatory cytokine release by monocytes and that Rel/NF-kappaB activation is the obligatory pathway.
