In human neutrophils TNF-elicited O2? production requires adherence and integrin activation. p47phox phosphorylation indicating a role for δ-PKC in regulating O2? production at the level of p47phox. Activation of ERK and p38 MAPK is also required for TNF-elicited O2? generation. TNF-mediated ERK but not p38 MAPK recruitment to p47phox was δ-PKC-dependent. δ-PKC activity is controlled through serine/threonine phosphorylation and phosphorylation of δ-PKC (Ser643) and δ-PKC (Thr505) was increased significantly by TNF in adherent cells via a PI3K-dependent process. Thus signaling for TNF-elicited O2? generation is regulated by δ-PKC. Adherence-dependent cooperative signaling activates PI3K signaling δ-PKC phosphorylation and δ-PKC recruitment to p47phox. δ-PKC activates p47phox by serine phosphorylation or indirectly through control of ERK recruitment to p47phox. < 0.05. RESULTS TNF-mediated O2? generation is δ-PKC-dependent TNF elicited O2? generation in neutrophils requires adherence and is mediated via the TNFR-1 complex [9 10 12 41 Adherence of human neutrophils to ECM proteins such as FN produces significant alterations in the kinetics of oxygen radical production in response to soluble mediators. There is a significant delay lasting from 15 to 60 min followed by O2? generation which is enhanced significantly as compared with nonadherent neutrophil responses to the same stimuli [9]. To determine whether δ-PKC is a regulator of TNF-elicited O2? generation in FN-adherent neutrophils human neutrophils were pretreated with the selective cell-permeant δ-PKC TAT inhibitory peptide a TAT carrier control peptide or buffer alone. Previous studies demonstrated that this dominant-negative δ-PKC TAT peptide inhibits TNF-mediated activation of δ-PKC in neutrophils [17 18 When stimulated with TNF (25 ng/ml) FN-adherent neutrophils produced significant quantities of O2? over a 2-h time period (Fig. 1 and Table 1). In agreement with previous studies we observed a 30- to 45-min delay in O2? production following TNF stimulation of adherent neutrophils [9 10 42 43 Most of the O2? was produced within 90 min following the addition of TNF (Fig. 1 and Table 1). Pretreatment of human neutrophils with the dominant-negative δ-PKC TAT peptide resulted in a significant delay in the onset Nepicastat of TNF-mediated O2? generation but had no effect on the Vmax of the reaction (Fig. UBE2T 1 and Table 1). The delay in onset of O2? generation in response to TNF produced a 65% decrease of O2? generation at 60 min and a 25% decrease at 90 min (Fig. 1 and Table 1). However by 120 min there were no significant differences in the amount of O2? produced (Fig. 1 and Table 1). Conversely pretreatment with the TAT carrier alone had no significant effect on onset time Vmax or buy Nepicastat total O2? produced (Fig. 1 and Table 1). No significant O2? was generated by neutrophils in the absence of stimuli or by the addition of the TAT carrier or the δ-PKC TAT peptide alone (data not shown). Thus although inhibition of δ-PKC significantly delayed the onset time of O2? production and the time required to achieve maximal O2? it did not alter the level of maximal O2? generation in response to TNF. Similar to FN-adherent neutrophils pretreatment of neutrophils adherent to tissue culture-treated polystyrene with Nepicastat the δ-PKC TAT inhibitory peptide also delayed the onset time of TNF-elicited O2? production (onset time=43±3 min for buffer vs. 63±6 min for δ-PKC TAT peptide-treated neutrophils; n=4 donors in triplicates; P<0.01). These results indicate that the role for δ-PKC in TNF-elicited O2? production is not limited to neutrophils adherent to FN and is part of a more general mechanism. Thus δ-PKC is a positive regulator of TNF-elicited assembly and activation of the NADPH Nepicastat oxidase for O2? generation in adherent neutrophils. Figure 1. TNF-elicited O2? generation in adherent neutrophils is δ-PKC-dependent. TNF-mediated O2? generation was determined in FN-adherent neutrophils pretreated with the specific δ-PKC TAT peptide inhibitor (1 μM) TAT ... TABLE 1. TNF-Elicited O2? Generation Requires δ-PKC fMLP-elicited O2? generation is independent of δ-PKC To ascertain whether the regulatory role of δ-PKC in O2? generation was adherence- or ligand-dependent we determined the role of δ-PKC in O2? generation triggered by the bacterial peptide fMLP in adherent and nonadherent neutrophils. As shown in Figure 2A and Table 2 in nonadherent neutrophils in the presence of cytochlasin B.