p38 Mitogen‐activated protein kinase and phosphatidylinositol 3‐kinase activities have opposite effects on human neutrophil apoptosis

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Abstract
Neutrophil apoptosis is essential for resolution of inflammatory reactions, but there is only limited information regarding the involvement of protein phosphorylation events in the regulation of human neutrophil apoptosis. Therefore, our specific objective was to investigate the role of two apoptosis/survival-associated protein kinases—p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI 3-K)—in the regulation of Fas-mediated neutrophil apoptosis. Western blot analysis with a specific antibody (Ab) recognizing the active form of p38 MAPK revealed constitutive activity of the enzyme in cells isolated by two different methods and immediately analyzed. Such an activity has previously been reported in human neutrophils and freshly isolated thymocytes. Here we made the novel finding that this activity was transiently lost but subsequently regained during spontaneous and Fas-induced apoptosis. This transient loss of p38 MAPK activity was independent of the absence or presence of fetal calf serum in the cell culture medium. To further confirm our observation of a transient inhibition of p38 MAPK activity during neutrophil apoptosis, we analyzed its activity by an in vitro kinase assay in which the p38 MAPK-induced phosphorylated form of activating transcription factor 2 (ATF2)19–96 is detected by Western blot. The results were confirmed by this approach, which again revealed a transient and early inhibition of p38 MAPK activity (Fig. 1 ⤻ , representative blot and filled circles in the diagram). Even more interesting was the observation that during the transient inhibition of p38 MAPK activity (at 60 min) there is a statistically significant (PA⤻ ). The activation of PI 3-K was accompanied by a subsequent translocation of p85 PI 3-K to a membrane fraction where the enzyme remained even after its activity had declined (Fig. 2B⤻ ). Note that the peak in PI 3-K activity occurred at the same time the activity of p38 MAPK was transiently impaired (Fig. 1⤻ ; for comparison, indicated as a dashed line in Fig. 2A⤻ ). The opposite behavior of PI 3-K and p38 MAPK activities was matched by their effects on neutrophil apoptosis (Fig. 2C, D⤻ ). We found that two different inhibitors of PI 3-K (wortmannin and LY294002) significantly inhibited Fas-mediated PS exposure and chromatin condensation. We investigated a possible interdependence between PI 3-K and p38 MAPK signaling. Neither wortmannin nor LY294002 had any effect on transient inhibition of p38 MAPK activity during Fas-induced apoptosis (Fig. 2E⤻ ). In reversed experiments, we found no statistically significant effects of SB203580 on Fas-induced PI 3-K...
Funding Information
  • Medicinska Forskningsrådet (# 12871)