Bacterial phagocytosis activates extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades in human neutrophils.

The hypothesis that bacterial phagocytosis by human polymorphonuclear neutrophils (PMNs) stimulates MAPK cascades that regulate respiratory burst activation was tested. Extracellular response kinase (ERK) and p38 kinase, but not c-Jun NH2-terminal kinase, activities were increased within 5 min of phagocytosis of plasma-opsonized Staphylococcus aureus (S-SA), reached maximum at 20-30 min, and remained elevated through 60 min. The role of Fcy receptors was examined using gamma globulin-opsonized SA (IgG-SA), whereas CR3 receptors were activated by particulate beta-glucan. IgG-SA stimulated a maximal ERK activity at 30 min, whereas p38 activity was maximal at 5 min. Beta-glucan stimulated maximal ERK activity at 5 min and maximal p38 activity at 2 min. Non-opsonized bacteria were ingested at 10% of the level of S-SA and stimulated a minimal increase in ERK and p38 activity at 60 min. S-SA stimulation of ERK was inhibited by wortmannin, LY294002, and genistein, but not calphostin C; whereas p38 stimulation was inhibited by calphostin C and genistein, but not wortmannin and LY294002. Simultaneous measurement of phagocytosis and H2O2 production by flow cytometry was used to assess the role of ERKs and p38 kinase in phagocytosis. The MEK inhibitor PD098059 had no significant effect on phagocytosis or H2O2 production. The p38 kinase inhibitor SB203580 significantly attenuated H2O2 production, whereas phagocytosis was unaffected. In conclusion, bacterial phagocytosis stimulates ERK and p38 activation by distinct signal transduction pathways. Phagocytosis-stimulated p38 kinase activity is necessary for optimal H2O2 production.

Azotemia, TNF alpha, and LPS prime the human neutrophil oxidative burst by distinct mechanisms.

The oxidative burst of neutrophils from azotemic patients (AzoPMNs) is primed for an enhanced response compared to neutrophils from normal subjects (NorPMNs). The mechanism for this priming is unknown, although TNF alpha does not further prime AzoPMNs. The present study examines the hypothesis that azotemia and TNF alpha prime neutrophils by the same mechanism. Formyl peptide receptor expression and degranulation were not primed in AzoPMNs, but were primed by both LPS and TNF alpha. LPS was also able to prime the AzoPMN oxidative burst. Guanine nucleotide exchange by multiple guanine nucleotide binding proteins, including heterotrimeric G-proteins and low molecular weight GTP-binding proteins (LMWGs), was increased in AzoPMNs, as demonstrated by GTP gamma S binding and azidoanilide GTP photoaffinity labeling. The plasma membrane density of G-protein alpha i2, alpha i3, and alpha s subunits and the density in the cytosol of the LMWG, Rap1A, was present in significantly greater amounts on plasma membranes from AzoPMNs. FMet-Leu-Phe-stimulated phospholipase D activity, but not basal activity, was significantly greater in AzoPMNs. Finally, incubation of NorPMNs in plasma from azotemic patients resulted in a significant increase in basal GTP gamma S binding. These results demonstrate that priming of AzoPMNs is restricted to oxidative burst activity and that it occurs by a mechanism distinct from that utilized by TNF alpha and LPS. While the exact mechanism remains unknown, it appears to involve a plasma factor and changes in LMWG expression or activity.