Priming of the Neutrophil Respiratory Burst Involves p38 Mitogen-activated Protein Kinase-dependent Exocytosis of Flavocytochrome b 558-containing Granules

Abstract

The respiratory burst of human neutrophils is primed by a number of pro-inflammatory stimuli, including tumor necrosis factor-␣ (TNF␣) and lipopolysaccharide (LPS); however, the mechanism of priming remains unknown. LPS has been shown previously to increase membrane expression of flavocytochrome b 558 , a component of the NADPH oxidase. This study shows that TNF␣ also increases membrane expression of flavocytochrome b 558 . Mitogen-activated protein kinase (MAPK) modules have been implicated in the action of priming agents. Pharmacologic inhibitors of MAPKs, SB203580 and PD098059, revealed that priming of the respiratory burst and up-regulation of flavocytochrome b 558 are dependent on p38 MAPK but not on extracellular-signal regulated kinase (ERK). TNF␣ and LPS primed respiratory burst activity and increased membrane expression of CD35 and CD66b, specific markers of secretory vesicles and specific granules that contain flavocytochrome b 558 , with similar time courses and concentration dependences. These processes also required p38 MAPK but were independent of ERK. TNF␣ failed to prime respiratory burst activity or to increase membrane CD35 expression in enucleated neutrophil cytoplasts. These data suggest that one mechanism by which TNF␣ and LPS prime neutrophil respiratory burst activity is by increasing membrane expression of flavocytochrome b 558 through exocytosis of intracellular granules in a process regulated by p38 MAPK.

NADPH Oxidase Is Internalized by Clathrin-coated Pits and Localizes to a Rab27A/B GTPase-regulated Secretory Compartment in Activated Macrophages

“…Endosomal localization of NADPH oxidase may be highly relevant to redox signaling (9, 13, 48) and cytokine expression. Higher concentrations of ATP (1 mM) caused a more sustained up-regulation of cyt b 558 on the cell surface, and also pro-inflammatory mediators TNF-␣ and CD40L induced cyt b 558 expression on the surface in accord with their documented role in neutrophils (38). As a consequence, the fraction of total superoxide production released to the external environment after NADPH oxidase activation was expectedly increased, which in vivo should be relevant for the magnitude of oxidative tissue damage incurred during chronic inflammatory reactions.…”

A Rab8-specific GDP/GTP Exchange Factor Is Involved in Actin Remodeling and Polarized Membrane Transport

“…Bar, 5 m. ation, necrosis, respiratory burst, and adhesion (Beyaert and Fiers, 1994). TNF-alpha-and phorbol ester-induced respiratory burst is known to increase exocytosis of granules containing NADPH oxidase and adhesion molecules (Ward et al, 2000). The fact that phorbol induced actin reorganization is associated with translocation of both Rabin8 and Rab8-specific vesicles to the periphery of lamellipodia argues for a possible role of Rab8 in regulating respiratoryassociated exocytosis.…”

Myeloid-Related Protein-14 Is a p38 MAPK Substrate in Human Neutrophils

“…The p38 MAPK pathway participates in a number of neutrophil functions critical to generation and regulation of the inflammatory response, including chemotaxis, adherence, respiratory burst activity, degranulation, and cytoskeletal reorganization (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Understanding the molecular mechanisms by which p38 MAPK participates in these responses is hindered by the limited number of targets of p38 MAPK identified to date in neutrophils.…”

“…A number of neutrophil responses, including chemotaxis, granule exocytosis, respiratory burst activity, and production of chemokines, are dependent on the activation of the p38 MAPK pathway (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). MAPK pathways consist of three-kinase modules formed by the terminal MAPKs, which in turn are activated by dual-specificity serine-threonine/tyrosine kinases termed MAP2Ks, which in turn are activated by serine-threonine kinases termed MAP3Ks (13)(14)(15).…”