Mitogen-activated Protein Kinase in Neutrophils and Enucleate Neutrophil Cytoplasts

Abstract

We employed neutrophils and enucleate neutrophil cytoplasts to study the activation of the mitogen-activated protein kinases (MAPKs) p44 erk1 and p42 erk2 in neutrophils by inflammatory agonists that engage G protein-linked receptors. Formyl-methionyl-leucylphenylalanine (FMLP) rapidly and transiently activated MAPK in neutrophils and cytoplasts, consistent with a role in signaling for neutrophil functions. FMLP stimulated p21 ras activation in neutrophils and Raf-1 translocation from cytosol to plasma membrane in cytoplasts, with kinetics consistent with events upstream of MAPK activation. Insulin, a protein tyrosine kinase receptor (PTKR) agonist, stimulated neutrophil MAPK activation, demonstrating an intact system of PTKR signaling in these post-mitotic cells. FMLP-and insulin-stimulated MAPK activation in cytoplasts were inhibited by Bt 2 cAMP, consistent with signaling through Raf-1 and suggesting a mechanism for cAMP inhibition of neutrophil function. However, Bt 2 cAMP had no effect on FMLP-stimulated MAPK activation in neutrophils. The extent of MAPK activation by various chemoattractants correlated with their capacity to stimulate neutrophil and cytoplast homotypic aggregation. Consistent with its effects on MAPK, Bt 2 cAMP inhibited FMLP-stimulated aggregation in cytoplasts but not neutrophils. Insulin had no independent effect but primed neutrophils for aggregation in response to FMLP. Our studies support a p21 ras -, Raf-1-dependent pathway for MAPK activation in neutrophils and suggest that neutrophil adhesion may be regulated, in part, by MAPK.

Neutrophils: Molecules, Functions and Pathophysiological Aspects

“…Tyrosine kinases (mainly Lyn of the Srk-family) (Berton, 1999a;Ptasznik et al, 1996;Welch and Maridonneau-Parini, 1997) and the GTP-binding protein Ras (Worthen et al, 1994) are also activated. Ras activation triggers the MAPK/ERK cascade, which appears to be involved in various chemoattractant-induced neutrophil functions (Krump et al, 1997;Nick et al, 1997;Pillinger et al, 1996). Activation of small GTP-binding proteins of the Ras, Rac, and Rho families regulate actin-dependent processes such as membrane ruffling, formation of filopodia and stress fibers, mediating cell adhesion and motility (Benard et al, 1999;Cox et al, 1997;Nobes and Hall, 1999).…”

Signaling Pathways Involved in IL-8-Dependent Activation of Adhesion Through Mac-1

“…There is also evidence that IL-8 stimulates phospholipase D activity (27), phosphatidylinositol-3 kinase (PI3K) activity (28), and GTP loading of RhoA (29) in human neutrophils. IL-8 has been shown to activate mitogen-activated protein kinase (MAPK) in human neutrophils as well as in enucleate neutrophil cytoplasts (28,30,31). In addition, IL-8 has been shown to stimulate GTP loading of Ras and Raf activation in human neutrophils with a time course that indicates that these events are upstream of MAPK activation.…”

Cyclooxygenase-2-Derived E Prostaglandins Down-Regulate Matrix Metalloproteinase-1 Expression in Fibroblast-Like Synoviocytes via Inhibition of Extracellular Signal-Regulated Kinase Activation

“…However, ERK may also be activated in response to proinflammatory stimuli (22,23). For example, Vilcek and colleagues (24 -26) have demonstrated that in FS-4 fibroblasts, ERK undergoes activation in response to the cytokines IL-1␤ and TNF-␣, both of which are critical to the pathogenesis of RA.…”