Cytokine-Specific Activation of Distinct Mitogen-Activated Protein Kinase Subtype Cascades in Human Neutrophils Stimulated by Granulocyte Colony-Stimulating Factor, Granulocyte-Macrophage Colony-Stimulating Factor, and Tumor Necrosis Factor-

Abstract

To clarify the differences of the signaling pathways used by granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor- (TNF), we investigated activation of mitogen-activated protein kinase (MAPK) subtype cascades in human neutrophils stimulated by these cytokines. G-CSF exclusively tyrosine-phosphorylated extracellular signal-regulated kinase (ERK). GM-CSF tyrosine-phosphorylated ERK strongly and p38 MAPK weakly, whereas TNF tyrosine-phosphorylated p38 MAPK strongly and ERK weakly. Consistent with these findings, MEK, an upstream kinase of ERK, was phosphorylated by G-CSF, GM-CSF, and TNF, whereas MKK3/MKK6, an upstream kinase of p38 MAPK, was phosphorylated by GM-CSF and TNF, but not by G-CSF. The potency of these cytokines to phosphorylate ERK and MEK was GM-CSF > G-CSF > TNF, whereas that to phosphorylate p38 MAPK and MKK3/MKK6 was TNF > GM-CSF. C-Jun amino-terminal kinase (JNK) was not tyrosine-phosphorylated by any cytokine despite the existence of JNK proteins in human neutrophils, whereas it was tyrosine-phosphorylated by TNF in undifferentiated and all-trans retinoic acid-differentiated HL-60 cells. Increased phosphorylation of ERK or p38 MAPK was detected within 1 to 5 minutes after stimulation with each cytokine and was dependent on the concentrations of cytokines used. MEK inhibitor (PD98059) reduced tyrosine phosphorylation of ERK, but not p38 MAPK, induced by G-CSF, GM-CSF, or TNF. GM-CSF– or TNF-induced superoxide (O2−) release was inhibited by p38 MAPK inhibitor (SB203580) in a dose-dependent manner, suggesting the possible involvement of p38 MAPK in GM-CSF– or TNF-induced O2− release. The results indicate that G-CSF, GM-CSF, and TNF activate the overlapping but distinct MAPK subtype cascades in human neutrophils and suggest that the differential activation of ERK and p38 MAPK cascades may explain the differences of the effects of these cytokines on human neutrophil functions.

Regulation of matrix metalloproteinase-9 (MMP-9) in TNF-stimulated neutrophils: novel pathways for tertiary granule release

“…The MAPK mediate diverse functions such as cell division and degranulation in various cell types. TNF stimulation of neutrophils activates all three MAPK: ERK1/2, Jun N-terminal kinase, and p38 MAPK [24]. p38 MAPK and ERK1/2 have been shown to be involved in the degranulation of granulocytes [25,26]; however, we have previously demonstrated that inhibiting p38 with SB203580 has no effect on TNF-induced MMP-9 release [20].…”

Toll‐like receptor agonists stimulate human neutrophil migration via activation of mitogen‐activated protein kinases

“…These findings are consistent with our recent studies showing that ERK is selectively activated in human neutrophils stimulated by G-CSF, and ERK is involved in G-CSF-induced neutrophil migration. 3,14 The role of ERK in cell migration has also been demonstrated in various cell lines, and ERK-mediated activation of myosin light chain kinase and calpain 2 (m-calpain) is proposed to be involved in the stimulation of cell migration. 17,18 In human neutrophils, it has been recently reported that calpain 1 (l-calpain) functions as a negative regulator in cell migration by inhibiting the activation of Cdc42 and Rac, 19 and calpain 2 is a component of the frontness signal that promotes polarization during chemotaxis.…”

“…Human neutrophils were prepared from healthy adult donors, as described previously, 3 using dextran sedimentation, centrifugation with Conray-Ficoll and hypotonic lysis of contaminating erythrocytes. Neutrophil fractions contained more than 98% neutrophils.…”

Calpain-mediated regulation of the distinct signaling pathways and cell migration in human neutrophils

“…Human peripheral blood neutrophils were prepared from healthy adult donors as described previously [13], using dextran sedimentation, centrifugation with Conray-Ficoll, and hypotonic lysis of contaminating erythrocytes. Neutrophil fractions were suspended in HBSS containing 10 mM HEPES (pH 7.4) and contained more than 95% neutrophils, and contaminating cells were almost exclusively eosinophils.…”