MLK3 regulates fMLP-stimulated neutrophil motility

Abstract

Introduction: Mixed lineage kinase 3 (MLK3) is part of the intracellular regulatory system that connects extracellular cytokine or mitogen signals received through G-protein coupled receptors to changes in gene expression. MLK3 activation stimulates motility of epithelial cells and epithelial-derived tumor cells, but its role in mediating the migration of other cell types remains unknown. Since neutrophils play a crucial role in innate immunity and contribute to the pathogenesis of several diseases, we therefore examined whether MLK3 might regulate the motility of mouse neutrophils responding to a chemotactic stimulus, the model bacterial chemoattractant fMLP.

Methods: The expression of Mlk3 in mouse neutrophils was determined by immunocytochemistry and by RT-PCR. In vitro chemotaxis in a gradient of fMLP, fMLP-stimulated random motility, fMLP-stimulated F-actin formation were measured by direct microscopic observation using neutrophils pre-treated with a novel small molecule inhibitor of MLK3 (URMC099) or neutrophils obtained from Mlk3-/- mice. In vivo effects of MLK3 inhibition were measured by counting the fMLP-induced accumulation of neutrophils in the peritoneum following pre-treatment with URMC099 in wild-type C57Bl/6 or mutant Mlk3-/- mice.

Results: The expression of Mlk3 mRNA and protein was observed in neutrophils purified from wild-type C57Bl/6 mice but not in neutrophils from mutant Mlk3-/- mice. Chemotaxis by wild-type neutrophils induced by a gradient of fMLP was reduced by pre-treatment with URMC099. Neutrophils from C57Bl/6 mice pretreated with URMC099 and neutrophils from Mlk3-/- mice moved far less upon fMLP-stimulation and did not form F-actin as readily as untreated neutrophils from C57Bl/6 controls. In vivo recruitment of neutrophils into the peritoneum by fMLP was significantly reduced in wild-type mice treated with URMC099, as well as in untreated Mlk3-/- mice-thereby confirming the role of MLK3 in neutrophil migration.

Conclusions: Mlk3 mRNA is expressed in murine neutrophils. Genetic or pharmacologic inhibition of MLK3 blocks fMLP-mediated motility of neutrophils both in vitro and in vivo, suggesting that MLK3 may be a therapeutic target in human diseases characterized by exuberant neutrophil migration.

Keywords: AD; AKT; APC; CRIB; Chemotaxis; DMSO; EDTA; ERK; EtBr; FITC; G-protein coupled receptor; GAPDH; GPCR; GTP; HBSS; Hank's buffered salt solution; ICAM; JIP; JNK; JNK interacting protein; MKK; MLK3; Mixed lineage kinase 3; Mouse; N-formyl-l-methionyl-l-leucyl-l-phenylalanine; Neutrophil; PBS; PCR; PE; PEG; PI3K; RT-PCR; SH3; Src homology domain 3; TBS; TNF; accumulated distance; allophycocyanin; c-jun N-terminal protein kinase; cdc42/Rac-interactive binding; dimethyl sulfoxide; ethidium bromide; ethylenediaminotetraacetic acid; extracellular signal-related kinase; fMLP; fluorescein isothiocyanate; glyceraldehyde-3-phosphate dehydrogenase; guanosine triphosphate; intercellular adhesion molecule; mitogen-activated protein kinase kinase; mixed lineage kinase 3; phosphate-buffered saline; phosphoinositide-3-kinase; phycoerythrin; polyethyleneglycol; polymerase chain reaction; protein kinase B; reverse transcription-polymerase chain reaction; tris-buffered saline; tumor necrosis factor.