Pharmacological Inhibition of p38 Mitogen-Activated Protein Kinases Affects KC/CXCL1-Induced Intraluminal Crawling, Transendothelial Migration, and Chemotaxis of NeutrophilsIn Vivo

Abstract

p38 mitogen-activated protein kinase (MAPK) signalling is critical in the pathophysiology of a variety of inflammatory processes. Leukocyte recruitment to the site of inflammation is a multistep process governed by specific signalling cascades. After adhesion in the lumen, many leukocytes crawl to optimal sites at endothelial junctions and transmigrate to extravascular tissue in a Mac-1-dependent manner. The signalling mechanisms that regulate postadhesion steps of intraluminal crawling, transmigration, and chemotaxis in tissue remain incompletely understood. The present study explored the effect of p38 MAPK inhibitor SB203580 on various parameters of neutrophil recruitment triggered by chemokine KC (CXCL1) gradient. Neutrophil-endothelial interactions in microvasculature of murine cremaster muscle were determined using intravital microscopy and time-lapsed video analysis. SB203580 (100 nM) did not change leukocyte rolling but significantly attenuated neutrophil adhesion, emigration, and transmigration and impaired the initiation of neutrophil crawling and transmigration. In response to KC chemotactic gradient, SB203580 significantly reduced the velocity of migration and chemotaxis index of neutrophils in tissue. The upregulation of Mac-1 expression in neutrophils stimulated by KC was significantly blunted by SB203580 in vitro. Collectively, our findings demonstrate that pharmacological suppression of p38 MAPK significantly impairs multiple steps of neutrophil recruitment in vivo.

The role of p38 MAPK in neutrophil functions: single cell chemotaxis and surface marker expression

“…This might be due to 1) CD11b and CD66b being stored in different granules with different threshold for degranulation, 17, 36, 38, 52–55 2) our assessments were on neutrophils (both untreated and p38 MAPK-blocked), already adhered to a fibronectin-coated surface, 56–58 or 3) our separation of neutrophils from other blood components. In previous studies that have shown up-regulation of these adhesion molecules, stimulation was frequently applied in the presence of other cell type.…”

Endothelial LSP1 Modulates Extravascular Neutrophil Chemotaxis by Regulating Nonhematopoietic Vascular PECAM-1 Expression

“…The mouse cremaster muscle preparation was used to study dynamic leukocyte-endothelial interactions in microvasculature as described previously (6,7,11,33). For induction of neutrophil recruitment, a piece of agarose gel (∼1 mm 3 ) containing the optimal concentration of murine chemokine CXCL2 (0.5 mmol/l; R&D Systems, Minneapolis, MN) was placed and held on the surface of the cremaster muscle in a preselected area 350 mm from and parallel to the observed postcapillary venule.…”

“…For induction of neutrophil recruitment, a piece of agarose gel (∼1 mm 3 ) containing the optimal concentration of murine chemokine CXCL2 (0.5 mmol/l; R&D Systems, Minneapolis, MN) was placed and held on the surface of the cremaster muscle in a preselected area 350 mm from and parallel to the observed postcapillary venule. Leukocyte rolling velocity (mm/s), leukocyte rolling flux (cells/min), and the number of adherent (cells/100-mm venule) and emigrated neutrophils (cells/235 3 208 mm 2 field) were determined in the cremasteric postcapillary venule (25-40 mm diameter) using video-playback analysis (6,7,11,33). By using ImageJ software (National Institutes of Health) and timelapsed video analysis, neutrophil intraluminal crawling, transmigration time, and extravascular chemotaxis were quantified as described previously (11,33,34).…”

“…Leukocyte rolling velocity (mm/s), leukocyte rolling flux (cells/min), and the number of adherent (cells/100-mm venule) and emigrated neutrophils (cells/235 3 208 mm 2 field) were determined in the cremasteric postcapillary venule (25-40 mm diameter) using video-playback analysis (6,7,11,33). By using ImageJ software (National Institutes of Health) and timelapsed video analysis, neutrophil intraluminal crawling, transmigration time, and extravascular chemotaxis were quantified as described previously (11,33,34). Chemotaxis index was calculated by dividing the chemotaxis distance (distance in the direction of chemotactic gradient) by the total migration distance in tissue.…”

“…LSP1 serves as a major substrate for both protein kinase C (PKC) as well as MAPK-activated protein kinase 2, downstream of p38 MAPK signaling (9,10). These kinases are known to be involved in neutrophil migration and chemotaxis (11)(12)(13)(14).…”