The role of LSP1 in chemokine-induced neutrophil recruitment

Abstract

Leukocyte-specific protein 1 (LSP1) is an intracellular, F-actin- and Ca2+-binding, phosphoprotein expressed in leukocytes and endothelial cells (ECs). It is important in leukocyte motility and recruitment during inflammation. The role of LSP1 and its signaling mechanisms in chemokine-induced neutrophil transendothelial recruitment are not well understood. In this study, the role of neutrophil LSP1 (N-LSP1) and endothelial LSP1 (E-LSP1) in neutrophil-endothelial cell interactions in the post-capillary venules was investigated by using intravital microscopy and time-lapse video photography. This was determined in response to two very similar CXC chemokines keratinocyte-derived chemokine (KC, CXCL1) and macrophage inflammatory protein-2 (MIP-2, CXCL2). To induce neutrophil recruitment, one mm3 piece of agarose gel containing MIP-2 or KC was carefully placed on 350 μm from the observed post-capillary venule of the mouse cremaster muscle. Neutrophil intraluminal crawling, transmigration, and chemotaxis in the muscle were analyzed by the use of LSP1-deficient (Lsp1−/−) mice, wild-type (WT) 129/SvJ mice and their four types of chimeric mice (Lsp1−/−→Lsp1−/−, WT→Lsp1−/−, Lsp1−/−→WT and WT→WT)*. We observed that Lsp1−/− mice exhibited similar responses to MIP-2 or KC-induced neutrophil recruitment compared to the WT mice. Emigration in neutrophil recruitment was significantly inhibited in Lsp1−/− mice. Neutrophils in Lsp1−/− mice, compared to those in the WT mice, displayed longer crawling time, slower crawling velocities and had lower chemotaxis index during their intraluminal crawling in venules in response to MIP-2. The transmigration time of Lsp1−/− neutrophils was longer than that of WT neutrophils, although LSP1 did not appear to play a role during neutrophil detachment from endothelium in response to MIP-2. Substantial defects were observed in Lsp1−/− neutrophils during their chemotaxis in muscle tissue in response to MIP-2. For dissecting the role of LSP1 in neutrophils and ECs, we repeated these experiments using chimeric mice. We found that neutrophil emigration was significantly inhibited in WT→Lsp1−/− mice as well as in Lsp1−/−→Lsp1−/− mice but not in Lsp1−/−→WT mice or WT→WT mice. N-LSP1 promoted intraluminal crawling and transmigration while E-LSP1 played more important roles in MIP-2-induced neutrophil transmigration and chemotaxis in tissue. In addition, p38 mitogen-activated protein kinase (MAPK) inhibitor SKF86002 abolished neutrophil recruitment and chemotaxis in tissue of WT mice. These inhibitory effects were partially achieved through LSP1 in MIP-2-induced neutrophil recruitment. Taken together, these results suggest that both N-LSP1 and E-LSP1 are important in MIP-2-induced neutrophil intraluminal crawling and neutrophil transendothelial migration. These results also suggest that E-LSP1 may have a more important role in neutrophil transendothelial migration and in neutrophil chemotaxis both in the lumen and in extravascular tissues as well. Additionally, they demonstrate that MIP-2-induced neutrophil recruitment and chemotaxis in tissue is dependent on p38 MAPK activation. *, Four types of LSP1 chimeric mice were generated; Lsp1−/− mice reconstituted with Lsp1−/− neutrophils and WT neutrophils were indicated as Lsp1−/−→Lsp1−/− and WT→Lsp1−/−, respectively, and WT mice reconstituted with Lsp1−/− and WT neutrophils were indicated as Lsp1−/−→WT and WT→WT, respectively.