LSP1 is an endothelial gatekeeper of leukocyte transendothelial migration

Abstract

Leukocyte-specific protein 1 (LSP1), an F-actin binding protein and a major downstream substrate of p38 mitogen-activated protein kinase as well as protein kinase C, has been reported to be important in leukocyte chemotaxis. Although its distribution has been thought to be restricted to leukocytes, herein we report that LSP1 is expressed in endothelium and is essential to permit neutrophil emigration. Using intravital microscopy to directly visualize leukocyte rolling, adhesion, and emigration in postcapillary venules in LSP1-deficient (Lsp1 −/−) mice, we found that LSP1 deficiency inhibits neutrophil extravasation in response to various cytokines (tumor necrosis factor-α and interleukin-1β) and to neutrophil chemokine keratinocyte-derived chemokine in vivo. LSP1 deficiency did not affect leukocyte rolling or adhesion. Generation of Lsp1 −/− chimeric mice using bone marrow transplantation revealed that in mice with Lsp1 −/− endothelial cells and wild-type leukocytes, neutrophil transendothelial migration out of postcapillary venules is markedly restricted. In contrast, Lsp1 −/− neutrophils in wild-type mice were able to extravasate normally. Consistent with altered endothelial function was a reduction in vascular permeability to histamine in Lsp1 −/− animals. Western blot analysis and immunofluorescence microscopy examination confirmed the presence of LSP1 in wild-type but not in Lsp1 −/− mouse microvascular endothelial cells. Cultured human endothelial cells also stained positive for LSP1. Our results suggest that LSP1 expressed in endothelium regulates neutrophil transendothelial migration.

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

“…With specific transgenic mice, inhibitors and selective chemoattractants, the assay system helps us to reveal the functions of specific proteins in leukocyte recruitment. For example, this technique assisted us to identify the role for LSP1 in endothelial cells as the gatekeeper in the regulation of neutrophil transendothelial migration 10 , the role for Mac-1 (αMβ2 integrin) in neutrophil intraluminal crawling, an essential step for optimal transendothelial migration 3 , and the role for PI3Kγ in neutrophil chemotaxis in tissue 11 .…”

Downregulation of HOPX Controls Metastatic Behavior in Sarcoma Cells and Identifies Genes Associated with Metastasis

“…We subsequently validated the microarray results by qRT-PCR of several genes, ADCY7, ARGHDIB, BAMBI, FOXG1, FSCN1, FUCA1, GFPT2, ITPR3, ITGA4, LSP1, NCAM1, NUDT14, RGS20, RHOF, and RIAM, that have already been implicated in cell migration, adhesion, or angiogenesis (28,(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46). The results from qRT-PCR analysis (Fig.…”

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

“…On the contrary, however, nonhematopoietic endothelial-expressed LSP1 is localized primarily in the nucleus (6). In neutrophils, LSP1 is phosphorylated by the activation of p38 MAPK downstream of soluble chemoattractant receptor signaling and then colocalizes with F-actin, thus contributing to the stability of its polarization during chemotaxis (16,17).…”

“…Discordantly, however, LSP1-deficient neutrophils exhibited impaired chemotaxis speed and directionality, but enhanced superoxide production, in response to keratinocyte-derived chemokine (KC/CXCL1) gradient in vitro (23). By intravital microscopy of mouse cremasteric microvasculature in vivo, decreased transendothelial migration of neutrophils was demonstrated in LSP1-deficient mice in response to IL-1b or TNF-a, effects attributed to the supportive role of endothelialexpressed LSP1 in neutrophil extravasation (6). More recently, endothelial LSP1 was shown to regulate microvascular permeability by participating in endothelial dome formation during neutrophil transmigration (8).…”

“…Neutrophil chemotaxis is modulated by F-actin-binding proteins that dictate actin polymerization and cytoskeletal rearrangement (5). Previous studies have shown that the intracellular Ca 2+ -and F-actin-binding leukocyte-specific protein 1 (LSP1), expressed in both neutrophils and endothelial cells, is critical in regulating transendothelial migration of neutrophils and microvascular permeability (6)(7)(8). 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).…”