Mac-1 dependent crawling is a new step in the leukocyte recruitment cascade which follows LFA-1 dependent adhesion and precedes emigration. Neutrophil adhesion via LFA-1 has been shown to induce cytoskeletal reorganization through Vav1-dependent signaling, and the current study investigates the role of Vav1 in the leukocyte recruitment process in vivo with particular attention to the events immediately downstream of LFA-1 dependent adhesion. Intravital and spinning-disk-confocal microscopy was used to investigate intravascular crawling in relation to endothelial junctions in vivo in wild-type (WT) and Vav1−/− mice. Adherent WT neutrophils almost immediately began crawling perpendicular to or against blood flow via Mac-1 until they reached an endothelial junction where they often changed direction. This pattern of perpendicular, mechanotactic crawling was recapitulated in vitro when shear was applied. In sharp contrast, the movement of Vav1−/− neutrophils was always in the direction of flow, and appeared more passive as if the cells were dragged in the direction of flow in vivo and in vitro. More than 80% of Vav1−/− neutrophils moved independent of Mac-1 and could be detached with LFA-1 antibodies. An inability to release the uropod was frequently noted for Vav1−/− neutrophils, leading to greatly elongated tails. The Vav1−/− neutrophils failed to stop or follow junctions, and ultimately detached leading to fewer emigrated neutrophils. The Vav1−/− phenotype resulted in fewer neutrophils recruited in a relevant model of infectious peritonitis. Clearly, Vav1 is critical for the complex interplay between LFA-1 and Mac-1 that underlies the programmed intravascular crawling of neutrophils.

The endothelium actively participates in neutrophil migration out of the vasculature via dynamic, cytoskeleton-dependent rearrangements leading to the formation of transmigratory cups in vitro, and to domes that completely surround the leukocyte in vivo. Leukocyte-specific protein 1 (LSP1), an F-actin-binding protein recently shown to be in the endothelium, is critical for effective transmigration, although the mechanism has remained elusive. Herein we show that endothelial LSP1 is expressed in the nucleus and cytosol of resting endothelial cells and associates with the cytoskeleton upon endothelial activation. Two-photon microscopy revealed that endothelial LSP1 was crucial for the formation of endothelial domes in vivo in response to neutrophil chemokine keratinocyte-derived chemokine (KC) as well as in response to endogenously produced chemokines stimulated by cytokines (tumor necrosis factor ␣ [TNF␣] or interleukin-1␤ [IL-1␤]). Endothelial domes were significantly reduced in Lsp1 ؊/؊ compared with wildtype (WT) mice. Lsp1 ؊/؊ animals not only showed impaired neutrophil emigration after KC and TNF␣ stimulation, but also had disproportionate increases in vascular permeability. We demonstrate that endothelial LSP1 is recruited to the cytoskeleton in inflammation and plays an important role in forming endothelial domes thereby regulating neutrophil transendothelial migration. The permeability data may underscore the physiologic relevance of domes and the role for LSP1 in endothelial barrier integrity. IntroductionRecruitment of circulating neutrophils from the bloodstream to sites of tissue injury and infection is the hallmark feature of the inflammatory response. This process involves multiple, interdependent, regulated molecular interactions between the neutrophils and the vascular endothelium. 1,2 Initial tethering and rolling of leukocytes along the vessel wall is followed by firm adhesion to the vascular endothelium caused by chemokine activation of leukocyte integrins. 3,4 The neutrophils then crawl to sites where they migrate through the vascular endothelium, 5 a process known as transendothelial migration or diapedesis. A growing body of literature suggests that transendothelial migration is an interactive process between leukocytes and endothelial cells, in which endothelial cells are not passive bystanders but rather active participants that regulate this process. [6][7][8][9] There is also evidence that leukocyte adhesion molecules binding to endothelial adhesion molecules 2,10 induce intracellular signaling, stimulating the endothelial cytoskeleton and regulatory proteins to initiate transendothelial migration. 1,11,12 One such regulatory protein in endothelial cells is leukocytespecific protein 1 (LSP1), which was initially characterized in lymphocytes and thymocytes. 13,14 LSP1 has now been identified in monocytes, macrophages, dendritic cells, Langerhans cells, and neutrophils [15][16][17] and in both murine and human endothelium. 18 LSP1 is a cytoplasmic intracellular Ca 2ϩ -and F-actin-binding ...

Fps/Fes and Fer are members of a distinct subfamily of cytoplasmic protein tyrosine kinases that have recently been implicated in the regulation of innate immunity. Previous studies showed that mice lacking Fps/Fes are hypersensitive to systemic LPS challenge, and Fer-deficient mice displayed enhanced recruitment of leukocytes in response to local LPS challenge. This study identifies physiological, cellular, and molecular defects that contribute to the hyperinflammatory phenotype in Fps/Fes null mice. Plasma TNF-alpha levels were elevated in LPS challenged Fps/Fes null mice as compared with wild-type mice and cultured Fps/Fes null peritoneal macrophages treated with LPS showed increased TNF-alpha production. Cultured Fps/Fes null macrophages also displayed prolonged LPS-induced degradation of IkappaB-alpha, increased phosphorylation of the p65 subunit of NF-kappaB, and defective TLR4 internalization, compared with wild-type macrophages. Together, these observations provide a likely mechanistic basis for elevated proinflammatory cytokine secretion by Fps/Fes null macrophages and the increased sensitivity of Fps/Fes null mice to endotoxin. We posit that Fps/Fes modulates the innate immune response of macrophages to LPS, in part, by regulating internalization and down-regulation of the TLR4 receptor complex.

CanadaDuring an inflammatory response, endothelial cells undergo morphological changes to allow for the passage of neutrophils from the blood vessel to the site of injury or infection. Although endothelial cell junctions and the cytoskeleton undergo reorganization during inflammation, little is known about another class of cellular structures, the focal adhesions. In this study, we examined several focal adhesion proteins during an inflammatory response. We found that there was selective loss of paxillin and focal adhesion kinase (FAK) from focal adhesions in proximity to transmigrating neutrophils; in contrast the levels of the focal adhesion proteins b1-integrin and vinculin were unaffected. Paxillin was lost from focal adhesions during neutrophil transmigration both under static and flow conditions. Down-regulating endothelial paxillin with siRNA blocked neutrophil transmigration while having no effect on rolling or adhesion. As paxillin dynamics are regulated partly by FAK, the role of FAK in neutrophil transmigration was examined using two complementary methods. siRNA was used to down-regulate total FAK protein while dominant-negative, kinase-deficient FAK was expressed to block FAK signaling. Disruption of the FAK protein or FAK signaling decreased neutrophil transmigration. Collectively, these findings reveal a novel role for endothelial focal adhesion proteins paxillin and FAK in regulating neutrophil transmigration. IntroductionNeutrophils are essential mediators of host defense. During inflammation, neutrophils leave the bloodstream and traffic into the tissue in a well-described series of steps [1,2]. The molecular interactions governing the initial stages of tethering, rolling and firm adhesion have been well studied [1,2], whereas a clear picture of the latter steps underlying the passage of the leukocytes through the blood vessel has been slower to emerge [3][4][5].During transmigration endothelial cells undergo rapid changes in their structural organization [5,6]. For example, the junctional protein vascular endothelial cadherin (VE-cadherin) is phosphorylated and redistributes during neutrophil transmigration [7][8][9]. This results in the formation of transient gaps that 436allow for the passage of neutrophils [9]. Platelet endothelial cell adhesion molecule-1 (PECAM-1), another junctional protein, is regulated by the action of the kinesins and requires an intact microtubule cytoskeleton to enable PECAM-1 recycling from membrane compartments to the lateral junctions [10]. In the absence of PECAM-1 recycling, leukocyte transmigration is arrested [10]. Cortactin-mediated rearrangement of the actin cytoskeleton [7] and intermediate filaments [11] is also critical for successful transmigration. Overall, these results reveal that endothelial cells are active participants in transmigration.The importance of cytoskeletal and junctional proteins during leukocyte transmigration led us to ask whether another class of cellular structures, the focal adhesions, also play a role during this process. Focal a...