Abstract
Integrin activation is crucial for the regulation of leukocyte rolling, adhesion and trans-vessel migration during inflammation and occurs by engagement of myeloid cells through factors presented by inflamed vessels. However, endothelial-dependent mechanisms of myeloid cell recruitment are not fully understood. Here we show using an autoperfused flow chamber assay of whole blood neutrophils and intravital microscopy of the inflamed cremaster muscle that CD95 mediates leukocyte slow rolling, adhesion and transmigration upon binding of CD95-ligand (CD95L) that is presented by endothelial cells. In myeloid cells, CD95 triggers activation of Syk-Btk/PLCγ2/Rap1 signaling that ultimately leads to integrin activation. Excitingly, CD95-deficient myeloid cells exhibit impaired bacterial clearance in an animal model of sepsis induced by cecal ligation and puncture (CLP). Our data identify the cellular and molecular mechanisms underlying the chemoattractant effect of endothelial cell-derived CD95L in induction of neutrophil recruitment and support the use of therapeutic inhibition of CD95's activity in inflammatory diseases.
Highlights
Leukocyte recruitment comprises of a cascade with four major steps: slow rolling, firm adhesion, intraluminal crawling and trans-vessel migration (Ley et al, 2007)
In order to exclude the possibility that CD95 mediates neutrophil slow rolling via the CD95-induced chemokine production, which has been reported in various cell types (Park et al, 2003; Guo et al, 2005; Altemeier et al, 2007; Miwa et al, 1998), mouse blood was perfused through the flow chambers coated with E-selectin, ICAM1 and CD95L
Flow chamber coated with 50 mg/ml CD95L showed the strongest effect on slow rolling as compared to other coating concentrations (Figure 1—figure supplement 1B)
Summary
Leukocyte recruitment comprises of a cascade with four major steps: slow rolling, firm adhesion, intraluminal crawling and trans-vessel migration (Ley et al, 2007). E-selectin engagement with PSGL-1 and CD44 ligands induces activation of the Src family kinases (SFKs) Hck, Fgr and Lyn (Yago et al, 2010) which phosphorylate and activate immunoreceptor tyrosine-based activation motif (ITAM)-bearing adaptor protein Fc receptor common g signaling chain (FcRg) and DNAX activation protein of 12 kDa (DAP12) (Zarbock et al, 2008). These activated adaptor proteins recruit and phosphorylate spleen tyrosine kinase (Syk), which in turn activates Bruton’s tyrosine kinase (Btk) (Mueller et al, 2010; Yago et al, 2010). Btk further activates the phosphoinositide 3kinase (PI3K), phospholipase C g2 (PLCg2) and p38 mitogen-activated protein kinase (p38 MAPK) pathways that mediate the integrin signaling to induce slow rolling
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