Coordinated navigation within tissues is essential for cells of the innate immune system to reach the sites of inflammatory processes, but the signals involved are incompletely understood. Here we demonstrate that NG2(+) pericytes controlled the pattern and efficacy of the interstitial migration of leukocytes in vivo. In response to inflammatory mediators, pericytes upregulated expression of the adhesion molecule ICAM-1 and released the chemoattractant MIF. Arteriolar and capillary pericytes attracted and interacted with myeloid leukocytes after extravasating from postcapillary venules, 'instructing' them with pattern-recognition and motility programs. Inhibition of MIF neutralized the migratory cues provided to myeloid leukocytes by NG2(+) pericytes. Hence, our results identify a previously unknown role for NG2(+) pericytes as an active component of innate immune responses, which supports the immunosurveillance and effector function of extravasated neutrophils and macrophages.

We studied the mechanisms underlying the severely impaired wound healing associated with human leukocyte-adhesion deficiency syndrome-1 (LAD1) using a murine disease model. In CD18(-/-) mice, healing of full-thickness wounds was severely delayed during granulation-tissue contraction, a phase where myofibroblasts play a major role. Interestingly, expression levels of myofibroblast markers alpha-smooth muscle actin and ED-A fibronectin were substantially reduced in wounds of CD18(-/-) mice, suggesting an impaired myofibroblast differentiation. TGF-beta signalling was clearly involved since TGF-beta1 and TGF-beta receptor type-II protein levels were decreased, while TGF-beta(1) injections into wound margins fully re-established wound closure. Since, in CD18(-/-) mice, defective migration leads to a severe reduction of neutrophils in wounds, infiltrating macrophages might not phagocytose apoptotic CD18(-/-) neutrophils. Macrophages would thus be lacking their main stimulus to secrete TGF-beta1. Indeed, in neutrophil-macrophage cocultures, lack of CD18 on either cell type leads to dramatically reduced TGF-beta1 release by macrophages due to defective adhesion to, and subsequent impaired phagocytic clearance of, neutrophils. Our data demonstrates that the paracrine secretion of growth factors is essential for cellular differentiation in wound healing.

The receptor for advanced glycation end products (RAGE) contributes to the inflammatory response in many acute and chronic diseases. In this context, RAGE has been identified as a ligand for the ␤ 2 -integrin Mac-1 under static in vitro conditions. Because intercellular adhesion molecule (ICAM)-1 also binds ␤ 2 -integrins, we studied RAGE ؊/؊ , Icam1 ؊/؊ , and RAGE ؊/؊ Icam1 ؊/؊ mice to define the relative contribution of each ligand for leukocyte adhesion in vivo. We show that trauma-induced leukocyte adhesion in cremaster muscle venules is strongly dependent on RAGE and ICAM-1 acting together in an overlapping fashion. Additional in vivo experiments in chimeric mice lacking endothelium-expressed RAGE and ICAM-1 located the adhesion defect to the endothelial compartment. Using microflow chambers coated with P-selectin, CXCL1, and soluble RAGE (sRAGE) demonstrated that sRAGE supports leukocyte adhesion under flow conditions in a Mac-1-but not LFA-1-dependent fashion. A static adhesion assay revealed that wild-type and RAGE ؊/؊ neutrophil adhesion and spreading were similar on immobilized sRAGE or fibrinogen. These observations indicate a crucial role of endotheliumexpressed RAGE as Mac-1 ligand and uncover RAGE and ICAM-1 as a new set of functionally linked adhesion molecules, which closely cooperate in mediating leukocyte adhesion during the acute traumainduced inflammatory response in vivo. IntroductionLeukocyte recruitment into inflamed tissue follows a well-defined cascade of events, beginning with the capture of free-flowing leukocytes to the vessel wall and subsequent leukocyte rolling along and adhesion to the inflamed endothelial layer. 1,2 During rolling, leukocytes get into close contact with the endothelial surface, which allows endothelial bound chemokines to interact with their specific receptors on the leukocyte surface. This triggers the activation of integrins, which leads to firm leukocyte arrest on the endothelium. In addition, integrindependent signaling events induce cytoskeletal rearrangements and cell polarization, modifications necessary in helping to prepare the attached leukocyte to spread and crawl in search for its way out of the vasculature into tissue. [2][3][4][5][6] Recent evidence has shown that the ␤ 2 -integrin Mac-1 is crucially involved in transducing Syk-dependent signaling events necessary for sustained leukocyte adhesion. [7][8][9] The receptor for advanced glycation end products (RAGE) is a pattern recognition receptor that has been identified to be a major player in chronic inflammatory conditions. 10,11 This has been mainly attributed to its strong effects on perpetuating nuclear factor-B (NF-B) activation and NF-Bdependent signaling 10,11 and its ability to induce its own expression. 10,12 Besides its function as a signaling molecule, RAGE also binds to Mac-1, which has been demonstrated under in vitro conditions. 13 In addition, a reduction of leukocyte extravasation into the inflamed peritoneal cavity was found in RAGE Ϫ/Ϫ mice after intraperitoneal application of th...

IntroductionPolymorphonuclear neutrophils (PMNs) play an important role in host defense and inflammation. During the acute inflammatory response, PMNs extravasate from the blood into the tissue, where they migrate toward the sites of lesion by following a gradient of chemoattractants. 1,2 Site-directed migration (chemotaxis) requires 3 different processes 3 : (1) the periodical formation of lamellipodia; (2) the establishment of cell polarity that is characterized by the formation of a posterior pole (uropod) and an anterior pole (leading edge) that forms the lamellipodium with enhanced sensitivity for chemoattractants; and (3) the translation of the external chemotactic gradient into an internal signaling gradient. This process of directional sensing is due to an asymmetric distribution of signaling components within the cell. 3 Accordingly, neutrophil-like differentiated cells, an established model for PMNs, orient their polarity in response to a chemoattractant gradient and perform site-directed migration. 4 Chemoattractants signal via G-protein-coupled receptors that are responsible for directional sensing and the establishment of the polarized sensitivity within the cell. 3 However, ordered leukocyte polarization not only requires the presence of chemoattractants, but also depends on adhesive cell-matrix interactions that are mediated mainly by the leukocyte adhesion molecules of the integrin family. 5 ␤ 2 integrins (CD11/CD18) especially are critically involved in firm adhesion and migration of PMNs. 6 Recently, we and others have shown that the non-receptor tyrosine kinase Syk is constitutively associated with CD18, the ␤ subunit of the ␤ 2 integrins in human PMNs and in HL-60 cells. [7][8][9] Syk is widely expressed in hematopoietic cells and fulfills essential roles in hematopoietic cell functions like Fc receptor 10 and cytokine receptor signaling, 11 phagocytosis, 12 lymphocyte development, 13,14 and platelet activation. 15,16 Syk and ZAP-70, the other member of this protein family, contain 2 Src homology 2 (SH2) domains in a tandem repeat that are separated by the interdomain A. The C-terminal kinase domain is separated from the 2 SH2 domains by the interdomain B. 17 Outside-in signaling of Mac-1 (CD11b/CD18), the most abundant ␤ 2 integrin on PMNs, occurs upon ligand binding to immobilized fibrinogen or the intercellular adhesion molecules-1 and -2. 18 Mac-1-mediated adhesion induces tyrosine phosphorylation of different signaling components, including Syk. 7 A physiologic role for Syk has been demonstrated previously for multiple ␤ 2 integrin-mediated functions of murine PMNs, including the respiratory burst, degranulation, and spreading. 19 Recently, we have shown that endogenous Syk is enriched at the leading edge of both murine PMNs and HL-60 cells, where Syk plays a role in the stabilization of the leading edge of the polarized cell and colocalizes with CD18. 19,20 Syk has several downstream targets, including Pyk2, and Vav. 17,21 Vav is a guanine exchange factor for the Rho family GTPases Ra...