Little is known about the factors that enable the mobilisation of human mesenchymal stem cells (MSC) from the bone marrow into the blood stream and their recruitment to and retention in the tumour. We found specific migration of MSC towards growth factors present in pancreatic tumours, such as PDGF, EGF, VEGF and specific inhibitors Glivec, Erbitux and Avastin interfered with migration. Within a few hours, MSC migrated into spheroids consisting of pancreatic cancer cells, fibroblasts and endothelial cells as measured by time-lapse microscopy. Supernatant from subconfluent MSC increased sprouting of HUVEC due to VEGF production by MSC itself as demonstrated by RT-PCR and ELISA. Only few MSCs were differentiated into endothelial cells in vitro, whereas in vivo differentiation was not observed. Lentiviral GFP-marked MSCs, injected in nude mice xenografted with orthotopic pancreatic tumours, preferentially migrated into the tumours as observed by FACS analysis of green fluorescent cells. By immunofluorescence and intravital microscopic studies, we found the interaction of MSC with the endothelium of blood vessels. Mesenchymal stem cells supported tumour angiogenesis in vivo, that is CD31 þ vessel density was increased after the transfer of MSC compared with siVEGF-MSC. Our data demonstrate the migration of MSC toward tumour vessels and suggest a supportive role in angiogenesis.

β2 integrins and Fcγ receptors are critically involved in neutrophil activation at the site of inflammation. Both receptor types trigger a receptor-proximal tyrosine phosphorylation cascade through Src family kinases and Syk, but further downstream signaling events are poorly understood. We show that phospholipase C (PLC) γ2 is phosphorylated downstream of Src family kinases and Syk during integrin or Fc receptor-mediated activation of neutrophils. PLCγ2−/− neutrophils are completely defective in β2 integrin or Fcγ receptor-mediated functional responses such as respiratory burst, degranulation, or cell spreading in vitro and show reduced adhesion/spreading in inflamed capillary venules in vivo. However, PLCγ2−/− neutrophils respond normally to various other agonists, including chemokines, bacterial formyl peptides, Toll-like receptor ligands, or proinflammatory cytokines, and migrate normally both in vitro and in vivo. To confirm the in vivo relevance of these observations, the effect of the PLCγ2−/− mutation was tested in the K/B×N serum transfer arthritis model, which is known to require β2 integrins, Fcγ receptors, and neutrophils. PLCγ2 deficiency completely protected mice from clinical signs and histological features of arthritis as well as from arthritis-induced loss of articular function. These results identify PLCγ2 as a critical player of integrin and Fc receptor-mediated neutrophil functions and the neutrophil-mediated effector phase of autoimmune arthritis.

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...