For multiple analyses of variance, the Kruskal-Wallis test was used with Dunns post-test. in leukocyte trafficking from blood into tissues. Chemokine-mediated integrin activation leads to firm adhesion of leukocytes to the endothelium, essential Sildenafil Mesylate for tranendothelial migration. In contrast, leukocyte interstitial migration is usually thought to be largely integrin-independent2. Migration studies using artificial collagen matrices or confinement chambers suggest that the dense three-dimensional ECM scaffold supports the use of nonadhesive, actin-based traction mechanisms by leukocytes3, 4. In the lymph node (LN), T cells migrate along networks of fibroblastic reticular cells (FRCs) that express integrin Sildenafil Mesylate ligands ICAM-1 and VCAM-1 and chemokines CCL19 and CCL21. However, motility is usually driven by chemokine-mediated chemotaxis/haptotaxis directly and does not require integrins for adhesion5. Similarly, genetic ablation of all known integrins in DCs failed to perturb DC migration in the LN or skin6. Thus, under a variety of conditions, integrins appear to be largely dispensable for extravascular trafficking of leukocytes. Inflammatory mediators and pathogens themselves change ECM density and composition in peripheral tissues which may dictate new requirements for leukocyte motility at sites of inflammation7. T cells expressing distinct integrins accumulate in particular inflammatory settings and in discrete tissues where integrin expression is thought Sildenafil Mesylate to play a role in tissue-specific homing8 and retention within the tissue9. In contrast to the situation in LNs, where collagen fibers are coated by FRCs, T cells in non-lymphoid tissues such as the skin and lung are directly exposed to collagen fibers and associated ECM components that could act as guidance cues for movement through the interstitial space. The use of multiphoton microscopy has enabled the visualization of leukocyte motility within peripheral tissues with studies highlighting a close association between Sildenafil Mesylate T cells and matrix fibers in skin, brain and tumors10C16. Lymphocyte movement in the skin during delayed type hypersensitivity showed a correlation between collagen-binding integrins on T cells and their migration along fibers requiring calcium signaling by Kv1.3 potassium-channels14. During contamination with Toxoplasma in the brain, effector T cells also migrate along reticular fibers, although the brain was largely devoid of infection-induced increases in collagen13. In many studies, blockade of G protein-coupled receptor signaling attenuated leukocyte Rabbit polyclonal to CDK4 interstitial motility, but it is not clear if this is due to blockade of chemotaxis/kinesis or the absence of chemokine-induced integrin activation. Although neutrophil interstitial migration in the mesentery was shown to be integrin dependent17, there have been no studies that directly test the role of integrins in T cell motility within inflamed peripheral tissues. Given the critical role that physical confinement plays in three-dimensional cell motility4 and the extent of inflammation-driven ECM remodeling, we addressed mechanisms of interstitial motility of effector CD4+ T lymphocytes in the inflamed dermis. Inflammation was associated with a reduction in the density of collagen fibers and with extensive fibronectin deposition. Using intravital multiphoton microscopy (IV-MPM) we found that effector CD4+ T cell movement within the inflamed dermis is usually integrin-dependent and the expression of v-integrins is essential for interstitial motility and pathogen clearance. Thus, movement through inflamed interstitial tissue requires the coordinate expression of specific integrins on effector T cells with ECM changes in the tissue. Results Inflammation-induced migration along ECM fibers Analysis of T cell interstitial migration was performed in the inflamed and non-inflamed dermis using intravital multiphoton microscopy (IV-MPM). Dermal location was assessed using the second harmonic generation (SHG) to distinguish the collagen-rich dermis from collagen-replete epidermis and from blood vessels using intravenous Texas-red dextran (Fig 1a, b). Sildenafil Mesylate Cells were tracked over time in three-dimensional space using semi-automated software. T cells in the non-inflamed dermis were identified by using CD4-Cre+ Stop-floxed YFP mice (mice) (86% + T cells, the majority being CD4+ and Foxp3?, Supplementary Physique 1). In the constant state non-inflamed dermis T cells were observed at low frequencies and were uniformly non-motile (Fig 1c). Average velocities for dermal CD4+ T cells (predominantly T cells) were 0.803 0.05 SEM m/min; noticeably less than that observe for actively surveying dermal T cells in the constant state (common speeds of 2C5 m/min)18, 19. For T cell migration in the inflamed dermis, we followed a populace of antigen-specific CD4+ T cells. Na?ve CD4+ T cells from WT15 T cell receptor (TCR)-transgenic mice20 were primed under TH1 differentiation.