Selectin-mediated tethering and rolling initiates the multi-step process of leukocyte extravasation which is crucial for the formation of an inflammatory infiltrate. We studied the impact of platelets on this process in the skin. Using intravital microscopy, we analyzed platelet interactions with cutaneous post-capillary venules of mouse ears and observed an increase in platelet rolling if platelets were activated (41.6+/-20.2% vs. 13.1+/-8.5% rolling of resting platelets). Experiments with P-selectin deficient mice and antibodies blocking either P-selectin, GPIIb/IIIa or GPIb showed that rolling depends on platelet PSGL-1 and GPIIb/IIIa on one hand, and endothelial P-selectin on the other. Next, formation of platelet-leukocyte aggregates was demonstrated by simultaneous observation of platelets and leukocytes in vivo utilizing a newly developed two-color technique. Aggregates increased overall leukocyte rolling (leukocytes alone: 27.4+/-11.2%, leukocytes with resting platelets: 25.3+/-10.2%, leukocytes with activated platelets 38.1+/-11.8%). To investigate if activated platelets may contribute to the pathogenesis of chronic cutaneous inflammation, platelet P-selectin expression was studied in 8 patients with psoriasis. A correlation between platelet P-selectin expression and disease severity was established. In summary, we show that activated, not resting, platelets increase leukocyte rolling in murine skin. This increased rolling is due to the aggregate formation of platelets with leukocytes. We also provide evidence for a potential role of this mechanism in the pathogenesis of chronic inflammatory skin diseases.

Inflammation induced by circulating immunoglobulin G-immune complexes (ICs) characterizes many immune-mediated diseases. In this work, the molecular requirements for the deposition of circulating ICs and subsequent acute leukocyte recruitment in mice were elucidated. We show that after intravenous injection, preformed soluble ICs are rapidly deposited in the postcapillary venules of the cremaster microcirculation, secondary to increased vascular permeability. This deposition is dependent on complement C1q. IC deposition is associated with leukocyte recruitment. Leukocyte rolling, which is mediated by P-selectin in the exteriorized cremaster muscle, is not further increased in response to ICs. In contrast, leukocyte rolling velocity is significantly decreased and leukocyte adhesion is significantly increased in the presence of ICs. The IC-mediated slow leukocyte rolling velocity and subsequent adhesion and emigration are dependent on Fcgamma receptors (FcgammaRs), particularly FcgammaRIII, with complement C3 and C5 having no detectable role. These studies suggest a regulatory mechanism of IC deposition and leukocyte trafficking in IC-mediated inflammation requiring C1q and FcgammaRs in sequential, noninteracting roles.

Chemokines compose a sophisticated communication system used by all our cell types, including immune cells. Chemokine messages are decoded by specific receptors that initiate signal transduction events leading to a multitude of cellular responses, leukocyte chemotaxis and adhesion in particular. Critical determinants of the in vivo activities of chemokines in the immune system include their presentation by endothelial cells and extracellular matrix molecules, as well as their cellular uptake via "silent" chemokine receptors (interceptors) leading either to their transcytosis or to degradation. These regulatory mechanisms of chemokine histotopography, as well as the promiscuous and overlapping receptor specificities of inflammation-induced chemokines, shape innate responses to infections and tissue damage. Conversely, the specific patterns of homeostatic chemokines, where each chemokine is perceived by a single receptor, are charting lymphocyte navigation routes for immune surveillance. This review presents our current understanding of the mechanisms that regulate the cellular perception and pathophysiologic meaning of chemokines.

Changes in chromatin structure underlie the activation or silencing of genes during development. The chromatin remodeler Mi-2beta is highly expressed in thymocytes and is presumed to be a transcriptional repressor because of its presence in the nucleosome remodeling deacetylase (NuRD) complex. Using conditional inactivation, we show that Mi-2beta is required at several steps during T cell development: for differentiation of beta selected immature thymocytes, for developmental expression of CD4, and for cell divisions in mature T cells. We further show that Mi-2beta plays a direct role in promoting CD4 gene expression. Mi-2beta associates with the CD4 enhancer as well as the E box binding protein HEB and the histone acetyltransferase (HAT) p300, enabling their recruitment to the CD4 enhancer and causing histone H3-hyperacetylation to this regulatory region. These findings provide important insights into the regulation of CD4 expression during T cell development and define a role for Mi-2beta in gene activation.

Blood-borne lymphocyte trafficking to peripheral lymph nodes (PLNs) depends on the successful initiation of rolling interactions mediated by L-selectin binding to sialomucin ligands in high endothelial venules (HEVs). Biochemical analysis of purified L-selectin ligands has identified posttranslational modifications mediated by Core2GlcNAcT-I and high endothelial cell GlcNAc-6-sulfotransferase (HECGlcNAc6ST). Consequently, lymphocyte migration to PLNs of C2GlcNAcT-I(-/-) and HEC-GlcNAc6ST(-/-) mice was reduced; however, B-cell homing was more severely compromised than T-cell migration. Accordingly, intravital microscopy (IVM) of PLN HEVs revealed a defect in B-cell tethering and increased rolling velocity (V(roll)) in C2GlcNAcT-I(-/-) mice that was more pronounced than it was for T cells. By contrast, B- and T-cell tethering was normal in HEC-GlcNAc6ST(-/-) HEVs, but V(roll) was accelerated, especially for B cells. The increased sensitivity of B cells to glycan deficiencies was caused by lower expression levels of L-selectin; L-selectin(+/-) T cells expressing L-selectin levels equivalent to those of B cells exhibited intravascular behavior similar to that of B cells. These results demonstrate distinct functions for C2GlcNAcT-I and HEC-GlcNAc6ST in the differential elaboration of HEV glycoproteins that set a threshold for the amount of L-selectin needed for lymphocyte homing.

Central memory CD8(+) T cells (T(CM)) confer superior protective immunity against infections compared with other T cell subsets. T(CM) recirculate mainly through secondary lymphoid organs, including peripheral lymph nodes (PLNs). Here, we report that T(CM), unlike naive T cells, can home to PLNs in both a CCR7-dependent and -independent manner. Homing experiments in paucity of lymph node T cells (plt/plt) mice, which do not express CCR7 ligands in secondary lymphoid organs, revealed that T(CM) migrate to PLNs at approximately 20% of wild-type (WT) levels, whereas homing of naive T cells was reduced by 95%. Accordingly, a large fraction of endogenous CD8(+) T cells in plt/plt PLNs displayed a T(CM) phenotype. Intravital microscopy of plt/plt subiliac lymph nodes showed that T(CM) rolled and firmly adhered (sticking) in high endothelial venules (HEVs), whereas naive T cells were incapable of sticking. Sticking of T(CM) in plt/plt HEVs was pertussis toxin sensitive and was blocked by anti-CXCL12 (SDF-1alpha). Anti-CXCL12 also reduced homing of T(CM) to PLNs in WT animals by 20%, indicating a nonredundant role for this chemokine in the presence of physiologic CCR7 agonists. Together, these data distinguish naive T cells from T(CM), whereby only the latter display greater migratory flexibility by virtue of their increased responsiveness to both CCR7 ligands and CXCL12 during homing to PLN.

Selective recruitment of blood-borne leukocytes to tissues and their proper positioning within them is crucial for the many integrated functions of the immune system. Intravital microscopy (IVM) techniques have been employed for more than a century to study these events at the single-cell level in living animals. Conventional video-based IVM allows the visualization of extremely rapid adhesion events at the interface between blood and tissue. Multiphoton IVM is a relatively new tool for imaging the slower dynamics of cell migration and cell-cell interactions in the extravascular space in three dimensions. Fueled by the burgeoning development of sophisticated fluorescent markers and increasingly powerful imaging tools, we are currently witnessing the emergence of a new field in immuno-imaging, in which leukocyte function and cell-cell communication is explored in a truly physiological context.

Recent advances in photonics, particularly multi-photon microscopy (MPM) and new molecular and genetic tools are empowering immunologists to answer longstanding unresolved questions in living animals. Using intravital microscopy (IVM) investigators are dissecting the cellular and molecular underpinnings controlling immune cell motility and interactions in tissues. Recent IVM work showed that T cell responses to antigen in lymph nodes are different from those observed in vitro and appear dictated by factors uniquely relevant to intact organs. Other IVM models, particularly in the bone marrow, reveal how different anatomic contexts regulate leukocyte development, immunity, and inflammation. This article will discuss the current state of the field and outline how IVM can generate new discoveries and serve as a "reality check" for areas of research that were formerly the exclusive domain of in vitro experimentation.

T cell priming by dendritic cells (DC) from gut-associated lymphoid tissues gives rise to effector cells with pronounced gut tropism. The mechanism for DC-dependent imprinting of gut specificity has remained unknown. New findings point to retinoic acid, which is uniquely produced by intestinal DC, but not by DC from other lymphoid organs.

In vivo, beta(2) integrins and particularly alpha(L)beta(2) (LFA-1) robustly support firm adhesion of leukocytes, but can also cooperate with other molecules in supporting rolling adhesion. Strikingly, a small molecule alpha/beta I-like allosteric antagonist, XVA143, inhibits LFA-1-dependent firm adhesion, while at the same time it enhances adhesion in shear flow and rolling both in vitro and in vivo. XVA143 appears to induce the extended conformation of integrins as shown by increased activation epitope exposure. Fab to the beta(2) I-like domain converts firm adhesion to rolling adhesion, but does not enhance adhesion. Residue alpha(L)-Glu-310 in the linker following the I domain is critical for communication to the beta(2) I-like domain, rolling, integrin extension, and activation by Mn(2+) of firm adhesion. The results demonstrate the importance of integrin extension in rolling, and suggest that rolling and firm adhesion are mediated by extended conformations of alpha(L)beta(2) that differ in the affinity of the alpha(L) I domain for ICAM-1.

Primary T-cell responses in lymph nodes (LNs) require contact-dependent information exchange between T cells and dendritic cells (DCs). Because lymphocytes continually enter and leave normal LNs, the resident lymphocyte pool is composed of non-synchronized cells with different dwell times that display heterogeneous behaviour in mouse LNs in vitro. Here we employ two-photon microscopy in vivo to study antigen-presenting DCs and naive T cells whose dwell time in LNs was synchronized. During the first 8 h after entering from the blood, T cells underwent multiple short encounters with DCs, progressively decreased their motility, and upregulated activation markers. During the subsequent 12 h T cells formed long-lasting stable conjugates with DCs and began to secrete interleukin-2 and interferon-gamma. On the second day, coinciding with the onset of proliferation, T cells resumed their rapid migration and short DC contacts. Thus, T-cell priming by DCs occurs in three successive stages: transient serial encounters during the first activation phase are followed by a second phase of stable contacts culminating in cytokine production, which makes a transition into a third phase of high motility and rapid proliferation.

Interaction of the platelet receptor glycoprotein (GP) Ib-V-IX with von Willebrand factor exposed at a site of vascular injury is an essential step in the initiation of a hemostatic plug. Proteolytic cleavage (shedding) of the GPIbalpha subunit was first described >25 years ago, the protease mediating this event as well as its physiological function, however, have not been elucidated. We reported recently that shedding of GPIbalpha induced by platelet storage or mitochondrial injury involves a platelet-derived metalloproteinase(s). Here we show that GPIbalpha shedding in response to mitochondrial injury or physiological activation is inhibited in platelets obtained from chimeric mice, which express inactive tumor necrosis factor-alpha converting enzyme (TACE(DeltaZn/DeltaZn)) in blood cells only. Shedding was also inhibited in mouse and human platelets in the presence of 2 potent TACE inhibitors: TAP1 and TMI-1. Our data further suggest that TACE is important in the regulation of GPIbalpha expression in vivo because we observed an approximately 90% reduction in soluble GPIbalpha (glycocalicin) in plasma of TACE(DeltaZn/DeltaZn) chimeras as well as significantly increased levels of GPIbalpha on circulating platelets. In contrast, shedding of P-selectin from activated platelets was not affected by the mutation in TACE. Damaged TACE(DeltaZn/DeltaZn) platelets were further characterized by a markedly improved post-transfusion recovery and hemostatic function in mice. In conclusion, our data demonstrate that TACE is expressed in platelets and that it is the key enzyme mediating shedding of GPIbalpha.