In order to analyze the function of DcR3 for the regulation of cell adhesion and apoptosis in macrophages, we investigated the expression of decoy receptor 3 (DcR3) in THP-1 monocytes/macrophages. DcR3 was expressed in THP-1 and increased by phorbol 12-myristate 13-acetate (PMA). The formation of macrophage aggregates was observed when THP-1 cells were differentiated by PMA or stimulated with DcR3-Fc. Undifferentiated THP-1 cells were also induced to form aggregates by DcR3-Fc. The expression of integrin alpha4 was significantly increased by DcR3-Fc. CHX-induced apoptosis in THP-1 was inhibited by DcR3-Fc, of which inhibition against CHX-induced apoptosis and aggregate formation were ameliorated by anti-VLA4 antibody. DcR3 may play a significant role in macrophages not only by a decoy receptor but also by increasing alpha4 integrin.

Integrins coordinate spatial signaling events essential for cell polarity and directed migration. Such signals from alpha4 integrins regulate cell migration in development and in leukocyte trafficking. Here, we report that efficient alpha4-mediated migration requires spatial control of alpha4 phosphorylation by protein kinase A, and hence localized inhibition of binding of the signaling adaptor, paxillin, to the integrin. In migrating cells, phosphorylated alpha4 accumulated along the leading edge. Blocking alpha4 phosphorylation by mutagenesis or by inhibition of protein kinase A drastically reduced alpha4-dependent migration and lamellipodial stability. alpha4 phosphorylation blocks paxillin binding in vitro; we now find that paxillin and phospho-alpha4 were in distinct clusters at the leading edge of migrating cells, whereas unphosphorylated alpha4 and paxillin colocalized along the lateral edges of those cells. Furthermore, enforced paxillin association with alpha4 inhibits migration and reduced lamellipodial stability. These results show that topographically specific integrin phosphorylation can control cell migration and polarization by spatial segregation of adaptor protein binding.

Insights into sequential leukocyte-endothelial interactions during leukocyte trafficking have been obtained through experiments using human umbilical vein endothelial cells (HUVEC) under flow conditions. To investigate leukocyte-brain endothelial cell interactions, we developed a dynamic in vitro system, using Transfected Human Brain Microvascular Endothelial Cells (THBMEC) and a parallel plate flow chamber. Human peripheral blood mononuclear cells (PBMC) were perfused across confluent THBMEC cultures at a velocity that approximates the rate found in human brain capillaries. Leukocyte-THBMEC interactions were visualized by phase-contrast microscopy, and images were captured on a CCD camera. To simulate inflammatory conditions, we activated THBMEC with the inflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma), which up-regulated chemokine and adhesion molecule expression in THBMEC without affecting the distribution of immunoreactivity for tight junction-associated proteins. PBMC adhesion was enhanced by cytokine-mediated activation of THBMEC. G protein-coupled receptor (GPCR) activation was essential for leukocyte-THBMEC interaction, as pertussis toxin (PTX) treatment of PBMC abrogated PBMC adhesion to activated THBMEC. The anti-alpha4 integrin antibody, natalizumab, infused into MS patients, significantly reduced the adhesion of their ex vivo PBMC to activated THBMEC under flow conditions. Further study showed that alternatively spliced fibronectin containing the CS1 region (FN-CS1), but not Vascular Cell Adhesion Molecule type 1 (VCAM-1), was the ligand of alpha4 integrin on activated THBMEC. Blocking FN-CS1 abrogated PBMC adhesion on activated THBMEC, while anti-VCAM-1 antibodies had no effect. These results established a novel in vitro dynamic BBB model. We also demonstrated the dependence of leukocyte-endothelial interactions in this model on alpha4 integrins and FN-CS1.

The cell surface receptor alpha4 integrin plays a critical role in the homing, engraftment, and maintenance of hematopoietic progenitor cells (HPCs) in the bone marrow (BM). Down-regulation or functional blockade of alpha4 integrin or its ligand vascular cell adhesion molecule-1 mobilizes long-term HPCs. We investigated the role of alpha4 integrin in the mobilization and homing of BM endothelial progenitor cells (EPCs). EPCs with endothelial colony-forming activity in the BM are exclusively alpha4 integrin-expressing cells. In vivo, a single dose of anti-alpha4 integrin antibody resulted in increased circulating EPC counts for 3 d. In hindlimb ischemia and myocardial infarction, systemically administered anti-alpha4 integrin antibody increased recruitment and incorporation of BM EPCs in newly formed vasculature and improved functional blood flow recovery and tissue preservation. Interestingly, BM EPCs that had been preblocked with anti-alpha4 integrin ex vivo or collected from alpha4 integrin-deficient mice incorporated as well as control cells into the neovasculature in ischemic sites, suggesting that alpha4 integrin may be dispensable or play a redundant role in EPC homing to ischemic tissue. These data indicate that functional disruption of alpha4 integrin may represent a potential angiogenic therapy for ischemic disease by increasing the available circulating supply of EPCs.

Many prevalent diseases of advanced societies, such as obesity-induced type 2 diabetes, are linked to indolent mononuclear cell-dependent inflammation. We previously proposed that blockade of alpha4 integrin signaling can inhibit inflammation while limiting mechanism-based toxicities of loss of alpha4 function. Thus, we hypothesized that mice bearing an alpha4(Y991A) mutation, which blocks signaling, would be protected from development of high-fat diet-induced insulin resistance.

In this study, we examined the relationship between the endothelial selectins (P-selectin and E-selectin) and whether they are critical for alpha4-integrin-dependent leukocyte recruitment in inflamed (late phase response), cremasteric postcapillary venules. Animals were systemically sensitized and 2 wk later challenged intrascrotally with chicken ovalbumin. Leukocyte rolling flux, adhesion, and emigration were assessed at baseline and 4 and 8 h postantigen challenge. There was a significant increase in leukocyte rolling flux, adhesion, and emigration in sensitized and challenged mice at both 4 and 8 h. At 8 h, the increase in leukocyte rolling flux was approximately 50% inhibitable by an anti-alpha4-integrin antibody, 98% inhibitable by fucoidin (a selectin-binding carbohydrate), and 100% inhibitable by an anti-P-selectin antibody. P-selectin-deficient animals displayed no leukocyte rolling or adhesion at 8 h after challenge. However, at 8 h there were many emigrated leukocytes in the perivascular space suggesting P-selectin-independent rolling at an earlier time point. Indeed, at 4 h postantigen challenge in P-selectin-deficient mice, there was increased leukocyte rolling, adhesion, and emigration. The rolling in the P-selectin-deficient mice at 4 h was largely alpha4-integrin dependent. However, there was an essential E-selectin-dependent component inasmuch as an anti-E-selectin antibody completely reversed the rolling, and in E-selectin and P-selectin double deficient mice rolling, adhesion and emigration were completely absent. These results illustrate that P-selectin underlies all of the antigen-induced rolling with a brief transient contribution from E-selectin in the P-selectin-deficient animals. Finally, the antigen-induced alpha4-integrin-mediated leukocyte recruitment is entirely dependent upon endothelial selectins.

Th1 and Th17 cell migration into the central nervous system (CNS) is a fundamental process in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). Particularly, leptomeningeal vessels of the subarachnoid space (SAS) constitute a central route for T cell entry into the CNS during EAE. Once migrated into the SAS, T cells show an active motility behavior, which is a prerequisite for cell-cell communication, in situ reactivation and neuroinflammation. However, the molecular mechanisms selectively controlling Th1 and Th17 cell trafficking in the inflamed leptomeninges are not well understood. By using epifluorescence intravital microscopy, we obtained results showing that myelin-specific Th1 and Th17 cells have different intravascular adhesion capacity depending on the disease phase, with Th17 cells being more adhesive at disease peak. Inhibition of αLβ2 integrin selectively blocked Th1 cell adhesion, but had no effect on Th17 rolling and arrest capacity during all disease phases, suggesting that distinct adhesion mechanisms control the migration of key T cell populations involved in EAE induction. Blockade of α4 integrins affected myelin-specific Th1 cell rolling and arrest, but only selectively altered intravascular arrest of Th17 cells. Notably, selective α4β7 integrin blockade inhibited Th17 cell arrest without interfering with intravascular Th1 cell adhesion, suggesting that α4β7 integrin is predominantly involved in Th17 cell migration into the inflamed leptomeninges in EAE mice. Two-photon microscopy experiments showed that blockade of α4 integrin chain or α4β7 integrin selectively inhibited the locomotion of extravasated antigen-specific Th17 cells in the SAS, but had no effect on Th1 cell intratissue dynamics, further pointing to α4β7 integrin as key molecule in Th17 cell trafficking during EAE development. Finally, therapeutic inhibition of α4β7 integrin at disease onset by intrathecal injection of a blocking antibody attenuated clinical severity and reduced neuroinflammation, further demonstrating a crucial role for α4β7 integrin in driving Th17 cell-mediated disease pathogenesis. Altogether, our data suggest that a better knowledge of the molecular mechanisms controlling myelin-specific Th1 and Th17 cell trafficking during EAE delevopment may help to identify new therapeutic strategies for CNS inflammatory and demyelinating diseases.

Allylamine, a selective cardiovascular toxin that induces oxidative stress, is known to alter expression of extracellular matrix and cell adhesion proteins that are central to arterial remodeling. Our goals were to determine whether AAM treatment in rats modulates integrin/matrix-dependent arteriolar function, and to what extent integrin expression correlated to these alterations. Integrins are transmembrane proteins that facilitate mechanical and molecular signaling between the extracellular matrix and cytoskeleton, and so are suitable candidates for involvement in phenotypic and functional alterations of smooth muscle in response to oxidative stress. Arg-Gly-Asp (RGD) and Leu-Asp-Val (LDV), two integrin-binding motifs found in ECM proteins such as collagens and fibronectin, are known to interact with integrins alphavbeta3 and alpha4beta1, respectively. Previously, we found that RGD containing peptides induce vasodilation through alphavbeta3, while LDV containing peptides induce vasoconstriction through alpha4beta1 of normal rat cremasteric arterioles. In allylamine-treated rats (AAM), the vasomotor response to LDV, but not RGD, was attenuated in a dose-dependent manner. To determine whether changes in integrin subunit mRNA levels correlated with these functional changes, we performed reverse transcription and Real-time PCR for alpha4 and beta3 integrin subunits on RNA isolated from single, first-order cremasteric arterioles. AAM treatment caused a dose-dependent decrease in alpha4 mRNA expression, but not beta3 mRNA expression, suggesting that the changes in vasomotor activity to LDV peptides may be attributable in part to reduced alpha4 expression upon exposure to AAM. These data are supported by similar decreases in alpha4integrin cell surface protein expression in cultured vascular smooth muscle cells treated either in vivo and in vitro with AAM.

Leukocyte recruitment to target tissue is initiated by weak rolling attachments to vessel wall ligands followed by firm integrin-dependent arrest triggered by endothelial chemokines. We show here that immobilized chemokines can augment not only arrest but also earlier integrin-mediated capture (tethering) of lymphocytes on inflamed endothelium. Furthermore, when presented in juxtaposition to vascular cell adhesion molecule 1 (VCAM-1), the endothelial ligand for the integrin very late antigen 4 (VLA-4, alpha4beta1), chemokines rapidly augment reversible lymphocyte tethering and rolling adhesions on VCAM-1. Chemokines potentiate VLA-4 tethering within <0.1 s of contact through Gi protein signaling, the fastest inside-out integrin signaling events reported to date. Although VLA-4 affinity is not altered upon chemokine signaling, subsecond VLA-4 clustering at the leukocyte-substrate contact zone results in enhanced leukocyte avidity to VCAM-1. Endothelial chemokines thus regulate all steps in adhesive cascades that control leukocyte recruitment at specific vascular beds.

Based on the observation that antibodies directed against alpha4-integrin and its counterreceptor VCAM-1 inhibit inflammatory cell recruitment into the central nervous system (CNS) during experimental autoimmune encephalomyelitis (EAE), it has been concluded that alpha4-integrin/VCAM-1 interaction plays a critical role in T cell interaction with the blood-brain barrier (BBB) endothelium. In order to define the exact role of alpha4-integrin and VCAM-1 in T cell recruitment across the BBB endothelium we set up in vitro studies, where we investigated the interaction of freshly activated autoaggressive T cell blasts with the brain endothelial cell line bEnd5. A large panel of blocking antibodies directed against the alpha4-, beta1- and beta7-integrin subunits or against the alpha4beta7-heterodimer and against endothelial VCAM-1 significantly reduced adhesion of encephalitogenic T cell blasts to brain endothelium. However, the very same antibodies did not influence transendothelial migration of autoaggressive T cell blasts across a bEnd5 monolayer. Our in vitro observations, therefore, suggest that in vivo alpha4/VCAM-1 interactions are not involved in transendothelial migration of encephalitogenic T cells across the BBB but rather mediate earlier steps of T cell/BBB-interaction such as firm adhesion.

Regulatory CD4 T cells (Tregs) are critical in maintaining the homeostasis of the immune system. Quantitative or phenotypic alterations and functional impairment of Tregs have been associated with the development of pathologies including those of the central nervous system. Individuals with HIV-1/HTLV-1 co-infection are more prone to develop neurological complications. The aim of this study was to characterize phenotypically Treg cells in HIV-1/HTLV-1 co-infected Mozambican individuals presenting neurological symptoms.

Chemoattractants and chemokines induce arrest of rolling monocytes during emigration from blood into tissues. In this study, we demonstrated that alpha4 integrin affinity for vascular cell adhesion molecule (VCAM)-1 was upregulated rapidly and transiently by chemoattractants and stromal cell-derived factor (SDF)-1alpha and mediated monocyte arrest. alpha4 integrin affinity changes were detected and blocked using soluble VCAM-1/Fc (sVCAM-1/Fc). In a flow cytometry assay, markedly increased sVCAM-1/Fc binding to human blood monocytes or U937 cells transfected with formyl peptide (FP) receptor was detected 30 s after FP or SDF-1alpha treatment and declined after 2 min. In a parallel plate flow chamber assay, FP, C5a, platelet-activating factor, or SDF-1alpha coimmobilized with VCAM-1 induced leukocyte arrest, which was blocked by inclusion of sVCAM-1/Fc but not soluble nonimmune immunoglobulin G in the assay buffer.

The capacity of integrins to mediate adhesiveness is modulated by their cytoplasmic associations. In this study, we describe a novel mechanism by which alpha4-integrin adhesiveness is regulated by the cytoskeletal adaptor paxillin. A mutation of the alpha4 tail that disrupts paxillin binding, alpha4(Y991A), reduced talin association to the alpha4beta1 heterodimer, impaired integrin anchorage to the cytoskeleton, and suppressed alpha4beta1-dependent capture and adhesion strengthening of Jurkat T cells to VCAM-1 under shear stress. The mutant retained intrinsic avidity to soluble or bead-immobilized VCAM-1, supported normal cell spreading at short-lived contacts, had normal alpha4-microvillar distribution, and responded to inside-out signals. This is the first demonstration that cytoskeletal anchorage of an integrin enhances the mechanical stability of its adhesive bonds under strain and, thereby, promotes its ability to mediate leukocyte adhesion under physiological shear stress conditions.

In the retina, integrins in the beta1 family have been shown to be important in many phases of neuronal development, particularly neuroblast migration and axon outgrowth. However, the functions of specific integrin heterodimers are not well defined. In this study, we investigated the functions of beta1 integrins in developing chicken retina by expression of a dominant-negative beta1A construct using a replication-competent retrovirus. Inhibition of integrins using this approach resulted in alteration of cell morphology and increased apoptosis, but did not preclude migration and axon elongation. In an attempt to identify which specific beta1 heterodimer was important, expression and function of the alpha4beta1 heterodimer were also investigated. At early developmental stages, alpha4 protein and mRNA were detected in undifferentiated neuroblasts throughout the retina. At later stages, expression was confined to retinal ganglion cells (RGCs) and amacrine cells. A small molecule antagonist of alpha4 integrins was shown to inhibit neurite outgrowth on recombinant soluble vascular cell adhesion molecule-1 (VCAM-1), a known ligand of alpha4beta1. Introduction of alpha4 antagonist in vivo gave rise to increased apoptosis and led to a thinning of the retina and reduced numbers of retinal ganglion cells (RGCs). We conclude that the integrin alpha4beta1 is important for survival of developing retinal neurons, including RGCs.

Engagement of integrin receptors by the extracellular matrix (ECM) protein fibronectin (FN) activates intracellular signaling, cytoskeletal reorganization and cellular tension. The soluble factor lysophosphatidic acid (LPA) acts through Rho GTPase and its effector Rho kinase (ROCK) to enhance alpha5beta1 integrin-mediated cell spreading on the Arg-Gly-Asp (RGD) cell-binding domain of FN. A second cell-binding site for alpha4 integrins resides in the CS1 segment of the alternatively spliced V region of FN. We show here that LPA treatment of alpha4beta1-expressing CHOalpha4 cells on FN induced a significant decrease in spread cell area. LPA also decreased apoptosis induced by serum-deprivation in CHOalpha4 and human A375 melanoma cells in an alpha4beta1-dependent manner. Improvement in cell viability and changes in cell morphology were dependent on ROCK and on the number of substrate binding sites for alpha4beta1. LPA signaling combined with alpha4beta1-mediated adhesion appears to sustain cell viability in situations where FN matrix is limiting. Such cooperation may impact dynamic cellular events such as wound healing, fibrosis, and metastasis.

The fibronectin (FN)-binding integrins alpha4beta1 and alpha5beta1 confer different cell adhesive properties, particularly with respect to focal adhesion formation and migration. After analyses of alpha4+/alpha5+ A375-SM melanoma cell adhesion to fragments of FN that interact selectively with alpha4beta1 and alpha5beta1, we now report two differences in the signals transduced by each receptor that underpin their specific adhesive properties. First, alpha5beta1 and alpha4beta1 have a differential requirement for cell surface proteoglycan engagement for focal adhesion formation and migration; alpha5beta1 requires a proteoglycan coreceptor (syndecan-4), and alpha4beta1 does not. Second, adhesion via alpha5beta1 caused an eightfold increase in protein kinase Calpha (PKCalpha) activation, but only basal PKCalpha activity was observed after adhesion via alpha4beta1. Pharmacological inhibition of PKCalpha and transient expression of dominant-negative PKCalpha, but not dominant-negative PKCdelta or PKCzeta constructs, suppressed focal adhesion formation and cell migration mediated by alpha5beta1, but had no effect on alpha4beta1. These findings demonstrate that different integrins can signal to induce focal adhesion formation and migration by different mechanisms, and they identify PKCalpha signaling as central to the functional differences between alpha4beta1 and alpha5beta1.

Previous studies have shown that integrin alpha chain tails make strong positive contributions to integrin-mediated cell adhesion. We now show here that integrin alpha4 tail deletion markedly impairs static cell adhesion by a mechanism that does not involve altered binding of soluble vascular cell adhesion molecule 1 ligand. Instead, truncation of the alpha4 cytoplasmic domain caused a severe deficiency in integrin accumulation into cell surface clusters, as induced by ligand and/ or antibodies. Furthermore, alpha4 tail deletion also significantly decreased the membrane diffusivity of alpha4beta1, as determined by a single particle tracking technique. Notably, low doses of cytochalasin D partially restored the deficiency in cell adhesion seen upon alpha4 tail deletion. Together, these results suggest that alpha4 tail deletion exposes the beta1 cytoplasmic domain, leading to cytoskeletal associations that apparently restrict integrin lateral diffusion and accumulation into clusters, thus causing reduced static cell adhesion. Our demonstration of integrin adhesive activity regulated through receptor diffusion/clustering (rather than through altered ligand binding affinity) may be highly relevant towards the understanding of inside-out signaling mechanisms for beta1 integrins.

Simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) infection results in an early and enduring depletion of intestinal CD4(+) T cells. SIV and HIV bind integrin alpha4beta7, thereby facilitating infection of lymphocytes that home to the gut-associated lymphoid tissue (GALT). Using an ex vivo flow cytometry assay, we found that SIVmac239-infected cells expressed significantly lower levels of integrin alpha4beta7 than did uninfected cells. This finding suggested a potential viral effect on integrin alpha4beta7 expression. Using an in vitro model, we confirmed that integrin alpha4beta7 was downregulated on the surfaces of SIVmac239-infected cells. Further, modulation of integrin alpha4beta7 was dependent on de novo synthesis of viral proteins, but neither cell death, the release of a soluble factor, nor a change in activation state was involved. Downregulation of integrin alpha4beta7 may have an unappreciated role in the CD4 depletion of the mucosal-associated lymphoid compartments, susceptibility to superinfection, and/or immune evasion.

Mast cells (MCs) are centrally important in allergic inflammation of the airways, as well as in the intestinal immune response to helminth infection. A single lineage of bone marrow (BM)-derived progenitors emigrates from the circulation and matures into phenotypically distinct MCs in different tissues. Because the mechanisms of MC progenitor (MCp) homing to peripheral tissues have not been evaluated, we used limiting dilution analysis to measure the concentration of MCp in various tissues of mice deficient for candidate homing molecules. MCp were almost completely absent in the small intestine but were present in the lung, spleen, BM, and large intestine of beta7 integrin-deficient mice (on the C57BL/6 background), indicating that a beta7 integrin is critical for homing of these cells to the small intestine. MCp concentrations were not altered in the tissues of mice deficient in the alphaE integrin (CD103), the beta2 integrin (CD18), or the recombination activating gene (RAG)-2 gene either alone or in combination with the interleukin (IL)-receptor common gamma chain. Therefore, it is the alpha4beta7 integrin and not the alphaEbeta7 integrin that is critical, and lymphocytes and natural killer cells play no role in directing MCp migration under basal conditions. When MCp in BALB/c mice were eliminated with sublethal doses of gamma-radiation and then reconstituted with syngeneic BM, the administration of anti-alpha4beta7 integrin, anti-alpha4 integrin, anti-beta7 integrin, or anti-MAdCAM-1 monoclonal antibodies (mAbs) blocked the recovery of MCp in the small intestine. The blocking mAbs could be administered as late as 4 d after BM reconstitution with optimal inhibition, implying that the MCp must arise first in the BM, circulate in the vasculature, and then translocate into the intestine. Inasmuch as MCp are preserved in the lungs of beta7 integrin-deficient and anti-alpha4beta7 integrin-treated mice but not in the small intestine, alpha4beta7 integrin is critical for tissue specific extravasation for localization of MCp in the small intestine, but not the lungs.

Formaldehyde cross-linking of protein complexes combined with immunoprecipitation and mass spectrometry analysis is a promising technique for analysing protein-protein interactions, including those of transient nature. Here we used integrin beta1 as a model to describe the application of formaldehyde cross-linking in detail, particularly focusing on the optimal parameters for cross-linking, the detection of formaldehyde cross-linked complexes, the utility of antibodies, and the identification of binding partners. Integrin beta1 was found in a high molecular weight complex after formaldehyde cross-linking. Eight different anti-integrin beta1 antibodies were used for pull-down experiments and no loss in precipitation efficiency after cross-linking was observed. However, two of the antibodies could not precipitate the complex, probably due to hidden epitopes. Formaldehyde cross-linked complexes, precipitated from Jurkat cells or human platelets and analyzed by mass spectrometry, were found to be composed of integrin beta1, alpha4 and alpha6 or beta1, alpha6, alpha2, and alpha5, respectively.