Rami Hershkoviz

The cell-binding domain of intimin from enteropathogenic Escherichia coli binds to beta1 integrins

Bacteria interact with mammalian cells surface molecules, such as integrins, to colonize tissues and evade immunological detection. Herein, the ability of intimin, an outer membrane protein from enteropathogenic Escherichia coli, to bind β1 integrins was investigated. Solid-phase binding assays revealed binding of the carboxyl-terminal 280 amino acids of intimin (Int280) to α4β1 and α5β1 integrins. The binding required divalent ions (in particular, it was enhanced by Mn2+) and was inhibited by an RGD-containing peptide. Nonderivatized Int280, but not Int280CS (like Int280 but with Cys-937 replaced by Ser) blocked the binding of biotinylated Int280 to integrins. Int280 did not efficiently inhibit β1 integrin binding of invasin from Yersinia pseudotuberculosis. Both intimin and invasin, immobilized on plastic surfaces, mediated adherence of resting or phorbol 12-myristate 13-acetate-activated human CD4+ T cells, whereas fibronectin mediated the adherence of only activated T cells. T cell binding to intimin and invasin was integrin mediated because it was specifically blocked by an RGD-containing peptide and by antibodies directed against the integrin subunits β1, α4, and α5. These results demonstrate a specific integrin binding activity for intimin that is related to, but distinct from, that of invasin.

Human mast cells release metalloproteinase-9 on contact with activated T cells: juxtacrine regulation by TNF-alpha.

Mast cells, essential effector cells in allergic inflammation, have been found to be activated in T cell-mediated inflammatory processes in accordance with their residence in close physical proximity to T cells. We have recently reported that mast cells release granule-associated mediators and TNF-α upon direct contact with activated T cells. This data suggested an unrecognized activation pathway, where mast cells may be activated during T cell-mediated inflammation. Herein, we show that this cell-cell contact results in the release of matrix metalloproteinase (MMP)-9 and the MMP inhibitor tissue inhibitor of metalloproteinase 1 from HMC-1 human mast cells or from mature peripheral blood-derived human mast cells. The expression and release of these mediators, as well as of β-hexosaminidase and several cytokines, were also induced when mast cells were incubated with cell membranes isolated from activated, but not resting, T cells. Subcellular fractionation revealed that the mature form of MMP-9 cofractionated with histamine and tryptase, indicating its localization within the secretory granules. MMP-9 release was first detected at 6 h and peaked at 22 h of incubation with activated T cell membranes, while TNF-α release peaked after only 6 h. Anti-TNF-α mAb inhibited the T cell membrane-induced MMP-9 release, indicating a possible autocrine regulation of MMP release by mast cell TNF-α. This cascade of events, whereby mast cells are activated by T cells to release cytokines and MMP-9, which are known to be essential for leukocyte extravasation and recruitment to affected sites, points to an important immunoregulatory function of mast cells within the context of T cell-mediated inflammatory processes.

Specific inhibition of T‐cell adhesion to extracellular matrix and proinflammatory cytokine secretion by human recombinant galectin‐1

The migration of immune cells through the extracellular matrix (ECM) towards inflammatory sites is co‐ordinated by receptors recognizing ECM glycoproteins, chemokines and proinflammatory cytokines. In this context, galectins are secreted to the extracellular milieu, where they recognize poly‐N‐acetyllactosamine chains on major ECM glycoproteins, such as fibronectin and laminin. We investigated the possibility that galectin‐1 could modulate the adhesion of human T cells to ECM and ECM components. T cells were purified from human blood, activated with interleukin‐2 (IL‐2), labelled, and incubated further with intact immobilized ECM and ECM glycoproteins in the presence of increasing concentrations of human recombinant galectin‐1, or its more stable, related, C2‐S molecule obtained by site‐directed mutagenesis. The presence of galectin‐1 was shown to inhibit T‐cell adhesion to intact ECM, laminin and fibronectin, and to a lesser extent to collagen type IV, in a dose‐dependent manner. This effect was specifically blocked by anti‐galectin‐1 antibody and was dependent on the lectin’s carbohydrate‐binding properties. The inhibition of T‐cell adhesion by galectin‐1 correlates with the ability of this molecule to block the re‐organization of the activated cell’s actin cytoskeleton. Furthermore, tumour necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ) production was markedly reduced when IL‐2‐activated T cells were incubated with galectin‐1 or its mutant. This effect was prevented by β‐galactoside‐related sugars. The present study reveals an alternative inhibitory mechanism for explaining the suppressive properties of the galectin‐1 subfamily on inflammatory and autoimmune processes.

Combinatorial signals by inflammatory cytokines and chemokines mediate leukocyte interactions with extracellular matrix

On their extravasation from the vascular system into inflamed tissues, leukocytes must maneuver through a complex insoluble network of molecules termed the extracellular matrix (ECM). Leukocytes navigate toward their target sites by adhering to ECM glycoproteins and secreting degradative enzymes, while constantly orienting themselves in response to specific signals in their surroundings. Cytokines and chemokines are key biological mediators that provide such signals for cell navigation. Although the individual effects of various cytokines have been well characterized, it is becoming increasingly evident that the mixture of cytokines encountered in the ECM provides important combinatorial signals that influence cell behavior. Herein, we present an overview of previous and ongoing studies that have examined how leukocytes integrate signals from different combinations of cytokines that they encounter either simultaneously or sequentially within the ECM, to dynamically alter their navigational activities. For example, we describe our findings that tumor necrosis factor (TNF)‐α acts as an adhesion‐strengthening and stop signal for T cells migrating toward stromal cell‐derived factor‐1α, while transforming growth factor‐β down‐regulates TNF‐α‐induced matrix metalloproteinase‐9 secretion by monocytes. These findings indicate the importance of how one cytokine, such as TNF‐α, can transmit diverse signals to different subsets of leukocytes, depending on its combination with other cytokines, its concentration, and its time and sequence of exposure. The combinatorial effects of multiple cytokines thus affect leukocytes in a step‐by‐step manner, whereby cells react to cytokine signals in their immediate vicinity by altering their adhesiveness, directional movement, and remodeling of the ECM.

Intravenous immunoglobulin treatment of experimental T cell-mediated autoimmune disease. Upregulation of T cell proliferation and downregulation of tumor necrosis factor alpha secretion.

It has been reported previously that intravenous administration of normal human immunoglobulins (IVIg) to human patients can suppress the clinical signs of certain autoimmune diseases. However, the mechanism(s) by which normal Ig interferes with the various disorders and the scheduling of treatment have been poorly delineated. To study these questions, we examined IVIg treatment of two experimentally induced T cell autoimmune diseases in rats: experimental autoimmune encephalomyelitis (EAE) and adjuvant arthritis (AA). We now report that IVIg treatment (0.4 g/kg) inhibited the active induction of both EAE and AA, and that this treatment did not affect the acquisition of resistance to reinduction of EAE. The importance of the site of administration and schedule of treatment were studied in the AA model. Ig was effective when given intravenously, but not when administrated subcutaneously or intraperitoneally. IVIg treatment was effective when given daily from immunization to outbreak of disease; but it was also effective when given once at the time of immunization or once 2 wk after induction of AA, just at the clinical outbreak of disease. Administration of IVIg between immunization and outbreak of AA was less effective. Prevention of disease by IVIg occurred despite the presence of T cell reactivity to the specific antigens in the disease. In fact, IVIg administrated to naive rats activated T cell reactivity to some self-antigens. Nevertheless, IVIg treatment led to decreased production of the inflammatory cytokine TNF alpha. Thus, IVIg treatment may exert its therapeutic power not by inhibiting T cell recognition of self-antigens, but by inhibiting the biological consequences of T cell recognition.

Heat Shock Protein 60 Inhibits Th1-Mediated Hepatitis Model via Innate Regulation of Th1/Th2 Transcription Factors and Cytokines

Extracellular heat shock protein 60 (HSP60) has been considered a proinflammatory danger signal. Yet, HSP60 can also down-regulate experimental immune arthritis and diabetes models by specific inhibition of Th1-like responses. We now report that HSP60 in vitro differentially modulates the expression of Th1/Th2 transcription factors in human T cells: HSP60 down-regulates T-bet, NF-κB, and NFATp and up-regulates GATA-3, leading to decreased secretion of TNF-α and IFN-γ and enhanced secretion of IL-10. These effects depended on TLR2 signaling and could not be attributed to LPS or to other contaminants. In BALB/c mice, HSP60 in vivo inhibited the clinical, histological, and serological manifestations of Con A-induced hepatitis associated with up-regulated T cell expression of suppressor of cytokine signaling 3 and GATA-3 and down-regulated T-bet expression. These results provide a molecular explanation for the effects of HSP60 treatment on T cell inflammation via innate regulation of the inflammatory response.

Invariant chain induces B cell maturation in a process that is independent of its chaperonic activity

Early stages of B cell development take place in the bone marrow, resulting in formation of immature B cells, which migrate to the spleen for their final differentiation into mature cells. This final maturation step is essential for B cells to become responsive to antigens and to participate in the immune response. Previously, we showed that the MHC class II chaperone, invariant chain (Ii), controls the differentiation of B cells from the immature to the mature stage. In this study, by generating transgenic mice expressing truncated Ii lacking its luminal domain, we could dissect the chaperonin activity of Ii from its role in B cell maturation. We demonstrate in vivo that Ii N-terminal domain is directly involved in the maturation of B cells and is sufficient to promote B cell differentiation.

TNF-α Associated with Extracellular Matrix Fibronectin Provides a Stop Signal for Chemotactically Migrating T Cells

The migration of T cells into extravascular sites of inflammation is regulated by information derived from the molecular structure of the invaded tissue and from chemokine and cytokine gradients in the context of the extracellular matrix (ECM). Although recent studies have highlighted the role of particular chemoattractants in leukocyte migration, to date little is known about how specific combinations of contextual signals control the migration of leukocytes and their localization at sites of inflammation. Here we studied the interplay between a pleiotropic cytokine, TNF-α, and two prototypic chemoattractants, RANTES and stromal cell-derived factor-1α (SDF-1α), on human CD45RO+ T cells migrating within an ECM-like context. For this purpose, we used a newly constructed three-dimensional gel system designed to follow, in real time, the migration of individual leukocytes along chemotactic gradients in vitro. We found that TNF-α, which binds the ECM protein fibronectin and lacks adhesion- and migration-promoting effects of its own, can act as a proadhesive cytokine on T cells exposed to RANTES and SDF-1α. Furthermore, fibronectin-complexed TNF-α provided anchorage signals to the T cells as they moved directionally along chemoattractive gradients. This effect of TNF-α required an intact TNF-α receptor II subtype on the migrating T cells. The anchoring effect of TNF-α appears to be specific; IL-2, an integrin-activating proadhesive cytokine, does not transmit stoppage signals to T cell migration induced by RANTES. Thus, TNF-α present in the ECM at sites of inflammation may function to anchor T cells recruited to these sites by chemotactic signals.

Enzymatically Quiescent Heparanase Augments T Cell Interactions with VCAM-1 and Extracellular Matrix Components under Versatile Dynamic Contexts

During their migration into inflammatory sites, immune cells, such as T cells, secrete extracellular matrix (ECM)-degrading enzymes, such as heparanase, which, under mildly acidic conditions, degrade heparan sulfate proteoglycans (HSPG). We have previously shown that at pH 7.2, human placental heparanase loses its enzymatic activity, while retaining its ability to bind HSPG and promote T cell adhesion to unfractionated ECM. We now demonstrate that the 65-kDa recombinant human heparanase, which is devoid of enzymatic activity, but can still bind HSPG, captures T cells under shear flow conditions and mediates their rolling and arrest, in the absence or presence of stromal cell-derived factor 1α (SDF-1α; CXCL12), in an α4β1-VCAM-1-dependent manner. Furthermore, heparanase binds to and induces T cell adhesion to key ECM components, like fibronectin and hyaluronic acid, in β1 integrin- and CD44-specific manners, respectively, via the activation of the protein kinase C and phosphatidylinositol 3-kinase intracellular signaling machineries. Although the nature of the putative T cell heparanase-binding moiety is unknown, it appears that heparanase exerts its proadhesive activity by interacting with the T cells’ surface HSPG, because pretreatment of the cells with heparinase abolished their subsequent response to heparanase. Also, heparanase augmented the SDF-1α-triggered phosphorylation of Pyk-2 and extracellular signal-regulated kinase-2 implicated in integrin functioning. Moreover, heparanase, which had no chemotactic effect on T cells on its own, augmented the SDF-1α-induced T cell chemotaxis across fibronectin. These findings add another dimension to the known versatility of heparanase as a key regulator of T cell activities during inflammation, both in the context of the vasculature and at extravascular sites.

Cutting Edge: Anti-Inflammatory Properties of Low Levels of IFN-γ

Activation of naive T and B cells occurs only within the context of organized lymphoid tissue. Thus, the continuous recirculation of mature lymphocytes is crucial for the development of primary immune response to foreign Ags. We have previously shown that low levels of IFN-γ inhibit homing of B cells to the secondary lymphoid organs. In this study, we demonstrate that similarly low doses of IFN-γ down-regulate integrin-mediated adhesion and migration of naive T and Th2 cells, and have a profound effect on the in vivo homing of naive T cells to the lymph nodes. Moreover, we show that these low doses of IFN-γ have anti-inflammatory effects in an in vivo asthma model. Thus, in contrast to the proinflammatory effects of IFN-γ at relatively high concentrations, low dose IFN-γ appears to exert global suppressory effects on T cell trafficking and may have clinical application as an anti-inflammatory agent.