The chaperonin CCT controls T cell receptor-driven 3D configuration of centrioles.

T lymphocyte activation requires the formation of immune synapses (IS) with antigen-presenting cells. The dynamics of membrane receptors, signaling scaffolds, microfilaments, and microtubules at the IS determine the potency of T cell activation and subsequent immune response. Here, we show that the cytosolic chaperonin CCT (chaperonin-containing TCP1) controls the changes in reciprocal orientation of the centrioles and polarization of the tubulin dynamics induced by T cell receptor in T lymphocytes forming an IS. CCT also controls the mitochondrial ultrastructure and the metabolic status of T cells, regulating the de novo synthesis of tubulin as well as posttranslational modifications (poly-glutamylation, acetylation, Δ1 and Δ2) of αß-tubulin heterodimers, fine-tuning tubulin dynamics. These changes ultimately determine the function and organization of the centrioles, as shown by three-dimensional reconstruction of resting and stimulated primary T cells using cryo-soft x-ray tomography. Through this mechanism, CCT governs T cell activation and polarity.

Mechanisms of polarized cell-cell communication of T lymphocytes.

Cell-cell communication comprises a variety of molecular mechanisms that immune cells use to respond appropriately to diverse pathogenic stimuli. T lymphocytes polarize in response to different stimuli, such as cytokines, adhesion to specific ligands and cognate antigens presented in the context of MHC. Polarization takes different shapes, from migratory front-back polarization to the formation of immune synapses (IS). The formation of IS between a T cell and an antigen-presenting cell involves early events of receptor-ligand interaction leading to the reorganization of the plasma membrane and the cytoskeleton to orchestrate vesicular and endosomal traffic and directed secretion of several types of mediators, including cytokines and nanovesicles. Cell polarization involves the repositioning of many subcellular organelles, including the endosomal compartment, which becomes an effective platform for the shuttling of molecules as vesicular cargoes that lately will be secreted to transfer information to antigen-presenting cells. Overall, the polarized interaction between a T cell and APC modifies the recipient cell in different ways that are likely lineage-dependent, e.g. dendritic cells, B cells or even other T cells. In this review, we will discuss the mechanisms that mediate the polarization of different membrane receptors, cytoskeletal components and organelles in T cells in a variety of immune contexts.

Immune cells from patients with psoriasis are defective in inducing indoleamine 2,3-dioxygenase expression in response to inflammatory stimuli.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO) is an inducible enzyme that suppresses the immune response. The role of IDO as a negative regulator of inflammatory responses has been documented in several experimental autoimmune diseases. OBJECTIVES: To explore the regulation of IDO by immune cells in psoriasis and its relation with disease severity. METHODS: The expression and activity of IDO were assessed by reverse-transcriptase polymerase chain reaction, flow cytometry and high-performance liquid chromatography in peripheral blood of patients with moderate-to-severe plaque-type psoriasis. The ability of immune cells to express IDO in response to inflammatory stimuli was studied. The functional role of IDO expression was evaluated in a regulatory T cell (Treg) differentiation assay, using cocultures of immature monocyte-derived dendritic cells with autologous peripheral CD4+ T cells. RESULTS: Analysis of the kynurenine-to-tryptophan ratio in serum samples indicated higher IDO activity in patients with psoriasis than in healthy controls. However, correlation studies showed lower IDO activity in those patients with higher Psoriasis Area and Severity Index (PASI). Although myeloid dendritic cells from patients with psoriasis expressed higher levels of IDO than those from healthy controls, these cells did not upregulate IDO in response to a combination of tumour necrosis factor-α, interleukin (IL)-1ß and IL-6 cytokines. The defective expression of IDO correlated with PASI. Immature monocyte-derived dendritic cells from patients with psoriasis also expressed low levels of IDO and induced CD4+ Treg differentiation poorly. CONCLUSIONS: Immune cells from patients with psoriasis have a defect in upregulating IDO in response to inflammation associated with the severity of psoriasis.

CD81 controls sustained T cell activation signaling and defines the maturation stages of cognate immunological synapses.

In this study, we investigated the dynamics of the molecular interactions of tetraspanin CD81 in T lymphocytes, and we show that CD81 controls the organization of the immune synapse (IS) and T cell activation. Using quantitative microscopy, including fluorescence recovery after photobleaching (FRAP), phasor fluorescence lifetime imaging microscopy-Föster resonance energy transfer (phasorFLIM-FRET), and total internal reflection fluorescence microscopy (TIRFM), we demonstrate that CD81 interacts with ICAM-1 and CD3 during conjugation between T cells and antigen-presenting cells (APCs). CD81 and ICAM-1 exhibit distinct mobilities in central and peripheral areas of early and late T cell-APC contacts. Moreover, CD81-ICAM-1 and CD81-CD3 dynamic interactions increase over the time course of IS formation, as these molecules redistribute throughout the contact area. Therefore, CD81 associations unexpectedly define novel sequential steps of IS maturation. Our results indicate that CD81 controls the temporal progression of the IS and the permanence of CD3 in the membrane contact area, contributing to sustained T cell receptor (TCR)-CD3-mediated signaling. Accordingly, we find that CD81 is required for proper T cell activation, regulating CD3ζ, ZAP-70, LAT, and extracellular signal-regulated kinase (ERK) phosphorylation; CD69 surface expression; and interleukin-2 (IL-2) secretion. Our data demonstrate the important role of CD81 in the molecular organization and dynamics of the IS architecture that sets the signaling threshold in T cell activation.

Reduced expression of galectin-1 and galectin-9 by leucocytes in asthma patients.

Accumulating evidence shows that galectins play roles in the initiation and resolution phases of inflammatory responses by promoting anti- or proinflammatory effects. This study investigated the presence of three members of the galectin family (galectin-1, -3 and -9) in induced sputum samples of asthma patients, as well as their possible implication in the immunopathogenesis of human asthma. Levels of interleukin (IL)-5, IL-13, and galectins were determined in leucocytes isolated from induced sputum samples by reverse transcription-polymerase chain reaction (RT-PCR) immunofluorescence and flow cytometry. High levels of IL-5 and IL-13 mRNA were detected in sputum cells from asthma patients. In parallel, immunoregulatory proteins galectin-1 and galectin-9 showed a reduced expression on macrophages from sputum samples compared with cells from healthy donors. In-vitro immunoassays showed that galectin-1 and galectin-9, but not galectin-3, are able to induce the production of IL-10 by peripheral blood mononuclear cells from healthy donors. These findings indicate that macrophages from sputum samples of asthma patients express low levels of galectin-1 and galectin-9, favouring the exacerbated immune response observed in this disease.

Inhibition of tumour necrosis factor and IL-17 production by leflunomide involves the JAK/STAT pathway.

OBJECTIVE: To study the effects of different disease-modifying antirheumatic drugs (DMARD) on different events mediated by IL-15-activated lymphocytes. METHODS: Peripheral blood lymphocytes (PBL) were isolated from healthy donors and activated with IL-15 after exposure to different DMARD: leflunomide, cyclosporin A, methotrexate, mycophenolic acid, FK-506, sulphasalazine and sodium aurothiomalate. The expression of different surface molecules on the PBL was then determined by flow cytometry. Cells were also co-cultured with the monocytic cell line THP-1 and the tumour necrosis factor (TNF) concentration in the supernatant was measured after 24 h using an immunoenzyme assay. The effect of the aforementioned drugs on IL-17 production by IL-15-activated PBL was also studied. RESULTS: Treatment of PBL with leflunomide, cyclosporin A and FK-506 inhibited the IL-15-induced expression of both CD54 and CD69 by PBL, as well as TNF production in co-cultures of activated PBL and THP-1 cells. The downregulation of CD54 and CD69 in PBL was correlated with the inhibition of TNF production. Likewise, leflunomide, cyclosporin A and FK-506 all inhibited IL-17 production in IL-15-activated PBL. Interestingly, the effect of leflunomide was not reverted by the presence of uridine in the medium. In addition, leflunomide inhibited the phosphorylation of STAT6 in vitro. CONCLUSION: Inhibition of the JAK/STAT pathway may represent an additional effect of leflunomide in chronic polyarthritis because it impairs certain events that control proinflammatory TNF and IL-17 cytokine production.

Embryonic implantation and leukocyte transendothelial migration: different processes with similar players?

A clear parallelism between the different steps in human embryo-endometrial apposition/adhesion/invasion and leukocyte-endothelium rolling/adhesion/extravasation can be established. During human implantation and leukocyte trafficking, a first wave of soluble mediators regulates the expression and functional activity of adhesion molecules such as L-selectin and integrins, which mediate both processes. Apical surfaces of human endometrial epithelium and endothelium are key elements for the initiation of molecular interactions to capture the blastocyst or leukocyte, respectively. Subsequently, the blastocyst and the leukocyte migrate through the epithelium and endothelium toward their final destination, the endometrial stroma, to initiate placentation or the inflammatory foci as part of the immune response. Similarities between the intermediate molecular mechanisms of these two physiologically unrelated processes are discussed.

[Intercellular adhesion molecules and chemotaxic factors in the pathogenesis of multiple sclerosis]. / Moléculas de adhesión intercelular y factores quimiotácticos en la patogenia de la esclerosis múltiple.

INTRODUCTION: Multiple sclerosis (MS) is an inflammatory immune disorder of the central nervous system characterized by the destruction of myelin sheaths and the cells which make them, the oligodendrocytes. Experimental allergic encephalitis (EAE) is an autoimmune condition mainly induced by the myelin basic protein (MBP) that is a very useful model for the study of demyelinating inflammatory diseases, particularly MS. METHOD: Cellular adhesion molecules are a wide group of membrane receptors which mediate adhesion processes, both cell to cell and between cells and the extracellular matrix. These molecules play an essential role in inflammatory phenomena, including EAE/MS. Integrins of the b1 subfamily (mainly a4b1), as well as leukocyte integrins and adhesion receptors of the immunoglobulin superfamily (ICAM 1, VCAM 1) are the main molecules involved. Chemokines also have an important role in MS, since they are able to attract and activate leukocytes, essential phenomena in the inflammatory reaction. An increased expression of chemokines CC or beta (e.g., RANTES, MIP 1a and b, MCP 1, etc.) has been found in EAE/MS, and it is very likely that they are involved in the migration of lymphocytes and monocytes towards the MS inflammatory lesions. CONCLUSION: The pharmacological blockade of adhesion molecules and chemokines is a promising and novel therapeutic approach in MS.

Regulatory role of tetraspanin CD9 in tumor-endothelial cell interaction during transendothelial invasion of melanoma cells.

Heterotypic interaction among tumor cells (TCs) and endothelial cells (ECs) may play a critical role during the vascular dissemination of neoplastic cells and during pathologic angiogenesis in tumors. To identify molecules involved in these processes, the distribution of vascular junctional proteins was first studied by immunofluorescence at sites of heterologous intercellular contact using TC-EC mosaic monolayers grown on 2-dimensional collagen. Several members of the tetraspanin superfamily, including CD9, CD81, and CD151, were found to localize at the TC-EC contact area. The localization of tetraspanins to the TC-EC heterologous contact area was also observed during the active transmigration of TCs across EC monolayers grown onto 3-dimensional collagen matrices. Dynamic studies by time-lapse immunofluorescence confocal microscopy showed an active redistribution of endothelial CD9 to points of melanoma insertion. Anti-CD9 monoclonal antibodies were found to specifically inhibit the transendothelial migration of melanoma cells; the inhibitory effect was likely caused by a strengthening of CD9-mediated heterotypic interactions of TCs to the EC monolayer. These data support a novel mechanism of tetraspanin-mediated regulation of TC transcellular migration independent of TC motility and growth during metastasis and a role for these molecules in the formation of TC-EC mosaic monolayers during tumor angiogenesis.

Thyrocytes from autoimmune thyroid disorders produce the chemokines IP-10 and Mig and attract CXCR3+ lymphocytes.

To better understand the selective migration of lymphocytes in autoimmune thyroid disorders (AITDs), we analyzed thyroid samples and demonstrated an enhanced expression of the chemokines interferon (IFN)-inducible protein (Ip)-10 and regulated on activation normal T lymphocyte expressed and secreted (RANTES) in thyroids from AITD patients. Ip-10 and monokine induced by IFN-gamma (Mig) were expressed in vivo in thyroid follicular cells (TFCs) from AITD thyroids. Interestingly, Ip-10 mRNA, although not basally detected in cultured TFCs, was strongly induced by IFN-gamma and synergistically increased by TNF-alpha addition. Furthermore, high levels of Ip-10 protein were detected in the supernatants of IFN-gamma-stimulated TFCs. Likewise, Mig protein was strongly induced in TFCs by the same stimuli as Ip-10. Unlike Ip-10 and Mig, the expression of RANTES was induced mainly by TNF-alpha. In addition, intrathyroidal lymphocytes from AITD patients showed higher expression of CXCR3, CCR2, and CCR5 chemokine receptors than autologous peripheral blood lymphocytes. T lymphoblasts expressing CXCR3 showed an increased migration to supernatants from stimulated TFCs, which was abolished by specific antibodies to the chemokines Ip-10 and Mig, as well as to their receptor CXCR3. Taken together, these data suggest a potential role of TFCs, through the production of the chemokines Ip-10, Mig and RANTES, in regulating the recruitment of specific subsets of activated lymphocytes in AITDs.

Rho and Rho-associated kinase modulate the tyrosine kinase PYK2 in T-cells through regulation of the activity of the integrin LFA-1.

We have examined the role of the small GTPase Rho and its downstream effector, the Rho-associated kinase (ROCK), in the control of the adhesive and signaling function of the lymphocyte function-associated antigen-1 (LFA-1) integrin in human T-lymphocytes. Inhibition of Rho (either by treatment with C3-exoenzyme or transfection with a dominant-negative form of Rho (N19Rho)) or ROCK (by treatment with Y-27632) results in the following: (a) partial disorganization and aggregation of cortical filamentous actin (F-actin); (b) induction of LFA-1-mediated cellular adhesion to the LFA-1 ligand intercellular adhesion molecule-1 (ICAM-1) through a mechanism involving clustering of LFA-1 molecules, rather than alterations in the level of expression or in the affinity state of this integrin; and (c) induction of cellular polarization and activation of the tyrosine kinase PYK2. Transfection of T-cells with a constitutively active form of Rho (V14Rho) blocks the clustering of LFA-1 on the membrane and the LFA-1-mediated activation of PYK2. Importantly, the activation of PYK2 caused by inhibition of Rho or ROCK takes place only when the T-cells are plated onto ICAM-1 but not when they are either prevented from interacting with ICAM-1 with anti-LFA-1 blocking antibodies or when they are plated on the nonspecific poly-l-lysine substrate. These results indicate that the small GTPase Rho regulates the tyrosine kinase PYK2 in T-cells through the F-actin-mediated control of the activity of the integrin LFA-1. These findings represent a novel paradigm for the regulation of the activity of a cytoplasmic tyrosine kinase by the small GTPase Rho.

Tetraspanins and intercellular interactions.

The superfamily of tetraspanins comprises a group of polypeptides with four transmembrane domains that form large supramolecular structures in the plasma membrane through their associations to multiple integral membrane proteins. They are involved in homo- and heterotypic intercellular interactions in different processes such as hematopoiesis, lymphocyte activation, cancer metastasis, and fertilization. Intercellularly located tetraspanins regulate the juxtacrine activity of growth factors, cell fusion, and myelin formation. On the other hand, in motile cells they relocalize from cell-cell junctions to actin-based structures such as filopodia or growth cones and regulate cell motility in wound healing and angiogenesis processes.

Membrane type 1-matrix metalloproteinase is activated during migration of human endothelial cells and modulates endothelial motility and matrix remodeling.

Matrix metalloproteinases are thought to play an important role in endothelial cell migration and matrix remodeling. We have used an in vitro wound healing migration model and newly generated anti-membrane type 1-matrix metalloproteinase (MT1-MMP) monoclonal antibodies (mAbs) to characterize the role of MT1-MMP during this process. First, the expression and shedding of MT1-MMP are up-regulated upon induction of migration in endothelial cells, as demonstrated by flow cytometry and Western blot analysis. Furthermore, MT1-MMP is concentrated at discrete areas in migrating endothelial cells, in contrast to the diffuse pattern observed in confluent cells. Interestingly, migration of endothelial cells results in the stimulation of MT1-MMP activity, as shown by its ability to process pro-MMP-2 and to degrade fibrinogen assessed by zymography. Moreover, MT1-MMP-mediated gelatin degradation is enriched at migration sites. mAbs generated against the MT1-MMP catalytic domain are shown to inhibit MT1-MMP enzymatic activity and to impair both phorbol 12-myristate 13-acetate-induced endothelial migration and invasion of collagen and fibrin gels. Furthermore, a reduction in the formation of capillary tubes in Matrigel is also observed when endothelial cells are pretreated with the blocking anti-MT1-MMP mAbs. Altogether, these data demonstrate that MT1-MMP plays an important role during endothelial cell migration, and its activity can modulate endothelial migration, invasion, and formation of capillary tubes during the angiogenic response.

The hepatitis B virus HBx protein induces adherens junction disruption in a src-dependent manner.

Chronic hepatitis B virus infection is strongly associated with the development of hepatocellular carcinoma (HCC). Epithelial tumors are frequently characterized by loss of cadherin expression or function. Cadherin-dependent adhesion prevents the acquisition of a migratory and invasive phenotype, and loss of its function is itself enough for the progression from adenoma to carcinoma. The HBx protein of hepatitis B virus is thought to contribute to the development of the carcinoma, however, its role in the oncogenic and metastatic processes is far from being fully understood. We report herein the ability of HBx to disrupt intercellular adhesion in three different cell lines stably transfected with an inducible HBx expression vector. The linkage between the actin cytoskeleton and cadherin complex, which is essential for its function, is disrupted in the presence of HBx, as indicated by detergent solubility and immunoprecipitation experiments. In addition, beta-catenin was tyrosine phosphorylated in HBx-expressing cells. Inhibition of the src family of tyrosine kinases resulted in the prevention of the disruption of adherens junctions. These results suggest that HBx is able to disrupt intercellular adhesion in a src-dependent manner, and provide a novel mechanism by which HBx may contribute to the development of HCC.

The hepatitis B virus X protein (HBx) induces a migratory phenotype in a CD44-dependent manner: possible role of HBx in invasion and metastasis.

The hepatitis B virus X protein (HBx) of the hepatitis B virus (HBV) has been involved in the development of hepatocellular carcinoma (HCC). However, its possible contribution to the metastatic spreading of liver tumors has not been explored so far. We report here the ability of HBx to enhance cell motility, both alone and in synergy with growth factors, and to induce a migratory phenotype in transformed cells. HBx altered the cellular morphology by inducing the formation of pseudopodial protrusions and cytoskeletal rearrangements, which was accompanied by the polarization of cell-surface adhesion molecules, including the hyaluronan (HA) receptor, CD44. Furthermore, HBx induced the redistribution to the pseudopodial tips of F-actin-binding proteins of the ezrin/radixin/moesin (ERM) family in a Rho- and Rac-dependent manner and increased the association of CD44 with moesin. The migration of HBx-bearing cells in response to HA and growth factors was impaired by a blocking anti-CD44 monoclonal antibody (mAb), suggesting that the HBx-induced cell motility is partially mediated by CD44. Interestingly, HBx-bearing cells showed increased HA-interaction efficiency as assessed under laminar flow conditions, which was the result, at least in part, of an enhanced binding affinity of CD44. HBx may therefore contribute to the acquisition of metastatic properties by modifying the migratory behavior of transformed hepatocytes and by increasing their ability to bind HA in the outer margin of the tumors or in secondary target organs.

Drugs, inflammation and cell adhesion receptors.

The cell adhesion receptors that participate in the extravasation and migration of leucocytes towards inflammatory foci mainly include the selectins and different members of the integrin and immunoglobulin superfamilies. These adhesion receptors mediate the sequential steps of leucocyte-endothelial cell interaction and, together with chemoattractant molecules (e.g., chemokines), direct the influx of inflammatory cells and define the characteristics of the cell infiltrate. Many different drugs, including non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, rheumatoid arthritis disease-modifying agents and phosphodiesterase inhibitors, interfere with the expression and/or function of cell adhesion receptors and this effect accounts for, at least in part, their anti-inflammatory activity. In recent years, novel approaches for the modulation of the cell membrane receptors involved in inflammation have been active areas in pharmaceutical research. Upgraded synthetic blocking compounds, chimeric monoclonal antibodies or improved antisense oligonucleotides represent important advances in this field. The proper development of these novel approaches, as well as other alternative strategies, will allow a better and more specific pharmacological modulation of the inflammatory phenomenon.

Effect of the hepatitis B virus HBx protein on integrin-mediated adhesion to and migration on extracellular matrix.

BACKGROUND/AIMS: The hepatitis B virus HBx protein is associated with the development of hepatocellular carcinoma (HCC). However, its possible contribution to tumor spreading has not been explored. The migration of tumor cells through the extracellular matrix (ECM) represents a crucial step in tumor metastasis. Our aim was to study the effect of HBx on the integrin-mediated cell-ECM interaction, and its possible consequences for cell migration. METHODS: Cell-ECM interaction was evaluated by static adhesion experiments, using blocking and stimulating anti-beta1 integrin mAbs. ECM receptor expression was analyzed by flow cytometry. The cellular distribution of the activated beta1 integrin subunit was determined by immunofluorescence analysis, and cell motility was determined by wound-healing assays. RESULTS: HBx-bearing cells showed decreased adhesion to fibronectin, which correlated with a decreased expression of the alpha5 integrin subunit. The activated beta1 subunit was redistributed to the tips of pseudopodial protrusions of HBx-bearing cells, whereas it was evenly localized in the control cells. HBx-induced cell migration was abrogated by irreversible stimulation of beta1 integrins. CONCLUSIONS: These results suggest that HBx might play a role in tumor spreading by modulating the adhesion-deadhesion balance of the cells in the primary tumor site and favoring integrin-mediated cell migration.

CXCR3 chemokine receptor distribution in normal and inflamed tissues: expression on activated lymphocytes, endothelial cells, and dendritic cells.

Using new human CXCR3 chemokine receptor-specific monoclonal antibodies, we studied human CXCR3 tissue distribution in lymphoid and nonlymphoid organs, as well as in inflammatory conditions, including rheumatoid arthritis, Hashimoto's thyroiditis, and dermal vasculitis. CXCR3 was expressed by certain dendritic cell subsets, specifically myeloid-derived CD11c positive cells, not only in those present in normal lymphoid organs, but also in germinal centers generated in inflammatory conditions. CXCR3 expression was also detected in some lymphocyte subsets such as intraepithelial lymphocytes of secondary lymphoid organs and infiltrating lymphocytes in inflammatory conditions. In addition, CXCR3 was constitutively expressed by endothelial cells (EC) of vessels of medium and large caliber but not in small vessels from different organs. Finally, enhanced CXCR3 expression was found in EC and in infiltrating lymphocytes with an activated phenotype in inflammatory diseases. The CXCR3 chemokine receptor may play a role in the regulation of leukocyte migration to inflammatory sites.