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.

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.

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.

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.

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.

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.

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.

Tetraspanins in intercellular adhesion of polarized epithelial cells: spatial and functional relationship to integrins and cadherins.

The subcellular distribution of tetraspanin molecules and their functional relationship with integrins in cell-cell adhesion was studied in detail in different polarized epithelial cell models. CD9, CD81 and CD151 tetraspanins were localized at lateral cell-cell contact sites in a similar distribution to E-cadherin. Interestingly, CD9 was partially localized at the apical microvillae of Madin-Darby canine kidney cells forming multimolecular complexes distinct from those found on the basolateral membrane, suggesting the coexistence of differential tetraspanin webs with different subcellular localization. We found that tetraspanin-associated beta1 integrins at cell-to-cell contacts were in a low-affinity conformational state, and that their localization at intercellular contacts was independent of cadherin expression and adhesion. Furthermore, integrin-tetraspanin complexes were functionally relevant in cell-cell adhesion in a cadherin-independent manner, without requiring a conformational change of the integrin moiety. Nevertheless, the integrin alpha3beta1 was ligand-binding competent and this binding did not disrupt association to tetraspanins. Moreover, Chinese hamster ovary cells treated with anti-tetraspanin mAbs or activatory anti-beta1 integrin mAbs were able to develop tubule-like structures. Together, these data support tetraspanin association as a new regulatory mechanism of integrin function and suggest a role for tetraspanins-integrin complexes in providing the cell with the spatial cues necessary for their proper polarization.

Crystal structure of the C-type lectin-like domain from the human hematopoietic cell receptor CD69.

CD69, one of the earliest specific antigens acquired during lymphoid activation, acts as a signal-transducing receptor involved in cellular activation events, including proliferation and induction of specific genes. CD69 belongs to a family of receptors that modulate the immune response and whose genes are clustered in the natural killer (NK) gene complex. The extracellular portion of these receptors represent a subfamily of C-type lectin-like domains (CTLDs), which are divergent from true C-type lectins and are referred to as NK-cell domains (NKDs). We have determined the three-dimensional structure of human CD69 NKD in two different crystal forms. CD69 NKD adopts the canonical CTLD fold but lacks the features involved in Ca(2+) and carbohydrate binding by C-type lectins. CD69 NKD dimerizes noncovalently, both in solution and in crystalline state. The dimer interface consists of a hydrophobic, loosely packed core, surrounded by polar interactions, including an interdomain beta sheet. The intersubunit core shows certain structural plasticity that may facilitate conformational rearrangements for binding to ligands. The surface equivalent to the binding site of other members of the CTLD superfamily reveals a hydrophobic patch surrounded by conserved charged residues that probably constitutes the CD69 ligand-binding site.

Down-regulation of L-selectin expression in neutrophils by nonsteroidal anti-inflammatory drugs: role of intracellular ATP concentration.

L-selectin is an adhesion molecule that plays an essential role in the early events of the inflammatory response. Our group has recently described that several nonsteroidal anti-inflammatory drugs (NSAIDs) are able to induce both in vivo and in vitro the shedding of L-selectin in neutrophils through an unknown mechanism. In this work, we have studied potential mechanisms involved in the shedding of L-selectin induced by NSAIDs. This effect of NSAIDs did not involve any detectable intracellular calcium flux. Pretreatment of neutrophils either with Ro 31-8220 and H7, 2 specific inhibitors of protein kinase C (PKC), or with inhibitors of protein tyrosine kinases such as tyrphostin A25 or herbimycin A did not prevent the NSAID-mediated L-selectin shedding. However, the KD-IX-73-4, an inhibitor of L-selectin proteolysis was able to block the effect of NSAIDs on L-selectin expression. Remarkably, NSAIDs caused a variable reduction in the neutrophil intracellular ATP concentration that highly correlated with the differential ability of NSAIDs to trigger L-selectin shedding (r = 0.8, P <.01). In agreement with this finding, azide plus 2-deoxy-D-glucose, 2 metabolic blockers, also induced a rapid L-selectin shedding (65% +/- 8%) without affecting the neutrophil viability, activation, or expression level of other surface molecules with soluble isoforms such as CD16 and CD59. These data indicate that the maintenance of L-selectin on the neutrophil surface requires energy consumption, which suggests that L-selectin is shed in neutrophils by default. Interestingly, NSAIDs seem to cause the shedding of L-selectin, at least in part, through the reduction of the intracellular ATP concentration.

Functional analysis of ligand-binding and signal transduction domains of CD69 and CD23 C-type lectin leukocyte receptors.

CD69 and CD23 are leukocyte receptors with distinctive pattern of cell expression and functional features that belong to different C-type lectin receptor subfamilies. To assess the functional equivalence of different domains of these structurally related proteins, a series of CD69/CD23 chimeras exchanging the carbohydrate recognition domain, the neck region, and the transmembrane and cytoplasmic domains were generated. Biochemical analysis revealed the importance of the neck region (Cys68) in the dimerization of CD69. Functional analysis of these chimeras in RBL-2H3 mast cells and Jurkat T cell lines showed the interchangeability of structural domains of both proteins regarding Ca2+ fluxes, serotonin release, and TNF-alpha synthesis. The type of the signal transduced mainly relied on the cytoplasmic domain and was independent of receptor oligomerization. The cytoplasmic domain of CD69 transduced a Ca2+-mediated signaling that was dependent on the extracellular uptake of Ca2+. Furthermore, a significant production of TNF-alpha was induced through the cytoplasmic domain of CD69 in RBL-2H3 cells, which was additive to that promoted via FcepsilonRI, thus suggesting a role for CD69 in the late phase of reactions mediated by mast cells. Our results provide new important data on the functional equivalence of homologous domains of these two leukocyte receptors.

Tetraspanins are localized at motility-related structures and involved in normal human keratinocyte wound healing migration.

We have described previously that beta1 integrins, which mediate keratinocyte cell adhesion and migration, are in ligand-occupied conformation at the basal surface but not at the lateral and apical surfaces of keratinocytes. This led us to study the cellular localization and function of tetraspanin molecules, which have been postulated to modulate integrin activity. We found that CD9 and CD81 are highly expressed by keratinocytes clearly delineating filopodia at lateral and apical surfaces. CD63 and CD151 are largely expressed in the intracellular compartment, although some membrane expression is observed. We found accumulation of CD9, CD81, and CD151 together with alpha3 and beta1 integrins at intercellular junctions. In low calcium medium, this intercellular space is crossed by a zipper of filopodia enriched in alpha3beta1 and tetraspanin proteins. Interestingly, the expression of CD9, CD81, and beta1 and alpha3 integrins was detected in the footprints and rippings of motile keratinocytes, suggesting their role in both adhesion to extracellular matrix and keratinocyte motility. beta1 integrins were only partially activated in the rips, whereas cytoskeleton-linking proteins such as talin were completely absent. On the other hand, antitetraspanin antibodies did not stain focal adhesions, which contain talin. The involvement of tetraspanins in keratinocyte motility was assessed in a wound healing migration assay. Inhibition of cell migration was observed with antibodies to CD9, CD81, beta1, and alpha3, and, to a lesser extent, to CD151. Together these results indicate that tetraspanin-integrin complexes might be involved in transient adhesion and integrin recycling during keratinocyte migration, as well as in intercellular recognition.

The tyrosine kinase PYK-2/RAFTK regulates natural killer (NK) cell cytotoxic response, and is translocated and activated upon specific target cell recognition and killing.

The compartmentalization of plasma membrane proteins has a key role in regulation of lymphocyte activation and development of immunity. We found that the proline-rich tyrosine kinase-2 (PYK-2/RAFTK) colocalized with the microtubule-organizing center (MTOC) at the trailing edge of migrating natural killer (NK) cells. When polyclonal NK cells bound to K562 targets, PYK-2 translocated to the area of NK-target cell interaction. The specificity of this process was assessed with NK cell clones bearing activatory or inhibitory forms of CD94/NKG2. The translocation of PYK-2, MTOC, and paxillin to the area of NK-target cell contact was regulated upon specific recognition of target cells through NK cell receptors, controlling target cell killing. Furthermore, parallel in vitro kinase assays showed that PYK-2 was activated in response to signals that specifically triggered its translocation and NK cell mediated cytotoxicity. The overexpression of both the wt and a dominant-negative mutant of PYK-2, but not ZAP-70 wt, prevented the specific translocation of the MTOC and paxillin, and blocked the cytotoxic response of NK cells. Our data indicate that subcellular compartmentalization of PYK-2 correlates with effective signal transduction. Furthermore, they also suggest an important role for PYK-2 on the assembly of the signaling complexes that regulate the cytotoxic response.

Phenotypic and functional characteristics of hematopoietic cell lineages in CD69-deficient mice.

AIM/CD69 is the earliest leukocyte activation antigen and is expressed mainly by activated T, B, and natural killer (NK) cells. It is also constitutively expressed by platelets, by bone marrow myeloid precursors, and by small subsets of resident lymphocytes in the secondary lymphoid tissues. The engagement of CD69 by specific antibodies induces intracellular signals, including Ca(++) flux, cytokine synthesis, and cell proliferation. To investigate the physiological relevance of CD69, we generated mice deficient in CD69 (CD69-/-) by gene targeting in embryonic stem cells. CD69 (-/-) mice showed largely normal hematopoietic cell development and normal T-cell subpopulations in thymus and periphery. Furthermore, studies of negative- and positive-thymocyte selection using a T-cell receptor transgenic model demonstrated that these processes were not altered in CD69 (-/-) mice. In addition, natural killer and cytotoxic T lymphocyte cells from CD69-deficient mice displayed cytotoxic activity similar to that of wild-type mice. Interestingly, B-cell development was affected in the absence of CD69. The B220(hi)IgM(neg) bone marrow pre-B cell compartment was augmented in CD69 (-/-) mice. In addition, the absence of CD69 led to a slight increase in immunoglobulin (Ig) G2a and IgM responses to immunization with T-dependent and T-independent antigens. Nevertheless, CD69-deficient lymphocytes had a normal proliferative response to different T-cell and B-cell stimuli. Together, these observations indicate that CD69 plays a role in B-cell development and suggest that the putative stimulatory activity of this molecule on bone marrow-derived cells may be replaced in vivo by other signal transducing receptors.