RESUMEN
The inflammatory response is a self-limiting process which involves the sequential activation of signaling pathways leading to the production of both pro- and anti-inflammatory mediators. Galectin-1 (Gal-1), an endogenous lectin found in peripheral lymphoid organs and inflammatory sites, elicits a broad spectrum of biological functions predominantly by acting as a potent anti-inflammatory factor and as a suppressive agent for T-cell responses. However, the molecular pathways underlying Gal-1 expression and function remain poorly understood. Here we identified a regulatory loop linking Gal-1 expression and function to NF-κB activation. NF-κB-activating stimuli increased Gal-1 expression on T cells, an effect which could be selectively prevented by inhibitors of NF-κB signaling. Accordingly, transient transfection of the p65 subunit of NF-κB was sufficient to induce high Gal-1 expression. Using in silico studies and chromatin immunoprecipitation analysis we have identified a functional NF-κB binding site within the first intron of the LGALS1 gene. In addition, our results show that exogenous Gal-1 can attenuate NF-κB activation, as shown by inhibition of IκB-α degradation induced by pro-inflammatory stimuli, higher cytoplasmic retention of p65, lower NF-κB DNA binding activity and impaired transcriptional activation of target genes. The present study suggest a novel regulatory loop by which NF-κB induces expression of Gal-1, which in turn may lead to negative control of NF-κB signaling.
Asunto(s)
Galectina 1/biosíntesis , Regulación de la Expresión Génica/inmunología , FN-kappa B/metabolismo , Transducción de Señal/inmunología , Sitios de Unión , Western Blotting , Células Cultivadas , Inmunoprecipitación de Cromatina , Ensayo de Cambio de Movilidad Electroforética , Retroalimentación Fisiológica/fisiología , Galectina 1/genética , Galectina 1/inmunología , Expresión Génica , Humanos , Microscopía Confocal , FN-kappa B/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunología , Linfocitos T/metabolismo , TransfecciónRESUMEN
Most tumors grow in immunocompetent hosts despite expressing NKG2D ligands (NKG2DLs) such as the MHC class I chain-related genes A and B (MICA/B). However, their participation in tumor cell evasion is still not completely understood. Here we demonstrate that several human melanomas (cell lines and freshly isolated metastases) do not express MICA on the cell surface but have intracellular deposits of this NKG2DL. Susceptibility to NK cell-mediated cytotoxicity correlated with the ratio of NKG2DLs to HLA class I molecules but not with the amounts of MICA on the cell surface of tumor cells. Transfection-mediated overexpression of MICA restored cell surface expression and resulted in an increased in vitro cytotoxicity and IFN-gamma secretion by human NK cells. In xenografted nude mice, these melanomas exhibited a delayed growth and extensive in vivo apoptosis. Retardation of tumor growth was due to NK cell-mediated antitumor activity against MICA-transfected tumors, given that this effect was not observed in NK cell-depleted mice. Also, mouse NK cells killed MICA-overexpressing melanomas in vitro. A mechanistic analysis revealed the retention of MICA in the endoplasmic reticulum, an effect that was associated with accumulation of endoH-sensitive (immature) forms of MICA, retrograde transport to the cytoplasm, and degradation by the proteasome. Our study identifies a novel strategy developed by melanoma cells to evade NK cell-mediated immune surveillance based on the intracellular sequestration of immature forms of MICA in the endoplasmic reticulum. Furthermore, this tumor immune escape strategy can be overcome by gene therapy approaches aimed at overexpressing MICA on tumor cells.
Asunto(s)
Citotoxicidad Inmunológica/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Péptidos y Proteínas de Señalización Intercelular/inmunología , Células Asesinas Naturales/inmunología , Melanoma/inmunología , Línea Celular Tumoral , Proliferación Celular , Proteínas Ligadas a GPI , Humanos , Melanoma/patología , Melanoma/ultraestructura , Microscopía Inmunoelectrónica , Sensibilidad y EspecificidadRESUMEN
MICA is a stress-regulated molecule recognized by the NK cell-activating receptor NKG2D. Previously, we demonstrated that MICA is induced on activated T cells but regulation by mitogenic cytokines and its biological consequences remain unexplored. Here, we show that IL-2, IL-4, and IL-15 but not TNF-alpha or IFN-alpha induced MICA expression in T lymphocytes present in peripheral blood mononuclear cells (PBMCs), as assessed by Western blot. IL-2 effect involved Jak3/STAT5, p38 MAPK, p70(56) kinase, Lck/fyn kinases, and NF-kappaB. MICA expression was also observed in Th1 and Th2 cells. However, surface expression was not detected. T lymphocytes present in PBMCs and isolated CD4+ T lymphocytes stimulated with phorbol-12-myristate-13-acetate and ionomycin also induced MICA expression as assessed by Western blot, but only low levels were expressed at the cell surface. Activated but not resting CD4+ T lymphocytes were lysed by IL-15- or IL-2-stimulated NK cells, and susceptibility was increased when HLA class I molecules were blocked. Also, cytokine-stimulated NK cells produced more IFN-gamma after culture with activated CD4+ T lymphocytes. However, the participation of MICA in these responses, if any, was marginal. Confocal microscopy revealed that MICA is retained mostly inside activated CD4+ T cells. Our results suggest that low surface expression of MICA on activated CD4+ T lymphocytes might be a safeguard mechanism to protect them from NK cells in an inflammatory, virus-infected, or tumor microenvironment, where NK and activated CD4+ T cells are recruited.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Antígenos de Histocompatibilidad Clase I/biosíntesis , Células Asesinas Naturales/fisiología , Linfocitos T CD4-Positivos/efectos de los fármacos , Supervivencia Celular , Citocinas/metabolismo , Citotoxicidad Inmunológica , Humanos , Ionomicina/farmacología , Activación de Linfocitos , Microscopía Confocal , Proteínas Quinasas/metabolismo , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacología , Células TH1/efectos de los fármacos , Células TH1/metabolismo , Células Th2/efectos de los fármacos , Células Th2/metabolismoRESUMEN
MHC class I-related chain A gene (MICA) is a stress-regulated, HLA-related molecule which exhibits a restricted pattern of expression. MICA protein is up-regulated on different tumor cells, and is recognized by the lectin-like NKG2D molecule expressed by cytotoxic gammadelta T lymphocytes, CD8+ alphabeta T lymphocytes, and NK cells. Although MICA is not expressed on resting lymphocytes, we demonstrated that it is induced on activated T cells. Because NF-kappaB is actively involved in T cell activation, and is constitutively activated in many tumors, here we investigated whether NF-kappaB may modulate MICA expression. Treatment with the NF-kappaB inhibitor sulfasalazine (Sz) resulted in a dose-dependent inhibition of MICA expression in anti-CD3- and anti-CD28/PMA-activated T lymphocytes, as assessed by Western blot and RT-PCR analysis. Moreover, Sz also down-regulated MICA expression on epithelial tumor HeLa cells. MICA expression was accompanied by a Sz-sensitive IkappaBalpha degradation. EMSA with nuclear extracts from anti-CD3- and anti-CD28/PMA-stimulated T lymphocytes demonstrated the binding of a potential NF-kappaB family transcription factor to a MICA gene intron 1-derived oligonucleotide that contains a putative kappaB binding site. Supershift assays demonstrated the presence of p65(RelA)/p50 heterodimers and p50/p50 homodimers in the NF-kappaB complexes bound to the kappaB-MICA oligonucleotide. Transient transfection of HeLa cells with p65(RelA) up-regulated MICA expression, as assessed by Western blot and flow cytometry analysis. Hence, we conclude that NF-kappaB regulates MICA expression on activated T lymphocytes and HeLa tumor cells, by binding to a specific sequence in the long intron 1 of the MICA gene. This constitutes the first description of a transcription factor that regulates MICA gene expression.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Antígenos de Histocompatibilidad Clase I/biosíntesis , Antígenos de Histocompatibilidad Clase I/genética , Activación de Linfocitos/genética , FN-kappa B/fisiología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Anticuerpos Monoclonales/farmacología , Antígenos CD28/inmunología , Complejo CD3/inmunología , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/inmunología , Ensayo de Cambio de Movilidad Electroforética , Células HeLa , Humanos , Intrones/genética , Activación de Linfocitos/efectos de los fármacos , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Unión Proteica/genética , Unión Proteica/inmunología , Sulfasalazina/farmacología , Subgrupos de Linfocitos T/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , TransfecciónRESUMEN
Superantigens (SAgs) are a class of disease-causing and immunostimulatory proteins of bacterial or viral origin that activate a large number of T-cells through interaction with the Vbeta domain of T-cell receptors (TCRs). In this study, recombinant TCR beta chains were constructed with human variable domains Vbeta5.2, Vbeta1 and Vbeta2.1, expressed as inclusion bodies, refolded and purified. The Streptococcus pyogenes SAg SSA-1 was cloned and expressed as a soluble periplasmic protein. SSA-1 was obtained both as a monomer and a dimer that has an intermolecular disulfide bond. We analyzed the biological activity of the recombinant SAgs by proliferation assays. The results suggest that SSA dimerization occludes the TCR interaction site. Naturally occurring SSA dimerization was also observed in supernatants of S. pyogenes isolates. An SSA mutant [SSA(C26S)] was produced to eliminate the Cys responsible for dimerization. Affinity assays using a resonant biosensor showed that both the mutant and monomeric wild type SSA have affinity for human Vbeta5.2 and Vbeta1 with Kd of 9-11 microm with a fast kass and a moderately fast kdiss. In spite of the reported stimulation of Vbeta2.1 bearing T-cells by SSA, we observed no measurable interaction.
Asunto(s)
Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Streptococcus pyogenes/inmunología , Superantígenos/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/química , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Clonación Molecular , Dimerización , Relación Dosis-Respuesta Inmunológica , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Humanos , Immunoblotting , Activación de Linfocitos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Superantígenos/química , Superantígenos/inmunología , Superantígenos/farmacología , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Recent evidence has implicated galectins and their carbohydrate ligands as novel regulators of T-cell homeostasis. Galectin-1 (Gal-1), a member of this family, inhibits clonal expansion, induces apoptosis of antigen-primed T lymphocytes and suppresses the development of T-cell-mediated autoimmune diseases in vivo. Because the beta-galactoside-binding protein is expressed in activated but not resting T cells, it has been hypothesized that Gal-1-induced apoptosis may constitute an autocrine suicide mechanism to eliminate activated T cells contributing to the termination of an effector immune response. We undertook this study to investigate the signals and intracellular pathways leading to Gal-1 expression during T-cell activation. When T cells were stimulated either with anti-CD3 or anti-CD28 monoclonal antibody plus PMA in the presence of accessory cells, a sustained up-regulation of Gal-1 was observed, reaching a plateau between days 3 and 5 following CD3 engagement or costimulation through CD28. Investigation of the signal transduction events involved in this process revealed a role for Lck and Fyn kinases, since the Src kinase inhibitor PP1 inhibited the up-regulated expression of Gal-1 following T-cell activation. Downstream signaling routes involve mitogen-activated protein kinase (MAPK) kinase (MEK)1/extracellular signal-regulated kinase (ERK) and p38 MAPK, as Gal-1 expression was prevented by U0126 and SB202190. In addition, expression of Gal-1 involves interleukin (IL)-2-dependent signaling routes triggered by p70S6 kinase, as it could be inhibited by rapamycin. This is the first demonstration of the intracellular pathways that control activation-induced expression of Gal-1, which may reveal potential targets for immune intervention to modulate expression of this beta-galactoside-binding protein in pathological disorders.
Asunto(s)
Galectina 1/metabolismo , Regulación de la Expresión Génica/inmunología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , MAP Quinasa Quinasa 1/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Linfocitos T/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Familia-src Quinasas/metabolismo , Anticuerpos Monoclonales/farmacología , Western Blotting , Butadienos/farmacología , Proliferación Celular , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Galectina 1/genética , Humanos , Imidazoles/farmacología , Interleucina-2/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Activación de Linfocitos , Modelos Biológicos , Nitrilos/farmacología , Proteínas Proto-Oncogénicas c-fyn , Piridinas/farmacología , Transducción de Señal , Sirolimus/farmacología , Acetato de Tetradecanoilforbol/farmacología , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Major histocompatibility complex class I-related chain (MICA) is a cell stress-regulated molecule recognized by cytotoxic cells expressing the NKG2D molecule. MICA can be induced on T cells after CD3 or CD28 engagement. Here, we investigated the intracellular pathways leading to activation-induced expression of MICA. The Src kinase inhibitor PP1 inhibited up-regulated expression of MICA on anti-CD3-stimulated T cells. Downstream signaling routes involved mitogen-activated protein kinase (MAPK) kinase (MEK)1/extracellular signal-regulated kinase (ERK), p38 MAPK, and calcineurin, as MICA expression was prevented by U0126, SB202190, cyclosporin A, and FK506. Also, Lck and Fyn as well as MEK1/ERK and p38 MAPK were found to regulate MICA expression in anti-CD28/phorbol 12-myristate 13-acetate-stimulated T cells. Expression of MICA on activated T cells involved interleukin-2-dependent signaling routes triggered by Janus tyrosine kinases/signal transducer and activators of transcription and p70(S)(6) kinase, as it could be inhibited by AG490 and rapamycin. This is the first demonstration of the intracellular pathways involved in activation-induced expression of MICA, which may reveal potential targets for immune intervention to modulate MICA expression in pathological disorders.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/biosíntesis , Sistema de Señalización de MAP Quinasas , Linfocitos T/enzimología , Linfocitos T/inmunología , Regulación hacia Arriba , Calcineurina/metabolismo , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Humanos , Interleucina-2/fisiología , Activación de Linfocitos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/fisiología , MAP Quinasa Quinasa 1 , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-fyn , Proteínas Quinasas S6 Ribosómicas 70-kDa/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas 70-kDa/fisiología , Transducción de Señal , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Seborrhoeic dermatitis is a disease of unknown etiopathogenesis that affects 5% of the population. In this study, we investigated expression of mRNA for IL-1 alpha, IL-6, IL-4, IFN-gamma, and the stress-inducible MICA molecule in skin biopsies from 12 patients with moderate to severe seborrhoeic dermatitis and 2 healthy volunteers by RT-PCR and hybridization with specific probes. Eight patients expressed INF-gamma, 2 expressed IL-6, 8 expressed IL-1 alpha, and 2 expressed IL-4 (1 with moderate disease). Eight patients expressed inflammatory cytokines (IL-1 alpha, IL-6, and/or IFN-gamma) in healthy skin. Higher cytokine mRNA in damaged vs healthy skin was also observed, suggesting the existence of an inflammation that predisposes healthy skin to develop overt disease. Up-regulated expression of MICA mRNA was observed in 8 patients. Although the pathogenesis of seborrhoeic dermatitis remains to be elucidated, expression of cytotoxicity-activating ligands (MICA), recruitment of NK cells, and a local pro-inflammatory microenvironment may facilitate the development of tissue injury.
Asunto(s)
Citocinas/inmunología , Dermatitis Seborreica/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Piel/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biopsia , Dermatitis Seborreica/fisiopatología , Femenino , Antígenos de Histocompatibilidad Clase I/biosíntesis , Humanos , Masculino , Persona de Mediana Edad , Piel/patología , Regulación hacia Arriba/inmunologíaRESUMEN
MICA is an HLA-related cell stress-regulated antigen recognized by cytotoxic cells expressing the NKG2D molecule. Although resting lymphocytes do not express MICA, it can be induced on PHA-activated T cells. Here, we demonstrate by Western blot that MICA is induced on allogeneic-activated CD4(+) and CD8(+) T lymphocytes. Blocking activation with anti-HLA class I, anti-HLA-DR, or anti-CD86 mAb affected the expression of MICA slightly. When T cells were stimulated with anti-CD3 or anti-CD28 mAb plus PMA, a sustained up-regulation of MICA was observed by Western blot, RT-PCR, and flow cytometry. The expression of MICA reached a plateau at day 4 after CD3 engagement and at day 3 after anti-CD28/PMA stimulation. Conversely, the proliferative response reached a peak at day 4. Hence, CD3 or CD28 engagement induces MICA expression on T lymphocytes. This activation-induced expression might participate in NKG2D-mediated cytotoxicity toward activated T cells to maintain homeostasis during an ongoing immune response.