RESUMEN
During clinical trials of a tumour necrosis factor (TNF)-R1 domain antibody (dAb™) antagonist (GSK1995057), infusion reactions consistent with cytokine release were observed in healthy subjects with high levels of a novel, pre-existing human anti-VH (HAVH) autoantibody. In the presence of HAVH autoantibodies, GSK1995057 induced cytokine release in vitro due to binding of HAVH autoantibodies to a framework region of the dAb. The epitope on GSK1995057 was characterized and dAbs with reduced binding to HAVH autoantibodies were generated; pharmacological comparability was determined in human in-vitro systems and in-vivo animal experiments. A Phase I clinical trial was conducted to investigate the safety and tolerability of the modified dAb (GSK2862277). A significant reduction in HAVH binding was achieved by adding a single alanine residue at the C-terminus to create GSK2862277. Screening a pool of healthy donors demonstrated a reduced frequency of pre-existing autoantibodies from 51% to 7%; in all other respects, GSK2862277 and the parent dAb were comparable. In the Phase I trial, GSK2862277 was well tolerated by both the inhaled and intravenous routes. One subject experienced a mild infusion reaction with cytokine release following intravenous dosing. Subsequently, this subject was found to have high levels of a novel pre-existing antibody specific to the extended C-terminus of GSK2862277. Despite the reduced binding of GSK2862277 to pre-existing HAVH autoantibodies, adverse effects associated with the presence of a novel pre-existing antibody response specific to the modified dAb framework were identified and highlight the challenge of developing biological antagonists to this class of receptor.
Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Autoanticuerpos/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/inmunología , Receptores Tipo I de Factores de Necrosis Tumoral/inmunología , Administración por Inhalación , Administración Intravenosa , Adulto , Anciano , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/sangre , Especificidad de Anticuerpos/inmunología , Autoanticuerpos/sangre , Línea Celular , Línea Celular Tumoral , Epítopos/inmunología , Femenino , Humanos , Interleucina-8/inmunología , Interleucina-8/metabolismo , Macaca fascicularis , Masculino , Persona de Mediana Edad , Unión Proteica/inmunología , Receptores Tipo I de Factores de Necrosis Tumoral/antagonistas & inhibidores , Factores de Tiempo , Adulto JovenAsunto(s)
Enfermedad Pulmonar Obstructiva Crónica/sangre , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Enfermedad Pulmonar Obstructiva Crónica/terapia , Factores de Necrosis Tumoral/sangre , Anciano , Biomarcadores/sangre , Composición Corporal , Estudios de Casos y Controles , Ensayos Clínicos como Asunto , Estudios de Cohortes , Citocina TWEAK , Ejercicio Físico , Femenino , Humanos , Inflamación , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/mortalidad , Músculo Cuádriceps/patologíaRESUMEN
Asthma is a disease characterized, in part, by reversible airflow obstruction, hyperresponsiveness and inflammation. Traditional concepts concerning airway inflammation have focused on leukocyte trafficking and on the effects of inflammatory mediators, cytokines and chemokines secreted by these cells. Airway smooth muscle, the major effector cell responsible for bronchomotor tone, has been thought of as a passive tissue that responds to neurohumoral control and inflammatory mediators. New evidence, however, suggests that airway smooth muscle may secrete cytokines and chemokines and express cell adhesion molecules that are important in modulating submucosal airway inflammation. The cellular and molecular mechanisms that regulate the immunomodulatory functions of airway smooth muscle may offer new and important therapeutic targets in treating this common lung disease.
Asunto(s)
Adyuvantes Inmunológicos/fisiología , Antiasmáticos/farmacología , Antiasmáticos/uso terapéutico , Asma/tratamiento farmacológico , Músculo Liso/efectos de los fármacos , Músculo Liso/inmunología , Sistema Respiratorio/efectos de los fármacos , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Asma/inmunología , Asma/patología , Humanos , Músculo Liso/patología , Sistema Respiratorio/citologíaRESUMEN
VCAM-1 is a member of the Ig superfamily of receptors the expression of which is up-regulated on human airway smooth muscle (ASM) cells following stimulation with inflammatory mediators. The function of these receptors in adhesion is well known, but there is growing recognition that they also possess "outside-in" signaling functions, such as cytoskeletal reorganization, calcium mobilization, and cytokine release. The present study examined the activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase (PI3K) in ASM cells following VCAM-1 engagement. VCAM-1 ligation activated extracellular signal-regulated kinase 2 and resulted in increased expression of cyclin D1, yet there was neither p27(kip1) degradation nor an increase in smooth muscle cell DNA synthesis. VCAM-1 ligation, however, augmented the proliferative response to submitogenic concentrations of epidermal growth factor. VCAM-1 engagement also stimulated a rapid increase in PI3K activity. This was associated with phosphorylation of the adapter protein p120(Cbl) and an increase in Cbl-associated PI3K activity. These studies suggest that VCAM-1 is linked to multiple signaling pathways in human ASM cells and may function to augment growth factor-induced responses.
Asunto(s)
Músculo Liso/enzimología , Músculo Liso/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Tráquea/enzimología , Tráquea/inmunología , Ubiquitina-Proteína Ligasas , Molécula 1 de Adhesión Celular Vascular/fisiología , División Celular/inmunología , Células Cultivadas , Sinergismo Farmacológico , Activación Enzimática/inmunología , Humanos , Interleucina-4/farmacología , Ligandos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Músculo Liso/citología , Músculo Liso/metabolismo , Fosforilación , Fosfotirosina/metabolismo , Proteínas Proto-Oncogénicas c-cbl , Tráquea/citología , Tráquea/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Molécula 1 de Adhesión Celular Vascular/inmunología , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Although 3':5' cyclic adenosine monophosphate (cAMP) is known to modulate cytokine production in a number of cell types, little information exists regarding cAMP-mediated effects on this synthetic function of human airway smooth-muscle (HASM) cells. We examined the effect of increasing intracellular cAMP concentration ([cAMP](i)) on tumor necrosis factor (TNF)-alpha-induced regulated on activation, normal T cells expressed and secreted (RANTES) and interleukin (IL)-6 secretion from cultured HASM cells. Pretreatment of HASM with prostaglandin (PG) E(2), forskolin, or dibutyryl cAMP inhibited TNF-alpha-induced RANTES secretion but increased TNF-alpha-induced IL-6 secretion. Moreover, stimulation with PGE(2), forskolin, or dibutyryl cAMP alone increased basal IL-6 secretion in a concentration-dependent manner. SB 207499, a specific phosphodiesterase type 4 inhibitor, augmented the inhibitory effects of PGE(2) and forskolin on TNF-alpha-induced RANTES. Collectively, these data demonstrate that increasing [cAMP](i) in HASM effectively increases IL-6 secretion but reduces RANTES secretion promoted by TNF-alpha. Reverse transcriptase/polymerase chain reaction and ribonuclease protection assays suggested that these opposite effects of increased [cAMP](i) on TNF-alpha- induced IL-6 and RANTES secretion may occur at the transcriptional level. Accordingly, we examined the effects of TNF- alpha and cAMP on the regulation of nuclear factor (NF)-kappaB, a transcription factor known to modulate cytokine synthesis in numerous cell types. Stimulation of HASM cells with TNF-alpha increased NF-kappaB DNA-binding activity. However, increased [cAMP](i) in HASM neither activated NF-kappaB nor altered TNF-alpha- induced NF-kappaB DNA-binding activity. These results were confirmed using a NF-kappaB-luciferase reporter assay. Together, our data suggest that TNF-alpha-induced IL-6 and RANTES secretion may be associated with NF-kappaB activation, and that inhibition of TNF-alpha-stimulated RANTES secretion and augmentation of IL-6 secretion by increased [cAMP](i) in HASM cells occurs via an NF-kappaB-independent mechanism.
Asunto(s)
Quimiocina CCL5/metabolismo , Interleucina-6/metabolismo , Músculo Liso/efectos de los fármacos , Tráquea/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Bucladesina/farmacología , Células Cultivadas , Quimiocina CCL5/genética , Colforsina/farmacología , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Ácidos Ciclohexanocarboxílicos/farmacología , Dinoprostona/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Interleucina-6/genética , Músculo Liso/citología , Músculo Liso/metabolismo , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Nitrilos , Inhibidores de Fosfodiesterasa/farmacología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Tiempo , Tráquea/citología , Tráquea/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Tumor necrosis factor-alpha (TNFalpha) stimulates the expression of intercellular adhesion molecule-1 (ICAM-1) by activating the transcription factor nuclear factor-kappaB (NF-kappaB) in human airway smooth muscle (ASM) cells. This study characterizes the receptor involved as well as critical downstream signaling events mediating cytokine-induced NF-kappaB activation and ICAM-1 expression. TNFalpha stimulation for 1 to 4 h induced ICAM-1 expression in human ASM cells. This rapid TNFalpha-induced ICAM-1 expression enhanced T-lymphocyte adhesion to ASM cells, which was inhibited by anti-ICAM-1 antibodies. Using immunostaining, we demonstrated that TNFalpha receptors TNFR1 and TNFR2 are expressed on native human tracheal smooth muscle. Treatment of cells with htr-9, an antibody that specifically activates TNFR1, also stimulated expression of ICAM-1 mRNA and protein. Utr-1, a blocking antibody to TNFR2, did not affect TNFalpha-mediated ICAM-1 expression. Both TNFalpha and htr-9 increased luciferase activity in ASM cells transfected with a NF-kappaB reporter plasmid. Overexpression of a dominant negative TNF receptor-associated factor 2 construct, lacking the NH(2)-terminal RING finger, completely abrogated both TNFalpha- and htr-9-mediated increases in NF-kappaB reporter activity. Thapsigargin, an agent that depletes intracellular calcium stores, abrogated both cytokine-mediated NF-kappaB-dependent ICAM-1 mRNA transcription and protein expression but had no effect on IkappaB degradation. In addition, chelating cytosolic calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid acetoxymethyl ester also inhibited cytokine TNFalpha-induced ICAM-1 expression. These data suggest that TNFR1, through a TNF receptor-associated factor 2-NF-kappaB signaling pathway, mediates TNFalpha-induced expression of ICAM-1 on ASM cells by involving a thapsigargin-sensitive signaling pathway.
Asunto(s)
Antígenos CD/fisiología , Molécula 1 de Adhesión Intercelular/biosíntesis , Músculo Liso/metabolismo , Proteínas/fisiología , Receptores del Factor de Necrosis Tumoral/fisiología , Antígenos CD/metabolismo , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Activación de Linfocitos , FN-kappa B/fisiología , Proteínas/metabolismo , ARN Mensajero/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral , Linfocitos T/inmunología , Linfocitos T/metabolismo , Factor 2 Asociado a Receptor de TNF , Tapsigargina/farmacología , Tráquea/metabolismo , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Cell adhesion molecules (CAMs) have been implicated in many cellular functions, including development, tumor metastasis, leukocyte activation, homing, and transendothelial migration, and can also serve as viral receptors. The role of CAMs on leukocytes and endothelial cells, and the interactions between these cell types, have been extensively characterized. The current model of leukocyte recruitment and homing involves the expression and activation of a cascade of CAMs, as well as the local production of chemoattractants, leading to leukocyte adhesion and transmigration into lymph nodes and sites of inflammation (1). Most studies have focused on the mechanisms that control leukocyte adhesion and transendothelial migration, yet the subsequent interactions of infiltrating leukocytes with other cell types in the submucosa, and with the extracellular matrix, may also be important for sustaining the inflammatory response.
RESUMEN
Although the precise mechanisms by which steroids mediate their therapeutic effects remain unknown, steroids have been reported to abrogate cytokine-induced activation of the transcription factor NF-kappa B. In some cell types, NF-kappa B activation is necessary to regulate cytokine-mediated cellular functions. However, compelling evidence suggests that the steroid inhibition of NF-kappa B is complex and cell specific. Using EMSA, we show that stimulation with TNF-alpha or IL-1 beta induces NF-kappa B DNA-binding activity in human airway smooth muscle cells. TNF-alpha and IL-1 beta also increased luciferase activity in airway smooth muscle cells transfected with a reporter plasmid containing kappa B enhancer elements. Cytokines activated NF-kappa B by rapidly degrading its cytosolic inhibitor I kappa B alpha, which was then regenerated after 60 min. Cytokine-mediated I kappa B alpha reappearance was completely blocked by the protein synthesis inhibitor cycloheximide. Inhibition of cytokine-mediated I kappa B alpha proteolysis using the protease inhibitors N-tosyl-L -phenylalanine chloromethyl ketone and N-acetyl-L -leucinyl-L -leucinyl-norleucinal also inhibited cytokine-mediated early expression of ICAM-1. Although dexamethasone partially inhibited IL-1 beta- and TNF-alpha-induced up-regulation of ICAM-1 at 4 h, dexamethasone had no effect on cytokine-induced ICAM-1 expression at 18-24 h. In addition, neither cytokine-induced degradation or resynthesis of I kappa B alpha nor NF-kappa B DNA-binding activity were affected by dexamethasone. In cells transfected with the luciferase reporter, dexamethasone did not affect TNF-alpha-induced NF-kappa B-dependent transcription. Interestingly, cytokine-mediated expression of cyclooxygenase-2 was completely abrogated by dexamethasone at 6 h. Together, these data demonstrate that cytokine-mediated NF-kappa B activation and ICAM-1 expression involve activation of a steroid-insensitive pathway.
Asunto(s)
Citocinas/fisiología , Dexametasona/farmacología , Proteínas I-kappa B , Molécula 1 de Adhesión Intercelular/metabolismo , Músculo Liso/metabolismo , FN-kappa B/fisiología , Transducción de Señal/efectos de los fármacos , Tráquea/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Citocinas/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Interleucina-1/fisiología , Isoenzimas/biosíntesis , Proteínas de la Membrana , Músculo Liso/citología , Músculo Liso/enzimología , Músculo Liso/inmunología , Inhibidor NF-kappaB alfa , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Transducción de Señal/inmunología , Tráquea/citología , Tráquea/enzimología , Tráquea/inmunología , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/fisiología , Regulación hacia Arriba/inmunologíaRESUMEN
CD40 is a member of the TNF receptor family that was initially described on the surface of B cells. Recently, CD40 has also been described on mesenchymal cells, such as endothelial cells and fibroblasts, where engagement by its ligand CD40 ligand can lead to up-regulation of costimulatory and cell adhesion molecules, as well as secretion of proinflammatory cytokines. Since airway inflammation potentially involves cell-cell interactions of T cells and eosinophils (which express CD40 ligand) with airway smooth muscle (ASM) cells, we postulated that ASM may express CD40 and that engagement of ASM CD40 may modulate smooth muscle cell function. We demonstrate that CD40 is expressed on cultured human ASM and that expression can be increased by treatment with TNF-alpha or IFN-gamma. Cross-linking CD40 on ASM resulted in enhanced IL-6 secretion and an increase in intracellular calcium concentrations, which were dependent on calcium influx. We show that CD40-mediated signaling events include protein tyrosine phosphorylation and activation of NF-kappaB. Pretreatment of ASM with the tyrosine kinase inhibitors genistein or herbimycin inhibited the rapid mobilization of calcium induced via CD40, suggesting that calcium mobilization was coupled to activation of protein tyrosine kinases. In addition, inhibition of calcium influx inhibited both CD40-mediated NF-kappaB activation and enhancement of IL-6 secretion. These results delineate a potentially important CD40-mediated signal-transduction pathway in ASM, involving protein tyrosine kinase-dependent calcium mobilization, NF-kappaB activation, and IL-6 production. Together, these results suggest a mechanism whereby T cell/smooth muscle cell interactions may potentiate airway inflammation.
Asunto(s)
Antígenos CD40/fisiología , Músculo Liso/inmunología , Transducción de Señal/inmunología , Tráquea/inmunología , Antígenos CD40/biosíntesis , Antígenos CD40/metabolismo , Ligando de CD40 , Calcio/metabolismo , Calcio/fisiología , Células Cultivadas , Espacio Extracelular/metabolismo , Espacio Extracelular/fisiología , Humanos , Interferón gamma/farmacología , Interleucina-6/biosíntesis , Interleucina-6/metabolismo , Líquido Intracelular/metabolismo , Ligandos , Glicoproteínas de Membrana/fisiología , Músculo Liso/citología , Músculo Liso/enzimología , Músculo Liso/metabolismo , FN-kappa B/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Tráquea/citología , Tráquea/enzimología , Tráquea/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/inmunologíaRESUMEN
The current model of lymphocyte extravasation into areas of inflammation involves the sequential engagement of multiple cell adhesion molecules (CAMs) expressed on lymphocytes and endothelial cells. In addition, the expression of CAMs and the elaboration of matrix by subendothelial/submucosal cells may contribute to the retention and stimulation of infiltrating cells in an inflammatory lesion. We previously demonstrated that mitogen-activated T cells adhered to airway smooth muscle (ASM) in an integrin-dependent fashion. ASM are MHC class II-negative and expressed low basal levels of intercellular adhesion molecule-1 (ICAM-1). In this study, we demonstrate that anti-CD3-stimulated peripheral blood T cells also adhere to ASM and markedly upregulate ICAM-1 expression and induce the expression of MHC class II on ASM. The induction of HLA-DR was completely inhibited, and the induction of ICAM-1 partially inhibited, by neutralizing antibody against interferon-gamma. Furthermore, in studies with bronchoalveolar lavage-derived T cells isolated from atopic donors following local antigen challenge, we observed adhesion to ASM and upregulation of ASM expression of ICAM-1 and HLA-DR similar to that seen with in vitro-activated T cells. Finally, we found that despite expression of ICAM-1 and HLA-DR, ASM could not present alloantigen to CD4+ T cells. These findings suggest that the interaction of activated T cells with parenchymal cells of the lung such as airway smooth muscle affects the phenotype of myocytes and thus may have significant implications for inflammatory diseases such as asthma or transplant rejection.
Asunto(s)
Antígenos HLA/biosíntesis , Molécula 1 de Adhesión Intercelular/biosíntesis , Músculo Liso/inmunología , Linfocitos T/inmunología , Tráquea/inmunología , Presentación de Antígeno , Líquido del Lavado Bronquioalveolar/citología , Complejo CD3/inmunología , Linfocitos T CD4-Positivos/inmunología , Adhesión Celular , Células Cultivadas , Técnicas de Cocultivo , Humanos , Hipersensibilidad Inmediata/inmunología , Interferón gamma/farmacología , Activación de Linfocitos , Músculo Liso/citología , Músculo Liso/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Interleucina-2/biosíntesis , Tráquea/citología , Tráquea/metabolismo , Regulación hacia Arriba , Molécula 1 de Adhesión Celular Vascular/biosíntesisRESUMEN
Asthma is a disease of airway inflammation and hyper-reactivity associated with lymphocytic infiltration in the bronchial submucosa. We recently demonstrated that human airway smooth muscle (ASM) cells express the cell adhesion molecules ICAM-1 and VCAM-1, which are up-regulated by cytokines such as TNF-alpha, and which mediate binding of activated T lymphocytes. In this study, we examined whether an increase in [cAMP]i, presumably via activation of cAMP-dependent protein kinase, modulates TNF-alpha-induced ICAM-1 and VCAM-1 on ASM. We found that treatment of ASM with either forskolin, which directly activates adenylyl cyclase, or with cholera toxin, which activates the heterotrimeric GTP-binding protein, Gs, inhibited TNF-alpha-induced cell adhesion molecule expression. In addition, treatment with either isoproterenol or prostaglandin E2, which activates receptors coupled to Gs and increases [cAMP]i, also inhibited TNF-alpha-induced expression of ICAM-1 and VCAM-1 on ASM. Furthermore, adhesion of activated T cells to TNF-alpha-stimulated ASM was inhibited by treating the ASM cells with either forskolin or PGE2. These data suggest that cAMP-dependent protein kinase activation decreases cytokine-induced expression of cell adhesion molecules on ASM cells, modulates T cell binding to airway myocytes and, thus, suggests novel therapeutic approaches to airway inflammation.
Asunto(s)
AMP Cíclico/metabolismo , Músculo Liso/metabolismo , Tráquea/metabolismo , Asma/inmunología , Asma/metabolismo , Adhesión Celular , Moléculas de Adhesión Celular/biosíntesis , Células Cultivadas , Colforsina/farmacología , Dinoprostona/farmacología , Proteínas de Unión al GTP/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Isoproterenol/farmacología , Activación de Linfocitos , Músculo Liso/efectos de los fármacos , Músculo Liso/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Tráquea/efectos de los fármacos , Tráquea/inmunología , Factor de Necrosis Tumoral alfa/farmacología , Molécula 1 de Adhesión Celular VascularRESUMEN
CD44 is a cell surface adhesion molecule that plays a role in leukocyte extravasation, leukopoiesis, T lymphocyte activation, and tumor metastasis. The principal known ligand for CD44 is the glycosaminoglycan hyaluronate, (HA), a major constituent of extracellular matrices. CD44 expression is required but is not sufficient to confer cellular adhesion to HA, suggesting that the adhesion function of the receptor is regulated. We recently demonstrated that CD44 in primary leukocytes is phosphorylated in a cell type- and activation state-dependent fashion. In this study we demonstrate that serines 325 and 327 within the cytoplasmic domain of CD44 are required for the constitutive phosphorylation of CD44 in T cells. Furthermore, we demonstrate that cells expressing mutated CD44 containing a serine to glycine substitution at position 325 or a serine to alanine substitution at amino acid 327 are defective in HA binding, CD44-mediated adhesion of T cells to smooth muscle cells, as well as ligand-induced receptor modulation. The effect of these mutations can be partially reversed by a monoclonal anti-CD44 antibody that enhances CD44-mediated HA binding.
Asunto(s)
Proteínas Portadoras/metabolismo , Ácido Hialurónico/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Mensajeros de Linfocitos/metabolismo , Linfocitos T/metabolismo , Secuencia de Aminoácidos , Animales , Modulación Antigénica , Secuencia de Bases , Adhesión Celular , Endocitosis , Receptores de Hialuranos , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Fosfoserina/metabolismo , Relación Estructura-ActividadRESUMEN
Asthma is a disease of airway inflammation and hyperreactivity that is associated with a lymphocytic infiltrate in the bronchial submucosa. The interactions between infiltrating T lymphocytes with cellular and extracellular matrix components of the airway and the consequences of these interactions have not been defined. We demonstrate the constitutive expression of CD44 on human airway smooth muscle (ASM) cells in culture as well as in human bronchial tissue transplanted into severe combined immunodeficient mice. In contrast, basal levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expression are minimal but are induced on ASM by inflammatory mediators such as tumor necrosis factor alpha (TNF-alpha). Activated, but not resting T cells, adhere to cultured ASM; stimulation of the ASM with TNF-alpha enhanced this adhesion. Adhesion was partially blocked by monoclonal antibodies (mAb) specific for lymphocyte function-associated antigen 1 (LFA-1) and very late antigen 4 (VLA-4) on T cells and ICAM-1 and VCAM-1 on ASM cells. The observed integrin-independent adhesion was mediated by CD44/hyaluronate interactions as it was inhibited by anti-CD44 mAb 5F12 and by hyaluronidase. Furthermore, the adhesion of activated T lymphocytes induced DNA synthesis in growth-arrested ASM cells. Thus, the interaction between T cells and ASM may provide insight into the mechanisms that induce bronchial inflammation and possibly ASM cell hyperplasia seen in asthma.
Asunto(s)
Bronquios/citología , Proteínas Portadoras/fisiología , ADN/biosíntesis , Integrinas/fisiología , Músculo Liso/metabolismo , Receptores de Superficie Celular/fisiología , Receptores Mensajeros de Linfocitos/fisiología , Linfocitos T/fisiología , Tráquea/citología , Animales , Bronquios/metabolismo , Adhesión Celular , Moléculas de Adhesión Celular/análisis , Comunicación Celular , Células Cultivadas , Humanos , Receptores de Hialuranos , Molécula 1 de Adhesión Intercelular , Ratones , Ratones SCID , Tráquea/metabolismo , Molécula 1 de Adhesión Celular VascularRESUMEN
The transposon Tn5 consists of inverted repeats, called IS50R and IS50L, each of which encode two proteins. We show here that the larger protein encoded on IS50R, protein 1, is absolutely required for transposition. Deletion or insertion mutants that fail to make this protein fail to promote gene movement. In addition, his protein acts in cis preferentially. We also show that the smaller protein encoded on IS50R, protein 2, is competent to inhibit transposition of a Tn5 freshly introduced into the cell on a lambda phage. In contrast, the proteins from IS50L possess neither of these two activities. By assaying expression of proteins that are hybrids between beta-galactosidase and IS50R proteins, we find that the regulation of transposition cannot be due to the inhibitor repressing synthesis of Tn5 proteins. Control experiments, in which we assay synthesis of IS50 proteins synthesized from a lambda::IS50R that has been infected into cells carrying the transposition inhibitor, confirm this conclusion.