RESUMEN
BACKGROUND: Very late antigen-4 (VLA(4)) plays a key role in the recruitment of eosinophils in allergic responses in animal studies. OBJECTIVE: We investigated whether pretreatment with multiple doses of a VLA(4) receptor antagonist, HMR 1031, protects against allergen-induced airway responses and airway inflammation in humans. METHODS: Fourteen asthmatics (7F/7M), 18-49 years, PC(20) forced expiratory volume in 1 s (FEV(1)) methacholine (M) (<8 mg/mL; FEV(1) 82.3-116.1% predicted) with dual responses to inhaled allergen participated in a double-blind, placebo-controlled, cross-over study. Each treatment period consisted of 9 days, separated by >or=2 weeks. Exhaled nitric oxide (eNO), PC(20)FEV(1)(M) and hypertonic saline-induced sputum was obtained on Days 1, 7 and 9. Subjects inhaled HMR 1031 (20 mg b.i.d.) or placebo (P) on Days 1--8. On Day 8, an allergen bronchoprovocation test was performed, the airway response was measured by FEV(1), and expressed as %fall from baseline. Data from 12 evaluable subjects are presented here. RESULTS: Both treatments were well tolerated. There was no significant difference between HMR 1031 and P in the early asthamatic response: mean AUC (0-3 h)+/-SEM (%fall h): 26.01+/-4.26 and 17.41+/-4.26, respectively (P=0.18), nor in the late response: mean AUC (3-9 h)+/-SEM (%fall h): 97.09+/-8.63 and 97.61+/-8.63, respectively, P=0.97. This corresponded to the absence of significant allergen-induced changes in PC(20)FEV(1)(M), eNO, sputum eosinophils and soluble inflammation markers between both treatment periods. CONCLUSIONS: Treatment with multiple inhaled doses of the VLA(4) antagonist, HMR 1031, did not result in detectable protection against allergen-induced airway responses or airway inflammation in asthma.
Asunto(s)
Alérgenos/inmunología , Asma/inmunología , Imidazoles/inmunología , Integrina alfa4beta1/inmunología , Propionatos/inmunología , Receptores de Antígeno muy Tardío/antagonistas & inhibidores , Administración por Inhalación , Adolescente , Adulto , Bronquios/inmunología , Pruebas de Provocación Bronquial/métodos , Broncoespirometría/métodos , Estudios Cruzados , Método Doble Ciego , Femenino , Volumen Espiratorio Forzado/inmunología , Humanos , Imidazoles/administración & dosificación , Masculino , Cloruro de Metacolina/inmunología , Persona de Mediana Edad , Óxido Nítrico/inmunología , Propionatos/administración & dosificación , Receptores de Antígeno muy Tardío/inmunología , Esputo/inmunologíaRESUMEN
A micropipette technique was adopted to investigate the effect of blockade of integrin betal on adhesion of hepatocellular carcinoma (HCC) cells onto type IV collagen (Col IV) coated surfaces and pseudopod protrusion of HCC cells in response to Col IV stimulation. Adhesion strength was expressed as an adhesion force, which was defined as the product of the cross sectional area and critical negative pressure needed to detach single cell away from the substrate. Chemotactic pseudopod protrusion of an HCC cell was evaluated using a dual-pipette set-up, in which two pipettes filled with Col IV solution were positional in close contact with the same cell and pseudopod protrusion into each pipette was viewed dynamically and recorded with a tape recorder. The lengths of pseudopods were measured and plotted against time to obtain a pseudopod growth curve. The integrin beta1 subunit on the surfaces of HCC cells was analyzed by flow cytometry. The results showed that the adhesion forces for HCC cells adhering on 5 microg/ml Col IV coated surfaces were 932 +/- 134 (x 10(-10) N, n = 60). Upon treatment of HCC cells with Anti-CD29 in a protein concentration of 5 microg/ml and 10 microg/ml, the value decreased significantly to 449 +/- 119 (x 10(-10) N, n = 60) and 220 +/- 78 (x 10(-10) N, n = 55), respectively. In dual pipette chemotaxis experiment, when the two pipettes were filled with Col IV in an identical concentration of 600 microg/ml, pseudopods extended from the HCC cell into each of the pipettes nearly symmetrically, i.e., with nearly identical maximum pseudopod length and similar pseudopod growth curves. Upon addition of Anti-CD29 to one of the pipettes in a protein concentration of 20 microg/ml, pseudopod protrusion was blocked nearly completely while protrusion into the opposite pipette became more evidently, with larger maximum length. Expression of integrin beta1 was up to 95.78% to cells chosen in the experiment. These results suggested that integrin beta1 subunit was important constituent receptor subunit for mediating HCC cell adhesion and chemotactic pseudopod protrusion to Col IV.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Quimiotaxis/inmunología , Colágeno Tipo IV/metabolismo , Integrina beta1/biosíntesis , Neoplasias Hepáticas/metabolismo , Anticuerpos Monoclonales/farmacología , Carcinoma Hepatocelular/patología , Adhesión Celular , Humanos , Integrina beta1/inmunología , Neoplasias Hepáticas/patología , Invasividad Neoplásica , Receptores de Antígeno muy Tardío/biosíntesis , Receptores de Antígeno muy Tardío/inmunología , Células Tumorales CultivadasRESUMEN
El Factor Inhibidor de la Locomoción de Monocitos (FILM) es un pentapéptido (Met-Gln-Cys-Asn-Ser) termoestable producido por E. histolytica cultivada en medio axénico. Este factor posee diversas propiedades antiinflamatorias in vitro e in vivo (i.e. inhibición de la locomoción de monocitos y del estallido respiratorio de monocitos y neutrófilos, inhibición de la hipersensibilidad retardada cutánea al dinitrocluorobenceno (DNCB) en cobayos y gerbos, retraso de leucocitos mononucleares en ventanas de Rebuck en humanos, con abatimiento de las moléculas de adhesión VLA-4, VCAM en el endotelio poscapilar y monocitos, etc). Desconocemos el mecanismo de acción molecular del FILM. La multiplicidad de sus efectos en diversas células nos llevó a considerar una acción sobre la red de citocinas inflamatorias del huésped (humano), en particular de las quimiocinas y sus receptores. El FILM inhibe la expresión inducida de las CC-quimiocinas MIP-1alfa, MIP1beta, I-309 y del receptor CCR1. Estudios preliminares sugieren que el FILM incide en la cascada de señalización NF-kappaB en monocitos humanos, aumentando la translocación del homodímero p50/p50 y alterando la dinámica del heterodímero p65/p50. Igualmente, corriente arriba el pentapéptido inhibe la síntesis de la proteína acopladora MyD88 y altera su translocación membranal. Las citocinas inflamatorias IL-1beta e IL-6 (involucradas en esta vía de señalización) aparentemente se abaten en tanto que se incrementa la síntesis de IL-10, la citocina antiinflamatoria por excelencia. Estos resultados contribuyen a la elucidación del mecanismo de acción molecular del FILM, que parece alterar la regulación de la inmunidad innata en su matriz NF-kappaB (AU)
Asunto(s)
Humanos , Entamoeba histolytica/inmunología , Movimiento Celular/inmunología , Inflamación/inmunología , Dinitroclorobenceno/inmunología , Citocinas/inmunología , Antiinflamatorios , Receptores de Antígeno muy Tardío/inmunología , Molécula 1 de Adhesión Celular Vascular/inmunología , Proteínas Inflamatorias de Macrófagos/inmunologíaRESUMEN
The integrin alpha1beta1 (very late antigen-1; CD49a/CD29) is a major adhesion receptor for collagen I, IV, and VI, and its induced expression on activated monocytes and lymphocytes plays a central role in their retention and activation at inflammatory sites in autoimmune pathologies. However, the role of alpha1beta1 in allergic settings has not been explored. In this study, we show that a single 45-mg dose of aerosolized monoclonal antibody AQC2 to the alpha1 chain of human and sheep very late antigen-1, given 30 minutes before challenge, blocks both the allergen-induced late response and the associated airway hyperresponsiveness, functional indicators of allergen-induced inflammation, in sheep. AQC2 does not affect the early response. Consistent with these effects, AQC2 tended to reduce the cell response associated with local antigen instillation. An isotype-matched control antibody had no protective effects. Two humanized versions of AQC2, a wild-type IgG1 and an aglycosyl form of the same monoclonal antibody, which has reduced Fc receptor-mediated effector functions, are equally effective in blocking the antigen-induced late response and airway hyperresponsiveness in the sheep model. These data suggest that mononuclear leukocyte adhesion-dependent pathologies contribute to allergic lung disease and provide proof-of-concept that antagonists of alpha1 integrins may be useful in preventing these events.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/prevención & control , Integrinas/antagonistas & inhibidores , Receptores de Antígeno muy Tardío/inmunología , Administración por Inhalación , Resistencia de las Vías Respiratorias/efectos de los fármacos , Animales , Pruebas de Provocación Bronquial , Modelos Animales de Enfermedad , Femenino , Integrinas/fisiología , Masculino , Probabilidad , Valores de Referencia , Sensibilidad y Especificidad , Oveja DomésticaRESUMEN
This study tested the hypothesis that very late antigen (VLA)-4 mediates CD18-independent neutrophil emigration into the airspaces induced by either Streptococcus pneumoniae, a stimulus that induces primarily CD18-independent neutrophil emigration, or Escherichia coli, toward which only 20-30% of the total number of neutrophils emigrate through CD18-independent pathways. In wild-type (WT) mice, VLA-4 expression was less on neutrophils that emigrated into the airspaces than on circulating neutrophils. Vascular cell adhesion molecule-1 (VCAM-1) mRNA, the major endothelial cell ligand for VLA-4, increased more in E. coli than in S. pneumoniae pneumonia. VCAM-1 protein expression was not detected in capillaries, the major site of neutrophil emigration. Neutrophil emigration during E. coli or S. pneumoniae pneumonia was similar in mice given antibodies against both CD18 and VLA-4 compared with mice given the anti-CD18 antibody and a control antibody. However, in hematopoietically reconstituted mice with both WT and CD18-deficient neutrophils in their blood, the migration of CD18-deficient neutrophils in response to S. pneumoniae was slightly but significantly less in animals pretreated with the anti-VLA-4 antibody than in those receiving a control antibody. These data suggest that VLA-4 plays a small role in CD18-independent neutrophil emigration, but the majority of CD18-independent neutrophil emigration induced by bacteria in the lungs occurs through VLA-4-independent mechanisms.
Asunto(s)
Integrinas/metabolismo , Neutrófilos/metabolismo , Neumonía Bacteriana/inmunología , Receptores Mensajeros de Linfocitos/metabolismo , Receptores de Antígeno muy Tardío/metabolismo , Enfermedad Aguda , Animales , Anticuerpos Antiidiotipos/inmunología , Antígenos CD18 , Infecciones por Escherichia coli/inmunología , Inmunoglobulina G/inmunología , Inflamación/inmunología , Integrina alfa4beta1 , Integrinas/inmunología , Ratones , Ratones Endogámicos C57BL , Neutrófilos/inmunología , Neumonía Neumocócica/inmunología , Receptores Mensajeros de Linfocitos/inmunología , Receptores de Antígeno muy Tardío/inmunología , Molécula 1 de Adhesión Celular Vascular/inmunología , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Antibodies against integrins have been shown to inhibit allergic airway responses. The purpose of this study was to test the hypothesis that the beta1 integrin, very late antigen-4 (VLA-4), is involved in mast cell activation triggered by allergen exposure in sensitized animals. To do this we studied Brown Norway rats that were sensitized to ovalbumin (OA; 1 mg subcutaneously) using Bordetella pertussis as an adjuvant. Two weeks later rats were challenged with OA, pulmonary resistance (RL) was determined, and the concentrations of histamine and tryptase in bronchoalveolar lavage fluid and N-acetyl-leukotriene (LT)E4 in bile were measured. Pretreatment with a monoclonal antibody against VLA-4 (TA-2) attenuated the early response after OA challenge (342.9 +/- 24.4% baseline RL versus 153.3 +/- 19.4%; p < 0.01). There were significantly lower concentrations of histamine (67.11 +/- 11.90 microgram/ml versus 26.69 +/- 1.84; p < 0.01) and tryptase (0.143 +/- 0. 035 microgram/ml versus 0.053 +/- 0.022 microgram/ml; p < 0.01) in TA-2-treated animals. The increases in the concentrations of biliary N-acetyl-LTE4 after OA challenge were also significantly lower in TA-2-treated animals. These data suggest that a selective anti-VLA-4 monoclonal antibody prevents early responses through inhibition of mast cell activation.
Asunto(s)
Antígenos CD/inmunología , Moléculas de Adhesión Celular/inmunología , Degranulación de la Célula/inmunología , Integrinas/inmunología , Mastocitos/inmunología , Receptores Mensajeros de Linfocitos/inmunología , Hipersensibilidad Respiratoria/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Resistencia de las Vías Respiratorias/inmunología , Alérgenos/administración & dosificación , Alérgenos/inmunología , Animales , Antialérgicos/inmunología , Anticuerpos Monoclonales/uso terapéutico , Bilis/química , Líquido del Lavado Bronquioalveolar/química , Quimasas , Histamina/análisis , Inmunización , Integrina alfa4 , Integrina alfa4beta1 , Integrina beta1/inmunología , Leucotrieno E4/análogos & derivados , Leucotrieno E4/análisis , Masculino , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Ratas , Ratas Endogámicas BN , Receptores de Antígeno muy Tardío/inmunología , Serina Endopeptidasas/análisis , Triptasas , Factores de Virulencia de Bordetella/administración & dosificaciónRESUMEN
A prominent feature of Lyme disease is the perivascular accumulation of mononuclear leukocytes. Incubation of human umbilical vein endothelial cells (HUVEC) cultured on amniotic tissue with either interleukin-1 (IL-1) or Borrelia burgdorferi, the spirochetal agent of Lyme disease, increased the rate at which human monocytes migrated across the endothelial monolayers. Very late antigen 4 (VLA-4) and CD11/CD18 integrins mediated migration of monocytes across HUVEC exposed to either B. burgdorferi or IL-1 in similar manners. Neutralizing antibodies to the chemokine monocyte chemoattractant protein 1 (MCP-1) inhibited the migration of monocytes across unstimulated, IL-1-treated, or B. burgdorferi-stimulated HUVEC by 91% +/- 3%, 65% +/- 2%, or 25% +/- 22%, respectively. Stimulation of HUVEC with B. burgdorferi also promoted a 6-fold +/- 2-fold increase in the migration of human CD4(+) T lymphocytes. Although MCP-1 played only a limited role in the migration of monocytes across B. burgdorferi-treated HUVEC, migration of CD4(+) T lymphocytes across HUVEC exposed to spirochetes was highly dependent on this chemokine. The anti-inflammatory cytokine IL-10 reduced both migration of monocytes and endothelial production of MCP-1 in response to B. burgdorferi by approximately 50%, yet IL-10 inhibited neither migration nor secretion of MCP-1 when HUVEC were stimulated with IL-1. Our results suggest that activation of endothelium by B. burgdorferi may contribute to formation of the chronic inflammatory infiltrates associated with Lyme disease. The transendothelial migration of monocytes that is induced by B. burgdorferi is significantly less dependent on MCP-1 than is migration induced by IL-1. Selective inhibition by IL-10 further indicates that B. burgdorferi and IL-1 employ distinct mechanisms to activate endothelial cells.
Asunto(s)
Grupo Borrelia Burgdorferi/inmunología , Quimiotaxis de Leucocito , Endotelio Vascular/inmunología , Interleucina-1/inmunología , Monocitos/inmunología , Amnios/citología , Antígenos CD11/inmunología , Antígenos CD18/inmunología , Linfocitos T CD4-Positivos/inmunología , Inhibición de Migración Celular , Quimiocina CCL2/metabolismo , Técnicas de Cocultivo , Endotelio Vascular/citología , Humanos , Integrina alfa4beta1 , Integrinas/inmunología , Interleucina-10/inmunología , Receptores Mensajeros de Linfocitos/inmunología , Receptores de Antígeno muy Tardío/inmunologíaAsunto(s)
Adhesión Celular/inmunología , Fibronectinas/antagonistas & inhibidores , Rechazo de Injerto/inmunología , Rechazo de Injerto/prevención & control , Trasplante de Corazón/inmunología , Integrinas/antagonistas & inhibidores , Fragmentos de Péptidos/farmacología , Receptores Mensajeros de Linfocitos/antagonistas & inhibidores , Trasplante de Piel/inmunología , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Adhesión Celular/efectos de los fármacos , Fibronectinas/biosíntesis , Fibronectinas/química , Variación Genética , Integrina alfa4beta1 , Integrinas/inmunología , Masculino , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Reacción en Cadena de la Polimerasa , Ratas , Ratas Endogámicas BN , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Receptores Mensajeros de Linfocitos/inmunología , Receptores de Antígeno muy Tardío/inmunología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Trasplante Heterotópico , Trasplante HomólogoRESUMEN
Experimental autoimmune encephalomyelitis (EAE) is a term given to describe a collection of animal models representing the human disease multiple sclerosis (MS). Although not fully understood, the involvement of cytokines and the immune system in either EAE or human MS is well established. Past efforts have shown that inhibition of proinflammatory cytokines tumor necrosis factor (TNF-alpha) or interleukin-1 (IL-1) result in amelioration of acute EAE in Lewis rats. The present study examined this model for the effect of concomitant inhibition of both TNF-alpha and IL-1, which resulted in a modest but significant therapeutic effect that was superior to inhibition of either single agent alone with respect to four of the five variables used to follow the progression of disease in this model, i.e., clinical severity, frequency of disease, loss of body weight, and day of onset. These results are in accordance with the idea that combination treatments are likely to prove superior to single agent therapy in the treatment of autoimmune inflammatory disease.
Asunto(s)
Encefalomielitis Autoinmune Experimental/terapia , Receptores del Factor de Necrosis Tumoral/uso terapéutico , Sialoglicoproteínas/uso terapéutico , Animales , Encéfalo/inmunología , Encéfalo/patología , Dimerización , Esquema de Medicación , Quimioterapia Combinada , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/fisiopatología , Femenino , Humanos , Inyecciones Intravenosas , Inyecciones Subcutáneas , Integrina alfa4beta1 , Integrinas/biosíntesis , Molécula 1 de Adhesión Intercelular/biosíntesis , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/antagonistas & inhibidores , Antígeno-1 Asociado a Función de Linfocito/biosíntesis , Polietilenglicoles , Ratas , Ratas Endogámicas Lew , Receptores Mensajeros de Linfocitos/biosíntesis , Receptores del Factor de Necrosis Tumoral/administración & dosificación , Receptores de Antígeno muy Tardío/inmunología , Sialoglicoproteínas/administración & dosificación , Médula Espinal/inmunología , Médula Espinal/patología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
Our previous study showed that the cross-linking of very late antigen (VLA)/beta1 with anti-CD29 monoclonal antibody (MoAb), or interactions with extracellular matrix (ECM) proteins through VLA/beta1, failed to induce T-cell costimulation via the CD3/T cell receptor (TCR) pathway for over 1 year after allogeneic bone marrow transplantation (allo-BMT), although normal CD29 and CD3 expression was observed after 3 months following allo-BMT. Molecular analysis revealed altered tyrosine phosphorylation of cellular proteins by the solid-phase cross-linking of VLA/beta1 molecules in T cells from patients after allo-BMT. In T cells from early allo-BMT patients (<4 months), various sizes of highly tyrosine phosphorylated proteins were observed as high background even without the stimulation through VLA/beta1 integrin. The high tyrosine phosphorylation pattern gradually disappeared and it was finally returned to normal tyrosine phosphorylation patterns by 2 years after BMT. Interestingly, poor expression of focal adhesion kinase (pp125FAK), a VLA/beta1-mediated signaling molecule, was observed within 1 year after BMT. These results suggest that these molecular defects appear to be implicated in the impaired VLA/beta1-mediated signaling in T cells from patients after allo-BMT, and it could explain, in part, the persistent immunoincompetent state after allo-BMT at least 1 year.
Asunto(s)
Trasplante de Médula Ósea , Integrinas/inmunología , Receptores Mensajeros de Linfocitos/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Adolescente , Adulto , Células Cultivadas , Humanos , Integrina alfa4beta1 , Integrina beta1/inmunología , Persona de Mediana Edad , Fosforilación , Receptores de Antígeno muy Tardío/inmunología , Trasplante Homólogo , TirosinaRESUMEN
Polymorphonuclear leucocyte (PMNL) accumulation in extravascular tissues and inflammatory exudates is dependent on their migration through blood vessel endothelium and then through connective tissue. Previously we utilized a barrier of human synovial and dermal fibroblasts (HSF or HDF) grown on microporous filters, as a model of PMNL migration through connective tissue. Those studies showed that beta 2 (CD18) and the beta 1 integrins, very late activation antigen-5 (VLA-5) and VLA-6, in part mediate this PMNL migration. Here we report that VLA-4, which can also be expressed at low levels on activated PMNL, is also involved in PMNL migration induced by C5a through fibroblast (HSF and HDF) barriers, because monoclonal antibody (mAb) to VLA-4 significantly inhibited (by 20-30%) PMNL migration. Blocking the function of CD18, VLA-5 or VLA-6 was not required for detection of the VLA-4-mediated migration. Combination treatment with mAb to VLA-4 and with mAb to VLA-5 or to VLA-6 further inhibited PMNL migration, irrespective of whether CD11/CD18 mechanisms were blocked with anti-CD18 mAb or not. Treatment of PMNL with a peptide based on the VLA-4-binding domain in the CS-1 fragment of fibronectin, but not a control peptide, inhibited PMNL migration to a comparable extent to treatment with mAb to VLA-4. A low level of VLA-4 was expressed on C5a-activated PMNL, detected by immunofluorescence flow cytometry. These results suggest that VLA-4 can be mobilized by human peripheral blood PMNL and can, in addition to VLA-5, VLA-6 and CD11/CD18 integrins, mediate PMNL migration through connective tissue. This is in marked contrast to PMNL transendothelial migration, where beta 1 integrins appear to play no significant role.
Asunto(s)
Tejido Conectivo/inmunología , Integrinas/inmunología , Neutrófilos/inmunología , Receptores Mensajeros de Linfocitos/inmunología , Receptores de Antígeno muy Tardío/inmunología , Antígenos CD18/inmunología , Proteínas Portadoras/farmacología , Técnicas de Cultivo de Célula , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Fibroblastos/inmunología , Humanos , Integrina alfa4beta1 , Oligopéptidos/farmacología , Receptores de Antígeno muy Tardío/metabolismo , Piel/inmunología , Membrana Sinovial/inmunologíaRESUMEN
The in vitro transendothelial migration of circulating filarial antigen-specific T-cells was examined in Wuchereria banerofti infection. Circulating T-cells from individuals with filaria-induced lymphatic pathology (LP) had significantly greater migration through unstimulated HUVEC monolayers than did T-cells from asymptomatic infected (MF) individuals (P = 0.04). In contrast to the MF individuals where no effect was seen, transendothelial migration of 48-hr filarial antigen stimulated T-cells from LP individuals was significantly (P = 0.01) greater than migration of 48-hr media-stimulated T-cells. In six of seven patients examined, inhibition of the VLA-4/VCAM-1 pathway resulted in greater than 50% inhibition of transendothelial migration of T-cells.
Asunto(s)
Filariasis Linfática/patología , Linfocitos T/citología , Wuchereria bancrofti , Adulto , Animales , Anticuerpos Monoclonales/fisiología , Unión Competitiva , Movimiento Celular/efectos de los fármacos , Endotelio Vascular/citología , Femenino , Humanos , Integrina alfa4beta1 , Integrinas/fisiología , Masculino , FN-kappa B/antagonistas & inhibidores , Receptores Mensajeros de Linfocitos/fisiología , Receptores de Antígeno muy Tardío/inmunología , Venas Umbilicales/citología , Molécula 1 de Adhesión Celular Vascular/farmacologíaRESUMEN
In this study, we investigated the involvement of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), very late activation antigen-4 (VLA-4), lymphocyte function-associated antigen 1 (LFA-1) and macrophage antigen-1 (Mac-1) on eosinophil infiltration in the cutaneous late phase response (LPR) in OVA-sensitized Balb/c mice by two approaches, immunostaining and inhibition assays with each monoclonal antibody. The eosinophil infiltration into the skin reached a peak at 12 hr after an intradermal challenge with OVA. Infiltrated eosinophils and mononuclear cells in the skin expressed Mac-1 (eosinophils: 38.9 +/- 1.55%, mononuclear cells: 51.2 +/- 2.15%), LFA-1 (eosinophils: 33.3 +/- 0.95%, mononuclear cells: 23.1 +/- 1.07%) and VLA-4 (eosinophils: 14.3 +/- 1.6%, mononuclear cells: 17.2 +/- 1.38%) at 12 h. Intraperitoneal administration of anti-mouse ICAM-1, VCAM-1, and VLA-4 monoclonal antibodies (mAb) before the challenge decreased the eosinophil infiltration by 66.2%, 61.0%, and 54.0%, respectively. On the other hand, pretreatment with anti-mouse LFA-1 mAb or Mac-1 mAb did not significantly decrease the infiltration. These results suggest that VCAM-1/VLA-4 interaction and ICAM-1 play important roles in eosinophil infiltration in cutaneous LPR.
Asunto(s)
Moléculas de Adhesión Celular/inmunología , Dermatitis/inmunología , Eosinófilos/inmunología , Ovalbúmina/inmunología , Inhibidores de Serina Proteinasa/inmunología , Piel/inmunología , Animales , Anticuerpos Monoclonales , Colorantes , Dermatitis/patología , Eosinófilos/patología , Femenino , Inmunización , Inmunohistoquímica , Inyecciones Intradérmicas , Integrina alfa4beta1 , Integrinas/inmunología , Molécula 1 de Adhesión Intercelular/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/patología , Antígeno-1 Asociado a Función de Linfocito/inmunología , Antígeno de Macrófago-1/inmunología , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/efectos adversos , Receptores Mensajeros de Linfocitos/inmunología , Receptores de Antígeno muy Tardío/inmunología , Inhibidores de Serina Proteinasa/efectos adversos , Piel/patología , Molécula 1 de Adhesión Celular Vascular/inmunologíaAsunto(s)
Empalme Alternativo , Fibronectinas/biosíntesis , Trasplante de Corazón/inmunología , Fragmentos de Péptidos/farmacología , Linfocitos T/inmunología , Animales , Fibronectinas/química , Fibronectinas/inmunología , Variación Genética , Trasplante de Corazón/patología , Terapia de Inmunosupresión/métodos , Integrina alfa4beta1 , Integrinas/antagonistas & inhibidores , Integrinas/fisiología , Ratas , Ratas Endogámicas Lew , Ratas Endogámicas , Receptores Mensajeros de Linfocitos/antagonistas & inhibidores , Receptores Mensajeros de Linfocitos/fisiología , Receptores de Antígeno muy Tardío/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/patología , Factores de Tiempo , Trasplante HomólogoRESUMEN
The mechanisms by which T lymphocytes are transformed from passively transported cells during circulation in the vascular system to actively migrating cells during extravasation are unknown. Therefore, the possibility that lymphocyte receptors are capable of inducing motility was investigated using a modified Boyden chamber assay. Cross-linking of alphaL beta2 and alpha4 beta1 on human T lymphocytes (T cell line and peripheral blood T cells) with immobilized mAbs induced motile behavior on fibronectin, laminin, collagen type IV, and poly-L-lysine. This induction of T cell migration was very potent and in most cases more efficient than pretreatment of the cells with phorbol esters. In contrast, control Abs to several other integrin- and non-integrin molecules present on T lymphocytes did not induce T cell migration. Anti-CD3 Abs themselves did not trigger motile behavior. However, anti-CD3 promoted T cell migration in the Boyden chamber system if present simultaneously with 40-kDa alpha4 beta1 binding fibronectin fragments or alphaL beta2 binding intercellular adhesion molecule-1/hIgG1Fc fusion proteins on the upper side of the filter. Abs to other surface components on T cells did not trigger motility when presented together with the 40-kDa fibronectin fragments or the intercellular adhesion molecule-1/hIgG1Fc fusion proteins. The induction of motile behavior could be blocked if the T cells were pretreated with Genistein and Calphostin C, indicating the involvement of a protein tyrosine kinase and protein kinase C-dependent signaling pathway in triggering of T cell motility via integrins. These results indicate that alphaL beta2 and alpha4 beta1 on T lymphocytes can selectively trigger motile behavior when cross-linked by their endothelial or extracellular matrix ligands. Furthermore, these data indicate that cross-linking of CD3 facilitates ligand binding and subsequent triggering of a motile phenotype by alphaL beta2 and alpha4 beta1.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Movimiento Celular/inmunología , Integrinas/inmunología , Integrinas/metabolismo , Antígeno-1 Asociado a Función de Linfocito/inmunología , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Receptores Mensajeros de Linfocitos/inmunología , Receptores Mensajeros de Linfocitos/metabolismo , Linfocitos T/inmunología , Adulto , Complejo CD3/inmunología , Complejo CD3/metabolismo , Adhesión Celular/inmunología , Línea Celular , Células Clonales , Humanos , Integrina alfa4beta1 , Integrinas/biosíntesis , Ligandos , Antígeno-1 Asociado a Función de Linfocito/biosíntesis , Unión Proteica/inmunología , Receptores Mensajeros de Linfocitos/biosíntesis , Receptores de Antígeno muy Tardío/biosíntesis , Receptores de Antígeno muy Tardío/inmunología , Receptores de Antígeno muy Tardío/metabolismo , Linfocitos T/metabolismoAsunto(s)
Anticuerpos/uso terapéutico , Ciclosporina/uso terapéutico , Supervivencia de Injerto/inmunología , Inmunosupresores/uso terapéutico , Integrinas/inmunología , Intestino Delgado/trasplante , Receptores Mensajeros de Linfocitos/inmunología , Trasplante Homólogo/inmunología , Animales , Sinergismo Farmacológico , Integrina alfa4beta1 , Prueba de Cultivo Mixto de Linfocitos , Ratas , Ratas Endogámicas BN , Ratas Endogámicas Lew , Receptores de Antígeno muy Tardío/inmunología , Factores de Tiempo , Trasplante HeterotópicoRESUMEN
Accumulating evidence suggests that the VLA/CD29 molecule plays an important role in T-cell costimulation, and CD4+CD29/VLA+ memory T cells play a key role in induction of CD8 killer effector T cells which are considered to be a major population involved in graft rejection. To target limited elements of the T-lymphocyte population, we have described the preparation of a bispecific antibody-toxin conjugate designed to target CD4+CD29+ memory T cells. We also showed that the solid-phase crosslinking of VLA-4 by the antibody against this molecule or by its ligand, the CS-1 region of fibronectin, stimulates tyrosine phosphorylation of 140, 120-105, 80-70, 60-55, 50 and 45 kilodalton proteins. In addition, we identified the pp140 protein as PLC gamma, pp120 protein as pp125FAK, pp70 and pp50 proteins as paxillin, and pp60-55 proteins as pp59fyn and pp56lck, and pp45 as MAP kinase, respectively. Moreover, we demonstrated that pp125FAK is directly associated with paxillin. The paxillin binding domain of pp125FAK is homologous to the paxillin binding domain of vinculin. Mutations in the conserved amino acid residues between pp125FAK and vinculin result in the loss of paxillin-binding activity. Because VLA/CD29 is preferentially expressed on CD4 memory T cells, the above described system will be used to develop a novel drug design for providing selective immunosuppression useful for organ transplantation.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Linfocitos T CD8-positivos/inmunología , Rechazo de Injerto/prevención & control , Integrina beta1/inmunología , Receptores de Antígeno muy Tardío/inmunología , Anticuerpos Monoclonales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/citología , Moléculas de Adhesión Celular , Proteínas del Citoesqueleto/uso terapéutico , Diseño de Fármacos , Rechazo de Injerto/inmunología , Humanos , Integrina beta1/genética , Peso Molecular , Trasplante de Órganos , Paxillin , Fosfoproteínas/uso terapéutico , Fosforilación , Receptores de Antígeno muy Tardío/efectos de los fármacos , Receptores de Antígeno muy Tardío/genética , Vinculina/uso terapéuticoAsunto(s)
Anticuerpos Monoclonales/uso terapéutico , Trasplante de Córnea/inmunología , Rechazo de Injerto/prevención & control , Supervivencia de Injerto/inmunología , Inmunosupresores/uso terapéutico , Integrinas/inmunología , Receptores Mensajeros de Linfocitos/inmunología , Animales , Trasplante de Córnea/patología , Integrina alfa4beta1 , Linfocitos/inmunología , Linfocitos/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Receptores de Antígeno muy Tardío/inmunología , Trasplante Homólogo , Trasplante IsogénicoRESUMEN
Blood neutrophils contribute to joint injury in human and experimental models of arthritis. Neutrophil migration out of the blood in joint inflammation involves both the CD18 (beta2) integrins and a CD18 integrin-independent pathway. To investigate this migration, radiolabeled rat blood neutrophils were used to measure neutrophil accumulation in the inflamed joints of rats with adjuvant arthritis and the role of leukocyte integrins in migration to these joints and to dermal inflammation was determined. Neutrophils migrated rapidly (<2 h) to the inflamed joints 14-18 d after immunization with adjuvant. Blocking monoclonal antibodies (mAbs) to both LFA-1 and Mac-1 together, as well as a mAb to CD18, inhibited neutrophil accumulation in the inflamed joints by 50-75%. However, migration to dermal inflammation induced by C5a(des Arg)' tumor necrosis factor alpha, lipopolysaccharide, and poly-inosine:cytosine was inhibited by approximately 90%. Flow cytometry revealed the expression of low levels of very late antigen 4 (VLA-4) on nearly all rat blood neutrophils. Treatment with anti-VLA-4 plus anti-LFA-1 but neither mAb alone, strongly (60-75%) inhibited neutrophil accumulation in arthritic joints. This mAb combination also inhibited neutrophil migration to dermal inflammatory reactions by 30-70%. Blocking VLA-4 together with the CD18 integrins inhibited neutrophil accumulation by 95-99%, virtually abolishing neutrophil accumulation in cutaneous inflammation. A similar blockade of VLA-4 and CD18 decreased neutrophil accumulation in the inflamed joints by 70-83%, but a significant portion of the neutrophil accumulation to these joints still remained. In conclusion, rat blood neutrophils express functional VLA-4 that can mediate neutrophil migration to both inflamed joints and dermal inflammatory sites. VLA-4 appears to be able to substitute for LFA-1 in this migration and is particularly important for accumulation in inflamed joints. However, there exists an additional CD18- and VLA-4-independent pathway of neutrophil migration to arthritic joints that is not involved in acute dermal inflammation.
Asunto(s)
Artritis Experimental/fisiopatología , Quimiotaxis de Leucocito , Integrinas/biosíntesis , Articulaciones/fisiopatología , Neutrófilos/inmunología , Receptores Mensajeros de Linfocitos/biosíntesis , Animales , Anticuerpos Monoclonales/farmacología , Artritis Experimental/inmunología , Antígenos CD18/inmunología , Antígenos CD18/fisiología , Humanos , Inflamación , Integrina alfa4beta1 , Articulaciones/inmunología , Antígeno-1 Asociado a Función de Linfocito/inmunología , Antígeno-1 Asociado a Función de Linfocito/fisiología , Antígeno de Macrófago-1/inmunología , Antígeno de Macrófago-1/fisiología , Masculino , Mycobacterium/inmunología , Neutrófilos/fisiología , Ratas , Ratas Endogámicas Lew , Receptores de Antígeno muy Tardío/inmunología , Piel/inmunologíaRESUMEN
The beta 1-integrins (VLA family) are cellular adhesion molecules (CAM) that play a major role in cell-cell and cell-matrix interactions. The expression pattern of CAM was studied in 5 clinically normal volunteers with healthy gingiva and in 18 patients with clinically different stages of periodontitis. In healthy human gingiva alpha 2, alpha 3 and alpha 6 integrin chains were found in a characteristic distribution, showing a broad continuous expression on the junctional and sulcular epithelium sites. The expression of these integrins was demonstrated primarily on the basal cell layers and in some cells of the stratum spinosum. Inflammatory stages of periodontitis revealed further upregulation of alpha 2, alpha 3 and alpha 6 integrins into the junctional and sulcular epithelial cells, which correlated with the stage of the periodontitis and the extent of the cellular infiltration. alpha 4 and alpha 6 were found to be the predominant beta 1 integrin chains on inflammatory cells. The amount of alpha 4 and alpha 6 positive infiltrative cells increased with the number of inflammatory cells. VCAM-1, the corresponding cell-cell ligand of VLA-4 (alpha 4) was present on the majority of subepithelial vessels in all stages of gingivitis and periodontitis. The alpha 5 subunit was expressed on both endothelium and gingival connective tissue cells. Samples from advanced periodontitis cases showed a higher number of alpha 5 positive mononuclear cells. In comparison to normal epidermis, human gingival epithelial cells express higher levels of integrins. This expression is further upregulated in advanced stages of periodontitis, indicating changes of the beta 1 integrin organization.