RESUMO
Multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) cells must attach to the bone marrow (BM) microvasculature before lodging in the BM microenvironment. Using intravital microscopy (IVM) of the BM calvariae we demonstrate that the α4ß1 integrin is required for MM and CLL cell firm arrest onto the BM microvasculature, while endothelial P-selectin and E-selectin mediate cell rolling. Talin, kindlin-3 and ICAP-1 are ß1-integrin-binding partners that regulate ß1-mediated cell adhesion. We show that talin and kindlin-3 cooperatively stimulate high affinity and strength of α4ß1-dependent MM and CLL cell attachment, whereas ICAP-1 negatively regulates this adhesion. A functional connection between talin/kindlin-3 and Rac1 was found to be required for MM cell attachment mediated by α4ß1. Importantly, IVM analyses with talin- and kindlin-3-silenced MM cells indicate that these proteins are needed for cell arrest on the BM microvasculature. Instead, MM cell arrest is repressed by ICAP-1. Moreover, MM cells silenced for talin and kindlin-3, and cultured on α4ß1 ligands showed higher susceptibility to bortezomib-mediated cell apoptosis. Our results highlight the requirement of α4ß1 and selectins for the in vivo attachment of MM and CLL cells to the BM microvasculature, and indicate that talin, kindlin-3 and ICAP-1 differentially control physiological adhesion by regulating α4ß1 activity.
Assuntos
Medula Óssea/patologia , Adesão Celular , Endotélio Vascular/patologia , Integrina alfa4beta1/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Mieloma Múltiplo/patologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Apoptose , Western Blotting , Medula Óssea/metabolismo , Movimento Celular , Proliferação de Células , Citoplasma/metabolismo , Selectina E/genética , Selectina E/metabolismo , Endotélio Vascular/metabolismo , Citometria de Fluxo , Humanos , Integrina alfa4beta1/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microscopia Intravital , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Microvasos , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Selectina-P/genética , Selectina-P/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Talina/genética , Talina/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
La mayoría de los antígenos que entran en contacto con el sistema inmune durante la vida de un ser vivo lo hacen a través de la superficie de la mucosa de los tractos respiratorio, gastrointestinal y urogenital. Ocupan una superficie de 400 m2 y forman el área de mayor tamaño en contacto directo con el ambiente externo. Las mucosas separan el ambiente externo del ambiente interno, estéril, y representan una primera línea de defensa. Esta barrera está en contacto tanto con patógenos que han desarrollado mecanismos eficaces para la colonización de epitelios e invasión de mucosas, como con antígenos inocuos, tales como comida, o la flora bacteriana comensal. En el primer caso se necesita una respuesta inmune eficaz y robusta, mientras que en el segundo se requiere una respuesta caracterizada por ignorancia o supresión activa. En estas condiciones, las mucosas han desarrollado un complejo sistema inmune, con características anatómicas y funcionales particulares, capaz de generar rigurosas respuestas frente a antígenos patogénicos, mientras mantiene una situación de ignorancia o supresión activa frente a antígenos no patogénicos (AU)
Assuntos
Humanos , Mucosa/imunologia , Imunidade nas Mucosas/imunologia , Antígenos de Histocompatibilidade Classe II , Células Epiteliais/imunologia , Células Dendríticas/imunologia , Enterócitos/imunologia , Tecido Linfoide/imunologia , Imunoglobulina A/metabolismo , Nódulos Linfáticos Agregados/imunologiaRESUMO
BACKGROUND: T lymphocytes play a crucial role in the pathogenesis of inflammatory bowel disease. Achieving stable T-cell lines, rather than continuous bleeding of patients, is desirable in order to dissect their implication in the disease. METHODS: Long-lasting T-cell lines from patients with Crohn disease and ulcerative colitis and from healthy volunteers have been obtained by transformation of T lymphocytes using the lymphotropic Herpesvirus saimiri. Lines were subjected to phenotypic and functional analyses, and the results compared with freshly isolated peripheral blood mononuclear cells. RESULTS: Fresh cells revealed only minor differences between patients and controls, with regard to phenotype and proliferative capacity. In contrast, the use of T-cell lines showed that cells from Crohn disease patients, but not ulcerative colitis patients, over-responded to several membrane or cytoplasmic stimuli when compared to control T-cell lines. Thus, higher responses were found when stimulated with alphaCD3 and IL2, alphaCD3 and alphaCD28, IL2 alone, phorbol esters (PMA) and alphaCD3 and, finally, PMA and alphaCD2 (P < 0.05 in all instances). Further, lines from patients with Crohn disease responded more vigorously to alphaCD3 and alphaCD28 or alphaCD3 and PMA when compared to ulcerative colitis (P < 0.05 in both instances). CONCLUSIONS: The data obtained with these lines suggest that T cells from patients with Crohn disease differ in vivo in their proliferative capacity, as compared with those from ulcerative colitis patients, a finding that may reflect the clear Th-1 phenotype found in the former and absent in the latter.