RESUMO
The dissemination of T cell hybridomas to multiple nonhematopoietic tissues is blocked by pertussis toxin, suggesting the involvement of a chemokine. To study whether this chemokine is SDF-1, we employed a strategy proposed previously for gene therapy of AIDS, whereby the SDF-1 receptor CXCR4 (also a coreceptor for HIV) is retained in the endoplasmic reticulum (ER) and fails to reach the cell surface. We transfected SDF-1, carrying an ER retention sequence, into a T cell hybridoma. This altered chemokine is retained in the ER, where it binds CXCR4 and prevents the latter protein from reaching the surface. These cells failed to migrate toward SDF-1 or to invade fibroblast monolayers, although they could still migrate toward thymus and activation-regulated chemokine (TARC) and invade TARC-treated monolayers. Furthermore, the ability of the transfected cells to disseminate to multiple organs upon intravenous injection into mice was abolished. This dissemination reflects the in vivo migration patterns of activated and memory T cells into nonhematopoietic tissues, which is thus likely to depend on CXCR4. Attempts to block CXCR4 function as a therapy for AIDS may affect this migration with consequences for T cell function. Our results also suggest a decisive role for CXCR4 in the dissemination of hematopoietic malignancies expressing this receptor.
Assuntos
Retículo Endoplasmático/metabolismo , Hibridomas/metabolismo , Receptores CXCR4/metabolismo , Linfócitos T/metabolismo , Animais , Membrana Celular/metabolismo , Transplante de Células , Quimiocina CCL17 , Quimiocina CXCL12 , Quimiocinas CC/genética , Quimiocinas CC/farmacologia , Quimiocinas CXC/química , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Terapia Genética/métodos , Células-Tronco Hematopoéticas/metabolismo , Hibridomas/citologia , Hibridomas/imunologia , Memória Imunológica , Camundongos , Camundongos Nus , Mutação , Metástase Neoplásica , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Sinais Direcionadores de Proteínas/genética , Receptores CXCR4/análise , Receptores CXCR4/antagonistas & inibidores , Transdução de Sinais , Linfócitos T/imunologia , TransfecçãoRESUMO
Dissemination of T cell hybridomas in mice, a model for in vivo migration of memory T cells and for T lymphoma metastasis, depends on the chemokine stromal cell-derived factor-1 (SDF-1) and the integrin LFA-1 and correlates well with invasion into fibroblast cultures. In addition to the known role of the pertussis toxin-sensitive heterotrimeric GTPase G(i), we show that also the pertussis toxin-insensitive GTPase G(q/11) is required for dissemination and invasion. Furthermore, we show that the small GTPases, Cdc42 and RhoA, are involved, and that invasion is blocked by inhibitors of actinomyosin contraction. G(q/11), RhoA, and contraction are specifically required for LFA-1 activation, since 1) they are essential for LFA-1-dependent migration toward low SDF-1 concentrations through ICAM-1-coated filters, but not for migration toward high SDF-1 levels, which is LFA-1 independent; 2) G protein (AlF(4)(-))-induced adhesion to ICAM-1 requires RhoA and contraction; 3) constitutively active G(q) induces aggregation, mediated by LFA-1. We previously reported that binding of this activated LFA-1 to ICAM-1 triggers a signal, transduced by the zeta-associated protein 70 tyrosine kinase, that activates additional LFA-1 molecules. This amplification of LFA-1 activation is essential for invasion. We show here that zeta-associated protein 70-induced LFA-1 activation requires neither Cdc42 and RhoA nor contraction and is thus quite different from that induced by SDF-1. We conclude that two modes of LFA-1 activation, with distinct underlying mechanisms, are required for the in vivo migration of T cell hybridomas.