RESUMEN
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.
Asunto(s)
Movimiento Celular/inmunología , Quimiocinas CXC/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Proteínas de Unión al GTP Heterotriméricas/fisiología , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Miosinas/fisiología , Linfocitos T/metabolismo , Proteína de Unión al GTP cdc42/fisiología , Proteína de Unión al GTP rhoA/fisiología , Animales , Inhibición de Migración Celular , Movimiento Celular/genética , Quimiocina CXCL12 , Quimiocinas CXC/antagonistas & inhibidores , Dactinomicina/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Proteínas Activadoras de GTPasa/fisiología , Vectores Genéticos/síntesis química , Vectores Genéticos/inmunología , Proteínas de Unión al GTP Heterotriméricas/genética , Hibridomas/citología , Hibridomas/efectos de los fármacos , Hibridomas/metabolismo , Ratones , Células del Estroma/fisiología , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Transfección , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP rhoA/genéticaRESUMEN
The migration of leukocytes into tissues is regulated by chemokines and other chemotactic factors that act on receptors that signal through Gi proteins. It seems likely that the colonization of tissues during dissemination of hematopoietic tumor cells is similarly regulated. In fact, dissemination of a T-cell hybridoma, a model for T lymphoma, was blocked when Gi proteins were inactivated by the S1 catalytic subunit of pertussis toxin that had been transfected into those cells. Pertussis toxin S1 blocked dissemination of MDAY-D2 murine myeloid leukemia cells to the liver and spleen, as in T-cell hybridoma cells, but it did not prevent bone marrow colonization. In contrast, overexpression of a function-defective mutant of the Gq/11 protein blocked dissemination to the bone marrow and also prevented Gq/11 dissemination to the liver and spleen. This indicates that the influx of these myeloid cells into all tissues requires the Gq/11 protein in addition to the Gi protein in the liver and spleen. (Blood. 2000;96:691-698)
Asunto(s)
Médula Ósea/patología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Proteínas de Unión al GTP/fisiología , Leucemia Mieloide/patología , Infiltración Leucémica , Hígado/patología , Bazo/patología , Animales , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Expresión Génica , Ratones , Ratones Endogámicos DBA , Trasplante de Neoplasias , Toxina del Pertussis , Transfección , Células Tumorales Cultivadas , Factores de Virulencia de Bordetella/genética , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
The ZAP-70 tyrosine kinase is essential for T cell activation by the T cell receptor. We show that ZAP-70 is also required for migration of T cells that is dependent on the integrin LFA-1. Invasion of TAM2D2 T cell hybridoma cells into fibroblast monolayers, which is LFA-1-dependent, was blocked by overexpression of dominant-negative ZAP-70 and by piceatannol but not by herbimycin A. The Syk inhibitor piceatannol blocks the Syk homologue ZAP-70, which is expressed by TAM2D2 cells, with the same dose dependence as the inhibition of invasion. Dominant-negative ZAP-70 completely inhibited the extensive metastasis formation of TAM2D2 cells to multiple organs upon i.v. injection into mice. Migration of TAM2D2 cells through filters coated with the LFA-1 ligand ICAM-1, induced by 1 ng/ml of the chemokine SDF-1, was blocked by anti-LFA-1 mAb and also abrogated by dominant-negative ZAP-70 and piceatannol. In contrast, migration induced by 100 ng/ml SDF-1 was independent of both LFA-1 and ZAP-70. LFA-1 cross-linking induced tyrosine phosphorylation, which was blocked by dominant-negative ZAP-70 and piceatannol. We conclude that LFA-1 engagement triggers ZAP-70 activity that is essential for LFA-1-dependent migration.
Asunto(s)
Movimiento Celular/fisiología , Antígeno-1 Asociado a Función de Linfocito/fisiología , Proteínas Tirosina Quinasas/fisiología , Linfocitos T/metabolismo , Animales , Benzoquinonas , Quimiocina CXCL12 , Quimiocinas CXC/farmacología , Fibroblastos , Expresión Génica/genética , Humanos , Hibridomas/metabolismo , Integrinas/fisiología , Molécula 1 de Adhesión Intercelular/metabolismo , Lactamas Macrocíclicas , Ratones , Metástasis de la Neoplasia/fisiopatología , Fosfotirosina/metabolismo , Quinonas/farmacología , Ratas , Rifabutina/análogos & derivados , Estilbenos/farmacología , Factores de Virulencia de Bordetella/farmacología , Proteína Tirosina Quinasa ZAP-70RESUMEN
HT-29 colon carcinoma cells form liver metastases upon intrasplenic injection, and adhesion to fibronectin under the liver microvascular liver endothelium is likely to be important for metastasis formation. We have therefore studied the integrins involved in fibronectin adhesion. This was not affected by blocking antibodies against the beta1, alpha3, and alpha5 integrin subunits, but it was blocked by an RGD-containing peptide, indicating involvement of RGD-dependent non-beta1 alphaV integrins. Both alphaVbeta5 and alphaVbeta6 were detected on HT-29 cells. Blocking mAb against alphaV, but not against alphaVbeta5, abolished adhesion. From a HT-29 cell lysate, only alphaVbeta6 bound to a fibronectin-Sepharose column. Thus, alphaVbeta6 is the main fibronectin receptor on HT-29 cells, despite the very low levels of alphaVbeta6 and the much higher levels of alphaVbeta5. The HT29 cells did not spread on fibronectin in the absence of serum, not even after a three- to fourfold increase in alphaVbeta6 levels, induced by interleukin 4. The cells did spread on vitronectin. Using immunofluorescence we observed that both on vitronectin and on fibronectin alphaVbeta5 was arranged in a striped pattern, aligned with actin fibers, and not in focal adhesions. On fibronectin, but not on vitronectin, alphaVbeta6 was concentrated in a punctate pattern at the periphery of cell islands.