RESUMO
Sphingosine-1-phosphate receptor 1 (S1P(1)) was recently shown to be required for lymphocyte egress from lymphoid organs. Here we have examined the relationship between S1P(1) abundance on the cell and egress efficiency. Using an integrin neutralization approach to separate the processes of entry and exit, we show that pertussis toxin treatment reduces lymphocyte egress from lymph nodes. Retrovirally mediated S1P(1) overexpression is sufficient to reduce B cell accumulation in the splenic white pulp and to promote egress of activated T cells from lymph nodes, whereas S1P(1)(+/-) cells have reduced lymph node exit efficiency. Furthermore, lymphocyte S1P(1) is down-regulated in the blood, up-regulated in lymphoid organs, and down-regulated again in the lymph. We propose that cyclical ligand-induced modulation of S1P(1) on circulating lymphocytes contributes to establishing their lymphoid organ transit time.
Assuntos
Linfócitos B/metabolismo , Tecido Linfoide/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Linfócitos T/metabolismo , Animais , Cadeias alfa de Integrinas/metabolismo , Cadeias beta de Integrinas/metabolismo , Linfonodos/metabolismo , Tecido Linfoide/citologia , Camundongos , Receptores de Lisoesfingolipídeo/genética , Baço/citologiaRESUMO
The steps involved in lymphocyte homing to the white pulp cords of the spleen are poorly understood. We demonstrate here that the integrins lymphocyte function associated (LFA)-1 and alpha 4 beta 1 make essential and mostly overlapping contributions necessary for B cell migration into white pulp cords. T cell entry to the white pulp is also reduced by blockade of LFA-1 and alpha 4 beta 1. The LFA-1 ligand, intercellular adhesion molecule 1 is critical for lymphocyte entry and both hematopoietic cells and radiation-resistant cells contribute to this requirement. Vascular cell adhesion molecule 1 contributes to the alpha 4 beta 1 ligand requirement and a second ligand, possibly fibronectin, also plays a role. By contrast with the entry requirements, antigen-induced movement of B cells from follicles to the outer T zone is not prevented by integrin blocking antibodies. Comparison of the distribution of integrin-blocked B cells and B cells treated with the G alpha i inhibitor, pertussis toxin, early after transfer reveals in both cases reduced accumulation in the inner marginal zone. These observations suggest that chemokine receptor signaling and the integrins LFA-1 and alpha 4 beta 1 function together to promote lymphocyte transit from the marginal zone into white pulp cords.
Assuntos
Integrinas/metabolismo , Linfócitos/citologia , Linfócitos/imunologia , Baço/citologia , Baço/imunologia , Transferência Adotiva , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Movimento Celular , Integrina alfa4beta1/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Ligantes , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores de Quimiocinas/metabolismo , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
Adaptive immunity depends on T-cell exit from the thymus and T and B cells travelling between secondary lymphoid organs to survey for antigens. After activation in lymphoid organs, T cells must again return to circulation to reach sites of infection; however, the mechanisms regulating lymphoid organ exit are unknown. An immunosuppressant drug, FTY720, inhibits lymphocyte emigration from lymphoid organs, and phosphorylated FTY720 binds and activates four of the five known sphingosine-1-phosphate (S1P) receptors. However, the role of S1P receptors in normal immune cell trafficking is unclear. Here we show that in mice whose haematopoietic cells lack a single S1P receptor (S1P1; also known as Edg1) there are no T cells in the periphery because mature T cells are unable to exit the thymus. Although B cells are present in peripheral lymphoid organs, they are severely deficient in blood and lymph. Adoptive cell transfer experiments establish an intrinsic requirement for S1P1 in T and B cells for lymphoid organ egress. Furthermore, S1P1-dependent chemotactic responsiveness is strongly upregulated in T-cell development before exit from the thymus, whereas S1P1 is downregulated during peripheral lymphocyte activation, and this is associated with retention in lymphoid organs. We find that FTY720 treatment downregulates S1P1, creating a temporary pharmacological S1P1-null state in lymphocytes, providing an explanation for the mechanism of FTY720-induced lymphocyte sequestration. These findings establish that S1P1 is essential for lymphocyte recirculation and that it regulates egress from both thymus and peripheral lymphoid organs.
Assuntos
Movimento Celular , Linfócitos/citologia , Linfócitos/metabolismo , Tecido Linfoide/citologia , Lisofosfolipídeos , Receptores Acoplados a Proteínas G/metabolismo , Esfingosina/análogos & derivados , Timo/citologia , Transferência Adotiva , Animais , Movimento Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Quimera/metabolismo , Regulação para Baixo/efeitos dos fármacos , Cloridrato de Fingolimode , Deleção de Genes , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Tecido Linfoide/efeitos dos fármacos , Tecido Linfoide/imunologia , Camundongos , Camundongos Knockout , Propilenoglicóis/farmacologia , Receptores Acoplados a Proteínas G/genética , Receptores de Lisofosfolipídeos , Esfingosina/farmacologia , Timo/efeitos dos fármacos , Timo/imunologiaRESUMO
Wnt proteins are conserved axon guidance cues that control growth cone navigation. However, the intracellular signaling mechanisms that mediate growth cone turning in response to Wnts are unknown. We previously showed that Wnt-Frizzled signaling directs spinal cord commissural axons to turn anteriorly after midline crossing through an attractive mechanism. Here we show that atypical protein kinase C (aPKC), is required for Wnt-mediated attraction of commissural axons and proper anterior-posterior (A-P) pathfinding. A PKCzeta pseudosubstrate, a specific blocker of aPKC activity, and expression of a kinase-defective PKCzeta mutant in commissural neurons resulted in A-P randomization in "open-book" explants. Upstream of PKCzeta, heterotrimeric G-proteins and phosphatidylinositol-3-kinases (PI3Ks), are also required for A-P guidance, because pertussis toxin, wortmannin, and expression of a p110gamma kinase-defective construct all resulted in A-P randomization. Overexpression of p110gamma, the catalytic subunit of PI3Kgamma, caused precocious anterior turning of commissural axons before midline crossing in open-book explants and caused dissociated precrossing commissural axons, which are normally insensitive to Wnt attraction, to turn toward Wnt4-expressing cells. Therefore, we propose that atypical PKC signaling is required for Wnt-mediated A-P axon guidance and that PI3K can act as a switch to activate Wnt responsiveness during midline crossing.
Assuntos
Axônios/fisiologia , Neurônios/citologia , Fosfatidilinositol 3-Quinases/fisiologia , Transdução de Sinais/fisiologia , Medula Espinal/embriologia , Proteínas Wnt/metabolismo , Animais , Axônios/efeitos dos fármacos , Células COS , Chlorocebus aethiops , Eletroporação/métodos , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Mutação/fisiologia , Técnicas de Cultura de Órgãos , Proteína Quinase C/genética , Medula Espinal/citologia , Transfecção , Proteínas Wnt/farmacologia , Proteína Wnt4RESUMO
The mechanism by which sphingosine-1-phosphate receptor-1 (S1P1) acts to promote lymphocyte egress from lymphoid organs is not defined. Here, we showed that CCR7-deficient T cells left lymph nodes more rapidly than wild-type cells did, whereas CCR7-overexpressing cells were retained for longer. After treatment with FTY720, an agonist that causes downmodulation of lymphocyte S1P1, CCR7-deficient T cells were less effectively retained than wild-type T cells. Moreover, treatment with pertussis toxin to inactivate signaling via G alpha i-protein-coupled receptors restored egress competence to S1P1-deficient lymphocytes. We also found that T cell accumulation in lymph node cortical sinusoids required intrinsic S1P1 expression and was antagonized by CCR7. These findings suggest a model where S1P1 acts in the lymphocyte to promote lymph node egress by overcoming retention signals mediated by CCR7 and additional G alpha i-coupled receptors. Furthermore, by simultaneously upregulating S1P1 and downregulating CCR7, T cells that have divided multiple times switch to a state favoring egress over retention.
Assuntos
Quimiotaxia de Leucócito/imunologia , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Linfonodos/imunologia , Receptores de Lisoesfingolipídeo/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Transferência Adotiva , Animais , Citometria de Fluxo , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/imunologia , Imuno-Histoquímica , Linfonodos/citologia , Linfonodos/metabolismo , Camundongos , Camundongos Transgênicos , Receptores CCR7/imunologia , Receptores CCR7/metabolismo , Receptores de Lisoesfingolipídeo/imunologia , Linfócitos T/metabolismoRESUMO
Activated Cdc42-associated kinase-2 (ACK2) is a non-receptor tyrosine kinase that serves as a specific effector for Cdc42, a Rho family small G-protein. Recently, we have found that ACK2 directly interacts with clathrin heavy chain through a clathrin-binding motif that is conserved in all endocytic adaptor proteins and regulates clathrin assembly, suggesting that ACK2 plays a role in clathrin-coated vesicle endocytosis (Yang, W., Lo, C. G., Dispenza, T., and Cerione, R. A. (2001) J. Biol. Chem. 276, 17468-17473). Here we report the identification of another binding partner for ACK2 that has previously been implicated in endocytosis, namely the sorting nexin protein SH3PX1 (sorting nexin 9). The interaction occurs between a proline-rich domain of ACK2 and the Src homology 3 domain (SH3) of SH3PX1. Co-immunoprecipitation studies indicate that ACK2, clathrin, and SH3PX1 form a complex in cells. Epidermal growth factor (EGF) stimulated the tyrosine phosphorylation of SH3PX1, whereas co-transfection of ACK2 with SH3PX1 resulted in the constitutive phosphorylation of SH3PX1. However, co-transfection of the kinase-dead mutant ACK2(K158R) with SH3PX1 blocked EGF-induced tyrosine phosphorylation of SH3PX1, indicating that the EGF-stimulated phosphorylation of SH3PX1 is mediated by ACK2. EGF receptor levels were significantly decreased following EGF stimulation of cells co-expressing ACK2 and SH3PX1, thus highlighting a novel role for ACK2, working together with SH3PX1 to promote the degradation of the EGF receptor.