Carlos Martínez-A

Segregation of leading-edge and uropod components into specific lipid rafts during T cell polarization

Redistribution of specialized molecules in migrating cells develops asymmetry between two opposite cell poles, the leading edge and the uropod. We show that acquisition of a motile phenotype in T lymphocytes results in the asymmetric redistribution of ganglioside GM3- and GM1-enriched raft domains to the leading edge and to the uropod, respectively. This segregation to each cell pole parallels the specific redistribution of membrane proteins associated to each raft subfraction. Our data suggest that raft partitioning is a major determinant for protein redistribution in polarized T cells, as ectopic expression of raft-associated proteins results in their asymmetric redistribution, whereas non-raft-partitioned mutants of these proteins are distributed homogeneously in the polarized cell membrane. Both acquisition of a migratory phenotype and SDF-1α-induced chemotaxis are cholesterol depletion-sensitive. Finally, GM3 and GM1 raft redistribution requires an intact actin cytoskeleton, but is insensitive to microtubule disruption. We propose that membrane protein segregation not only between raft and nonraft domains but also between distinct raft subdomains may be an organizational principle that mediates redistribution of specialized molecules needed for T cell migration.

The chemokine SDF-1α triggers CXCR4 receptor dimerization and activates the JAK/STAT pathway

The chemokine stromal cell‐derived factor (SDF‐1α), the ligand for the CXCR4 receptor, induces a wide variety of effects that include calcium mobilization, chemotactic responses, bone marrow myelopoiesis, neuronal patterning, and prevention of HIV‐1 infection. Nonetheless, little is known of the biochemical pathways required to achieve this variety of responses triggered after receptor—chemokine interaction. We developed a set of monoclonal antibodies that specifically recognize the CXCR4 receptor and used them to identify the signaling pathway activated after SDF‐1α binding in human T cell lines. Here we demonstrate that SDF‐1α activation promotes the physical association of Gαi with the CXCR4. Furthermore, within seconds of SDF‐1α activation, the CXCR4 receptor becomes tyrosine phosphorylated through the activation and association with the receptor of JAK2 and JAK3 kinases. After SDF‐1α binding, JAK2 and JAK3 associate with CXCR4 and are activated, probably by transphosphorylation, in a Gαi‐independent manner. This activation enables the recruitment and tyrosine phosphorylation of several members of the STAT family of transcription factors. Finally, we have also observed SDF‐1α‐induced activation and association of the tyrosine phosphatase Shpl with the CXCR4 in a Gαi‐dependent manner. As occurs with the cytokine receptors in response to cytokines, the CXCR4 undergoes receptor dimerization after SDF‐1α binding and is a critical step in triggering biological responses. We present compelling evidence that the chemokines signal through mechanisms similar to those activated by cytokines.—Vila‐Coro, A. J., Rodríguez‐Frade, J. M., Martín de Ana, A., Moreno‐Ortíz, M. C., Martínez‐A., C., Mellado, M. The chemokine SDF‐1 α triggers CXCR4 receptor dimerization and activates the JAK/STAT pathway. FASEB J. 13, 1699–1710 (1999)

Disrupted cardiac development but normal hematopoiesis in mice deficient in the second CXCL12/SDF-1 receptor, CXCR7

Chemotactic cytokines (chemokines) attract immune cells, although their original evolutionary role may relate more closely with embryonic development. We noted differential expression of the chemokine receptor CXCR7 (RDC-1) on marginal zone B cells, a cell type associated with autoimmune diseases. We generated Cxcr7−/− mice but found that CXCR7 deficiency had little effect on B cell composition. However, most Cxcr7−/− mice died at birth with ventricular septal defects and semilunar heart valve malformation. Conditional deletion of Cxcr7 in endothelium, using Tie2-Cre transgenic mice, recapitulated this phenotype. Gene profiling of Cxcr7−/− heart valve leaflets revealed a defect in the expression of factors essential for valve formation, vessel protection, or endothelial cell growth and survival. We confirmed that the principal chemokine ligand for CXCR7 was CXCL12/SDF-1, which also binds CXCR4. CXCL12 did not induce signaling through CXCR7; however, CXCR7 formed functional heterodimers with CXCR4 and enhanced CXCL12-induced signaling. Our results reveal a specialized role for CXCR7 in endothelial biology and valve development and highlight the distinct developmental role of evolutionary conserved chemokine receptors such as CXCR7 and CXCR4.

Eosinophil recruitment to the lung in a murine model of allergic inflammation. The role of T cells, chemokines, and adhesion receptors.

Eosinophil accumulation is a distinctive feature of lung allergic inflammation. Here, we have used a mouse model of OVA (ovalbumin)-induced pulmonary eosinophilia to study the cellular and molecular mechanisms for this selective recruitment of eosinophils to the airways. In this model there was an early accumulation of infiltrating monocytes/macrophages in the lung during the OVA treatment, whereas the increase in infiltrating T-lymphocytes paralleled the accumulation of eosinophils. The kinetics of accumulation of these three leukocyte subtypes correlated with the levels of mRNA expression of the chemokines monocyte chemotactic peptide-1/JE, eotaxin, and RANTES (regulated upon activation in normal T cells expressed and secreted), suggesting their involvement in the recruitment of these leukocytes. Furthermore, blockade of eotaxin with specific antibodies in vivo reduced the accumulation of eosinophils in the lung in response to OVA by half. Mature CD4+ T-lymphocytes were absolutely required for OVA-induced eosinophil accumulation since lung eosinophilia was prevented in CD4+-deficient mice. However, these cells were neither the main producers of the major eosinophilic chemokines eotaxin, RANTES, or MIP-1alpha, nor did they regulate the expression of these chemokines. Rather, the presence of CD4+ T cells was necessary for enhancement of VCAM-1 (vascular cell adhesion molecule-1) expression in the lung during allergic inflammation induced by the OVA treatment. In support of this, mice genetically deficient for VCAM-1 and intercellular adhesion molecule-1 failed to develop pulmonary eosinophilia. Selective eosinophilic recruitment during lung allergic inflammation results from a sequential accumulation of certain leukocyte types, particularly T cells, and relies on the presence of both eosinophilic chemoattractants and adhesion receptors.

PATHOGENS: RAFT HIJACKERS

Throughout evolution, organisms have developed immune-surveillance networks to protect themselves from potential pathogens. At the cellular level, the signalling events that regulate these defensive responses take place in membrane rafts — dynamic microdomains that are enriched in cholesterol and glycosphingolipids — that facilitate many protein–protein and lipid–protein interactions at the cell surface. Pathogens have evolved many strategies to ensure their own survival and to evade the host immune system, in some cases by hijacking rafts. However, understanding the means by which pathogens exploit rafts might lead to new therapeutic strategies to prevent or alleviate certain infectious diseases, such as those caused by HIV-1 or Ebola virus.

Chemokine receptor homo‐ or heterodimerization activates distinct signaling pathways

Chemokine receptors of both the CC and CXC families have been demonstrated to undergo a ligand‐mediated homodimerization process required for Ca2+ flux and chemotaxis. We show that, in the chemokine response, heterodimerization is also permitted between given receptor pairs, specifically between CCR2 and CCR5. This has functional consequences, as the CCR2 and CCR5 ligands monocyte chemotactic protein‐1 (MCP‐1) and RANTES (regulated upon activation, normal T cell‐expressed and secreted) cooperate to trigger calcium responses at concentrations 10‐ to 100‐fold lower than the threshold for either chemokine alone. Heterodimerization results in recruitment of each receptor‐associated signaling complex, but also recruits dissimilar signaling path ways such as Gq/11 association, and delays activation of phosphatidyl inositol 3‐kinase. The consequences are a pertussis toxin‐resistant Ca2+ flux and trig gering of cell adhesion rather than chemotaxis. These results show the effect of heterodimer formation on increasing the sensitivity and dynamic range of the chemokine response, and may aid in understanding the dynamics of leukocytes at limiting chemokine concentrations in vivo.

Membrane raft microdomains mediate lateral assemblies required for HIV-1 infection

HIV‐1 infection triggers lateral membrane diffusion following interaction of the viral envelope with cell surface receptors. We show that these membrane changes are necessary for infection, as initial gp120–CD4 engagement leads to redistribution and clustering of membrane microdomains, enabling subsequent interaction of this complex with HIV‐1 co‐receptors. Disruption of cell membrane rafts by cholesterol depletion before viral exposure inhibits entry by both X4 and R5 strains of HIV‐1, although viral replication in infected cells is unaffected by this treatment. This inhibitory effect is fully reversed by cholesterol replenishment of the cell membrane. These results indicate a general mechanism for HIV‐1 envelope glycoprotein‐mediated fusion by reorganization of membrane microdomains in the target cell, and offer new strategies for preventing HIV‐1 infection.

PI3Kgamma inhibition blocks glomerulonephritis and extends lifespan in a mouse model of systemic lupus.

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease generated by deregulation of T cell–mediated B-cell activation, which results in glomerulonephritis and renal failure. Disease is treated with immunosuppressants and cytostatic agents that have numerous side effects. Here we examine the use of inhibitors of phosphoinositide 3-kinase (PI3K) γ, a lipid kinase that regulates inflammation, in the MRL-lpr mouse model of SLE. Treatment reduced glomerulonephritis and prolonged lifespan, suggesting that P13Kγ may be a useful target in the treatment of chronic inflammation.

Membrane raft microdomains mediate front-rear polarity in migrating cells.

The acquisition of spatial and functional asymmetry between the rear and the front of the cell is a necessary step for cell chemotaxis. Insulin‐like growth factor‐I (IGF‐I) stimulation of the human adenocarcinoma MCF‐7 induces a polarized phenotype characterized by asymmetrical CCR5 chemokine receptor redistribution to the leading cell edge. CCR5 associates with membrane raft microdomains, and its polarization parallels redistribution of raft molecules, including the raft‐associated ganglioside GM1, glycosylphosphatidylinositol‐anchored green fluorescent protein and ephrinB1, to the leading edge. The non‐raft proteins transferrin receptor and a mutant ephrinB1 are distributed homogeneously in migrating MCF‐7 cells, supporting the raft localization requirement for polarization. IGF‐I stimulation of cholesterol‐depleted cells induces projection of multiple pseudopodia over the entire cell periphery, indicating that raft disruption specifically affects the acquisition of cell polarity, but not IGF‐I‐induced protrusion activity. Cholesterol depletion inhibits MCF‐7 chemotaxis, which is restored by replenishing cholesterol. Our results indicate that initial segregation between raft and non‐raft membrane proteins mediates the necessary redistribution of specialized molecules for cell migration.

Forkhead transcription factors contribute to execution of the mitotic programme in mammals

Cell cycle progression is a process that is tightly controlled by internal and external signals. Environmental cues, such as those provided by growth factors, activate early signals that promote cell cycle entry. Cells that have progressed past the restriction point become independent of growth factors, and cell cycle progression is then controlled endogenously. The phosphatidylinositol 3OH kinase (PI(3)K)/protein kinase B (PKB) pathway must be activated in G1 to inactivate forkhead transcription factors (FKH-TFs) and allow cell cycle entry. Here we show that subsequent attenuation of the PI(3)K/PKB pathway is required to allow transcriptional activation of FKH-TF in G2. FKH-TF activity in G2 controls mammalian cell cycle termination, as interference with FKH transcriptional activation by disrupting PI(3)K/PKB downregulation, or by expressing a transcriptionally inactive FKH mutant, induces cell accumulation in G2/M, defective cytokinesis, and delayed transition from M to G1 of the cell cycle. We demonstrate that FKH-TFs regulate expression of mitotic genes such as cyclin B and polo-like kinase (Plk). Our results support the important role of forkhead in the control of mammalian cell cycle completion, and suggest that efficient execution of the mitotic programme depends on downregulation of PI(3)K/PKB and consequent induction of FKH transcriptional activity.