Martín de Ana

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)

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.

Chemokine control of HIV-1 infection

Chemokines are proinflammatory cytokines that attract and activate specific types of leukocyte. There are two main chemokine families, based on the position of the first two cysteine residues: the CC and the CXC chemokines. Chemokines mediate their effects through interactions with seven-transmembrane-spanning glyco-protein receptors coupled to a G-protein signalling pathway. Chemokine receptors normally undergo a ligand-mediated homodimerization process, which is required for Ca2+ flux and chemotaxis. Here we show that in the chemokine response it is possible for heterodimerization, rather than homodimerization, to occur between a mutant form of the CCR2 receptor (the CCR2V64I receptor), which helps to delay the development of AIDS in HIV-1-infected individuals, and the CCR5 or CXCR4 chemokine receptor, which are used by HIV to gain entry into cells. These results may explain why AIDS takes longer to develop in HIV-1-infected individuals carrying the CCR2V64I mutation.

Chemokines integrate JAK/STAT and G-protein pathways during chemotaxis and calcium flux responses

The JAK/STAT (Janus kinase / signaling transducer and activator of transcription) signaling pathway is implicated in converting stationary epithelial cells to migratory cells. In mammals, migratory responses are activated by chemoattractant proteins, including chemokines. We found that by binding to seven‐transmembrane G‐protein‐coupled receptors, chemokines activate the JAK/STAT pathwayto trigger chemotactic responses. We show that chemokine‐mediated JAK/STAT activation is critical for G‐protein induction and for phospholipase C‐β dependent Ca2+ flux; in addition, pharmacological inhibition of JAK or mutation of the JAK kinase domain causes defects in both responses. Furthermore, Gαi association with the receptor is dependent on JAK activation, andthe chemokine‐mediated Ca2+ flux that requires phospholipase C‐β activity takes place downstream of JAK kinases. The chemokines thus employ a mechanism that links heterologous signaling pathways — G proteins and tyrosine kinases — in a network that may be essential for mediating their pleiotropic responses.

Blocking HIV-1 infection via CCR5 and CXCR4 receptors by acting in trans on the CCR2 chemokine receptor

The identification of chemokine receptors as HIV‐1 coreceptors has focused research on developing strategies to prevent HIV‐1 infection. We generated CCR2‐01, a CCR2 receptor‐specific monoclonal antibody that neither competes with the chemokine CCL2 for binding nor triggers signaling, but nonetheless blocks replication of monotropic (R5) and T‐tropic (X4) HIV‐1 strains. This effect is explained by the ability of CCR2‐01 to induce oligomerization of CCR2 with the CCR5 or CXCR4 viral coreceptors. HIV‐1 infection through CCR5 and CXCR4 receptors can thus be prevented in the absence of steric hindrance or receptor downregulation by acting in trans on a receptor that is rarely used by the virus to infect cells.

Functional inactivation of CXC chemokine receptor 4-mediated responses through SOCS3 up-regulation

Hematopoietic cell growth, differentiation, and chemotactic responses require coordinated action between cytokines and chemokines. Cytokines promote receptor oligomerization, followed by Janus kinase (JAK) kinase activation, signal transducers and transactivators of transcription (STAT) nuclear translocation, and transcription of cytokine-responsive genes. These include genes that encode a family of negative regulators of cytokine signaling, the suppressors of cytokine signaling (SOCS) proteins. After binding their specific receptors, chemokines trigger receptor dimerization and activate the JAK/STAT pathway. We show that SOCS3 overexpression or up-regulation, stimulated by a cytokine such as growth hormone, impairs the response to CXCL12, measured by Ca2+ flux and chemotaxis in vitro and in vivo. This effect is mediated by SOCS3 binding to the CXC chemokine receptor 4 receptor, blocking JAK/STAT and Gαi pathways, without interfering with cell surface chemokine receptor expression. The data provide clear evidence for signaling cross-talk between cytokine and chemokine responses in building a functional immune system.

A potential immune escape mechanism by melanoma cells through the activation of chemokine-induced T cell death

The immune system attempts to prevent or limit tumor growth, yet efforts to induce responses to tumors yield minimal results, rendering tumors virtually invisible to the immune system [1]. Several mechanisms may account for this subversion, including the triggering of tolerance to tumor antigens [2, 3], TGF-alpha or IL-10 production, downregulation of MHC molecules, or upregulation of FasL expression [4, 5]. Melanoma cells may in some instances use FasL expression to protect themselves against tumor-infiltrating lymphocytes (TIL) [4, 5]. Here, we show another, chemokine-dependent mechanism by which melanoma tumor cells shield themselves from immune reactions. Melanoma-inducible CCL5 (RANTES) production by infiltrating CD8 cells activates an apoptotic pathway in TIL involving cytochrome c release into the cytosol and activation of caspase-9 and -3. This process, triggered by CCL5 binding to CCR5, is not mediated by TNFalpha, Fas, or caspase-8. The effect is not unique to CCL5, as other CCR5 ligands such as CCL3 (MIP-1alpha) and CCL4 (MIP-1beta) also trigger TIL cell death, nor is it limited to melanoma cells, as it also operates in activated primary T lymphocytes. The model assigns a role to the CXC chemokine CXCL12 (SDF-1alpha) in this process, as this melanoma cell-produced chemokine upregulates CCL5 production by TIL, initiating TIL cell death.

Chemokine Receptor 2 Blockade Prevents Asthma in a Cynomolgus Monkey Model

The pathophysiology of asthma is characterized by accumulation and activation of several cell types in the lung, which correlates with coordinated production of specific cytokines and chemokines. To study the effect of selective CCR2 chemokine receptor blockade on leukocyte recruitment to the lung and on bronchial function, we used a nonhuman primate model of allergic airway disease that closely resembles human asthma. Allergic cynomolgus monkeys were treated with the antagonist anti-CCR2 (CCR2-05) monoclonal antibody and then challenged with Ascaris suum antigen; the effect of antibody treatment on macrophage and eosinophil infiltration was determined. Pulmonary function was calculated by measurement of lung resistance and dynamic compliance. Local inflammatory responses were analyzed after intradermal challenge with A. suum antigen. CCL2 up-regulation in bronchoalveolar lavage (BAL) was analyzed by enzyme-linked immunosorbent assay, and in vitro CCR2-05 antagonistic activity was tested in monkey peripheral blood mononuclear cells using chemotaxis and calcium mobilization assays. The results show that neutralization of CCR2 reduces antigen-induced bronchial hyper-responsiveness and attenuates macrophage and eosinophil accumulation in the BAL of asthmatic monkeys. The results confirm that selective blockade of a single chemokine receptor involved in early stages of asthma can condition later disease stages and suggest the utility of anti-CCR2-neutralizing monoclonal antibodies in the treatment of asthma in man.