José Miguel Rodríguez-Frade

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.

Identification of amino acid residues crucial for chemokine receptor dimerization.

Chemokines coordinate leukocyte trafficking by promoting oligomerization and signaling by G protein–coupled receptors; however, it is not known which amino acid residues of the receptors participate in this process. Bioinformatic analysis predicted that Ile52 in transmembrane region-1 (TM1) and Val150 in TM4 of the chemokine receptor CCR5 are key residues in the interaction surface between CCR5 molecules. Mutation of these residues generated nonfunctional receptors that could not dimerize or trigger signaling. In vitro and in vivo studies in human cell lines and primary T cells showed that synthetic peptides containing these residues blocked responses induced by the CCR5 ligand CCL5. Fluorescence resonance energy transfer showed the presence of preformed, ligand-stabilized chemokine receptor oligomers. This is the first description of the residues involved in chemokine receptor dimerization, and indicates a potential target for the modification of chemokine responses.

CXCR3 Chemokine Receptor Distribution in Normal and Inflamed Tissues: Expression on Activated Lymphocytes, Endothelial Cells, and Dendritic Cells

Using new human CXCR3 chemokine receptor–specific monoclonal antibodies, we studied human CXCR3 tissue distribution in lymphoid and nonlymphoid organs, as well as in inflammatory conditions, including rheumatoid arthritis, Hashimotos thyroiditis, and dermal vasculitis. CXCR3 was expressed by certain dendritic cell subsets, specifically myeloid-derived CD11c positive cells, not only in those present in normal lymphoid organs, but also in germinal centers generated in inflammatory conditions. CXCR3 expression was also detected in some lymphocyte subsets such as intraepithelial lymphocytes of secondary lymphoid organs and infiltrating lymphocytes in inflammatory conditions. In addition, CXCR3 was constitutively expressed by endothelial cells (EC) of vessels of medium and large caliber but not in small vessels from different organs. Finally, enhanced CXCR3 expression was found in EC and in infiltrating lymphocytes with an activated phenotype in inflammatory diseases. The CXCR3 chemokine receptor may play a role in the regulation of leukocyte migration to inflammatory sites.

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.

Ligand stabilization of CXCR4/δ-opioid receptor heterodimers reveals a mechanism for immune response regulation

The CXCR4 chemokine receptor and the delta opioid receptor (DOR) are pertussis toxin‐sensitive G protein‐coupled receptors (GPCR). Both are widely distributed in brain tissues and immune cells, and have key roles in inflammation processes and in pain sensation on proximal nerve endings. We show that in immune cells expressing CXCR4 and DOR, simultaneous addition of their ligands CXCL12 and [D‐Pen2, D‐Pen5]enkephalin does not trigger receptor function. This treatment does not affect ligand binding or receptor expression, nor does it promote heterologous desensitization. Our data indicate that CXCR4 and DOR form heterodimeric complexes that are dynamically regulated by the ligands. This is compatible with a model in which GPCR oligomerization leads to suppression of signaling, promoting a dominant negative effect. Knockdown of CXCR4 and DOR signaling by heterodimerization might have repercussions on physiological and pathological processes such as inflammation, pain sensation and HIV‐1 infection.

Chemokine receptor dimerization: two are better than one

The chemokines participate in an exceptional range of physiological and pathological processes, including the control of lymphocyte trafficking, tumor growth, wound healing, allograft rejection, regulation of T-cell differentiation, asthma, infection with HIV and atherosclerosis. This vast array of activities is triggered by the interaction of nearly 50 different chemokines with a relatively modest number of 20 G-protein-coupled receptors. The asymmetry between the number of receptors and ligands suggests an underlying, shared control mechanism activated at a very early stage of the response. One of the first events triggered by the binding of chemokines is the homo- and hetero-dimerization of their receptors; here, we outline these events and their consequences in chemokine signaling.

Leukocyte attraction through the CCR5 receptor controls progress from insulitis to diabetes in non-obese diabetic mice

Lymphocyte infiltration to pancreatic islets is associated to chemoattraction, as are other inflammatory autoimmune processes. We examined whether development of insulitis and diabetes dependson chemoattraction of lymphocytes via the CCR5 chemokine receptor. In non‐obese diabetic (NOD) mice, a substantial fraction of peripheral T cells and virtually all B cells expressed high CCR5 levels. CCR5 expression characterized the effector T cell phenotype, suggesting their potential involvement in disease development. In view of these findings and the CCL5 (RANTES, the CCR5 ligand) expression by pancreatic islets, we treated NOD mice with a neutralizing anti‐CCR5 antibody. This did not influence peri‐insulitis advancement, but inhibited β‐cell destruction and diabetes. These data demonstrate a role of CCR5‐dependent chemoattraction in insulitis progression to islet destruction, suggesting the potential value of therapeutic intervention by CCR5 targeting.

The Chemokine Stromal Cell-Derived Factor-1α Modulates α4β7 Integrin-Mediated Lymphocyte Adhesion to Mucosal Addressin Cell Adhesion Molecule-1 and Fibronectin

The interaction between the integrin α4β7 and its ligand, mucosal addressin cell adhesion molecule-1, on high endothelial venules represents a key adhesion event during lymphocyte homing to secondary lymphoid tissue. Stromal cell-derived factor-1α (SDF-1α) is a chemokine that attracts T and B lymphocytes and has been hypothesized to be involved in lymphocyte homing. In this work we show that α4β7-mediated adhesion of CD4+ T lymphocytes and the RPMI 8866 cell line to mucosal addressin cell adhesion molecule-1 was up-regulated by SDF-1α in both static adhesion and cell detachment under shear stress assays. Both naive and memory phenotype CD4+ T cells were targets of SDF-1α-triggered increased adhesion. In addition, SDF-1α augmented α4β7-dependent adhesion of RPMI 8866 cells to connecting segment-1 of fibronectin. While pertussis toxin totally blocked chemotaxis of CD4+ and RPMI 8866 cells to SDF-1α, enhanced α4β7-dependent adhesion triggered by this chemokine was partially inhibited, indicating the participation of Gαi-dependent as well as Gαi-independent signaling. Accordingly, we show that SDF-1α induced a rapid and transient association between its receptor CXCR4 and Gαi, whereas association of pertussis toxin-insensitive Gα13 with CXCR4 was slower and of a lesser extent. SDF-1α also activated the small GTPases RhoA and Rac1, and inhibition of RhoA activation reduced the up-regulation of α4β7-mediated lymphocyte adhesion in response to SDF-1α, suggesting that activation of RhoA could play an important role in the enhanced adhesion. These data indicate that up-regulation by SDF-1α of lymphocyte adhesion mediated by α4β7 could contribute to lymphocyte homing to secondary lymphoid tissues.

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.