Cerebral malaria is a significant cause of global mortality, causing an estimated two million deaths per year, mainly in children. The pathogenesis of this disease remains incompletely understood. Chemokines have been implicated in the development of cerebral malaria, and the IFN-inducible CXCR3 chemokine ligand IP-10 (CXCL10) was recently found to be the only serum biomarker that predicted cerebral malaria mortality in Ghanaian children. We show that the CXCR3 chemokine ligands IP-10 and Mig (CXCL9) were highly induced in the brains of mice with murine cerebral malaria caused by Plasmodium berghei ANKA. Mice deficient in CXCR3 were markedly protected against cerebral malaria and had far fewer T cells in the brain compared with wild-type mice. In competitive transfer experiments, CXCR3-deficient CD8 ؉ T cells were 7-fold less efficient at migrating into the infected brains than wild-type CD8 ؉ T cells. Adoptive transfer of wild-type CD8 ؉ effector T cells restored susceptibility of CXCR3-deficient mice to cerebral malaria and also restored brain proinflammatory cytokine and chemokine production and recruitment of T cells, independent of CXCR3. Mice deficient in IP-10 or Mig were both partially protected against cerebral malaria mortality when infected with P. berghei ANKA. Brain immunohistochemistry revealed Mig staining of endothelial cells, whereas IP-10 staining was mainly found in neurons. These data demonstrate that CXCR3 on CD8 ؉ T cells is required for T cell recruitment into the brain and the development of murine cerebral malaria and suggest that the CXCR3 ligands Mig and IP-10 play distinct, nonredundant roles in the pathogenesis of this disease.
CXCR3 is a G-protein-coupled seven-transmembrane domain chemokine receptor that plays an important role in effector T-cell and NK cell trafficking. Three gamma interferon-inducible chemokines activate CXCR3: CXCL9 (Mig), CXCL10 (IP-10), and CXCL11 (I-TAC). Here, we identify extracellular domains of CXCR3 that are required for ligand binding and activation. We found that CXCR3 is sulfated on its N terminus and that sulfation is required for binding and activation by all three ligands. We also found that the proximal 16 amino acid residues of the N terminus are required for CXCL10 and CXCL11 binding and activation but not CXCL9 activation. In addition, we found that residue R216 in the second extracellular loop is required for CXCR3-mediated chemotaxis and calcium mobilization but is not required for ligand binding or ligand-induced CXCR3 internalization. Finally, charged residues in the extracellular loops contribute to the receptor-ligand interaction. These findings demonstrate that chemokine activation of CXCR3 involves both high-affinity ligand-binding interactions with negatively charged residues in the extracellular domains of CXCR3 and a lower-affinity receptor-activating interaction in the second extracellular loop. This lower-affinity interaction is necessary to induce chemotaxis but not ligand-induced CXCR3 internalization, further suggesting that different domains of CXCR3 mediate distinct functions.Chemokines, or chemoattractant cytokines, are a family of small (8-to 10-kDa) secreted proteins that play an important role in the recruitment and activation of leukocytes (29). Approximately 50 chemokines have been described, and these chemokines interact with some redundancy with 20 G-proteincoupled chemokine receptors. The chemokine system, with its ability to control the migration of leukocytes, plays a critical role in both innate and adaptive immunity.CXCR3 is a chemokine receptor that is expressed on the surface of a number of cell types, including activated CD4 ϩ and CD8 ϩ T cells, NK and NK-T cells, plasmacytoid dendritic cells, and some B cells (26,27,38,45). CXCR3 is activated by three related chemokines: CXCL9, CXCL10, and CXCL11 (9,26,27,38,45). Each of these ligands is induced by gamma interferon and is produced in Th1-type immune responses (9, 14, 31). CXCR3 has been localized to infiltrating effector T cells in a wide variety of human inflammatory diseases, including atherosclerosis (32), rheumatoid arthritis (38), multiple sclerosis (3, 43), heart and lung transplant rejection (1, 22, 50), and psoriasis (17). The CXCR3 ligands have similarly been identified in these same lesions (1,17,20,32,36), leading to the hypothesis that this receptor-ligand system plays an important role in the recruitment of effector T cells into these lesions, resulting in T-cell-mediated inflammation. Supporting this hypothesis, CXCR3-deficient mice were protected from heart allograft transplant rejection and autoimmune type 1 diabetes mellitus in murine models (19,23). Likewise, using CXCL10-deficient mice and inhi...
Chemokines and other chemoattractants direct leukocyte migration and are essential for the development and delivery of immune and inflammatory responses. To probe the molecular mechanisms that underlie chemoattractant-guided migration, we did an RNA-mediated interference screen that identified several members of the synaptotagmin family of calcium-sensing vesicle-fusion proteins as mediators of cell migration: SYT7 and SYTL5 were positive regulators of chemotaxis, whereas SYT2 was a negative regulator of chemotaxis. SYT7-deficient leukocytes showed less migration in vitro and in a gout model in vivo. Chemoattractant-induced calcium-dependent lysosomal fusion was impaired in SYT7-deficient neutrophils. In a chemokine gradient, SYT7-deficient lymphocytes accumulated lysosomes in their uropods and had impaired uropod release. Our data identify a molecular pathway required for chemotaxis that links chemoattractant-induced calcium flux to exocytosis and uropod release. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests. NIH Public Access Author ManuscriptNat Immunol. Author manuscript; available in PMC 2011 June 1. Published in final edited form as:Nat Immunol. 2010 June ; 11(6): 495-502. doi:10.1038/ni.1878. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptChemoattractant-directed cell migration is critical for the generation and delivery of immune and inflammatory responses 1 . Defining the molecular mechanisms that control directed cell movement is therefore essential for understanding the function of cells of the immune response, the host response to infection and tumors, and immune-mediated auto-immune and inflammatory diseases. Chemokines and other classical chemoattractants, such as formyl-MetLeu-Phe (fMLP), C5a and leukotriene B 4 , induce directed cell migration through the activation of seven-transmembrane-spanning G protein-coupled receptors 2 . Chemoattractant receptors form a related subfamily of ~50 G protein-coupled receptors that all couple to the pertussis toxin-sensitive cAMP-inhibitory heterotrimeric guanine nucleotide-binding protein G i .Chemokine receptors and other chemoattractant receptors are found on all leukocyte lineages. Activation of chemoattractant receptors transforms a chemical signal in the form of a gradient into a biophysical program that results in leukocyte shape change and directed cell movement 3 . A leading edge called the lamellopodia and a trailing edge called the uropod characterize the polarized migrating leukocyte that develops after activation of chemoattractant receptors. These complex changes involve cycles of membrane protrusions and contractions, polymerization and depolymerization of F-actin, and adhesion and de-adhesion. These processes are coordinated through the activation of multiple signaling pathways that are only partially understood 4,5 .After chemokines and chemoattractants bind to the extracellular domains of their cognate G protein-coupled receptor, the Gα and Gβγ subunits of G i are liberate...
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