Synaptotagmin-mediated vesicle fusion regulates cell migration.

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.

Chemokine receptor CXCR3 and its ligands CXCL9 and CXCL10 are required for the development of murine cerebral malaria.

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 requires tyrosine sulfation for ligand binding and a second extracellular loop arginine residue for ligand-induced chemotaxis.

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.

Development of a novel chemokine-mediated in vivo T cell recruitment assay.

Trafficking of leukocytes to sites of inflammation is an important step in the establishment of an immune response. Chemokines are critical regulators of leukocyte trafficking and are widely studied molecules for their important role in disease and for their potential as new therapeutic targets. The ability of chemokines to induce leukocyte recruitment has been mainly measured by in vitro chemotaxis assays, which lack many components of the complex biological process of leukocyte migration and therefore provide incomplete information about chemokine function in vivo. In vivo assays to study the activity of chemokines to induce leukocyte recruitment have been difficult to establish. We describe here the development of a robust in vivo recruitment assay for CD8(+) and CD4(+) T lymphocytes induced by the CXCR3 ligands IP-10 (CXCL10) and I-TAC (CXCL11). For this assay, in vitro activated T lymphocytes were adoptively transferred into the peritoneum of naïve mice. Homing of these transferred T lymphocytes into the airways was measured following intratracheal instillation of chemokines. High recruitment indices were achieved that were dependent on chemokine concentration and CXCR3 expression on the transferred lymphocytes. Recruitment was also inhibited by antibodies to the chemokine. The assay models the natural condition of chemokine-mediated lymphocyte migration into the airways as chemokines are expressed in the airways during inflammation. The nature of this model allows flexibility to study wildtype and mutant chemokines and chemokine receptors and the ability to evaluate chemokine antagonists and antibodies in vivo. This assay will therefore help elucidate a deeper understanding of the chemokine system in vivo.

Oligomerization of CXCL10 is necessary for endothelial cell presentation and in vivo activity.

The chemokine IFN-gamma-inducible protein of 10 kDa (IP-10; CXCL10) plays an important role in the recruitment of activated T lymphocytes into sites of inflammation by interacting with the G protein-coupled receptor CXCR3. IP-10, like other chemokines, forms oligomers, the role of which has not yet been explored. In this study, we used a monomeric IP-10 mutant to elucidate the functional significance of oligomerization. Although monomeric IP-10 had reduced binding affinity for CXCR3 and heparin, it was able to induce in vitro chemotaxis of activated T cells with the same efficacy as wild-type IP-10. However, monomeric IP-10 was unable to induce recruitment of activated CD8+ T cells into the airways of mice after intratracheal instillation. Use of a different IP-10 mutant demonstrated that this inability was due to lack of oligomerization rather than reduced CXCR3 or heparin binding. Molecular imaging demonstrated that both wild-type and monomeric IP-10 were retained in the lung after intratracheal instillation. However, in vitro binding assays indicated that wild-type, but not monomeric, IP-10 was retained on endothelial cells and could induce transendothelial chemotaxis of activated T cells. We therefore propose that oligomerization of IP-10 is required for presentation on endothelial cells and subsequent transendothelial migration, an essential step for lymphocyte recruitment in vivo.