RESUMO
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.
Assuntos
Movimento Celular/fisiologia , Sinaptotagminas/metabolismo , Animais , Quimiocina CXCL12/metabolismo , Quimiotaxia , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase , Receptores CXCR4/metabolismo , Sinaptotagmina II/metabolismo , Sinaptotagminas/genética , Linfócitos T/imunologiaRESUMO
Leukotriene B4 is a lipid mediator that recently has been shown to have potent chemotactic activity for effector T lymphocytes mediated through its receptor, BLT1. Here, we developed a novel murine model of acute lung rejection to demonstrate that BLT1 controls effector CD8+ T cell trafficking into the lung and that disruption of BLT1 signaling in CD8+ T cells reduces lung inflammation and mortality in the model. In addition, we used BLT1-deficient mice and a BLT1 antagonist in two tracheal transplant models of lung transplantation to demonstrate the importance of BLT1 for the recruitment of T cells into tracheal allografts. We also show that BLT1-mediated CD8+ T cell recruitment plays an important role in the development of airway fibroproliferation and obliteration. Finally, in human studies of lung transplant recipients, we found that BLT1 is up-regulated on T lymphocytes isolated from the airways of patients with obliterative bronchiolitis. These data demonstrate that BLT1 contributes to the development of lung rejection and obliterative bronchiolitis by mediating effector T lymphocyte trafficking into the lung. This is the first report that describes a pathologic role for BLT1-mediated T lymphocyte recruitment in disease and identifies BLT1 as a potential therapeutic target after lung transplantation.
Assuntos
Bronquiolite Obliterante/etiologia , Linfócitos T CD8-Positivos/fisiologia , Rejeição de Enxerto/etiologia , Transplante de Pulmão/efeitos adversos , Receptores do Leucotrieno B4/fisiologia , Animais , Sequência de Bases , Bronquiolite Obliterante/imunologia , Bronquiolite Obliterante/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Colágeno/metabolismo , Modelos Animais de Doenças , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Humanos , Transplante de Pulmão/imunologia , Transplante de Pulmão/patologia , Transplante de Pulmão/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina/imunologia , Receptores do Leucotrieno B4/deficiência , Receptores do Leucotrieno B4/genética , Traqueia/transplanteRESUMO
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.
Assuntos
Quimiocina CXCL10/imunologia , Quimiocina CXCL9/imunologia , Malária Cerebral/imunologia , Malária Cerebral/patologia , Receptores CXCR3/imunologia , Animais , Encéfalo/parasitologia , Encéfalo/patologia , Complexo CD3/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/parasitologia , Movimento Celular , Quimiocina CXCL10/genética , Quimiocina CXCL9/genética , Ligantes , Malária Cerebral/parasitologia , Malária Cerebral/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasmodium berghei/imunologia , Receptores de Citocinas/deficiência , Baço/metabolismo , Baço/patologia , Taxa de Sobrevida , Regulação para Cima/genéticaRESUMO
Poxviruses encode a number of secreted virulence factors that modulate the host immune response. The vaccinia virus A41 protein is an immunomodulatory protein with amino acid sequence similarity to the 35-kDa chemokine binding protein, but the host immune molecules targeted by A41 have not been identified. We report here that the vaccinia virus A41 ortholog encoded by ectromelia virus, a poxvirus pathogen of mice, named E163 in the ectromelia virus Naval strain, is a secreted 31-kDa glycoprotein that selectively binds a limited number of CC and CXC chemokines with high affinity. A detailed characterization of the interaction of ectromelia virus E163 with mutant forms of the chemokines CXCL10 and CXCL12alpha indicated that E163 binds to the glycosaminoglycan binding site of the chemokines. This suggests that E163 inhibits the interaction of chemokines with glycosaminoglycans and provides a mechanism by which E163 prevents chemokine-induced leukocyte migration to the sites of infection. In addition to interacting with chemokines, E163 can interact with high affinity with glycosaminoglycan molecules, enabling E163 to attach to cell surfaces and to remain in the vicinity of the sites of viral infection. These findings identify E163 as a new chemokine binding protein in poxviruses and provide a molecular mechanism for the immunomodulatory activity previously reported for the vaccinia virus A41 ortholog. The results reported here also suggest that the cell surface and extracellular matrix are important targeting sites for secreted poxvirus immune modulators.
Assuntos
Quimiocinas/metabolismo , Vírus da Ectromelia/fisiologia , Glicoproteínas/metabolismo , Glicosaminoglicanos/metabolismo , Proteínas Virais/metabolismo , Animais , Sítios de Ligação , Quimiocinas/genética , Humanos , Camundongos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação ProteicaRESUMO
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.
Assuntos
Arginina/metabolismo , Quimiotaxia de Leucócito/fisiologia , Estrutura Secundária de Proteína , Receptores de Quimiocinas/química , Receptores de Quimiocinas/metabolismo , Proteínas Recombinantes/química , Tirosina/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Quimiocina CXCL10 , Quimiocina CXCL11 , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Humanos , Ligantes , Camundongos , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Receptores CXCR3 , Receptores de Quimiocinas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
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.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Quimiocinas CXC/metabolismo , Quimiotaxia de Leucócito , Receptores de Quimiocinas/metabolismo , Animais , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Quimiocinas CXC/imunologia , Inflamação/imunologia , Inflamação/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Quimiocinas/imunologiaRESUMO
CXCL10 (or Interferon-inducible protein of 10 kDa, IP-10) is an interferon-inducible chemokine with potent chemotactic activity on activated effector T cells and other leukocytes expressing its high affinity G protein-coupled receptor CXCR3. CXCL10 is also active on other cell types, including endothelial cells and fibroblasts. The mechanisms through which CXCL10 mediates its effects on non-leukocytes is not fully understood. In this study, we focus on the anti-proliferative effect of CXCL10 on endothelial cells, and demonstrate that CXCL10 can inhibit endothelial cell proliferation in vitro independently of CXCR3. Four main findings support this conclusion. First, primary mouse endothelial cells isolated from CXCR3-deficient mice were inhibited by CXCL10 as efficiently as wildtype endothelial cells. We also note that the proposed alternative splice form CXCR3-B, which is thought to mediate CXCL10's angiostatic activity, does not exist in mice based on published mouse CXCR3 genomic sequences as an in-frame stop codon would terminate the proposed CXCR3-B splice variant in mice. Second, we demonstrate that human umbilical vein endothelial cells and human lung microvascular endothelial cells that were inhibited by CXL10 did not express CXCR3 by FACS analysis. Third, two different neutralizing CXCR3 antibodies did not inhibit the anti-proliferative effect of CXCL10. Finally, fourth, utilizing a panel of CXCL10 mutants, we show that the ability to inhibit endothelial cell proliferation correlates with CXCL10's glycosaminoglycan binding affinity and not with its CXCR3 binding and signaling. Thus, using a very defined system, we show that CXCL10 can inhibit endothelial cell proliferation through a CXCR3-independent mechanism.
Assuntos
Proliferação de Células , Quimiocina CXCL10/metabolismo , Regulação para Baixo , Células Endoteliais/citologia , Receptores CXCR3/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Quimiocina CXCL10/genética , Células Endoteliais/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Splicing de RNA , Receptores CXCR3/genéticaRESUMO
Pulmonary fibrosis is a progressive, dysregulated response to injury culminating in compromised lung function due to excess extracellular matrix production. The heparan sulfate proteoglycan syndecan-4 is important in mediating fibroblast-matrix interactions, but its role in pulmonary fibrosis has not been explored. To investigate this issue, we used intratracheal instillation of bleomycin as a model of acute lung injury and fibrosis. We found that bleomycin treatment increased syndecan-4 expression. Moreover, we observed a marked decrease in neutrophil recruitment and an increase in both myofibroblast recruitment and interstitial fibrosis in bleomycin-treated syndecan-4-null (Sdc4-/-) mice. Subsequently, we identified a direct interaction between CXCL10, an antifibrotic chemokine, and syndecan-4 that inhibited primary lung fibroblast migration during fibrosis; mutation of the heparin-binding domain, but not the CXCR3 domain, of CXCL10 diminished this effect. Similarly, migration of fibroblasts from patients with pulmonary fibrosis was inhibited in the presence of CXCL10 protein defective in CXCR3 binding. Furthermore, administration of recombinant CXCL10 protein inhibited fibrosis in WT mice, but not in Sdc4-/- mice. Collectively, these data suggest that the direct interaction of syndecan-4 and CXCL10 in the lung interstitial compartment serves to inhibit fibroblast recruitment and subsequent fibrosis. Thus, administration of CXCL10 protein defective in CXCR3 binding may represent a novel therapy for pulmonary fibrosis.
Assuntos
Glicosaminoglicanos/metabolismo , Pulmão/metabolismo , Fibrose Pulmonar/metabolismo , Sindecana-4/metabolismo , Animais , Bleomicina/imunologia , Bleomicina/metabolismo , Bleomicina/farmacologia , Matriz Extracelular/imunologia , Matriz Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/metabolismo , Fibrose/patologia , Glicosaminoglicanos/imunologia , Glicosaminoglicanos/farmacologia , Proteoglicanas de Heparan Sulfato/imunologia , Proteoglicanas de Heparan Sulfato/metabolismo , Proteoglicanas de Heparan Sulfato/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Doenças Pulmonares Intersticiais/imunologia , Doenças Pulmonares Intersticiais/metabolismo , Doenças Pulmonares Intersticiais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibrose Pulmonar/genética , Fibrose Pulmonar/imunologiaRESUMO
The ability of chemokines to induce the migration of cells expressing their cognate G-protein-coupled receptor is a characteristic property of chemokine function. To study this important function, in vitro chemotaxis assays are most often used, which, although useful, lack many components of the complex in vivo trafficking process. Reliable in vivo recruitment assays have been very difficult to establish. We describe a robust in vivo T-cell recruitment assay for adoptively transferred T lymphocytes in mice. Instillation of the CXCR3 chemokine ligands IP-10/CXCL10 or I-TAC/CXCL11 into the airways results in robust recruitment of transferred T lymphocytes. The assay thereby models the natural environment of chemokine function, as chemokines are expressed in the airways during inflammation, inducing selective leukocyte homing. This assay is particularly useful for the analysis of chemokine and chemokine receptor mutants in structure function studies and for testing the in vivo efficacy of inhibitory chemokine and chemokine receptor antibodies and small molecule antagonists.
Assuntos
Quimiocinas/farmacologia , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Quimiocina CXCL10/farmacologia , Quimiocina CXCL11/genética , Quimiocina CXCL11/metabolismo , Quimiocina CXCL11/farmacologia , Quimiocinas/genética , Quimiocinas/metabolismo , Camundongos , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/fisiologiaRESUMO
IP-10 secretion is induced by pro-inflammatory cytokines and mediates the migration of CXCR3+ cells. Its elevation in clinical samples has been associated with multiple inflammatory diseases and its antagonism has been reported to be effective in several animal models of inflammatory disease. We generated a mouse anti-mouse IP-10 monoclonal antibody (mAb; Clone 20A9) that specifically bound murine IP-10 with high affinity and inhibited in vitro IP-10 induced BaF3/mCXCR3 cell migration with an IC(50) of approximately 4 nM. The 20A9 mAb was completely absorbed in vivo and had dose proportional pharmacokinetic exposure with a serum half life of 2.4-6 days. The 20A9 mAb inhibited IP-10 mediated T-cell recruitment to the airways, indicating that it is effective in vivo. However, administration of the 20A9 mAb had no significant effect on disease in mouse models of delayed type hypersensitivity, collagen induced arthritis, cardiac allograft transplantation tolerance, EAE or CD4+ CD45RBHi T-cell transfer-induced IBD. These data suggest that the 20A9 mAb can antagonize IP-10 mediated chemotaxis in vitro and in vivo and that this is insufficient to cause a therapeutic benefit in multiple mouse models of inflammatory disease.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL10/antagonistas & inibidores , Quimiocina CXCL10/imunologia , Animais , Anticorpos Monoclonais/farmacocinética , Artrite Experimental/patologia , Artrite Experimental/terapia , Líquido da Lavagem Broncoalveolar/citologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Movimento Celular/imunologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/terapia , Feminino , Rejeição de Enxerto/prevenção & controle , Transplante de Coração/imunologia , Inflamação/patologia , Inflamação/terapia , Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/terapia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Camundongos Endogâmicos , Camundongos SCID , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/efeitos dos fármacos , Resultado do TratamentoRESUMO
Aberrant wound-healing responses to injury have been implicated in the development of pulmonary fibrosis, but the mediators directing these pathologic responses have yet to be fully identified. We show that lysophosphatidic acid levels increase in bronchoalveolar lavage fluid following lung injury in the bleomycin model of pulmonary fibrosis, and that mice lacking one of its receptors, LPA1, are markedly protected from fibrosis and mortality in this model. The absence of LPA1 led to reduced fibroblast recruitment and vascular leak, two responses that may be excessive when injury leads to fibrosis rather than to repair, whereas leukocyte recruitment was preserved during the first week after injury. In persons with idiopathic pulmonary fibrosis, lysophosphatidic acid levels in bronchoalveolar lavage fluid were also increased, and inhibition of LPA1 markedly reduced fibroblast responses to the chemotactic activity of this fluid. LPA1 therefore represents a new therapeutic target for diseases in which aberrant responses to injury contribute to fibrosis, such as idiopathic pulmonary fibrosis.
Assuntos
Fibroblastos/metabolismo , Lesão Pulmonar , Receptores de Ácidos Lisofosfatídicos/fisiologia , Animais , Bleomicina/farmacologia , Líquido da Lavagem Broncoalveolar , Feminino , Leucócitos/metabolismo , Pulmão/patologia , Lisofosfolipídeos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Fibrose Pulmonar/patologia , Receptores de Ácidos Lisofosfatídicos/metabolismoRESUMO
Th1 and Th2 effector CD4+ T cells orchestrate distinct counterregulatory biological responses. To deliver effective tissue Th1- and Th2-type responses, Th1 and Th2 cell recruitment into tissue must be differentially regulated. We show that tissue-derived STAT1 controls the trafficking of adoptively transferred, Ag-specific, wild-type Th1 cells into the lung. Trafficking of Th1 and Th2 cells is differentially regulated as STAT6, which regulates Th2 cell trafficking, had no effect on the trafficking of Th1 cells and STAT1 deficiency did not alter Th2 cell trafficking. We demonstrate that STAT1 control of Th1 cell trafficking is not mediated through T-bet. STAT1 controls the recruitment of Th1 cells through the induction of CXCL9, CXCL10, CXCL11, and CXCL16, whose expression levels in the lung were markedly decreased in STAT1-/- mice. CXCL10 replacement partially restored Th1 cell trafficking in STAT1-deficient mice in vivo, and deficiency in CXCR3, the receptor for CXCL9, CXCL10, and CXCL11, impaired the trafficking of adoptively transferred Th1 cells in wild-type mice. Our work identifies that STAT1 in peripheral tissue regulates the homing of Ag-specific Th1 cells through the induction of a distinct subset of chemokines and establishes that Th1 and Th2 cell trafficking is differentially controlled in vivo by STAT1 and STAT6, respectively.
Assuntos
Fator de Transcrição STAT1/metabolismo , Células Th1/imunologia , Células Th2/imunologia , Transferência Adotiva , Animais , Antígenos , Movimento Celular/imunologia , Proliferação de Células , Quimiocina CXCL10 , Quimiocina CXCL11 , Quimiocina CXCL16 , Quimiocina CXCL6 , Quimiocina CXCL9 , Quimiocinas CXC/metabolismo , Pulmão/citologia , Pulmão/imunologia , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina/imunologia , Receptores CXCR3 , Receptores de Quimiocinas/deficiência , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Receptores Depuradores/metabolismo , Fator de Transcrição STAT1/deficiência , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT6/deficiência , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismo , Células Th1/citologia , Células Th2/citologiaRESUMO
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.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Quimiocinas CXC/química , Quimiocinas CXC/fisiologia , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Animais , Apresentação de Antígeno/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/transplante , Adesão Celular/imunologia , Linhagem Celular , Linhagem Celular Transformada , Linhagem Celular Tumoral , Quimiocina CXCL10 , Quimiocinas CXC/administração & dosagem , Quimiocinas CXC/genética , Quimiotaxia de Leucócito/genética , Quimiotaxia de Leucócito/imunologia , Proteínas do Ovo/imunologia , Proteínas do Ovo/metabolismo , Endotélio Vascular/citologia , Humanos , Intubação Intratraqueal , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina/imunologia , Ovalbumina/metabolismo , Fragmentos de PeptídeosRESUMO
Epithelial cells play an important role in orchestrating mucosal immune responses. In allergic-type inflammation, epithelial cells control the recruitment of eosinophils into the mucosa. Th2-type cytokine-driven release of eosinophil-active chemokines from epithelial cells directs eosinophil migration into the mucosal epithelium. CCR3, the main eosinophil chemokine receptor, regulates this process; however, the respective contribution of individual CCR3 ligands in eosinophil transepithelial migration is less well understood. Using an in vitro transepithelial chemotaxis system, we found that eotaxin-3 produced by IL-4-stimulated airway epithelial cells and CCR3 on eosinophils exclusively mediate eosinophil transepithelial migration. Eotaxin-3 protein levels were also increased in the nasal mucosal epithelium recovered from allergic patients as compared to non-allergic patients. Surprisingly, eotaxin-3 in IL-4-stimulated airway epithelial cells was predominantly cell surface bound, and the cell surface form was critical for eosinophil transepithelial migration. Eotaxin-3 cell surface association was partially glycosaminoglycan (GAG) dependent, but was completely protein dependent, suggesting that eotaxin-3 associates with both GAG and cell surface proteins. We thus provide evidence that cell surface-associated eotaxin-3 is the critical IL-4-dependent chemotactic signal mediating eosinophil transepithelial migration in the setting of allergic inflammation.
Assuntos
Quimiocinas CC/metabolismo , Quimiotaxia de Leucócito/imunologia , Eosinófilos/metabolismo , Células Epiteliais/metabolismo , Hipersensibilidade/imunologia , Interleucina-4/metabolismo , Northern Blotting , Western Blotting , Linhagem Celular , Membrana Celular/metabolismo , Quimiocina CCL26 , Quimiocinas CC/imunologia , Ensaio de Imunoadsorção Enzimática , Eosinófilos/imunologia , Células Epiteliais/imunologia , Glicosaminoglicanos/metabolismo , Humanos , Imuno-Histoquímica , Mucosa Nasal/citologia , Mucosa Nasal/imunologia , Mucosa Nasal/metabolismo , Receptores CCR3 , Receptores de Quimiocinas/imunologia , Receptores de Quimiocinas/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismoRESUMO
Dengue virus is an arthropod-borne flavivirus that causes a mild febrile illness, dengue fever, or a potentially fatal syndrome, dengue hemorrhagic fever/dengue shock syndrome. Chemokines primarily orchestrate leukocyte recruitment to the areas of viral infection, which makes them critical mediators of immune and inflammatory responses. In the present study, we investigated the induction and function of chemokines in mice early after infection with dengue virus in vivo. We found that CXCL10/IFN-gamma-inducible protein 10 (IP-10) expression was rapidly and transiently induced in liver following infection. The expressed CXCL10/IP-10 likely mediates the recruitment of activated NK cells, given that anti-CXCL10/IP-10-treated mice showed diminished NK cell infiltration and reduced hepatic expression of effector molecules in activated NK cells after dengue virus infection. Of particular interest, we found that CXCL10/IP-10 also was able to inhibit viral binding to target cells in vitro. Further investigation revealed that various CXCL10/IP-10 mutants, in which the residues that mediate the interaction between the chemokine and heparan sulfate were substituted, failed to exert the inhibitory effect on dengue binding, which suggests that CXCL10/IP-10 competes with dengue virus for binding to heparan sulfate on the cell surface. Moreover, subsequent plaque assays showed that this inhibition of dengue binding blocked viral uptake and replication. The inhibitory effect of CXCL10/IP-10 on the binding of dengue virus to cells may represent a novel contribution of this chemokine to the host defense against viral infection.
Assuntos
Quimiocinas CXC/metabolismo , Vírus da Dengue/fisiologia , Dengue/metabolismo , Dengue/virologia , Heparitina Sulfato/metabolismo , Animais , Fusão Celular , Linhagem Celular , Quimiocina CXCL10 , Quimiocina CXCL11 , Quimiocina CXCL9 , Quimiocinas CXC/genética , Culicidae , Dengue/genética , Dengue/imunologia , Regulação da Expressão Gênica , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The chemokine IP-10 (interferon-inducible protein of 10 kDa, CXCL10) binds the G protein-coupled receptor CXCR3, which is found mainly on activated T cells and NK cells, and plays an important role in Th1-type inflammatory diseases. IP-10 also binds to glycosaminoglycans (GAGs), an interaction thought to be important for its sequestration on endothelial and other cells. In this study, we performed an extensive mutational analysis to identify the CXCR3 and heparin binding sites of murine IP-10. The mutants were characterized for heparin binding, CXCR3 binding, and the ability to induce chemotaxis, Ca(2+) flux, and CXCR3 internalization. Double mutations neutralizing adjacent basic residues at the C terminus did not lead to a significant reduction in heparin binding, indicating that the main heparin binding site of IP-10 is not along the C-terminal alpha helix. Alanine exchange of Arg-22 had the largest effect on heparin binding, with residues Arg-20, Ile-24, Lys-26, Lys-46, and Lys-47 further contributing to heparin binding. A charge change mutation of Arg-22 resulted in further reduction in heparin binding. The N-terminal residue Arg-8, preceding the first cysteine, was critical for CXCR3 signaling. Mutations of charged and uncharged residues in the loop regions of residues 20-24 and 46-47, which caused reduced heparin binding, also resulted in reduced CXCR3 binding and signaling. CXCR3 expressing GAG-deficient Chinese hamster ovary cells revealed that GAG binding was not required for IP-10 binding and signaling through CXCR3, which suggests that the CXCR3 and heparin binding sites of IP-10 are partially overlapping.
Assuntos
Quimiocinas CXC/análise , Heparina/metabolismo , Receptores de Quimiocinas/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Quimiocina CXCL10 , Quimiocinas CXC/química , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Quimiotaxia , Heparina/química , Humanos , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Conformação Proteica , Receptores CXCR3 , Receptores de Quimiocinas/químicaRESUMO
The chemokine receptor CXCR3 is a G protein-coupled receptor found predominantly on T cells that is activated by three ligands as follows: CXCL9 (Mig), CXCL10 (IP-10), and CXCL11 (I-TAC). Previously, we have found that of the three ligands, CXCL11 is the most potent inducer of CXCR3 internalization and is the physiologic inducer of CXCR3 internalization after T cell contact with activated endothelial cells. We have therefore hypothesized that these three ligands transduce different signals to CXCR3. In light of this hypothesis, we sought to determine whether regions of CXCR3 are differentially required for CXCL9, CXCL10, and CXCL11 function. Here we identified two distinct domains that contributed to CXCR3 internalization. The carboxyl-terminal domain and beta-arrestin1 were predominantly required by CXCL9 and CXCL10, and the third intracellular loop was predominantly required by CXCL11. Chemotaxis and calcium mobilization induced by all three CXCR3 ligands were dependent on the CXCR3 carboxyl terminus and the DRY sequence in the third trans-membrane domain. Our findings demonstrate that distinct domains of CXCR3 mediate its functions and suggest that the differential requirement of these domains contributes to the complexity of the chemokine system.
Assuntos
Quimiocinas CXC/química , Peptídeos e Proteínas de Sinalização Intercelular/química , Receptores de Quimiocinas/química , Sequência de Aminoácidos , Arrestinas/metabolismo , Sítios de Ligação , Western Blotting , Cálcio/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Quimiocina CXCL10 , Quimiocina CXCL11 , Quimiocina CXCL9 , Quimiotaxia , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Dinaminas/metabolismo , Endotélio/metabolismo , Humanos , Concentração Inibidora 50 , Ligantes , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Receptores CXCR3 , Proteínas Recombinantes/metabolismo , Transfecção , beta-ArrestinasRESUMO
Pulmonary fibrosis is an enigmatic and devastating disease with few treatment options, now thought to result from abnormal wound healing in the lung in response to injury. We have previously noted a role for the chemokine interferon gamma-inducible protein of 10 kD (IP-10)/CXC chemokine ligand 10 in the regulation of cutaneous wound healing, and consequently investigated whether IP-10 regulates pulmonary fibrosis. We found that IP-10 is highly expressed in a mouse model of pulmonary fibrosis induced by bleomycin. IP-10-deficient mice exhibited increased pulmonary fibrosis after administration of bleomycin, suggesting that IP-10 limits the development of fibrosis in this model. Substantial fibroblast chemoattractant and proliferative activities were generated in the lung after bleomycin exposure. IP-10 significantly inhibited fibroblast responses to the chemotactic, but not the proliferative activity generated, suggesting that IP-10 may attenuate fibroblast accumulation in bleomycin-induced pulmonary fibrosis by limiting fibroblast migration. Consistent with this inhibitory activity of IP-10 on fibroblast migration, fibroblast accumulation in the lung after bleomycin exposure was dramatically increased in IP-10-deficient mice compared with wild-type mice. Conversely, transgenic mice overexpressing IP-10 were protected from mortality after bleomycin exposure, and demonstrated decreased fibroblast accumulation in the lung after challenge compared with wild-type mice. Our findings suggest that interruption of fibroblast recruitment may represent a novel therapeutic strategy for pulmonary fibrosis, which could have applicability to a wide range of fibrotic illnesses.