ABSTRACT
BACKGROUND & AIMS: Hepatitis B virus (HBV) infection is difficult to cure owing to the persistence of covalently closed circular viral DNA (cccDNA). We performed single-cell transcriptome analysis of newly established HBV-positive and HBV-negative hepatocellular carcinoma cell lines and found that dedicator of cytokinesis 11 (DOCK11) was crucially involved in HBV persistence. However, the roles of DOCK11 in the HBV lifecycle have not been clarified. METHODS: The cccDNA levels were measured by Southern blotting and real-time detection polymerase chain reaction in various hepatocytes including PXB cells by using an HBV-infected model. The retrograde trafficking route of HBV capsid was investigated by super-resolution microscopy, proximity ligation assay, and time-lapse analysis. The downstream molecules of DOCK11 and underlying mechanism were examined by liquid chromatography-tandem mass spectrometry, immunoblotting, and enzyme-linked immunosorbent assay. RESULTS: The cccDNA levels were strongly increased by DOCK11 overexpression and repressed by DOCK11 suppression. Interestingly, DOCK11 functionally associated with retrograde trafficking proteins in the trans-Golgi network (TGN), Arf-GAP with GTPase domain, ankyrin repeat, and pleckstrin homology domain-containing protein 2 (AGAP2), and ADP-ribosylation factor 1 (ARF1), together with HBV capsid, to open an alternative retrograde trafficking route for HBV from early endosomes (EEs) to the TGN and then to the endoplasmic reticulum (ER), thereby avoiding lysosomal degradation. Clinically, DOCK11 levels in liver biopsies from patients with chronic hepatitis B were significantly reduced by entecavir treatment, and this reduction correlated with HBV surface antigen levels. CONCLUSIONS: HBV uses a retrograde trafficking route via EEs-TGN-ER for infection that is facilitated by DOCK11 and serves to maintain cccDNA. Therefore, DOCK11 is a potential therapeutic target to prevent persistent HBV infection.
Subject(s)
Hepatitis B, Chronic , Hepatitis B , Humans , Hepatitis B virus/genetics , trans-Golgi Network/metabolism , Hepatitis B/metabolism , Lysosomes/metabolismABSTRACT
Populations of CD8+ lung-resident memory T (TRM) cells persist in the interstitium and epithelium (airways) following recovery from respiratory virus infections. While it is clear that CD8+ TRM cells in the airways are dynamically maintained via the continuous recruitment of new cells, there is a vigorous debate about whether tissue-circulating effector memory T (TEM) cells are the source of these newly recruited cells. Here we definitively demonstrate that CD8+ TRM cells in the lung airways are not derived from TEM cells in the circulation, but are seeded continuously by TRM cells from the lung interstitium. This process is driven by CXCR6 that is expressed uniquely on TRM cells but not TEM cells. We further demonstrate that the lung interstitium CD8+ TRM cell population is also maintained independently of TEM cells via a homeostatic proliferation mechanism. Taken together, these data show that lung memory CD8+ TRM cells in the lung interstitium and airways are compartmentally separated from TEM cells and clarify the mechanisms underlying their maintenance.
Subject(s)
Alveolar Epithelial Cells/immunology , CD8-Positive T-Lymphocytes/immunology , Immunologic Memory , Parenchymal Tissue/immunology , Animals , CD8-Positive T-Lymphocytes/metabolism , Immunophenotyping , Lung/immunology , Lung/metabolism , Lymphocyte Activation/immunology , Mice , Models, Biological , Parenchymal Tissue/metabolism , Receptors, CXCR6/metabolism , Respiratory Mucosa/immunology , Respiratory Mucosa/metabolismABSTRACT
Resident memory T cells (TRM cells) are an important first-line defense against respiratory pathogens, but the unique contributions of lung TRM cell populations to protective immunity and the factors that govern their localization to different compartments of the lung are not well understood. Here, we show that airway and interstitial TRM cells have distinct effector functions and that CXCR6 controls the partitioning of TRM cells within the lung by recruiting CD8 TRM cells to the airways. The absence of CXCR6 significantly decreases airway CD8 TRM cells due to altered trafficking of CXCR6-/- cells within the lung, and not decreased survival in the airways. CXCL16, the ligand for CXCR6, is localized primarily at the respiratory epithelium, and mice lacking CXCL16 also had decreased CD8 TRM cells in the airways. Finally, blocking CXCL16 inhibited the steady-state maintenance of airway TRM cells. Thus, the CXCR6/CXCL16 signaling axis controls the localization of TRM cells to different compartments of the lung and maintains airway TRM cells.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Immunologic Memory , Immunomodulation , Receptors, CXCR6/metabolism , Respiratory Mucosa/immunology , Respiratory Mucosa/metabolism , Animals , Gene Expression , Humans , Immunophenotyping , Mice , Mice, Knockout , Protein Binding , Receptors, CXCR6/genetics , T-Cell Antigen Receptor SpecificityABSTRACT
BACKGROUND & AIMS: The KRAS gene is mutated in most pancreatic ductal adenocarcinomas (PDAC). Expression of this KRAS oncoprotein in mice is sufficient to initiate carcinogenesis but not progression to cancer. Activation of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) is required for KRAS for induction and maintenance of PDAC in mice. The somatostatin receptor subtype 2 (sst2) inhibits PI3K, but sst2 expression is lost during the development of human PDAC. We investigated the effects of sst2 loss during KRAS-induced PDAC development in mice. METHODS: We analyzed tumor growth in mice that expressed the oncogenic form of KRAS (KRAS(G12D)) in pancreatic precursor cells, as well as sst2+/- and sst2-/-, and in crossed KRAS(G12D);sst2+/- and KRAS(G12D);sst2-/- mice. Pancreatic tissues and acini were collected and assessed by histologic, immunoblot, immunohistochemical, and reverse-transcription polymerase chain reaction analyses. We also compared protein levels in paraffin-embedded PDAC samples from patients vs heathy pancreatic tissues from individuals without pancreatic cancer. RESULTS: In sst2+/- mice, PI3K was activated and signaled via AKT (PKB; protein kinase B); when these mice were crossed with KRAS(G12D) mice, premalignant lesions, tumors, and lymph node metastases developed more rapidly than in KRAS(G12D) mice. In crossed KRAS(G12D);sst2+/- mice, activation of PI3K signaling via AKT resulted in activation of nuclear factor-κB (NF-κB), which increased KRAS activity and its downstream pathways, promoting initiation and progression of neoplastic lesions. We found this activation loop to be mediated by PI3K-induced production of the chemokine CXCL16. Administration of a CXCL16-neutralizing antibody to KRAS(G12D) mice reduced activation of PI3K signaling to AKT and NF-κB, blocking carcinogenesis. Levels of CXCL16 and its receptor CXCR6 were significantly higher in PDAC tissues and surrounding acini than in healthy pancreatic tissues from mice or human beings. In addition, expression of sst2 was progressively lost, involving increased PI3K activity, in mouse lesions that expressed KRAS(G12D) and progressed to PDAC. CONCLUSIONS: Based on analyses of mice, loss of sst2 from pancreatic tissues activates PI3K signaling via AKT, leading to activation of NF-κB, amplification of oncogenic KRAS signaling, increased expression of CXCL16, and pancreatic tumor formation. CXCL16 might be a therapeutic target for PDAC.
Subject(s)
Carcinoma, Pancreatic Ductal/enzymology , Cell Proliferation , Chemokine CXCL6/metabolism , Mutation , Pancreatic Neoplasms/enzymology , Phosphatidylinositol 3-Kinase/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Receptors, Somatostatin/deficiency , Signal Transduction , Animals , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/immunology , Carcinoma, Pancreatic Ductal/secondary , Case-Control Studies , Cell Line, Tumor , Chemokine CXCL16 , Chemokines, CXC/metabolism , Disease Models, Animal , Genetic Predisposition to Disease , Humans , Lymphatic Metastasis , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , NF-kappa B/metabolism , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/pathology , Phenotype , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Scavenger/metabolism , Receptors, Somatostatin/genetics , Receptors, Somatostatin/metabolism , Time Factors , Transfection , Tumor Burden , Up-RegulationABSTRACT
Pulmonary fibrosis is characterized by accumulation of activated fibroblasts that produce excessive amounts of extracellular matrix components such as collagen type I. However, the dynamics and activation signatures of fibroblasts during fibrogenesis remain poorly understood, especially in vivo. We examined changes in lung tissue cell populations and in the phenotype of activated fibroblasts after acute injury in a model of bleomycin-induced pulmonary fibrosis. Despite clustering of collagen type I-producing fibroblasts in fibrotic regions, flow cytometry-based quantitative analysis of whole lungs revealed that the number of fibroblasts in the lungs remained constant. At the peak of inflammation, fibroblast proliferation and apoptosis were both increased, suggesting that the clustering was not merely a result of proliferation, but also of fibroblast migration from nearby alveolar walls. Parabiosis experiments demonstrated that fibroblasts were not supplied from the circulation. Comprehensive gene expression analysis of freshly isolated fibroblasts revealed a detailed activation signature associated with fibrogenesis, including changes in genes responsible for migration and extracellular matrix construction. The Spp1 gene, which encodes osteopontin, was highly up-regulated and was an identifying characteristic of activated fibroblasts present at the sites of remodeling. Osteopontin may serve as a useful marker of profibrotic fibroblasts. These results provide insights into the cellular and molecular mechanisms underlying pulmonary fibrosis and provide a foundation for development of specific antifibrotic therapies.
Subject(s)
Fibroblasts/metabolism , Fibroblasts/pathology , Pulmonary Fibrosis/metabolism , Pulmonary Fibrosis/pathology , Animals , Apoptosis , Biomarkers/metabolism , Bleomycin , Bone Marrow Cells/pathology , Cell Proliferation , Collagen Type I/metabolism , Gene Expression Profiling , Gene Ontology , Green Fluorescent Proteins/metabolism , Lung/metabolism , Lung/pathology , Mice , Mice, Inbred C57BL , Osteopontin/metabolism , Recombinant Fusion Proteins/metabolism , Up-Regulation/geneticsABSTRACT
CD4(+) T cells play a central role in the development of inflammatory bowel disease (IBD) via high-level production of effector cytokines such as IFN-γ and TNF-α. To better characterize the colitogenic CD4(+) T cells, we examined their expression of CXCR6, a chemokine receptor that is expressed by T cells upon activation and is upregulated in several inflammatory diseases. We found that 80% of colonic lamina propria CD4(+) T cells expressed CXCR6 in the CD45RB(high) T cell-transferred colitis model. CXCR6 expression was similarly upregulated in inflamed mucosa of patients with Crohn's disease. Although surface marker analysis demonstrated that both CXCR6(+) and CXCR6(-) CD4(+) T-cell subsets consist of the cells with effector and effector-memory cells, the more cells in the CXCR6(+) subset produced IFN-γ and TNF-α compared to CXCR6(-) subset, and only the CXCR6(+) subset produced IL-17A. Nevertheless, adoptive retransfer of lamina propria CXCR6(+) T cells into Rag1 (-/-) recipients failed to induce the disease due to limited expansion of the transferred cells. By contrast, retransfer of CXCR6(-) cells evoked colitis similar to that observed in CD4(+)CD45RB(high) T cell-transferred mice, and resulted in their conversion into CXCR6(+) cells. Collectively, these observations suggest that the CXCR6(+)CD4(+) T-cell subset consists of terminally differentiated effector cells that serve as the major source of effector cytokines in the inflamed tissue, whereas CXCR6(-)CD4(+) T-cell subset serves as a colitogenic memory compartment that retains the ability to proliferate and differentiate into CXCR6(+)CD4(+) T cells.
Subject(s)
CD4-Positive T-Lymphocytes/physiology , Colitis/immunology , Receptors, Chemokine/metabolism , Receptors, Virus/metabolism , Adult , Aged , Animals , CD4-Positive T-Lymphocytes/metabolism , Chronic Disease , Colitis/metabolism , Colitis/pathology , Crohn Disease/immunology , Crohn Disease/metabolism , Crohn Disease/pathology , Female , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Knockout , Middle Aged , Receptors, CXCR6 , Young AdultABSTRACT
BACKGROUND AND AIMS: Inflammatory bowel disease (IBD) is initiated and perpetuated by a dysregulated immune response to unknown environmental antigens such as luminal bacteria in genetically susceptible hosts. SR-PSOX/CXCL16, a scavenger receptor that binds phosphatidylserine and oxidised lipoprotein, has both phagocytic activity and chemotactic properties. The aim of this study was to investigate the role of SR-PSOX/CXCL16 in patients with IBD and experimental murine colitis. METHODS: The serum levels of SR-PSOX/CXCL16 were measured in patients with IBD. The roles of SR-PSOX/CXCL16 in phagocytosis of bacterial components and cytokine production by macrophages from wild-type (WT) and SR-PSOX/CXCL16 knockout (KO) mice were assessed. Colitis was induced by administering dextran sulfate sodium (DSS) to WT and SR-PSOX/CXCL16 KO mice. Colonic inflammation was analysed by clinical, histological and immunological parameters. Finally, the effect of a monoclonal antibody (mAb) to SR-PSOX/CXCL16 on DSS-induced colitis and trinitrobenzene sulfonic acid-induced colitis models was evaluated. RESULTS: Serum levels of SR-PSOX/CXCL16 correlated significantly with the disease activity of patients with IBD. Ex vivo experiments showed that SR-PSOX/CXCL16 was involved in both phagocytosis of bacterial antigens and the T helper 1 immune response through the production of interleukin 12 and interferon γ. In vivo murine experiments demonstrated the upregulated gene expression of SR-PSOX/CXCL16 in inflamed colonic tissues and the predominant expression of SR-PSOX/CXCL16 on macrophages. SR-PSOX/CXCL16 KO mice were less susceptible to colonic inflammation than were their WT littermates. Administration of SR-PSOX/CXCL16 mAb ameliorated the condition in the two different experimental colitis models. CONCLUSIONS: SR-PSOX/CXCL16 plays a critical role in colonic inflammation and could be a potential therapeutic target for patients with IBD.
Subject(s)
Chemokine CXCL6/physiology , Chemokines, CXC/physiology , Inflammatory Bowel Diseases/immunology , Receptors, Scavenger/physiology , Adult , Animals , Chemokine CXCL16 , Colon/pathology , Cytokines/metabolism , Disease Progression , Female , Gene Expression/immunology , Humans , Inflammatory Bowel Diseases/physiopathology , Interleukin-12/biosynthesis , Interleukin-12/immunology , Macrophages, Peritoneal/immunology , Male , Mice , Mice, Knockout , Mucous Membrane/cytology , Phagocytosis/physiology , Young AdultABSTRACT
Invariant NKT (iNKT) cells can induce potent antitumor responses in vivo. However, the mechanisms that regulate the effects of iNKT cells are unclear. The chemokine receptor CXCR6, and its ligand CXCL16, have been shown to play critical roles in iNKT cell homeostasis and activation. Thus we investigated the role of CXCR6 in protection against experimental metastasis of B16-F10 melanoma (B16) and Lewis lung carcinoma (LLC) cells to the liver and lungs. Wild-type and CXCR6(-/-) mice exhibited no differences in tumor cell metastasis to the lungs. However, metastasis of LLC and B16 tumor cells to the liver was enhanced in CXCR6(-/-) mice. Liver metastasis was also increased in wild-type mice treated with a CXCL16 neutralizing Ab. As Ab treatments did not alter iNKT cell numbers, this implicates a direct role for CXCR6/CXCL16 in regulating antitumor immunity. Cytokine induction was significantly attenuated in CXCR6(-/-) mice upon systemic iNKT cell activation with the glycolipid Ags alpha-galactosylceramide (alpha-GalCer), alpha-C-GalCer (a Th1 polarizing derivative), or OCH (a Th2 polarizing derivative). Despite differences in the levels of cytokine production, liver and lung metastasis were inhibited significantly in both wild-type and CXCR6(-/-) mice treated with glycolipids. Single doses of alpha-GalCer, alpha-C-GalCer, or OCH were sufficient to prevent liver metastasis and subsequent doses failed to elicit optimal cytokine responses. Our findings implicate a role for CXCR6 in natural immunosurveillance against liver metastasis. However, CXCR6 deficiency could be overcome by systemic iNKT cell activation, demonstrating that even suboptimal iNKT cell activation can protect against metastasis.
Subject(s)
Carcinoma, Lewis Lung/immunology , Liver Neoplasms, Experimental/immunology , Lung Neoplasms/immunology , Lymphocyte Activation/immunology , Melanoma, Experimental/immunology , Natural Killer T-Cells/immunology , Natural Killer T-Cells/metabolism , Receptors, CXCR/antagonists & inhibitors , Animals , Carcinoma, Lewis Lung/metabolism , Carcinoma, Lewis Lung/pathology , Cell Line, Tumor , Liver Neoplasms, Experimental/pathology , Liver Neoplasms, Experimental/secondary , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Natural Killer T-Cells/pathology , Receptors, CXCR/deficiency , Receptors, CXCR/genetics , Receptors, CXCR6ABSTRACT
Tumor growth is associated with aberrant myelopoiesis, including the accumulation of CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs) that have the potential to promote tumor growth. However, the identity, growth, and migration of tumor-associated MDSCs remain undefined. We demonstrate herein that MDSCs at tumor site were composed primarily of bone marrow-derived CD11b(+)Gr-1(hi)Ly-6C(int) neutrophils and CD11b(+)Gr-1(int/dull)Ly-6C(hi) macrophages. Unexpectedly, in vivo bromodeoxyuridine (BrdU) labeling and parabiosis experiments revealed that tumor-infiltrating macrophages were replenished more rapidly than neutrophils. CCR2 deficiency caused striking conversion of infiltrating cellular dominance from macrophages to neutrophils in the tumor with the excessive production of CXCR2 ligands and granulocyte-colony stimulating factor in the tumor without affecting tumor growth. Overall, our data established the identity and dynamics of MDSCs in a tumor-bearing host mediated by chemokines and elucidated unexpected effects of the paucity of macrophages on tumor development.
Subject(s)
Carcinoma, Lewis Lung/pathology , Macrophages/pathology , Myeloid Cells/pathology , Myelopoiesis/physiology , Neutrophils/pathology , Receptors, Chemokine/genetics , Animals , CD11b Antigen/metabolism , CX3C Chemokine Receptor 1 , Carcinoma, Lewis Lung/immunology , Cell Line, Tumor , Female , Green Fluorescent Proteins/genetics , Immunophenotyping , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Myeloid Cells/metabolism , Neoplasm Transplantation , Neutrophils/metabolism , Receptors, CCR1/genetics , Receptors, CCR2/genetics , Receptors, CCR5/genetics , Receptors, Chemokine/metabolism , T-Lymphocytes/metabolism , T-Lymphocytes/pathologyABSTRACT
The transmembrane chemokine CXCL 16 (CXCL16), which is the same molecule as the scavenger receptor that binds phosphatidylserine and oxidized lipoprotein (SR-PSOX), has been shown to mediate chemotaxis and adhesion of CXC chemokine receptor 6-expressing cells such as NKT and activated Th1 cells. We generated SR-PSOX/CXCL16-deficient mice and examined the role of this chemokine in vivo. The mutant mice showed a reduced number of liver NKT cells, and decreased production of IFN-gamma and IL-4 by administration of alpha-galactosylceramide (alphaGalCer). Of note, the alphaGalCer-induced production of IFN-gamma was more severely impaired than the production of IL-4 in SR-PSOX-deficient mice. In this context, SR-PSOX-deficient mice showed impaired sensitivity to alphaGalCer-induced anti-tumor effect mediated by IFN-gamma from NKT cells. NKT cells from wild-type mice showed impaired production of IFN-gamma, but not IL-4, after their culture with alphaGalCer and APCs from mutant mice. Moreover, Propionibacterium acnes-induced in vivo Th1 responses were severely impaired in SR-PSOX-deficient as well as NKT KO mice. Taken together, SR-PSOX/CXCL16 plays an important role in not only the production of IFN-gamma by NKT cells, but also promotion of Th1-inclined immune responses mediated by NKT cells.
Subject(s)
Chemokine CXCL6/physiology , Interferon-gamma/metabolism , Killer Cells, Natural/immunology , Th1 Cells/immunology , Animals , Chemokine CXCL16 , Chemokine CXCL6/genetics , Galactosylceramides/administration & dosage , Gram-Positive Bacterial Infections/immunology , Liver/immunology , Mice , Mice, Knockout , Propionibacterium acnes/immunologyABSTRACT
PURPOSE: The accumulation of macrophages is known to be involved in the pathogenesis of age-related macular degeneration (AMD), but the reasons why macrophages accumulate in AMD lesions have not been determined. Because the histopathology of AMD has some factors common with those of atherosclerosis, the authors hypothesized that macrophages accumulate to take up oxidized lipoproteins in the eyes of patients with AMD, as has been demonstrated in atherosclerosis. METHODS: Immunohistochemistry was performed on 10 surgically excised choroidal neovascular (CNV) membranes from eyes with AMD. An antibody against oxidized lipoprotein and antibodies against the scavenger receptors SR-PSOX and LOX-1 were used. Antibodies against cytokeratin, CD68, and von Willebrand factor were used to identify retinal pigment epithelium (RPE), macrophages, and vascular endothelial cells, respectively. RT-PCR was performed to detect the mRNAs of the scavenger receptors in the CNV membranes. RESULTS: Oxidized lipoproteins were immunohistochemically detected in the CNV membranes. Intense immunostaining was observed at the surface of the CNV membranes with the SR-PSOX antibody, whereas LOX-1 immunostaining was weak. Cells expressing scavenger receptors were found to be predominantly macrophages with a minority of RPE. Both SR-PSOX and LOX-1 mRNAs were detected in CNV membranes. CONCLUSIONS: Oxidized lipoproteins are present in AMD lesions. Macrophages and RPE in the CNV membranes express cell surface scavenger receptors for oxidized lipoproteins. These findings suggest that macrophages may accumulate to take up oxidized lipoproteins in AMD and that the control of oxidative stress and macrophage responses may therefore be potential treatments for AMD.
Subject(s)
Chemokines, CXC/metabolism , Lipoproteins, LDL/metabolism , Macular Degeneration/metabolism , Receptors, Scavenger/metabolism , Scavenger Receptors, Class E/metabolism , Aged , Aged, 80 and over , Antigens, CD , Antigens, Differentiation, Myelomonocytic , Chemokine CXCL16 , Chemokines, CXC/genetics , Choroidal Neovascularization/metabolism , Choroidal Neovascularization/surgery , Female , Humans , Immunoenzyme Techniques , Keratins , Macrophages/metabolism , Male , Middle Aged , Pigment Epithelium of Eye/metabolism , RNA, Messenger/metabolism , Receptors, Scavenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Scavenger Receptors, Class E/genetics , von Willebrand FactorABSTRACT
Coincidence of the beneficial graft-vs.-tumor (GVT) effects and the detrimental graft-vs.-host disease (GVHD) remains the major obstacle against the widespread use of allogeneic bone marrow transplantation (BMT) as tumor immunotherapy. We here demonstrate that intervention of MAdCAM-1 (mucosal vascular addressin cell adhesion molecule-1) or fractalkine/CX3CL1 after the expansion of allo-reactive donor CD8 T cells selectively inhibits the recruitment of effector donor CD8 T cells to the intestine and alleviates the graft-vs.-host reaction (GVHR) associated intestinal injury without impairing GVT effects. In a nonirradiated acute GVHD model, donor CD8 T cells up-regulate the expression of intestinal homing receptor alpha4beta7 and chemokine receptors CXCR6 and CX3CR1, as they differentiate into effector cells and subsequently infiltrate into the intestine. Administration of anti-MAdCAM-1 antibody or anti-fractalkine antibody, even after the expansion of alloreactive donor CD8 T cells, selectively reduced the intestine-infiltrating donor CD8 T cells and the intestinal crypt cell apoptosis without affecting the induction of donor derived anti-host CTL or the infiltration of donor CD8 T cells in the hepatic tumor. Moreover, in a clinically relevant GVHD model with myeloablative conditioning, these antibodies significantly improved the survival and loss of weight without impairing the beneficial GVT effects. Thus, interruption of alpha4beta7-MAdCAM-1 or CX3CR1-fractalkine interactions in the late phase of GVHD would be a novel therapeutic approach for the separation of GVT effects from GVHR-associated intestinal injury.
Subject(s)
Cell Adhesion Molecules/physiology , Chemokines, CX3C/physiology , Graft vs Host Disease/pathology , Graft vs Host Reaction/immunology , Graft vs Tumor Effect/immunology , Intestines/pathology , Membrane Proteins/physiology , Animals , Antibodies/pharmacology , Biological Transport , CD8-Positive T-Lymphocytes/immunology , Chemokine CX3CL1 , Chemokines, CX3C/immunology , Intestines/immunology , Membrane Proteins/immunology , Mice , Mice, Inbred C57BL , MucoproteinsABSTRACT
The recently identified CXCL16 has dual functions as a transmembrane adhesion molecule and a soluble chemokine. In this study we found that CXCL16 mRNA and protein were expressed constitutively on the follicle-associated epithelium covering Peyer's patches (PPs), isolated lymphoid follicles, and cecal patches, but minimally on the villous epithelium in the murine gastrointestinal tract. The CXCL16 receptor CXCR6/Bonzo was constitutively expressed on subpopulations of CD4+ and CD8+ T cells isolated from PPs. The expression of CXCR6/Bonzo on the PP T cells was up-regulated after stimulation with anti-CD3 and anti-CD28 mAbs. The activated PP T cells showed chemotactic migration in response to the soluble N-terminal chemokine domain of CXCL16. Furthermore, the activated PP T cells selectively adhered to cells expressing murine CXCL16. To determine the physiological role of CXCL16 in GALT, we first carefully analyzed T cell distribution in PPs. T cells localized not only in the interfollicular region but also at a lesser frequency in the subepithelial dome (SED) and in the germinal center of lymphoid follicles. Consistently, the majority of the adoptive transferred activated T cells migrated into the SED and the interfollicular region. However, the neutralization of CXCL16 specifically reduced the migration of the adoptive, transferred, activated T cells into the SED of PPs. These data suggest that CXCL16 expressed on the follicle-associated epithelium plays an important role in the recruitment and retention of activated T cells in the SED and should, at least partially, be responsible for lymphocyte compartmentalization in GALT.
Subject(s)
Chemokines, CXC/biosynthesis , Gastric Mucosa/immunology , Intestinal Mucosa/immunology , Receptors, Scavenger/biosynthesis , T-Lymphocytes/immunology , Adoptive Transfer , Animals , CD4 Antigens/immunology , CD8 Antigens/immunology , Chemokine CXCL16 , Chemokine CXCL6 , Chemokines/immunology , Chemokines, CXC/immunology , Chemotaxis, Leukocyte/immunology , Flow Cytometry , Gene Expression , Gene Expression Profiling , Immunohistochemistry , In Situ Hybridization , Mice , Peyer's Patches/cytology , Peyer's Patches/immunology , Polymerase Chain Reaction , RNA, Messenger/analysis , Receptors, Scavenger/immunology , T-Lymphocytes/cytologyABSTRACT
OBJECTIVE: Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with massive T cell infiltration into the synovium. The accumulated T cells express type 1 cytokines, such as interferon-gamma (IFNgamma) and tumor necrosis factor alpha, and activated markers of inflammation, such as CD154 and inducible costimulator (ICOS). It is thought that chemokines contribute to T cell accumulation in the synovium. In this study, we examined the role of CXCL16 and CXCR6 in T cell migration and stimulation in RA synovium. METHODS: Expression of CXCL16 and CXCR6 was analyzed by immunohistochemistry, reverse transcription-polymerase chain reaction, Western blotting, and/or flow cytometry. Migration activity was assessed using a chemotaxis chamber. IFNgamma production was analyzed by enzyme-linked immunosorbent assay. The effect of anti-CXCL16 monoclonal antibody on murine collagen-induced arthritis (CIA) was evaluated. RESULTS: CXCL16 was expressed in RA synovium. CXCR6 was expressed more frequently on synovial T cells than in peripheral blood. Moreover, CXCR6-positive synovial T cells more frequently expressed CD154 and ICOS than did CXCR6-negative T cells. Stimulation with interleukin-15 (IL-15) up-regulated the expression of CXCR6 on peripheral blood T cells, and then stimulation with CXCL16 induced migration of IL-15-stimulated T cells and enhanced IFNgamma production. Furthermore, anti-CXCL16 monoclonal antibody significantly reduced the clinical arthritis score and reduced infiltration of inflammatory cells and bone destruction in the synovium of mice with CIA. CONCLUSION: Our results indicate that CXCL16 plays an important role in T cell accumulation and stimulation in RA synovium and suggest that CXCL16 could be a target molecule in new therapies for RA.
Subject(s)
Arthritis, Rheumatoid/immunology , Chemokines, CXC/immunology , Membrane Proteins/immunology , Receptors, Cytokine/immunology , Receptors, G-Protein-Coupled/immunology , Receptors, Immunologic/immunology , Receptors, Virus/immunology , Aged , Animals , Antibodies, Monoclonal/pharmacology , Arthritis, Rheumatoid/etiology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Chemokine CXCL16 , Chemokine CXCL6 , Chemokines, CXC/metabolism , Female , Humans , Male , Membrane Proteins/metabolism , Mice , Mice, Inbred DBA , Middle Aged , Receptors, CXCR6 , Receptors, Chemokine , Receptors, Cytokine/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Immunologic/metabolism , Receptors, Scavenger , Receptors, Virus/metabolism , Synovial Membrane/cytology , Synovial Membrane/immunologyABSTRACT
It is well-documented that certain chemokines or their receptors play important roles in the graft rejection. However, the roles of chemokines and their receptors in the maintenance of transplantation tolerance remain unclear. In this study, we demonstrate that blocking of the interaction between the chemokine receptor, CXCR6, highly expressed on V alpha14+ NKT cells and its ligand, CXCL16, resulted in the failure to maintain graft tolerance and thus in the induction of acceleration of graft rejection. In a mouse transplant tolerance model, the expression of CXCL16 was up-regulated in the tolerated allografts, and anti-CXCL16 mAb inhibited intragraft accumulation of NKT cells. In vitro experiments further showed that blocking of CXCL16/CXCR6 interaction significantly affected not only chemotaxis but also cell adhesion of NKT cells. These results demonstrate the unique role of CXCL16 and CXCR6 molecules in the maintenance of cardiac allograft tolerance mediated by NKT cells.
Subject(s)
Chemokines, CXC/physiology , Chemotaxis, Leukocyte/immunology , Killer Cells, Natural/immunology , Receptors, Chemokine/physiology , Receptors, Scavenger/physiology , T-Lymphocyte Subsets/immunology , Transplantation Tolerance/immunology , Animals , Antibodies, Blocking/administration & dosage , CHO Cells , Cell Adhesion/genetics , Cell Adhesion/immunology , Cell Aggregation/genetics , Cell Aggregation/immunology , Chemokine CXCL16 , Chemokines, CXC/antagonists & inhibitors , Chemokines, CXC/metabolism , Chemotaxis, Leukocyte/genetics , Cricetinae , Graft Rejection/genetics , Graft Rejection/immunology , Heart Transplantation/immunology , Heart Transplantation/pathology , Killer Cells, Natural/cytology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Receptors, CXCR , Receptors, CXCR6 , Receptors, Chemokine/antagonists & inhibitors , Receptors, Scavenger/antagonists & inhibitors , Receptors, Scavenger/metabolism , T-Lymphocyte Subsets/cytology , Transplantation Tolerance/geneticsABSTRACT
CXCL16 is a unique chemokine with characteristics as a receptor for phosphatidylserine and oxidized low density lipoproteins in macrophages, and is involved in the accumulation of cellular cholesterol during atherosclerotic lesion development. In this study, we report a new function of CXCL16 as a novel angiogenic factor in human umbilical vein endothelial cells (HUVEC). CXCL16 stimulated proliferation and chemotaxis of HUVEC in a dose-dependent manner, reaching a maximum at 1 nM. CXCL16 also significantly induced tube formation of HUVEC on Matrigel. Further, exposure of HUVEC to CXCL16 led to a time- and dose-dependent activation of mitogen-activated protein kinase (ERK1/2), which was completely inhibited by a mitogen-activated protein kinase kinase inhibitor, PD98059. Proliferation and tube formation in response to CXCL16 were also blocked by the pretreatment with PD98059, but not CXCL16-induced chemotaxis. Thus, our data indicate that CXCL16 may act as a novel angiogenic factor for HUVEC and that ERK is involved as an important signaling molecule to mediate its angiogenic effects.
Subject(s)
Chemokines, CXC/physiology , Endothelium, Vascular/cytology , Membrane Proteins/physiology , Neovascularization, Physiologic/physiology , Receptors, Immunologic/physiology , Umbilical Veins/cytology , Cell Movement/physiology , Cell Proliferation , Cells, Cultured , Chemokine CXCL16 , Humans , Receptors, ScavengerABSTRACT
Dendritic cells (DCs) coordinate T cell responses by producing T cell-attracting chemokines and by inducing the expression of chemokine receptors on T cells. Scavenger receptor for phosphatidylserine and oxidized lipoprotein (SR-PSOX)/CXC chemokine ligand 16 (CXCL16) is a unique chemokine that also functions as an endocytic receptor and an adhesion molecule in its membrane-bound form. SR-PSOX/CXCL16 is the only known ligand of CXC chemokine receptor 6 (CXCR6) that is expressed on activated T cells and thus, may play an important role in enhancing effector functions of T cells. Here, we investigated the expression of SR-PSOX/CXCL16 on human DC subsets and that of CXCR6 on T cell subpopulations to elucidate the dynamics of CXCL16/CXCR6 interaction in DC/T cell responses. Membrane-bound SR-PSOX/CXCL16 was expressed on macrophages, monocyte-derived DCs, and blood myeloid DCs, and the expression increased after DC maturation. Myeloid antigen-presenting cells constitutively secreted SR-PSOX/CXCL16 for an extended period, suggesting the involvement of CXCL16 in peripheral and lymphoid tissues. Plasmacytoid DCs hardly expressed SR-PSOX/CXCL16 on their surfaces but secreted significant amounts of SR-PSOX/CXCL16. A subset of CD4+ effector memory T (T(EM)) cells constitutively expressed CXCR6, whereas central memory T cells (T(CM)) and naive T cells did not. Upon stimulation with mature DCs, however, the expression of CXCR6 on T(CM) cells was markedly up-regulated, whereas the expression on naive T cells was induced only weakly. These results suggest that the interaction between SR-PSOX/CXCL16 and CXCR6 plays an important role in enhancing T(CM) cell responses by mature DCs in lymphoid tissues and in augmenting T(EM) cell responses by macrophages in peripheral inflamed tissues.
Subject(s)
CD4-Positive T-Lymphocytes/metabolism , Chemokines, CXC/metabolism , Dendritic Cells/metabolism , Membrane Proteins/metabolism , Receptors, Cytokine/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Immunologic/metabolism , Receptors, Virus/metabolism , Antigen Presentation , CD4-Positive T-Lymphocytes/immunology , Cell Communication/immunology , Cell Differentiation/immunology , Chemokine CXCL16 , Dendritic Cells/cytology , Dendritic Cells/immunology , Humans , Kinetics , Receptors, CXCR6 , Receptors, Chemokine , Receptors, ScavengerABSTRACT
The scavenger receptor that binds phosphatidylserine and oxidized lipoprotein (SR-PSOX)/CXCL16 is a chemokine expressed on macrophages and dendritic cells, while its receptor expresses on T and NK T cells. We investigated the role of SR-PSOX/CXCL16 on acute and adoptive experimental autoimmune encephalomyelitis (EAE), which is Th1-polarized T cell-mediated autoimmune disease of the CNS. Administration of mAb against SR-PSOX/CXCL16 around the primary immunization decreased disease incidence of acute EAE with associated reduced infiltration of mononuclear cells into the CNS. Its administration was also shown to inhibit elevation of serum IFN-gamma level at primary immune response, as well as subsequent generation of Ag-specific T cells. In adoptive transfer EAE, treatment of recipient mice with anti-SR-PSOX/CXCL16 mAb also induced not only decreased clinical disease incidence, but also diminished traffic of mononuclear cells into the CNS. In addition, histopathological analyses showed that clinical development of EAE correlates well with expression of SR-PSOX/CXCL16 in the CNS. All the results show that SR-PSOX/CXCL16 plays important roles in EAE by supporting generation of Ag-specific T cells, as well as recruitment of inflammatory mononuclear cells into the CNS.
Subject(s)
Chemokines, CXC/physiology , Encephalomyelitis, Autoimmune, Experimental/etiology , Membrane Proteins/physiology , Receptors, Immunologic/physiology , Acute Disease , Adoptive Transfer , Amino Acid Sequence , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/therapeutic use , Chemokine CXCL16 , Chemokine CXCL6 , Chemokines, CXC/deficiency , Chemokines, CXC/genetics , Chemokines, CXC/immunology , Chemotaxis, Leukocyte , Demyelinating Diseases , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/pathology , Encephalomyelitis, Autoimmune, Experimental/prevention & control , Glycoproteins/immunology , Immunization , Inflammation , Lipoproteins, LDL/metabolism , Membrane Proteins/deficiency , Membrane Proteins/genetics , Membrane Proteins/immunology , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Myelin-Oligodendrocyte Glycoprotein , Peptide Fragments/immunology , Phosphatidylserines/metabolism , Receptors, Immunologic/deficiency , Receptors, Immunologic/genetics , Receptors, Immunologic/immunology , Receptors, Scavenger , Spinal Cord/immunology , Spinal Cord/pathology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/transplantationABSTRACT
Chemokines are a family of cytokines that induce directed migration of various types of leukocytes through specific interactions with a group of seven transmembrane receptors. Scavenger receptors are a heterogenous family of transmembrane molecules that commonly bind and uptake oxidized low density lipoprotein and bacteria. Here, we show that not only CXC chemokine 16 (CXCL16)/SR-PSOX, a transmembrane chemokine with scavenger receptor activity, but also 12 out of 15 chemokines examined efficiently bound scavenger receptor ligands in competition with cells expressing their specific chemokine receptors. Furthermore both the chemotactic and scavenger receptor activities of SR-PSOX/CXCL16 were similarly impaired in a series of mutants altered in the chemokine domain, indicating that SR-PSOX/CXCL16 binds scavenger receptor ligands as well as CXCR6 using highly overlapping binding motifs. Taken together, chemokines generally have scavenger receptor-like activity through their receptor-binding domain, suggesting a close evolutionary relationship between chemokines and scavenger receptors.
Subject(s)
Chemokines/metabolism , Receptors, Immunologic/metabolism , Amino Acid Substitution , Animals , B-Lymphocytes/cytology , B-Lymphocytes/metabolism , COS Cells , Cell Line , Chemokine CXCL16 , Chemokine CXCL6 , Chemokines/antagonists & inhibitors , Chemokines/chemistry , Chemokines/genetics , Chemokines, CXC/metabolism , Chemotaxis, Leukocyte , Chlorocebus aethiops , Escherichia coli/metabolism , Humans , Jurkat Cells , Lipoproteins, LDL/metabolism , Lipoproteins, LDL/pharmacology , Membrane Proteins/metabolism , Mice , Phylogeny , Protein Binding , Protein Structure, Tertiary , Receptors, Scavenger , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Staphylococcus aureus/metabolismABSTRACT
OBJECTIVE: SR-PSOX/CXCL16 is a transmembrane chemokine and is implicated in activated CD8+ T cell trafficking. In the present study, we examined the expression pattern of SR-PSOX/CXCL16 in the heart and investigated a potential role of SR-PSOX/CXCL16 in inflammatory valvular heart disease. METHODS AND RESULTS: Initial expression of SR-PSOX/CXCL16 in murine embryos was detected in endothelial cells lining endocardial cushions in the forming heart at E11.5. From mid-gestation to adult, expression of this gene in the heart was exclusively observed in valvular endothelial cells. Examination of SR-PSOX/CXCL16 expression in human cardiac valves demonstrated that SR-PSOX/CXCL16 was strongly expressed in valvular and neocapillary endothelial cells in patients with infective endocarditis. SR-PSOX/CXCL16 expression in neocapillary endothelial cells was also observed in patients with rheumatic and atherosclerotic valvular disease. Moreover, CD8+ T cells were distributed closely to endothelial cells expressing SR-PSOX/CXCL16. In vitro adhesion assays showed that SR-PSOX/CXCL16 induced adhesion of activated CD8+ T cells to vascular cell adhesion molecule-1 (VCAM-1) through very late antigen-4 (VLA-4) activation. Furthermore, SR-PSOX/CXCL16 stimulated interferon-gamma (IFN-gamma) production by CD8+ T cells. CONCLUSIONS: SR-PSOX/CXCL16 may be involved in CD8+ T cell recruitment through VLA-4 activation and stimulation of IFN-gamma production by CD8+ T cells during inflammatory valvular heart disease.