RESUMO
BACKGROUND: Osteoporosis is a devastating skeletal disease responsible for bone fragility and fracture. CX3C chemokine ligand 1 (CX3CL1) is an inflammatory chemokine which has been identified to possess increased expression in the serum of postmenopausal osteoporotic patients. This paper was to illuminate the impacts of CX3CL1 on inflammation, apoptosis and osteogenic differentiation, mineralization in LPS-treated osteoblasts and investigate the regulatory mechanism. METHODS: The viability of MC3T3-E1 cells exposed to elevating doses of LPS was detected by CCK-8 assay. CX3CL1 and C-X3-C motif chemokine receptor 1 (CX3CR1) expression were detected by RT-qPCR and western blot. CX3CR1 expression was examined again following CX3CL1 depletion. The binding of CX3CL1 with CX3CR1 was testified through Co-IP assay. In MC3T3-E1 cells co-transduced with CX3CL1 interference and CX3CR1 overexpression plasmids following LPS exposure, cell activity and inflammation were separately estimated via CCK-8 assay and RT-qPCR. Apoptosis was measured by TUNEL assay and western blot. Osteoblast differentiation was evaluated by ALP activity assay, RT-qPCR and western blot. Osteoblast mineralization was assessed by ARS staining, RT-qPCR and western blot. Results The experimental data presented that LPS attenuated the viability and enhanced CX3CL1 and CX3CR1 expression in MC3T3-E1 cells in a dose-dependent manner. CX3CR1 interacted with CX3CL1 and was positively modulated by CX3CL1. The suppressive role of CX3CL1 absence in LPS-evoked viability decrease, inflammation and apoptosis in MC3T3-E1 cells was reversed by CX3CR1 elevation. Besides, CX3CR1 reversed the promoted osteoblast differentiation and mineralization imposed by CX3CL1 interference. CONCLUSIONS: CX3CL1 knockdown eased inflammation, apoptosis and promoted osteogenic differentiation, mineralization in MC3T3-E1 cells upon LPS exposure through down-regulating CX3CR1.
Assuntos
Calcinose , Quimiocinas CX3C , Humanos , Osteogênese , Lipopolissacarídeos/toxicidade , Ligantes , Linhagem Celular , Diferenciação Celular , Osteoblastos , Apoptose , Quimiocinas CX3C/metabolismo , Receptor 1 de Quimiocina CX3C/metabolismo , Quimiocina CX3CL1/metabolismoRESUMO
Respiratory syncytial virus (RSV) belongs to the family Paramyxoviridae and is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. Through a CX3C chemokine motif (182CWAIC186) in the G protein, RSV binds to the corresponding chemokine receptor, CX3CR1. Since RSV binding to CX3CR1 contributes to disease pathogenesis, we investigated whether a mutation in the CX3C motif by insertion of an alanine, A186, within the CX3C motif, mutating it to CX4C (182CWAIAC187), which is known to block binding to CX3CR1, might decrease disease. We studied the effect of the CX4C mutation in two strains of RSV (A2 and r19F) in a mouse challenge model. We included RSV r19F because it induces mucus production and airway resistance, two manifestations of RSV infection in humans, in mice. Compared to wild-type (wt) virus, mice infected with CX4C had a 0.7 to 1.2 log10-fold lower virus titer in the lung at 5 days postinfection (p.i.) and had markedly reduced weight loss, pulmonary inflammatory cell infiltration, mucus production, and airway resistance after challenge. This decrease in disease was not dependent on decrease in virus replication but did correspond to a decrease in pulmonary Th2 and inflammatory cytokines. Mice infected with CX4C viruses also had higher antibody titers and a Th1-biased T cell memory response at 75 days p.i. These results suggest that the CX4C mutation in the G protein could improve the safety and efficacy of a live attenuated RSV vaccine.IMPORTANCE RSV binds to the corresponding chemokine receptor, CX3CR1, through a CX3C chemokine motif (182CWAIC186) in the G protein. RSV binding to CX3CR1 contributes to disease pathogenesis; therefore, we investigated whether a mutation in the CX3C motif by insertion of an alanine, A186, within the CX3C motif, mutating it to CX4C (182CWAIAC187), known to block binding to CX3CR1, might decrease disease. The effect of this mutation and treatment with the F(ab')2 form of the anti-RSV G 131-2G monoclonal antibody (MAb) show that mutating the CX3C motif to CX4C blocks much of the disease and immune modulation associated with the G protein and should improve the safety and efficacy of a live attenuated RSV vaccine.
Assuntos
Quimiocinas CX3C/metabolismo , Proteínas de Ligação ao GTP/genética , Mutação , Infecções por Vírus Respiratório Sincicial/imunologia , Vacinas contra Vírus Sincicial Respiratório/efeitos adversos , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Quimiocinas CX3C/genética , Quimiocinas CX3C/imunologia , Feminino , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/imunologia , Humanos , Memória Imunológica , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Domínios e Motivos de Interação entre Proteínas , Vacinas contra Vírus Sincicial Respiratório/química , Vacinas contra Vírus Sincicial Respiratório/genética , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/fisiologia , Células Th1 , Células Th2 , Vacinas Atenuadas/química , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Replicação ViralRESUMO
The extracellular domain of plasma membrane integrin αvß3 contains a receptor for thyroid hormone (L-thyroxine, T4; 3,5,3'-triiodo-L-thyronine, T3); this receptor also binds tetraiodothyroacetic acid (tetrac), a derivative of T4. Tetrac inhibits the binding of T4 and T3 to the integrin. Fractalkine (CX3CL1) is a chemokine relevant to inflammatory processes in the CNS that are microglia-dependent but also important to normal brain development. Expression of the CX3CL1 gene is downregulated by tetrac, suggesting that T4 and T3 may stimulate fractalkine expression. Independently of its specific receptor (CX3CR1), fractalkine binds to αvß3 at a site proximal to the thyroid hormone-tetrac receptor and changes the physical state of the integrin. Tetrac also affects expression of the genes for other CNS-relevant chemokines, including CCL20, CCL26, CXCL2, CXCL3, and CXCL10. The chemokine products of these genes are important to vascularity of the brain, particularly of the choroid plexus, to inflammatory processes in the CNS and, in certain cases, to neuroprotection. Thyroid hormones are known to contribute to regulation of each of these CNS functions. We propose that actions of thyroid hormone and hormone analogues on chemokine gene expression contribute to regulation of inflammatory processes in brain and of brain blood vessel formation and maintenance.
Assuntos
Quimiocinas/metabolismo , Tiroxina/análogos & derivados , Tiroxina/metabolismo , Animais , Quimiocinas/química , Quimiocinas/genética , Quimiocinas C/metabolismo , Quimiocinas CC/metabolismo , Quimiocinas CX3C/metabolismo , Quimiocinas CXC/metabolismo , Humanos , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismoRESUMO
Respiratory syncytial virus (RSV) is a primary cause of severe lower respiratory tract disease in infants, young children, and the elderly worldwide, and despite decades of effort, there remains no safe and effective vaccine. RSV modifies the host immune response during infection by CX3C chemokine mimicry adversely affecting pulmonary leukocyte chemotaxis and CX3CR1+ RSV-specific T-cell responses. In this study we investigated whether immunization of mice with RSV G protein polypeptides from strain A2 could induce antibodies that block G protein-CX3CR1 interactions of both RSV A and B strains. The results show that mice immunized with RSV A2 G polypeptides generate antibodies that block binding of RSV A2 and B1 native G proteins to CX3CR1, and that these antibodies effectively cross-neutralize both A and B strains of RSV. These findings suggest that vaccines that induce RSV G protein-CX3CR1 blocking antibodies may provide a disease intervention strategy in the efforts to develop safe and efficacious RSV vaccines.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Quimiocinas CX3C/metabolismo , Ligação Proteica/efeitos dos fármacos , Receptores de Quimiocinas/metabolismo , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Receptor 1 de Quimiocina CX3C , Linhagem Celular , Reações Cruzadas , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/classificação , Vacinação , Proteínas Virais de Fusão/administração & dosagemRESUMO
The CX3C chemokine family is composed of only one member, CX3CL1, also known as fractalkine, which in mice is the sole ligand of the G protein-coupled, 7-transmembrane receptor CX3CR1. Unlike classic small peptide chemokines, CX3CL1 is synthesized as a membrane-anchored protein that can promote integrin-independent adhesion. Subsequent cleavage by metalloproteases, either constitutive or induced, can generate shed CX3CL1 entities that potentially have chemoattractive activity. To study the CX3C interface in tissues of live animals, we generated transgenic mice (CX3CL1cherry:CX3CR1gfp), which express red and green fluorescent reporter genes under the respective control of the CX3CL1 and CX3CR1 promoters. Furthermore, we performed a structure/function analysis to differentiate the in vivo functions of membrane-tethered versus shed CX3CL1 moieties by comparing their respective ability to correct established defects in macrophage function and leukocyte survival in CX3CL1-deficient mice. Specifically, expression of CX3CL1(105Δ), an obligatory soluble CX3CL1 isoform, reconstituted the formation of transepithelial dendrites by intestinal macrophages but did not rescue circulating Ly6Clo CX3CR1hi blood monocytes in CX3CR1gfp/gfp mice. Instead, monocyte survival required the full-length membrane-anchored CX3CL1, suggesting differential activities of tethered and shed CX3CL1 entities.
Assuntos
Quimiocina CX3CL1/química , Quimiocina CX3CL1/genética , Quimiocina CX3CL1/fisiologia , Animais , Células Cultivadas , Quimiocina CX3CL1/metabolismo , Quimiocinas CX3C/química , Quimiocinas CX3C/genética , Quimiocinas CX3C/metabolismo , Quimiocinas CX3C/fisiologia , Feminino , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/fisiologia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Relação Estrutura-AtividadeRESUMO
Respiratory syncytial virus (RSV) infection causes substantial morbidity and some deaths in the young and elderly worldwide. There is no safe and effective vaccine available, although it is possible to reduce the hospitalization rate for high-risk children by anti-RSV antibody prophylaxis. RSV has been shown to modify the immune response to infection, a feature linked in part to RSV G protein CX3C chemokine mimicry. This study determined if vaccination with G protein polypeptides or peptides spanning the central conserved region of the G protein could induce antibodies that blocked G protein CX3C-CX3CR1 interaction and disease pathogenesis mediated by RSV infection. The results show that mice vaccinated with G protein peptides or polypeptides containing the CX3C motif generate antibodies that inhibit G protein CX3C-CX3CR1 binding and chemotaxis, reduce lung virus titers, and prevent body weight loss and pulmonary inflammation. The results suggest that RSV vaccines that induce antibodies that block G protein CX3C-CX3CR1 interaction may offer a new, safe, and efficacious RSV vaccine strategy.
Assuntos
Anticorpos Antivirais/sangue , Quimiocinas CX3C/metabolismo , Receptores de Quimiocinas/metabolismo , Infecções por Vírus Respiratório Sincicial , Vacinas contra Vírus Sincicial Respiratório , Proteínas Virais de Fusão/imunologia , Animais , Anticorpos Antivirais/imunologia , Receptor 1 de Quimiocina CX3C , Linhagem Celular , Quimiocinas CX3C/imunologia , Quimiotaxia de Leucócito/imunologia , Feminino , Humanos , Inflamação/imunologia , Inflamação/prevenção & controle , Pulmão/imunologia , Pulmão/fisiopatologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/síntese química , Peptídeos/química , Peptídeos/imunologia , Receptores de Quimiocinas/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/fisiopatologia , Infecções por Vírus Respiratório Sincicial/virologia , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Vírus Sincicial Respiratório Humano/patogenicidade , Vírus Sincicial Respiratório Humano/fisiologia , Vacinação , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo , Replicação ViralRESUMO
BACKGROUND: The chemokine/chemokine receptor pair CX(3)C-L/CX(3)C-R is suspected to play a role in renal fibrogenesis. The aim of this study was to investigate their function in an animal model of slowly progressive chronic renal failure. METHODS: Functional data were analysed in folic acid nephropathy (FAN) at different time points (up to day 142 after induction). Immunostaining for CX(3)C-L, CD3, S100A4, collagen type I, fibronectin, alpha-smooth muscle actin, Tamm-horsfall protein, aquaporin 1 and 2 as well as quantitative real-time PCR (qRT-PCR) for CX(3)C-L, CX(3)C-R and fibroblast-specific protein 1 (FSP-1) were performed. Additionally, regulatory mechanisms and functional activity of CX(3)C-L in murine proximal and distal tubular epithelial cells as well as in fibroblasts were investigated. RESULTS: CX(3)C-L/GAPDH ratio was upregulated in FAN 3.4-fold at day 7 further increasing up to 7.1-fold at day 106. The expression of mRNA CX(3)C-L correlated well with CX(3)C-R (R(2) = 0.96), the number of infiltrating CD3+ cells (R(2) = 0.60) and the degree of tubulointerstitial fibrosis (R(2) = 0.56) and moderately with FSP-1 (R(2) = 0.33). Interleukin-1beta, tumour necrosis factor-alpha, transforming growth factor-beta as well as the reactive oxygen species (ROS) H(2)O(2) were identified by qRT-PCR as inductors of CX(3)C-L/fractalkine (FKN) in tubular epithelial cells. Functionally, CX(3)C-L/FKN chemoattracts peripheral blood mononuclear cells, activates several aspects of fibrogenesis and induces the mitogen-activated protein kinases in renal fibroblasts. CONCLUSIONS: In FAN, there is a good correlation between the expression of CX(3)C-L with markers of interstitial inflammation and fibrosis which may result from upregulation by pro-inflammatory and pro-fibrotic cytokines as well as by ROS in tubular epithelial cells. The FKN system may promote renal inflammation and renal fibrogenesis.
Assuntos
Quimiocina CX3CL1/metabolismo , Quimiocinas CX3C/metabolismo , Progressão da Doença , Túbulos Renais Distais/metabolismo , Túbulos Renais Proximais/metabolismo , Insuficiência Renal/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Fibrose , Ácido Fólico/efeitos adversos , Humanos , Túbulos Renais Distais/patologia , Túbulos Renais Proximais/patologia , Camundongos , Camundongos Endogâmicos , Espécies Reativas de Oxigênio/metabolismo , Insuficiência Renal/induzido quimicamente , Proteína A4 de Ligação a Cálcio da Família S100 , Proteínas S100RESUMO
OBJECTIVE: To examine effects of in vitro exposure to solutions of hay dust, lipopolysaccharide (LPS), or beta-glucan on cytokine expression in pulmonary mononuclear cells isolated from healthy horses and horses with recurrent airway obstruction (RAO). ANIMALS: 8 RAO-affected and 7 control horses (experiment 1) and 6 of the RAO-affected and 5 of the control horses (experiment 2). PROCEDURES: Bronchoalveolar lavage cells were isolated from horses that had been stabled and fed dusty hay for 14 days. Pulmonary mononuclear cells were incubated for 24 (experiment 1) or 6 (experiment 2) hours with PBS solution or solutions of hay dust, beta-glucan, or LPS. Gene expression of interleukin (IL)-17, IL-23(p19 and p40 subunits), IL-8, IL-1beta, and chemokine (C-X-C motif) ligand 2 (CXCL2) was measured with a kinetic PCR assay. RESULTS: Treatment with the highest concentration of hay dust solution for 6 or 24 hours increased expression of IL-23(p19 and p40), IL-8, and IL-1beta in cells from both groups of horses and increased early expression of IL-17 and CXCL2 in RAO-affected horses. Lipopolysaccharide upregulated early expression of IL-23(p40) and IL-8 in cells from both groups of horses but only late expression of these cytokines in cells from RAO-affected horses. Treatment with beta-glucan failed to increase cytokine expression at 6 or 24 hours. CONCLUSIONS AND CLINICAL RELEVANCE: Cells from RAO-affected horses were not more responsive to the ligands tested than were cells from control horses, which suggests a minimal role of mononuclear cells in propagation of airway neutrophilia in horses with chronic RAO.
Assuntos
Quimiocinas CX3C/metabolismo , Interleucina-17/metabolismo , Interleucina-1beta/metabolismo , Interleucina-23/metabolismo , Interleucina-9/metabolismo , Doenças Respiratórias/veterinária , Animais , Células Cultivadas , Quimiocinas CX3C/genética , Poeira , Regulação da Expressão Gênica , Doenças dos Cavalos/metabolismo , Cavalos , Interleucina-17/genética , Interleucina-1beta/genética , Interleucina-23/genética , Interleucina-9/genética , Leucócitos Mononucleares/metabolismo , Pulmão/citologiaRESUMO
BACKGROUND: The membrane-bound chemokine fractalkine (CX3CL1) is expressed on various cell types such as activated endothelial cells and has been implicated in the inflammatory process of atherosclerosis. The aim of the present study was to dissect the role of fractalkine in leukocyte recruitment to inflamed endothelium under arterial shear forces. METHODS AND RESULTS: With the use of immunofluorescence and laminar flow assays, the present study shows that human umbilical vein endothelial cells stimulated with tumor necrosis factor-alpha and interferon-gamma abundantly express CX3CL1 and promote substantial leukocyte accumulation under arterial flow conditions. In the presence of high shear, firm adhesion of leukocytes to inflamed endothelial cells is reduced by approximately 40% by a function-blocking anti-fractalkine antibody or by an antibody directed against the fractalkine receptor (CX3CR1). With the use of intravital video-fluorescence microscopy we demonstrate that inhibition of fractalkine signaling attenuates leukocyte adhesion to the atherosclerotic carotid artery of apolipoprotein E-deficient mice, which suggests that the CX3CL1-CX3CR1 axis is critically involved in leukocyte adhesion to inflamed endothelial cells under high shear forces both in vitro and in vivo. Surprisingly, platelets were strictly required for fractalkine-induced leukocyte adhesion at high shear rates. Correspondingly, specific inhibition of platelet adhesion to inflamed endothelial cells also significantly reduced leukocyte accumulation. We show that both soluble and membrane-bound fractalkine induces platelet degranulation and subsequent surface expression of P-selectin, which thereby promotes direct platelet-leukocyte interaction. CONCLUSIONS: Fractalkine expressed by inflamed endothelial cells triggers P-selectin exposure on adherent platelets, which thereby initiates the local accumulation of leukocytes under arterial shear, an essential step in the development of atherosclerotic lesions.
Assuntos
Aterosclerose/imunologia , Plaquetas/metabolismo , Quimiocinas CX3C/metabolismo , Quimiotaxia de Leucócito , Endotélio Vascular/imunologia , Proteínas de Membrana/metabolismo , Selectina-P/metabolismo , Ativação Plaquetária , Animais , Circulação Sanguínea , Plaquetas/fisiologia , Células CHO , Adesão Celular , Degranulação Celular , Células Cultivadas , Quimiocina CX3CL1 , Quimiocinas CX3C/farmacologia , Cricetinae , Cricetulus , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Endotélio Vascular/citologia , Humanos , Inflamação/imunologia , Mediadores da Inflamação/farmacologia , Leucócitos/imunologia , Proteínas de Membrana/farmacologia , CamundongosRESUMO
OBJECTIVE: Fibroblast-like synoviocytes (FLS) are a major constituent of the hyperplastic synovial pannus that aggressively invades cartilage and bone during the course of rheumatoid arthritis (RA). Fractalkine (FKN/CX(3)CL1) expression is up-regulated in RA synovium and RA synovial fluid. While RA FLS express the FKN receptor, CX(3)CR1, the pathophysiologic relevance of FKN stimulation of RA FLS is not understood. This study was undertaken to better characterize the relationship between FKN and the RA FLS that both produce it and express its receptor. METHODS: RA FLS were subjected to chemotaxis and proliferation assays, Western blotting, enzyme-linked immunosorbent assays, and filamentous actin staining to characterize the relationship between FKN and RA FLS. RESULTS: FKN secretion by RA FLS was regulated mainly by tumor necrosis factor alpha. Stimulation of RA FLS with FKN led to significant cytoskeletal rearrangement but no proliferation. Chemotaxis assays revealed that FKN was a novel chemoattractant for RA FLS. Stimulation of RA FLS with FKN resulted in activation of MAP kinases and Akt. JNK, ERK-1/2, and Akt (at both Ser-473 and Thr-308) were each up-regulated in a time-dependent manner. Inhibition of ERK-1/2-mediated signaling, but not JNK or Akt, significantly repressed FKN-induced RA FLS migration. CONCLUSION: These findings indicate a novel role of FKN in regulating RA FLS cytoskeletal structure and migration. FKN specifically induces RA FLS phosphorylation of the MAP kinases JNK and ERK-1/2, as well as full activation of Akt.
Assuntos
Artrite Reumatoide/metabolismo , Quimiocinas CX3C/metabolismo , Fatores Quimiotáticos/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Membrana Sinovial/metabolismo , Citoesqueleto de Actina/metabolismo , Adulto , Idoso , Artrite Reumatoide/patologia , Western Blotting , Células Cultivadas , Quimiocina CX3CL1 , Quimiocinas CX3C/farmacologia , Fatores Quimiotáticos/farmacologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Proteínas de Membrana/farmacologia , Pessoa de Meia-Idade , Proteínas Recombinantes , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/patologiaRESUMO
Neointimal cushion formation (NCF) is an important vascular remodeling for anatomical closure of the ductus arteriosus (DA). Inflammatory responses to vascular injury or atherosclerosis are known to be associated with the pathogenesis of NCF. We found that the expression of interleukin (IL)-15 mRNA was significantly higher in rat DA than in the aorta. IL-15 immunoreactivity was detected predominantly in the internal elastic laminae (IEL) and to a lesser extent in smooth muscle cells (SMCs) in rat DA. Prostaglandin E (PGE) increased the expression of IL-15 mRNA in cultured DA SMCs. IL-15 significantly attenuated the platelet-derived growth factor (PDGF)-BB-mediated SMC proliferation, but did not change SMC migration. IL-15 significantly attenuated PGE1-induced hyaluronic acid (HA) production in a dose-dependent manner, which is a potent stimulator of NCF. Accordingly, IL-15 might have an inhibitory effect on the physiologic vascular remodeling processes in closing the DA.
Assuntos
Aorta/metabolismo , Proliferação de Células , Canal Arterial/metabolismo , Ácido Hialurônico/metabolismo , Interleucina-15/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Alprostadil/metabolismo , Animais , Aorta/embriologia , Becaplermina , Receptor 1 de Quimiocina CX3C , Movimento Celular , Células Cultivadas , Quimiocina CX3CL1 , Quimiocinas CX3C/metabolismo , Relação Dose-Resposta a Droga , Canal Arterial/embriologia , Retroalimentação Fisiológica , Regulação da Expressão Gênica no Desenvolvimento , Interleucina-15/genética , Interleucina-15/farmacologia , Proteínas de Membrana/metabolismo , Éteres Metílicos/farmacologia , Músculo Liso Vascular/embriologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-sis , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de Quimiocinas/metabolismo , Receptores de Interleucina-15/metabolismo , Receptores de Prostaglandina E/metabolismo , Receptores de Prostaglandina E Subtipo EP4RESUMO
Chemokines (chemotactic cytokines) are important in the recruitment of leukocytes to injured tissues and, as such, play a pivotal role in arteriogenesis and the tissue response to ischemia. Hind limb ischemia represents a complex model with arteriogenesis (collateral artery formation) occurring in tissues with normal perfusion while areas exhibiting ischemic necrosis undergo angiogenesis and skeletal muscle regeneration; monocytes and macrophages play an important role in all three of these processes. In addition to leukocyte trafficking, chemokines are produced by and chemokine receptors are present on diverse cell types, including myoblasts, endothelial, and smooth muscle cells. Thus, the chemokine system may have direct effects as well as inflammatory-mediated effects on arteriogenesis, angiogenesis, and skeletal muscle regeneration. This article reviews the complexity of the hind limb ischemia model and the role of the chemokine system in arteriogenesis and the tissue response to ischemia. Special emphasis will be placed on the roles of monocytes/macrophages and CCL2/monocyte chemotactic protein-1 (MCP-1) in these processes.
Assuntos
Quimiocinas/metabolismo , Inflamação/metabolismo , Isquemia/metabolismo , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Receptores de Quimiocinas/metabolismo , Regeneração , Animais , Artérias/metabolismo , Artérias/fisiopatologia , Quimiocina CCL2/metabolismo , Quimiocinas CX3C/metabolismo , Modelos Animais de Doenças , Membro Posterior , Inflamação/fisiopatologia , Isquemia/fisiopatologia , Macrófagos/metabolismo , Camundongos , Monócitos/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Transdução de SinaisRESUMO
CX3CL1 (fractalkine) and CXCL16 are unique members of the chemokine family because they occur not only as soluble, but also as membrane-bound molecules. Expressed as type I transmembrane proteins, the ectodomain of both chemokines can be proteolytically cleaved from the cell surface, a process known as shedding. Our previous studies showed that the disintegrin and metalloproteinase 10 (ADAM10) mediates the largest proportion of constitutive CX3CL1 and CXCL16 shedding, but is not involved in the phorbolester-induced release of the soluble chemokines (inducible shedding). In this study, we introduce the calcium-ionophore ionomycin as a novel, very rapid, and efficient inducer of CX3CL1 and CXCL16 shedding. By transfection in COS-7 cells and ADAM10-deficient murine embryonic fibroblasts combined with the use of selective metalloproteinase inhibitors, we demonstrate that the inducible generation of soluble forms of these chemokines is dependent on ADAM10 activity. Analysis of the C-terminal cleavage fragments remaining in the cell membrane reveals multiple cleavage sites used by ADAM10, one of which is preferentially used upon stimulation with ionomycin. In adhesion studies with CX3CL1-expressing ECV-304 cells and cytokine-stimulated endothelial cells, we demonstrate that induced CX3CL1 shedding leads to the release of bound monocytic cell lines and PBMC from their cellular substrate. These data provide evidence for an inducible release mechanism via ADAM10 potentially important for leukocyte diapedesis.
Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Adesão Celular , Quimiocinas CX3C/metabolismo , Quimiocinas CXC/metabolismo , Desintegrinas/metabolismo , Leucócitos/imunologia , Proteínas de Membrana/metabolismo , Receptores Depuradores/metabolismo , Proteínas ADAM/genética , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/genética , Animais , Células COS , Membrana Celular/metabolismo , Quimiocina CX3CL1 , Quimiocina CXCL16 , Chlorocebus aethiops , Humanos , Metaloproteinase 10 da Matriz/metabolismo , Proteínas de Membrana/genética , Camundongos , TransfecçãoRESUMO
BACKGROUND: Fractalkine (Fkn) is a chemokine that affects cells expressing its receptor, CX3CR1, including CD16-positive (CD16+) monocytes/macrophages (CD16+ Mos). The relationship of levels of glomerular Fkn expression and infiltration by CD16+ Mos with the severity and diversity of glomerular lesions in human lupus nephritis is not known. STUDY DESIGN: Retrospective cross-sectional analysis of variables observed in biopsy specimens. SETTINGS & PARTICIPANTS: 88 patients with systemic lupus erythematosus. PREDICTOR: Histological class and severity of lupus nephritis according to the International Society of Nephrology/Renal Pathology Society and clinicopathologic factors. OUTCOMES: Outcome variables are assays related to the degree of glomerular Fkn expression and CD16+ Mo infiltration. MEASUREMENTS: Immunohistological grading of Fkn staining, number of CD16+ Mos, and messenger RNA levels of Fkn and CD16 in glomeruli. RESULTS: Patients with proliferative lupus nephritis (class III and IV glomeruli) showed significantly greater glomerular Fkn expression and CD16+ Mo counts than those with other classes. Infiltrating CD16+ Mos within glomeruli expressed CX3CR1. Moreover, glomerular Fkn expression significantly correlated with the histopathologic activity index and CD16+ Mo counts, and CD16+ Mo counts significantly correlated with serum levels of blood urea nitrogen, complement (CH50), and anti-DNA antibody; estimated glomerular filtration rate; and urinary protein excretion. Glucocorticoid therapy had a tendency to decrease both glomerular Fkn expression and CD16+ Mo counts. LIMITATIONS: Only frozen biopsy specimens (from 49 patients) were analyzed for the evaluation of glomerular Fkn expression. CONCLUSION: Disease activity and proliferative glomerular lupus nephritis lesions are associated with the glomerular Fkn expression and CD16+ Mo accumulation.
Assuntos
Quimiocinas CX3C/metabolismo , Glomérulos Renais/metabolismo , Nefrite Lúpica/metabolismo , Proteínas de Membrana/metabolismo , Monócitos/metabolismo , Adolescente , Adulto , Idoso , Quimiocina CX3CL1 , Estudos Transversais , Feminino , Humanos , Glomérulos Renais/imunologia , Glomérulos Renais/patologia , Contagem de Leucócitos , Nefrite Lúpica/imunologia , Nefrite Lúpica/patologia , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Receptores de IgG/metabolismo , Estudos RetrospectivosRESUMO
1. The aim of the present study was to examine if and how rat hypoxia-induced astrocytes affect the migration of neural progenitor cells (NPC) and to investigate the expression patterns of some chemokines, such as vascular endothelial growth factor (VEGF), stem cell factor (SCF), stromal-derived factor-1alpha (SDF-1alpha), fractalkine and monocyte chemoattractant protein-1 (MCP-1) in hypoxia-induced astrocytes and their contribution to NPC migration in vitro. 2. Costar Transwell inserts were used for the chemotaxis assay and quantified changes in the chemokines mRNA for between 0 h and 24 h posthypoxia were tested using real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The results showed that the chemotaxis of astrocyte cells exposed to hypoxia for 18 h reached a peak value, whereas the chemotaxis of astrocytes exposed to hypoxia for 24 h began to decrease compared with those exposed to hypoxia for 18 h. Hypoxia upregulated chemokine VEGF, SCF, SDF-1alpha and MCP-1 expression in a time-dependent manner but downregulated fractalkine expression in astrocytes. In addition, the time points of the peak expressions for VEGF, SCF, SDF-1alpha and MCP-1 were similar to the time point of maximum NPC migration. 3. Specific inhibitors that block the binding of specific chemokines to its receptors were used for analysing the contribution of the chemokine to NPC migration. When VEGF, SCF, SDF-1alpha and MCP-1 were each inhibited independently, NPC migration was reduced. When they were inhibited together, NPC migration was obviously inhibited compared with both the control and single-block cultures, which implies that the migratory effect of hypoxia-induced astrocytes was synergetic by several chemokines. 4. In conclusion, we demonstrated the time-dependent manner of NPC migration promotion by hypoxia-induced astrocytes. We also provide evidence that soluble factors, such as VEGF, SCF, SDF-1alpha and MCP-1, released from astrocytes, direct the migration of NPC under hypoxic circumstances. Given that astrocytes were activated to all hypoxia-ischaemia diseases, these results indicate an important role for astrocytes in directing NPC replacement therapy in the central nervous system.
Assuntos
Astrócitos/metabolismo , Quimiocina CCL2/metabolismo , Quimiocinas CXC/metabolismo , Quimiotaxia , Neurônios/metabolismo , Fator de Células-Tronco/metabolismo , Células-Tronco/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Animais Recém-Nascidos , Diferenciação Celular , Hipóxia Celular , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Quimiocina CCL2/genética , Quimiocina CX3CL1 , Quimiocina CXCL12 , Quimiocinas CX3C/metabolismo , Quimiocinas CXC/genética , Técnicas de Cocultura , Meios de Cultivo Condicionados/metabolismo , Regulação para Baixo , Proteínas de Membrana/metabolismo , Comunicação Parácrina , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fator de Células-Tronco/genética , Fatores de Tempo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
CD16+ monocytes, identified as a minor population of monocytes in human peripheral blood, have been implicated in several inflammatory diseases, including rheumatoid arthritis (RA). Fractalkine (FKN, CX3CL1), a member of the CX3 C subfamily, is induced by pro-inflammatory cytokines, while a receptor for FKN, CX3CR1, is capable of mediating both leukocyte migration and firm adhesion. Here, we investigated the role of FKN and CX3CR1 in activation of CD16+ monocytes and their recruitment into synovial tissues in RA patients. High levels of soluble FKN were detected in the synovial fluid and sera of RA patients. Circulating CD16+ monocytes showed a higher level of CX3CR1 expression than CD16- monocytes in both RA patients and healthy subjects. High level expression of CX3CR1 was also seen in CD16+ monocytes localized to the lining layer in RA synovial tissue. In the in vitro culture experiments, IL-10 induced CX3CR1 expression on the surface of monocytes, and TNFalpha induced membrane-bound FKN as well as soluble FKN expression in synovial fibroblasts. Moreover, soluble FKN was capable of inducing IL-1beta and IL-6 by activated monocytes. These results suggest that FKN might preferentially mediate migration and recruitment of CD16+ monocytes, and might contribute to synovial tissue inflammation.
Assuntos
Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Quimiocinas CX3C/metabolismo , Proteínas de Membrana/metabolismo , Monócitos/metabolismo , Monócitos/patologia , Receptores de Quimiocinas/metabolismo , Receptores de IgG/metabolismo , Idoso , Artrite Reumatoide/sangue , Receptor 1 de Quimiocina CX3C , Membrana Celular/metabolismo , Células Cultivadas , Quimiocina CX3CL1 , Quimiocinas CX3C/sangue , Quimiocinas CX3C/farmacologia , Células Endoteliais/metabolismo , Feminino , Humanos , Interleucina-10/farmacologia , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Proteínas de Membrana/sangue , Proteínas de Membrana/farmacologia , Monócitos/efeitos dos fármacos , Osteoartrite/sangue , Osteoartrite/metabolismo , Osteoartrite/patologia , Proteínas Recombinantes/farmacologia , Líquido Sinovial/metabolismo , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Over the past 2 decades, significant effort has been dedicated to the development of adeno-associated virus (AAV) as a vector for human gene therapy. However, understanding of the virus with respect to the functional domains of the capsid remains incomplete. In this study, the goal was to further examine the role of the unique Vp1 N terminus, the N terminus plus the recently identified nuclear localization signal (NLS) (J. C. Grieger, S. Snowdy, and R. J. Samulski, J. Virol 80:5199-5210, 2006), and the virion pore at the fivefold axis in infection. We generated two Vp1 fusion proteins (Vp1 and Vp1NLS) linked to the 8-kDa chemokine domain of rat fractalkine (FKN) for the purpose of surface exposure upon assembly of the virion, as previously described (K. H. Warrington, Jr., O. S. Gorbatyuk, J. K. Harrison, S. R. Opie, S. Zolotukhin, and N. Muzyczka, J. Virol 78:6595-6609, 2004). The unique Vp1 N termini were found to be exposed on the surfaces of these capsids and maintained their phospholipase A2 (PLA2) activity, as determined by native dot blot Western and PLA2 assays, respectively. Incorporation of the fusions into AAV type 2 capsids lacking a wild-type Vp1, i.e., Vp2/Vp3 and Vp3 capsid only, increased infectivity by 3- to 5-fold (Vp1FKN) and 10- to 100-fold (Vp1NLSFKN), respectively. However, the surface-exposed fusions did not restore infectivity to AAV virions containing mutations at a conserved leucine (Leu336Ala, Leu336Cys, or Leu336Trp) located at the base of the fivefold pore. EM analyses suggest that Leu336 may play a role in global structural changes to the virion directly impacting downstream conformational changes essential for infectivity and not only have local effects within the pore, as previously suggested.
Assuntos
Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Dependovirus , Vírion/metabolismo , Sequência de Aminoácidos , Animais , Proteínas do Capsídeo/genética , Linhagem Celular , Quimiocina CX3CL1 , Quimiocinas CX3C/genética , Quimiocinas CX3C/metabolismo , Dependovirus/metabolismo , Dependovirus/patogenicidade , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Sinais de Localização Nuclear , Estrutura Quaternária de Proteína , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vírion/genética , Vírion/ultraestruturaRESUMO
The chemokines CX3CL1/Fractalkine and CXCL16 are expressed as transmembrane molecules and can mediate cell-cell-adhesion. By proteolytic processing, CX3CL1 and CXCL16 are released from the cell surface by proteolytic shedding resulting in the generation of soluble chemoattractants. This ectodomain release is mediated by the alpha-secretase-like activity of the two disintegrins and metalloproteinases ADAM10 and ADAM17. Using CX3CL1 and CXCL16 constructs C-terminally fused to two Z-domains of Protein A (2Z-tag) we detect C-terminal fragments (CTFs) of both chemokines resulting from ADAM10-mediated cleavages at multiple sites as examined by inhibitor studies. Furthermore, inhibitor studies as well as genetic studies using presenilin 1/2-deficient cell lines suggest the involvement of gamma-secretase-but not beta-secretase-like activity in the processing of transmembrane chemokines. The combination of alpha- and gamma-secretase and proteasomal inhibitors points towards a sequential processing of transmembrane chemokines by first ADAM10 and then gamma-secretases and possible further degradation. This proteolytic processing cascade of transmembrane chemokines is similar to that described for Notch and E-cadherin where CTFs generated by gamma-secretase serve as intracellular signal transmitters.
Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Quimiocinas CX3C/metabolismo , Quimiocinas CXC/metabolismo , Proteínas de Membrana/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores Depuradores/metabolismo , Proteínas ADAM/antagonistas & inibidores , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Linhagem Celular , Quimiocina CX3CL1 , Quimiocina CXCL16 , Clonagem Molecular , Humanos , Proteínas de Membrana/antagonistas & inibidores , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/genética , Presenilina-2/metabolismo , Estrutura Terciária de ProteínaRESUMO
NK cells are cytotoxic lymphocytes that also secrete regulatory cytokines and can therefore influence adaptive immune responses. NK cell function is largely controlled by genes present in a genomic region named the NK complex. It has been shown that the NK complex is a genetic determinant of murine cerebral malaria pathogenesis mediated by Plasmodium berghei ANKA. In this study, we show that NK cells are required for cerebral malaria disease induction and the control of parasitemia. NK cells were found infiltrating brains of cerebral malaria-affected mice. NK cell depletion resulted in inhibition of T cell recruitment to the brain of P. berghei-infected animals. NK cell-depleted mice displayed down-regulation of CXCR3 expression and a significant reduction of T cells migrating in response to IFN-gamma-inducible protein 10, indicating that this chemokine pathway plays an essential role in leukocyte trafficking leading to cerebral disease and fatalities.
Assuntos
Encéfalo/imunologia , Células Matadoras Naturais/imunologia , Malária Cerebral/imunologia , Plasmodium berghei , Receptores de Quimiocinas/metabolismo , Subpopulações de Linfócitos T/imunologia , Animais , Encéfalo/patologia , Quimiocina CXCL10 , Quimiocinas CX3C/metabolismo , Quimiocinas CX3C/farmacologia , Quimiocinas CXC/metabolismo , Quimiocinas CXC/farmacologia , Modelos Animais de Doenças , Regulação para Baixo , Depleção Linfocítica , Malária Cerebral/patologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores CXCR3 , Receptores de Quimiocinas/análise , Baço/imunologia , Subpopulações de Linfócitos T/química , Subpopulações de Linfócitos T/efeitos dos fármacosRESUMO
MSCs are nonhematopoietic stem cells capable of differentiating into various mesoderm-type cells. MSCs have been considered to be a potential vehicle for cell-based gene therapy because MSCs are relatively easily expanded in vitro and have the propensity to migrate to and proliferate in the tumor tissue after systemic administration. Here, we demonstrated the tropism of mouse MSCs to tumor cells in vitro and multiple tumor tissues in the lung after i.v. injection of green fluorescent protein-positive MSCs in vivo. We transduced CX3CL1 (fractalkine), an immunostimulatory chemokine, to the mouse MSCs ex vivo using an adenoviral vector with the Arg-Gly-Asp-4C peptide in the fiber knob. Intravenous injection of CX3CL1-expressing MSCs to the mice bearing lung metastases of C26 and B16F10 cells strongly inhibited the development of lung metastases and thus prolonged the survival of these tumor-bearing mice. This antitumor effect depended on both innate and adaptive immunity. These results suggest that MSCs can be used as a vehicle for introducing biological agents into multiple lung tumor tissues. Disclosure of potential conflicts of interest is found at the end of this article.