RESUMEN
Extracellular nucleotides are released as constitutive danger signals by various cell types and activate nucleotide (P2) receptors such as P2Y6 receptor. P2Y6 activation on monocytes induces the secretion of the chemokine CXCL8 which may propagate intestinal inflammation. Also, P2Y6 expression is increased in infiltrating T cells of Crohn's disease patients. As inflammatory bowel disease (IBD) is associated with immune cell recruitment, we hypothesised that P2Y6 would participate to the establishment of inflammation in this disease. To address this, we used P2Y6 deficient (P2ry6--/-) mice in the dextran sodium sulfate (DSS) murine model of IBD. In disagreement with our hypothesis, P2Y6 deficient mice were more susceptible to inflammation induced by DSS than WT mice. DSS treated-P2ry6-/- mice showed increased histological damage and increased neutrophil and macrophage infiltration that correlated with increased mRNA levels of the chemokines KC and MCP-1. DSS treated-P2ry6-/- mice exhibited also higher levels of Th17/Th1 lymphocytes in their colon which correlated with increased levels of IFN-γ and IL-17A in the sera as well as increased mRNA levels of IFN-γ, IL-17A, IL-6, IL-23 and IL-1ß in P2ry6-/- colons. This inflammation was also accompanied by a decreased cell proliferation and goblet cell number. Importantly, injection of anti-IL-17 intraperitoneally partially protected P2ry6-/- mice from DSS-induced colitis. Taken together, in the absence of P2Y6, an exacerbated intestinal inflammation to DSS was observed which correlated with increased recruitment of Th17/Th1 lymphocytes. These data suggest a protective role of P2Y6 expressed on leukocytes in intestinal inflammation.
Asunto(s)
Inflamación/metabolismo , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Células Th17/metabolismo , Animales , Proliferación Celular , Quimiocina CCL2/metabolismo , Quimiocinas/metabolismo , Colitis/inducido químicamente , Colitis/metabolismo , Colon/metabolismo , Citocinas/sangre , Citocinas/metabolismo , Sulfato de Dextran/efectos adversos , Modelos Animales de Enfermedad , Femenino , Inflamación/inducido químicamente , Inflamación/patología , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Interleucina-8/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células TH1/metabolismo , Células Th17/inmunología , TranscriptomaRESUMEN
ß1 integrins are critical for T cell migration, survival and costimulation. The integrin α2ß1, which is a receptor for collagen, also named VLA-2, is a major costimulatory pathway of effector T cells and has been implicated in arthritis pathogenesis. Herein, we have examined its ability to promote methotrexate (MTX) resistance by enhancing effector T cells survival. Our results show that attachment of anti-CD3-activated human polarized Th17 cells to collagen but not to fibronectin or laminin led to a significant reduction of MTX-induced apoptosis. The anti-CD3+collagen-rescued cells still produce significant amounts of IL-17 and IFNγ upon their reactivation indicating that their inflammatory nature is preserved. Mechanistically, we found that the prosurvival role of anti-CD3+collagen involves activation of the MTX transporter ABCC1 (ATP Binding Cassette subfamily C Member 1). Finally, the protective effect of collagen/α2ß1 integrin on MTX-induced apoptosis also occurs in memory CD4+ T cells isolated from rheumatoid arthritis (RA) patients suggesting its clinical relevance. Together these results show that α2ß1 integrin promotes MTX resistance of effector T cells, and suggest that it could contribute to the development of MTX resistance that is seen in RA.
Asunto(s)
Apoptosis/efectos de los fármacos , Integrina alfa2beta1/inmunología , Metotrexato/farmacología , Células Th17/efectos de los fármacos , Apoptosis/inmunología , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/inmunología , Células Cultivadas , Colágeno/farmacología , Fibronectinas/farmacología , Humanos , Integrina alfa2beta1/metabolismo , Laminina/farmacología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Sustancias Protectoras/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Células Th17/inmunologíaRESUMEN
T cell migration across extracellular matrix (ECM) is an important step of the adaptive immune response but is also involved in the development of inflammatory autoimmune diseases. Currently, the molecular mechanisms regulating the motility of effector T cells in ECM are not fully understood. Activation of p38 MAPK has been implicated in T cell activation and is critical to the development of immune and inflammatory responses. In this study, we examined the implication of p38 MAPK in regulating the migration of human Th17 cells through collagen. Using specific inhibitor and siRNA, we found that p38 is necessary for human Th17 migration in three-dimensional (3D) collagen and that 3D collagen increases p38 phosphorylation. We also provide evidence that the collagen receptor, discoidin domain receptor 1 (DDR1), which promotes Th17 migration in 3D collagen, is involved in p38 activation. Together, our findings suggest that targeting DDR1/p38 MAPK pathway could be beneficial for the treatment of Th17-mediated inflammatory diseases. J. Cell. Biochem. 118: 2819-2827, 2017. © 2017 Wiley Periodicals, Inc.
Asunto(s)
Movimiento Celular/inmunología , Colágeno/química , Sistema de Señalización de MAP Quinasas/inmunología , Células Th17/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología , Receptor con Dominio Discoidina 1/inmunología , Activación Enzimática/inmunología , HumanosRESUMEN
Effector T cell migration through the tissue extracellular matrix (ECM) is an important step of the adaptive immune response and in the development of inflammatory diseases. However, the mechanisms involved in this process are still poorly understood. In this study, we addressed the role of a collagen receptor, the discoidin domain receptor 1 (DDR1), in the migration of Th17 cells. We showed that the vast majority of human Th17 cells express DDR1 and that silencing DDR1 or using the blocking recombinant receptor DDR1:Fc significantly reduced their motility and invasion in three-dimensional (3D) collagen. DDR1 promoted Th17 migration by activating RhoA/ROCK and MAPK/ERK signaling pathways. Interestingly, the RhoA/ROCK signaling module was required for MAPK/ERK activation. Finally, we showed that DDR1 is important for the recruitment of Th17 cells into the mouse dorsal air pouch containing the chemoattractant CCL20. Collectively, our results indicate that DDR1, via the activation of RhoA/ROCK/MAPK/ERK signaling axis, is a key pathway of effector T cell migration through collagen of perivascular tissues. As such, DDR1 can contribute to the development of Th17-dependent inflammatory diseases.
Asunto(s)
Movimiento Celular/fisiología , Receptor con Dominio Discoidina 1/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Células Th17/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/fisiologíaRESUMEN
Th17 cells are critical effectors in inflammation and tissue damage such as bone erosion, but the mechanisms regulating their activation in this process are not fully understood. In this study, we considered the cooperation between cytokine receptors and integrin pathways in Th17-osteoclast function. We found that human Th17 cells coexpress IL-7R and the collagen-binding integrin α2ß1 (CD49b), and IL-7 increases their adhesion to collagen via α2ß1 integrin. In addition, coengagement of the two receptors in human Th17 cells cooperatively enhanced their IL-17 production and their osteoclastogenic function. The functional cooperation between IL-7R and α2ß1 integrin involves activation of the JAK/PI3K/AKT (protein kinase B) and MAPK/ERK pathways. We also showed that IL-7-induced bone loss in vivo is associated with Th17 cell expansion. Moreover, blockade of α2ß1 integrin with a neutralizing mAb inhibited IL-7-induced bone loss and osteoclast numbers by reducing Th17 cell numbers in the bone marrow and reducing the production of IL-17 and the receptor activator of NF-κB ligand. Thus, the cooperation between IL-7R and α2ß1 integrin can represent an important pathogenic pathway in Th17-osteoclast function associated with inflammatory diseases.
Asunto(s)
Resorción Ósea/etiología , Integrina alfa2beta1/fisiología , Receptores de Interleucina-7/fisiología , Células Th17/fisiología , Adhesión Celular , Polaridad Celular , Colágeno/farmacología , Humanos , Activación de Linfocitos , Sistema de Señalización de MAP Quinasas , Osteoclastos/fisiología , Osteogénesis , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiologíaRESUMEN
Th17 cells play a critical role in the pathogenesis of rheumatoid arthritis (RA), but the mechanisms by which these cells regulate the development of RA are not fully understood. We have recently shown that α2ß1 integrin, the receptor of type I collagen, is the major collagen-binding integrin expressed by human Th17 cells. In this study, we examined the role of α2ß1 integrin in Th17-mediated destructive arthritis in the murine model of collagen-induced arthritis (CIA). We found that α2ß1 integrin is expressed on synovial Th17 cells from CIA mice and its neutralization with a specific mAb significantly reduced inflammation and cartilage degradation, and protected the mice from bone erosion. Blockade of α2ß1 integrin led to a decrease in the number of Th17 cells in the joints and to a reduction of IL-17 levels in CIA mice. This was associated with an inhibition of receptor activator of NF-κB ligand levels and osteoclast numbers, and reduction of bone loss. We further show that α2ß1 integrin is expressed on synovial Th17 cells from RA patients, and that its ligation with collagen costimulated the production of IL-17 by polarized human Th17 cells by enhancing the expression of retinoic acid receptor-related orphan receptor C through ERK and PI3K/AKT. Our findings provide the first evidence, to our knowledge, that α2ß1 integrin is an important pathway in Th17 cell activation in the pathogenesis of CIA, suggesting that its blockade can be beneficial for the treatment of RA and other Th17-associated autoimmune diseases.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Artritis Experimental/terapia , Artritis Reumatoide/metabolismo , Integrina alfa2beta1/fisiología , Osteólisis/prevención & control , Receptores de Colágeno/fisiología , Células Th17/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Especificidad de Anticuerpos , Artritis Experimental/inmunología , Artritis Experimental/metabolismo , Artritis Reumatoide/inmunología , Cartílago Articular/patología , Colágeno/farmacología , Cricetinae , Regulación hacia Abajo , Femenino , Humanos , Inflamación , Integrina alfa2beta1/antagonistas & inhibidores , Interleucina-17/sangre , Activación de Linfocitos , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Endogámicos DBA , FN-kappa B/fisiología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/biosíntesis , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Osteoclastos/patología , Osteólisis/etiología , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Ligando RANK/sangre , Receptores de Colágeno/antagonistas & inhibidores , Transducción de Señal , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Células Th17/fisiologíaRESUMEN
The mechanisms by which ß1 integrins regulate chemoresistance of cancer cells are still poorly understood. In this study, we report that collagen/ß1 integrin signaling inhibits doxorubicin-induced apoptosis of Jurkat and HSB2 leukemic T-cells by up-regulating the expression and function of the ATP-binding cassette C 1 (ABCC1) transporter, also known as multidrug resistance-associated protein 1. We find that collagen but not fibronectin reduces intracellular doxorubicin content and up-regulates the expression levels of ABCC1. Inhibition and knockdown studies show that up-regulation of ABCC1 is necessary for collagen-mediated reduction of intracellular doxorubicin content and collagen-mediated inhibition of doxorubicin-induced apoptosis. We also demonstrate that activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase signaling pathway is involved in collagen-induced reduction of intracellular doxorubicin accumulation, collagen-induced up-regulation of ABCC1 expression levels, and collagen-mediated cell survival. Finally, collagen-mediated up-regulation of ABCC1 expression and function also requires actin polymerization. Taken together, our results indicate for the first time that collagen/ß1 integrin/ERK signaling up-regulates the expression and function of ABCC1 and suggest that its activation could represent an important pathway in cancer chemoresistance. Thus simultaneous targeting of collagen/ß1 integrin and ABCC1 may be more efficient in preventing drug resistance than targeting each pathway alone.
Asunto(s)
Colágeno/metabolismo , Resistencia a Antineoplásicos , Integrina beta1/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Doxorrubicina/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Células Jurkat , Leucemia de Células T , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Transporte de ProteínasRESUMEN
The role and the mechanisms by which ß1 integrins regulate the survival and chemoresistance of T cell acute lymphoblastic leukemia (T-ALL) still are poorly addressed. In this study, we demonstrate in T-ALL cell lines and primary blasts, that engagement of α2ß1 integrin with its ligand collagen I (ColI), reduces doxorubicin-induced apoptosis, whereas fibronectin (Fn) had no effect. ColI but not Fn inhibited doxorubicin-induced mitochondrial depolarization, cytochrome c release, and activation of caspase-9 and -3. ColI but not Fn also prevented doxorubicin from down-regulating the levels of the prosurvival Bcl-2 protein family member Mcl-1. The effect of ColI on Mcl-1 occurred through the inhibition of doxorubicin-induced activation of c-Jun N-terminal kinase (JNK). Mcl-1 knockdown experiments showed that the maintenance of Mcl-1 levels is essential for ColI-mediated T-ALL cell survival. Furthermore, activation of MAPK/ERK, but not PI3K/AKT, is required for ColI-mediated inhibition of doxorubicin-induced JNK activation and apoptosis and for ColI-mediated maintenance of Mcl-1 levels. Thus, our study identifies α2ß1 integrin as an important survival pathway in drug-induced apoptosis of T-ALL cells and suggests that its activation can contribute to the generation of drug resistance.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Integrina alfa2beta1/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 9/genética , Caspasa 9/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Citocromos c/genética , Citocromos c/metabolismo , Activación Enzimática/genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Femenino , Fibronectinas/genética , Fibronectinas/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Integrina alfa2beta1/genética , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Células Jurkat , Sistema de Señalización de MAP Quinasas/genética , Masculino , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
The expression and function of discoidin domain receptor 1 (DDR1) in T cells are still poorly explored. We have recently shown that activation of primary human T cells via their T cell receptor leads to increased expression of DDR1, which promoted their migration in three-dimensional collagen. In the present study, we provide evidence that activated T cells bind collagen through DDR1. We found that the DDR1:Fc blocking molecule significantly reduced the ability of activated T cells to bind soluble biotinylated collagen. However, DDR1:Fc had no impact on the adhesion of activated T cells to collagen and overexpression of DDR1 in Jurkat T cells did not enhance their adhesion. Together, our results indicate that DDR1 can promote T cell migration without enhancing adhesion to collagen, suggesting that it can contribute to the previously described amoeboid movement of activated T cells in collagen matrices. Our results also show that CD28, in contrast to IL-2 expression, did not costimulate the expression of DDR1 in primary human T cells. Using specific inhibitors, we demonstrated that TCR-induced expression of DDR1 in T cells is regulated by the Ras/Raf/ERK MAP Kinase and PKC pathways but not by calcium/calcineurin signaling pathway or the JNK and P38 MAP Kinases. Thus, our study provides additional insights into the physiology of DDR1 in T cells and may therefore further our understanding of the regulatory mechanisms of T cell migration.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Mitogénicos/metabolismo , Linfocitos T/metabolismo , Señalización del Calcio , Receptores con Dominio Discoidina , Humanos , Células Jurkat , Activación de Linfocitos , Reacción en Cadena de la PolimerasaRESUMEN
Cytohesin-1 is a guanine nucleotide exchange factor for ADP ribosylation factor 6 (Arf6) in human blood neutrophils and differentiated PLB-985 neutrophil-like cells. Cytohesin-1 regulates adhesion and the transendothelial migration of monocytes, dendritic cells and T lymphocytes through activation of the ß2 integrin LFA-1. In this study we investigated the role of cytohesin-1 in neutrophil and neutrophil-like cell adhesion to HUVECs, immobilized ICAM-1, and the α4ß1 and α5ß1 integrin extracellular matrix ligand fibronectin. We show that cytohesin-1 knockdown or inhibition with secinH3 inhibits fMLF-mediated cell adhesion to HUVECs and immobilized ICAM-1, whereas cytohesin-1 over-expression has the opposing effect. Binding of PLB-985 cells to HUVECs correlated with expression of the high-affinity ß2 integrin epitope recognized by mAb24. Adhesion to HUVECs was inhibited by soluble ICAM-1, anti-ICAM-1, anti-CD11a and anti-CD18, but not anti-CD11b, blocking antibodies. We also demonstrate that cytohesin-1 knockdown promotes fMLF-mediated cell adhesion to fibronectin whereas cytohesin-1 over-expression has the opposing effect. Crosstalk between ß1 and ß2 integrins also exists since inhibition of ß1 integrin functions with blocking antibodies enhanced adhesion of PLB-985 over-expressing cytohesin-1 to ICAM-1. We suggest that cytohesin-1 is a key regulator of neutrophil adhesion to endothelial cells and to components of extracellular matrix, which may influence cell emigration through its dual opposing effect on ß2 and ß1 integrin activation.
Asunto(s)
Antígenos CD18/metabolismo , Adhesión Celular , Células Endoteliales/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Neutrófilos/fisiología , Factor 6 de Ribosilación del ADP , Adulto , Anticuerpos Monoclonales , Antígenos CD11/inmunología , Antígenos CD18/inmunología , Adhesión Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Células Endoteliales/inmunología , Femenino , Fibronectinas/metabolismo , Técnicas de Silenciamiento del Gen , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Molécula 1 de Adhesión Intercelular/inmunología , Molécula 1 de Adhesión Intercelular/metabolismo , Masculino , Persona de Mediana Edad , ARN Interferente Pequeño , Triazoles/farmacología , Arterias UmbilicalesRESUMEN
The nucleotide exchange factor cytohesin-1 was previously reported to interact with the cytoplasmic domains of the integrin ß-chain common to all ß(2) integrins such as LFA-1 and Mac-1. We show here that cytohesin-1, which contributes to fMLF-induced functional responses in PMNs through activation of Arf6, restrains the activation of the ß(2) integrin Mac-1 (αMß(2)) in PMNs or dcAMP-differentiated PLB-985 cells. We found that the cytohesin-1 inhibitor SecinH3 or siRNA increased cell adhesion to immobilized fibrinogen and fMLF-mediated conformational changes of Mac-1, monitored using mAb CBRM1/5, specific for the activation epitope of the αM subunit. In contrast, PLB-985 cells overexpressing cytohesin-1 showed little adhesion to fibrinogen. The use of SecinH3 and siRNA also revealed that interference with cytohesin-1 signaling also enhanced phagocytosis of zymosan particles and chemotaxis toward fMLF in transwell migration assays. These increments of phagocytosis and chemotaxis in cells treated with SecinH3 and cytohesin-1 siRNA were reversed by a blocking mAb to the integrin-αM subunit. We provide evidence for increased polymerized cortical actin in cells treated with SecinH3 and that altered signaling through cytohesin-1 increased cell surface expression of FPRL-1 and impairs the late calcium mobilization response elicited by fMLF. The data provide evidence that stimulation with fMLF initiates a signaling cascade that restrains Mac-1 activation in PMNs. Such crosstalk between FPRL-1 and Mac-1 involves cytohesin-1. We suggest that cytohesin-1 may coordinate activation of the ß(2) integrins to regulate PMN adhesion, phagocytosis, and chemotaxis.
Asunto(s)
Antígenos CD18/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Antígeno de Macrófago-1/química , Antígeno de Macrófago-1/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Actinas/metabolismo , Adulto , Western Blotting , Calcio/metabolismo , Adhesión Celular , Diferenciación Celular , Movimiento Celular , Quimiotaxis , Fibrinógeno/metabolismo , Citometría de Flujo , Humanos , Inmunoprecipitación , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Activación Neutrófila/efectos de los fármacos , Fagocitosis , Conformación Proteica/efectos de los fármacosRESUMEN
Although phosphatidic acid (PA) regulates a wide variety of physiological processes, its targets remain poorly characterized in human neutrophils. By co-sedimentation with PA-containing vesicles we identified several PA-binding proteins including vesicle amine transport protein-1 (VAT-1), Annexin A3 (ANXA3), Rac2, Cdc42 and RhoG in neutrophil cytosol. Except for ANXA3, protein binding to PA-containing liposomes was calcium-independent. Cdc42 and RhoG preferentially interacted with PA whereas VAT-1 bound to PA or phosphatidylserine with the same affinity. VAT-1 translocated to neutrophil membranes upon N-formyl-methionyl-leucyl-phenylalanine (fMLF) stimulation. Inhibition of fMLF-induced PLD activity with the Src kinase inhibitor PP2, the selective inhibitor of PLD FIPI, or of PA formation with primary alcohols reduced VAT-1 translocation. In contrast, inhibition of PA hydrolysis with propranolol enhanced fMLF-mediated VAT-1 recruitment to membranes. PMA also redistributed VAT-1 to membranes in a PKC- and PLD-dependent manner. Though fMLF and PMA increased VAT-1 phosphorylation, different kinases appear to be involved. Cell fractionation revealed that a pool of VAT-1 was co-localized with primary, secondary and tertiary granules and plasma membrane markers in resting neutrophils. Stimulation with fMLF enhanced VAT-1 co-localization with CD32a, a plasma membrane marker. Confocal microscopy revealed that VAT-1 decorates granular structures at the cell periphery and double labeling with VAT-1/lactoferrin antibodies showed a partial co-localization with secondary granules in control and fMLF-stimulated cells. Characterization of these putative PA-binding proteins constitutes another step forward for a better understanding of the role of PLD-derived PA in neutrophil physiology.
Asunto(s)
Membrana Celular/metabolismo , Fosfolipasa D/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Secuencia de Aminoácidos , Calcio/metabolismo , Humanos , Liposomas , Datos de Secuencia Molecular , Ácidos Fosfatidicos/metabolismo , Unión Proteica , Transporte de Proteínas , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Proteína RCA2 de Unión a GTPRESUMEN
Polymorphonuclear neutrophil (PMN) stimulation with fMLP stimulates small G proteins such as ADP-ribosylation factors (Arfs) Arf1 and Arf6, leading to phospholipase D (PLD) activation and functions such as degranulation and the oxidative burst. However, the molecular links between fMLF receptors and PLD remain unclear. PMNs express cytohesin-1, an Arf-guanine exchange factor that activates Arfs, and its expression is strongly induced during the acquisition of the neutrophilic phenotype by neutrophil-like cells. The role of cytohesin-1 in the activation of the fMLF-Arf-PLD signaling axis, and the accomplishment of superoxide anion production, and degranulation was investigated in PMNs using the selective inhibitor of cytohesin, Sec 7 inhibitor H3 (secinH3). Cytohesin-1 inhibition with secinH3 leads to Arf6 but not Arf1 inhibition, demonstrating the specificity for Arf6, and fMLF-mediated activation of PLD and of the oxidative burst as well. We observed a decrease in fMLF-mediated protein secretion and expression of cell surface markers corresponding to primary (CD63/myeloperoxidase), secondary (CD66/lactoferrin), and tertiary (matrix metalloproteinase-9) granules in PMNs incubated with secinH3. Similarly, silencing cytohesin-1 or Arf6 in PLB-985 cells negatively affected fMLF-induced activation of PLD, superoxide production, and expression of granule markers on the cell surface. In contrast, stable overexpression of cytohesin-1 in PLB-985 cells enhanced fMLF-induced activation of Arf6, PLD, and NADPH oxidase. The results of this study provide evidence for an involvement of cytohesin-1 in the regulation of the functional responses of human PMNs and link these events, in part at least, to the activation of Arf6.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Factores de Intercambio de Guanina Nucleótido/fisiología , Neutrófilos/metabolismo , Fosfolipasa D/metabolismo , Superóxidos/metabolismo , Factor 6 de Ribosilación del ADP , Degranulación de la Célula/efectos de los fármacos , Células Cultivadas , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacología , NADPH Oxidasas/metabolismo , Triazoles/farmacologíaRESUMEN
Exocytosis of neutrophil granules is a major event that converts circulating neutrophils into fully activated cells capable of chemotaxis, phagocytosis and destruction of pathogens. The PLB-985 cell line is a suitable neutrophilic cellular model which is utilised to study the different functional responses of neutrophils. In this study, we characterised the differentiation of PLB-985 cells toward the granulocytic pathway, using three different inducing agents: dbcAMP, DMSO and DMF. The differentiation efficiency was monitored by observation of cell morphology with electron microscopy, and by analysis of the expression of receptors such as FPRL1 and FcgammaRIIA, the distribution or release of granule markers, phagocytic capacity, as well as measurement of fMLF-induced calcium fluxes. Exocytosis and phagocytosis in differentiated cells were weaker as compared to neutrophils. fMLF stimulated primary granule exocytosis in cells differentiated with dbcAMP, DMSO and DMF, whereas the release of the contents of tertiary granules, as well as that of secretory vesicles, was only observed in dbcAMP-differentiated cells. DMSO-differentiated cells exhibited the highest phagocytic capacity. Altogether our results reinforce the fact that depending on the differentiating agent used, PLB-985 cells represent a useful model to study neutrophil functions and to bypass difficulties inherent to these primary cells.