RESUMEN
Phagocytosis of Yersinia pseudotuberculosis occurs through interaction of the bacterial protein invasin with beta1-integrins. Here we report that N-WASP plays a role in internalisation of an invasin-expressing, avirulent strain of Y. pseudotuberculosis. Ectopic expression of N-WASP mutants, which affect recruitment of the Arp2/3 complex to the phagosome, reduces uptake of Yersinia. In addition, expression of the Cdc42/Rac-binding (CRIB) region of N-WASP has an inhibitory effect on uptake. Using GFP-tagged Rho GTPase mutants, we provide evidence that Rac1, but not Cdc42, is important for internalisation. Furthermore, activated Rac1 rescues Toxin B, CRIB and Src family kinase inhibitor PP2-mediated impairment of uptake. Our observations indicate that invasin-mediated phagocytosis occurs via a Src and WASP family-dependent mechanism(s), involving the Arp2/3 complex and Rac, but does not require Cdc42.
Asunto(s)
Adhesinas Bacterianas , Proteínas Bacterianas/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/fisiología , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Mutación , Proteínas del Tejido Nervioso/metabolismo , Fagocitosis , Plásmidos/metabolismo , Unión Proteica , Transducción de Señal , Transfección , Proteína Neuronal del Síndrome de Wiskott-Aldrich , Yersinia pseudotuberculosis/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Complement-opsonised particles are readily ingested by human neutrophils through a complement receptor-mediated process leading to phagolysosome fusion and production of oxidative metabolites. To investigate the complement receptor 3 (CR3)-associated signal system involved, cells were challenged with protein A-positive, heat-killed Staphylococcus aureus to which antibodies with specificity for the subunits of the beta(2)-integrins, i.e. anti-CD11b (the alpha subunit of CR3) and anti-CD18 (the beta subunit of CR3), were bound through their Fc moiety. Despite not being ingested by the neutrophils, the surface associated anti-CD18- and anti-CD11b-coated particles were able to activate the neutrophil NADPH-oxidase. Also anti-CD11a- (the alpha subunit of LFA-1) and to a lesser extent anti-CD11c- (the alpha subunit of CR4) coated particles were able to trigger the NADPH-oxidase. The NADPH-oxidase was activated without extracellular release of reactive oxygen species. The activity was inhibited by cytochalasin B, suggesting a necessary role for the cytoskeleton in the signalling pathway that activates the oxidase. We show that particle-mediated cross-linking of beta(2)-integrins on the neutrophil surface initiates a signalling cascade, involving cytoskeletal rearrangements, leading to an activation of the NADPH-oxidase without phagosome formation or extracellular release of reactive oxygen species.
Asunto(s)
Antígenos CD18/metabolismo , Antígeno de Macrófago-1/inmunología , Neutrófilos/metabolismo , Proteína Estafilocócica A/inmunología , Anticuerpos/inmunología , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , Antígenos CD11/inmunología , Antígenos CD18/inmunología , Citocalasina B/farmacología , Activación Enzimática , Células HL-60 , Humanos , NADPH Oxidasas/análisis , NADPH Oxidasas/metabolismo , Oxidación-Reducción , Fagocitosis , Transducción de Señal/inmunologíaRESUMEN
The tyrosine phosphatase YopH is an essential virulence effector of pathogenic Yersinia spp. YopH, which is translocated from extracellularly located bacteria into interacting target cells, blocks phagocytosis by professional phagocytes. We show here that immunoprecipitation of YopH from lysates of J774 cells infected with Y. pseudotuberculosis expressing an inactive form of YopH resulted in co-precipitation of certain phosphotyrosine proteins. The association between the inactive YopH and phosphotyrosine proteins in the 120 kDa range was rapid and could be detected after 2 min of infection. The proteins were identified as the docking proteins Cas and Fyn-binding protein (FYB). Upon infection of J774 cells with Y. pseudotuberculosis lacking YopH expression both of these proteins became tyrosine phosphorylated. Moreover, this infection caused recruitment of Cas to peripheral focal complexes, and FYB was relocalized to areas surrounding these structures. Both Cas and FYB became dephosphorylated upon infection with Y. pseudotuberculosis expressing active YopH, and this was associated with disruption of focal complexes. With regard to the previous identification of Cas and focal complexes as targets of YopH in HeLa cells, the present study supports an important role for these targets in a general mechanism of bacterial uptake.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Yersinia pseudotuberculosis/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Proteínas Portadoras/aislamiento & purificación , Cateninas , Moléculas de Adhesión Celular/aislamiento & purificación , Línea Celular , Técnica del Anticuerpo Fluorescente , Macrófagos , Fosfoproteínas/aislamiento & purificación , Plásmidos/administración & dosificación , Mutación Puntual , Pruebas de Precipitina , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/aislamiento & purificación , Factores de Tiempo , Catenina deltaRESUMEN
The protein tyrosine phosphatase YopH, produced by the pathogen Yersinia pseudotuberculosis, is an essential virulence determinant involved in antiphagocytosis. Upon infection, YopH is translocated into the target cell, where it recognizes focal complexes. Genetic analysis revealed that YopH harbours a region that is responsible for specific localization of this PTPase to focal complexes in HeLa cells and professional phagocytes. This region is a prerequisite for blocking an immediate-early Yersinia-induced signal within target cells. The region is also essential for antiphagocytosis and virulence, illustrating the biological significance of localization of YopH to focal complexes during Yersinia infection. These results also indicate that focal complexes play a role in the general phagocytic process.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Tirosina Fosfatasas/genética , Yersiniosis/microbiología , Yersinia pseudotuberculosis/enzimología , Animales , Proteínas de la Membrana Bacteriana Externa/metabolismo , Calcio/metabolismo , Modelos Animales de Enfermedad , Células HeLa , Humanos , Macrófagos/microbiología , Ratones , Modelos Moleculares , Mutación , Neutrófilos/microbiología , Fagocitosis/inmunología , Proteínas Tirosina Fosfatasas/metabolismo , Transducción de Señal , Vinculina/metabolismo , Virulencia/genética , Yersiniosis/inmunología , Yersinia pseudotuberculosis/patogenicidadRESUMEN
BACKGROUND & AIMS: An ability to invade host cells could be a means for Helicobacter pylori to achieve resistance to antibiotic therapy. The aim of this study was to investigate the mechanisms involved in adherence and entry of H. pylori into cultured cells. METHODS: Coinfection with Yersinia expressing mutant or wild-type YopH tyrosine phosphatase was used. Genistein and cytochalasin D were used as inhibitors of adherence and entry; entry was monitored by a gentamicin-protection assay. Target cells were AGS cells and a beta1-integrin-deficient cell line with its corresponding beta1-integrin-expressing transfectant. RESULTS: H. pylori induced phosphorylation of 125-130-kilodalton proteins, similar in size to the target proteins of Yersinia YopH. Adherence of H. pylori was inhibited by Yersinia organisms expressing enzymatically active YopH but not by inactive YopH. Adherence and entry of H. pylori was considerably higher with beta1-integrin-transfected cells than with beta1-integrin-deficient cells. Antibodies directed against alpha5- and beta1-integrin chains reduced adherence to the alpha5beta1-integrin-expressing gastric cell line AGS. Entry was inhibited by both cytochalasin D and genistein. Entry, but not adherence, was higher for 2 type I strains than for a type II isolate. CONCLUSIONS: Invasion of gastric epithelium via an integrin-mediated pathway could contribute to the ability of H. pylori to establish persistent infection.
Asunto(s)
Adhesión Bacteriana , Helicobacter pylori/fisiología , Integrina beta1/fisiología , Transducción de Señal , Estómago/citología , Estómago/microbiología , Western Blotting , Línea Celular , Citometría de Flujo , Humanos , Immunoblotting , Integrinas , Fosforilación , Proteínas Tirosina Quinasas , Transfección , YersiniaRESUMEN
Pathogenic species of the genus Yersinia evade the bactericidal functions of phagocytes. This evasion is mediated through their virulence effectors, Yops, which act within target cells. In this study we investigated the effect of Yersinia pseudotuberculosis on Ca2+ signaling in polymorphonuclear neutrophils. The intracellular free calcium concentration in single adherent human neutrophils was monitored during bacterial infection and, in parallel, the encounter between the bacteria and cells was observed. When a plasmid-cured strain was used for infection, adherence of a single bacterium to the cellular surface induced a beta1 integrin-dependent transient increase in the intracellular concentration of free calcium. This was, however, not seen with Yop-expressing wild-type bacteria, which adhered to the cell surface without generating any Ca2+ signal. Importantly, the overall Ca2+ homeostasis was not affected by the wild-type strain; the Ca2+ signal mediated by the G-protein-coupled formyl-methionyl-leucyl-phenylalanine receptor was still functioning. Hence, the blocking effect was restricted to certain receptors and their signaling pathways. The use of different Yop mutant strains revealed that the protein tyrosine phosphatase YopH was responsible for the inhibition. This virulence determinant has previously been implicated in very rapid Yersinia-mediated effects on target cells as the key effector in the blockage of phagocytic uptake. The present finding, that Y. pseudotuberculosis, via YopH, specifically inhibits a self-induced immediate-early Ca2+ signal in neutrophils, offers more-detailed information concerning the effectiveness of this virulence effector and implies an effect on Ca2+-dependent, downstream signals.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/fisiología , Señalización del Calcio/fisiología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Proteínas Tirosina Fosfatasas/fisiología , Yersinia pseudotuberculosis/patogenicidad , Adhesión Bacteriana , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de Unión al GTP/fisiología , Humanos , Técnicas In Vitro , Integrina beta1/fisiología , Mutación , Fagocitosis , Plásmidos/genética , Proteínas Tirosina Fosfatasas/genética , Receptores de Superficie Celular/fisiología , Virulencia/genética , Virulencia/fisiología , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/fisiologíaRESUMEN
Preventing the early host immune defense allows pathogenic Yersinia to proliferate in lymphatic tissue. This ability depends on signaling that occurs between the bacteria and the host cells. Following intimate contact with the target cell a signal is generated within the bacterium that results in increased expression of virulence-associated proteins that are subsequently delivered into the infected cell. These proteins, designated Yops, interfere with the host-cell signaling pathways that are normally activated to eliminate infectious agents.
Asunto(s)
Sistema Linfático/citología , Sistema Linfático/microbiología , Transducción de Señal/fisiología , Yersiniosis/microbiología , Yersinia/fisiología , HumanosRESUMEN
Pathogenic Yersinia resist uptake by eukaryotic cells by a mechanism involving the virulence protein YopH, a protein tyrosine phosphatase. We show that p130Cas and FAK are phosphorylated and recruited to peripheral focal complexes during bacterial uptake in HeLa cells. The inactive form of YopH interacts with the tyrosine phosphorylated forms of FAK and p130Cas and co-localizes with these proteins in focal adhesions. On the other hand, the presence of active YopH results in inhibition of uptake, dephosphorylation of p130Cas and FAK, and disruption of peripheral focal complexes. We suggest that p130Cas and FAK are substrates for YopH and that the dephosphorylation of these proteins impairs the uptake of Yersinia pseudotuberculosis into HeLa cells.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Moléculas de Adhesión Celular/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas , Receptor de Insulina/metabolismo , Proteína de Retinoblastoma/metabolismo , Tirosina/metabolismo , Yersinia pseudotuberculosis/metabolismo , Proteína Sustrato Asociada a CrK , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Células HeLa , Humanos , Integrina beta1/metabolismo , Peso Molecular , Fosforilación , Proteína p130 Similar a la del Retinoblastoma , Yersinia pseudotuberculosis/enzimologíaRESUMEN
Introduction of anti-host factors into eukaryotic cells by extracellular bacteria is a strategy evolved by several Gram-negative pathogens. In these pathogens, the transport of virulence proteins across the bacterial membranes is governed by closely related type III secretion systems. For pathogenic Yersinia, the protein transport across the eukaryotic cell membrane occurs by a polarized mechanism requiring two secreted proteins, YopB and YopD. YopB was recently shown to induce the formation of a pore in the eukaryotic cell membrane, and through this pore, translocation of Yop effectors is believed to occur (Håkansson et al., 1996b). We have previously shown that YopK of Yersinia pseudotuberculosis is required for the development of a systemic infection in mice. Here, we have analysed the role of YopK in the virulence process in more detail. A yopK-mutant strain was found to induce a more rapid YopE-mediated cytotoxic response in HeLa cells as well as in MDCK-1 cells compared to the wild-type strain. We found that this was the result of a cell-contact-dependent increase in translocation of YopE into HeLa cells. In contrast, overexpression of YopK resulted in impaired translocation. In addition, we found that YopK also influenced the YopB-dependent lytic effect on sheep erythrocytes as well as on HeLa cells. A yopK-mutant strain showed a higher lytic activity and the induced pore was larger compared to the corresponding wild-type strain, whereas a strain overexpressing YopK reduced the lytic activity and the apparent pore size was smaller. The secreted YopK protein was found not to be translocated but, similar to YopB, localized to cell-associated bacteria during infection of HeLa cells. Based on these results, we propose a model where YopK controls the translocation of Yop effectors into eukaryotic cells.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Yersinia pseudotuberculosis/metabolismo , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Línea Celular , Membrana Celular/metabolismo , Perros , Células Eucariotas/metabolismo , Expresión Génica , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Yersinia pseudotuberculosis/genéticaAsunto(s)
Médicos de Familia , Servicios de Salud Rural , Historia del Siglo XX , Humanos , Suecia , Recursos HumanosRESUMEN
Integrin receptors on human neutrophils mediate adhesion and phagocytosis. These functions are linked to a signal-transduction cascade that rearranges the cytoskeleton. The intention of this study was to clarify how activation of phospholipase D (PLD) is coupled to the complement receptor three (CR3, CD18/CD11b)-mediated ingestion process. Carbobenzyloxy-leucine-tyrosine-choloromethylketone (zLYCK) inhibited PLD activation induced by complement-opsonized yeast particles (COYP) by 39%. Phagocytosis of these particles was reduced by zLYCK to the same extent. Anti-CD18-antibodies bound to protein A-positive Staphylococcus aureus bacteria induced a significant PLD activation. These particles were not ingested which implicates that CR-mediated ingestion per se is not required to induce PLD activity. Cytochalasin B-treatment, which blocks actin reorganization, partly reduced COYP-mediated PLD activity, but had no effect on activity caused by anti-CD18-coated particles. This excludes activation of PLD to be a secondary event, but rather an early signal in the phagocytic uptake prior to actin reorganization. These data suggest an important and early role for PLD in integrin-mediated phagocytosis.
Asunto(s)
Antígeno de Macrófago-1/fisiología , Neutrófilos/enzimología , Fagocitosis/fisiología , Fosfolipasa D/metabolismo , Transducción de Señal/fisiología , Actinas/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Calcio/fisiología , Citocalasina B/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/ultraestructura , Activación Enzimática/efectos de los fármacos , Humanos , Neutrófilos/fisiología , Proteínas Opsoninas , Saccharomyces cerevisiae , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The PTPase YopH of Yersinia is essential to the ability of these bacteria to block phagocytosis. Wild-type Yersinia pseudotuberculosis, but not the yopH mutant strain, resisted phagocytosis by J774 cells. Ingestion of a yopH mutant was dependent on tyrosine kinase activity. Transcomplementation with wild-type yopH restored the anti-phagocytic effect, whereas introduction of the gene encoding the catalytically inactive yopHC403A was without effect. The PTPase inhibitor orthovanadate impaired the anti-phagocytic effect of the wild-type strain, further demonstrating the importance of bacteria-derived PTPase activity for this event. The ability to resist phagocytosis indicates that the effect of the bacterium is immediately exerted when it becomes associated with the phagocyte. Within 30 s after the onset of infection, wild-type Y. pseudotuberculosis caused a YopH-dependent dephosphorylation of phosphotyrosine proteins in J774 cells. Furthermore, interaction of the cells with phagocytosable strains led to a rapid and transient increase in tyrosine phosphorylation of paxillin and some other proteins, an event dependent on the presence of the bacterial surface-located protein invasin. Co-infection with the phagocytosable strain and the wild-type strain abolished the induction of tyrosine phosphorylation. Taken together, the present findings demonstrate an immediate YopH-mediated dephosphorylation of macrophage phosphotyrosine proteins, suggesting that this PTPase acts by preventing early phagocytosis-linked signalling in the phagocyte.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Fosfotirosina/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Transducción de Señal/fisiología , Yersinia pseudotuberculosis/metabolismo , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Línea Celular , Ratones , Fagocitosis/fisiología , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Yersinia pseudotuberculosis/genéticaRESUMEN
Nonopsonized as well as immunoglobulin-G (IgG)-opsonized Yersinia pseudotuberculosis resists phagocytic uptake by the macrophage-like cell line J774 by a mechanism involving the plasmid-encoded proteins Yops. The tyrosine phosphatase YopH was of great importance for the antiphagocytic effect of the bacteria. YopH-negative mutants did not induce antiphagocytosis; instead, they were readily ingested, almost to the same extent as that of the translocation mutants YopB and YopD and the plasmid-cured strain. The bacterial determinant invasin was demonstrated to mediate phagocytosis of nonopsonized bacteria by these cells. In addition to inhibiting uptake of itself, Y. pseudotuberculosis also interfered with the phagocytic uptake of other types of prey: J774 cells that had been exposed to virulent Y. pseudotuberculosis exhibited a reduced capacity to ingest IgG-opsonized yeast particles. This effect was impaired when the bacterium-phagocyte interaction occurred in the presence of gentamicin, indicating a requirement for in situ bacterial protein synthesis. The Yersinia-mediated antiphagocytic effect on J774 cells was reversible: after 18 h in the presence of gentamicin, the phagocytic capacity of Yersinia-exposed J774 cells was completely restored. Inhibition of the uptake of IgG-opsonized yeast particles was dependent on the Yops in a manner similar to that seen for blockage of Yersinia phagocytosis. This similarity suggests that the pathogen affected a general phagocytic mechanism. Despite a marked reduction in the capacity to ingest IgG-opsonized yeast particles, no effect was observed on the binding of the prey. Taken together, these results demonstrate that Yop-mediated antiphagocytosis by Y. pseudotuberculosis affects regulatory functions downstream of the phagocytic receptor and thereby extends to other types of phagocytosis.
Asunto(s)
Macrófagos/inmunología , Fagocitosis , Yersinia pseudotuberculosis/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Bacterianas/biosíntesis , Gentamicinas/farmacología , Inmunoglobulina G/inmunología , Técnicas In Vitro , Ratones , Proteínas Opsoninas , Fagocitosis/efectos de los fármacos , Receptores Fc/inmunología , Yersinia pseudotuberculosis/efectos de los fármacos , Infecciones por Yersinia pseudotuberculosis/inmunologíaRESUMEN
Human neutrophils preincubated with antibodies against complement receptor type 1 (CR1) (anti-CD35) and/or complement receptor type 3 (CR3) (anti-CD11b or anti-CD18) exhibited a reduced ability to engulf complement-opsonized yeast particles, whereas cellular adhesion of these particles was reduced only in the presence of anti-CD35 antibodies. These data support the idea that CR1 primarily promotes the adhesion of particles and CR3 mediates the subsequent engulfment. However, the effects of anti-CR1 and anti-CR3 antibodies on particle-induced diglyceride production correlate with the effects of these antibodies on the cellular uptake of the particles. Hence, it seems reasonable to suggest that CR1 also participates in mediating the signal(s) that induce particle uptake. This idea is further supported by the findings that cross-linking surface-bound anti-CD11b, anti-CD18 as well as anti-CD35 antibodies results in activation of phospholipase D (PLD), a signal closely associated with phagocytosis of complement-opsonized yeast particles in human neutrophils. The signaling property of CR1 was further revealed by the observation that cross-linking of surface-bound anti-CD35 triggered a rapid and transient mobilization of intracellular Ca2+, a signal most likely involved in the phagosome-lysosome fusion that occurs after the uptake of a particle. Pretreatment with PMA, which positively modulates CR-mediated engulfment of particles, was found to potentiate the CR3- and CR1-induced activation of PLD but impair the activation of phospholipase C, giving added support to the idea that PLD activation is the principal signal for the engulfment process. The activation of PLD was also increased by stimulating the cells with anti-CD18 or anti-CD35 antibodies prefixed on Staphylococcus aureus particles, instead of cross-linking cellular-bound antibodies, suggesting that the form of ligand presentation is a critical parameter of phagocytic signaling. Taken together, the present results demonstrate that both CR1 and CR3 can initiate transmembrane signaling in human neutrophils and, in particular, activation of PLD. This activation was also further recognized as an important signal regulating the engulfment of complement-opsonized particles.
Asunto(s)
Proteínas del Sistema Complemento/fisiología , Antígeno de Macrófago-1/fisiología , Neutrófilos/fisiología , Fagocitosis , Receptores de Complemento 3b/fisiología , Diglicéridos/metabolismo , Humanos , Técnicas In Vitro , Fosfolipasa D/metabolismo , Transducción de Señal , Levaduras/inmunologíaRESUMEN
The generation of oxygen radicals by polymorphonuclear leukocytes (PMNL) plays a pivotal role for host defense. Since ethanol reduced FMLP- but not PMA-induced superoxide ion (O2-) formation by PMNL, the effects of ethanol on second messenger systems in PMNL were studied. FMLP induced a biphasic rise in cytosolic calcium concentrations, [Ca2+]i. Ethanol treatment abolished the second phase (believed to reflect Ca2+ influx), an effect also observed in PMNL treated with La3+ or suspended in Ca(2+)-free buffer. The FMLP-induced inositol trisphosphate generation was unaffected by ethanol, whereas diacylglycerol formation was, as expected, markedly reduced. Propranolol, an inhibitor of diacylglycerol formation from phosphatidic acid, caused a prolonged transmembrane influx of Ca2+ and partially reversed the inhibitory effect of ethanol on FMLP-induced O2- production. Thus, the ability of ethanol to inhibit FMLP-induced O2- generation in neutrophils seems to be due to both impaired influx of Ca2+ across the plasma membrane and reduced phospholipase D-mediated generation of phosphatidic acid.
Asunto(s)
Etanol/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Superóxidos/metabolismo , Calcio/metabolismo , Diglicéridos/metabolismo , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Neutrófilos/metabolismo , Propranolol/farmacologíaRESUMEN
Complement receptor (CR)-mediated phagocytosis is associated with an increased accumulation of diglyceride (sn-1,2-diacylglycerol and/or 1-O-alkyl-2-acyl-glycerol) in human neutrophils. The C3bi-mediated increase in diglyceride (5-20 min) was only partially impaired when phosphoinositide-specific phospholipase C (PLC) activity was abolished by reduction of cytosolic free Ca2+. At an early time point (1 min), however, diglyceride production was barely detectable in control cells, whereas production was considerable in cells with a reduced cytosolic free Ca2+ concentration. C3bi stimulation of 32P-labeled neutrophils caused a rapid and significant breakdown of [32P]phosphatidylcholine (PC) which was not affected by inhibition of Ca(2+)-dependent phosphoinositide-specific PLC. Thus, PC hydrolysis could be involved in C3bi-induced diglyceride formation. Stimulation of cells labeled with [3H]1-O-alkyl-lyso-PC ([3H]alkyl-lyso-PC), resulted in an increased formation of [3H]1-O-alkyl-phosphatidic acid ([3H]alkyl-PA) and a later and slower formation of [3H]1-O-alkyl-diglyceride ([3H]alkyl-diglyceride); this suggests activation of phospholipase D (PLD). When these labeled cells were stimulated in the presence of 0.5% ethanol a marked accumulation of [3H]1-O-alkyl-phosphatidylethanol ([3H]alkyl-PEt) was observed in both controls and calcium-reduced cells, further strengthening the suggested involvement of PLD activity. In parallel with the sustained increase in diglyceride formation, CR-mediated phagocytosis was also associated with phosphorylation of a cellular protein kinase C substrate (MARCKS). Therefore it seems reasonable to suggest a causal relationship between C3bi-induced PLD activation, which results in diglyceride formation, and activation of protein kinase C. In electropermeabilized cells which were incapable of ingesting particles, C3bi particles were still able to activate PLD and induce formation of diglyceride. This signaling event must therefore be triggered by binding of particles to the cell and not by the engulfment process. Most importantly, introduction of the protein kinase C inhibitor peptides, PKC(19-36) and PKC(19-31), into these permeabilized cells resulted in a clear reduction of the C3bi-induced production of diglyceride, indicating that CR-mediated activation of protein kinase C directly triggers a positive feedback mechanism for additional diglyceride formation. Taken together, these data further clarify the mechanisms of CR-mediated diglyceride formation and give added support to the concept that protein kinase C plays an important role in the phagocytic process.
Asunto(s)
Diglicéridos/metabolismo , Neutrófilos/metabolismo , Fagocitosis , Fosfolipasa D/metabolismo , Proteína Quinasa C/metabolismo , Receptores de Complemento/metabolismo , Western Blotting , Calcio/metabolismo , Activación Enzimática , Retroalimentación , Humanos , Técnicas In Vitro , Neutrófilos/inmunología , Fosfatidilcolinas/metabolismo , Fosforilación , Especificidad por SustratoRESUMEN
Auranofin, an antiarthritic gold compound, modulates a number of chemotactic factor-induced inflammatory responses in human neutrophils. In order to unravel the mechanism involved, the present study investigated the effects of auranofin on early signal transduction events in these cells. Auranofin did not affect the chemotactic peptide (fMetLeuPhe)-induced formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), neither in the presence nor in the absence of extracellular calcium ions. In contrast, there was a progressive inhibition by auranofin on the fMet-Leu-Phe-induced mobilization of intracellular calcium. This demonstrates that auranofin can dissociate the generation of Ins(1,4,5)P3 from the subsequent release of intracellular calcium, perhaps by interfering with the intracellular binding of Ins(1,4,5)P3 to its receptor. In experiments performed in electro-permeabilized cells, however, a relatively high concentration of the drug failed to abolish the specific binding of Ins(1,4,5)P3. In addition, in the same system, auranofin also failed to abolish the Ins(1,4,5)P3-induced release of Ca2+. Consequently, auranofin-mediated dissociation of fMLP-induced Ins(1,4,5)P3 formation and intracellular calcium release can not be explained merely by an antagonistic effect of auranofin on the Ins(1,4,5)P3 receptor. Instead the interaction between auranofin and the plasma membrane seems to be an initial and important part of the mechanism by which this drug interferes with the transduction signalling system.
Asunto(s)
Auranofina/farmacología , Canales de Calcio , Calcio/sangre , Inositol 1,4,5-Trifosfato/sangre , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/metabolismo , Receptores Citoplasmáticos y Nucleares , Aminoquinolinas , Ácido Egtácico/farmacología , Colorantes Fluorescentes , Humanos , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/biosíntesis , Receptores de Inositol 1,4,5-Trifosfato , Cinética , Neutrófilos/efectos de los fármacos , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/metabolismoRESUMEN
Most ligand-receptor interactions result in an immediate generation of various second messengers and a subsequent association of the ligand-receptor complex to the cytoskeleton. Depending on the receptor involved, this linkage to the cytoskeleton has been suggested to play a role in the termination of second messenger generation and/or the endocytic process whereby the ligand-receptor complex is internalized. We have studied how the binding of chemotactic peptide-receptor complexes to the cytoskeleton of human neutrophils is accomplished. As much as 76% of the tritiated formylmethionyl-leucyl-phenylalanine (fMet-Leu-[3H]Phe) specifically bound to intact cells, obtained by a 30-s stimulation with 20 nM fMet-Leu-[3H]Phe, still remained after Triton X-100 extraction. Preincubating intact cells with dihydrocytochalasin B (dhCB) or washing the cytoskeletal preparation with a high concentration of potassium, reduced the binding of ligand-receptor complexes to the cytoskeleton by 46% or more. Inhibition of fMet-Leu-Phe-induced generation of second messengers by ADP-ribosylating the alpha-subunit of the receptor-coupled G-protein with pertussis toxin, did not reduce the binding of ligand-receptor complexes to the cytoskeleton. However, using guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) to prevent the dissociation of the fMet-Leu-Phe-associated G-protein within electrically permeabilized cells, led to a pronounced reduction (62%) of the binding between ligand-receptor complexes and the cytoskeleton. In summary, in human neutrophils the rapid association between chemotactic peptide-receptor complexes and the cytoskeleton is dependent on filamentous actin. This association is most likely regulated by the activation and dissociation of the fMet-Leu-Phe-associated G-protein.
Asunto(s)
Actinas/sangre , Proteínas de Unión al GTP/sangre , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Neutrófilos/metabolismo , Receptores Inmunológicos/metabolismo , Anticuerpos Monoclonales , Citocalasina B/análogos & derivados , Citocalasina B/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Proteínas de Unión al GTP/fisiología , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacología , Humanos , Técnicas In Vitro , Cinética , Ligandos , Microscopía Electrónica de Rastreo , Neutrófilos/inmunología , Toxina del Pertussis , Cloruro de Potasio/farmacología , Receptores de Formil Péptido , Receptores Inmunológicos/efectos de los fármacos , Tionucleótidos/farmacología , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Phagocytosis of C3bi- or IgG-opsonized yeast particles in human neutrophils was found to be associated with an increased formation of inositol phosphates and diacylglycerol. Pertussis toxin only marginally affected phagocytosis of IgG- and C3bi-opsonized particles and the associated formation of second messengers. Forskolin, which induced a threefold rise of cellular cAMP, however, markedly inhibited both C3bi- and IgG-mediated phagocytosis as well as the particle-induced formation of inositol phosphates and diacylglycerol. These observations are in contrast to what was found to occur with chemotactic factors and indicate that chemotactic and phagocytic signaling can be regulated independently in human neutrophils. Since C3bi-mediated phagocytosis has been shown to occur at vanishingly low cytosolic free calcium levels, calcium-depleted cells were used to study the importance of the inositol cycle for the engulfment of C3bi-opsonized particles. Despite a total lack of receptor-induced formation of inositol phosphates, a significantly increased accumulation of diacylglycerol accompanied the ingestion of C3bi-opsonized particles. These data show that the engulfment of C3bi-opsonized particles can occur independently of both a calcium transient and an increased inositol phosphate production. However, the observed accumulation of diacylglycerol, not derived from phosphoinositides, suggests that this second messenger play a role in the control of the engulfment process.