RESUMEN
Apoptosis occurred in human neutrophils within an hour of exposure to viable serum-opsonized Staphylococcus aureus, as indicated by appearance of cells with condensed nuclei, fragmented DNA, and increased phosphatidylserine exposure. In contrast, serum-opsonized, heat-killed S. aureus did not induce apoptosis. This discrepancy could not be explained by differences in bacterial uptake or total NADPH-oxidase activity. Suppressing phagocytosis by pretreating the neutrophils with cytochalasin b or by using nonopsonized bacteria did not prevent apoptosis. A supernatant from bacteria grown for 2 h in nutrient broth had a strong proapoptotic influence that was abrogated by heat treatment. Exposure to viable S. aureus or supernatant also led to activation of p38-mitogen-activated protein kinase in the neutrophils. Inhibition of this kinase with SB203580 reduced the apoptosis-inducing capacity of both bacteria and supernatant. We conclude that S. aureus activates p38-mitogen-activated protein kinase in neutrophils and induces apoptosis, probably mediated by a bacteria-derived soluble factor(s).
Asunto(s)
Apoptosis , Proteínas Quinasas Activadas por Mitógenos/fisiología , Neutrófilos/inmunología , Staphylococcus aureus/patogenicidad , Núcleo Celular/ultraestructura , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Imidazoles/farmacología , Cinética , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , NADPH Oxidasas/fisiología , Neutrófilos/citología , Neutrófilos/ultraestructura , Fagocitosis , Piridinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
For reasons unknown, certain Escherichia coli strains become highly virulent when injected with hemoglobin or other soluble iron sources. Two clinical isolates (virulent and nonvirulent) showed equivalent hemoglobin-mediated growth acceleration in vitro. However, when injected intraperitoneally into mice without hemoglobin, the virulent strain was cleared more slowly (t(1/2), >4 h vs. <30 min). The virulent E. coli strain had a polysialic acid-containing capsule, whereas the nonvirulent strain did not. Virulent E. coli grown at 20 degrees C (which blocks polysialylation) were cleared as rapidly as nonvirulent organisms. In another virulent E. coli strain having abundant outer membrane polysialic acid, targeted deletion of the polysialyltransferase accelerated host clearance and blocked iron-dependent virulence. The iron-dependent virulence of certain E. coli strains may represent the combined effect of slow in vivo clearance-associated, in this case, with outer membrane polysialylation coupled with accelerated growth permitted by iron compounds.
Asunto(s)
Escherichia coli/clasificación , Escherichia coli/patogenicidad , Compuestos de Hierro/metabolismo , Peritonitis/microbiología , Ácidos Siálicos/metabolismo , Azul Alcián/metabolismo , Animales , Colorantes/metabolismo , Escherichia coli/crecimiento & desarrollo , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/metabolismo , Cinética , Masculino , RatonesRESUMEN
OBJECTIVE AND DESIGN: To examine the hypothesis that an accelerated rate of neutrophil apoptosis occurs following beta2-integrin activation, and further investigate the signal transduction pathways involved. MATERIAL: Human polymorphonuclear neutrophils. TREATMENT: Neutrophils were challenged with pansorbins coated with antibodies towards the beta2-integrin subunit CD18 in a proportion of 1:100 with or without the inhibitors diphenylene iodonium (10 M), cytochalasin B (5 microg/ml), genistein (10 nM), herbimycin A (10 M) and Z-VAD-FMK (10 microM). METHODS: Measurement of phosphatidylserine exposure and DNA fragmentation in flow cytometry and assessment of H2O2-production through spectrofluorometry. The results were analysed using Mann Whitney U test and Kruskal Wallis. RESULTS: Pansorbins coated with antibodies to CD18 induce apoptosis in neutrophils (p<0.01), and activate the production of reactive oxygen species (p<0.01). Pre-treatment with the inhibitors have no effect on anti-CD 18 induced apoptosis. CONCLUSION: Anti-CD18 pansorbins induce apoptosis in neutrophils through an alternative pathway not involving reactive oxygen species and independent of tyrosine phosphorylation, cytoskeletal reorganisation and caspases.
Asunto(s)
Apoptosis , Antígenos CD18/fisiología , Neutrófilos/fisiología , Transducción de Señal , Adulto , Citoesqueleto/fisiología , Humanos , Fagocitosis , Fosforilación , Especies Reactivas de Oxígeno , Tirosina/metabolismoRESUMEN
An increase in cytosolic Ca(2+)concentration periphagosomally is critical for phagolysosomal formation and neutrophil elimination of microbes. The Ca(2+)increase could be achieved through release of Ca(2+)from mobilized intracellular stores. Alternatively, Ca(2+)that passively enter the phagosome during phagocytosis could be provided by the phagosome. Intraphagosomal Ca(2+)changes in single human neutrophils was measured during phagocytosis of serum opsonized Fura-2-conjugated zymosan particles, using a digital image processing system for microspectrofluorometry. A decrease in phagosomal Ca(2+)down to nanomolar concentrations was seen within minutes following phagosomal closure. Blockage of plasma membrane Ca(2+)channels by econazole abolished this decrease. The fluorescence properties of Fura-2 zymosan were retained after phagocytosis and stable to pH changes, reactive oxygen species, and proteolytic enzymes. We suggest that Ca(2+)ions present in the phagosome enter the cell cytosol through Ca(2+)channels in the phagosomal membrane, achieving a localized Ca(2+)rise that is important for phagosome processing.
Asunto(s)
Calcio/metabolismo , Neutrófilos/metabolismo , Fagosomas/metabolismo , Calcio/química , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Fraccionamiento Celular , Gránulos Citoplasmáticos/metabolismo , Fluorescencia , Fura-2 , Humanos , Peróxido de Hidrógeno/farmacología , Concentración de Iones de Hidrógeno , Membranas Intracelulares/metabolismo , Microscopía por Video/métodos , Neutrófilos/efectos de los fármacos , Percloratos/farmacología , Fagosomas/ultraestructura , Zimosan/metabolismoRESUMEN
Oxidative stress induces apoptosis in many types of cells, including human neutrophils. Our objective was to determine whether reactive oxygen species (ROS) produced by activated neutrophils are associated with accelerated apoptosis. Exposing neutrophils to ionomycin or phorbol myristate acetate (PMA) induced intracellular H2O2 production and rapid onset of apoptosis, measured as condensed chromatin, cellular shrinkage, and DNA fragmentation. Neutrophils activated with formyl-methionyl-leucyl-phenylalanine (fMLP) generated mainly extracellular H2O2 and did not undergo apoptosis. Exogenously added H2O2, together with the catalase blocker sodium azide, induced apoptosis to the same extent and with similar kinetics as PMA and ionomycin. Adenosine inhibited ionomycin-induced intracellular H2O2 production and apoptosis. Neither PMA nor ionomycin caused apoptosis in dimethyl sulfoxide-differentiated HL-60 cells, which are incapable of intracellular H2O2 production, whereas H2O2 induced apoptosis more efficiently in these cells than in neutrophils. We propose that activated neutrophils use intracellularly formed H2O2 to commit suicide.