RESUMEN
Phagocytosis and killing of microorganisms are complex processes that involve tightly regulated membrane traffic events. Because many signaling molecules associate with membrane rafts and because these structures can be found on azurophilic granules, we decided to investigate raft recruitment and the signaling requirements for azurophilic granule secretion during phagosome maturation. At the site of phagocytosis of immunoglobulin G-opsonized prey in human neutrophils, we found that early secretion of azurophilic granules was both raft- and calcium-dependent. Subsequently, rafts at the phagocytic site were internalized with the prey. At the fully formed phagosome, the fusion of azurophilic granules was no longer dependent on rafts or calcium. These findings were found to be true also when using Streptococcus pyogenes bacteria as prey, and depletion of calcium affected the kinetics of bacterial intracellular survival. These findings suggest that the mechanisms for delivery of azurophilic content to nascent and sealed phagosomes, respectively, differ in their dependence on calcium and membrane rafts.
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Gránulos Citoplasmáticos/metabolismo , Fusión de Membrana , Fagosomas/metabolismo , Antígenos CD/metabolismo , Calcio/metabolismo , Colesterol/metabolismo , Gangliósido G(M1)/metabolismo , Humanos , Inmunoglobulina G/metabolismo , Fagocitosis , Glicoproteínas de Membrana Plaquetaria/metabolismo , Streptococcus pyogenes/inmunología , Fracciones Subcelulares/metabolismo , Tetraspanina 30RESUMEN
Targeting mechanisms of neutrophil elastase (NE) and other luminal proteins stored in myeloperoxidase (MPO)-positive secretory lysosomes/primary granules of neutrophils are unknown. These granules contain an integral membrane protein, CD63, with an adaptor protein-3-dependent granule delivery system. Therefore, we hypothesized that CD63 cooperates in granule delivery of the precursor of NE (proNE). Supporting this hypothesis, an association was demonstrated between CD63 and proNE upon coexpression in COS cells. This also involved augmented cellular retention of proNE requiring intact large extracellular loop of CD63. Furthermore, depletion of CD63 in promyelocytic HL-60 cells with RNA interference or a CD63 mutant caused reduction of cellular NE. However, the proNE steady-state level was similar to wild type in CD63-depleted clones, making it feasible to examine possible effects of CD63 on NE trafficking. Thus, depletion of CD63 led to reduced processing of proNE into mature NE and reduced constitutive secretion. Furthermore, CD63-depleted cells showed a lack of morphologically normal granules, but contained MPO-positive cytoplasmic vacuoles with a lack of proNE and NE. Collectively, our data suggest that granule proteins may cooperate in targeting; CD63 can be involved in ER or Golgi export, cellular retention, and granule targeting of proNE before storage as mature NE.
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Antígenos CD/fisiología , Elastasa de Leucocito/metabolismo , Peroxidasa/metabolismo , Glicoproteínas de Membrana Plaquetaria/fisiología , Animales , Antígenos CD/metabolismo , Células COS , Chlorocebus aethiops , Citoplasma/metabolismo , Gránulos Citoplasmáticos/metabolismo , Aparato de Golgi/metabolismo , Células HL-60 , Humanos , Modelos Biológicos , Neutrófilos/metabolismo , Plásmidos/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Interferencia de ARN , Tetraspanina 30RESUMEN
BACKGROUND: There is a growing awareness of the importance of intracellular events in determining the outcome of infectious disease. To improve the understanding of such events, like phagosome maturation, we set out to develop a versatile technique for phagosome isolation that is rapid and widely applicable to different pathogens. RESULTS: We developed two different protocols to isolate phagosomes containing dead or live bacteria modified with small magnetic particles, in conjunction with a synchronized phagocytosis protocol and nitrogen cavitation. For dead bacteria, we performed analysis of the phagosome samples by microscopy and immunoblot, and demonstrated the appearance of maturation markers on isolated phagosomes. CONCLUSION: We have presented detailed protocols for phagosome isolation, which can be adapted for use with different cell types and prey. The versatility and simplicity of the approach allow better control of phagosome isolation, the parameters of which are critical in studies of host-bacteria interaction and phagosome maturation.
Asunto(s)
Investigación Biomédica/métodos , Fraccionamiento Celular/métodos , Magnetismo , Fagosomas/microbiología , Técnicas Bacteriológicas , InmunofenotipificaciónRESUMEN
Proteinase 3 (PR3) is found in granules of all neutrophils but also on the plasma membrane of a subset of neutrophils (mPR3). CD177, another neutrophil protein, also displays a bimodal surface expression. In this study, we have investigated the coexpression of these two molecules, as well as the effect of cell activation on their surface expression. We can show that CD177 is expressed on the same subset of neutrophils as mPR3. Experiments show that the expression of mPR3 and CD177 on the plasma membrane is increased or decreased in parallel during cell stimulation or spontaneous apoptosis. Furthermore, we observed a rapid internalization and recirculation of mPR3 and plasma membrane CD177, where all mPR3 is replaced within 30 min. Our findings suggest that the PR3 found on the plasma membrane has its origin in the same intracellular storage as CD177, i.e., secondary granules and secretory vesicles and not primary granules. PR3- and CD177-expressing neutrophils constitute a subpopulation of neutrophils with an unknown role in the innate immune system, which may play an important role in diseases such as Wegener's granulomatosis and polycythemia vera.
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Membrana Celular/inmunología , Isoantígenos/biosíntesis , Glicoproteínas de Membrana/biosíntesis , Mieloblastina/biosíntesis , Neutrófilos/inmunología , Receptores de Superficie Celular/biosíntesis , Proteínas Ligadas a GPI , Humanos , Neutrófilos/citologíaRESUMEN
In this chapter, we describe methods to study membrane traffic during phagosome formation and maturation. Although it is convenient to define events as occurring either before (i.e., during formation) or after (during maturation) the creation of a sealed phagosome, it might not be correct to assume that this is reflected by a sudden change in the membrane traffic events involved. Nevertheless, formation events are studied by approaches different from those of maturation events. Before closure of a phagosome, methods relying on immunochemistry and fluorescent markers are employed. Once phagosomes have formed, these can be isolated and additional methods can be used for their characterization. Here, we describe (1) methods to study membrane traffic during phagosome formation and (2) magnetic purification of bacteria-containing phagosomes for maturation studies.
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Bioquímica/métodos , Membrana Celular/metabolismo , Neutrófilos/metabolismo , Fagocitosis/fisiología , Fagosomas/metabolismo , Transporte Biológico , Fraccionamiento Celular/métodos , Electroforesis en Gel de Poliacrilamida , Humanos , Magnetismo , Modelos Biológicos , Neutrófilos/fisiología , PinocitosisRESUMEN
Mature human neutrophils contain small amounts of interleukin-8 [CXC chemokine ligand 8 (CXCL-8)], which upon proinflammatory activation, increases significantly. It has been suggested that the CXCL-8 content of resting human neutrophils is stored in the secretory vesicles. Here, we have used a fractionation technique, which allows isolation of these vesicles, and we find that CXCL-8 neither colocalizes with the secretory vesicles nor with markers of any of the classical neutrophil granules. To increase resolution in the system, we induced CXCL-8 production by lipopolysaccharide. After 8 h of stimulation, CXCL-8 was visualized within the cell using immunoelectron microscopy. The images revealed CXCL-8-containing stuctures resembling neutrophil granules, and these were distinct from all known neutrophil organelles, as shown by double immunostaining. Further, the CXCL-8 organelle was present in nonstimulated neutrophil cytoplasts, entities lacking all other known granules and secretory vesicles. Upon fractionation of the cytoplasts, CXCL-8 was found to partly cofractionate with calnexin, a marker for endoplasmic reticulum (ER). Thus, part of CXCL-8 may be localized to the ER or ER-like structures in the neutrophil.
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Gránulos Citoplasmáticos/metabolismo , Retículo Endoplásmico/metabolismo , Interleucina-8/metabolismo , Neutrófilos/metabolismo , Orgánulos/metabolismo , Vesículas Secretoras/metabolismo , Biomarcadores/metabolismo , Calnexina/metabolismo , Células Cultivadas , Gránulos Citoplasmáticos/ultraestructura , Retículo Endoplásmico/ultraestructura , Histocitoquímica , Humanos , Interleucina-8/inmunología , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Lipopolisacáridos , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Neutrófilos/ultraestructura , Orgánulos/ultraestructura , Vesículas Secretoras/ultraestructura , Fracciones SubcelularesRESUMEN
In this experiment, we explored the potential of secretory lysosomes of hematopoietic cells to act as vehicles for immunomodulatory protein delivery at an inflammation site. We investigated whether exogenous soluble TNF-receptor 1 (sTNFR1) could be expressed in primary hematopoietic progenitor cells and become targeted for storage and secretion during granulopoietic differentiation. An sTNFR1 construct with a transmembrane domain (tm) and a cytosol sorting signal (Y) taken from CD63, was retrovirally transduced to lineage-negative murine hematopoietic bone marrow stem/progenitor cells. This process was followed by cytokine-driven granulopoietic maturation. The sTNFR1-tm-Y was found to be synthesized in precursor cells and to persist in mature granulocytes and monocytes/macrophages. Immunofluorescence-localization studies showed a granule pattern of sTNFR1-tm-Y in both precursor and mature granulocytes and secretion to phagosomes after ingestion of bacteria. Immunoelectron microscopy revealed co-localization between the sTNFR1-tm-Y and the primary (azurophil) granule marker myeloperoxidase. Collectively, our results demonstrated granule targeting, storage, and secretion of exogenous sTNFR1-tm-Y constitutively expressed during normal granulopoietic differentiation. These findings support the concept of using storage organelles of circulating hematopoietic cells as vehicles for targeting sites of inflammation with immunoregulatory agents.
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Células de la Médula Ósea/metabolismo , Diferenciación Celular/fisiología , Gránulos Citoplasmáticos/metabolismo , Granulocitos/citología , Inmunoglobulina G/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Animales , Células Cultivadas , Etanercept , Granulocitos/metabolismo , RatonesAsunto(s)
Proteínas Bacterianas/metabolismo , Fagocitosis/fisiología , Streptococcus pyogenes/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Leucocitos/inmunología , Leucocitos/metabolismo , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Streptococcus pyogenes/metabolismoRESUMEN
BACKGROUND: Death of smooth muscle cells in the atherosclerotic plaques makes the plaques more prone to rupture, which can initiate an acute ischemic event. The development of atherosclerosis includes the migration of immune cells e.g. monocytes/macrophages and T lymphocytes into the lesions. Immune cells can release antimicrobial peptides. One of these, human cathelicidin antimicrobial peptide hCAP-18, is cleaved by proteinase 3 generating a 4.5 kDa C-terminal fragment named LL-37, which has been shown to be cytotoxic. The aim of the study was to explore a potential role of LL-37 in the pathophysiology of atherosclerosis. METHODS: We investigated the presence of LL-37 in human atherosclerotic lesions obtained at autopsy using immunohistochemistry. The direct effects of LL-37 on cultured vascular smooth muscle cells and isolated neutrophil granulocytes were investigated with morphological, biochemical and flow cytometry analysis. RESULTS: The neointima of atherosclerotic plaques was found to contain LL-37-like immunoreactivity, mainly in macrophages. In cultured smooth muscle cells, LL-37 at 30 mug/ml caused cell shrinkage, membrane blebbing, nuclear condensation, DNA fragmentation and an increase in caspase-3 activity as studied by microscopy, ELISA and enzyme activity assay, respectively. Flow cytometry demonstrated that LL-37 in a subset of the cells caused a small but rapidly developing increase in membrane permeability to propidium iodide, followed by a gradual development of FITC-annexin V binding. Another cell population stained heavily with both propidium iodide and FITC-annexin V. Neutrophil granulocytes were resistant to these effects of LL-37. CONCLUSION: This study shows that LL-37 is present in atherosclerotic lesions and that it induces death of vascular smooth muscle cells. In a subset of cells, the changes indicate the development of apoptosis triggered by an initial mild perturbation of plasma membrane integrity. The findings suggest a role for LL-37 as a mediator of immune cell-induced death of vascular smooth muscle cells in atherosclerosis.
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Péptidos Catiónicos Antimicrobianos/análisis , Aorta/citología , Apoptosis/fisiología , Aterosclerosis/fisiopatología , Lipopolisacáridos/análisis , Músculo Liso Vascular/citología , Animales , Anexina A5 , Apoptosis/inmunología , Caspasa 3/metabolismo , Permeabilidad de la Membrana Celular/fisiología , Células Cultivadas , Fragmentación del ADN , Citometría de Flujo , Fluoresceína-5-Isotiocianato/análogos & derivados , Humanos , Inmunohistoquímica , L-Lactato Deshidrogenasa/metabolismo , Músculo Liso Vascular/metabolismo , Neutrófilos , Propidio , Ratas , CatelicidinasRESUMEN
During phagocytosis and phagosome maturation, complex membrane traffic events must be coordinated. We have observed, using fluorescent fluid-phase and membrane markers, that in the human neutrophil, internalization of nonopsonized, Gram-positive bacteria, but not of latex beads, is accompanied by a rapid and localized formation of pinosomal structures. This pinocytic response is calcium-dependent but insensitive to actin cytoskeleton disruption and wortmannin treatment. Contrary to what we observe, endosomal structures usually are considered to participate in phagosome formation by providing necessary membrane to forming phagosomes. Instead, our results show a coupling between neutrophil secretory and membrane-retrieval processes during phagosome maturation, and we suggest that the observed, localized pinocytic response is linked to the secretion of azurophilic granules toward nascent phagosomes. Accordingly, M and M-like protein-expressing Streptococcus pyogenes bacteria, which are able to survive inside neutrophil phagosomes, inhibit both the secretion of azurophilic granules to phagosomes and pinosome formation.
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Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Portadoras/inmunología , Membranas Intracelulares/inmunología , Neutrófilos/inmunología , Neutrófilos/microbiología , Fagocitosis/fisiología , Streptococcus pyogenes/inmunología , Antígenos Bacterianos/biosíntesis , Proteínas de la Membrana Bacteriana Externa/biosíntesis , Proteínas Portadoras/biosíntesis , Células Cultivadas , Gránulos Citoplasmáticos/inmunología , Regulación hacia Abajo/inmunología , Endocitosis/inmunología , Endosomas/inmunología , Humanos , Membranas Intracelulares/metabolismo , Modelos Inmunológicos , Fagocitosis/inmunología , Fagosomas/inmunología , Pinocitosis/inmunología , Transporte de Proteínas/inmunología , Streptococcus pyogenes/metabolismoRESUMEN
The present study demonstrates that the secretion of azurophilic granules occurring during Fc receptor-mediated attachment and spreading of neutrophils is highly localized to the adhering region of the cell. In contrast, the secretion of specific granules occurs in a nonpolarized way. This implies that unique signals are involved in the regulation of azurophilic degranulation. Assembly of actin filaments, as visualized by staining with rhodamine phalloidin, neither hindered nor facilitated degranulation. Further, the azurophilic secretory response remained localized in the presence of cytochalasin B. Release of azurophilic-granule content was inhibited by genistein and erbstatin, inhibitors of tyrosine kinases, and by GF109203X, a protein kinase C (PKC) inhibitor. We could also demonstrate a relative enrichment of syk tyrosine kinase and the PKC isoforms alpha and beta1 in adherent plasma membranes.
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Complejo Antígeno-Anticuerpo/fisiología , Degranulación de la Célula , Neutrófilos/fisiología , Actinas/metabolismo , Adhesión Celular , Citocalasina B/farmacología , Precursores Enzimáticos/análisis , Humanos , Péptidos y Proteínas de Señalización Intracelular , Isoenzimas/análisis , Proteína Quinasa C/análisis , Proteína Quinasa C/fisiología , Proteínas Tirosina Quinasas/análisis , Proteínas Tirosina Quinasas/fisiología , Quinasa SykRESUMEN
Hematopoietic cells contain secretory lysosomes that degranulate at sites of inflammation. We envisage that secretory granules can act as vehicles for targeting inflammatory sites, including malignancies, and thereafter, locally release therapeutically active agents to these sites. Exogenous proteins, such as the soluble tumor necrosis factor receptor 1 (sTNFR1), have been shown previously to be targeted to secretory lysosomes [1]. In this work, we asked whether exogenous, secretory lysosome-targeted proteins were subject to regulated secretion. sTNFR1-transmembrane (tm)-cytosol-sorting signal (Y) and sTNFR1-tm-Y-enhanced green fluorescent protein (egfp) were expressed in rat basophilic leukemia cell clones having different secretory capacities. sTNFR1-tm-Y was targeted directly from the Golgi to secretory lysosomes, followed by generation of membrane-free sTNFR1, whose secretion could be triggered by a Ca2+ ionophore or immunoglobulin E receptor activation. In contrast, sTNFR1-tm-Y-egfp was targeted to the plasma membrane and then subjected to endocytosis and presumably, secretory lysosome targeting, as judged by results from antibody ligation and cell-surface biotinylation. Activation of protein kinase C with phorbol ester promoted ectodomain shedding at the cell surface, resulting in sTNFR1 release from sTNFR1-tm-Y-egfp. These results support a concept for using the storage organelles of hematopoietic cells as vehicles for targeting sites of inflammation with therapeutically active agents.
Asunto(s)
Antígenos CD/metabolismo , Gránulos Citoplasmáticos/metabolismo , Exocitosis/fisiología , Leucemia Basofílica Aguda/metabolismo , Lisosomas/metabolismo , Procesamiento Proteico-Postraduccional , Receptores del Factor de Necrosis Tumoral/metabolismo , Animales , Antígenos CD/genética , Biotinilación , Membrana Celular/metabolismo , Endocitosis , Activación Enzimática/efectos de los fármacos , Aparato de Golgi/metabolismo , Proteínas Fluorescentes Verdes , Sistema Hematopoyético , Leucemia Basofílica Aguda/patología , Proteínas Luminiscentes/metabolismo , Ésteres del Forbol/farmacología , Proteína Quinasa C/metabolismo , Señales de Clasificación de Proteína , Transporte de Proteínas , Ratas , Receptores de IgE/metabolismo , Receptores del Factor de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral , Fracciones Subcelulares , Células Tumorales Cultivadas/metabolismoRESUMEN
Eosinophils participate in allergic inflammation, where expression of T helper cell type 2 (Th2) cytokines such as interleukin (IL)-4 and IL-5 are seen. However, eosinophils sometimes accumulate during disease with expression of Th1 cytokines [i.e., interferon-gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and IL-1beta]. In this study, we investigated whether eosinophils can respond with expression of the IFN-inducible C-X-C chemokines monokine induced by IFN-gamma [MIG; CXC chemokine ligand 9 (CXCL9)], IFN-gamma-inducible protein (IP-10/CXCL10), and IFN-inducible T cell alpha chemoattractant (I-TAC/CXCL11). These chemokines share the ability to recruit and activate T cells and natural killer cells to sites of inflammation. We found that IFN-gamma induced rapid and sustained gene expression of MIG, IP-10, and I-TAC in eosinophils, as detected by quantitative reverse transcriptase-polymerase chain reaction. During incubation, IFN-gamma-stimulated eosinophils released MIG and IP-10, as detected by enzyme-linked immunosorbent assay, while I-TAC could not be detected in the medium. TNF-alpha but not IL-1beta enhanced the IFN-gamma-induced production of MIG and IP-10. Conversely, addition of the Th2 cytokine IL-4 down-regulated IFN-gamma-induced synthesis of MIG and IP-10 in eosinophils. Crohn's disease is characterized by a Th1-polarized inflammation and presence of eosinophils. In lesions from this disease, MIG was detected in eosinophils by immunohistochemistry. Taken together, the results point to immunoregulatory roles for eosinophils during some diseases with Th1-polarized inflammation.
Asunto(s)
Quimiocinas CXC/inmunología , Quimiotaxis de Leucocito/inmunología , Eosinófilos/inmunología , Péptidos y Proteínas de Señalización Intercelular/inmunología , Interferón gamma/farmacología , Linfocitos T/inmunología , Línea Celular , Quimiocina CXCL10 , Quimiocina CXCL11 , Quimiocina CXCL9 , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Quimiotaxis de Leucocito/efectos de los fármacos , Enfermedad de Crohn/inmunología , Enfermedad de Crohn/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/inmunología , Sinergismo Farmacológico , Eosinófilos/efectos de los fármacos , Eosinófilos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Interferón gamma/inmunología , Interleucina-4/farmacología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Células TH1/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Secretory lysosomes of natural killer (NK) cells combine storage, regulated secretion and lysosomal activity. We asked whether one could target exogenous proteins to the secretory lysosomes of NK-cells for final delivery into a tumor site upon degranulation. cDNAs for both soluble and transmembrane (tm) proteins were expressed in the human YT-Indy NK-cell line. Targeting of a soluble TNF receptor (sTNFR1) was achieved by expressing a cDNA construct with a transmembrane sequence to facilitate ER-export and by incorporating a cytosolic sorting signal (Y) from CD63 to overcome constitutive secretion. The resulting sTNFR1-tm-Y was targeted to secretory lysosomes as confirmed by results from biosynthetic radiolabeling in combination with subcellular fractionation, immunoelectron microscopy, and immunofluorescence microscopy. A soluble sTNFR1 form was generated in the secretory lysosome by endogenous proteolytic activity. Expression of exogenous normally secretory non-membrane proteins, such as alpha1-microglobulin (alpha1-m) and alpha1-antitrypsin (alpha1-at) resulted mostly in constitutive secretion although a small amount of alpha1-microglobulin was targeted to secretory lysosomes. Our results suggest a potential for delivery of pharmacologically active agents into tumor sites by use of the NK-cell secretory lysosome as a carrier.
Asunto(s)
Células Asesinas Naturales/metabolismo , Lisosomas/metabolismo , Proteínas/metabolismo , Animales , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/ultraestructura , Microscopía Electrónica , Transporte de Proteínas/fisiologíaRESUMEN
Cytokines play an important role in the regulation of homeostasis and inflammation. A de-regulated cytokine function can subsequently promote chronic inflammation. This is supported by clinical evidence showing the beneficial effect of inhibiting TNF-alpha through injection of antibodies and soluble receptor in disorders such as rheumatoid arthritis and Crohn's disease. Systemic anti-TNF-alpha therapy however is associated with infectious complications. We therefore suggest a concept for the local deposition of therapeutically active agents into areas of inflammation or malignancy, based on the use of hematopoietic storage and secretory granules as delivery vehicles. Hematopoietic cells are induced to express the therapeutically active protein and to store it in the secretory lysosomes. The cells migrate into a tumour or site of inflammation, where the cells become activated and release the contents of their secretory lysosomes resulting in the local delivery of the therapeutically active protein. In support of this concept, gene transfer and granule loading can be achieved using the soluble TNF-alpha receptor (sTNFR1) after cDNA expression in hematopoietic cell lines. Endoplasmic reticulum (ER)-export can be facilitated by the addition of a transmembrane domain, and constitutive secretion can be prevented by incorporating a cytosol-sorting signal resulting in secretory lysosome targeting. The sTNFR1 is released from the transmembrane domain by proteolytic cleavage and finally, regulated sTNFR1-secretion can be triggered by a calcium signal. In vivo investigations are currently determining the feasibility of local protein delivery at sites of inflammation.
Asunto(s)
Células de la Médula Ósea/ultraestructura , Citocinas/administración & dosificación , Inflamación/metabolismo , Receptores de Citocinas/administración & dosificación , Vesículas Secretoras , Animales , HumanosRESUMEN
Streptococcus pyogenes, a significant bacterial pathogen in humans, interferes with the membrane traffic of human neutrophils and survives following phagocytosis. The mechanism(s) behind this property is not known, but in contrast to wild-type bacteria, mutant bacteria lacking virulence factors regulated by the transcriptional regulator Mga, are phagocytosed and killed. In the present work we investigated whether differences in phagosomal acidification may contribute to this difference. Phagosomal pH in neutrophil-differentiated HL-60 cells was studied by fluorescence ratio imaging, and phagosomes containing wild-type S. pyogenes bacteria of the M1 serotype exhibited little or no acidification, whereas Mga mutant bacteria were found in more acidic phagosomes. With phagosomes containing these bacteria, proton delivery was inhibited by adding folimycin, a vacuolar-type adenosine triphosphatase (V-ATPase) inhibitor. This inhibitor had no effect on phagosomes containing wild-type bacteria, indicating either inactivation or removal of V-ATPases by the bacteria. Analysis of isolated bacteria-containing phagosomes confirmed the latter scenario and showed a more efficient delivery of V-ATPases to phagosomes containing Mga mutant bacteria. The results demonstrate that V-ATPase-mediated phagosomal proton delivery is reduced during phagocytosis of wild-type S. pyogenes, leading to impaired acidification, and that surface proteins of the mga regulon are responsible for this effect.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/metabolismo , Fagocitosis/fisiología , Fagosomas/microbiología , Streptococcus pyogenes/patogenicidad , ATPasas de Translocación de Protón Vacuolares/metabolismo , Proteínas Bacterianas/química , Células HL-60 , Humanos , Concentración de Iones de Hidrógeno , Macrólidos/farmacología , Proteínas de la Membrana/química , Microscopía Fluorescente , Neutrófilos/metabolismo , Neutrófilos/microbiología , Fagocitosis/efectos de los fármacos , Fagosomas/metabolismo , Estadísticas no Paramétricas , Streptococcus pyogenes/química , Streptococcus pyogenes/metabolismo , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidoresRESUMEN
The neutrophil is a key player in immunity, and its activities are essential for the resolution of infections. Neutrophil-pathogen interactions usually trigger a large arsenal of antimicrobial measures that leads to the highly efficient killing of pathogens. In neutrophils, the phagocytic process, including the formation and maturation of the phagosome, is in many respects very different from that in other phagocytes. Although the complex mechanisms that coordinate the membrane traffic, oxidative burst, and release of granule contents required for the microbicidal activities of neutrophils are not completely understood, it is evident that they are unique and differ from those in macrophages. Neutrophils exhibit more rapid rates of phagocytosis and higher intensity of oxidative respiratory response than do macrophages. The phagosome maturation pathway in macrophages, which is linked to the endocytic pathway, is replaced in neutrophils by the rapid delivery of preformed granules to nonacidic phagosomes. This review describes the plasticity and dynamics of the phagocytic process with a special focus on neutrophil phagosome maturation.
Asunto(s)
Neutrófilos/inmunología , Fagocitosis/inmunología , Fagosomas/inmunología , Humanos , InmunidadRESUMEN
During phagocytosis, neutrophils kill microorganisms by delivering antimicrobial substances to the phagosome. For this, the intracellular targeting and fusion of granules must be strictly regulated and a dependence on the cytosolic concentration of free calcium has been suggested. New evidence show that different mechanisms regulate early and late stages of Fc receptor-mediated phagocytosis. The early fusion events are dependent on calcium but this is not the case for the fusion of azurophilic granules with phagosomes at later stages. Certain pathogens target the granule-phagosome fusion machinery in order to survive intracellularly; a deeper understanding of intracellular membrane traffic processes could allow new approaches for the eradication of pathogens that are harbored inside the cells of our immune system.
RESUMEN
BACKGROUND: New experimental approaches to the study of the neutrophil phagosome and bacterial killing prompted a reassessment of the usefulness of all-trans retinoic acid (ATRA)-differentiated HL-60 cells as a neutrophil model. HL-60 cells are special in that they possess azurophilic granules while lacking the specific granules with their associated oxidase components. The resulting inability to mount an effective intracellular respiratory burst makes these cells more dependent on other mechanisms when killing internalized bacteria. METHODOLOGY/PRINCIPAL FINDINGS: In this work phagocytosis and phagosome-related responses of ATRA-differentiated HL-60 cells were compared to those earlier described in human neutrophils. We show that intracellular survival of wild-type S. pyogenes bacteria in HL-60 cells is accompanied by inhibition of azurophilic granule-phagosome fusion. A mutant S. pyogenes bacterium, deficient in M-protein expression, is, on the other hand, rapidly killed in phagosomes that avidly fuse with azurophilic granules. CONCLUSIONS/SIGNIFICANCE: The current data extend our previous findings by showing that a system lacking in oxidase involvement also indicates a link between inhibition of azurophilic granule fusion and the intraphagosomal fate of S. pyogenes bacteria. We propose that differentiated HL-60 cells can be a useful tool to study certain aspects of neutrophil phagosome maturation, such as azurophilic granule fusion.