RESUMEN
Scavenger receptors have a broad range of functions that include pathogen clearance, and identification of the scavenger receptor family has been of great benefit to the field of physiology. The shuttling-protein nucleolin has recently been shown to possess scavenger receptor-like activity. We therefore investigated whether or not nucleolin is a receptor for maleylated-bovine serum albumin (maleylated-BSA), which is a common ligand for scavenger receptors. Binding and phagocytosis of native control-BSA by thioglycollate-elicited mouse peritoneal macrophages was weak, but that of maleylated-BSA was strong. Surface plasmon-resonance analysis revealed that nucleolin strongly associated with maleylated-BSA but not control-BSA or maleic anhydride. Further, co-treatment of macrophages with anti-nucleolin antibody, but not control-immunoglobulin G, inhibited binding of maleylated-BSA. In addition, antineoplastic guanine rich oligonucleotide (AGRO), a nucleolin-specific oligonucleotide aptamer, inhibited binding of maleylated-BSA. Further, binding of maleylated-BSA to nucleolin-transfected HEK293 cells was higher than that by control HEK cells. These results indicate that nucleolin is a receptor that enables macrophages to recognize maleylated-BSA.
Asunto(s)
Macrófagos Peritoneales/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Albúmina Sérica Bovina/metabolismo , Animales , Células HEK293 , Humanos , Macrófagos Peritoneales/fisiología , Masculino , Ratones , Fagocitosis , Fosfoproteínas/genética , Proteínas de Unión al ARN/genética , NucleolinaRESUMEN
BACKGROUND: Photodynamic therapy (PDT) induces selective cell death of neoplastic tissue and connecting vasculature by combining photosensitizers with light. We have previously reported that PDT induces apoptotic cell death in glioma cells when the photosensitizer talaporfin sodium (NPe6) is used. Here, we investigated the combined effect of NPe6-PDT with temozolomide, a DNA-alkylating drug used in glioma therapy. METHODS: Human glioblastoma T98G cells and human glioma U251 cells were used as glioma cells. Cell viability was evaluated by WST-8 assay. Apoptosis was evaluated by measurement of caspase-3 activity and DNA-fragmentation. Intracellular reactive oxygen species were evaluated by dihydrorhodamine assay. RESULTS: While the degree of NPe6-PDT induced cell death unchanged in T98G and U251 cells when temozolomide treatment was adjuvant, it was dose-dependently increased by concomitant treatment with temozolomide. Further, concomitantly administered temozolomide dose-dependently increased caspase-3 activity and DNA-fragmentation, while adjuvant-temozolomide did not. These results are suggesting that concomitantly administered temozolomide potentiates the effect of NPe6-PDT to facilitate apoptotic cell death. Additionally, concomitantly administered temozolomide increased intracellular NPe6-fluorescence and reactive oxygen species, suggesting that the augmentation effect of combined treatment may be due to increased intracellular accumulation of NPe6. CONCLUSION: These results suggest that concomitant treatment with NPe6-PDT and temozolomide is a potentially useful therapy for glioma.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Dacarbazina/análogos & derivados , Glioma/tratamiento farmacológico , Fotoquimioterapia/métodos , Porfirinas/administración & dosificación , Antineoplásicos Alquilantes/administración & dosificación , Apoptosis/efectos de la radiación , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Dacarbazina/administración & dosificación , Quimioterapia Combinada , Glioma/patología , Humanos , Fármacos Fotosensibilizantes/administración & dosificación , Temozolomida , Resultado del TratamientoRESUMEN
Advanced glycosylation end-products (AGEs) are non-enzymatically glycosylated proteins that play an important role in several diseases and aging processes, including angiopathy, renal failure, diabetic complications, and some neurodegenerative diseases. In particular, glyceraldehyde (GCA)- and glycolaldehyde (GOA)-derived AGEs are deemed toxic AGEs, due to their cytotoxicity. Recently, the shuttling-protein nucleolin has been shown to possess scavenger receptor-activity. Here, we investigated whether or not macrophages recognize toxic AGEs through nucleolin receptors expressed on their surface. Free amino acid groups and arginine residues found in bovine serum albumin (BSA) were time-dependently modified by incubation with GCA and GOA. In addition, average molecular size was increased by incubation with GCA and GOA. While GCA-treated BSA (GCA-BSA) and GOA-treated BSA (GOA-BSA) were recognized by thioglycollate-elicited mouse peritoneal macrophages in proportion to their respective aldehyde-modification ratios, aldehyde-untreated control-BSA was not. Surface plasmon-resonance analysis revealed that nucleolin strongly associated with GCA-BSA and GOA-BSA, but not with control-BSA. Further, pretreating macrophages with anti-nucleolin antibody, but not control-Immunoglobulin G, inhibited recognition of GCA-BSA and GOA-BSA by macrophages. Additionally, AGRO, a nucleolin-specific oligonucleotide aptamer, inhibited recognition of GCA-BSA and GOA-BSA. Moreover, nucleolin-transfected HEK293 cells recognized more GCA-BSA and GOA-BSA than control HEK cells did. Binding of nucleolin and GCA-BSA/GOA-BSA was also blocked by anti-nucleolin antibody at molecular level. These results indicate that nucleolin is a receptor that allows macrophages to recognize toxic AGEs.
Asunto(s)
Productos Finales de Glicación Avanzada/metabolismo , Macrófagos/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Acetaldehído/análogos & derivados , Acetaldehído/metabolismo , Animales , Aptámeros de Nucleótidos , Bovinos , Gliceraldehído/metabolismo , Células HEK293 , Humanos , Inmunoglobulina G/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Masculino , Ratones , Oligodesoxirribonucleótidos/farmacología , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Albúmina Sérica Bovina/metabolismo , Transfección , NucleolinaRESUMEN
Amyloid-beta peptide 1-42 (Aß42) plays a key role in the neurotoxicity found in Alzheimer's disease. Mononuclear phagocytes in the brain (microglia), can potentially clear Aß via phagocytosis. Recently, the shuttling-protein nucleolin has been shown to possess scavenger receptor-activity. Here, we investigated whether this receptor interacts specifically with Aß type 1-42 and mediates its phagocytosis by microglia. While monomeric and fibril Aß42 were phagocytosed by mouse microglial EOC2 cells, amyloid ß peptide 1-40 (Aß40) was only weakly phagocytosed. Surface plasmon-resonance analysis revealed that nucleolin strongly associates with Aß42, but only weakly associates with Aß40. Immunofluorescence staining of anti-nucleolin antibody revealed that EOC2 cells and rat primary microglia express nucleolin on their cell surfaces. Further, pretreating EOC2 cells with anti-nucleolin antibody, but not immunoglobulin G (IgG), inhibited phagocytosis of monomeric Aß42 by microglia. Additionally, nucleolin-transfected HEK293 cells phagocytosed monomeric and fibril Aß42 but not monomeric and fibril Aß40. Moreover, AGRO, a nucleolin-specific oligonucleotide aptamer, inhibited phagocytosis of monomeric and fibril Aß42, but not monomeric and fibril Aß40. These results indicate that nucleolin is a receptor that allows microglia to recognize monomeric and fibril Aß42.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Microglía/metabolismo , Sistema Mononuclear Fagocítico , Fagocitosis , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Enfermedad de Alzheimer/inmunología , Animales , Línea Celular , Células HEK293 , Humanos , Ratones , Ratas , NucleolinaRESUMEN
OBJECTIVE: To investigate the kinetics of cell death in human glioma cell lines induced by photodynamic therapy (PDT) with the second-generation photosensitizer talaporfin sodium (TS) and a 664-nm diode laser. MATERIALS AND METHODS: Three human glioma cell lines (T98G, A172, U251) were studied. After incubation of the cell lines with various concentrations of TS for 4 h, PDT using diode laser irradiation at 33 mW/cm² and 10 J/cm² was performed. Cell viability and changes in cell morphology were examined by the Cell Counting Kit-8 assay and phase-contrast microscopy, respectively. In addition, to evaluate the pathology of cell death, changes in cell viability after treatment with a caspase activation inhibitor and an autophagy inhibitor were also examined. RESULTS: In all 3 human glioma cell lines, TS induced dose-dependent cell death. However, the 50% lethal dose of TS varied among these cell lines. The main morphological feature of cell death was shrinkage of the cell body, and the number of cells with this morphological change increased in a time-dependent manner, resulting in cell death. In addition, a dose-dependent improvement in cell viability by the caspase inhibitor Z-VAD-fmk was observed. CONCLUSION: PDT with TS induces dose-dependent apoptosis in human glioma cell lines. However, the sensitivity to PDT varied among the cell lines, indicating a possible difference in the intracellular content of TS, or a difference in the susceptibility to the intracellular oxidative stress caused by PDT.
Asunto(s)
Apoptosis/efectos de los fármacos , Glioma/tratamiento farmacológico , Glioma/patología , Fotoquimioterapia/métodos , Porfirinas/administración & dosificación , Apoptosis/efectos de la radiación , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Fármacos Sensibilizantes a Radiaciones/administración & dosificación , Resultado del TratamientoRESUMEN
Macrophages remove deteriorating cells (those undergoing apoptosis and oxidation) via poly-N-acetyllactosaminyl chains on CD43 caps, a major cell-surface glycoprotein. Unusually high intracellular calcium levels are also deteriorating for cells and tissue. Here we artificially elevated calcium levels in cells and examined the mechanism by which this elevation was resolved by macrophages. Results showed that treatment with the calcium ionophore A23187 and ionomycin induces capping of CD43 on Jurkat cells, which are subsequently recognized and phagocytosed by macrophages, indicating that macrophages regard cells with elevated calcium as targets for removal. Further tests showed that A23187- and ionomycin-treated Jurkat cells did not induce apoptotic changes such as DNA fragmentation or phosphatidylserine expression, indicating that these cells were removed despite still being viable. Jurkat cells pretreated with anti-CD43 antibody or those with poly-N-acetyllactosaminyl chains containing oligosaccharides inhibited macrophage binding, indicating that macrophages recognize the poly-N-acetyllactosaminyl chains on CD43. Binding was also inhibited by treating macrophages with anti-nucleolin antibody, indicating that recognition occurs through nucleolin, a cell-surface receptor. Further, nucleolin-transfected HEK293 cells bound A23187-treated cells, and this binding was inhibited by in the presence of oligosaccharides. Taken together, these results show that elevated calcium levels induce CD43 capping, and macrophages remove the cells if their nucleolin receptors can bind to the poly-N-acetyllactosaminyl chains of capped CD43.
Asunto(s)
Apoptosis , Calcio/farmacología , Leucosialina/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/genética , Calcimicina/farmacología , Calcio/metabolismo , Carbohidratos/química , Fragmentación del ADN/efectos de los fármacos , Células HEK293 , Humanos , Ionomicina/farmacología , Células Jurkat , Leucosialina/química , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Oligosacáridos/metabolismo , Unión Proteica/efectos de los fármacos , NucleolinaRESUMEN
Photodynamic therapy (PDT) induces selective cell death of neoplastic tissue and connecting vasculature by combining photosensitizers with light. Here we clarified the types of cell death induced by PDT in combination with the photosensitizer talaporfin sodium (mono-L-aspartyl chlorine e6, NPe6) in order to evaluate the potential of this therapy as a treatment for glioma. PDT with NPe6 (NPe6-PDT) induces dose-dependent cell death in human glioblastoma T98G cells. Specifically, cell death modalities were observed in NPe6-PDT treated T98G cells, including signs of apoptosis (activation of caspase-3, expression of phosphatidylserine, and DNA fragmentation) and necrosis (stainability of propidium iodide). In addition, high doses of NPe6-PDT decreased the proportion of apoptotic cell death, while increasing necrosis. Closer examination of apoptotic characteristics revealed release of cytochrome-c from mitochondria as well as activation of both caspse-9 and caspase-3 in cells treated with low doses of NPe6-PDT. Benziloxycarbonyl-Leu-Gln(OMe)-His-Asp(OMe)-fluoromethyl-ketone (Z-LEHD-fmk), a caspase-9 specific inhibitor, and benziloxycarbonyl-Asp(OMe)-Gln-Met-Asp(OMe)-fluoromethyl-ketone (Z-DQMD-fmk), a caspase-3 specific inhibitor, showed dose-dependent prevention of cell death in NPe6-PDT treated cells, indicating that mitochondrial apoptotic pathway was a factor in the observed cell death. Further, the cell morphology was observed after PDT. Time- and NPe6-dose dependent necrotic features were increased in NPe6-PDT treated cells. These results suggest that NPe6-PDT could be an effective treatment for glioma if used in mild doses to avoid the increased necrosis that may induce undesirable obstacles.
Asunto(s)
Antineoplásicos/administración & dosificación , Neoplasias Encefálicas/tratamiento farmacológico , Glioma/tratamiento farmacológico , Fotoquimioterapia , Fármacos Fotosensibilizantes/administración & dosificación , Porfirinas/administración & dosificación , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Citocromos c/metabolismo , Fragmentación del ADN , Glioma/metabolismo , Glioma/patología , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Necrosis/inducido químicamente , Necrosis/metabolismo , Necrosis/patologíaRESUMEN
The mechanism was investigated for macrophage recognition of cells oxidized by diamide, a thiol group-specific oxidizing reagent. Jurkat cells exposed to various concentrations of diamide were recognized by macrophages, the cells exposed to 25 µM diamide being best recognized. CD43, a major glycoprotein on the Jurkat cell surface, tended to form clusters upon diamide oxidization, and pretreating Jurkat cells with the anti-CD43 antibody inhibited macrophage binding. This indicates that macrophages appeared to recognize CD43. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and a Western blot analysis of CD43 of the diamide-oxidized cells showed no increase in the amount of cross-linked CD43 compared with control cells, indicating that cross-linking of CD43 by a disulphide bond was not involved in the clustering. Both CD43 clustering and binding of the oxidized cells to macrophages was prevented by the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD-fmk), suggesting that the oxidized and macrophage-bound cells were undergoing apoptosis. A closer examination revealed that the caspase-3 activity, chromatin condensation, and DNA fragmentation in Jurkat cells were all increased by oxidation. The macrophage receptor involved in the binding appeared to be the cell-surface protein, nucleolin; an anti-nucleolin antibody treatment inhibited the binding. These results suggest that thiol group-oxidized cells underwent early apoptosis and were recognized by nucleolin on macrophages as early apoptotic cells.
Asunto(s)
Apoptosis , Metabolismo de los Hidratos de Carbono , Macrófagos/citología , Macrófagos/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Apoptosis/efectos de los fármacos , Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Diamida/farmacología , Humanos , Células Jurkat , Ligandos , Macrófagos/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacos , Unión Proteica/efectos de los fármacos , NucleolinaRESUMEN
Apoptotic cells must be recognized early for phagocytosis to ensure that their toxic contents do not damage neighboring cells. In some cases this is achieved via CD43-capped membrane glycoproteins, the sialylpolylactosaminyl chains of which serve as ligands for phagocytosis by macrophages. However, because many additional changes occur during apoptosis, determining exactly which events are responsible for signaling macrophages to initiate phagocytosis remains a challenge. Here, we examined one clearance mechanism in detail and determined that capping of CD43 alone is sufficient to initiate phagocytosis. We induced macrophage-mediated phagocytosis by using cytochalasin B to artificially cap CD43 on healthy (non-apoptotic) Jurkat cells. Additional experiments confirmed that sialylpolylactosaminyl chains formed through this capping method are a prerequisite for removal, and that nucleolin is the macrophage receptor responsible for their detection. These findings strongly suggest that capping of CD43 presents a sufficient signal for phagocytosis without any additional membrane changes.
Asunto(s)
Citocalasina B/farmacología , Leucosialina/inmunología , Macrófagos/inmunología , Fagocitosis/inmunología , Apoptosis , Células HEK293 , Humanos , Células Jurkat , Fosfoproteínas/inmunología , Proteínas de Unión al ARN/inmunología , NucleolinaRESUMEN
Nucleolin is an abundant non-ribosomal protein found in nucleolus and a major component of silver-stained nucleolar organizer region (AgNOR), a histopathological marker of cancer which is highly elevated in cancer cells. We recently reported that nucleolin on the cell surface of mouse gastric cancer cells acts as a receptor for tumor necrosis factor-alpha-inducing protein (Tipalpha), a new carcinogenic factor of Helicobacter pylori. In this study, we first examined the localization of nucleolin on cell surface of five gastric cancer cell lines by cell fractionation and flow cytometry: We found that large amounts of nucleolin were present on surface of MKN-45, KATOIII, MKN-74, and AGS cells, with smaller amounts on surface of MKN-1 cells. The membrane fraction of normal epithelial cells of mouse glandular stomach did not contain much nucleolin, suggesting that translocation of nucleolin to the cell surface occurs during carcinogenesis, making for easier binding with Tipalpha. AS1411, a nucleolin targeted DNA aptamer, inhibited growth of gastric cancer cell lines in this order of potency: MKN-45>KATOIII>AGS>MKN-74=MKN-1, associated with induction of S-phase cell cycle arrest. Fluorescein isothiocyanate (FITC)-AS1411 was more rapidly incorporated into MKN-45 and AGS than into MKN-1 cells, based on varying amounts of cell surface nucleolin. We think that AS1411 first binds to nucleolin on the cell surface and that the binding complex is then incorporated into the cells. All results indicate that nucleolin on the cell surface is a new and promising therapeutic target for treatment of gastric cancer.
Asunto(s)
Antineoplásicos/farmacología , Membrana Celular/metabolismo , Células Epiteliales/metabolismo , Mucosa Gástrica/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptores de Superficie Celular/metabolismo , Neoplasias Gástricas/metabolismo , Animales , Antineoplásicos/uso terapéutico , Aptámeros de Nucleótidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Transporte Biológico , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/microbiología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/microbiología , Fluoresceína/metabolismo , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/microbiología , Helicobacter pylori/metabolismo , Helicobacter pylori/patogenicidad , Humanos , Isotiocianatos/metabolismo , Ratones , Ratones Endogámicos BALB C , Oligodesoxirribonucleótidos/farmacología , Fase S/efectos de los fármacos , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/microbiología , NucleolinaRESUMEN
PURPOSE: Tumor necrosis factor-alpha inducing protein (Tipalpha) is a unique carcinogenic factor released from Helicobacter pylori (H. pylori). Tipalpha specifically binds to cells and is incorporated into cytosol and nucleus, where it strongly induces expression of TNF-alpha and chemokine genes mediated through NF-kappaB activation, resulting in tumor development. To elucidate mechanism of action of Tipalpha, we studied a binding protein of Tipalpha in gastric epithelial cells. METHODS: Tipalpha binding protein was found in cell lysates of mouse gastric cancer cell line MGT-40 by FLAG-pull down assay and identified to be cell surface nucleolin by flow cytometry using anti-nucleolin antibody. Incorporation of Tipalpha into the cells was determined by Western blotting and expression of TNF-alpha gene was quantified by RT-PCR. RESULTS: Nucleolin was co-precipitated with Tipalpha-FLAG, but not with del-Tipalpha-FLAG (an inactive mutant). After treatment with Tipalpha-FLAG, incorporated Tipalpha was co-immunoprecipitated with endogenous nucleolin using anti-nucleolin antibody. The direct binding of Tipalpha to recombinant His-tagged nucleolin fragment (284-710) was also confirmed. Although nucleolin is an abundant non-ribosomal protein of the nucleolus, we found that nucleolin is present on the cell surface of MGT-40 cells. Pretreatment with anti-nucleolin antibody enhanced Tipalpha-incorporation into the cells through nucleolin internalization. In addition, pretreatment with tunicamycin, an inhibitor of N-glycosylation, decreased the amounts of cell surface nucleolin and inhibited both internalization of Tipalpha and expression of TNF-alpha gene. CONCLUSIONS: All the results indicate that nucleolin acts as a receptor for Tipalpha and shuttles Tipalpha from cell surface to cytosol and nuclei. These findings provide a new mechanistic insight into gastric cancer development with Tipalpha.
Asunto(s)
Proteínas Bacterianas/metabolismo , Carcinógenos/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Factor de Necrosis Tumoral alfa/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Cromatografía Liquida , Regulación hacia Abajo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Inmunoprecipitación , Espectrometría de Masas , Ratones , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Receptores de Superficie Celular/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/genética , NucleolinaRESUMEN
The mechanism of macrophage recognition of oxidatively damaged cells was investigated. Jurkat T cells exposed to various concentrations of H(2)O(2) were bound and phagocytosed by macrophages. The cells exposed to 0.1 mM H(2)O(2) were best bound. The cell-surface ligands recognized by macrophages were suggested to be sialylpolylactosaminyl sugar chains of a major sialoglycoprotein CD43 because 1) the cell binding was inhibited by oligosaccharides containing sialylpolylactosaminyl chains, and their inhibitory activity was destroyed by a polylactosamine-cleaving enzyme endo-beta-galactosidase, and by neuraminidase; 2) the oxidized Jurkat cells pretreated with either glycosidase or with anti-CD43 antibody were not bound. The macrophage receptor involved in the binding was suggested to be cell-surface nucleolin because 1) anti-nucleolin antibody inhibited the binding; 2) nucleolin-transfected HEK293 cells bound the oxidized cells; and 3) this binding was inhibited by anti-nucleolin antibody and by anti-CD43 antibody. CD43 on oxidized Jurkat cells tended to form clusters in good accordance with their susceptibility to the macrophage binding. CD43 clustering and the oxidized-cell binding to macrophages were prevented by a caspase inhibitor Z-VAD-fmk, suggesting that the oxidized and bound cells were undergoing apoptosis. Indeed, caspase-3 activity of Jurkat cells increased by the oxidation. These results suggest that moderately oxidized cells undergo apoptosis and are recognized by macrophages as early apoptotic cells.
Asunto(s)
Apoptosis/fisiología , Glicoproteínas/química , Macrófagos/fisiología , Estrés Oxidativo/fisiología , Fosfoproteínas/química , Proteínas de Unión al ARN/química , Caspasas/metabolismo , Línea Celular , Núcleo Celular/fisiología , Núcleo Celular/ultraestructura , Citosol/fisiología , Citosol/ultraestructura , Humanos , Células Jurkat , Leucosialina/química , Proteínas de la Membrana/química , Oxidación-Reducción , NucleolinaRESUMEN
Human erythrocytes exposed to appropriate concentrations of H(2)O(2) for 1h became susceptible to the binding and phagocytosis by macrophages. The binding was inhibited by anti-band 3 serum and prevented by pretreatment of erythrocytes with a polylactosamine-cleaving enzyme endo-beta-galactosidase, indicating that polylactosaminyl sugar chains of band 3 are recognized by macrophages. The macrophage receptor involved was suggested to be nucleolin, a recently identified macrophage surface protein recognizing sialylpolylactosaminyl-chain clusters on early apoptotic cells, because anti-nucleolin antibody and a soluble form of recombinant nucleolin blocked the recognition. Treatment of erythrocytes with caspase inhibitors Z-VAD-fmk or Z-DQMD-fmk (caspase 3 selective) before the oxidation resulted in lowered binding of the oxidized erythrocytes to macrophages, suggesting that actions of caspases, particularly those of caspase 3, are prerequisite for the membrane changes leading to band 3 aggregation. Moreover, the cytosolic caspase 3 was found to be activated by H(2)O(2), and the extent of the activation correlated well with the susceptibility of the oxidized erythrocytes to the macrophage recognition. These results suggest that oxidative stress renders the erythrocytes susceptible to clearance by macrophages through activation of caspases leading to band 3 aggregation.
Asunto(s)
Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Caspasas/metabolismo , Eritrocitos/fisiología , Peróxido de Hidrógeno/administración & dosificación , Macrófagos/fisiología , Fagocitosis/fisiología , Transducción de Señal/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Eritrocitos/efectos de los fármacos , Humanos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Tasa de Depuración Metabólica/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Fagocitosis/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Early apoptotic Jurkat T cells undergo capping of CD43, and its polylactosaminyl saccharide chains serve as ligands for phagocytosis by macrophages. This suggests the presence of a polylactosaminoglycan-binding receptor on macrophages. Here we show that this receptor is nucleolin, a multifunctional shuttling protein present in nucleus, cytoplasm, and on the surface of some types of cells. Nucleolin was detected at the surface of macrophages, and anti-nucleolin antibody inhibited the binding of the early apoptotic cells to macrophages. Nucleolin-transfected HEK293 cells expressed nucleolin on the cell surface and bound the early apoptotic cells but not phosphatidylserine-exposing late apoptotic cells. This binding was inhibited by anti-nucleolin antibody, by polylactosamine-containing oligosaccharides, and by anti-CD43 antibody. Deletion of the antibody binding region of nucleolin resulted in loss of the apoptotic cell-binding ability. Moreover, truncated recombinant nucleolin in solution containing this region blocked the apoptotic cell binding to macrophages, and the blocking effect was cancelled by the oligosaccharides. These results indicate that nucleolin is a macrophage receptor for apoptotic cells.
Asunto(s)
Apoptosis/fisiología , Macrófagos/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , ADN Complementario/genética , Humanos , Técnicas In Vitro , Células Jurkat , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Peso Molecular , Monocitos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , NucleolinaRESUMEN
At an early stage of apoptosis, Jurkat cells transiently become susceptible to binding and phagocytosis by macrophages through the polylactosamine-type carbohydrate chains of CD43 [J. Biol. Chem. 279 (2004) 5967]. Susceptibility of apoptotic Jurkat cells to macrophage recognition was studied over an extended time range of 0-24 h including a later stage. Jurkat cells incubated with appropriate concentrations of apoptosis-inducing agents etoposide or anti-Fas antibody became susceptible to macrophage-binding at 2 h, and the susceptibility fell to the control level at 4 or 6 h. However, it increased again at later hours (6-24 h). Flow cytometric analyses of CD43 and phosphatidylserine (PS) on the apoptotic cells indicated that CD43 began to degrade at around 4 h, and PS is externalized significantly at 4 or 6 h. The macrophage-binding at 2 h was prevented by glycosidase treatment of Jurkat cells, but not by annexin V. Conversely, the later binding at 12 or 18 h was not prevented by glycosidase treatment, but was done so by annexin V. These results suggest that Jurkat cells become susceptible to phagocytic removal at an early stage of apoptosis by the carbohydrate-mediated mechanism, and at a later stage by the PS-mediated mechanism.
Asunto(s)
Antígenos CD/metabolismo , Apoptosis/fisiología , Metabolismo de los Hidratos de Carbono , Macrófagos/fisiología , Fagocitosis/fisiología , Fosfatidilserinas/metabolismo , Sialoglicoproteínas/metabolismo , Transducción de Señal/fisiología , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales de Origen Murino , Apoptosis/efectos de los fármacos , Células Cultivadas , Etopósido/farmacología , Humanos , Células Jurkat , Leucosialina , Macrófagos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
A novel mechanism of phagocytic recognition of apoptotic cells was found and characterized. Jurkat cells incubated with appropriate concentrations of etoposide or anti-Fas antibody transiently became susceptible to binding and phagocytosis by THP-1 cell-derived macrophages at 2 h. The bound Jurkat cells showed no chromatin condensation, but the binding was prevented by a caspase inhibitor, indicating that they were recognized at an early stage of apoptosis. The ligands recognized on the apoptotic cells were sialylpolylactosaminyl sugar chains because 1) the binding was inhibited by an oligosaccharide preparation of erythrocyte membrane, and its inhibitory activity was destroyed by polylactosaminoglycan-specific endo-beta-galactosidase or neuraminidase; 2) Jurkat cells pretreated with endo-beta-galactosidase or neuraminidase failed to be recognized; and 3) treatment of the apoptotic cells with polylactosaminoglycan-binding Datura stramonium agglutinin prevented recognition. The sialylpolylactosaminyl chains involved were most likely those of a major sialoglycoprotein CD43 because anti-CD43 antibody inhibited recognition. CD43 on apoptotic Jurkat cells was found to form a cap at 2 h, and the cap disappeared at 4 h. This transient capping of CD43 coincided with the transient increase in the susceptibility of the cells to macrophage recognition, suggesting that CD43 capping is responsible for generation of the carbohydrate ligands for recognition. Furthermore, microscopic observation suggested that the apoptotic cells were recognized at the CD43 cap. Taken together, we conclude that apoptotic Jurkat cells transiently undergo CD43 capping at an early stage of apoptosis and are recognized by macrophages through the cluster of sialylpolylactosaminyl chains of the capped CD43.
Asunto(s)
Antígenos CD , Apoptosis , Carbohidratos/química , Sialoglicoproteínas/química , Línea Celular , Cromatina/metabolismo , Relación Dosis-Respuesta a Droga , Glicoproteínas/química , Humanos , Células Jurkat , Lectinas , Leucosialina , Ligandos , Macrófagos/metabolismo , Oligosacáridos/química , Fagocitosis , Factores de TiempoRESUMEN
Increased production of advanced glycosylation end products (AGEs) and augmented oxidative stress may contribute to vascular complications in diabetes. Little is known about the formation and accumulation of AGEs in young patients with type 1 diabetes. The aim of the present study was to investigate whether AGE production and oxidative stress are augmented in young patients with type 1 diabetes at early clinical stages of the disease. Urine samples of 38 patients with type 1 diabetes [mean age (+/-SD), 12.8 +/- 4.5 y; diabetes duration, 5.7 +/- 4.3 y; HbA1c, 8.0 +/- 1.6%; urinary albumin excretion, 12.6 +/- 14.4 mg/g creatinine (Cr)] and those of 60 age-matched healthy control subjects were assayed for AGEs, pentosidine and pyrraline, and markers of oxidative stress, 8-hydroxy-2'-deoxyguanosine (8-OHdG) and acrolein-lysine. Of these four markers, urinary concentrations of pentosidine, 8-OHdG, and acrolein-lysine were significantly higher in the patients with diabetes than in the healthy control subjects. For the patient group, pentosidine correlated significantly with 8-OHdG and acrolein-lysine, and pyrraline correlated significantly with acrolein-lysine. Urinary pentosidine, 8-OHdG, and acrolein-lysine but not pyrraline correlated significantly with urinary albumin excretion. Patients with microalbuminuria (> or =15 mg/g Cr) showed significantly higher levels of all four markers than did normoalbuminuric patients and control subjects. The present study indicates that accumulation of AGEs, whose formation is closely linked to oxidative stress, and resultant endothelial dysfunction may start early in the course of type 1 diabetes. This means that the risk of vascular complications may be present at an early age and that the best possible glycemic control should be emphasized from the diagnosis of diabetes.
Asunto(s)
Arginina/análogos & derivados , Desoxiguanosina/análogos & derivados , Diabetes Mellitus Tipo 1/orina , Productos Finales de Glicación Avanzada/metabolismo , Lisina/análogos & derivados , Norleucina/análogos & derivados , Estrés Oxidativo , 8-Hidroxi-2'-Desoxicoguanosina , Acroleína/química , Acroleína/orina , Adolescente , Adulto , Arginina/orina , Niño , Preescolar , Creatinina/orina , Desoxiguanosina/orina , Femenino , Humanos , Lisina/química , Lisina/orina , Masculino , Norleucina/orina , Pirroles/orina , Análisis de RegresiónRESUMEN
Arachidonic acid cascade inhibitors, including phospholipase A2 inhibitors, dexamethasone and quinacrine (mepacrine), cyclooxygenase inhibitors, indomethacin and aspirin, and lipoxygenase inhibitor AA861, prevented foam cell formation and cholesterol accumulation in the incubation of thioglycollate-induced mouse peritoneal macrophages with oxidized low density lipoprotein (LDL) at 37 degrees C for 24 h. These inhibitors similarly prevented foam cell formation of fibronectin- and Ca ionophore A23187-stimulated macrophages. Binding and/or uptake of Dil (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine)-acetyl LDL by macrophages at 37 degrees C for 3h and arachidonic acid release from macrophages at 37 degrees C for 4h were inhibited by dexamethasone. Exogenously added phospholipase A2 of bee venom and Crotalus adamanteous venom increased arachidonic acid release during incubation for 2 h, and increased macrophage binding and/or uptake of Dil-acetyl LDL at 37 degrees C for 3 h, and foam cell formation at 37 degrees C for 24 h. Protein kinase inhibitors, ML-9 and staurosporine, that inhibited macrophage binding and/or uptake of Dil-acetyl LDL did not inhibit arachidonic acid release, indicating that protein phosphorylation was not involved in the arachidonic acid pathway in the macrophage scavenger receptor activation. Nordihydroguaiaretic acid that inhibited arachidonic acid release prevented binding and/or uptake of Dil-acetyl LDL. The release of arachidonic acid was not enhanced by fibronectin-stimulation, indicating that Ca influx-dependent stimulation of macrophage activity was not through the activation of phospholipase A2. These results indicate that, as well as the fibronectin-stimulated Ca influx pathway and protein phosphorylation pathway, the arachidonic acid pathway participated in the activation of macrophages to bind and take up oxidized LDL.
Asunto(s)
Ácido Araquidónico/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Animales , Supervivencia Celular , Colesterol/metabolismo , Inhibidores de la Ciclooxigenasa/farmacología , Dexametasona/farmacología , Inhibidores Enzimáticos/farmacología , Fibronectinas/metabolismo , Fibronectinas/farmacología , Técnicas In Vitro , Masculino , Masoprocol/farmacología , Ratones , Oxidación-Reducción , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A/metabolismo , Fosfolipasas A2 , FosforilaciónRESUMEN
The involvement of intracellular protein phosphorylation in macrophages in the binding and uptake of oxidized low density lipoprotein (oxLDL) was investigated. The treatment of fibronectin-unstimulated and stimulated mouse thioglycolate-induced macrophages with inhibitors of myosin light chain kinase, protein kinase C and protein tyrosine kinase resulted in decreased macrophage binding of oxLDL, macrophage foam cell formation, and whole intracellular protein phosphorylation. The treatment of fibronectin-unstimulated and stimulated macrophages with inhibitors of protein serine/threonine and tyrosine phosphatases caused enhanced macrophage binding of oxLDL, macrophage foam cell formation, and whole intracellular protein phosphorylation. Fibronectin, which stimulates macrophage activity, enhanced macrophage intracellular protein phosphorylation. Myosin light chain phosphorylation may be involved in the fibronectin stimulation of macrophages. Treatment of fibronectin-unstimulated and stimulated macrophages with thiophosphate, which forms thiophosphate esters of intracellular proteins that are not so susceptible to protein phosphatases, enhanced macrophage binding of oxLDL. The above results indicate that intracellular protein phosphorylation maintains and enhances macrophage binding and the uptake of oxLDL.