RESUMEN
Two of the major cancerous diseases associated with asbestos exposure are malignant pleural mesothelioma (MPM) and lung cancer (LC). In addition to asbestos exposure, genetic factors have been suggested to be associated with asbestos-related carcinogenesis and lung genotoxicity. While genetic factors involved in the susceptibility to MPM were reported, to date the influence of individual genetic variations on asbestos-related lung cancer risk is still poorly understood. Since inflammation and disruption of iron (Fe) homeostasis are hallmarks of asbestos exposure affecting the pulmonary tissue, this study aimed at investigating the association between Fe-metabolism and inflammasome gene variants and susceptibility to develop LC or MPM, by comparing an asbestos-exposed population affected by LC with an "asbestos-resistant exposed population". A retrospective approach similar to our previous autopsy-based pilot study was employed in a novel cohort of autoptic samples, thus giving us the possibility to corroborate previous findings obtained on MPM by repeating the analysis in a novel cohort of autoptic samples. The protective role of HEPH coding SNP was further confirmed. In addition, the two non-coding SNPs, either in FTH1 or in TF, emerged to exert a similar protective role in a new cohort of LC exposed individuals from the same geographic area of MPM subjects. No association was found between NLRP1 and NLRP3 polymorphisms with susceptibility to develop MPM and LC. Further research into a specific MPM and LC "genetic signature" may be needed to broaden our knowledge of the genetic landscape attributed to result in MPM and LC.
Asunto(s)
Amianto/toxicidad , Inflamasomas/genética , Hierro/metabolismo , Neoplasias Pulmonares/epidemiología , Mesotelioma/epidemiología , Neoplasias Pleurales/epidemiología , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Italia , Neoplasias Pulmonares/inducido químicamente , Masculino , Mesotelioma/inducido químicamente , Mesotelioma Maligno , Neoplasias Pleurales/inducido químicamente , Prevalencia , Estudios Retrospectivos , Factores de RiesgoRESUMEN
The presence of asbestos bodies (ABs) in lung parenchyma is considered a histopathologic hallmark of past exposure to asbestos fibers, of which there was a population of longer fibers. The mechanisms underlying AB formation are complex, involving inflammatory responses and iron (Fe) metabolism. Thus, the responsiveness to AB formation is variable, with some individuals appearing to be poor AB formers. The aim of this study was to disclose the possible role of genetic variants of genes encoding inflammasome and iron metabolism proteins in the ability to form ABs in a population of 81 individuals from North East Italy, who died after having developed malignant pleural mesothelioma (MPM). This study included 86 genetic variants distributed in 10 genes involved in Fe metabolism and 7 genetic variants in two genes encoding for inflammasome molecules. Genotypes/haplotypes were compared according to the number of lung ABs. Data showed that the NLRP1 rs12150220 missense variant (H155L) was significantly correlated with numbers of ABs in MPM patients. Specifically, a low number of ABs was detected in individuals carrying the NLRP1 rs12150220 A/T genotype. Our findings suggest that the NLRP1 inflammasome might contribute in the development of lung ABs. It is postulated that the NLRP1 missense variant may be considered as one of the possible host genetic factors contributing to individual variability in coating efficiency, which needs to be taken when assessing occupational exposure to asbestos.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Reguladoras de la Apoptosis/genética , Amianto/toxicidad , Neoplasias Pulmonares/genética , Pulmón/patología , Mesotelioma/genética , Exposición Profesional/efectos adversos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Anciano , Anciano de 80 o más Años , Proteínas Reguladoras de la Apoptosis/metabolismo , Carga Corporal (Radioterapia) , Femenino , Variación Genética , Humanos , Italia , Pulmón/efectos de los fármacos , Neoplasias Pulmonares/metabolismo , Masculino , Mesotelioma/metabolismo , Mesotelioma Maligno , Persona de Mediana Edad , Proteínas NLRRESUMEN
BACKGROUND: Mast cells (MCs) and eosinophils (Eos) are the key effector cells of the allergic reaction. Although classically associated with different stages of the response, the cells co-exist in the inflamed tissue in the late and chronic phases in high numbers and are likely to cross-talk. While some mediators of MCs are known to affect Eos biology and vice versa, paracrine and physical interplay between the two cells has not been described yet. We aimed to investigate whether intercellular MC-Eos communication could take place in the allergic response and exert functional bidirectional changes on the cells. METHODS: Tissue sections from various allergic disorders were specifically stained for both cells. Human cord blood-derived MCs and peripheral blood Eos, co-cultured under different conditions, were studied by advanced microscopy and flow cytometry. RESULTS: Several co-localized MC-Eos pairs were detected in human nasal polyps and asthmatic bronchi, as well in mouse atopic dermatitis. In vitro, MCs and Eos formed stable conjugates at high rates, with clear membrane contact. In the presence of MCs, Eos were significantly more viable under several co-culture conditions and at both IgE-activated and steroid-inhibited settings. MC regulation of Eos survival required communication through soluble mediators but was even more dependent on physical cell-cell contact. CONCLUSIONS: Our findings provide the first evidence for a complex network of paracrine and membrane interactions between MCs and Eos. The prosurvival phenotype induced by this MC-Eos interplay may be critical for sustaining chronic allergic inflammation.
Asunto(s)
Eosinófilos/metabolismo , Mastocitos/metabolismo , Animales , Antígenos CD/metabolismo , Antígeno CD48 , Comunicación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Técnicas de Cocultivo , Citocinas/metabolismo , Dexametasona/farmacología , Eosinófilos/citología , Humanos , Hipersensibilidad/inmunología , Hipersensibilidad/fisiopatología , Inmunoglobulina E/inmunología , Mastocitos/citología , Ratones , Comunicación Paracrina/efectos de los fármacos , Receptores Inmunológicos/metabolismo , Familia de Moléculas Señalizadoras de la Activación LinfocitariaRESUMEN
OBJECTIVE: A neuropathic basis has been suggested for burning mouth syndrome (BMS) and an altered concentration of neuropeptides has been reported in lingual oral mucosa and saliva in this disease. The aims of this study were to compare the levels of nerve growth factor (NGF), substance P (SP) and degranulation products from mast cells and neutrophils in the saliva of BMS subjects with those of control subjects. MATERIAL AND METHODS: Salivary flow rate, protein concentration, NGF peptide and mRNA, SP, mast cells tryptase, neutrophil myeloperoxidase and calprotectin were analyzed in saliva of 20 BMS subjects and of 20 age- and gender-matched healthy subjects. RESULTS AND CONCLUSIONS: NGF peptide and tryptase activity were shown to be significantly and persistently higher in saliva of BMS subjects, with respect to control values. Conversely the salivary levels of SP were shown to be significantly lower, while neutrophil markers didn't show any change. We conclude that the neuropathic origin of the disease is confirmed at salivary level. Furthermore, the higher tryptase activity indicates a possible involvement of mast cells. The salivary neuropeptide concentration in BMS subjects, together with mast cell derived compounds, could be useful biomarkers for diagnosis and monitoring of this disease.
Asunto(s)
Síndrome de Boca Ardiente/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Saliva/metabolismo , Sustancia P/metabolismo , Triptasas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/metabolismo , Síndrome de Boca Ardiente/inmunología , Estudios de Casos y Controles , Degranulación de la Célula/inmunología , Femenino , Humanos , Complejo de Antígeno L1 de Leucocito/metabolismo , Masculino , Mastocitos/enzimología , Mastocitos/inmunología , Análisis por Apareamiento , Persona de Mediana Edad , Factor de Crecimiento Nervioso/genética , Neutrófilos/enzimología , Neutrófilos/inmunología , Peroxidasa/metabolismo , ARN Mensajero/análisis , Valores de ReferenciaRESUMEN
Newborn rat oligodendrocyte cultures were investigated by scanning near-field optical microscope (SNOM), a versatile new tool able to map cell membranes in 3D and simultaneously obtain images of the cytoplasm. Topography, error, transmission and reflection signals were acquired to describe cell morphology with nanometer-scale resolution. Oligodendrocytes were studied as a model because their extensive membrane processes (typical of their physiological role in myelination) made them particularly suitable to test the sensitivity of the new method. Furthermore, we combined a classical histochemical method with SNOM, to identify specific intracellular proteins at high definition. In particular, with this technique, cytoskeleton elements of oligodendrocytes, such as microtubules, were observed with tubulin antibodies. Images obtained with SNOM were also compared with those from conventional scanning electron microscopy (SEM) and optical microscopy. Our results showed that SNOM allowed to observe cell nanostructures otherwise undetectable all together with other microscopies. In conclusion, SNOM, combined with rapid and non-invasive methods of specimen preparation, appears to be a powerful tool that can offer new possibilities in the field of neuroscience imaging at nano-scale level.
Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Microscopía/métodos , Oligodendroglía/ultraestructura , Animales , Animales Recién Nacidos , Células Cultivadas , Citoesqueleto/ultraestructura , Microscopía Electrónica de Rastreo , Microtúbulos/ultraestructura , Ratas , Fijación del Tejido , Tubulina (Proteína)/ultraestructuraRESUMEN
Imidazolium trans-imidazoledimethylsulphoxidetrachlororuthenate (NAMI-A) was tested in vitro on the pro-adhesive properties, evaluated as resistance to trypsin treatment, which is a bona fide measure of adhesion strength, of KB and HeLa carcinoma cell lines and on human polymorphonuclear neutrophils (HPMN). NAMI-A increased the pro-adhesive activity of KB cells at 0.001 mM concentration, after few minutes incubation and this effect was not influenced by the vehicle used for cell challenge, neither did it depend on NAMI-A concentration or on temperature. The same effect occurred on HeLa cells at 0.01 mM NAMI-A. This effect, detected at concentrations up to 100 times lower than those necessary to block cells at the G(2)-M premitotic phase of cell cycle, or to inhibit matrix metalloproteinase release or cell invasion, was not related to ruthenium uptake by tumour cells. HeLa cells and healthy HPMN, following short exposure to 0.1 mM NAMI-A, assumed a different shape, with the extrusion of filopodia (HeLa) and of large lamellopodia (HPMN), which increased their interactions with the substrate. This effect was attributed to stabilisation, altered turnover and sensitivity to cytochalasin D of actin filaments. Provided that adhesion is associated with cell motility and invasion, these data suggest that NAMI-A may exert antimetastatic properties at concentrations lower than those observed in the lungs at the end of a conventional intraperitoneal treatment in vivo.
Asunto(s)
Antineoplásicos/farmacología , Dimetilsulfóxido/análogos & derivados , Dimetilsulfóxido/farmacología , Neoplasias/tratamiento farmacológico , Neutrófilos/efectos de los fármacos , Compuestos Organometálicos/farmacología , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/ultraestructura , Anticuerpos Bloqueadores/farmacología , Antineoplásicos/análisis , Adhesión Celular/efectos de los fármacos , Línea Celular , Dimetilsulfóxido/análisis , Células HeLa , Humanos , Integrinas/inmunología , Microscopía Confocal , Microscopía Electrónica de Rastreo , Neoplasias/patología , Neutrófilos/química , Neutrófilos/ultraestructura , Compuestos Organometálicos/análisis , Rutenio/análisis , Compuestos de Rutenio , TripsinaRESUMEN
We have isolated a new cell line (metGM) obtained from the spontaneous lung metastases of the mouse MCa mammary carcinoma. MetGM is a stable cell line which, after one year from its isolation, grows in vitro in suspension, forming cell aggregates, with cells that show irregular blabbing borders, active protein synthesis and convoluted nuclei and which have the capacity of invading matrigel membranes on which they give rise to a network of branching colonies. The preliminary study of the effects of the anti-metastasis ruthenium complex NAMI-A on metGM showed no direct cytotoxicity, with a mild reduction of cell proliferation, independent of the concentration of the ruthenium complex and not evident before 24 hours from treatment. A 10% DNA fragmentation was also measured on metGM cells 24 hours after challenge for 1 hour with 10(-5)M NAMI-A, suggesting that this compound is probably capable of apoptosis in a metastasis-derived cell line. Besides these effects on a limited percent of the cell population, NAMI-A changed the shape of the metGM cells and these alterations might account for the non-cytotoxic anti-metastatic properties of this innovative ruthenium complex. Thus MetGM appears to be a novel cell line suitable for the in vitro study of compounds endowed with anti-metastatic properties and for the development of new drugs with this activity.
Asunto(s)
Antineoplásicos/farmacología , Dimetilsulfóxido/análogos & derivados , Dimetilsulfóxido/farmacología , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/patología , Compuestos Organometálicos/farmacología , Células Tumorales Cultivadas/patología , Animales , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Neoplasias Pulmonares/prevención & control , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Ratones , Ratones Endogámicos CBA , Compuestos de Rutenio , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
The growth capacity and adaptation of TS/A and TS/A-IL4 lines on laminin, fibronectin, collagens I and IV and matrigel compared to plastics were studied by flow cytometry. On plastic plates, TS/A-IL4 grows in vitro more slowly than the TS/A line and shows a more differentiated phenotype. TS/A-IL4 cells loose the capacity to bind lymphocytes and peroxidase positive cells obtained from mice implanted with the same tumour. The ratio between fibroblast- and epithelial-like cells of TS/A adenocarcinoma is subjected to marked modifications depending on the substrate on which the two cell lines are grown. IL4 release per cell unit is increased by collagen I as is the number of CD54 positive cells, suggesting that, at least in part, the in vivo rejection of TS/A-IL4 tumor might be ascribed to the stimulatory effect of the tissue on the IL4 release by tumor cells. The overall result is that gene modified TS/A-IL4 line shows marked changes of behaviour, most of them depending on the substrate on which tumor cells are growing.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Interleucina-4/genética , Neoplasias Mamarias Experimentales/patología , Animales , Adhesión Celular , Técnicas de Cultivo de Célula/instrumentación , Ciclo Celular , Diferenciación Celular , Células Clonales/metabolismo , Células Clonales/patología , Técnicas de Cocultivo , Colágeno , Combinación de Medicamentos , Células Epiteliales/patología , Femenino , Fibroblastos/patología , Fibronectinas , Rechazo de Injerto , Molécula 1 de Adhesión Intercelular/análisis , Interleucina-4/metabolismo , Laminina , Activación de Linfocitos , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteínas de Neoplasias/análisis , Trasplante de Neoplasias , Plásticos , Proteoglicanos , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patologíaRESUMEN
Neutrophils and macrophages express on their membrane molecules which may, in principle, interact with each other, promote specific cell to cell adhesion, affect cell function and finally, as a consequence, modulate the progression of the inflammatory process. We tested therefore if human neutrophils specifically adhere to human monocyte-derived macrophage monolayer (MDMM). Our findings show that neutrophils significantly adhere to 4-day old MDMM and that the extent of adhesion is increased by LPS-activation of MDMM. The specificity of the interaction was shown by the very low extent of adhesion of neutrophils either to freshly prepared monocyte or other types of cell monolayers and by the low percent of adhesion showed by eosinophils exposed to 7-day old MDMM. A role for beta2 integrins, CD31 and PAF-receptor in the mechanism of neutrophil-MDMM interaction is suggested by specific antagonists. We suggest that the adhesion between the two cell types could lead to an increase in concentration of neutrophil- or macrophage released factors in the interaction site and in a mutual modulation of phagocyte functions.
Asunto(s)
Macrófagos/fisiología , Monocitos/citología , Neutrófilos/fisiología , Receptores de Superficie Celular , Receptores Acoplados a Proteínas G , Antígenos CD18/fisiología , Cationes Bivalentes/farmacología , Adhesión Celular/fisiología , Células Cultivadas , Medios de Cultivo/química , Humanos , Molécula 1 de Adhesión Intercelular/fisiología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/ultraestructura , Neutrófilos/ultraestructura , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/fisiología , Glicoproteínas de Membrana Plaquetaria/fisiologíaRESUMEN
The in vitro/in vivo growth capacity and phenotype of TS/A and the IL4-transfected TS/A-IL4 cell lines were studied by cell cycle analysis, expression of ICAM-1/CD54, transferrin receptor/CD71 and E-cadherin and by histology of the primary tumors. TS/A-IL4, unlike the TS/A line, shows in vitro a marked increase in the fibroblastoid cell type and a decreased E-cadherin expression. Administration of conditioned medium containing IL4 obtained from the TS/A-IL4 cell line, stimulates CD54 expression in the TS/A cell line. TS/A-IL4 tumors grow more slowly in vivo and are ultimately rejected. These processes are accompanied by a marked increase in collagen and extracellular matrix proteins and increased recruitment and degranulation of mast cells. The paracrine effect of IL4, released by the transfected tumor cells, might be responsible for the reduced in vivo growth of the TS/A cell line in the presence of TS/A-IL4 cells.
Asunto(s)
Adenocarcinoma/terapia , Interleucina-4/genética , Neoplasias Mamarias Animales/terapia , Adenocarcinoma/patología , Animales , Ciclo Celular , División Celular , Femenino , Terapia Genética , Interleucina-4/uso terapéutico , Neoplasias Mamarias Animales/patología , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Comunicación Paracrina , Fenotipo , Transfección , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
We investigated the antimycobacterial role of myeloperoxidase (MPO), one of the most abundant granule proteins in human neutrophils. Our data indicate that purified MPO, in the presence of hydrogen peroxide, exerts a consistent killing activity against Mycobacterium tuberculosis H37Rv and against a clinical isolate. The activity is time and dose dependent and requires the presence of chloride ions in the assay medium.
Asunto(s)
Antibacterianos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Peroxidasa/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Peróxido de Hidrógeno/farmacología , Factores de TiempoRESUMEN
The mutual interaction between monocytes and low density lipoprotein (LDL) in atherogenesis prompted a test of the hypothesis that LDL-apheresis could reduce the adhesive properties of monocytes to endothelium; and therefore interfere with a key mechanism in atheroma formation. Five patients affected by heterozygous familial hypercholesterolemia were studied. All patients received LDL-apheresis treatment with selective adsorption of LDL-cholesterol on dextran-sulphate columns. Low density lipoprotein particles were isolated by sequential preparative ultracentrifugation and subfractionated by ion exchange high performance liquid chromatography. Thiobarbituric acid reacting products of lipid peroxidation were measured fluorometrically. Vitamin E was estimated by high performance liquid chromatographic technique. Monocytes were isolated from patients blood before and 1 day after LDL-apheresis by Percoll gradient. The blood samples for monocyte adhesion were drawn from control subjects for 2 consecutive days. The adhesion of monocytes to an endothelial monolayer was evaluated by assaying the peroxidase content of the adherent monocytes. Low density lipoprotein-apheresis reduced total cholesterol (-65%; p < 0.01), LDL-cholesterol (-75%; p < 0.01), triglycerides (-51%; p < 0.05), and fibrinogen (-28%; p < 0.01). With LDL-apheresis treatment, a reduction of 54% in oxidized LDLs was observed; vitamin E concentration significantly increased in LDLs (+ 14.2%; p < 0.05). The monocyte adhesion decreased by approximately 61% after apheresis; the variation became statistically significant (-65%; p < 0.01) when endothelial cells were stimulated by lipopolysaccaride.
Asunto(s)
Eliminación de Componentes Sanguíneos , Hiperlipoproteinemia Tipo II/terapia , Lipoproteínas LDL/aislamiento & purificación , Anciano , Arteriosclerosis/sangre , Arteriosclerosis/etiología , Arteriosclerosis/prevención & control , Adhesión Celular , Células Cultivadas , Endotelio Vascular/fisiología , Humanos , Hiperlipoproteinemia Tipo II/sangre , Hiperlipoproteinemia Tipo II/complicaciones , Técnicas In Vitro , Lipoproteínas LDL/sangre , Lipoproteínas LDL/fisiología , Masculino , Persona de Mediana Edad , Monocitos/fisiología , Oxidación-ReducciónRESUMEN
The evidence that small GTPases of the Rab family are regulators of vesicle traffic which can influence various cell functions prompted us to investigate the potential role of one of these proteins, Rab5a, in human neutrophils. In this paper we show that a large amount of Rab5a is present in the cytosol of peripheral blood mature neutrophils. The remaining protein was found to be membrane and azurophilic granule associated. Upon neutrophil challenge with PMA for 10 min the amount of membrane-associated Rab5a was upregulated while the cytosolic content of the protein concomitantly decreased. These findings support the hypothesis that Rab5a could be involved in the mechanism of neutrophil activation by modulating the rate of endocytosis and/or vesicle fusion.
Asunto(s)
GTP Fosfohidrolasas/análisis , Proteínas de Unión al GTP/análisis , Neutrófilos/enzimología , Fracciones Subcelulares/enzimología , Gránulos Citoplasmáticos/química , Gránulos Citoplasmáticos/enzimología , Citosol/química , Citosol/enzimología , GTP Fosfohidrolasas/efectos de los fármacos , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/efectos de los fármacos , Proteínas de Unión al GTP/metabolismo , Humanos , Activación de Linfocitos/fisiología , Proteínas de la Membrana/análisis , Microscopía Electrónica , Neutrófilos/química , Neutrófilos/ultraestructura , Fracciones Subcelulares/química , Fracciones Subcelulares/ultraestructura , Acetato de Tetradecanoilforbol/farmacología , Proteínas de Unión al GTP rab5RESUMEN
BACKGROUND: Atopic dermatitis is associated with skin and blood eosinophilia, but the role of eosinophils in the pathogenesis of the skin lesions is poorly understood. METHODS: To determine whether eosinophils play a role in the pathogenesis of the skin lesions in atopic dermatitis, we studied the relationship between the severity of the disease and both the number and the extent of activation of eosinophils in 15 patients with food-sensitive atopic dermatitis. Furthermore, this relationship was re-evaluated in eight of these patients who, after a period of elemental diet or total parenteral nutrition, showed significant clinical improvement. RESULTS: A clear relationship was found between the number of light-density eosinophils and the severity of the disease both during the active disease and after clinical improvement. Furthermore, we describe an adhesion-stimulating activity for eosinophils in patients' plasma, which does not change after recovery. CONCLUSIONS: Taken together, these observations strongly indicate that eosinophils play a pivotal role in the pathogenesis of the skin lesions in atopic dermatitis. In particular, the light-density phenotype seems to be an essential feature of eosinophils involved in this process. The adhesion-promoting activity that we observed in the patients' plasma could be important in the recruitment of eosinophils from the blood into the skin.
Asunto(s)
Dermatitis Atópica/etiología , Eosinófilos/fisiología , Hipersensibilidad a los Alimentos/etiología , Ribonucleasas , Adulto , Proteínas Sanguíneas/análisis , Niño , Preescolar , Proteínas en los Gránulos del Eosinófilo , Femenino , Humanos , Lactante , MasculinoRESUMEN
Inhibition of growth or eradication of experimentally induced tumors has been shown to be accompanied by infiltration of eosinophils and macrophages into the tumor mass. Since macrophages are important mediators of host antitumor activity, the possibility arises that a collaboration may exist between these two cell types in the control of tumor growth. In this study, we report the effect of eosinophil peroxidase (EPO), a basic protein contained in eosinophils that binds to several cell types including macrophages, on tumor necrosis factor (TNF) production and hydrogen peroxide release by human monocyte-derived macrophages. After incubation with EPO, the macrophages produced large amounts of TNF and displayed an enhanced phorbol 12-myristate 13-acetate-triggered hydrogen peroxide release. These effects were accompanied by an increased cell protein content and by morphologic changes leading the large, round macrophages of the control cultures to become elongated, pear-like or spindle shaped cells after treatment with EPO. The stimulatory effect of EPO on hydrogen peroxide release was insensitive to addition of exogenous catalase, a H2O2-degrading enzyme, suggesting that an extracellular catalytic activity of EPO was not involved. In addition, myeloperoxidase, the homologous peroxidase of neutrophils with a catalytic activity similar to that of EPO, was ineffective. The EPO-induced effects differed in several aspects from the effects of lipopolysaccaride and interferon-gamma, two well-known macrophage activators. These findings provide supportive evidence for a functional interrelationship between eosinophils and macrophages that may be physiologically relevant in the tumoricidal activity of macrophages.
Asunto(s)
Peróxido de Hidrógeno/metabolismo , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Monocitos/efectos de los fármacos , Peroxidasas/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Catalasa/farmacología , Tamaño de la Célula , Células Cultivadas , Medios de Cultivo Condicionados , Peroxidasa del Eosinófilo , Humanos , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Linfoma de Células B/patología , Macrófagos/metabolismo , Macrófagos/ultraestructura , Ratones , Monocitos/citología , Peroxidasa/farmacología , Polimixina B/farmacología , Biosíntesis de Proteínas , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
The role of glycosylation in modulating the activity of recombinant human granulocyte colony-stimulating factor (rHuG-CSF) on polymorphonuclear leukocytes (PMNs) was investigated. We addressed this study by comparing the effects of lenograstim (glycosylated rHuG-CSF) and its deglycosylated counterpart on superoxide production by PMNs on fibronectin. When the triggering activity of the cytokine was evaluated, no O2- release was elicited from neutrophils treated with either preparation of rHuG-CSF. Instead, a clear potentiation of both fMLP- and TNF-induced respiratory burst was produced by preincubating the cells with rHuG-CSF. Such effect was found to be significantly increased when glycosylated versus deglycosylated preparation was used, leading to the conclusion that the sugar moiety of the molecule could be of importance in improving the priming activity exerted by rHuG-CSF on PMN metabolic response.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Superóxidos/sangre , Sinergismo Farmacológico , Glicosilación , Humanos , Neutrófilos/metabolismo , Oxidación-Reducción , Proteínas Recombinantes , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
As the carbohydrate lacto-N-fucopentaose III (CD15 antigen or X-determinant) and its sialylated derivative sialyl-Lewis X are involved in the adhesion of cells rolling along the surface of endothelial cells, experiments were done to study the presence of these molecules on human eosinophils from patients with the idiopathic hypereosinophilic syndrome. Normal-density eosinophils from some patients showed higher levels of expression for lacto-N-fucopentaose III than light-density eosinophils. In contrast, sialyl-Lewis X was highly expressed by light-density eosinophils. Activation of normal-density eosinophils with calcium ionophore A23187 resulted in increased expression of these molecules for a short time. Monoclonal antibodies to these carbohydrates stimulated eosinophils to secrete eosinophil cationic protein, but not eosinophil peroxidase, and acted as costimulatory signals for C3b-induced degranulation of eosinophil cationic protein. It was suggested that CD15 and sialyl-Lewis X might contribute to eosinophil-mediated tissue injury in patients with the idiopathic hypereosinophilic syndrome.
Asunto(s)
Eosinófilos/inmunología , Síndrome Hipereosinofílico/inmunología , Antígeno Lewis X/sangre , Ribonucleasas , Ácidos Siálicos/sangre , Adulto , Anticuerpos Monoclonales/inmunología , Proteínas Sanguíneas/metabolismo , Calcimicina/inmunología , Proteínas en los Gránulos del Eosinófilo , Peroxidasa del Eosinófilo , Eosinófilos/metabolismo , Citometría de Flujo , Humanos , Interleucina-5/inmunología , Ácido N-Acetilneuramínico , Peroxidasa/sangre , Peroxidasas/sangre , Estallido Respiratorio/inmunologíaRESUMEN
Five eosinophil peroxidase (EPO)-deficient subjects were identified from 131,000 peripheral blood samples examined for routine automated analysis. The EPO-deficient eosinophils of these subjects met the main criteria established for EPO deficiency: absent or strongly decreased reaction for peroxidase, absent or strongly decreased staining with Sudan Black, and an increased ratio of the granule core volume to the total granule volume. In this report we show that this granule alteration is caused mainly by a decrease of its volume, particularly of the matrix, and that two other matrix proteins, eosinophil cationic protein and eosinophil derived neurotoxin, appear to be present in normal amounts in the EPO-deficient granules.
Asunto(s)
Gránulos Citoplasmáticos/enzimología , Eosinófilos/enzimología , Peroxidasas/deficiencia , Adulto , Anciano , Gránulos Citoplasmáticos/ultraestructura , Peroxidasa del Eosinófilo , Eosinófilos/patología , Eosinófilos/ultraestructura , Femenino , Humanos , Masculino , Microscopía Electrónica , Microscopía Inmunoelectrónica , Persona de Mediana Edad , Valores de ReferenciaRESUMEN
The degranulation of neutrophils and eosinophils is frequently monitored by assaying myeloperoxidase (MPO) and eosinophil peroxidase (EPO) activity in the cell-free supernatant of degranulating cells, after removal of the cells by centrifugation. This procedure leads to underestimation of the extent of degranulation, since both peroxidases tend to stick to cell surfaces, to test tube walls, and to particulate stimuli used to elicit degranulation, because of their highly cationic nature. In this paper we describe a method for assaying MPO and EPO secretion in whole cell suspensions that avoids separation of the cells from the incubation medium. The least toxic and thus safest among the sensitive peroxidase substrates, 3,3',5,5'-tetramethylbenzidine (TMB), was employed for peroxidase assay. The method we describe here is applied to the detection of peroxidase release by neutrophil and eosinophil cell suspensions incubated in either polypropylene test tubes or flat-bottomed microtiter plate wells. Because of the omission of the centrifugation step, the TMB method offers two major advantages over the currently used techniques: (1) higher estimates of degranulation, which permits the use of a smaller number of cells (in the microassay version, 150,000 neutrophils and 50,000 eosinophils) and smaller amounts of the secretagogues, and (2) rapidity, since the degranulation assay can be performed immediately on completion of the cell incubation with the secretagogue.