RESUMEN
Cyclo-oxygenase is the rate-limiting enzyme in the prostanoid pathway. Although expression of the inducible isoform of cyclo-oxygenase (COX-2) is associated with cytokine-mediated inflammation, recent evidence suggests a homeostatic role for epithelial COX-2 in the gastrointestinal tract. The aim of this study was to examine the expression and localization of COX-2 in human airway epithelium both in vivo and in vitro. Human airway specimens from patients undergoing lung resection surgery for primary lung tumours (n=10) or nasal mucosal resection for non-inflammatory nasal obstruction (n=5) were examined for COX-2 expression by in situ hybridization and immunohistochemistry. COX-2 expression was also studied in two human airway epithelial cell lines (BEAS-2B and A549) using reverse transcription polymerase chain reaction and Northern and Western blot analysis. COX-2 messenger ribonucleic acid (mRNA) and protein were localized to individual columnar epithelial cells and to airway resident inflammatory cells in 9/10 lower and 5/5 upper airway specimens. Expression of COX-2 did not correlate with evidence of airway inflammation. Focal expression of COX-2 mRNA and protein was observed in bronchus-associated lymphoid tissue. Both COX-2 mRNA and protein were detected in BEAS-2B and A549 cells cultured under standard conditions. In conclusion, expression of COX-2 in human airway epithelium occurs in the upper and lower airways, is widespread in airway epithelial and airway resident inflammatory cells in the absence of overt airway inflammation, and is detectable in cultured human airway epithelial cells in the absence of inflammatory cytokine stimulation. These data suggest a potentially important homeostatic role for COX-2 in the regulation of human airway contractility, inflammation and immune responses.
Asunto(s)
Isoenzimas/metabolismo , Pulmón/enzimología , Mucosa Nasal/enzimología , Peroxidasas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Adulto , Anciano , Northern Blotting , Western Blotting , Células Cultivadas , Ciclooxigenasa 2 , Células Epiteliales/enzimología , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Pulmón/citología , Masculino , Proteínas de la Membrana , Persona de Mediana Edad , Mucosa Nasal/citología , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: The pathogenesis of nasal polyp disease is poorly understood. Recent evidence has suggested that nitric oxide (NO), an endogenous soluble gas vasodilator and inflammatory mediator, may be synthesised within the nasal cavity. Three nitric oxide synthase isoforms have been identified in humans, with the inducible isoform (iNOS) generally expressed in the setting of inflammation. OBJECTIVE: The aim of this study was to detect and localize iNOS expression in nasal polyp tissue, and compare these findings with normal nasal turbinate tissue. METHODS: We examined the expression and localisation of inducible nitric oxide synthase (iNOS) in human nasal airway specimens from patients undergoing elective nasal turbinectomy (n = 5) or nasal polypectomy (n = 5). iNOS mRNA expression was determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) followed by Southern blot analysis and localised by in situ hybridization. Densitometric data were analysed using Student's unpaired t-test. Adjacent sections were also examined for iNOS protein expression by immunohistochemistry. RESULTS: Semi-quantitative RT-PCR/Southern analysis of RNA obtained from the 10 surgical specimens demonstrated that iNOS mRNA expression was significantly increased in the five nasal polyps (P < 0.05). In situ hybridization studies revealed strong iNOS mRNA signal localized to the respiratory epithelium of nasal polyps, but not nasal turbinates. This pattern was confirmed by immunohistochemistry. Localization to inflammatory cells or other subepithelial structures was not seen. CONCLUSIONS: We conclude that iNOS expression is upregulated in nasal polyp disease, and is localized to the polyp epithelial layer. These data reinforce the concept that the epithelial layer may be important in the pathogenesis of nasal disease, and suggest a potential role for NO in the formation of nasal polyps.
Asunto(s)
Isoenzimas/biosíntesis , Pólipos Nasales/enzimología , Óxido Nítrico Sintasa/biosíntesis , Epitelio/enzimología , Humanos , Inmunohistoquímica , Hibridación in Situ , Isoenzimas/genética , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisisRESUMEN
Asthma has been reported to be associated with a reduction in the activity of glutathione peroxidase (GSH-Px), an important antioxidant enzyme. However, the expression of GSH-Px enzyme activity has not previously been investigated in human eosinophils, which are important inflammatory cells involved in asthma. Reverse transcriptase-polymerase chain reaction and Southern blotting demonstrated that eosinophils express GSH-Px mRNA and the relative expression of GSH-Px was greater in eosinophils than in neutrophils for both asthmatic and non-asthmatic subjects. The presence of GSH-Px protein in eosinophil and neutrophil lysates was confirmed by size exclusion chromatography and by Western blotting. GSH-Px enzyme activity as measured by a spectrophotometric assay was greater in eosinophil (48.4+/-1.6 micromol NADPH oxidized x min(-1) x g(-1) protein) than in neutrophil lysates (18.1+/-0.4, n = 24, P < 0.0001). GSH-Px activities of eosinophils and neutrophils from asthmatic subjects did not differ from those of non-asthmatic subjects. Eosinophil GSH-Px activity was correlated with peripheral blood eosinophil count only in asthmatic subjects (rs = 0.59, n = 12, P = 0.04). Increased GSH-Px expression in eosinophils compared with neutrophils of asthmatic patients may provide antioxidant protection against the greater amounts of reactive oxygen species generated by these cells and may enhance the survival of eosinophils at sites of inflammation in asthma.
Asunto(s)
Asma/enzimología , Eosinófilos/enzimología , Glutatión Peroxidasa/metabolismo , Neutrófilos/enzimología , Adulto , Western Blotting , Separación Celular , Femenino , Glutatión Peroxidasa/genética , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , ARN Mensajero/genéticaRESUMEN
Nitric oxide (NO) synthesized by airway epithelium may be important in the regulation of airway inflammation and reactivity. As such, the expression and localization of nitric oxide synthase (NOS) isoforms was assessed in human airway tissue obtained following thoracotomy, and in cultured human airway epithelial (BEAS-2B) cells. NOS expression was determined by reverse transcription-polymerase chain reaction (RT-PCR)/Southern blot analysis, and localized by in situ hybridization and immunohistochemistry. No synthesis by cell cultures was detected by nitrite assay. Endothelial and neuronal constitutive NOS mRNAs were not detected in airways or cell cultures. Inducible NOS (iNOS) mRNA was detected in 5 of 6 airway specimens, and in situ hybridization demonstrated iNOS mRNA expression in columnar epithelial cells. This was confirmed by immunohistochemistry using an iNOS specific antibody. BEAS-2B cell cultures were stimulated with (I) combinations of tumor necrosis factor alpha (TNF alpha) (50-2,000 U/ml)/interferon gamma (IFN gamma) (20-500 U/ml)/lipopolysaccharide (LPS) (10 micrograms/ml) or (2) histamine (10(-3) M-10(-5) M). Cell cultures treated with TNF alpha/IFN gamma/LPS in combination expressed iNOS mRNA, and this was associated with increases in supernatant nitrite concentrations over 24 h; however, this response diminished with successive passage of cells. Histamine treatment did not result in iNOS mRNA expression or detectable NO synthesis. We conclude that iNOS in human airway tissue is localized to the airway epithelium. Cytokine/ LPS stimulation, but not histamine, results in iNOS mRNA expression and NO synthesis in BEAS-2B cells. BEAS-2B cells may not be a suitable model for the study of NO biology in airway epithelium.
Asunto(s)
Bronquios/citología , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Northern Blotting , Southern Blotting , Bronquios/cirugía , Carcinoma Broncogénico , Línea Celular Transformada , Clonación Molecular , Citocinas/farmacología , Inhibidores Enzimáticos/farmacología , Células Epiteliales , Epitelio/enzimología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Histamina/farmacología , Humanos , Hibridación in Situ , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitritos/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , ToracotomíaRESUMEN
Malignant mesothelioma (MM) is an asbestos-associated cancer that is increasing in incidence worldwide and is refractory to conventional therapy. MM cells are potent sources of a number of cytokines, some of which have recently been shown to be directly involved in the aggressive growth and spread of MM tumors. Emerging data also suggest involvement of MM-derived cytokines in systemic paraneoplastic syndromes including immunosuppression, thrombocytosis, cachexia, amyloidosis, and hypoglycemia. Additional characterization of the expression of cytokines and cytokine receptors in situ in MM tumors may provide a more complete picture of the autocrine and paracrine processes which occur in MM. Improved therapy of MM, particularly cytokine-based approaches, is likely to benefit from further elucidation of the patterns and regulation of cytokine expression associated with MM tumors.