RESUMEN
BACKGROUND: Nitric oxide (NO) has been associated with airway inflammation in asthma. Our previous work suggests that NO functions in an anti-inflammatory capacity through downregulation of stimulated cytokine secretion by normal human alveolar macrophages. Functional differences between alveolar macrophages and blood monocytes are thought to be related to maturation. OBJECTIVE: The purpose of this study was to determine the effect of NO on stimulated cytokine production by monocytes from asthmatics and normal healthy controls. METHODS: Monocytes and alveolar macrophages were obtained from normal volunteers (n = 13) and asthmatics with atopy (n = 7). Monocyte and alveolar macrophage cultures were stimulated with 0.5 microgram/mL lipopolysaccharide +/- 1.0 mM DETA NONOate (releases NO in culture with t1/2 = 20 hours at 37 degrees C) and incubated for 24 hours. Cell-free supernatants were collected and assayed by ELISA for tumor necrosis factor-alpha (TNF) and granulocyte macrophage colony stimulating factor (GM-CSF). RESULTS: Nitric oxide did not inhibit TNF production in monocytes of asthmatics and normals (mean +/- SEM % TNF stimulation = 19.6 +/- 9.7). Similar to previous results, NO did inhibit alveolar macrophages (% TNF suppression = 60.6 +/- 4.4). To determine whether this differential effect of NO on the two cell populations was related to maturation, monocytes were matured by culture for 7 days. The in vitro matured monocytes demonstrated 51.7 +/- 7.9% suppression of TNF. For each cell population, the responses of the asthmatics and healthy controls were not different. The differential effect is not cytokine specific since similar results were obtained with GM-CSF. CONCLUSION: These results demonstrate a differential effect of NO on monocyte and alveolar macrophages cytokine regulation and this effect may be related to the state of maturation.
Asunto(s)
Asma/inmunología , Citocinas/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Macrófagos Alveolares/efectos de los fármacos , Monocitos/efectos de los fármacos , Óxido Nítrico/farmacología , Adolescente , Adulto , Asma/genética , Asma/patología , Líquido del Lavado Bronquioalveolar , Quimiotaxis , Citocinas/genética , Femenino , Volumen Espiratorio Forzado , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Hipersensibilidad Inmediata/genética , Hipersensibilidad Inmediata/inmunología , Hipersensibilidad Inmediata/patología , Lipopolisacáridos/farmacología , Macrófagos Alveolares/metabolismo , Masculino , Persona de Mediana Edad , Monocitos/metabolismoRESUMEN
Alveolar macrophages regulate the inflammatory and immune responses within the lung through cytokine production. Nuclear factor kappa B (NF-kappaB), a transcription factor, controls the synthesis of cytokines such as interleukin 1beta, tumour necrosis factor alpha, and interleukin 8. In quiescent cells, NF-kappaB is located in the cytosol as a dimer of protein components (p50, p65) bound to an inhibitor (IkappaB). Upon activation, NF-kappaB translocates to the nucleus and binds to DNA. To determine the constitutive level of NF-kappaB activation in non-smoking normal volunteers, immunohistochemical analysis of alveolar macrophages from 29 subjects was performed with antibody directed against the p65 component of NF-kappaB. These results were confirmed in four subjects by electrophoretic mobility shift assay (EMSA). A human monocytic cell line, THP-1 with and without endotoxin stimulation was used as positive and negative controls, respectively. The mean number of positive cells was 4.1%+/-0.8. EMSA performed on whole cell extracts from four normal volunteers demonstrated minimal constitutive binding compared to the positive control. Supershift assay revealed the presence of the p65 dimer. By both immunohistochemistry and EMSA, alveolar macrophages from healthy non-smoking individuals demonstrate minimal NF-kappaB activation. Immunohistochemistry is a sensitive and quantifiable technique requiring only a minimal number of cells, and this technique may be useful in monitoring small changes in NF-kappaB activation in inflammatory diseases of the lung.
Asunto(s)
Macrófagos Alveolares/metabolismo , FN-kappa B/metabolismo , Citocinas/metabolismo , Humanos , Inmunohistoquímica , Activación de Macrófagos , FN-kappa B/análisisRESUMEN
OBJECTIVE: To determine whether inflammatory cytokine production by stimulated human alveolar macrophages is affected by perflubron exposure. DESIGN: Controlled laboratory investigation of alveolar macrophage function in vitro. SETTING: Research laboratory. SUBJECTS: Cultured alveolar macrophages obtained by bronchoalveolar lavage from eleven normal volunteers. INTERVENTIONS: Endotoxin-stimulated alveolar macrophages were treated with perflubron. MEASUREMENTS AND MAIN RESULTS: Alveolar macrophages were stimulated for 1 hr with lipopolysaccharide and then treated with perflubron for 23 hrs. Cell-free supernatants were collected and cytokines were assayed by enzyme-linked immunosorbent assay. Tumor necrosis factor-alpha, interleukin-1, and interleukin-6 were stimulated by lipopolysaccharide (endotoxin) and all of these cytokines were significantly (p < .05) inhibited by perflubron. Cell viability was not affected by perflubron. Basal cytokine concentrations from unstimulated alveolar macrophages were not altered by perflubron. CONCLUSIONS: Exposure of stimulated human alveolar macrophages to perflubron in vitro decreases cytokine production. This observation suggests that perflubron may have anti-inflammatory activity.
Asunto(s)
Medios de Contraste/farmacología , Citocinas/biosíntesis , Fluorocarburos/farmacología , Macrófagos Alveolares/efectos de los fármacos , Lavado Broncoalveolar , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Humanos , Hidrocarburos Bromados , Macrófagos Alveolares/metabolismoRESUMEN
High levels of nitric oxide (NO) have been reported in exhaled air of asthmatic individuals. Because alveolar macrophages (AM) are major producers of cytokines, and bronchoalveolar lavage fluid (BALF) from asthmatic individuals contains increased levels of inflammatory cytokines, this study was undertaken to determine whether NO modified the production of inflammatory cytokines by human AM. AM were obtained from normal volunteers by fiberoptic bronchoscopy. Tumor necrosis factor-alpha (TNF-alpha) production stimulated by lipopolysaccharide (LPS; 0.5 microg/ml) was measured with an enzyme-linked immunosorbent assay (ELISA). NO generated from 2,2-(hydroxynitrosohydrazono)-bis-ethanamine (DETA NONOate) (0.1 to 1.0 mM) inhibited TNF-alpha secretion in a dose-dependent manner. At 1 mM DETA NONOate, mean inhibition (+/- SEM) of TNF-alpha secretion was 56 +/- 4% (P = 0.002). To determine whether this effect was cytokine specific, interleukin-1beta (IL-1beta) and macrophage inflammatory protein-1alpha (MIP-1alpha) were evaluated, and DETA NONOate was also found to inhibit both of these cytokines. Basal cytokine levels from unstimulated AM were unaffected by NO. These findings indicate that NO is a potent inhibitor of cytokine production by stimulated human AM.