RESUMEN
Cytokine gene expression in T lymphocytes is a strictly regulated process, involving both stimulatory and inhibitory signals. beta-Adrenoceptor (betaAR) agonists are widely used in the treatment of asthma and are able to induce an inhibitory signal on immunological responses after binding to their specific receptors. In this study, the characterization of betaAR subtype(s) (beta1, beta2, and beta3) involved in the regulation of interleukin (IL)-3, IL-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon-gamma (IFN-gamma) mRNA accumulation was studied by using various betaAR agonists and antagonists. Concanavalin A (Con A)-induced IFN-gamma, GM-CSF, and IL-3 mRNAs are dose-dependently inhibited by the nonselective betaAR agonist isoproterenol and by the selective beta2AR agonist fenoterol. IL-4 mRNA accumulation was not susceptible to betaAR stimulation. The observed inhibition on IFN-gamma, GM-CSF, and IL-3 mRNA was blocked by the selective beta2AR antagonist ICI 118,551 (10(-6) M) and by timolol (10(-6) M), a nonselective antagonist. The selective beta1AR antagonist atenolol (0.3 x 10(-6) M) did not have any effect. Secretion of GM-CSF protein in the presence of increasing concentrations of isoproterenol followed a similar pattern as observed for GM-CSF mRNA. In addition, the betaAR-mediated inhibition of IFN-gamma, GM-CSF, and IL-3 mRNA accumulation and GM-CSF protein secretion were related to the accumulation of intracellular cyclic adenosine monophosphate (cAMP) levels. Although beta3AR mRNA was detectable in Con A-activated T lymphocytes, we could not demonstrate a functional activity in the regulation of cytokine expression: the beta3AR agonist BRL 37344 had no effect on the accumulation of the studied cytokine mRNAs, and did not significantly affect cellular cAMP levels. These data demonstrate that beta-agonist-induced inhibition of IFN-gamma, GM-CSF, and IL-3 mRNA accumulation is solely mediated by beta2-adrenoceptors.
Asunto(s)
ARN Mensajero/antagonistas & inhibidores , Receptores Adrenérgicos beta 2/fisiología , Linfocitos T/fisiología , Concanavalina A/farmacología , AMP Cíclico/metabolismo , Etanolaminas/farmacología , Fenoterol/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Humanos , Interferón gamma/genética , Interleucina-3/genética , Interleucina-4/metabolismo , Isoproterenol/farmacología , Timolol/farmacologíaRESUMEN
BACKGROUND: Dysfunction of the beta-adrenoceptor (betaAR)/adenylyl cyclase (AC) system can impair the response of different cell types, including lymphocytes. In asthma, impairment of this system as well as changes in cytokine production by lymphocytes have been described. Because the severity of asthma can change over the year, a circannual pattern of the betaAR/AC system activity may also exist. OBJECTIVES: We set out to examine the activity of this betaAR/AC signal transduction system in peripheral blood mononuclear cells (PBMCs) of allergic asthmatics to asses whether differences existed between seasons. We investigated whether changes were associated with asthma severity and circannual changes in serum cortisol levels. METHODS: During 19 months, 41 allergic asthmatics (mean age 28 years) with nocturnal airway obstruction were enrolled in the study. AC activity was measured by cyclic AMP production. Resting, stimulated and potentiated AC activities and their relationships with clinical parameters, seasonal influences and serum cortisol levels were assessed. RESULTS: The AC activity in resting, stimulated and potentiated cells varied during the year. AC activity was relatively low in the periods June-August and September-November, and higher in December-February and March-May. Receptor-mediated and potentiated responses expressed as percentage of the resting response were equivalent throughout the year. Serum cortisol levels were positively related to AC activity. No relationships were found between clinical parameters and AC activity or serum cortisol levels. CONCLUSIONS: These results indicate that AC activity in PBMCs of allergic asthmatics shows a seasonal variation. However, seasonal differences in AC activity seems to be unrelated with clinical parameters. Other factors such as serum cortisol levels may have a modulating influence on AC activity. Future studies of AC systems in blood cells of asthmatic patients need to take into account these seasonal influences.
Asunto(s)
Asma/metabolismo , AMP Cíclico/biosíntesis , Leucocitos Mononucleares/metabolismo , Estaciones del Año , Adenilil Ciclasas/sangre , Adulto , Obstrucción de las Vías Aéreas , Femenino , Humanos , Hidrocortisona/sangre , Activación de Linfocitos , Masculino , Receptores Adrenérgicos beta/sangre , Pruebas de Función RespiratoriaRESUMEN
BACKGROUND: In asthmatic inflammation, TH2 cells play an important role. TH2 cells specifically secrete cytokines like IL-4 and IL-5. IL-4 stimulates IgE production and IL-5 is involved in hemopoiesis, chemotaxis, priming and activation of eosinophils. IFNgamma, produced by TH1 cells, has an inhibitory action on IgE production. OBJECTIVES: To investigate the TH1/TH2-cell pattern in the cytokine production of peripheral blood of asthmatic children. We determined IL-4, IFNgamma and IL-5 in serum and in supernatants of unstimulated and stimulated (24 h with Concanavaline A) cultures of peripheral blood mononuclear cells (PBMCs) in 22 children with moderate asthma (mean age 9.3 years) and in 17 healthy controls (mean age 10.3 years). All children visited the out-patient department (OPD) where history taking, physical examination and blood sampling took place. Children younger than 8 years of age performed symptom and peak flow registration during 1 week after the visit to the OPD. RESULTS: The number of eosinophils were significantly higher in children with asthma, compared with healthy controls. The concentration of IFNgamma in supernatants of cultures of stimulated PBMCs was significantly lower and the ratio of IL-4/IFNgamma was significantly higher in children with asthma compared with healthy controls. The FEV1 was directly and IgE was inversely related to the concentration of IFNgamma in supernatants of cultures of stimulated PBMCs. CONCLUSION: IFNgamma may play an important role in the pathophysiology of childhood atopic asthma.
Asunto(s)
Asma/sangre , Interferón gamma/sangre , Interleucina-4/sangre , Interleucina-5/sangre , Leucocitos Mononucleares/inmunología , Adolescente , Niño , Preescolar , Eosinófilos/inmunología , Femenino , Volumen Espiratorio Forzado , Humanos , Inmunoglobulina E/sangre , Masculino , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
Using a newly developed guinea-pig model of asthma, characterized by allergen-induced early and late phase asthmatic reactions, bronchial hyperreactivity (BHR) and airway inflammation, the importance of eosinophil activation for the development of BHR to inhaled histamine was investigated at 6 h (after the early reaction) and 24 h (after the late reaction) after allergen provocation. Eosinophil activation was assessed by a sensitive kinetic assay for eosinophil peroxidase (EPO) activity, suitable for bronchoalveolar lavage (BAL) analysis. A significant 2.9-fold (P < 0.01) increase in bronchial reactivity to histamine was observed at 6 h after allergen exposure, which was associated with a 2.9-fold increase in the number of eosinophils (P < 0.05) and a 6.7-fold increase in EPO activity (P < 0.01) in the BAL fluid. At 24 h after allergen exposure the bronchial reactivity to histamine was lower (1.7-fold), but still significantly enhanced (P < 0.01). By contrast, the number of eosinophils was further increased compared with 6 h after provocation (3.8-fold, P < 0.05), while the EPO activity remained stable at 6 h levels. The number of eosinophils was significantly correlated with EPO activity at 6 h (r = 0.62; P < 0.05), but not at 24 h after provocation. No significant correlation was observed between the number of eosinophils in the BAL fluid and BHR to histamine at either time point.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Asma/fisiopatología , Hiperreactividad Bronquial/inmunología , Líquido del Lavado Bronquioalveolar/citología , Eosinófilos/inmunología , Animales , Asma/inmunología , Hiperreactividad Bronquial/fisiopatología , Pruebas de Provocación Bronquial , Peroxidasa del Eosinófilo , Eosinófilos/enzimología , Cobayas , Recuento de Leucocitos , Masculino , Peroxidasas/análisis , Pruebas de Función RespiratoriaRESUMEN
The isoprenaline-induced production of cAMP in human peripheral blood mononuclear cells (PBMC) was potentiated significantly by incubating PBMC with isoprenaline in the presence of phytohaemagglutinin (PHA), Concanavalin A (Con A) or A23187. This potentiation, that proved to be dependent on the concentration of PHA, Con A or A23187, increased the maximal response but did not cause a change in the potency of isoprenaline. Potentiation could not be induced by the phorbol ester phorbol-myristate acetate, suggesting that protein kinase C-dependent pathways are not likely to be involved in potentiation of adenylyl cyclase. Potentiation could be inhibited by chelating extracellular Ca2+ with EGTA and also by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamine, an inhibitor of calmodulin. Potentiation could not be induced by preincubation of PBMC with PHA, suggesting that transient biochemical changes are involved. It was concluded from these results that potentiation in PBMC probably involves the activation of Ca2+/calmodulin-dependent adenylyl cyclase subtypes. Potentiation of the adenylyl cyclase activity could be an important physiological mechanism in vivo preventing cells from becoming "over stimulated".
Asunto(s)
Adenilil Ciclasas/sangre , Isoproterenol/farmacología , Monocitos/enzimología , Calcimicina/farmacología , Concanavalina A/farmacología , AMP Cíclico/análisis , Sinergismo Farmacológico , Ácido Egtácico/farmacología , Activación Enzimática/efectos de los fármacos , Histamina/farmacología , Humanos , Fitohemaglutininas/farmacología , Prostaglandinas E/farmacología , Sulfonamidas/farmacologíaRESUMEN
We wanted to determine whether changes in bronchial hyperresponsiveness (BHR) following allergen challenge show a time relationship with inflammatory events in the airways of allergic asthmatic subjects. Lavage was performed and endobronchial biopsies were taken via the fiberoptic bronchoscope, before, and 3 and 24 h after, allergen challenge, on separate occasions, in nine dual asthmatic responders. The numbers of activated eosinophils, identified by immunohistochemistry, using the monoclonal anti-eosinophil cationic protein antibody, EG2, were significantly increased both at 3 h and at 24 h in the submucosa and bronchial lavage. A significant negative correlation was found between the number of EG2+ cells in the submucosa and in the bronchial lavage 24 h after the allergen challenge (r = -0.70). At 24 h, the amount of eosinophil cationic protein (ECP) was increased in the bronchial lavage. A significant correlation was observed between the amount of ECP at 3 h and the log provocative dose of house dust mite producing a 20% fall in forced expiratory volume in one second (PD20 HDM) (r = -0.63). The results suggest a recruitment of activated eosinophils to the submucosa and, further, to the epithelial lining, followed by degranulation. This process has already started 3 h after allergen challenge, and lasts for at least 24 h, which may result in mucosal damage and subsequent allergen-induced increase in BHR, before and after the late asthmatic reaction.
Asunto(s)
Asma/inmunología , Bronquios/inmunología , Hiperreactividad Bronquial/inmunología , Eosinófilos/inmunología , Adulto , Asma/patología , Asma/fisiopatología , Bronquios/patología , Hiperreactividad Bronquial/patología , Hiperreactividad Bronquial/fisiopatología , Pruebas de Provocación Bronquial , Líquido del Lavado Bronquioalveolar , Broncoscopía , Eosinófilos/fisiología , Femenino , Humanos , Inmunohistoquímica , Recuento de Leucocitos , Masculino , Membrana Mucosa/inmunología , Membrana Mucosa/patología , Pruebas de Función Respiratoria , Factores de TiempoRESUMEN
Human lymphocytes were labeled with biotinylated anti-lymphocyte-directed monoclonal antibodies, to which streptavidin and subsequently biotinylated dextran-magnetite particles were coupled. This labeling resulted in a strong and selective negative contrast enhancement of lymphocyte suspensions at 2.0 T, caused predominantly by the specific increase of R2 with a small but significant specific increase of R1. The R1 was found to decrease with increasing field strength. The immunolabeling procedure described here may be used for the selective signal depletion of target cells in MR imaging.