RESUMEN
BACKGROUND: Lung fibroblasts may have a pivotal role in airway inflammation as they are involved in continuous cycles of mediator secretion, proliferation, activation and cross-talk with recruited inflammatory cells. The role of fibroblasts as intermediate participants in the inflammatory network suggests that they could represent an important target for drugs commonly used in asthma; thus, we investigated the effects of triamcinolone acetonide (TAA) on primary human lung fibroblasts. METHODS: The in vitro activity of increasing concentrations (10(-9) to 10(-7) M) of TAA in fibroblast cultures was evaluated as regards the following parameters: proliferation, extracellular matrix (ECM) release, cytokine/chemokine secretion and surface antigen expression. RESULTS: All concentrations of TAA decreased fetal calf serum (FCS)-induced fibroblast proliferation, whereas in the presence of FCS plus basic fibroblast growth factor TAA was only effective at 10(-8) and 10(-7) M. TAA failed to decrease ECM, whereas at 10(-8) and 10(-7) M it decreased IL-6 and IL-8 secretion to different extents. In the presence of IFN-gamma the drug was able to reduce VCAM-1 expression at all of the tested concentrations; on the other hand, in TGF-beta 1-driven cultures a decrease in CD54 expression was detected with TAA at 10(-8) and 10(-7) M. CONCLUSIONS: TAA acts on some functional properties of human lung fibroblasts that make these cells active participants in the inflammatory network. The ability of TAA to inhibit lung fibroblast proliferation may prevent or even reverse some of the histological changes that characterize airway remodeling in chronic inflammatory diseases; moreover, IL-6, IL-8 and surface molecule decreases by TAA may suggest a direct anti-inflammatory effect of the drug by suppression of resident lung cell function.
Asunto(s)
Glucocorticoides/farmacología , Pulmón/efectos de los fármacos , Triamcinolona Acetonida/farmacología , División Celular/efectos de los fármacos , División Celular/inmunología , Colágeno/inmunología , Colágeno/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Fibroblastos/metabolismo , Fibronectinas/inmunología , Fibronectinas/metabolismo , Glucocorticoides/inmunología , Glucocorticoides/uso terapéutico , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/inmunología , Interferón gamma/inmunología , Interleucina-6/biosíntesis , Interleucina-6/inmunología , Interleucina-8/biosíntesis , Interleucina-8/inmunología , Pulmón/citología , Pulmón/inmunología , Pulmón/metabolismo , Masculino , Persona de Mediana Edad , Triamcinolona Acetonida/inmunología , Triamcinolona Acetonida/uso terapéutico , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Molécula 1 de Adhesión Celular Vascular/inmunologíaRESUMEN
Asthma is characterized by an irreversible subepithelial fibrosis with the appearance of myofibroblasts, which can be now considered important early participants in inflammatory responses as well as potential targets for anti-inflammatory drugs. In this study, we show that fluticasone propionate (FP), a powerful inhaled corticosteroid (ICS), displays novel anti-inflammatory effects on human lung fibroblasts during their myofibroblastic differentiation. Indeed, FP inhibits in lung myofibroblasts, at a very early stage of differentiation, the activation of Janus kinase/STAT pathways induced by IL-13 (tyrosine kinase 2, STAT1, STAT3, STAT6, mitogen-activated protein kinase). Contrarily, in mildly or fully differentiated myofibroblastic cultures, FP still displays a potential anti-inflammatory activity even if it only inhibits tyrosine kinase 2 phosphorylation. Moreover, FP inhibits constitutive and TGF-beta-induced expression of alpha-smooth muscle actin, the main marker of myofibroblastic differentiation, both in very early and in mild differentiated myofibroblasts. Finally, FP displays an additional powerful anti-inflammatory effect, decreasing nuclear translocation of NF-kappaB independent of the degree of myofibroblastic differentiation. These data 1) suggest that myofibroblasts are priority targets for ICS, which is able to revert them to a normal phenotype even if they appear to be already engaged in their differentiation, and 2) may help to explain why asthma is improved by an early ICS treatment, whereas advanced asthma is more resistant to these drugs.
Asunto(s)
Androstadienos/farmacología , Antiinflamatorios/farmacología , Pulmón/efectos de los fármacos , Proteínas Tirosina Quinasas , Actinas/análisis , Administración por Inhalación , Adulto , Androstadienos/administración & dosificación , Diferenciación Celular , Células Cultivadas , Proteínas de Unión al ADN/fisiología , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Fluticasona , Humanos , Interleucina-13/farmacología , Interleucina-4/farmacología , Pulmón/citología , Microscopía Confocal , FN-kappa B/metabolismo , Proteínas/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3 , TYK2 Quinasa , Transactivadores/fisiologíaRESUMEN
We studied whether different bronchial responses to allergen in asthma and rhinitis are associated with different bronchial inflammation and remodeling or airway mechanics. Nine subjects with mild asthma and eight with rhinitis alone underwent methacholine and allergen inhalation challenges. The latter was preceded and followed by bronchoalveolar lavage and bronchial biopsy. The response to methacholine was positive in all asthmatic but in only two rhinitic subjects. The response to allergen was positive in all asthmatic and most, i.e., five, rhinitic subjects. No significant differences between groups were found in airway inflammatory cells or basement membrane thickness either at baseline or after allergen. The ability of deep inhalation to dilate methacholine-constricted airways was greater in rhinitis than in asthma, but it was progressively reduced in rhinitis during allergen challenge. We conclude that 1) rhinitic subjects may develop similar airway inflammation and remodeling as the asthmatic subjects do and 2) the difference in bronchial response to allergen between asthma and rhinitis is associated with different airway mechanics.
Asunto(s)
Asma/inmunología , Hiperreactividad Bronquial/inmunología , Broncoconstricción/inmunología , Rinitis/inmunología , Adulto , Alérgenos , Membrana Basal/inmunología , Membrana Basal/patología , Biopsia , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Broncoconstricción/efectos de los fármacos , Broncoconstrictores , Volumen Espiratorio Forzado , Humanos , Masculino , Cloruro de Metacolina , Análisis de Regresión , Capacidad VitalRESUMEN
BACKGROUND: Mizolastine is a potent, peripherally acting, selective H1-receptor antagonist with potential anti-inflammatory properties. The aim of the study was to evaluate the in vitro effects of mizolastine on the expression of adhesion molecules by primary human airway epithelial and stromal cultures; moreover, the activity of mizolastine on parameters which reflect the immune response efficacy was investigated. METHODS: Airway epithelial and stromal cells were collected from hypereosinophilic subjects by enzymatic digestion of polyps or turbinates. Cells were stimulated with interferon (IFN)-gamma (500 IU/ml) in the presence of various mizolastine concentrations (6 x 10(-8)-6 x 10(-6) M) for 24 h and the expression of CD106, CD54, CD58 and HLA class I was evaluated. Peripheral blood mononuclear cells from healthy volunteers were incubated with 1% phytohemagglutinin or anti-CD3 monoclonal antibody (20 ng/ml) in the presence of mizolastine, then T lymphocyte proliferation, HLA-DR expression and T cell subpopulations were evaluated. RESULTS: Both in epithelial and stromal cultures, IFN-gamma significantly upregulated all of the tested surface molecules (p<0.05). The highest dose of mizolastine (6 x 10(-6) M), corresponding to 10-fold the peak plasma level after a single oral administration of 10 mg, was able to act on fibroblasts, significantly downregulating the expression of CD54 (p<0.05). Regarding T lymphocyte proliferation, the addition of mizolastine did not induce any significant change; furthermore, mizolastine was ineffective at all of the tested concentrations on both HLA-DR expression and CD4+/CD8+ ratio. CONCLUSIONS: This study demonstrated that mizolastine is able to selectively downregulate CD54 expression on stimulated stromal but not epithelial cells without impairing the immune system effectors. The possible clinical significance of these results are an antiallergic property and CD54 modulation on fibroblasts with a good safety profile as far as the lymphocyte response is concerned.
Asunto(s)
Bencimidazoles/farmacología , Antagonistas de los Receptores Histamínicos H1/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Mucosa Nasal/efectos de los fármacos , Adolescente , Adulto , Anciano , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Humanos , Molécula 1 de Adhesión Intercelular/análisis , Interferón gamma/farmacología , Leucocitos Mononucleares/inmunología , Activación de Linfocitos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Mucosa Nasal/inmunología , Pólipos Nasales/tratamiento farmacológico , Pólipos Nasales/inmunología , Linfocitos T/efectos de los fármacosRESUMEN
beta2-adrenoreceptor agonists have the ability to downregulate in vitro the proliferative response of peripheral blood mononuclear cells (BMCs). This activity could be related to a variety of beta2-adrenoreceptor-mediated functions, including induction of cell apoptosis in activated T-cells. To test this hypothesis, BMCs from atopic subjects, sensitized to house dust mites (Dermatophagoides [Der p]) and/or to Parietaria were incubated with fenoterol (10(-8)-10(-5) M) in the presence of (a) purified allergen extracts (Der p [5 microg/mL] or Parietaria [5 microg/mL]) or (b) antigens (tetanus toxoid [1 microg/mL] or Candida albicans [5 x 10(5) bodies/mL]). The BMC proliferation was assessed by [3H] thymidine incorporation and cell apoptosis was assessed by evaluating DNA fragmentation by a fluorescence technique, using propidium iodide. In cultures stimulated with Der p or with Parietaria, fenoterol induced a dose-dependent inhibition of BMC proliferation, significant also at the lowest concentration tested (10(-8) M) (p < 0.05, each comparison). In contrast, the inhibitory activity of the drug on tetanus-toxoid-stimulated BMCs was significant only at the highest dose tested (10(-5)M) (p < 0.05), whereas no effect was seen when BMCs were stimulated with C. albicans extract (p > 0.05). The different inhibitory efficacy of fenoterol appeared to be related to the degree of activation of beta2-adrenoreceptors on the different BMC populations that responded to the different stimuli. Indeed, in the presence of fenoterol (10(-6) and 10(-5)M), a significant increase in cyclic adenosine monophosphate (cAMP) levels was seen in Der p- or Parietaria-stimulated cells (p < 0.05; each comparison), but not in cell cultures stimulated with tetanus toxoid or with C. albicans extracts (p > 0.05; each comparison). Finally, the percentage of cells with fragmented DNA was lower in cultures stimulated with Der p or Parietaria than in those stimulated with tetanus toxoid or C. albicans, and the presence of fenoterol did not modify cell apoptosis (p > 0.05; each comparison). Thus, the different inhibitory activity of fenoterol on BMCs activated by allergens (Der p or Parietaria) or by antigens (tetanus toxoid or C. albicans) seems to be related to differences in beta2-adrenoreceptor expression and/or function in the different antigen-specific T-cell subsets, but it is not influenced by changes in cell apoptosis.
Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Alérgenos/inmunología , Fenoterol/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Adulto , Animales , Apoptosis/efectos de los fármacos , Candida albicans/inmunología , AMP Cíclico/metabolismo , Femenino , Humanos , Técnicas In Vitro , Leucocitos Mononucleares/inmunología , Activación de Linfocitos , Masculino , Ácaros/inmunología , Receptores Adrenérgicos beta 2/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Linfocitos T/inmunología , Toxoide Tetánico/inmunologíaRESUMEN
Recently, airway fibroblasts captured the attention of both allergists and basic scientists since they are no longer considered as mere bystanders, as far as allergic airway diseases are concerned. The aim of the present study was to assess the effects of different Cetirizine (Cet) concentrations (0.01, 0.05, 0.1 mg/ml) on human airway fibroblast proliferation and on CD54 expression. By means of flow cytometry analysis, we evaluated CD54 expression by airway fibroblasts in basal conditions or after gammaIFN stimulation in the presence of Cetirizine; we also evaluated the effect of the drug on cell proliferation by a [3H]thymidine incorporation assay. All of the tested doses of Cetirizine were able to significantly reduce CD54 upregulation induced by gammaIFN; concerning the fibroblast proliferation, we observed a dose-dependent inhibition of [3H]thymidine incorporation. These results show that Cetirizine exerts a biologic effect directly on human airway fibroblasts, suggesting a new rationale in the use of this compound.
Asunto(s)
Antialérgicos/farmacología , Cetirizina/farmacología , Regulación hacia Abajo , Fibroblastos/efectos de los fármacos , Antagonistas de los Receptores Histamínicos H1/farmacología , Mucosa Nasal/citología , División Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Fibroblastos/citología , Fibroblastos/inmunología , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Interferón gamma/farmacologíaRESUMEN
OBJECTIVE: To investigate whether the state of activation of circulating T-cells in childhood asthma could be related to serum IgE levels and/or to blood eosinophilia. METHODS: Seventeen atopic asthmatic children, sensitized to Dermatophagoides pteronyssinus (Der p), in stable condition at the time of the study and 15 sex-matched and age-matched controls were studied. The expression of activation surface markers (HLA-DR and CD25) on peripheral blood mononuclear cells (PBMCs) was tested by monoclonal antibodies and FACS analysis, while the PBMC proliferative response to Der p antigens was measured by tritiated thymidine (3HTdR) incorporation. RESULTS: As compared to controls, atopic children showed higher eosinophil counts (P < .01), similar lymphocyte counts (P > .1, each comparison) but higher proportion of HLA-DR+ and CD25+ T-lymphocytes (P < .05, each comparison). A significant Der p allergen-induced PBMC proliferation was observed in atopic children (P < .01) but not in controls (P > .1). Both in controls and in atopic children, no correlations were found between lymphocyte counts and eosinophil counts or total or allergen-specific IgE levels (P > .1, each comparison). In contrast, weak correlations were detected between the degree of allergen-induced PBMC proliferation and: a) allergen-specific IgE levels in serum (P < .05) and b) eosinophil counts (P < .05). CONCLUSION: These data support the concept that the degree of activation of allergen-specific T-lymphocytes in blood may reflect the intensity of allergic sensitization in childhood asthma.
Asunto(s)
Asma/inmunología , Eosinofilia , Glicoproteínas/inmunología , Hipersensibilidad Inmediata/inmunología , Inmunoglobulina E/sangre , Linfocitos T/inmunología , Alérgenos/inmunología , Antígenos Dermatofagoides , Niño , Preescolar , Femenino , Antígenos HLA-DR/metabolismo , Humanos , Activación de Linfocitos , Recuento de Linfocitos , Masculino , Receptores de Interleucina-2/metabolismo , Pruebas Cutáneas , Subgrupos de Linfocitos T , Árboles/inmunologíaRESUMEN
Patients with chronic obstructive lung disorders often show increased susceptibility to airway infections. As beta 2-adrenoceptor agonists, in addition to reversing the contractile response of bronchial smooth muscles, may inhibit a variety of inflammatory and immuno-effector cell functions, it is possible that these drugs interfere with host defence mechanisms. The present study was designed to test in vitro whether fenoterol, a short-acting beta 2-adrenoceptor agonist, could modify human blood neutrophil recruitment and antimicrobial activity. Pre-exposure to fenoterol significantly reduced neutrophil migration towards the complement component C5a, at concentrations ranging from 10(-7) M to 10(-5) M, or towards lipopolysaccharide, at a concentration of 10(-5) M (P < 0.05, each comparison). In contrast, the drug (10(-8)-10(-5) M) did not significantly modify the increased expression of lymphocyte function-associated antigen (LFA-1, i.e. CD11a/CD18) the macrophage antigen-1 (Mac-1, i.e. CD11b/CD18) induced by N-formylmethionylleucylphenylalanine (fMLP) (P > 0.05, each comparison). Finally, incubation of neutrophils with fenoterol (10(-8)-10(-5) M) did not significantly influence phagocytosis or intracellular killing of bacteria (Staphylococcus aureus) or H2O2 release induced by tetradecanoyl-phorbol-acetate (P > 0.1 for each comparison). These results suggest that short-acting beta 2-adrenoceptor agonists, such as fenoterol, are able partially to reduce neutrophil recruitment in the airways without interfering with the processes involved in phagocytic activity against bacteria.
Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Quimiotaxis de Leucocito/efectos de los fármacos , Fenoterol/farmacología , Enfermedades Pulmonares Obstructivas/inmunología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Adolescente , Adulto , Femenino , Humanos , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Antígeno de Macrófago-1/metabolismo , Masculino , Receptores Adrenérgicos/inmunologíaRESUMEN
BACKGROUND: Alveolar macrophages (AMs) are more efficient antigen-presenting cells in allergic individuals than in nonatopic subjects. OBJECTIVE: We studied whether this difference may be correlated to increased expression of membrane costimulatory molecules, such as the B7 molecules (CD80 and CD86). METHODS: Eleven subjects with allergic asthma sensitized to Dermatophagoides pteronyssinus and 5 healthy nonatopic volunteers underwent bronchoalveolar lavage, and the costimulatory molecule expression on AMs was evaluated. Peripheral blood T cells, either freshly isolated or as established D pteronyssinus -specific cell lines, were cultured with autologous monocytes or AMs as antigen-presenting cells. In vitro allergen-induced proliferation and cytokine production were evaluated in the presence of B7-blocking reagents. RESULTS: Allergic individuals had a significantly higher proportion of AMs expressing the CD80 molecule than control subjects (28.5% +/- 14.8% vs 1.4% +/- 1.2%; P <.001), whereas no difference was observed in CD86 expression (2.0% +/- 2.3% vs 1.1% +/- 0.6; P >.1). In a large proportion of the asthmatic subjects we studied, AMs were presenting soluble antigens (tetanus toxoid and streptolysin-O) to freshly isolated T cells more efficiently than AMs from nonatopic control subjects. Finally, both T-cell proliferation and cytokine production of D pteronyssinus- specific established T-cell lines were inhibited by a CD80-blocking antibody in a dose-dependent manner. CONCLUSION: Costimulation by means of CD80 expressed by AMs is probably involved in the amplification of the allergen-specific T-lymphocyte response in the airways of asthmatic subjects.
Asunto(s)
Células Presentadoras de Antígenos/fisiología , Asma/metabolismo , Antígeno B7-1/biosíntesis , Macrófagos Alveolares/metabolismo , Células Th2/inmunología , Adolescente , Adulto , Antígenos CD/biosíntesis , Antígeno B7-1/fisiología , Antígeno B7-2 , Líquido del Lavado Bronquioalveolar/citología , Citocinas/biosíntesis , Femenino , Humanos , Interleucina-4/biosíntesis , Interleucina-5/biosíntesis , Masculino , Glicoproteínas de Membrana/biosíntesis , Pruebas de Función RespiratoriaRESUMEN
Treatment of allergic asthma with inhaled corticosteroids results in local down-regulation of proinflammatory cytokine synthesis and in marked decrease in tissue eosinophilia. Blood concentrations of inhaled corticosteroids, although significantly lower than those measured in the lung, may still have antiinflammatory effects on circulating eosinophils, reducing their ability to migrate. The aim of our study was to evaluate in vitro the activity of budesonide on blood eosinophils by measuring their chemotactic response, eosinophil cationic protein (ECP) release, and hydrogen peroxide (H2O2) production in the presence of different drug concentrations similar to those obtained at airway level (10(-8) and 10(-7) M) and at blood level (10(-10) and 10(-9) M). Partially purified blood eosinophils, isolated from 23 asthmatic subjects, were used to evaluate the activity of budesonide on: (1) chemotaxis toward the activated fifth component of complement (C5a, 0.1 microg/ml) or recombinant human (rh) interleukin (IL)-5 (200 pg/ml), (2) ECP release by cells stimulated with tetradecanoylphorbol acetate (TPA) and (3) H2O2 production by TPA-activated cells. The chemotactic response to C5a was down-regulated significantly by budesonide only by the highest concentrations tested (10(-8) and 10(-7) M); differently, budesonide was effective in inhibiting eosinophil migration toward rhIL-5, at all concentrations tested (p < 0.01, each comparison). By contrast, no drug-induced modifications were observed in ECP release or in H2O2 production (p > 0.05, each comparison). We conclude that concentrations of budesonide similar to those obtained in vivo are effective in inhibiting eosinophil locomotion but not in down-regulating the release of reactive oxygen species and granule-associated proteins.
Asunto(s)
Antiinflamatorios/farmacología , Budesonida/farmacología , Eosinófilos/efectos de los fármacos , Ribonucleasas , Administración Tópica , Adolescente , Asma/sangre , Asma/inmunología , Proteínas Sanguíneas/metabolismo , Movimiento Celular/efectos de los fármacos , Regulación hacia Abajo , Proteínas en los Gránulos del Eosinófilo , Femenino , Glucocorticoides , Humanos , Peróxido de Hidrógeno/metabolismo , Técnicas In Vitro , Mediadores de Inflamación/metabolismo , MasculinoRESUMEN
In allergic asthma, there is convincing evidence that changes in eosinophil and lymphocyte state of activation in blood may reflect disease activity. We evaluated whether simple blood eosinophil or lymphocyte counts in atopic children with asthma could reflect the degree of allergic sensitization. Seventy-six asthmatic children, sensitized to house dust mites (HDM), in stable conditions at the time of the study, and 53 sex- and age-matched controls (CTR) were studied. As compared to CTR, allergic patients showed higher eosinophil numbers and percentages (p < 0.001) but similar lymphocyte numbers and proportions (p > 0.1). Both in CTR and in allergic patients, eosinophil counts did not correlate with lymphocyte counts (p > 0.05; each comparison) but positive correlations were observed between eosinophil numbers and percentages and paper radio immunosorbent test (PRIST) levels or radio-allergo sorbent test (RAST) classes (p < 0.001; each comparison). When allergic asthmatic individuals were subdivided according to their age into two subgroups (Gr), no differences were found in eosinophil and lymphocyte counts and in PRIST levels and RAST values between Gr1 (< or =5 years old [preschool children]) and Gr2 (>5 years old [school children]) (p > 0.05; each comparison). Interestingly, although positive correlations between eosinophil counts and PRIST levels were found in both subgroups (p < 0.05; each comparison), only in Gr2 did eosinophil counts correlate positively with RAST classes (p < 0.001). No correlations between lymphocyte counts and PRIST levels or RAST classes were demonstrated (p > 0.05; each comparison). These data suggest that although blood eosinophilia was similar in preschool and in allergic asthmatic school children sensitized to HDM, only in the oldest children did blood eosinophil counts appear to be related to the degree of HDM-specific sensitization.
Asunto(s)
Asma/inmunología , Eosinofilia/complicaciones , Ácaros/inmunología , Adolescente , Animales , Asma/complicaciones , Niño , Preescolar , Polvo , Eosinófilos , Femenino , Humanos , Hipersensibilidad Inmediata , Inmunoglobulina E/sangre , Lactante , Recuento de Leucocitos , MasculinoRESUMEN
Inflammatory airway disorders, such as asthma and chronic bronchitis, are characterized by overexpression of adhesion molecules on airway epithelial and endothelial cells. This phenomenon is associated with increased adherence and activation of polymorphonuclear leukocytes (PMNs). With the knowledge that beta2-adrenoceptor agonists demonstrate some anti-inflammatory activity in vitro, the present study was designed to evaluate whether fenoterol could interfere with adhesion molecule expression on airway epithelium. Human bronchial epithelial cells (HBECs), obtained by protease digestion from surgically resected bronchi, were stimulated with human recombinant interferon-gamma (rh IFN-gamma) in the presence of (a) fenoterol (10(-12)-10(-5) M); (b) dexamethasone (10(-12)-10(-5) M); and (c) fenoterol and dexamethasone. Because desensitization after high-dose exposure to agonists has been described for many membrane-associated receptors, in additional sets of experiments HBECs were preexposed to fenoterol and, as control, to dexamethasone for 8 hr, then washed and stimulated with rh IFN-gamma in the presence of fresh drugs. The cells were harvested after 24-hr culture and stained by specific monoclonal antibodies. The intensity of intercellular adhesion molecule-1 (ICAM-1) expression was then measured by flow cytometry analysis and expressed as mean fluorescence channel (mfc). The significant increase in ICAM-1 expression on HBECs induced by rh IFN-gamma was inhibited, in a dose-dependent manner, by the two drugs, but fenoterol was more efficient than dexamethasone at all of the concentrations tested (p < 0.05, all comparisons). In addition, the inhibitory activity of fenoterol was not enhanced by the simultaneous presence of dexamethasone in rh IFN-gamma-stimulated HBEC cultures (p > 0.05, all comparisons). Finally, preexposure to fenoterol or to dexamethasone did not induce any modification of the inhibitory effect of the two drugs on ICAM-1 expression (p > 0.05, all comparisons). These results suggest that clinical efficacy of fenoterol in patients with obstructive lung disease may include downregulation of adhesion molecule expression on airway epithelial cells.
Asunto(s)
Agonistas Adrenérgicos beta/uso terapéutico , Broncodilatadores/uso terapéutico , Fenoterol/uso terapéutico , Molécula 1 de Adhesión Intercelular/biosíntesis , Interferón gamma/farmacología , Antiinflamatorios/uso terapéutico , Células Cultivadas , Dexametasona/uso terapéutico , Regulación hacia Abajo , Sinergismo Farmacológico , Células Epiteliales/efectos de los fármacos , Humanos , Proteínas RecombinantesRESUMEN
The possibility that salmeterol could interfere with the inhibitory effect of glucocorticosteroids on allergen-induced activation of leukocytes was assessed in cultures of human blood mononuclear cells (BMCs). BMCs, recovered from 27 patients (21 +/- 4 years of age) sensitized to Dermatophagoides pteronyssinus (Der p), were incubated with a purified Der p extract (10 micrograms/ml) for 7 days in the presence of salmeterol (10(-8) and 10(-7) M) and/or dexamethasone (10(-10) and 10(-9) M). BMC proliferation and cytokine release were respectively tested by [3H]thymidine incorporation and EASIA. Stimulation with Der p extract induced a significant BMC proliferation (Der p 18.7 +/- 1.9 cpm x 10(3), control 1.8 +/- 0.3 cpm x 10(3), p < 0.01) which was significantly inhibited both by salmeterol and dexamethasone (p < 0.05, each comparison). The inhibition of BMC proliferation induced by dexamethasone (10(-9) M) was significantly enhanced by salmeterol 10(-7) M (p < 0.05). Evaluation of cytokine levels in BMC supernatants showed that Der p extract significantly increased the release of granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta and IL-2 (p < 0.001, each comparison). Salmeterol (10(-7) M) or dexamethasone (10(-9) M) induced a similar, statistically significant, downregulation of GM-CSF release (p < 0.05, each comparison) and induced a slight, not significant, decrease in the production of IFN-gamma, TNF-alpha, IL-1 beta and IL-2 (p > 0.05 each comparison). Interestingly, salmeterol (10(-7) M) significantly enhanced the inhibitory activity of dexamethasone (10(-9) M) on the release of GM-CSF, TNF-alpha, IL-1 beta and IL-2 (p < 0.05, each comparison) but not of IFN-gamma (p > 0.05). Thus beta 2-adrenoceptors may enhance the inhibitory activity of low doses of corticosteroids on allergen-induced BMC activation.
Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Albuterol/análogos & derivados , Citocinas/metabolismo , Dexametasona/farmacología , Glucocorticoides/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Adulto , Albuterol/farmacología , Alérgenos , División Celular , Células Cultivadas , Sinergismo Farmacológico , Femenino , Humanos , Leucocitos Mononucleares/inmunología , Masculino , Xinafoato de SalmeterolRESUMEN
Allergen exposure in atopic asthmatic patients is associated with recruitment and activation of eosinophils in the airways. Once activated, eosinophils release toxic products, including the eosinophil cationic protein (ECP), able to damage bronchial structures and to increase bronchial hyperresponsiveness. With this background, the present study was designed to evaluate whether ECP levels in bronchoalveolar lavage (BAL) fluid could reflect, better than BAL eosinophil counts, the cellular activation that follows allergen exposure in atopic asthmatics. Twenty-two atopic patients attended the laboratory on two separate days. On the 1st day, they underwent methacholine (MCh) inhalation challenge to detect the degree of nonspecific bronchial hyperresponsiveness. On the 2nd day, they underwent fiberoptic bronchoscopy and BAL, at baseline or 4-6 h after allergen inhalation challenge. In this latter patient group, MCh challenge was repeated 3-5 h after allergen challenge, 1 h before fiberoptic bronchoscopy. The analysis of the mean baseline FEV1 values and the degree of bronchial reactivity to MCh (MCh Pd20) on the 1st study day did not demonstrate differences between the two patient groups (p > 0.1, each comparison). In addition, in the allergen-challenged group, MCh Pd20 was decreased significantly after allergen challenge (151.4 micrograms/ml and 67.6 micrograms/ml, respectively, before and after challenge; p < 0.05). Evaluation of the different BAL cell types demonstrated that the proportions of eosinophils and epithelial cells were increased significantly in the allergen-challenged group compared with the group evaluated at baseline (p < 0.01 and p < 0.05, respectively). Moreover, ECP levels, corrected by the correspondent albumin levels (ECP/Alb), were higher in the allergen-challenged group compared with the group evaluated at baseline (p < 0.05). In addition, although a positive correlation was demonstrated between BAL eosinophil percentages and ECP/Alb values (r = 0.72, p < 0.05) in the group evaluated at baseline, no links were found between these parameters in the allergen-challenged group (p > 0.1). However, in this latter group, a weak positive correlation was demonstrated between eosinophil percentages and delta Mch, i.e., the increased non-specific bronchial reactivity, which is observed after allergen challenge (r = 0.55; p < 0.05). Thus, in stable asthmatic patients an ongoing activation of eosinophils parallels their migration, but this eosinophilic inflammation is not strictly related to bronchial reactivity to Mch. By contrast, after allergen inhalation challenge, eosinophil recruitment and activation seem to follow different temporal kinetics, and eosinophilic inflammation may be partially associated with the degree of airway hyperresponsiveness.
Asunto(s)
Alérgenos , Asma/fisiopatología , Hiperreactividad Bronquial/fisiopatología , Eosinofilia Pulmonar/etiología , Ribonucleasas , Adolescente , Adulto , Asma/inmunología , Proteínas Sanguíneas/metabolismo , Hiperreactividad Bronquial/inmunología , Pruebas de Provocación Bronquial , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Broncoconstrictores , Broncoscopía , Estudios de Casos y Controles , Proteínas en los Gránulos del Eosinófilo , Eosinófilos/metabolismo , Femenino , Humanos , Mediadores de Inflamación/metabolismo , Masculino , Cloruro de Metacolina , Eosinofilia Pulmonar/inmunologíaRESUMEN
Recently, much attention has been given to the possible role of lymphocytes and their soluble products in causing and maintaining allergic inflammation. The aim of this study was to assess the production of mRNAs for interleukins (IL) in bronchoalveolar lavage (BAL) cells obtained from allergic asthmatics after challenge with the relevant allergen in the period between early and late reactions. We evaluated BAL fluid cells obtained from six asthmatic subjects and four nonatopic controls. Challenge was performed with the relevant allergen. BAL fluid cells were obtained by fiberoptic bronchoscopy and bronchoalveolar lavage. To detect mRNA encoding each cytokine in BAL cells we used a reverse transcriptase polymerase chain reaction method. We evaluated IL-1 alpha, -2, -4, -5, -6, -13, and granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon-gamma (IFN-gamma). mRNAs for IL-1 alpha, -2, -4, -5, and IFN-gamma were detected in all of the atopic subjects; mRNAs for IL-6 and GM-CSF were found in five asthmatics; and mRNA for IL-13 was found in one patient only. In contrast, no mRNAs for IL-2, -4, -5, -6, -13, and GM-CSF were detected in the nonatopic healthy controls; mRNA for IL-1 alpha was found in one out of four normal subjects; and mRNA for IFN-gamma was evidenced in two of four subjects. The cellular environment in BAL fluids from allergic asthmatics before the clinical appearance of the late airway reaction shows an unrestricted expression of mRNA for cytokines. The local cytokine milieu could have an important role in the modulation of bronchial inflammation and in the appearance of allergic symptoms.
Asunto(s)
Asma/metabolismo , Líquido del Lavado Bronquioalveolar/química , Citocinas/genética , ARN Mensajero/biosíntesis , Adulto , Alérgenos/inmunología , Asma/inmunología , Líquido del Lavado Bronquioalveolar/citología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Humanos , Interferón gamma/genética , Interleucinas/genética , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Factores de TiempoRESUMEN
beta 2-adrenoceptor agonists are known to attenuate several functions of human mononuclear cells in response to activation. Since monocytes and lymphocytes play a major pathogenetic role in allergic asthma, this study was designed to evaluate the hypothesis that salmeterol, a beta 2-adrenoceptor agonist with a long duration of action, could inhibit in vitro the allergen-induced activation of blood mononuclear cells (BMCs). BMCs were collected from subjects sensitized to Dermatophagoides pteronyssinus (Der p) and incubated with a purified Der p allergen extract to evaluate the ability of salmeterol to downregulate; (a) BMC proliferation; (b) IL-2 receptors (r) and HLA-DR surface antigen (ag) expression; (c) cytokine release. Der p induced a significant BMC proliferation (P < 0.01), associated with increased expression of HLA-DRag and IL-2r (P < 0.05) and with enhanced release of IL-2, GM-CSF, IL-1 beta, TNF-alpha and IFN-gamma (P < 0.01, each comparison). Salmeterol (10(-8)-10(-6) M) significantly inhibited, in a dose dependent manner, the Der p-induced BMC proliferation, reducing (at 10(-7) M) HLA-DRag expression on monocytes and GM-CSF release (P < 0.05, each comparison). These data demonstrate that beta 2-adrenoceptor-mediated suppression of allergen-induced BMC activation is associated with inhibition of cytokine release and of surface molecule expression, which are involved in the interaction between T-lymphocytes and antigen-presenting cells.
Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Albuterol/análogos & derivados , Alérgenos/farmacología , Broncodilatadores/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Antígenos HLA-DR/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Adulto , Albuterol/farmacología , División Celular/efectos de los fármacos , Citocinas/metabolismo , Femenino , Humanos , Leucocitos Mononucleares/metabolismo , Masculino , Receptores de Interleucina-2 , Xinafoato de SalmeterolRESUMEN
To characterize the cellular inflammation at the bronchial and bronchoalveolar levels, we evaluated 43 patients with asthma who were sensitized to house dust mites. On 2 consecutive days patients underwent methacholine challenge and allergen bronchial challenge. In addition, 6, 24, or 72 h after allergen challenge, fiberoptic bronchoscopy with bronchial lavage (BL) and bronchoalveolar lavage (BAL) was performed. Patients belonging to the 6-h, 24-h, or 72-h group were divided further into two subgroups: those with isolated early response to allergen (LAR-), and those with dual response to allergen (LAR+). The percentage of eosinophils and of epithelial cells in BAL fluid was significantly higher in LAR+ than in LAR- patients in the 6-h group (p < 0.05, each comparison), but not 24 or 72 h after (p > 0.05, each comparison). Similarly, the proportion of BL eosinophils was also higher in LAR+ than in LAR- patients, both in the 6-h and in the 24-h group (p < 0.05, each comparison). In addition, increased proportions of BL neutrophils were present in the LAR+ patients belonging to the 24-h group (p < 0.05). Comparing "proximal" = BL vs "distal" = BAL data, we found a significantly higher proportion of epithelial cells in BL compared with BAL, in both LAR- and LAR+ subjects, either 6, or 24, or 72 h after challenge (p < 0.01, each comparison) and increased percentages of BL neutrophils and eosinophils in LAR+ patients (p < 0.05, each comparison), but not in LAR- patients, in the 24-h group. The percentages of BL or BAL macrophages and lymphocytes did not differ significantly among the different patient groups. These data indicate that the development of LAR after allergen inhalation challenge is associated with an early recruitment of eosinophils and with epithelial desquamation in the airways. In addition, after allergen challenge epithelial desquamation is more pronounced in the proximal than in the distal airways, independently of the type of bronchial response.