RESUMEN
BACKGROUND: Structural cells are an important reservoir of chemokines that coordinate the influx of various immune cells to the lungs of asthmatics. Airway smooth muscle cells (ASMC) are an important source of these chemokines. CCL15 is a recently described chemo-attractant for neutrophils, eosinophils, monocytes and lymphocytes. OBJECTIVE: To determine the production and the regulation of CCL15 by ASMC and to investigate its production in asthmatic airways. METHODS: Human ASMC were obtained from main bronchial airway segments of patients with mild, moderate and severe asthma. To induce chemokine production, cells were incubated with IL-4, IL-13, TNF-α or IFN-γ in presence or absence of dexamethasone, mithramycin A (SP-1 inhibitor) or the IKK-2 inhibitor, AS602868. CCL15 mRNA expression was evaluated by real-time PCR. Immunoreactive CCL15 was detected by immuno-fluorescence and CCL15 protein concentration in the supernatant was measured using ELISA. RESULTS: CCL15 is constitutively expressed in human ASMC and is strongly up-regulated by TNF-α. This up-regulation is inhibited by dexamethasone, mithramycin A and AS602868. TNF-α-induced CCL15 levels can be synergistically enhanced by the presence of IFN-γ, at both the transcriptional and translation level. This synergism is NF-κB-dependent. Asthmatic biopsies demonstrated higher expression of CCL15 compared with non-asthmatic controls. CONCLUSION AND CLINICAL RELEVANCE: Our results show that ASMC are a potent source of CCL15 in the airways and may directly participate in the recruitment of inflammatory cells to asthmatic airways. Targeting the production of CCL15 by ASMC might reduce the inflammatory response within the airways of asthmatic patients.
Asunto(s)
Asma/fisiopatología , Bronquios/citología , Quimiocinas CC/metabolismo , Proteínas Inflamatorias de Macrófagos/metabolismo , Miocitos del Músculo Liso/metabolismo , Regulación hacia Arriba , Adulto , Asma/inmunología , Biopsia , Quimiocinas CC/efectos de los fármacos , Quimiocinas CC/genética , Quimiocinas CC/inmunología , Femenino , Humanos , Interferón gamma/inmunología , Interferón gamma/farmacología , Proteínas Inflamatorias de Macrófagos/efectos de los fármacos , Proteínas Inflamatorias de Macrófagos/genética , Proteínas Inflamatorias de Macrófagos/inmunología , Masculino , Persona de Mediana Edad , Miocitos del Músculo Liso/inmunología , Reacción en Cadena en Tiempo Real de la Polimerasa , Índice de Severidad de la Enfermedad , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BACKGROUND: Occupational asthma (OA) may cause alterations of airways with inflammation and remodelling after cessation of exposure. Although the long-term clinical, functional and induced sputum sequelae have been examined in workers removed from exposure, the long-term pathological outcomes are unknown. OBJECTIVE: We aimed to investigate whether airway inflammation and remodelling were present in bronchial biopsies of subjects with prior OA but without evidence of persisting asthma at a mean interval of 14 years after cessation of exposure. METHODS: Ten clinically and functionally asymptomatic subjects with a prior diagnosis of OA were recruited and underwent bronchoscopy, bronchoalveolar lavage and bronchial biopsy. Comparisons were made with biopsies from normal control subjects. Epithelial detachment, epithelial metaplasia, mucous gland and airway smooth muscle (ASM) areas as well as the distance between the epithelium and ASM were measured by image analysis. The amount of collagen present was assessed by van Gieson staining. The numbers of TGF-beta1- and eosinophil cationic protein (ECP)-positive cells were evaluated by specific immunostaining. RESULTS: Statistically significant increases were found in the numbers of TGF-beta1- and ECP-positive cells and in the amount of subepithelial fibrosis present in the biopsies of subjects with prior OA compared with control biopsies. The distance between the epithelium and ASM was significantly reduced in the OA group. Increases in epithelial metaplasia, ASM mass, mucous gland numbers, collagen deposition and eosinophilia in the OA group were not statistically significant. There was no evidence of ongoing inflammation in the group with prior OA as assessed by the number of T lymphocytes present. CONCLUSION: Some aspects of airway inflammation and remodelling persist in subjects with prior OA long after cessation of exposure even in the absence of clinical, sputum and functional abnormalities. These findings are relevant to the assessment of long-term sequelae in subjects with OA when reviewed after cessation of exposure.
Asunto(s)
Contaminantes Ocupacionales del Aire/farmacología , Asma/inducido químicamente , Asma/patología , Adulto , Anciano , Asma/metabolismo , Asma/cirugía , Biopsia , Femenino , Estudios de Seguimiento , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Factores de Tiempo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
BACKGROUND: Toll-like receptor 4 (TLR4), part of the bacterial lipopolysaccharide (LPS) receptor, is an important bridge between innate and adaptive immunity. Our previous studies have indicated reduced expression of TLR4 and reduced responsiveness to LPS in nasal mucosa of atopic adults compared with non-atopic adults. IL-4 and signal transducer and activator of transcription 6 (STAT6), which are increased in atopic patients, may have a role in modulating TLR4. OBJECTIVE: To examine direct effects of IL-4 and STAT6 on TLR4 expression of U-937 monocytic cells. METHODS: LPS responsiveness, under different conditions of U-937 cells was measured by nuclear factor (NF)-kappaB activation of transcription. TLR4 mRNA was quantified by real-time PCR and TLR4 surface expression was measured by flow cytometry. The promoter and 4.3 kb of the upstream region of TLR4 were cloned into a plasmid vector and transiently transfected into U-937 cells. Transfected cells were incubated with IL-4 and transcriptional activity was assayed by the luciferase assay. STAT6 was transfected to evaluate overexpression of this transcription factor. Cells were also incubated with Tyrphostin AG490 to inhibit tyrosine kinases. RESULTS: NF-kappaB activation by LPS was inhibited by IL-4 pre-incubation but not when IL-4 was added at the same time as LPS. TLR4 mRNA expression was inhibited by IL-4 as early as 6 h but the effect was lost by 24 h. Surface expression of TLR4 was inhibited by IL-4 at 12 and 24 h, but returned to baseline at 48 h. IL-4 inhibited activity of the TLR4 promoter as early as 6 h, but, like the mRNA, these effects were transient. STAT6 overexpression enhanced the inhibition of the TLR4 promoter and prolonged it. Inhibition of TLR4 by IL-4 was abolished by pre-incubation with the tyrosine kinase inhibitor Tyrphostin AG490. CONCLUSION: Our findings demonstrate that IL-4, through STAT6, can modulate TLR4 expression and suggests that Th2 cytokines can impact on the LPS responsiveness of cells.
Asunto(s)
Regulación hacia Abajo , Monocitos/metabolismo , Factor de Transcripción STAT6/metabolismo , Receptor Toll-Like 4/metabolismo , Adulto , Línea Celular , Citometría de Flujo , Humanos , Interleucina-4/farmacología , Lipopolisacáridos , FN-kappa B/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 4/análisis , Receptor Toll-Like 4/genética , Transfección/métodos , Tirfostinos/farmacologíaRESUMEN
Prominent blood and tissue eosinophilia is clinically manifested in a number of inflammatory states, particularly in allergic diseases. Corticosteroids are the most effective anti-inflammatory drugs used in the treatment of eosinophilic disorders, including bronchial asthma. Their beneficial effects result, among others, from (i) the suppression of the synthesis and the effects of eosinophil survival factors, (ii) the direct induction of eosinophil apoptosis and (iii) the stimulation of their engulfment by professional phagocytic cells. Failure of steroids to propagate apoptotic signals and to promote eosinophil clearance may explain the corticoresistance observed in a proportion of asthmatic patients. Accordingly, studies on the intracellular mechanisms involved in eosinophil corticosensitivity and resistance may provide a valuable tool for identifying new and selective molecular targets to therapeutically resolve otherwise persistent eosinophilic inflammation. In this review, the intracellular cascade of events involved in corticosteroid-mediated eosinophil apoptotic death is discussed and compared to the signalling pathway governing this process in the established model of dexamethasone-induced thymocyte apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Eosinofilia/metabolismo , Eosinófilos/metabolismo , Animales , Asma/metabolismo , Asma/patología , Supervivencia Celular/fisiología , Eosinofilia/patología , Eosinófilos/efectos de los fármacos , Eosinófilos/ultraestructura , Genes bcl-2 , Glucocorticoides/metabolismo , Glucocorticoides/farmacología , Humanos , Mitocondrias/metabolismo , Fagocitos/fisiología , Fosfotransferasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
In this study, we examined the relative importance of caspases and mitochondria in Fas-mediated eosinophil apoptosis. Stimulation of human peripheral blood eosinophils with an agonistic anti-human Fas monoclonal antibody, but not with control IgM, induced a time-dependent increase in their apoptosis, which was associated with a loss in mitochondrial transmembrane potential (DeltaPsi(m)) and with caspase-8 and caspase-3 activation. Interleukin (IL)-5 and interferon (IFN)-gamma, two cytokines known to prolong eosinophil survival, inhibited Fas-mediated apoptosis and caspase activation but poorly affected the decrease in DeltaPsi(m). Eosinophil incubation with bongkrekic acid, an inhibitor of the mitochondrial permeability transition pore (MPTP) opening, failed to modify Fas-mediated loss in DeltaPsi(m), caspase activation, and apoptosis. In contrast, caspase inhibitors markedly reduced eosinophil apoptosis without significantly affecting DeltaPsi(m) dissipation. We conclude that caspase-8 and caspase-3 activation, but not MPTP opening, mediate Fas-induced eosinophil apoptosis and are the main targets for the protective effect of IL-5 and IFN-gamma.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasas/fisiología , Eosinófilos/efectos de los fármacos , Interferón gamma/farmacología , Interleucina-5/farmacología , Canales Iónicos , Glicoproteínas de Membrana/fisiología , Mitocondrias/fisiología , Receptor fas/fisiología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Anticuerpos Monoclonales/farmacología , Ácido Bongcréquico/farmacología , Caspasa 3 , Caspasa 8 , Caspasa 9 , Inhibidores de Caspasas , Inhibidores de Cisteína Proteinasa/farmacología , Grupo Citocromo c/metabolismo , Eosinófilos/citología , Eosinófilos/enzimología , Eosinófilos/ultraestructura , Proteína Ligando Fas , Humanos , Síndrome Hipereosinofílico/sangre , Inmunoglobulina M/farmacología , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/fisiología , Potenciales de la Membrana/efectos de los fármacos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/fisiología , Ratones , Mitocondrias/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial , Poro de Transición de la Permeabilidad Mitocondrial , Oligopéptidos/farmacología , Eosinofilia Pulmonar/inmunología , Eosinofilia Pulmonar/patología , Proteínas Recombinantes/farmacología , Receptor fas/inmunologíaRESUMEN
Airway inflammation and alterations in cellular turnover are histopathologic features of asthma. We show that the expression of peroxisome proliferator-activated receptor gamma (PPAR gamma), a nuclear hormone receptor involved in cell activation, differentiation, proliferation, and/or apoptosis, is augmented in the bronchial submucosa, the airway epithelium, and the smooth muscle of steroid-untreated asthmatics, as compared with control subjects. This is associated with enhanced proliferation and apoptosis of airway epithelial and submucosal cells, as assessed by the immunodetection of the nuclear antigen Ki67, and of the cleaved form of caspase-3, respectively, and with signs of airway remodeling, including thickness of the subepithelial membrane (SBM) and collagen deposition. PPAR gamma expression in the epithelium correlates positively with SBM thickening and collagen deposition, whereas PPAR gamma expressing cells in the submucosa relate both to SBM thickening and to the number of proliferating cells. The intensity of PPAR gamma expression in the bronchial submucosa, the airway epithelium, and the smooth muscle is negatively related to FEV(1) values. Inhaled steroids alone, or associated with oral steroids, downregulate PPAR gamma expression in all the compartments, cell proliferation, SBM thickness, and collagen deposition, whereas they increase apoptotic cell numbers in the epithelium and the submucosa. Our findings have demonstrated that PPAR gamma (1) is a new indicator of airway inflammation and remodeling in asthma; (2) may be involved in extracellular matrix remodeling and submucosal cell proliferation; (3) is a target for steroid therapy.
Asunto(s)
Apoptosis , Asma/inmunología , Asma/patología , Proteínas Nucleares/biosíntesis , Receptores Citoplasmáticos y Nucleares/biosíntesis , Factores de Transcripción/biosíntesis , Anciano , Asma/tratamiento farmacológico , Biopsia , Bronquios/química , División Celular , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas Nucleares/análisis , Receptores Citoplasmáticos y Nucleares/análisis , Factores de Transcripción/análisisRESUMEN
Eosinophils play a major role in the onset and maintenance of bronchial inflammation and tissue injury in asthma. Like other leukocytes, eosinophils present in excessive numbers in inflamed tissues are removed by apoptosis. This phenomenon, also called 'programmed cell death', allows elimination of dangerous or redundant cells, thereby ensuring maintenance of tissue homeostasis. It has been suggested that a defect in eosinophil apoptosis would participate in the development and persistence of allergic airways inflammation in asthma. Eosinophil apoptosis, as well as the expression and function of various molecules determining this process, are closely regulated by various stimuli, including cytokines, lipid mediators and growth factors released by various cell types and by the eosinophil itself, as well as exogenous molecules, such as glucocorticoids. These stimuli have been shown to alter the expression and function of different molecules involved in the cascade of events characterising the apoptotic process, particularly Bcl-2 family proteins and the pro-apoptotic membrane glycoprotein, Fas. These observations, together with a better understanding of the mechanisms underlying eosinophil apoptosis, will help to more clearly define the molecular events involved in accumulation of these cells in blood and tissues and to identify potential new targets for the treatment of allergic diseases.