RESUMEN
The inflammatory processes associated with pulmonary disorders remains incompletely understood. CCAAT/enhancer-binding protein (C/EBP)ß is implicated in inflammatory lung disorders as well as in ß(2)-adrenoceptor signaling. We hypothesized that C/EBPß in the lung epithelium contributes to lipopolysaccharide (LPS)-induced airway neutrophilia and expression of neutrophil chemoattractant chemokine (C-X-C) motif ligand (CXCL)1, as well as the suppressive effects of long-acting ß(2)-agonists (LABAs) and glucocorticoids (GCs). To investigate this, mice with a lung epithelial-specific deletion of C/EBPß (Cebpb(ΔLE)) and control littermates (Cebpb(fl/fl)) were pre-treated with a LABA, formoterol and/or a GC, budesonide, and challenged with LPS. Inflammatory cell recruitment in bronchoalveolar lavage (BAL) fluid and pulmonary expression of inflammatory mediators were investigated. In addition, the ability of formoterol to increase C/EBP transactivation was assessed in vitro. LPS-challenged Cebpb(ΔLE) mice exhibited fewer BAL neutrophils and lower pulmonary expression of CXCL1 versus Cebpb(fl/fl) mice. Suppression of LPS-induced neutrophilia by formoterol was impaired in Cebpb(ΔLE) mice and Cxcl1 expression was increased. However, suppression of the neutrophilia by budesonide with/without formoterol was preserved. Further studies indicated that C/EBP transactivation was increased by the cAMP elevating agent forskolin and formoterol in a ß(2)-adrenoceptor dependent manner. Thus, C/EBPß in the lung epithelium contributes to LPS-induced CXCL1 expression and airway neutrophilia as well as to the suppressive effects of formoterol. Reduced C/EBPß activity, observed in smokers with chronic obstructive pulmonary disease, may impair the responsiveness to LABAs when used without GCs.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2/farmacología , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Quimiocina CXCL1/biosíntesis , Etanolaminas/farmacología , Pulmón/metabolismo , Neumonía/metabolismo , Mucosa Respiratoria/metabolismo , Animales , Lavado Broncoalveolar , Proteína beta Potenciadora de Unión a CCAAT/genética , Quimiocina CXCL1/antagonistas & inhibidores , Fumarato de Formoterol , Glucocorticoides/farmacología , Humanos , Lipopolisacáridos/farmacología , Ratones , Ratones Mutantes , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Neutrófilos/patología , Mucosa Respiratoria/efectos de los fármacosRESUMEN
BACKGROUND: CCAAT/enhancer-binding protein (C/EBP)α is crucial for lung development and differentiation of the pulmonary epithelium. Conversely, no lung defects have been observed in C/EBPß-deficient mice, although C/EBPß trans-activate pulmonary genes by binding to virtually identical DNA-sequences as C/EBPα. Thus, the pulmonary phenotype of mice lacking C/EBPß could be explained by functional replacement with C/EBPα. We investigated whether C/EBPα and C/EBPß have overlapping functions in regulating lung epithelial differentiation during organogenesis. Epithelial differentiation was assessed in mice with a lung epithelial-specific (SFTPC-Cre-mediated) deletion of C/EBPα (Cebpa(ΔLE) ), C/EBPß (Cebpb(ΔLE) ), or both genes (Cebpa(ΔLE) ; Cebpb(ΔLE) ). RESULTS: Both Cebpa(ΔLE) mice and Cebpa(ΔLE) ; Cebpb(ΔLE) mice demonstrated severe pulmonary immaturity compared to wild-type littermates, while no differences in lung histology or epithelial differentiation were observed in Cebpb(ΔLE) mice. In contrast to Cebpa(ΔLE) mice, Cebpa(ΔLE) ; Cebpb(ΔLE) mice also displayed undifferentiated Clara cells with markedly impaired protein and mRNA expression of Clara cell secretory protein (SCGB1A1), compared to wild-type littermates. In addition, ectopic mucus-producing cells were observed in the conducting airways of Cebpa(ΔLE) ; Cebpb(ΔLE) mice. CONCLUSIONS: Our findings demonstrate that C/EBPα and C/EBPß play pivotal, and partly overlapping roles in determining airway epithelial differentiation, with possible implications for tissue regeneration in lung homeostasis and disease.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Diferenciación Celular/fisiología , Pulmón/embriología , Organogénesis/fisiología , Animales , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Pulmón/metabolismo , Ratones , Transducción de Señal/fisiología , Uteroglobina/genética , Uteroglobina/metabolismoRESUMEN
RATIONALE: Cigarette smoke is the major cause of chronic obstructive pulmonary disease and lung cancer. The mechanisms by which smoking induces pulmonary dysfunction are complex, involving stress from toxic components and inflammatory responses. Although CCCAAT/enhancer-binding protein (C/EBP)-ß is known as a key intracellular regulator of inflammatory signaling, its role in pulmonary inflammation has not been established. OBJECTIVES: To characterize the role of C/EBPß in the airway epithelial response to cigarette smoke. METHODS: mRNA expression in the airway epithelium of current, former, and never-smokers, and in in vitro cigarette smoke extract-treated primary human airway epithelial cells, was analyzed by microarray and quantitative real-time polymerase chain reaction, respectively. Mice with lung epithelial-specific inactivation of C/EBPß were generated and exposed to cigarette smoke for 4 or 11 days. Lung histology, bronchoalveolar lavage cell differentials, and expression of inflammatory and innate immune mediators in the lungs were assessed. MEASUREMENTS AND MAIN RESULTS: C/EBPß was significantly down-regulated in the airway epithelium of both current and former smokers compared with never-smokers, and in cigarette smoke-treated primary human airway epithelial cells in vitro. Cigarette smoke-exposed mice with a lung epithelial-specific inactivation of C/EBPß displayed blunted respiratory neutrophil influx and compromised induction of neutrophil chemoattractants growth-regulated oncogene-α, macrophage inflammatory protein-1γ, granulocyte colony-stimulating factor, and serum amyloid A 3 and proinflammatory cytokines tumor necrosis factor-α and interleukin-1ß, compared with smoke-exposed controls. Inhibition of C/EBPß in human airway cells in vitro caused a similarly compromised response to smoke. CONCLUSION: Our data suggest a previously unknown role for C/EBPß and the airway epithelium in mediating inflammatory and innate immune responses to cigarette smoke.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/inmunología , Células Epiteliales/inmunología , Inflamación/inmunología , Pulmón/inmunología , Fumar/inmunología , Animales , Western Blotting , Líquido del Lavado Bronquioalveolar/inmunología , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/efectos de los fármacos , Humanos , Inmunidad Innata/inmunología , Pulmón/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The transcription factor RUNX1 (AML1) is an important regulator of haematopoiesis, and an important fusion partner in leukaemic translocations. High-affinity DNA binding by RUNX1 requires the interaction of the RUNX1 Runt-Homology-Domain (RHD) with the core-binding factor beta protein (CBFbeta). To generate novel reagents for in vitro and in vivo studies of RUNX1 function, we have selected high-affinity RNA aptamers against a recombinant RHD-CBFbeta complex. Selection yielded two sequence families, each dominated by a single consensus sequence. Aptamers from each family disrupt DNA binding by the RUNX1 protein in vitro and compete with sequence-specific dsDNA binding. Minimal, high-affinity ( approximately 100-160 nM) active aptamer fragments 28 and 30 nts in length, consisting of simple short stem-loop structures, were then identified. These bind to the RHD subunit and disrupt its interaction with CBFbeta, which is consistent with reduced DNA affinity in the presence of aptamer. These aptamers represent new reagents that target a novel surface on the RHD required to stabilize the recombinant RHD-CBFbeta complex and thus will further aid exploring the functions of this key transcription factor.
Asunto(s)
Aptámeros de Nucleótidos/química , Subunidad alfa 2 del Factor de Unión al Sitio Principal/antagonistas & inhibidores , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Sitios de Unión , ADN/metabolismoRESUMEN
Six novel members of the IL-1 family of cytokines were recently identified, primarily through the use of DNA database searches for IL-1 homologues, and were named IL-1F5 to IL-1F10. In the present study, we investigated the effect of IL-1F8 on primary human joint cells, and examined the expression of the new IL-1 family members in human and mouse joints. Human synovial fibroblasts (hSFs) and human articular chondrocytes (hACs) expressed the IL-1F8 receptor (IL-1Rrp2) and produced pro-inflammatory mediators in response to recombinant IL-1F8. IL-1F8 mRNA expression was increased in hSFs upon stimulation with proinflammatory cytokines, whereas in hACs IL-1F8 mRNA expression was constitutive. However, IL-1F8 protein was undetectable in hSF and hAC culture supernatants. Furthermore, although IL-1beta protein levels were increased in inflamed human and mouse joint tissue, IL-1F8 protein levels were not. IL-1F8 levels in synovial fluids were similar to or lower than those in matched serum samples, suggesting that the joint itself is not a major source of IL-1F8. Serum levels of IL-1F8 were similar in healthy donors, and patients with rheumatoid arthritis, osteoarthritis and septic shock, and did not correlate with inflammatory status. Interestingly however, we observed high IL-1F8 levels in several serum samples in all groups. In conclusion, IL-1F8 exerts proinflammatory effects in primary human joint cells. Joint and serum IL-1F8 protein levels did not correlate with inflammation, but they were high in some human serum samples tested, including samples from patients with rheumatoid arthritis. It remains to be determined whether circulating IL-1F8 can contribute to joint inflammation in rheumatoid arthritis.
Asunto(s)
Artritis Reumatoide/inmunología , Condrocitos/inmunología , Fibroblastos/inmunología , Inflamación/inmunología , Interleucina-1/inmunología , Líquido Sinovial/citología , Animales , Artritis Reumatoide/sangre , Cartílago Articular/efectos de los fármacos , Cartílago Articular/inmunología , Condrocitos/efectos de los fármacos , Humanos , Interleucina-1/sangre , Articulaciones/inmunología , Ratones , Líquido Sinovial/inmunologíaRESUMEN
Six novel genes encoding proteins with the interleukin (IL)-1 fold have been identified recently. The classical family members are involved in inflammatory signaling. Previous work has placed the novel genes close to or within the same cluster as IL1A, IL1B, and IL1RN, which occupy an approximately 400-kb interval on chromosome 2. We have combined the incomplete public database sequence with our own sequence to generate a reference sequence and map that encompass all of the novel genes, allowing determination of the gene structures, precise localization of exons, and determination of distances between conventional SNP and microsatellite markers. Gene order from centromere to telomere is IL1A-IL1B-IL1F7-IL1F9-IL1F6-IL1F8-IL1F5-IL1F10-IL1RN, of which only IL1A, IL1B, and IL1F8 are transcribed towards the centromere. The gene order relates to the evolutionary relationship between the genes. Key features of exon boundaries are conserved. There is no evidence for other IL-1 family members within the cluster.