RESUMEN
Serine proteases released from neutrophils are central to the pathogenesis of cystic fibrosis lung disease and are considered to be obvious therapeutic targets. Neutrophil elastase digests key opsonins present in the lung and disrupts phagocytosis, allowing bacteria to persist despite established pulmonary inflammation. We have found that cathepsin G, an abundant serine protease found in human and murine neutrophils, has other roles in the development of suppurative lung diseases. Murine models of endobronchial inflammation indicate that cathepsin G inhibits airway defences and interferes with the host's ability to clear Pseudomonas aeruginosa from the lung with effects distinct from neutrophil elastase. We hypothesise that differences in bacterial killing are due to defects in innate defences created by proteolysis. Protein profiles of bronchoalveolar lavage of infected wild-type and cathepsin G-deficient mice were compared using two-dimensional polyacrylamide gel electrophoresis and tandem mass spectrometry. Four proteins in bronchoalveolar lavage were cleaved by cathepsin G. Serum amyloid P component leaked into the lung during acute infection and was digested by cathepsin G. Its cleavage products had greater binding to lipopolysaccharide and interfered with phagocytosis. These results indicate that cleaved serum amyloid P component acts as an anti-opsonin and interferes with bacterial clearance from the lung.
Asunto(s)
Catepsina G/química , Animales , Bronquios/microbiología , Lavado Broncoalveolar , Catepsina G/metabolismo , Electroforesis en Gel Bidimensional/métodos , Células HL-60 , Humanos , Pulmón/microbiología , Pulmón/patología , Ratones , Ratones Transgénicos , Neutrófilos/metabolismo , Proteínas Opsoninas/química , Fagocitosis , Componente Amiloide P Sérico/biosíntesis , Espectrometría de Masas en Tándem/métodosRESUMEN
We hypothesize that Type II epithelial cells, which line the distal airspaces of the lung, are early responders to invading pathogens and release a signal, which activates and alters the phenotype and phagocytosis properties of alveolar macrophages even at a distance. The T(7) cell line is a conditionally immortalized murine Type II epithelial cell line developed in our laboratory. Using an in vitro transwell model we have previously shown that UV-irradiated Escherichia coli (UVEC)-stimulated T(7) cells cultured in the lower transwell chamber, release a diffusible signal which activates MH-S cells (immortalized murine alveolar macrophages) cultured in the upper transwell chamber, to produce nitric oxide. Using scanning electron microscopy, we show that MH-S cells activated in this manner exhibit increased cell surface ruffling, numerous long filopodia, increased lamellipodia and cell flattening. DynaBead uptake studies show that these morphologic changes are accompanied by increased phagocytosis. These findings indicate that a diffusible signal released at a distance by UVEC-stimulated Type II epithelial cells initiates changes in morphology and phagocytosis reflective of macrophage activation concomitant with the functional activation we previously reported.
Asunto(s)
Macrófagos Alveolares/ultraestructura , Animales , Línea Celular Transformada , Técnicas de Cocultivo , Células Epiteliales/inmunología , Células Epiteliales/efectos de la radiación , Células Epiteliales/ultraestructura , Escherichia coli/inmunología , Activación de Macrófagos , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/efectos de la radiación , Ratones , Microscopía Electrónica de Rastreo , Óxido Nítrico/metabolismo , Fagocitosis , Seudópodos/ultraestructura , Rayos UltravioletaRESUMEN
OBJECTIVE AND DESIGN: To demonstrate a diffusible intercellular macrophage activation factor secreted by Type II alveolar epithelial cells (AECs) in transwell co-cultures. MATERIALS: T(7), our Type II conditionally immortalized AEC line; MH-S, an alveolar macrophage cell line; Lipopolysaccharide (LPS) or uv-killed Escherichia coli (UVEC) for antigen presentation. METHODS: LPS or UVEC stimulation of T(7) cells in the lower chamber was investigated for ability to activate MH-S cells in the upper chamber, as assayed by nitric oxide production and western blots for inducible nitric oxide synthase-2. RESULTS: Both transwell and UVEC-conditioned medium experiments showed secretion of an MH-S activation factor by T(7) cells. Many common inflammatory cytokines were ruled out as this immunoactivator. CONCLUSION: Demonstration of a diffusible activation factor produced by Type II AECs supports their potential role as first responders of innate immunity in the lung.
Asunto(s)
Células Epiteliales/inmunología , Escherichia coli/inmunología , Lipopolisacáridos/farmacología , Pulmón/inmunología , Activación de Macrófagos/inmunología , Macrófagos Alveolares/inmunología , Animales , Línea Celular , Técnicas de Cocultivo , Medios de Cultivo Condicionados/química , Difusión , Pulmón/citología , Ratones , Transducción de SeñalRESUMEN
We have permanently reversed the lethal phenotype in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-deficient (knockout) mouse after in utero gene therapy with an adenovirus containing the cftr gene. The gene transfer targeted somatic stem cells in the developing lung and intestine, and these epithelial surfaces demonstrated permanent developmental changes after treatment. The survival statistics from the progeny of heterozygote-heterozygote matings after in utero cftr gene treatment demonstrated an increased mortality in the homozygous normal pups, indicating that overexpression during development was detrimental. The lungs of these pups revealed accelerated secretory cell proliferation and differentiation. The extent of proliferation and differentiation in the secretory cells of the lung parenchyma after in utero transfer of the cftr gene was evaluated with morphometric and biochemical analyses. These studies provide further support of the regulatory role of the cftr gene in the development of the secretory epithelium.