Inducible expression of a TNF-alpha transgene in the murine lung: A mouse model of pulmonary inflammation and fibrosis
Description
Tumor necrosis factor alpha (TNF-alpha) is an important mediator of interstitial lung disease, but the exact mechanisms by which it promotes lung inflammation and fibrosis are unknown. In order to gain more information about the molecular mechanisms of TNF-alpha induced lung disease, transgenic mice were generated that express TNF-alpha in the lung under the control of a doxycycline inducible promoter. DNA constructs containing a mouse TNF-alpha cDNA downstream from tetracycline operator and minimal cytomegalovirus promoter sequences (TRE-TNF) and the reverse tetracycline transactivator downstream from the human surfactant protein-C promoter (SPC-rtTA) were prepared. Linear SPC-rtTA and TRE-TNF fragments were coinjected, resulting in 14 founder mice carrying both constructs. Offspring from founder animals were screened for inducible TNF-alpha expression by administering doxycycline at 0.5 mg/mL in the drinking water for 10 days and collecting lung tissue for Northern blot and histological analyses. Six of 12 lines examined exhibited TNF-alpha transgene expression in the lung that was inducible after treatment with doxycycline. Upregulation of the endogenous TNF-alpha message could be detected, suggesting activation of the wild type protein by the TNF-alpha transgene. The 320-1 line was selected for further experiments, because of its high level of transgene expression 320-1 transgenic mice exhibit perivascular, subpleural, and alveolar inflammation 10 days after induction of the transgene. An increase in neutrophils and lymphocytes in bronchoalveolar lavage fluid was seen seven days after TNF-alpha induction. Lung histology, after one month of transgene induction is characterized by massive inflammatory infiltrate, forming nodules concentrated in the vicinity of the airways and pleura. Alveolar septa in these lesions are thickened, with enlarged airspaces. At three months the phenotype becomes more severe, with focal fibrotic lesions that show thickened septa, enlarged airspaces, and architectural remodeling. Trichrome staining indicated an increase in peribronchiolar collagen. Due to the focal nature of the response, analysis for hydroxyproline, a major component of collagen, showed no significant difference between the nontransgenic and 320-1 mice at any time point. The inflammatory response in the 320-1 mice diminishes with greater lengths of TNF-alpha induction, with fewer nodules and less severe phenotypes at six and nine months In order to determine the potential interactions between TNF-alpha and transforming growth factor beta (TGF-beta), 320-1 mice were infected with an adenoviral vector carrying active TGF-beta sequences. In this model, it was hypothesized that TNF-alpha would promote inflammation, while TGF-beta caused deposition of extracellular matrix, two important events in the pathogenesis of interstitial pulmonary fibrosis. The 320-1 mice given the TGF-beta adenovirus show more severe inflammation and earlier onset of focal fibrotic lesions. However, this treatment is not sufficient to cause a widespread phenotype approaching the severity of disease seen in human patients The studies presented here indicate that TNF-alpha expression alone, or in conjuction with TGF-beta expression, is insufficient to cause severe interstitial lung disease. Although the focal areas of fibrosis resemble the phenotype seen in human patients, much of the lung remains unaffected, and the phenotype diminishes with time. The severe pathology seen in human patients is probably due to genetic interactions between many different factors