Increased and prolonged pulmonary fibrosis in surfactant protein C-deficient mice following intratracheal bleomycin.

Recent reports have linked mutations in the surfactant protein C gene (SFTPC) to familial forms of pulmonary fibrosis, but it is uncertain whether deficiency of mature SP-C contributes to disease pathogenesis. In this study, we evaluated bleomycin-induced lung fibrosis in mice with genetic deletion of SFTPC. Compared with wild-type (SFTPC+/+) controls, mice lacking surfactant protein C (SFTPC-/-) had greater lung neutrophil influx at 1 week after intratracheal bleomycin, greater weight loss during the first 2 weeks, and increased mortality. At 3 and 6 weeks after bleomycin, lungs from SFTPC-/- mice had increased fibroblast numbers, augmented collagen accumulation, and greater parenchymal distortion. Furthermore, resolution of fibrosis was delayed. Although remodeling was near complete in SFTPC+/+ mice by 6 weeks, SFTPC-/- mice did not return to baseline until 9 weeks after bleomycin. By terminal dUTP nick-end labeling staining, widespread cell injury was observed in SFTPC-/- and SFTPC+/+ mice 1 week after bleomycin; however, ongoing apoptosis of epithelial and interstitial cells occurred in lungs of SFTPC-/- mice, but not SFTPC+/+ mice, 6 weeks after bleomycin. Thus, SP-C functions to limit lung inflammation, inhibit collagen accumulation, and restore normal lung structure after bleomycin.

Characterization of fibroblast-specific protein 1 in pulmonary fibrosis.

Because fibroblasts produce collagen and other extracellular matrix components that are deposited during tissue fibrosis, defining the behavior of these cells is critical to understanding the pathogenesis of fibrotic diseases. We investigated the utility of fibroblast-specific protein 1 (FSP1), a member of the calmodulin S100 troponin C superfamily, for identifying lung fibroblasts in a murine model of pulmonary fibrosis induced by intratracheal administration of bleomycin. Protein and mRNA expression of FSP1 was minimal in untreated lungs, but increased by 1 week after bleomycin administration and remained increased at 2 and 3 weeks after treatment. By immunohistochemistry, the number of FSP1(+) cells increased in a dose-dependent manner in the lungs after bleomycin treatment. Colocalization of alpha1 procollagen and FSP1 in interstitial cells demonstrated that FSP1(+) fibroblasts contribute to the deposition of collagen after bleomycin administration. In primary lung cell cultures, lung fibroblasts, but not macrophages or type II alveolar epithelial cells, expressed FSP1. FSP1 also identified fibroblasts in lung biopsy specimens from patients with documented usual interstitial pneumonitis. Therefore, FSP1 is an improved marker for lung fibroblasts that could be useful for investigating the pathogenesis of pulmonary fibrosis.

Selective I kappa B kinase expression in airway epithelium generates neutrophilic lung inflammation.

To determine whether NF-kappaB activation is sufficient to generate lung inflammation in vivo, we selectively expressed a constitutively active form of IkappaB kinase 1 (cIKK1) or IkappaB kinase 2 (cIKK2) in airway epithelium. After intratracheal administration of adenoviral vectors expressing cIKK1 or cIKK2 to transgenic reporter mice that express Photinus luciferase under the control of an NF-kappaB-dependent promoter, we detected significantly increased luciferase activity over time (up to 96 h). Compared with control mice treated with adenoviral vectors expressing beta-galactosidase, lung bioluminescence and tissue luciferase activity were increased in NF-kappaB reporter mice treated with adenovirus (Ad)-cIKK1 or Ad-cIKK2. NF-kappaB activation in lungs of Ad-cIKK1- and Ad-cIKK2-treated mice was confirmed by immunoblots for RelA and EMSA from lung nuclear protein extracts. Mice treated with Ad-cIKK1 or Ad-cIKK2 showed induction of mRNA expression of several chemokines and cytokines in lung tissue. In lung lavage fluid, mice treated with Ad-cIKK1 or Ad-cIKK2 showed elevated concentrations of NF-kappaB-dependent chemokines macrophage-inflammatory protein 2 and KC and increased numbers of neutrophils. Coadministration of adenoviral vectors expressing a transdominant inhibitor of NF-kappaB with Ad-cIKK1 or Ad-cIKK2 resulted in abrogated NF-kappaB activation and other parameters of lung inflammation, demonstrating that the observed inflammatory effects of Ad-cIKK1 and Ad-cIKK2 were dependent on NF-kappaB activation by these kinases. These data show that selective expression of IkappaB kinases in airway epithelium results in NF-kappaB activation, inflammatory mediator production, and neutrophilic lung inflammation. Therapies targeted to NF-kappaB in lung epithelium may be beneficial in treating inflammatory lung diseases.