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.

No abstract

To determine whether NF-κB activation is sufficient to generate lung inflammation in vivo, we selectively expressed a constitutively active form of IκB kinase 1 (cIKK1) or IκB 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-κB-dependent promoter, we detected significantly increased luciferase activity over time (up to 96 h). Compared with control mice treated with adenoviral vectors expressing β-galactosidase, lung bioluminescence and tissue luciferase activity were increased in NF-κB reporter mice treated with adenovirus (Ad)-cIKK1 or Ad-cIKK2. NF-κB 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-κB-dependent chemokines macrophage-inflammatory protein 2 and KC and increased numbers of neutrophils. Coadministration of adenoviral vectors expressing a transdominant inhibitor of NF-κB with Ad-cIKK1 or Ad-cIKK2 resulted in abrogated NF-κB activation and other parameters of lung inflammation, demonstrating that the observed inflammatory effects of Ad-cIKK1 and Ad-cIKK2 were dependent on NF-κB activation by these kinases. These data show that selective expression of IκB kinases in airway epithelium results in NF-κB activation, inflammatory mediator production, and neutrophilic lung inflammation. Therapies targeted to NF-κB in lung epithelium may be beneficial in treating inflammatory lung diseases.

Ascorbate contributes to several metabolic processes including efficient hydroxylation of hydroxyproline in elastin, collagen, and proteins with collagenous domains, yet hydroxyproline in elastin has no known function. Prolyl hydroxylation is essential for efficient collagen production; in contrast, ascorbate has been shown to decrease elastin accumulation in vitro and to alter morphology of elastic tissues in vivo. Ascorbate doses that maximally stimulated collagen production (10-200 microM) antagonized elastin biosynthesis in vascular smooth muscle cells and skin fibroblasts, depending on a combination of dose and exposure time. Diminished elastin production paralleled reduced elastin mRNA levels, while collagen I and III mRNAs levels increased. We compared the stability of mRNAs for elastin and collagen I with a constitutive gene after ascorbate supplementation or withdrawal. Ascorbate decreased elastin mRNA stability, while collagen I mRNA was stabilized to a much greater extent. Ascorbate withdrawal decreased collagen I mRNA stability markedly (4.9-fold), while elastin mRNA became more stable. Transcription of elastin was reduced 72% by ascorbate exposure. Differential effects of ascorbic acid on collagen I and elastin mRNA abundance result from the combined, marked stabilization of collagen mRNA, the lesser stability of elastin mRNA, and the significant repression of elastin gene transcription.

We examined the effects of dexamethasone treatment on nuclear factor (NF)-kappa B activation and lung inflammation in transgenic reporter mice expressing photinus luciferase under the control of an NF-kappa B-dependent promoter (HLL mice). In vitro studies with bone marrow and peritoneal macrophages derived from these mice showed that treatment with dexamethasone blocked luciferase induction after treatment with Escherichia coli lipopolysaccharide (LPS); however, treatment of mice with intraperitoneal injection of dexamethasone at doses of 0.3 microg/g and 1 microg/g failed to inhibit NF-kappa B-dependent luciferase activity in the lungs. Furthermore, intraperitoneal treatment with 10 microg/g of dexamethasone prior to LPS paradoxically resulted in augmented luciferase activity as compared with that of mice treated with LPS alone. NF-kappa B-dependent luciferase expression in the lungs was detected by bioluminescence imaging and by measurement of luciferase activity in homogenized lung tissue. In these studies, there was an excellent correlation between indirect measurement of luciferase activity by bioluminescence in living mice and direct measurement of luciferase activity in lung tissue. Dexamethasone treatment did not affect LPS-induced neutrophilic influx or the concentration of macrophage inflammatory protein-2 in lung lavage fluid. These findings emphasize the potential error of extrapolating in vitro findings to complex in vivo events such as regulation of inflammation.