No abstract

The role of gender and sex hormones is unclear in host response to lung injury, inflammation, and fibrosis. To examine gender influence on pulmonary fibrosis, male and female rats were given endotracheal injections of either saline or bleomycin. Female rats showed higher mortality rates and more severe fibrosis than did male rats, as indicated by higher levels of lung collagen deposition and fibrogenic cytokine expression. To clarify the potential role of female sex hormones in lung fibrosis, female rats were ovariectomized and treated with either estradiol or vehicle plus endotracheal injections of either saline or bleomycin. The results showed diminished fibrosis in the ovariectomized, bleomycin-treated rats without hormone replacement. Estradiol replacement restored the fibrotic response to that of the intact female mice in terms of lung collagen deposition and cytokine expression, which was accompanied by higher plasma estradiol levels. Furthermore, fibroblasts from bleomycin-treated rats exhibited increased responsiveness to estradiol treatment, causing dose-dependent increases in procollagen 1 and transforming growth factor-␤ 1 mRNA expression relative to untreated controls. Taken together these findings suggest that female mice may have an exaggerated response to lung injury relative to male mice because of female sex hormones, which have direct fibrogenic activity on lung fibroblasts. This may provide a mechanism for a hormonally mediated intensification of pulmonary fibrosis.

We demonstrated that products of the L-arginine-dependent pathway of human alveolar macrophages (AM) effectively kill the Mycobacterium bovis bacillus Calmette-Guérin (BCG) in vitro. The formation of products was triggered by inoculation with BCG itself. Many reports have shown that activated rodent AM could produce an amount of nitric oxide (NO) sufficient to suppress the growth of mycobacteria. However, there have been no definitive results as to whether human AM might have the NO-dependent mechanism for the killing of mycobacteria. Therefore, we have undertaken some experiments to answer this question. Immunofluorescence assays showed an increased production of inducible nitric oxide synthase (iNOS) and peroxynitrite in BCGinoculated AM from patients with pulmonary fibrosis. Reverse transcriptase-PCR also revealed the higher expression of iNOS-coding mRNA. Colony assays demonstrated that these human AM effectively killed BCG in their cytoplasm. However, treatment of AM with N G-monomethyl-L-arginine monoacetate resulted in markedly reduced killing activity. These results clearly show that BCG-induced NO and its reactive product with the oxygen radical peroxynitrite could play an important role in the killing of BCG in human AM.

Fibroblasts from bleomycin-injured lungs express telomerase activity transiently during the period of active fibrosis, but the signal(s) responsible for its induction is (are) unknown. The objective of this study was to identify potential mediators capable of regulating telomerase activity induction in rat lung fibroblasts during pulmonary fibrosis. Lung fibroblasts from control (NRF) and bleomycin-treated (BRF) rats were isolated and treated in vitro with either basic fibroblast growth factor (bFGF) or interleukin-4 (IL-4). At selected time points after treatment, the cells were analyzed for telomerase activity, as well as telomerase reverse transcriptase (TERT) mRNA and protein by reverse transcriptase/polymerase chain reaction and Western blot, respectively. The results showed that bFGF could induce telomerase activity in NRF and stimulate further the induced activity in BRF. The bFGF effect was accompanied by increased TERT protein expression and a rapid but transient increase in TERT mRNA. In contrast, IL-4 inhibited the induced telomerase activity in BRF, which was accompanied by increased alpha-smooth muscle actin expression, an indicator of myofibroblast differentiation. These findings suggest that telomerase expression could be induced in rat lung fibroblasts by bFGF, but suppressed by IL-4, which promoted myofibroblast differentiation. The latter is consistent with the preferential expression of telomerase activity in fibroblasts relative to myofibroblasts.

Bleomycin-induced lung injury causes increased fibroblast numbers in the lung and pulmonary fibrosis. Studies of fibroblasts isolated from such injured lungs have revealed evidence of increased intrinsic proliferative capacity, but the mechanism is unknown. Telomerase catalyzes the addition of telomeric DNA repeats onto chromosomal ends, which is associated with increased cellular life span or immortality. To examine whether telomerase might play a role in regulating fibroblast proliferative capacity in pulmonary fibrosis, lung fibroblasts were isolated from rats treated with endotracheal injections of phosphate-buffered saline or bleomycin. At selected time points, the rats were killed and lung fibroblasts isolated. The isolated cells and lung tissue were then used in experiments for measurement of telomerase activity. The results show undetectable telomerase activity in fibroblasts isolated from control uninjured lungs, or in the control lung tissue extracts. Similar results were obtained in cells and lung tissue from Days 1, 3, and 28 bleomycin-injured lungs. However, significant telomerase activity was detected in fibroblasts and tissue extracts isolated from Days 7, 14, and 21 bleomycin-treated rat lungs, with maximal activity observed in the Day 14 samples. Analysis of the isolated cells for telomerase messenger RNA or reverse transcriptase expression, combined with alpha-smooth-muscle actin expression by immunohistochemistry, revealed that telomerase expression localized primarily to nonmyofibroblasts. These findings suggest that in addition to elevated growth factor expression, the injured lung fibroblast population may contain cells with increased life span, which could contribute to the observed overall increase in lung fibroblast numbers.