Abstract
To elucidate mechanisms involved in the regulation of lung collagen content we studied hamsters with bleomycin-induced pulmonary fibrosis. Lung collagen in this model is increased as the result of greatly increased lung collagen synthesis rates. However, collagen synthesis rates are subsequently restored to normal. Hamster lung explants from both normal and bleomycin-exposed hamsters were cultured, and the effects of explant conditioned medium (CM) on lung fibroblast (IMR-90) proliferation and collagen production in vitro were determined. Lung explant CM increased fibroblast prostaglandin (PG)E2 production and intracellular cAMP, and decreased both fibroblast proliferation and collagen production in a dose-dependent manner. Greater activity was observed with lung explant CM from bleomycin-exposed lungs. Incubation of fibroblasts with indomethacin prior to addition of CM blocked CM-mediated changes in PGE2 and cAMP and inhibited changes in fibroblast proliferation and collagen production. Exogenous PGE2 or dibutyryl cAMP also suppressed fibroblast proliferation and collagen production. The suppressive activity in lung-conditioned medium is nondialyzable, has an apparent molecular weight of 15,000-20,000 by gel filtration, and is heat-stable. It is not species-restricted since CM from hamster lung affected human and hamster lung fibroblasts similarly. Activity is present preformed in lung and bronchoalveolar lavage fluid, although bronchoalveolar macrophages produce a nondialyzable factor in culture which suppresses fibroblast proliferation. The suppressive activity identified in fibrotic lung may represent a means for limiting collagen accumulation following tumor injury.
Highlights
PRESENCE OF A FACTOR IN LUNG THAT INCREASES FIBROBLAST PROSTAGLANDIN E2 AND CAMPAND SUPPRESSES FIBROBLAST PROLIFERATION AND COLLAGEN PRODUCTION*
To elucidate mechanisms involved in the regulation change, and histologically by increased numbers of inflamof lung collagen content we studied hamsters withbleomycin-induced pulmonary fibrosis
Hamster lung fibroblasts were cultured for 24 h with basal medium or conditioned medium (CM) from normal hamster lung explants or bleomycin-exposedlung explants obtained 15 days after bleomycin instillation. ['HIdThd incorporation or [3H]hydroxyproline production was measured as described under "Experimental Procedures." Values are mean fS.D., n = 3 cultures
Summary
Radioimmunoassay was performed as described by Dray et al [25], using antibody specific for PGEZ (InstitutePasteur Production, Manes-la-Coquette,France), ['H]PGE2 probe (170Ci/mM; NewEnglandNuclear), and authentic PGE2 standard (The Upjohn Go.). Assays of each culture were performed in triplicate. Fibroblast CAMPLevels-After incubation of fibroblasts with CM, medium was removed, the cell layer rinsed once with Hanks' balanced salt solution, and ice-cold5%(w/v) trichloroacetic acid (1.0mi) added. After 30 min at 4 "C, the trichloroacetic acid supernatant was removed and extractedthree times with 5 ml of anhydrous ether. CM was fractionated on a column (40 X 2.5 cm) of Sephadex G75 (Pharmacia Fine Chemicals, Piscataway, N J ) calibrated with molecular weight standards and equilibrated with 0.5 M acetic acid. Dialyzed CM or basal medium (3 ml) was lyophilized and resuspended in water at one-third the original volume.
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