Idiopathic pulmonary fibrosis (IPF) is a chronic lung disorder characterized by fibroblast proliferation and extracellular matrix accumulation. However, studies on fibroblast growth rate and collagen synthesis have given contradictory results. Here we analyzed fibroblast growth rate by a formazan-based chromogenic assay; fibroblast apoptosis by in situ end labeling (ISEL) and propidium iodide staining; percent of alpha-smooth muscle actin (alpha-SMA) positive cells by fluorescence-activated cell sorter; and alpha1-(I) collagen, transforming growth factor (TGF)-beta1, collagenase-1, gelatinases A and B, and tissue inhibitor of metalloproteinase (TIMP)-1, -2, -3, and -4 expression by reverse transcriptase/polymerase chain reaction in fibroblasts derived from IPF and control lungs. Growth rate was significantly lower in IPF fibroblasts compared with controls (13.3 +/- 38.5% versus 294.6 +/- 57%, P < 0.0001 at 13 d). Conversely, a significantly higher percentage of apoptotic cells was observed in IPF-derived fibroblasts (ISEL: 31.9 +/- 7.0% versus 15.5 +/- 7.6% from controls; P < 0.008). alpha-SMA analysis revealed a significantly higher percentage of myofibroblasts in IPF samples (62.8 +/- 25.2% versus 14.8 +/- 11.7% from controls; P < 0.01). IPF fibroblasts were characterized by an increase in pro-alpha1-(I) collagen, TGF-beta1, gelatinase B, and all TIMPs' gene expression, whereas collagenase-1 and gelatinase A expression showed no differences. These results suggest that fibroblasts from IPF exhibit a profibrotic secretory phenotype, with lower growth rate and increased spontaneous apoptosis.

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Earlier work from this laboratory showed that abnormal fibroblast phenotypes isolated from fibrotic human lung produce factor(s) capable of inducing apoptosis and necrosis of alveolar epithelial cells in vitro [B. D. Uhal, I. Joshi, A. True, S. Mundle, A. Raza, A. Pardo, and M. Selman. Am. J. Physiol. 269 ( Lung Cell. Mol. Physiol. 13): L819–L828, 1995]. To determine whether epithelial cell death is associated with proximity to abnormal fibroblasts in vivo, the spatial distribution of epithelial cell loss, DNA fragmentation, and myofibroblasts was examined in the same tissue specimens used previously for fibroblast isolation. Paraffin sections of normal and fibrotic human lung were subjected to in situ end labeling (ISEL) of fragmented DNA and simultaneous immunolabeling of α-smooth muscle actin (α-SMA); replicate samples were subjected to electron microscopy and detection of collagens by the picrosirius red technique. Normal human lung exhibited very little labeling except for positive α-SMA immunoreactivity of smooth muscle surrounding bronchi and vessels. In contrast, fibrotic human lung exhibited moderate to heavy ISEL of interstitial, cuboidal epithelial, and free alveolar cells. ISEL of the alveolar epithelium was not distributed uniformly but was most intense immediately adjacent to underlying foci of α-SMA-positive fibroblast-like interstitial cells. Both electron microscopy and picrosirius red confirmed epithelial cell apoptosis, necrosis, and cell loss adjacent to foci of collagen accumulation surrounding fibroblast-like cells. These results demonstrate that the cuboidal epithelium of the fibrotic lung contains dying as well as proliferating cells and support the hypothesis that alveolar epithelial cell death is induced by abnormal lung fibroblasts in vivo as it is in vitro.