Anchorage-independent colony growth of pulmonary fibroblasts derived from fibrotic human lung tissue.

Fibroblast heterogeneity is known to exist in chronically inflamed tissue such as pulmonary fibrosis (IPF) and scleroderma. We have previously shown differences in proliferation rates in primary lines and cloned lines of fibroblasts derived from IPF tissue compared with normal lung. In this study, we report that cell lines derived from fibrotic tissue demonstrate anchorage-independent growth in soft agarose culture whereas normal lung fibroblast lines do not. We also show that fibroblast lines derived from neonatal lung tissue form colonies at about the same frequency as the fibrotic cells. Colonies from both fibrotic and neonatal lines were shown to be positive for vimentin, laminin, fibronectin, fibronectin receptor, beta-actin, and tropomyosin by immunohistochemistry but were negative for desmin, keratin, Factor VIII, alpha-smooth muscle cell actin, and tenascin. Treatment with cytokines TGF-beta and PDGF or with corticosteroid modified the colony-forming capacity of fibrotic and neonatal cell lines, however, none of these treatments induced normal lung cell lines to form colonies. The presence of cells in adult fibrotic tissue with growth characteristics similar to those exhibited by neonatal cells is further evidence of fibroblast heterogeneity and suggests newly differentiated fibroblasts may be prevalent in fibrotic tissue and contribute directly to the matrix disorder seen in this disease.

Modulation of the anchorage-independent phenotype of human lung fibroblasts obtained from fibrotic tissue following culture with retinoid and corticosteroid.

Fibroblast heterogeneity has been documented in fibrotic tissue from lung and skin. Differences have been demonstrated in proliferative rates in fibroblasts derived from fibrotic lung tissue as compared to normal. Fibroblast lines derived from adult fibrotic lung tissue and neonatal normal lung tissue exhibit colony growth in soft agarose culture, whereas fibroblast cell lines from normal adult lung tissue do not. The characteristic of anchorage-independent growth is consistent with the aggressive nature of the disease and with developmental lung growth. In this study, fibrotic lung fibroblasts were exposed to growth and differentiating factors to determine whether the anchorage-independent phenotype can be modulated. The results indicate that treatment of fibrotic lung fibroblasts with retinoic acid, known to modify matrix gene expression and induce differentiation, inhibits the cells ability to form colonies under soft agarose growth. Treatment with all-trans-retinoic acid yielded the greatest effect inhibiting both IPF and neonatal lung fibroblast anchorage-independent growth approximately 90% at 10(-6) M. Treatment of IPF fibroblasts with all-trans-retinoic acid also inhibited corticosteroid-induced colony growth. Modulation of the "fibrotic" fibroblast phenotype through retinoid therapy may prove beneficial as a potential therapeutic strategy.

Overexpression of RANTES using a recombinant adenovirus vector induces the tissue-directed recruitment of monocytes to the lung.

RANTES (regulated on activation, normal T cell expressed and secreted) is a member of the C-C superfamily of chemokines and is reported to function as a potent chemoattractant for monocytes, eosinophils, and a subpopulation of CD4+ T cells. Using a recombinant human type 5 adenovirus containing the murine RANTES cDNA (Ad5E3 mRANTES), which is capable of expressing biologically active cytokine upon infection, we initiated a study to characterize the biologic functions of RANTES cytokine in vivo. Intratracheal administration of Ad5E3 mRANTES targeted transient RANTES expression to the bronchial epithelium of the lung in Sprague-Dawley rats. Bronchoalveolar lavage fluids (BAL) collected at 24 h had increased chemotactic activity vs controls as measured in a murine CD4+ T cell Boyden chamber microchemotaxis assay. There was a dramatic increase in the number of cells (macrophage, monocytes, and neutrophils) recovered from BAL samples taken from Ad5E3 mRANTES-treated animals at 24 h, with a >50-fold increase in monocytes, indicating a proinflammatory effect for this cytokine in vivo. This effect on monocytes was transient, decreasing by 7 days, with evidence of increased eosinophils and lymphocytes at this time. Histologic examination of lung sections at 24 h revealed greatly increased numbers of mononuclear cells, primarily monocytes, within the lungs of Ad5E3 mRANTES-treated animals, with increased extravasation of monocytes around blood vessels, indicating an ongoing process of peripheral blood monocyte recruitment. This study provides further evidence for RANTES to be a monocyte chemoattractant in vivo.