Previous attempts to culture mouse alveolar type II (ATII) cells have been hampered by limited purity and cell recovery. We have now obtained culturable ATII cells from female C57BL/6 mice at a purity of 92% +/- 3 (mean +/- SD; n = 20), with viabilities of 96% +/- 2 and total yields of 5.1 +/- 0.7 X 10(6) cells per mouse. Crude lung cell suspensions were prepared by intratracheal instillation of Dispase and agarose followed by mechanical disaggregation of the lungs. Crude cell suspensions were purified by negative selection using a biotinylated-antibody, streptavidin-coated biomagnetic particle system. Cell purities were determined by Pap staining and confirmed ultrastructurally. Purified ATII cells were cultured on fibronectin-coated chamber slides and maintained for up to 5 days in DMEM with 10% fetal bovine serum. Cultures exhibited minimal contamination by Clara cells, mesenchymal cells, or endothelial cells, and the epithelial nature of the cultures was confirmed by positive cytokeratin staining in at least 97% of the cells through day 5. Day 3 cultures demonstrated osmium tetroxide/tannic acid-stained granules consistent with lamellar bodies in 76% +/- 3.6 of the cells. The cultures displayed features distinct from those previously described for adult rat ATII cells, including irregularly-shaped cells and the formation of numerous cytoplasmic projections in direct contact with other cells. These studies indicate that excellent yields of highly purified, culturable ATII cells can be obtained from genetically defined mice. These techniques may provide powerful new models for the study of parenchymal lung disease in vitro.

Connective tissue growth factor (CTGF) is a newly described 38-kDa peptide mitogen for fibroblasts and a promoter of connective tissue deposition in the skin. The CTGF gene promotor contains a transforming growth factor-β1 (TGF-β1) response element. Because TGF-β1 expression is upregulated in several models of fibroproliferative lung disease, we asked whether CTGF is also upregulated in a murine lung fibrosis model and whether CTGF could mediate some of the fibrogenic effects associated with TGF-β1. A portion of the rat CTGF gene was cloned and used to show that primary isolates of both murine and human lung fibroblasts express CTGF mRNA in vitro. There was a greater than twofold increase in CTGF expression in both human and murine lung fibroblasts 2, 4, and 24 h after the addition of TGF-β1 in vitro. A bleomycin-sensitive mouse strain (C57BL/6) and a bleomycin-resistant mouse strain (BALB/c) were given bleomycin, a known lung fibrogenic agent. CTGF mRNA expression was upregulated in the sensitive, but not in the resistant, mouse strain after administration of bleomycin. In vivo differences in the CTGF expression between the two mouse strains were not due to an inherent inability of BALB/c lung fibroblasts to respond to TGF-β1 because fibroblasts from untreated BALB/c mouse lung upregulated their CTGF message when treated with TGF-β1 in vitro. These data demonstrate that CTGF is expressed in lung fibroblasts and may play a role in the pathogenesis of lung fibrosis.

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