Precision-cut rat lung slices have been employed in combination with an extensive immunohistochemistry of paraffin-embedded slices for monitoring of early pathohistological changes after exposure to CdCl(2)/TGF-beta(1). Three days of CdCl(2) exposure in combination with TGF-beta(1) seem to be sufficient to induce lung injury with alterations similar to changes observed in early lung fibrogenesis: (1) extracellular matrix accumulation and myofibroblast transdifferentiation (Sirius red staining, collagen type IV, alpha-smooth muscle actin), (2) type I cell injury with loss of type I cell antigens (T1alpha antigen, aquaporin-5, RAGE), (3) increased apoptosis of pulmonary cells (active caspase-3, vimentin cleavage product V1 of caspase-9), and (4) activation of microvascular endothelial cells (podocalyxin, caveolin-1). Western blot analysis confirmed the increasing amount of alpha-smooth muscle actin, the loss of T1alpha antigen, and the increase in caveolin-1 immunoreactivity. The explant culture using CdCl(2)/TGF-beta(1) provides a suitable tool for the study of other factors involved in pulmonary pathology including transcription factors, cytokines, and other metabolites involved in early stages of fibrogenesis.

The study of function and regulation of the phenotype of alveolar type I (AT I) epithelial cells is limited by the rareness of suitable cell lines or primary cultures of this cell type. We describe in the present study the type I-like rat epithelial cell line R3/1. This cell line displays in vitro a phenotype with several characteristic features of AT I cells. R3/1 cells were analysed for mRNA and protein content of markers related to the AT I cell type (T1alpha, ICAM-1, connexin-43, caveolins-1 and -2) and AT II phenotypes [surfactant proteins (SPs) A, B, C and D]. The mRNAs for SPs were found to be at a low level. Moderate protein levels for SP-A and SP-B were found, and SP-C and SP-D proteins were not detectable. R3/1 cells are positive for CD44s, E-cadherin, cytokeratin, vimentin and RAGE, and bind the lectins BPA and SBA. For demonstration of the suitability of R3/1 cells for in vitro studies on epithelial injury, the cells were treated with bleomycin. As shown by real-time RT-PCR and immunoblotting, bleomycin-treatment of R3/1 cells resulted in a decrease in mRNA and protein for both caveolin-1 and caveolin-2 in comparison with controls. The AT I-like cell line R3/1 may serve as a promising tool for the study of lung cell biology.

Surfactant deficiency and bronchopulmonary dysplasia (BPD), major obstacles in preterm infants, are addressed with pre- and postnatal glucocorticoids which also evoke harmful catabolic side-effects. Keratinocyte growth factor (KGF) accelerates surfactant production in fetal type II pneumocytes (PN-II), protects epithelia from injury and is deficient in lungs developing BPD, highlighting its potential efficacy in neonates. Neonatal rats were treated with recombinant human (rh)KGF, betamethasone, or their combination for 48 hr prior to sacrifice after which body weight, surfactant, and tissue phosphatidylcholines (PC) were investigated at postnatal d3, d7, d15, and d21. Pneumocyte proliferation, surfactant protein (SP) expression and SP-B/C in lung lavage fluid (LLF) were also determined at d7 and d21 to identify broader surfactant changes occurring at the beginning and end of the initial alveolarization phase. While all treatments increased secreted surfactant PC, BM compromised animal growth whereas rhKGF did not. At d3 rhKGF was more effective in male compared to female rats. Single treatments became less effective towards d21. Neither treatment altered PC composition in LLF. BM inhibited PN-II proliferation and increased surfactant PCs at the expense of tissue PCs. rhKGF however increased surfactant PCs without decreasing other PC species. Whereas SP-B/C gene expression was induced by all treatments, the changes in secreted SP-B/C mirrored those observed for surfactant PC. Our results encourage investigation of the mechanisms by which rhKGF improves surfactant homoeostasis, and detailed examination of its efficacy in neonatal lung injury models with a view to implementing it as a non-catabolic surfactant-increasing therapeutic in neonatal intensive care.

SP-D accumulation is increased in this model of allergen-induced eosinophilia, both in upper and lower airways. The increase is unaffected by dexamethasone.

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