Histochem Cell Biol (2010) 134:205–213 211 Fig. 4 Representative prosurf- actant protein C (strong green, shown by arrow heads) and -SMA-stained (pink, shown by arrows) lung sections from HPS mice at postnatal ages of a 12, b 17 months with a Wbrotic area, Lung sections of WT c and CHS d mice at 17 months of age. Arrow heads showed representa- tive ATII cells which were stained strong green, and arrows showed representative myoW- broblasts which were stained pink. Highly positive -SMA cells were found in close prox- imity to aggregations of ATII cells at 12 months and 17 months of age in HPS double mutant mice, but this marker was not co-localized with that of prosurfactant C protein. Fewer myoWbroblasts were found in WT and CHS. Walls of bronchi- oles and blood vessels were also stained with -SMA marker found in ATII cells of mice during compensatory regrowth after single lung pneumonectomy (HoVman et al. 2010). The persistent presence of abnormally high levels of total TGF 1 which was seen in BAL samples and localized in AMs by IHC staining, is another preWbrotic condition. This cytokine was mainly localized to AMs, suggesting this cell type is its major source, which is in keeping with other studies using chemically induced animal models (Chen et al. 2009 Nishimura et al. 2007). Previous studies have shown that the deWciency of surfactant protein B in mice promoted AM activation (Ikegami et al. 2005). Thus, the blocked secretion of surfactant proteins in HPS mice (Guttentag et al. 2005) may increase TGF 1 secretion by activation of AMs. This hypothesis is also supported by the studies of CHS mouse strain, which also possesses enlarged lamellar bodies in ATII cells but exhibits normal surfactant lipid secretion of ATII cells (Prueitt et al. 1978) and, in this study, showed no increased levels of TGF 1 in BAL sam- ples with aging. On the basis of histological analyses, Tang et al. (2005) reported that CHS (Lystbg) mice exhibited a more robust Wbrosis than seen in the HPS1 mouse model, C57/B6 HPS1ep. Our results do not conWrm this observation regarding the appearance of pulmonary Wbrosis in the CHS strain. Intra-strain variation and environmental factors may account for this discrepancy. However, the similar levels of TGF 1 and hydroxyproline observed in the lungs of CHS compared with WT mice in our laboratory were consistent with the histological assessment of the lack of Wbrotic patches in the CHS animals at all ages tested. It is also consistent with CHS patients who are not at risk for lung Wbrosis (Intone et al. 2009). The appearance of TGF 1 in epithelial cells in lungs from HPS mice older than 1 year indicated a possible sec- ondary source of this cytokine. This change coincided with an increase of TGF 1 in BAL which suggests that addi- tional cell types contribute TGF 1 levels when HPS mice enter the accelerated deterioration period. Although the role of TGF 1 in the development of lung Wbrosis is well established, there are still questions about the importance of EMT in this process. For example, Kim et al. (2009) created a transgenic mouse strain that perma- nently expressed GFP in prosurfactant C positive cells, and found colocalization of -SMA and GFP after bleomycin treatment. KönigshoV et al. (2009) also found -SMA expression in ATII primary cell cultures from bleomycin treated mice. In this study, when single DAB staining of -SMA was used, some positively stained cells were observed with ATII cell morphology in HPS double mutant mice at old ages (data not shown). However, the Xuorescent double staining method did not support EMT in ATII cells. This is similar to the work of Yamada et al. (2008) who showed no evidence of EMT using dual IHC staining of epithelial markers and myoWbroblast markers, including -SMA, in bleomycin treated mice. These inconsistencies may be because either the sensitivity of antibody staining is aVected by double staining in animal tissues or that, in single staining, ATII cells from Wbrotic lungs absorb the reactive antibody and cause false positives. In this 123