212 Fig. 5 TGF 1 BAL levels and localization in lungs. Panel a shows average TGF 1 (§SD) levels in BAL samples of HPS, WT, and CHS mice from 2 to 17 months of age using ELISA. Samples of 4–10 mice were used for each time point and strain. SigniWcant diVerence P < 0.05: *HPS versus WT; #HPS versus CHS. No signiWcant diVerence between CHS and WT was found at any age tested. Panels b–e are representative TGF 1 IHC stained (brown precipitate) lung sections from HPS at (b) 9 and (c) 12 months of age and from (d) WT and (e) CHS at 12 months of age. AMs (intra- alveolar cells) were darkly stained in each of the mouse strains at all ages tested, and staining appeared in epithelial cells as well in older HPS mice but not in WT or CHS mice Histochem Cell Biol (2010) 134:205–213 investigation, the morphological changes observed in ATII cells of HPS mice with aging suggested that the cells under- went some conversion, but whether this involves EMT needs additional study. If this conversion is not due to EMT, these cells may diVerentiate into alveolar type I (ATI) cells in order to compensate for lung destruction. The abnormal prosurfactant C staining in HPS compared with WT mice may be due to diYculty in surfactant clearance in HPS ATII cells compared with WT. This hypothesis could be conWrmed by double staining of prosurfactant C and ATI cell markers in our future studies. The presence of Wbrotic patches and elevated levels of hydroxyproline with aging, and the persistently high levels of TGF- 1 in alveoli of these mice, versus the absence of these features in the WT control and CHS strain provide evidence for progressive HPSIP in the double mutant strain. As this has not been reported for either of the single HPS mutant animals from which the HPS double mutant strain was derived, it appears that the combination of two vesicle traYcking disorders aVecting separate pathways is 123 especially injurious to long-term maintenance of lung homeostasis. The lung cell abnormalities that are known in this strain include the presence of giant lamellar bodies in ATII cells (Lyerla et al. 2003) resulting in the block to secretion of surfactant protein (Guttentag et al. 2005) and, from this study, the high levels of alveolar TGF- 1 mainly due to activated AMs. It appears that the blocked secretion of ATII cells induces AM activation and lung stresses such as alveolar destruction. The lung stresses may trigger a compensatory response including ATII cell regrowth. The activated AMs, which secrete high levels of TGF 1, and ATII cells, which show morphological cell conversion, lead the lung into progressive HPSIP in this strain. Acknowledgments Partial support for this research was provided by NIH grant R15 HL091503-01, and is gratefully acknowledged. We thank Nuclea Biotechnologies, LLC, for the use of their equipment, and Edmund Sosnoski for his advice and assistance with automated histological processing. We are also indebted to the anonymous reviewers for their suggested corrections and changes in the original manuscript.