Hermansky-Pudlak syndrome (HPS) is a genetically heterogeneous inherited disease causing hypopigmentation and prolonged bleeding times. An additional serious clinical problem of HPS is the development of lung pathology, which may lead to severe lung disease and premature death. No cure for the disease exists, and previously, no animal model for the HPS lung abnormalities has been reported. A mouse model of HPS, which is homozygously recessive for both the Hps1 (pale ear) and Hps2 (pearl) genes, exhibits striking abnormalities of lung type II cells. Type II cells and lamellar bodies of this mutant are greatly enlarged, and the lamellar bodies are engorged with surfactant. Mutant lungs accumulate excessive autofluorescent pigment. The air spaces of mutant lungs contain age-related elevations of inflammatory cells and foamy macrophages. In vivo measurement of lung hysteresivity demonstrated aberrant lung function in mutant mice. All these features are similar to the lung pathology described in HPS patients. Morphometry of mutant lungs indicates a significant emphysema. These mutant mice provide a model to further investigate the lung pathology and therapy of HPS. We hypothesize that abnormal type II cell lamellar body structure/function may predict future lung pathology in HPS.

Hermansky Pudlak syndrome (HPS) is a heterogeneous recessive genetic disease with a tendency to develop lung fibrosis with aging. A mouse strain with two mutant HPS genes affecting separate vesicle trafficking pathways, C57BL/6-Hps1ep-Ap3b1pe, exhibits severe lung abnormalities at young ages, including enlarged alveolar type II (ATII) cells with giant lamellar bodies and foamy alveolar macrophages (AMs), which are readily identified histologically. In this study, the appearance of lung fibrosis in older animals was studied using classical histological and biochemical methods. The HPS double mutant mice, but not Chediak Higashi syndrome (C57BL/6-Lystbg-J-J, CHS) or C57BL/6J black control (WT) mice, were found to develop lung fibrosis at about 17 months of age using Masson trichrome staining, which was confirmed by hydroxyproline analysis. TGF β1 levels were elevated in bronchial alveolar lavage samples at all ages tested in the double mutant, but not WT or CHS mice, indicative of a prefibrotic condition in this experimental strain; and AMs were highly positive for this cytokine using immunohistochemistry staining. Prosurfactant protein C staining for ATII cells showed redistribution and dysmorphism of these cells with aging, but there was no evidence for epithelial-mesenchymal transition of ATII cells by dual staining for prosurfactant C protein and α-smooth muscle actin. This investigation showed that the HPS double mutant mouse strain develops interstitial pneumonia (HPSIP) past 1 year of age, which may be initiated by abnormal ATII cells and exacerbated by AM activation. With prominent prefibrotic abnormalities, this double mutant may serve as a model for interventive therapy in HPS.

Primary kidney cultures from C57BL/6J mice, 6 weeks of age or older, were produced using D-valine medium to select for epithelial cell growth. After allowing the cells to attach and proliferate for 1 week following plating, medium was changed once per week. Cells formed nearly confluent monolayers during the second week of culture. The cultured cells contained all of the glycosphingolipids seen in the adult kidney, analyzed by high performance liquid chromatography as their perbenzoyl derivatives. Glucosylceramide, however, was highly predominant in the cultured cells, whereas dihexosyl- and trihexosylceramides predominate in the intact kidney. Sex differences in glycolipid contents found in the intact kidney were also apparent in these cultured cells: The concentration of neutral glycolipids, in general, was higher in male cells than in those derived from females, and the male-specific glycolipid nonhydroxy fatty acid digalactosylceramide was high in male cells but very low in female cells. Neutral glycosphingolipids were labeled in 2-week-old cultures using [3H]palmitate. The [3H]palmitate was incorporated into all of the glycolipids within 2 hr of labeling. Hence, adult mouse kidney cells in D-valine medium retain their differentiated characteristics for a sufficient period of time to allow investigation of glycolipid syntheses in monolayer cultures of epithelial cells derived from this organ.

Primary cell cultures from twitcher (galactocerebrosidase deficient) mice were made by enzymatic dispersion and explantation of skin obtained from 3-d-old littermates of a twi+/twi X twi+/twi mating. Galactocerebrosidase activity remained deficient for two twitcher cell lines, TM-1 and TM-2, and both lines demonstrated an initial period of growth decline, followed by accelerated growth. The TM-2 line has been subcultured for more than 3.5 yr, has a modal chromosome number of 63, a doubling time of approximately 16 h, and has remained galactocerebrosidase deficient throughout its life span. These data indicate this to be an established twitcher cell line that can be continuously maintained in culture as a transformed galactocerebrosidase-deficient mouse cell line. This established line was rendered 6-thioguanine resistant so that the cells could be fused with control human fibroblasts and selected for hybrid lines in hypoxanthine-aminopterin-thymidine medium. Also, the established twitcher cells were crossed with neomycin-resistant control human fibroblasts and selected in G418 medium. Several of the hybrid lines from both crosses had higher than deficient levels of galactocerebrosidase activity initially, followed by a decrease to twitcher levels during subculture, whereas other lines retained high levels of activity. These results indicate that twitcher-human somatic cell hybrids will express galactocerebrosidase activity and thus may be useful for determining the human chromosome or chromosomes associated with this expression.