206 bodies of ATII cells (Lyerla et al. 2003), that are not as pronounced in the individual mutant strains (Feng et al. 2002). Thus, the mutant eVects of each gene on cellular secre- tion were exaggerated in this double homozygote condition, which resulted in abnormal perturbations of aVected cell types, particularly ATII cells. Subsequent investigations showed levels of phospholipid and surfactant proteins B and C were highly elevated in lung homogenates, but decreased in bronchial alveolar lavages (BAL, Guttentag et al. 2005), indi- cating accumulated storage and blocked secretion for both surfactant lipids and proteins of the ATII cells that accounted for the giant lamellar bodies seen in these mutant cells. The highly dysmorphic ATII cells in the HPS double mutants were similar to those observed in HPSIP patients (Nakatani et al. 2000), however, there was no evidence of lung Wbrosis at these early ages (Lyerla et al. 2003). Studies of lung morphology at the cellular and histological levels in older ages, then, were needed to determine the possible development of pulmonary Wbrosis with aging in this HPS double mutant, which could be attributed to the cellular defects in the secretory process caused by these mutations. Abnormal lamellar bodies in ATII cells are also found in another mouse mutant, beige (Lystbg, Lystbg), which is an animal model for Chediak Higashi syndrome (CHS) (Chi et al. 1975). This mouse strain exhibits accumulation of surfactant in ATII cells, but shows normal secretion of sur- factant phospholipids (Prueitt et al. 1978). Because of the shared dysmorphism of ATII cells in the HPS double mutant and CHS strains, the CHS mouse was also studied in these investigations. TGF 1 is considered to be the most important cytokine for lung Wbrosis because it links possible causative processes including inXammation and Wbroblast growth in the forma- tion of this disease. Previous studies found this cytokine is secreted from AMs in vitro (Nishimura et al. 2007) and stimulated the diVerentiation and growth of myoWbroblasts (Gu et al. 2007), the collagen producing cells in lung Wbro- sis. TGF 1 administration can also induce epithelial–mes- enchymal transition (EMT) (Kim et al. 2007; Liu 2004), a process that appears important for lung Wbrosis (Kim et al. 2009). For these reasons, investigations of the time course of TGF 1 secretion and localization of this cytokine in vivo were made in conjunction with the histochemical and cellu- lar analyses that assessed the development of pulmonary Wbrosis in the HPS double mutant mouse strain. Materials and methods Mice The breeding stock of C57BL/6-Hps1ep-Ap3b1pe, the dou- ble mutant strain homozygous for two Hermansky Pudlak 123 Histochem Cell Biol (2010) 134:205–213 Syndrome genes and referred to as the HPS double mutant, was kindly provided by Dr. Richard Swank of the Roswell Park Cancer Institute (BuValo, NY, USA). C57BL/6J (wild type, or WT), and C57BL/6-Lystbg-J-J, the beige mouse strain which is an animal model for Chediak Hig- ashi Syndrome (CHS), were purchased from Jackson Laboratories (Bar Harbor, ME, USA). All experimental procedures were approved by the Institutional Animal Care and Use Committee and followed the NIH Guidelines for Laboratory. Mouse samples from the three strains were free of infectious disorders as determined by Assessment Plus (Charles River Laboratories, Wilmington, MA, USA). For biochemical and histological analyses, mice of given ages were anesthetized with avertin, 1 mL of PBS bronchial alveolar lavage (BAL) Xuid was transfused into cannulated lungs three times, and 80% BAL Xuid was recovered. After the lavage, the right lobes were separated with surgical thread and snap frozen in liquid nitrogen for further analy- sis. The left lung was fully inXated and Wxed in 4% buVered formalin, embedded in paraYn, sectioned at 5 m, and mounted on positively charged slides. After centrifugation, the cell-free BAL supernatant was stored at ¡80°C for fur- ther studies. Hydroxyproline analysis The method of Lazo (1981) was used for hydroxyproline analyses from isolated right lobes. BrieXy, the lobes were homogenized in 2 ml deionized water using either a Dounce homogenizer or Polytron tissue grinder (Brinkman, Lucern, Switzerland), and samples prepared from trichloro- acetic acid precipitates were treated in 12 N HCl at 110°C for 16 h and dissolved in 2 ml deionized water. Samples and standards (50 l each) were incubated with Chloramine T reagent for 20 min at room temperature, and with Ehrlich’s reagent for 15 min at 65°C. The Wnal reaction solutions were measured for absorbance at 550 nm (UV/ VIS spectrophotometer UV-1650PC, Shimadzu ScientiWc Instruments, Columbia, MD, USA) and hydroxyproline levels obtained as microgram per lung. Alveolar size quantiWcation Mean linear intercept (Lm) was employed to assess for alveolar size according to Thurlbeck (1967). BrieXy, pho- tomicrographs of hematoxylin stained sections were divided into six identical squares by lines. Alveolar walls crossing each line were counted, the counts were summed for each print, and these were divided into the total length of the lines crossing the photos in order to provide the mean linear intercept value, Lm, for each photomicro- graph. For this analysis, three photomicrographs were