Aberrant lung structure, composition, and function in a murine model of Hermansky-Pudlak syndrome

Timothy A. Lyerla,1 Michael E. Rusiniak,2 Michael Borchers,3 Gerald Jahreis,2 Jian Tan,2 Patricia Ohtake,4 Edward K. Novak,2 and Richard T. Swank2

1Department of Biology, Clark University, Worcester, Massachusetts 01610; 2Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo 14263 and 4Department of Rehabilitation Science, State University of New York at Buffalo, Buffalo, New York 14214; and 3Department of Environmental Health, Division of Toxicology, University of Cincinnati Medical Center, Cincinnati, Ohio 45267

Submitted 24 January 2003 ; accepted in final form 21 May 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
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.

inherited lung dysfunction; inflammation; surfactant protein; lamellar body; secretion


HERMANSKY-PUDLAK SYNDROME (HPS; Mendelian inheritance in man 203300 [OMIM] ) is a genetically heterogeneous disease caused by abnormal trafficking to and among lysosome-related organelles such as melanosomes, platelet-dense granules, and lysosomes (25, 45). Six genetically distinct forms of HPS have been described in humans. HPS1 (39) is most prevalent and is caused by mutations in the novel HPS1 gene. HPS2 (AP3B1) (13) is caused by mutations in the {beta}3A-subunit of the adaptor protein-3 (AP-3) adaptor complex, which is well known to regulate endocytic vesicle trafficking (43). The remaining HPS forms (HPS3, HPS4, HPS5, and HPS6) (2, 48, 60) arise from mutations in novel vesicle-trafficking genes. The clinical consequences of HPS include hypopigmentation and loss of visual acuity and prolonged bleeding. However, the most serious clinical problem is lung fibrosis, which may lead to death in midlife (8, 25, 37). HPS lung disease presently has no cure, and its molecular cause(s) is unknown. The incidence and severity of pulmonary insufficiency varies widely among HPS patients. It appears greatest in HPS1 and HPS4, although there are relatively few relevant studies in the other genetic forms of HPS (3, 8, 26).

At least 16 mouse hypopigmentation mutants accurately model HPS (51, 52, 60). The corresponding mouse models for the various forms of human HPS are: HPS1/pale ear (16, 20), HPS2/pearl (18), HPS3/cocoa (49), HPS4/light ear (48), HPS5/ruby-eye-2 (60), and HPS6/ruby-eye (60). One group of mouse HPS genes, including mocha (Ap3d) (27), pearl (Ap3b1) (18), gunmetal (Rabggta) (14), ashen (Rab27a) (5, 38, 56), and buff (Vps33a) (50), encode the {delta}- and {beta}3A-subunits of the AP-3 adaptor complex, the {alpha}-subunit of rab geranylgeranyltransferase, Rab27a and Vps33a respectively. These genes encode proteins with well-established roles in vesicle trafficking. Another group of mouse HPS genes, including pallid (24), cocoa (49), muted (59), pale ear (16, 20), light ear (48), ruby-eye (60), and ruby-eye-2 (60), encode novel proteins whose detailed roles in vesicle trafficking are unknown, although evidence is accumulating that certain members of this group act together in protein complexes (15, 36, 48, 60). Some members of the "granule group" of Drosophila mutant genes are identical or closely related to HPS genes (29).

There is a limited number of studies of lung abnormalities in mouse HPS mutants. The most complete analyses involve the pallid mutant, which develops emphysema in older (>1 yr) mutants (11, 28). Bone marrow transplantation, which corrects the platelet defects in pallid mutants, does not remedy the lung deficiency (33). Preliminary studies of other mutant HPS mice have detected a decreased lifespan, accompanied by moderate lung morphological abnormalities in several mutants (34).

Double mutant HPS mice are useful because epi-static interactions between mutant gene products may amplify mutant physiological abnormalities. Mutants homozygous for both the pale ear (Hps1 or ep) and pearl (Ap3b1 or pe) HPS genes, produced in this laboratory (17), exhibit more severe mutant phenotypes for all the common lysosome-related organelles (melansomes, platelet-dense granules, and lysosomes), indicating that these genes cooperate in regulating different aspects of vesicle trafficking. An intriguing observation was that, unlike all other mouse HPS mutants, levels of lung lysosomal enzymes were significantly elevated in this ep/ep,pe/pe mutant, suggesting possible abnormalities of lung lysosome-related organelles, such as lamellar bodies (55) of type II cells. Here we describe prominent morphological and biochemical abnormalities in lung type II cells of ep/ep,pe/pe mouse mutants, similar to those reported in type II cells of Hermansky-Pudlak patients (37), together with aberrant lung function.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Mice. All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee and adhered to the principles of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All normal C57BL/6J mice and mutant Hps1/Hps1 (pale ear) and Ap3b1/Ap3b1 (pearl) mice were originally obtained from The Jackson Laboratory and were subsequently bred and maintained at Roswell Park Cancer Institute. The production, by appropriate breeding, of double mutant Hps1/Hps1,Ap3b1/Ap3b1 mice (referred to throughout this paper as ep/ep,pe/pe mutants) was previously described (17). All single and double mutant mice were maintained on the C57BL/6J inbred strain background. Animals of both sexes were utilized; we have observed no sex-specific effects among any of the parameters measured in this report. Serological tests (Missouri University Research Animal Diagnostic Laboratory, Columbia, MO) of three C57BL/6J and three ep/ep,pe/pe mice (3-5 mo of age) for parasites and for 14 common viral and bacterial antigens were negative as were tests of feces for five helicobacter species, tests of nasophyrnygeal swabs for Mycoplasma pulmonis and Pasteurella pneumotropica, and tests of cecum for Pseudomonas aeruginosa and Salmonella. Lung histopathology on normal and mutant mice was negative with the exception of the foamy macrophages in lungs of ep/ep,pe/pe mice described in this report.

Immunoblots. Lung tissues from each animal were weighed and then homogenized in a Polytron (PCU-2; Kinematica, Lucerne, Switzerland) at 1:10 dilution in 0.02 M imidazole buffer, pH 7.4, containing 0.1% Triton X-100 (vol/vol) and 0.25 M sucrose. Protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN) was added to each sample at 1:25 dilution, and samples were stored at -70°C. Proteins were separated by polyacrylamide gel electrophoresis (12% acrylamide gels in Tris-SDS buffer), and the proform of surfactant protein C detected on blots (polyvinylidene difluoride transfer membrane Hybond-P; Amersham Biosciences, Buckinghamshire, UK) using a goat polyclonal antibody (RD-1RTSURFCCabG; Research Diagnostics, Flanders, NJ). Equivalent loading and transfer were verified using actin (rabbit polyclonal antibody, cat. no. AAN01; Cytoskeleton, Denver, CO) as the internal standard. Blots were exposed to film for several different times to ensure that the density of bands was within the linear range.

Lung and lung cell preparations. Mice were anesthetized with a single dose of Avertin (300 mg tribromoethanol/kg body wt ip). When the animal was no longer responsive to a toe pinch, the trachea was exposed and cannulated with a blunt-tipped 18-gauge syringe needle that was affixed firmly with two 2-0 suture threads tied tightly around the trachea.

Lungs were lavaged with 0.02 M imidazole, 0.85% NaCl, pH 7.4, containing 5 mM EDTA, in three separate 1-ml volumes. The pooled bronchoalveolar lavage (BAL) fluid yielded at least 85% recovery of input lavage fluid. Cells were collected by centrifugation at 800 relative centrifugal force for 10 min at room temperature, the pellet was resuspended in PBS and 10 mM EDTA containing 0.15 M ammonium chloride erythrocyte lysis buffer, and the nonlysed nucleated cells were recovered by centrifugation. Cells were counted using a hemacytometer, and cell differentials were determined from stained cytospin preparations (Hema-3 stain set; Biochemical Sciences, Swedesboro, NJ).

Lungs were inflation-fixed in 10% buffered formalin for standard light microscopy. The cannulated preparation was infused with fixative to 20 cmH2O pressure, and the lung preparation was fixed for 24 h or longer. Fixed lungs were embedded in paraffin, sectioned at 5-µm thickness, and stained with hematoxylin and eosin (H&E) using standard methods. Sections were examined with a Nikon Optiphot microscope equipped with an RT Color digital camera (Diagnostics Instruments, Sterling Heights, MI).

For immunohistochemistry, slides were deparaffinized with a graded series of alcohols and quenched in 3% H2O2 for 20 min at room temperature. Antigen was exposed with proteinase K treatment (Invitrogen, Carlsbad, CA) at 20 µg/ml in PBS with 0.5 µl/l Tween 20 (PBST). Slides were processed for antibody staining using primary antibody to surfactant protein C (Research Diagnostics) at 2 µg/ml for 2 h after blocking with 0.03% casein in PBST for 30 min. Slides were treated with biotinylated donkey anti-goat secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in PBST for 20 min at 1:200 dilution, reacted with streptavidin peroxidase (Zymed, San Francisco, CA) for 30 min, counter-stained with hematoxylin for 7 min (Dako automation hematoxylin), and examined with the Nikon Optiphot research microscope.

Autofluorescence. Lungs were frozen by immersion in liquid nitrogen. Frozen lungs were embedded in cryogel (Instrumedics, Hackensack, NJ), and 8-µm sections were placed onto microscope glass slides that were kept at -70°C until they were examined for autofluorescence using the fluorescein filter set (Nikon Optiphot).

Hydroxyproline assays. Levels of lung hydroxyproline were determined according to the method of Reddy and Enwemeka (42). Briefly, samples were hydrolyzed in 2N NaOH, treated with chloramine T followed with Ehrlich's aldehyde reagent, and absorbance was compared with that of a standard curve at 550 nm.

Phospholipid analyses. A 10% lung homogenate in 0.25 M sucrose and 0.02 M imidazole, pH 7.4, was extracted with chloroform-methanol, and the lipid phase was used for colorimetric determination of released inorganic phosphate (6) with the Fiske-SubbaRow reagent (Sigma Chemical, St. Louis, MO). Dipalmitoylphosphatidylcholine (DPPC) was analyzed in lung homogenates and BAL surfactant after reaction with osmium tetroxide and isolation on neutral alumina columns, as described by Mason et al. (32). The resulting DPPC fraction comigrated on thin-layer chromatography with DPPC standards, and recoveries of standard DPPC from alumina columns ranged from 86% to 91%.

Electron microscopy. Excised lungs were infused with fresh fixative of 1.5% glutaraldehyde and 1.5% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.35, until filled. Blocks were postfixed in 1% osmium tetroxide, placed in cold 4% aqueous uranyl acetate solution, and dehydrated in a graded series of acetone. Thin sections were counterstained with lead citrate and examined on a Siemans 101 electron microscope.

Lung morphometry. The average interalveolar distance (Lm) (53) was determined on H&E-stained histological fields of normal single mutant and double mutant mice. For each normal or mutant mouse, 40 histological fields were evaluated, both vertically and horizontally, independently by each of two investigators. There were no significant differences in resulting measurements of the two investigators. Final Lm values reported were calculated from the combined measurements (n) of the two investigators. Large airways and blood vessels were excluded from measurements. The fields were taken from three separate C57BL/6J and ep/ep,pe/pe and two separate ep/ep and pe/pe mice.

Measurements of lung mechanics. Total respiratory system mechanics were assessed in mice according to the method of Gomes et al. (22). Mice were anesthetized with pentobarbital sodium (50 mg/kg ip Nembutal; Abbott, Chicago, IL). Tracheas were surgically accessed through a ventral midline incision and connected with a small animal ventilator (SAV; FlexiVent, SCIREQ, Montreal, Quebec) with a blunt 18-gauge needle. Mice were subsequently paralyzed with doxacurium chloride (0.5 mg/kg Nuromax; Catalytica, Greenville, NC) and ventilated at a frequency of 150 breath/min and at a volume of 6 ml/kg. The mice expired passively through the expiratory valve of the ventilator against a positive endexpiratory pressure of 3 cmH2O. The resistive and elastic properties of the respiratory system were determined at constant volume using forced oscillations by the SAV. The oscillations consisted of applying a small-amplitude volume perturbation (3 Hz) to the airway opening. Measurements of piston volume displacement and cylinder pressure were used to calculate the impedance of the respiratory system from which respiratory system resistance and elastance (1/compliance) values were derived using a single-compartment model of respiratory mechanics, as described in Pillow et al. (41). All data were analyzed with the flexiVent software.

Tissue hysteresivity is a dimensionless parameter that represents the coupling of dissipative and elastic properties of the lung (19). Hysteresivity was determined using forced oscillations by the SAV and consisted of applying small-amplitude volume perturbations (11 prime frequencies between 0.25 and 9.125 Hz) to the airway opening. The impedance values derived from the forced oscillations were fitted to a constant-phase model by using the flexiVent software as described in Pillow et al. (41). The flexiVent software calculates the tissue damping (G), tissue elastance (H), and hysteresivity (n = G/H).

Statistical analyses. Statistical comparisons utilized ANOVA for comparisons among more than two means and the Student's t-test for comparisons between means. Results are reported as averages ± SE.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Morphologically abnormal type II cells in double mutant mice. Light microscopic H&E analyses revealed that epithelial cells of ep/ep,pe/pe double mutant mice are grossly aberrant. The lung epithelium of double mutant cells contained large numbers of giant cells filled with a "foamy" material with the appearance of surfactant (Fig. 1). Although we have not quantitatively assessed the number and size of these aberrant cells, which appeared to be type II epithelial cells, it was evident that they were present throughout the mutant lung and were not visible in age-matched control C57BL/6J lungs. Furthermore, giant cells were apparent in all mutant sections examined. Typical type II cells were visible in lung epithelium of control lungs (Fig. 1), but they were less numerous, much smaller, and contained much less foamy material than the type II cells present in ep/ep,pe/pe lung.



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Fig. 1. Aberrant morphology of lung type II cells and increase in inflammatory cells in lungs of ep/ep,pe/pe mice. Lungs of 10-wk-old mice were inflation-fixed and stained by hematoxylin and eosin protocols. The normal type II cells of C57BL/6J (a and c) lungs (arrowheads), the morphologically aberrant type II cells (arrowheads) of ep/ep,pe/pe (b and d) lungs, and the infiltrating macrophages (arrows) of ep/ep,pe/pe lungs are indicated. At x200 magnification, the widespread accumulation of inflammatory cells (arrows) throughout the lung parenchyma of double mutants is apparent. Bar = 20 µm.

 

Immunohistochemical staining of mutant lung sections with an antibody to surfactant protein C, a specific component (55) of type II cells (Fig. 2), confirmed that these were type II cells. All foamy cells were strongly positive for surfactant protein C as were the less numerous and smaller type II cells of the control C57BL/6J. Furthermore, it was obvious, even by light microscopy, that the lamellar bodies of the ep/ep,pe/pe mutant type II cells were massive compared with lamellar bodies of control C57BL/6J. Similar sections (Fig. 2) of single mutant ep/ep and pe/pe exhibited an intermediate phenotype. Type II cells of single mutants were smaller and appeared to contain much less surfactant than type II cells of double mutants. However, type II cells of the pe/pe mutant appeared somewhat larger than those of C57BL/6J controls.



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Fig. 2. Morphologically aberrant granules of type II cells of ep/ep,pe/pe mutant mice are positive for surfactant protein C (SP-C). Antibody to SP-C was used to detect type II cells in lungs of 10-wk-old normal and mutant mice. Arrows indicate type II cells in normal and single mutant lung. Arrowheads indicate aberrant type II cells with massive lamellar bodies in lungs of ep/ep,pe/pe mutants. Macrophages are less numerous in these ep/ep,pe/pe sections than in those of Fig. 1 because these lungs were lavaged. Bar = 40 µm.

 

Another abnormality of ep/ep,pe/pe lungs was that many sections contained areas with large open air spaces (Figs. 1 and 2, magnification x200), a feature only occasionally observed in lungs of C57BL/6J or in either of the single mutant mice. Morphometric determinations of mean linear intercept values (Fig. 3) confirmed enlarged air spaces, or emphysema, throughout the lung parenchyma of double mutant mice. The mean linear intercept was 77% increased in ep/ep,pe/pe compared with C57BL/6J controls and was 25% increased in pe/pe compared with controls. There was no significant increase in ep/ep. Examination of ep/ep,pe/pe lungs (not shown) likewise revealed evidence of thickening in the submucosa throughout the conducting airways.



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Fig. 3. The mean linear intercept (µm ± SE) is increased in ep/ep,pe/pe and pe/pe mutants. Mean linear intercepts of alveolae septae were measured in the lungs of 3 mice of each genotype at 11-15 wk of age. All pairwise comparisons are significantly different at the P < 0.001 level (*) except for ep/ep vs. C57BL/6J.

 

Ultrastructural analyses (Fig. 4) confirmed that type II cells of the ep/ep,pe/pe mutants were highly aberrant. Mutant type II cells were greatly enlarged as the result of engorgement with extremely large and numerous abnormal lamellar bodies. Most lamellar bodies of C57BL/6J type II cells were 1.1-1.5 µm in diameter. In contrast, the majority of mutant lamellar bodies were 2.5-4.5 µm in diameter, and giant lamellar bodies up to 28 µm in diameter, which occupied nearly the entire cellular space, were occasionally observed.



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Fig. 4. Type II cells of ep/ep,pe/pe mutant mice are grossly abnormal at the ultrastructural level. Inflation-fixed lungs of 10-wk-old normal C57BL/6J (a) and mutant ep/ep,pe/pe (b) mice were analyzed by electron microscopy. Arrows show lamellar bodies in normal type II cells; arrowheads indicate abnormal lamellar bodies of ep/ep,pe/pe type II cells. Bar = 2 µm.

 

Altogether, the morphological and immunohistochemical analyses showed that type II cells of ep/ep,pe/pe HPS mutants are highly aberrant, containing massive quantities of giant, surfactant-filled lamellar bodies.

Inflammation in lungs of mutants. Another prominent feature of the ep/ep,pe/pe H&E lung sections (Fig. 1) was the presence of large numbers of inflammatory cells, which were not apparent in corresponding sections of control C57BL/6J or single mutant lungs. To further characterize this inflammatory infiltrate, lung lavages were analyzed both quantitatively and qualitatively. The number of cells in BALs of ep/ep,pe/pe and control C57BL/6J mice remained constant between 4 and 17 wk of age (Table 1). Throughout these ages, BALs of ep/ep,pe/pe mice had approximately twice the number of inflammatory cells as those of C57BL/6J. The inflammation in lungs of ep/ep,pe/pe mutants was considerably exacerbated in older (~1 yr) mice, at which point the number of inflammatory cells was 5.6-fold greater than that of comparably aged C57BL/6J. In contrast, no significant differences in BAL cell numbers were apparent among single mutant ep/ep or pe/pe mice or control C57BL/6J mice when mice of 11-17 wk of age were compared. Furthermore, there was no significant increase in BAL inflammatory cell numbers in lungs of aged (~1 yr) mice of any of these latter three genotypes.


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Table 1. Bronchoalveolar lavage (BAL) cell counts in control C57BL/6J and Hermansky-Pudlak syndrome (HPS) mutant mice

 

The inflammatory and morphological abnormalities observed in the ep/ep,pe/pe HPS mutant lungs appear to be mutant specific. We surveyed (not shown) BAL cell numbers in three to five aged (~1-yr-old) animals of each of 10 other mouse HPS mutants (51, 52, 60) including ashen, buff, cocoa, gunmetal, light ear, pallid, ruby-eye, ruby-eye-2, sandy, and subtle gray. No significant differences between mutant and control BAL cell numbers were observed in any case. Furthermore, no morphological abnormalities of type II cells comparable with those observed in the ep/ep,pe/pe mutant were apparent in H&E lung sections of any of these 10 mutants.

The great majority of BAL cells in both control C57BL/6J and ep/ep,pe/pe 8- to 15-wk-old mice were mononuclear macrophages (Table 2), although the percent mononuclear macrophages in double mutants (85%) was significantly lower than that (96%) in C57BL/6J. Furthermore, there were significantly higher percentages of lymphocytes, polymorphonuclear leukocytes, and binucleate macrophages in BALs of ep/ep,pe/pe mice (Table 2). Although BAL macrophages of 8- to 15-wk-old ep/ep,pe/pe mutants appeared normal (not shown), macrophages from these same mutants at 1 year of age were obviously enlarged and filled with foamy material (Fig. 5).


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Table 2. Cell differentials in BAL of control C57BL/6J and pale ear/pearl (ep/ep,pe/pe) mutant HPS mice

 


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Fig. 5. Macrophages of bronchoalveolar lavage (BAL) of double mutant mice are enlarged and foamy. BAL cells of 1-yr-old mice were cytocentrifuged and stained with a modified Wright-Giemsa (Hema-3) stain. Foamy macrophages are indicated by arrows. Bar = 25 µm.

 

Biochemical analyses of mutant lungs. Biochemical analyses confirmed that the aberrant morphology of ep/ep,pe/pe lamellar bodies was associated with accumulation of surfactant. A basic early observation was that the ratio of the wet weights of lungs to body weights of double mutant mice was significantly greater (34%) than that of control C57BL/6J (Table 3). Furthermore, a portion of this increase in wet weight of ep/ep,pe/pe lungs is apparently in nonproteinacious material since the milligram of protein/gram of wet weight ratio (Table 3) is greater (~10%) in control C57BL/6J lungs. A possible source for a portion of the increased wet weight of mutant lungs is lipid since surfactant, which is highly enriched in lipids (55), accumulates within mutant type II cells (Figs. 1 and 2). Indeed, two components of surfactant, total phospholipids (Table 4) and surfactant protein C, assayed by Western blotting (Fig. 6), are greatly elevated in lungs of ep/ep,pe/pe mice compared with control lungs. Phospholipid concentrations of lungs of ep/ep,pe/pe mutants are increased 3.3-fold over control C57BL/6J mice, and much smaller increases (25-34%) occur in single mutants (Table 4). Lungs of ep/ep,pe/pe mutants contain much higher surfactant protein C (Fig. 6) than single mutant ep/ep and pe/pe mice or controls. Surfactant protein C levels appear progressively higher in C57BL/6J controls followed by ep/ep and then pe/pe and then ep/ep,pe/pe (Fig. 6). As an additional test of the surfactant nature of the abnormal lipid in type II cells of mutant lung, the percentage of DPPC, which is highly enriched in surfactant (23), was determined (Table 5). Consistent with accumulation of surfactant, the percentage of DPPC was 2.5-fold elevated in double mutant lungs to levels equivalent, in fact, to those seen in lavage samples.


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Table 3. Lung wet weights and protein contents of control C57BL/6J and (ep/ep,pe/pe) HPS mutant mice

 

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Table 4. Phospholipids are elevated in mutant lungs

 


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Fig. 6. The quantity of SP-C is greatly increased in lungs of ep/ep,pe/pe mutants. Thirty micrograms of protein of lung homogenates of 12-wk-old normal C57BL/6J, ep/ep, pe/pe, and ep/ep,pe/pe mutants were electrophoresed under denaturing conditions and Western blotted with an antibody to SP-C.

 

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Table 5. DPPC is elevated in lungs of ep/ep,pe/pe mutants (11-12 wk of age)

 

There is a significant number of reports of increased autofluorescence in many tissues of HPS patients, due to accumulation of ceroid pigment (58), a protein/lipid material of undefined composition. We likewise noted (Fig. 7) an obvious increase in autofluorescence in lungs of ep/ep,pe/pe mutant mice. Consistent with the phospholipid analyses, autofluorescence in the HPS ep/ep,pe/pe mutant mice is greater than that of pearl (pe/pe) and pale ear (ep/ep) mutants, which in turn is greater than that of normal C57BL/6J controls.



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Fig. 7. Mutant ep/ep,pe/pe lungs exhibit high autofluorescence. Mice were 8-13 wk old. Bar = 80 µm.

 

ep/ep,pe/pe Mice exhibit aberrant respiratory system mechanics. Pulmonary mechanics of ep/ep,pe/pe double mutant mice and age-matched normal C57Bl/6J mice were measured to determine whether the biochemical and morphological changes correlated with functional changes in the lung. No significant differences in baseline measurements of resistance or compliance were observed between wild-type mice and ep/ep,pe/pe double mutant mice. However, significant differences were detected between wild-type and mutant mice in their baseline hysteresivity values (wild-type mice, 0.166 ± 0.006 vs. ep/ep,pe/pe, 0.131 ± 0.004; P < 0.05; Table 6).


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Table 6. Respiratory system mechanics of C57BL/6J and (ep/ep,pe/pe) HPS mutant mice

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The lung abnormalities of ep/ep,pe/pe mutant mice resemble, in most aspects, those of human HPS patients. In the most detailed microscopic morphological analysis of lungs of HPS patients thus far reported, Nakatani et al. (37) examined lung biopsy/autopsy samples from five Japanese HPS patients by light and electron microscopy. They described a "florid foamy swelling" of lung type II cells together with degenerative giant lamellar bodies within these cells. Both features are strikingly similar to the aberrant morphology observed in type II cells of ep/ep,pe/pe mutant mice in the present study. Type II cells of both HPS patients and mutant mice are greatly enlarged and contain correspondingly enlarged lamellar bodies, which are engorged with surfactant. The identification of aberrant lamellar bodies of type II cells was confirmed in each case by immunohistochemical demonstrations of surfactant protein within these organelles.

An additional important similarity between the lung abnormalities in the ep/ep,pe/pe mouse model and human HPS patients (37, 44) is that significant inflammation accompanies the aberrant morphology in both. Macrophage numbers in BAL fluid of lungs of HPS patients (44) are elevated two- to threefold over those in normal BAL fluid. Likewise, BAL fluid of ep/ep,pe/pe mice contains twice the number of inflammatory cells, largely macrophages, found in BAL of normal C57BL/6J controls. The lung inflammation expands in 1-yr-old mutants to approximately sixfold that of controls, and large foamy macrophages, similar to those described in lung lavages of HPS patients (44), become prevalent. Inflammation is likely important in the development of lung fibrosis (30) and emphysema (4). However, aberrant regulation of additional factors, including metalloproteinases and their inhibitors and/or elevated apoptosis, likely contribute given that clinical measures of inflammation do not always correlate with disease progression (10).

Nakatani et al. (37) have reported an overaccumulation of phospholipids, detected by histochemical staining, in lungs of HPS patients. Similarly, a large increase (to 3.5x control values) in phospholipids was observed in lungs of ep/ep,pe/pe mutants. This increase is consistent with observations, by light (Figs. 1 and 2) and electron (Fig. 4) microscopy, of surfactant-engorged lamellar bodies in ep/ep,pe/pe type II cells. Specific localization of surfactant protein C (Fig. 2) in these aberrant organelles confirms their identification as lamellar bodies. The increased levels of DPPC in lungs of ep/ep,pe/pe mutants are consistent with the presence of excess surfactant within their giant lamellar bodies. Surfactant is highly enriched in lipids (90-95%), and phospholipids comprise 78% of these lipids (23). This accumulation of lipids is a likely explanation for the increased lung weight and decreased protein content of ep/ep,pe/pe lungs (Table 3). Another lipid-related abnormality commonly reported in human HPS lungs (57) and other tissues is increased autofluorescence, thought to be due to accumulation of ceroid-related aging pigments, poorly defined lipid/protein complexes. The ep/ep,pe/pe mutants again mimic the human HPS lung pathology in this respect as their lungs exhibit significantly greater autofluorescence (Fig. 7) than control C57BL/6J or single mutant ep/ep or pe/pe mice. A reasonable possibility is that the increased autofluorescence of lungs of ep/ep,pe/pe mutants derives from the accumulations of surfactant and/or its derivatives in type II cells.

Our experiments confirm the importance of genetic constitution in the formation of lung abnormalities. Single mutant ep/ep or pe/pe mice exhibit modest lung morphological abnormalities, and lung inflammation is not apparent. Also, lung inflammation and/or abnormal type II cells were not apparent in lungs of 10 other HPS mouse mutants. In contrast, severe lung abnormalities, including massive lamellar bodies of type II cells and inflammation, are apparent in ep/ep,pe/pe mice maintained on the common C57BL/6J strain background. Clearly, the ep and pe genes cooperate synergistically in the lung, as they do in other tissues (17), to produce severe aberrations of lysosome-related organelles. Similarly, it is likely that the presence or absence of other susceptibility genes in individual HPS patients modifies the severity and age of onset of lung pathology.

Our analyses and related (37) studies suggest that the lung abnormalities of HPS are a consequence of abnormalities of lamellar bodies of type II cells. This suggestion is entirely consistent with the related facts that type II cell lamellar bodies are lysosome-related organelles (55) and that HPS is a disease of lysosome-related organelles (12, 25, 45, 51, 52). What is the explanation for the unusual lamellar body morphology in type II cells of the double mutant? A possible answer derives from the facts that 1) a major function of type II lamellar bodies is secretion of surfactant (55) and 2) most mouse HPS mutants (including pale ear and pearl) have decreased rates of secretion of the contents of lysosomes in other cells (47, 51, 52). A reasonable, although still unproven, hypothesis is that the contents of the lysosome-related organelle, the lamellar body, are secreted at decreased rates from ep/ep,pe/pe and HPS type II cells, leading to the observed [this study and Nakatani et al. (37)] accumulation of surfactant in type II cells. A related observation, given the close clinical and cell biological similarities of HPS and the Chediak-Higashi syndrome (22), is that giant lamellar bodies and increased lung phospholipids have also been observed in type II cells of the Chediak-Higashi syndrome (beige) mouse (9). Likewise, it is relevant to the above hypothesis of lysosomal secretion insufficiency in HPS type II cells that lysosomal enzymes are secreted at reduced rates in the beige mouse (7).

Although it was not examined in this study, there is no evidence of nervous system perturbations or, specifically, disruption of muscarinic receptor expression or function, in the airways in patients with HPS or in the mutant mouse models. Therefore, it is safe to assume that the functional changes observed in respiratory system mechanics are most likely due to the structural/pathological changes that develop in the lungs of ep/ep,pe/pe mutant mice. Several mechanisms may lead to a reduction in hysteresivity in these mice, most likely either disruption of the connective tissue network (41) or alterations in surfactant homeostasis (21). As the connective tissue network in the mutant mice is significantly disrupted, this functional abnormality is consistent with the morphological indications of emphysema observed in mutant mice. The decrease in hysteresivity in the mutant mice suggests a loss of tethering of the parenchymal tissue compared with the normal mechanical coupling of the tissue measured in the wild-type mice.

Another hallmark of lung pathology in human HPS, especially HPS1, is the presence of lung fibrosis in terminally ill patients (3, 8, 37). Pulmonary fibrosis has been documented in HPS1 (8) and HPS4 (1) patients where it is of variable severity and age of onset. It is apparently less prevalent in HPS2 and HPS3 patients (26), although relatively few of these patients have been analyzed. The lungs of ep/ep,pe/pe mutants contain a significant increase (~25%) in hydroxyproline content compared with controls (not shown). However, it is uncertain whether this represents incipient fibrosis, as hydroxyproline occurs not only in collagen but also in surfactant proteins A and D (23). Also, histological fibrosis was not apparent in mutants up to 1 yr of age. Additional studies on older mutant mice are required to determine whether they develop overt histological fibrosis; the 1-yr-old mice analyzed in these studies are relatively young compared with the 2.5- to 3-yr normal mouse lifespan. Our morphometry studies indicate significant emphysema in ep/ep,pe/pe double mutant mice. The cause of the emphysematous condition is unknown, although it may be relevant that another mouse HPS mutant, pallid (31), and the beige mouse (46), a model for the closely related Chediak-Higashi syndrome, develop emphysema-related lung pathologies. All three mouse models have defective trafficking of lysosome-related organelles, and two (ep/ep,pe/pe and beige) have greatly enlarged lamellar bodies of type II cells. Finally, it is noteworthy that some HPS patients with advanced disease have shown bullous emphysema (57). Abnormal lysosomal trafficking may be relevant to the disruption of lung parenchyma observed in emphysema in that intracellular vesicles have been implicated in repair of mechanically wounded cells (35), including alveolar cells (54), and rupture of enlarged lysosomes leads to plasma membrane disruption and cell death (40).

The combination of the ep and pe genes causes morphological abnormalities of type II cells to appear early in their lifespan compared with HPS patients in which lung abnormalities are commonly reported in the third to fourth decades. However, this difference between HPS patients and the mouse model may be more apparent than real. This is because analyses of lung abnormalities in HPS patients and HPS mouse models, with the exception of the report of Nakatani et al. (37), have employed very different techniques, whose results are difficult to compare directly. Patient lung analyses typically employ chest radiography, high-resolution computerized tomography, and/or pulmonary function tests instead of the biochemical and microscopic analyses utilized in the present studies. Also, there have been no detailed microscopic or biochemical analyses of lungs of young HPS patients. It is, therefore, possible that morphological abnormalities of lamellar bodies of type II cell pathology and/or incipient fibrosis occur in susceptible HPS patients before the midlife crisis of overt fibrotic lung disease. In turn, we speculate that biochemical/morphological analyses for abnormalities of lamellar bodies of type II cells and/or accompanying inflammation in lavages might be useful predictors of future susceptibility to fibrotic crises in young, at-risk HPS patients.

In summary the ep/ep,pe/pe mouse HPS mutant appears to model, in most respects, the morphological, biochemical, and inflammatory abnormalities observed in lungs of HPS patients. Lung pathology, including accumulation of surfactant in lamellar bodies of type II cells, lung inflammation, and functional lung impairment, develops in this model at an early age. In practical terms, the early onset of morphological and biochemical lung abnormalities in the ep/ep,pe/pe mutant should be advantageous for the study of mechanisms contributing to early stages of lung pathology and for therapeutic approaches for this deadly disease.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This work was supported in part by National Institutes of Health Grants HL-51480, HL-31698, and EY-12104 (R. T. Swank) and the American Lung Association - NY Affiliate (P. Ohtake). This research utilized core facilities supported in part by Roswell Park Cancer Institute's National Cancer Institute-funded Cancer Center Support Grant CA-16056.


    ACKNOWLEDGMENTS
 
We thank Madonna Reddington, Edward Hurley, Mary Vaughan, Diane Poslinski, Debra Tabaczynski, and Mary Kay Ellsworth for excellent technical assistance. We are indebted to Dr. Jennifer Black for expert advice on histochemical and ultrastructural procedures and to Dr. Arindam Sen for aid with lipid analyses.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. T. Swank, Dept. of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY 14263 (richard.swank{at}roswellpark.org).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
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 ABSTRACT
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 DISCUSSION
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