Am J Physiol Lung Cell Mol Physiol 285: L643–L653, 2003.
First published May 30, 2003; 10.1152/ajplung.00024.2003.
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
1
Department 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
inherited lung dysfunction; inflammation; surfactant protein; lamellar body; secretion
HERMANSKY-PUDLAK SYNDROME
(HPS; Mendelian inheritance in man 203300) 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 ␤3A-subunit of the
adaptor protein-3 (AP-3) adaptor complex, which is
Address for reprint requests and other correspondence: R. T.
Swank, Dept. of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY 14263 ([email protected]).
http://www.ajplung.org
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 ␦- and ␤3A-subunits of
the AP-3 adaptor complex, the ␣-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
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Lyerla, Timothy A., Michael E. Rusiniak, Michael
Borchers, Gerald Jahreis, Jian Tan, Patricia Ohtake,
Edward K. Novak, and Richard T. Swank. Aberrant lung
structure, composition, and function in a murine model of
Hermansky-Pudlak syndrome. Am J Physiol Lung Cell Mol
Physiol 285: L643–L653, 2003. First published May 30, 2003;
10.1152/ajplung.00024.2003.—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.
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MATERIALS AND METHODS
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 BioAJP-Lung Cell Mol Physiol • VOL
sciences, 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-␮m 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
␮g/ml in PBS with 0.5 ␮l/l Tween 20 (PBST). Slides were
processed for antibody staining using primary antibody to
surfactant protein C (Research Diagnostics) at 2 ␮g/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, counterstained 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-␮m 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 an-
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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 epistatic 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.
LUNG DEFECTS IN A MODEL OF HERMANSKY-PUDLAK SYNDROME
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 smallamplitude 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
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
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 ⫻200
magnification, the widespread accumulation of inflammatory cells (arrows) throughout the lung parenchyma
of double mutants is apparent. Bar ⫽
20 ␮m.
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alyzed 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 18gauge 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
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LUNG DEFECTS IN A MODEL OF HERMANSKY-PUDLAK SYNDROME
type II cells of the pe/pe mutant appeared somewhat
larger than those of C57BL/6J controls.
Another abnormality of ep/ep,pe/pe lungs was that
many sections contained areas with large open air
spaces (Figs. 1 and 2, magnification ⫻200), 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.
Ultrastructural analyses (Fig. 4) confirmed that type
II cells of the ep/ep,pe/pe mutants were highly aber-
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 ␮m.
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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.
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,
LUNG DEFECTS IN A MODEL OF HERMANSKY-PUDLAK SYNDROME
rant. 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 ␮m in diameter. In contrast, the majority of mutant lamellar bodies were 2.5–4.5 ␮m in diameter, and giant lamellar
bodies up to 28 ␮m in diameter, which occupied nearly
the entire cellular space, were occasionally observed.
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.
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
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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).
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 ␮m.
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Fig. 3. The mean linear intercept (␮m ⫾ 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.
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Table 1. Bronchoalveolar lavage (BAL) cell counts in
control C57BL/6J and Hermansky-Pudlak syndrome
(HPS) mutant mice
Genotype
Age, wk
n
Cell Number (⫻ 10⫺3)
ep/ep,pe/pe
ep/ep,pe/pe
ep/ep,pe/pe
ep/ep,pe/pe
C57BL/6J
C57BL/6J
C57BL/6J
C57BL/6J
pe/pe
pe/pe
ep/ep
ep/ep
4–5
7–8
11–17
48–96
4–5
7–8
11–17
52–136
11–17
56–60
11–17
50–56
9
9
22
10
8
10
23
12
5
5
5
5
393 ⫾ 53*
420 ⫾ 41*
440 ⫾ 40*
1,340 ⫾ 170†
196 ⫾ 27
214 ⫾ 24
179 ⫾ 21
239 ⫾ 32
244 ⫾ 45
274 ⫾ 29
196 ⫾ 26
250 ⫾ 32
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,
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 ␮m.
the percentage of DPPC was 2.5-fold elevated in double
mutant lungs to levels equivalent, in fact, to those seen
in lavage samples.
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.
Table 2. Cell differentials in BAL of control C57BL/6J and pale ear/pearl (ep/ep,pe/pe) mutant HPS mice
Genotype
Mononuclear
Macrophages
Binucleate
Macrophages
PMNs
Lymphocytes
Other
C57BL/6J n ⫽ 14
ep/ep,pe/pe n ⫽ 15
96.2 ⫾ 0.6
84.6 ⫾ 2.0*
1.7 ⫾ 0.2
5.7 ⫾ 0.9*
0.3 ⫾ 0.1
3.4 ⫾ 0.9*
1.2 ⫾ 0.4
5.8 ⫾ 0.9*
0.6 ⫾ 0.1
0.5 ⫾ 0.2
Values are average percentages of each cell type ⫾ SE from counts of 300–500 cells of cytospin preparations from BALs of 8- to 15-wk-old
mice. PMN, polymorphonuclear leukocytes. * Significantly different from C57B46J at P ⬍ 0.001.
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Values are means ⫾ SE. * Significantly different from C57BL/6J
and single mutants (pe/pe and ep/ep) at P ⬍ 0.05. † Significantly
different from C57BL/6J and single mutants at P ⬍ 0.001.
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Table 3. Lung wet weights and protein contents of control C57BL/6J and (ep/ep,pe/pe) HPS mutant mice
Genotype
Body Weight, g
Lung Weight, g
Lung/Body Weight (⫻103)
mg Protein/g Lung
C57BL/B6 (11)
ep/ep,pe/pe (11)
24.1 ⫾ 1.3
23.9 ⫾ 1.0
0.141 ⫾ 0.004
0.167 ⫾ 0.011*
6.32 ⫾ 0.23
8.49 ⫾ 0.82†
109 ⫾ 2.0
99.0 ⫾ 1.3*
Values represent the means ⫾ SE of eleven 8- to 15-wk-old mice each. * P ⬍ 0.01; † P ⬍ 0.02.
DISCUSSION
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 3.5⫻ 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 lipidrelated abnormality commonly reported in human
HPS lungs (57) and other tissues is increased autofluorescence, thought to be due to accumulation of ceroidrelated 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
Table 4. Phospholipids are elevated in mutant lungs
Genotype
C57BL/6J (10)
ep/ep (10)
pe/pe (10)
ep/ep,pe/pe (10)
␮mol P/mg protein
0.198 ⫾ 0.0056
0.248 ⫾ 0.0130*
0.265 ⫾ 0.0220†
0.654 ⫾ 0.0340‡
Values are means ⫾ SE of ten 8- to 15-wk-old mice in each group.
Total lipids were extracted from lungs with chloroform-methanol,
and phosphate (P) was estimated by the Fiske-SubbaRow reagent.
* P ⬍ 0.02; † P ⬍ 0.05; ‡ P ⬍ 0.001.
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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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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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LUNG DEFECTS IN A MODEL OF HERMANSKY-PUDLAK SYNDROME
Table 5. DPPC is elevated in lungs of ep/ep,pe/pe
mutants (11–12 wk of age)
Lung
Genotype
Lipid
Phosphate,
␮mol/mouse
Table 6. Respiratory system mechanics of C57BL/6J
and (ep/ep,pe/pe) HPS mutant mice
Genotype
Resistance,
cmH2O 䡠 ml⫺1 䡠 s⫺1
Compliance,
ml/cmH2O
Hysteresivity
C57BL/6J (n ⫽ 9)
ep/ep,pe/pe (n ⫽ 8)
0.87 ⫾ 0.07
0.97 ⫾ 0.11
0.027 ⫾ 0.004
0.026 ⫾ 0.002
0.166 ⫾ 0.006
0.131 ⫾ 0.004*
Lavage
% DPPC
C57BL/6J (8) 3.89 ⫾ 0.13 18.6 ⫾ 0.78
ep/ep,pe/pe (6) 12.2 ⫾ 1.08* 45.4 ⫾ 1.10*
Lipid
Phosphate,
␮mol/mouse
% DPPC
0.426 ⫾ 0.032
0.325 ⫾ 0.018
36.6 ⫾ 1.0
42.4 ⫾ 1.6
Values are means ⫾ SE of the number of mice in parentheses.
* P ⱕ 0.001. DPPC, dipalmitoylphosphatidylcholine.
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
Fig. 7. Mutant ep/ep,pe/pe lungs exhibit high autofluorescence. Mice were
8–13 wk old. Bar ⫽ 80 ␮m.
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(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 lysosomerelated organelles (12, 25, 45, 51, 52). What is the
Values represent means ⫾ SE. * Value significantly different from
C57BL/6J at P ⬍ 0.05.
LUNG DEFECTS IN A MODEL OF HERMANSKY-PUDLAK SYNDROME
AJP-Lung Cell Mol Physiol • VOL
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.
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.
DISCLOSURES
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.
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