Susceptibility of Hermansky-Pudlak Mice
to Bleomycin-Induced Type II Cell Apoptosis and Fibrosis
Lisa R. Young, Rajamouli Pasula, Peter M. Gulleman, Gail H. Deutsch, and Francis X. McCormack
Department of Medicine, Division of Pulmonary and Critical Care, University of Cincinnati; Department of Pediatrics, Division of Pulmonary
Medicine, and Department of Pathology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine,
Syndrome (HPS), a recessive disorder associated with intracellular
trafficking defects.We havepreviouslyreported that“Pearl” (HPS2)
and “Pale Ear” (HPS1) mouse models have pulmonary inflammatory
dysregulation and constitutive alveolar macrophage (AM) activa-
tion(YoungLRetal., J Immunol2006;176:4361–4368). Inthecurrent
study, weused these HPSmodels toinvestigate mechanisms oflung
fibrosis. Unchallenged HPS1 and HPS2 mice have subtle airspace
enlargement and foamy AMs, but little or no histologic evidence
of lung fibrosis. Seven days after intratracheal bleomycin (0.025
units), HPS1 and HPS2 mice exhibited increased mortality and dif-
sition, and reduced quasi-static and static compliance consistent
with a restrictive defect. The early airway and parenchymal cellular
inflammatory responses to bleomycin were similar in HPS2 and
WT mice. Greater elevations in levels of TGF-? and IL-12p40 were
produced in the lungs and AMs from bleomycin-challenged HPS
mice than in WT mice. TUNEL staining revealed apoptosis of type
II cells as early as 5 h after low-dose bleomycin challenge in HPS
mice, suggesting that type II cell susceptibility to apoptosis may
play a role in the fibrotic response. We conclude that the trafficking
abnormalities in HPS promote alveolar apoptosis and pulmonary
fibrosis in response to bleomycin challenge.
Keywords: alveolar type II cells; alveolar macrophage; lung fibrosis;
Hermansky-Pudlak; adaptor protein 3
Hermansky-Pudlak Syndrome (HPS) is a rare autosomal reces-
sive disordercharacterizedbyalbinism,platelet dysfunction, and
highly penetrant and frequently fatal pulmonary fibrosis (1, 2).
There are currently eight genetic loci known to be associated
in intracellular protein trafficking and in the biogenesis of lyso-
somes and lysosome-related organelles including melanosomes,
platelet dense granules, and lamellar bodies in alveolar type II
cells (4). Patients with HPS exhibit alveolar inflammation well
before fibrosis is apparent, with accumulation of activated alveo-
lar macrophages (AM) and elevated levels of proinflammatory
cytokines and chemokines in bronchoalveolar lavage (BAL)
fluid (5). Both the usual interstitial pneumonitis (UIP) pattern
(Received in original form December 20, 2006 and in final form February 27, 2007)
This work was funded byan American Thoracic Society/Hermansky-Pudlak Syndrome
Network grant (L.R.Y.), a Procter Scholar Award (L.R.Y.), HL68861 (F.X.M.), and
by the Department of Veterans Affairs (F.X.M.).
Correspondence and requests for reprints should be addressed to Francis X.
McCormack, M.D., University of Cincinnati, Pulmonary, Critical Care, and Sleep
Medicine, 231 Albert Sabin Way ML 0564, Cincinnati, OH 45267. E-mail: frank.
Am J Respir Cell Mol Biol
Originally Published in Press as DOI: 10.1165/rcmb.2006-0469OC on March 15, 2007
Internet address: www.atsjournals.org
Vol 37. pp 67–74, 2007
Hermansky-Pudlak Syndrome (HPS) is a highly penetrant
genetic disorder of pulmonary fibrosis. This study demon-
strates that bleomycin-challenged HPS mouse models have
increased levels of TGF-?, early type II cell apoptosis, and
marked pulmonary fibrosis.
and cellular nonspecific interstitial pneumonitis (NSIP) pattern
found in idiopathic pulmonary fibrosis (IPF) have been reported
in HPS, but ceroid accumulations in alveolar macrophages, large
eration, and lymphocytic and histiocytic infiltration of respira-
tory bronchioles are distinguishing features (6, 7). Clinically ap-
parentpulmonary fibrosisdevelopsinmostindividuals with HPS
in the fourth or fifth decades of life (8). Potential contributions
ofenvironmental factors inHPSlung diseaseare not known,and
it remains unclear how HPS trafficking abnormalities promote
Mouse models of HPS share many aspects of the human
disease phenotype and provide the opportunity to investigate
includingthe HPS1“PaleEar” andHPS2 “Pearl” mousemodels
used in this study, are naturally occurring and are maintained
as congenic mutants on the C57BL/6J inbred strain. The gene
product of HPS2 in Pearl mice and humans with HPS2 is the
?3A subunit of the adaptor protein-3 (AP-3) complex, a hetero-
oligomer of four subunits (?3a, ?3, ?, and ?3) that functions in
organelle biogenesis and protein trafficking (9). Mutations in
individual AP-3 subunits result in instability and ubiquitin-
mediated degradation of the entire AP-3 complex, which leads
to abnormalities in intracellular trafficking in a variety of cell
types and organ systems. The “Pale Ear” HPS1 mouse is the
model for the most common HPS subtype, and although the
precise function of the HPS1 gene product (HPS1) remains
unknown, it is also clearly critical for intracellular protein
Other human diseases provide evidence that abnormal pro-
tein trafficking in type II cells can cause interstitial lung disease
(ILD) (10, 11). Mutations in surfactant protein C (SP-C) cause
(ER) stress pathways (12), and the adaptation to the chronic
ER stress is associated with an increased susceptibility to RSV-
cassette family member of proteins, ABCA-3 gene, affect lipid
transport into lamellar bodies, and also cause ILD through type
II cell–dependent mechanisms (14, 15). Apoptosis of alveolar
type II cells is also a prominent feature of many types of ILD,
and correlates with disease progression in IPF and extent of
fibrosis in animal models (16, 17).
68 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 372007
Pearl and Pale Ear HPS mice have been shown to have
structural abnormalities in the alveolar compartment that are
similar to those observed in humans with HPS, including foamy
AMs and enlarged type II cells containing irregular dense inclu-
fibrils at the ultrastructural level (18). Others have shown that
double-mutant HPS1/HPS2 (ep/pe) mice have impaired lamellar
body secretion from type II cells (19), and that lung hydroxypro-
line content is significantly increased compared with controls
(20). Furthermore, silica-challenged Pale Ear mice develop a
persistent accumulation of activated macrophages and increased
collagen fibers in alveolar tissues (21). However, unchallenged
Pearl and Pale Ear mice do not spontaneously develophistologi-
cally significant pulmonary fibrosis by 1 yr of age, but instead
exhibit progressive airspace enlargement (20). Therefore, whether
the phenotype of HPS mouse models includes susceptibility to
pulmonary fibrosis has remained controversial.
We have previously demonstrated that Pearl and Pale Ear
mice have basal inflammatory dysregulation and constitutive
AM activation that parallels abnormalities reported in patients
with HPS (5, 22). We wondered whether HPS mice would de-
velop enhanced fibrosis in response to inhalation challenge with
bleomycin. Bleomycin is a chemotherapeutic agent known to
induce the release of cytokines, augment fibroblast proliferation
and activity, and to cause human and animal lung parenchymal
injury and subsequent lung fibrosis. Therefore, we investigated
the responses of HPS mice to intratracheal bleomycin challenge.
MATERIALS AND METHODS
mice that had been maintained on the C57BL/6J background were gifts
from Dr. R. Swank (Roswell Park Cancer Institute, Buffalo, NY).
C57BL/6J mice (Jackson, Bar Harbor, ME) were used as the wild-type
(WT) control for comparisons with HPS mice. Mice were housed in a
barrier facilityand studiedusingproceduresapprovedbythe University
of Cincinnati Institutional Animal Care and Use Committee. Sentinel
mice thatweretestedperiodically werefreeofknownviraland bacterial
pathogens. Male mice, age 8–14 wk, were used for all studies.
Mice were anesthetized to moderate depth with isofluorane (Forane;
Ohmeda Caribe, Guyama, Puerto Rico). After topical sterilization with
ethanol, the trachea was minimally exposed via a ventral midline neck
incision and cannulated with a sterile catheter. Bleomycin sulfate
(0.025–0.05 U/mouse; Sigma, St. Louis. MO) or saline control was in-
stilled intratracheally, the incision was closed with surgical adhesive,
and the mice were observed through recovery.
Lung Histology and Immunohistochemistry
Animals were killed by pentobarbital injection, and 0.9 ml of 10%
buffered formalin was instilled into the lungs via a tracheal cannula at
25 cm H2O. Lung tissues were paraffin embedded and cut into 5-?m
sections byconventionalmethods. Slideswerestainedwith hematoxylin
and eosin (H&E) or trichrome blue. Endogenous peroxidase activity
was blocked with immersion of deparaffinized sections in 3% H2O2for
10 min. For antigen retrieval, the sections were placed in 0.1 M citrate
buffer, pH 6.0, and treated with microwave for 10 min. Type II cells
were identified using a rabbit polyclonal antibody against human pro-
SP-C (kind gift of J. Whitsett, M.D., Cincinnati Children’s Hospital
Medical Center, Cincinnati, OH) (dilution 1:1500) and the Vectastain
ABC System (Vector Labs, Burlingame, CA) with DAB or NovaRED
(according to the manufacturer’s instructions). Immunostains were per-
formed withactive caspase 3goat polyclonal IgG (sc-1226, 1:50 dilution;
Santa Cruz Biotechnology, Santa Cruz, CA) using a similar protocol.
Sections were then lightly counterstained with hematoxylin.
Mice were anesthetized and tracheostomized, and respiratory mechan-
ics were measured using forced oscillation technique (0.25–20 Hz)
(Flexivent; SCIREQ, Montreal, PQ, Canada). Estimated total lung
compliance, airway resistance and elastance, and tissue damping were
obtained by fitting a model to each impedance spectrum.
Quantitation of Lung Collagen Content
Whole lungs were harvested, weighed, snap-frozen, and stored at
?80?C. Total soluble collagen was measured using the Sircol assay
(Biocolor; Accurate Chemical And Scientific Corporation, Westburg,
NY) according to the manufacturer’s instructions. Briefly, lungs were
homogenized in PBS, and Sircol dye was incubated with test samples
or collagen standard on a rocker for 30 min. Samples were then centri-
fuged, unbound dye solution was removed, and the remaining collagen-
bound dye pellet was solubilized. Absorbance was measured at 540 nm
and compared with a calibration curve generated using the collagen
standard provided by the manufacturer.
Evaluation of Airway and Tissue Cellular Recruitment
Mice were killed by lethal pentobarbital injection followed by transec-
tion of the abdominal aorta. BAL was performed as described, after
tracheostomy and intubation with a sterile 20-gauge adapter, by gentle
instillation and aspiration of three 1-ml aliquots of 0.9% saline with 5
mM Tris. After separation ofthe BAL cells by low-speedcentrifugation
(450 ? g, 10 min, 4?C), the cell-free BAL fluid was stored at ?80?C
until use. Cells were resuspended in PBS and counted using a Coulter
counter or hemacytometer. A cell aliquot was spun onto glass slides
using a cytocentrifuge (speed 300, 5 min; Shandon, Thermo Fisher
Scientific, Ontario, Canada), and cytospin preparations were stained
with DiffQuik (Sigma-Aldrich, St. Louis, MO). The percentages of
macrophages, neutrophils, lymphocytes, and eosinophils in BAL were
determined by differential counting of a minimum of 200 cells.
For analysis of tissue cellular composition, organs were individually
harvested in Hanks’ Buffered Salt Solution (HBSS) and teased apart
between glass slides. A cell suspension was obtained by aspiration
through a 21.5-ga. needle and filtering with 60-?m nylon mesh (Spec-
trum Laboratories, Inc., Rancho Dominguez, CA). The cell pellet was
collected by centrifugation (450 ? g, 5 min, 4?C) and resuspended in
HBSS. The lymphocyte population was then isolated using a 40–70%
Percoll (Pharmacia, Uppsala, Sweden) gradient (23). Cells were incubated
with allophyocyanin (APC) or fluorescein isothiocyanate–conjugated
Antibodies used to tag specific cell types included anti-F4/80 for macro-
phages (CalTag, Carlsbad, CA), anti-GR1 for neutrophils, and anti-
CD3 for lymphocytes (BD Biosciences, Bedford, MA). Cells were fixed
in 1% paraformaldehyde in PBS and stored at 4?C before analysis on
aFACSCalibur(Becton Dickinson,San Jose,CA).Datawereanalyzed
using FCS Express 2.0 software (De Novo Software, Thornhill, ON,
Enzyme-Linked Immunosorbent Assay Measurements of
Cytokines and Chemokines
Cytokine protein concentrations in cell-free BAL, lung homogenate,
and AM cell culture media supernatant were quantified by ELISA
(murine Quantikine kits; R&D Systems, Minneapolis, MN), using the
chromogenic substrate tetramethylbenzidine and analysis with a micro-
titer plate spectrophotometer at a wavelength of 450 nm.
Evaluation of Apoptosis
Terminal deoxynucleotidyl transferase-mediated dUTP nick end label-
ing (TUNEL) was performed on deparaffinized lung tissue sections
using the In Situ Cell Death detection kit (Roche Diagnostics,
Mannheim, Germany) according to the manufacturer’s instructions.
After permeabilization, tissue sections were incubated in TUNEL reac-
tion mixture (or the labeling solution without addition of TdT enzyme
as a negative control) for 1 h at 37?C. Fluorescence images were visual-
ized and collected with a digital camera mounted on an Olympus BX60
fluorescence microscope (Olympus America, Center Valley, PA), and
analyzed using SPOT RT Software v3.4 (SPOT Diagnostic, Sterling
Heights, MI). For immunofluorescent double-staining of alveolar type
Young, Pasula, Gulleman, et al.: Bleomycin-Induced Pulmonary Fibrosis in HPS Mice69
II cells and TUNEL, type II cells were identified by a rabbit polyclonal
antibody against human pro–SP-C (dilution 1:1,000), biotinylated anti-
rabbit IgG secondary antibody (dilution 1:1,000), and detection with
Cy3 red (dilution 1:3,000), immediately followed by TUNEL procedure
as above. For quantitative evaluation of TUNEL staining, the mean
number of cells with TUNEL-positive nuclei and pro–SP-C–positive
cells were recorded by two independent observers (L.R.Y. and P.M.G.)
from 10sequential, nonoverlapping high-power fields from each specimen
and expressed as a ratio.
Numeric data are presented as mean ? SEM. Parametric data were
evaluatedusing theStudent’sttest,or byone-wayANOVAforcompar-
ison of more than two groups (Prism 4; Graph Pad Software, Inc., San
Diego, CA). The log rank test was used for survival analysis. P values
? 0.05 were considered significant.
Pearl Mice Have Increased Mortality When Exposed to
Pearl mice were challenged with intratracheal bleomycin (or
saline control) at doses (0.025 and 0.05 U/mouse in volume of
50 ?l) that are considered sub-lethal for this mouse strain (24,
25). As shown in Figure 1, bleomycin challenge led to signifi-
cantly greater mortality in Pearl than WT mice. Bleomycin at a
dose of 0.05 U/mouse resulted in 100% mortality in Pearl mice
after 7 d (Figure 1B, P ? 0.001 by log-rank test), while a reduced
dose of 0.025 U/mouse resulted in 50% mortality in Pearl mice
at Day 10 and only 8% mortality in WT mice (Figure 1A,
P ? 0.001). No deaths occurred in Pearl or WT mice receiving
intratracheal saline as controls. Significantly greater weight loss
occurred in Pearl than WT mice by Day 7 after intratracheal
bleomycin challenge (not shown). Basedon these dose–response
data, all further bleomycin experiments were performed with
the lower dose of 0.025 U. Mortality for Pale Ear mice was 14%
within 7 d after intratracheal bleomycin challenge of 0.025 U.
HPS Mice Have Increased Fibrotic Susceptibility to
Significant histologic differences were observed in the lungs of
HPS and WT mice 7 d after intratracheal bleomycin. In WT
mice, very limited cellular inflammation and histologic evidence
of fibrosis was apparent, while Pearl and Pale Ear mice devel-
oped marked fibrosis with a varying extent of cellular interstitial
expansion (Figure 2). When challenged with 0.025 U of bleomy-
cin, histologic fibrosis did not become evident in WT mice until
after 14 d (not shown). The extent of fibrosis, as measured by
a colorimetric assay (Sircol red) of total lung collagen content,
was significantly greater in the lungs of Pearl and Pale Ear than
WT mice (Figure 3).
Figure 1. SurvivalrateofPearlandWTmiceafter
intratracheal bleomycin. WT (open squares) and
Pearl (circles) mice were challenged by intratra-
cheal instillation of a single bleomycin dose
(0.025 U/mouse in A or 0.05 U/mouse in B).
Mortality was monitored over 2 wk (n ? 12 per
group for 0.025 U and n ? 8 per group for
Pearl Mice Have Reduced Lung Compliance after
In order to determine if the observed histologic changes were
lation technique was used to assess pulmonary compliance in
Pearl mice. While no significant physiologic changes were de-
tected in bleomycin-treated WT mice at the 7-d point, quasi-
static and static compliance were significantly decreased in bleo-
mycin-challenged Pearl mice (0.046? 0.003 ml/cm H2O forPearl
versus 0.066 ? 0.006 ml/cm H2O for WT, P ? 0.05) after 1 wk
Airway and Pulmonary Inflammatory Cell Recruitment in
Pearl Mice Is Similar to That in WT Mice after
Intratracheal Bleomycin Challenge
Intratracheal bleomycin challenge is associated with a robust
early cellular inflammatory response (25, 26). We have pre-
viously reported that total BAL cell numbers and differentials
are not significantly different at baseline in Pearl, Pale Ear, and
WT mice (22). After intratracheal bleomycin challenge, total
BAL cell numbers were increased, and cell differentials were
similar in Pearl and WT mice at 1 and 3 d (Figures 5A and 5B).
Both WT and Pearl mice had a neutrophil-rich cellular influx
that peaked at approximately Day 3. Lymphocytes were also
recruited to the airspace. A modest influx of eosinophils also
occurred in both WT and Pearl mice (not shown). Using FACS
analysis, total lung leukocyte cell numbers and proportions of
neutrophils, macrophages, and lymphocytes were found to be
similar in Pearl and WT mice 3 d after intratracheal bleomycin
HPS Mice Have Elevated Levels of Profibrotic Mediators
after Bleomycin Challenge
Bleomycin-induced fibrosis in animal models has been shown to
be associated with production of profibrotic mediators including
TGF-?1 and IL-12 (27, 28). While the level of TGF-?1 was
similar in the pulmonary tissues of unchallenged Pearl, Pale Ear,
and WT mice, elevated levels of activated TGF-?1 were present
in the lungs of HPS mice after bleomycin challenge (Figure 6A).
This difference was not statistically significant until 3 d after
bleomycin challenge (4,215 ? 1,081 pg/ml for Pearl versus 2,792
? 805 pg/ml for WT, P ? 0.05). Higher levels of activated
TGF-?1 were also present in the lungs of Pale Ear mice 7 d
after bleomycin challenge (15,340 ? 1,118 pg/ml for Pale Ear
versus 5,278 ? 1,072 pg/ml for WT, P ? 0.05). IL-12/23p40
concentrations were significantly elevated in the lungs of unchal-
lenged Pearl versus WT mice (563 ? 61 pg/ml for Pearl versus
288 ? 32 pg/ml for WT, P ? 0.05), and bleomycin challenge
resultedinfurther time-dependentincreasesinthelungsof Pearl
mice (Figure 6B). Levels of IL-12/23p40 were also greater in
the lungs of Pale Ear compared with WT mice, both at baseline
70 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 372007
Figure 2. Increased evidence of pulmonary fibrosis in HPS
versus WT mice after bleomycin challenge.Mice were chal-
lenged with intratracheal bleomycin (0.025 U). After 7 d,
lungs were inflation-fixed with 10% buffered formalin at
25cm H2O. Representativelow-power (?10)(A) and high-
power (?40) (B) photomicrographs are shown for WT,
Pearl, and Pale Ear bleomycin-treated mice (n ? 4 per
group). Histologic analysis was performed with H&E stain-
(not shown) and 7 d after intratracheal bleomycin challenge
(1,820 ? 360 pg/ml for Pale Ear versus 1,086 ? 128 pg/ml for
WT, P ? 0.05). AMs isolated from bleomycin-challenged Pearl
and Pale Ear mice demonstrated more than 2-fold greater pro-
period in culture (Figure 6C). As we have previously reported,
unchallenged HPS AMs constitutively produce elevated levels
of IL-12/23p40 (22). Bleomycin challenge in vivo also led to
greater IL-12/23p40 production from AMs isolated from HPS
mice than from WT mice (Figure 6D). In contrast to the findings
for the profibrotic mediators TGF-? and IL-12/23p40, levels of
IL-12p70, IL-1?, TNF-?, and MIP1? were similar in BAL fluid
and lung tissue from Pearl and WT mice after bleomycin chal-
lenge (not shown).
Apoptosis Is Increased in the Lungs of HPS Mice after
To investigate mechanisms of the fibrotic susceptibility in HPS
mice, TUNEL assay was used to screen for apoptosis in pulmo-
nary cells after low dose intratracheal bleomycin challenge. Lim-
ited evidence of apoptosis was apparent in the lungs of unchal-
lenged Pearl and Pale Ear mice and in the lungs of WT mice
up to 7 d after bleomycin instillation (0.025 U) (not shown). In
contrast, increased apoptosis was detected in lung sections
from Pearl and Pale Ear mice as early as 5 h after intratracheal
bleomycin challenge (Figures 7A–7C). Co-immunostaining for
pro–SP-C was used to identify TUNEL-positive cells as alveolar
type II cells (Figures 7D–7F), and the ratio of TUNEL-positive
to SP-C–positive cells was quantitated from 10 consecutive ?40
microscopic fields from each of three animals at each timepoint
and group (Figure 7I). Positive immunostaining for active cas-
pase 3 was rarely seen in lung sections from HPS or WT mice
at baseline or Day 1 (not shown), but was more abundant in
Figure 3. Increased col-
lagen deposition in HPS
lungs in response to
Mice were challenged
with intratracheal bleo-
mycin (0.025 U) or an
equal volume of sterile
saline as a control. After
7 d, lungs were har-
lung collagen were assayed using the colorimetric Sircol assay. (Values
indicate totals for both lungs from each mouse; n ? 8 per group for
WT, n ? 4 per group for Pearl and Pale Ear, *P ? 0.01.)
the lungs of Pearl and Pale Ear than WT mice starting 3 d after
bleomycin challenge (Figures 7G and 7H).
We have previously reported that increased levels of soluble
Fas are present in BAL fluid from unchallenged Pearl mice (22),
and therefore investigated whether abnormalities in the Fas/
FasL system might be responsible for enhanced apoptosis of
Pearl type II cells via an extrinsic apoptotic pathway. ELISA of
lung homogenates and BAL revealed similarly elevated levels
of soluble Fas ligand (FasL) in the lungs of Pearl and WT mice
at Days 1 and 3 after bleomycin challenge. It was not until Day
7 after bleomycin challenge that significantly greater levels of
FasL were present in the lungs of Pearl versus WT mice (Figure
7H). Furthermore, immunohistochemistry for Fas receptor and
Fas ligandat baseline and at Days 1and 3 revealed no detectable
differences in extent or distribution of expression between Pearl
and WT mice (not shown).
These experiments provide a comprehensive assessment of the
response of Pearl mice to bleomycin challenge and demonstrate
that Pearl and Pale Ear HPS mouse models have a pulmonary
6J is a mouse strain known for susceptibility to bleomycin-
induced fibrosis (25, 29), the WT animals were only modestly
diffuse fibrosis and mortality in strain-matched HPS mice.
Figure 4. Reduced quasi-static pulmonary compliance in Pearl mice
after bleomycin challenge. Mice were challenged with intratracheal
bleomycin (0.025 U) or an equal volume of sterile saline as a control.
After 7 d, tracheostomized mice underwent physiologic testing with
the Scireq flexivent system (n ? 4 per group, *P ? 0.05 for WT versus
Pearl bleomycin and#P ? 0.01 for Pearl control versus Pearl bleomycin).
Open bars, wild type; solid bars, Pearl.
Young, Pasula, Gulleman, et al.: Bleomycin-Induced Pulmonary Fibrosis in HPS Mice71
Figure 5. BAL cell populations after bleomycin challenge. Mice were
challenged with intratracheal bleomycin (0.025 U), and BAL was per-
formed at various time points as indicated. (A) Total BAL cell counts at
baseline, and Days 1 and 3 after bleomycin challenge (P ? ns for Pearl
versus WT at each time point). (B) BAL cell differentials at baseline and
days 1 and 3 after bleomycin challenge (P ? ns for Pearl versus WT for
all comparisons). Open bars, WT; solid bars, Pearl.
loss, histologic evidence of fibrosis, and biochemical evidence
of collagen deposition compared with strain-matched WT mice.
These findings in Pearl mice were associated with reduced pul-
monary compliance consistent with a restrictive physiologic de-
fect. These data support the use of HPS mice as models of
Figure 6. ElevatedTGF-?andIL-12p40levels
in Pearl mice after bleomycin challenge. (A)
TGF-?1 levels in the lungs of Pearl mice at
were challenged with intratracheal bleomy-
cin (0.025 U). After 0, 1, 3, or 7 d, lungs were
harvested and homogenized. Acid-activated
TGF-?1 was measured by ELISA (n ? 4 per
group, *P ? 0.05 for WT versus Pearl at Day
3, **P ? 0.001 for WT versus Pearl at Day 7).
(B) IL-12/23p40 levels in the lungs of Pearl
mice at baseline and after bleomycin chal-
lenge. Mice were challenged with intratra-
cheal bleomycin (0.025 U) and levels of IL-
12/23p40 in lung homogenate were mea-
sured by ELISA (n ? 4 per group, *P ? 0.05
for WT versus Pearl at all time points). (C)
TGF-?1 production fromAM after bleomycin
challenge in vivo. AMs were isolated from
Pearl, Pale Ear, and WT mice at baseline
and 24 h after bleomycin challenge. Acid-
activatedTGF-?1 was measured fromthe cell
media supernatant after 24 h in culture (n ?
5 per group for WT, n ? 3 per group for
Pearl and Pale Ear, *P ? 0.05 for Pearl and Pale Ear versus WT). (D) IL-12/23p40 production from AMs after bleomycin challenge in vivo. AMs
were isolated from Pearl, Pale Ear, and WT mice at baseline and 24 h after bleomycin challenge. IL-12/23p40 was measured from the cell media
supernatant after 24 h in culture (n ? 5 per group for WT, n ? 3 per group for Pearl and Pale Ear, *P ? 0.05 for Pearl and Pale Ear versus WT).
Open bars, WT; solid bars, Pearl; shaded bars, Pale Ear.
HPS ranks among the most penetrant genetic disorders of
pulmonary fibrosis in adults, as virtually all patients with HPS
who survive to adulthood develop fibrotic lung disease (1, 30).
HPS1 is the most common human genotype, and accounts for
the majority of ILD cases reported in HPS. Although the eight
reported HPS2 patients are youngerin age than HPS1 and HPS4
patients with pulmonary fibrosis, mild pulmonary fibrosis was
described in two HPS2 cases (31, 32). In this study, key findings
of bleomycin-induced histologic fibrosis and alveolar type II cell
apoptosis in the HPS2 mice were also confirmed in Pale Ear
HPS1 mice, the more common genetic background associated
The role of cellular inflammation in ILD pathogenesis re-
mains unclear. While idiopathic pulmonary fibrosis (IPF) is in-
creasingly recognized as a pauci-immune disorder with fibro-
blastic proliferationbut relatively minimalleukocyte infiltration,
robust cellular interstitial inflammation precedes fibrosis in mul-
tiple other forms of ILD such as hypersensitivity pneumonitis,
sarcoidosis, desquamative interstitial pneumonitis, respiratory
bronchiolitis, and ILD associated with mutations in SP-C and
connective tissue disease (33–35). Alveolar inflammation also
precedes pulmonary fibrosis in patients with HPS (5), but the
direct role of inflammation in the pathogenesis of HPS lung
disease has not been determined. We have previously reported
that Pearl and Pale Ear mice exhibit pulmonary inflammatory
dysregulation, with constitutive AM activation and exaggerated
model of pulmonary fibrosis, inflammation induces the release
of extracellular matrix (26, 36). In this context, we postulated
that Pearl mice would have an aberrant early and exaggerated
elevations in selected pro-inflammatory cytokines and chemo-
recruitment, occurred in Pearl and WT mice after bleomycin
challenge. Further studies are needed to determine whether pul-
monary inflammation and fibrosis are causally linked in HPS.
72 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 372007
Figure 7. Increased apoptosis in lung sections
from HPS mice after intratracheal bleomycin
challenge.Atindicatedtime pointsafter intratra-
cheal bleomycin (0.025 U), lungs were inflation-
fixed in formalin as previously described. TUNEL
assay (Roche) was performed on paraffin-
embedded sections. (A–C) Representative fluo-
rescent micrographs(?40) for (A) WT, (B) Pearl,
and (C) Pale Ear at 5 h after bleomycin challenge
are shown. (D–F) Representative co-immunoflu-
orescence for TUNEL (D) and pro–SP-C (E) iden-
tifies apoptotic alveolar type II cells (F, merge)
from a Pearl lung section, 5 h after intratracheal
bleomycin challenge. (G and H) Representative
photomicrographs (?40) of active caspase 3
staining in lung sections from (G) WT, (H) Pearl,
or (I) Pale Ear mice, 3 d after intratracheal bleo-
itive for active caspase 3 were present in the
lungs of Pearl and Pale mice. Arrows indicate
examples of positive cells. (J) Quantitative as-
sessment of percentage of alveolar type II cells
which are TUNEL-positive. Alveolar wall TUNEL-
positive and SP-C–positive cells were counted
from 10 high-power (?40) fields (n ? 3 each at
baseline and 5 h, n ? 4 each at 24 h, *P ? 0.05).
(K) Levels of Fas ligand in the lungs of Pearl mice
after intratracheal bleomycin challenge. Mice
were challenged with intratracheal bleomycin
(0.025 U). Mice were killed at various time
points, lungs were harvested, and Fas ligand
(FasL) levels were assessed by ELISA (n ? 4 per
group, *P ? 0.05 for Pearl versus WT, Day 7).
ProfibroticcytokinessuchasTGF-? causefibroblast transfor-
mation and proliferation, leading to deposition of extracellular
matrix (37, 38), and multiple studies have demonstrated that
TGF-? is a key factor in fibrosis in the lung and in other tissues.
In animal models, adenoviral-driven overexpression of TGF-?1
results in pulmonary fibrosis, and blocking the action of TGF-?
attenuates fibrosis (28, 37, 39). Our studies revealed greater
elevations in levels of TGF-? in the lungs of bleomycin-
challenged HPS than WT mice. Further, isolated murine HPS
AMs produced excess quantities of TGF-? compared with WT
AMs after bleomycin challenge. In addition, IL-12/23p40, but
not IL-12p70, was constitutively produced by HPS AMs and
found in elevated levels in the lungs of HPS versus WT mice,
both at baseline and after bleomycin challenge. Previous studies
have demonstratedthat theprofibroticIL-12p40subunit,but not
the IL-12p70 heterodimer, is overproduced during experimental
induction of fibrosis, and that AMs are the predominant source
(40). Further, IL-12p40?/? mice are resistant to silica-induced
fibrosis (41), and a monoclonal antibody to IL12 was protective in
the bleomycin model (27). Together, our findings of increased IL-
12p40 and TGF-? production from HPS AMs suggest a possible
link between macrophage dysfunction and fibrogenesis in HPS.
In addition to cytokine/chemokine imbalance, many studies
support the concept that alveolar epithelial cell apoptosis is a
dominant feature of human fibrotic lung disease and pulmonary
fibrosis in the bleomycin model (42). In lung biopsies from pa-
tients with IPF, alveolar epithelial cell apoptosis has been found
in regions adjacent to myofibroblasic proliferation and collagen
deposition (16, 43). Among the fibrogenic factors we assessed,
the earliest distinguishing finding between HPS and WT mice
after bleomycin challenge was apoptosis of type II cells. TUNEL
staining revealed apoptosis of alveolar type II cells as early as
5 h after bleomycin challenge in HPS mice, and the subsequent
increase in caspase-3 activation indicates that the initial DNA
fragmentation and cellular injury was followed by apoptotic cell
death. Our data suggest that type II cell susceptibility to apopto-
sis may play a role in the fibrotic response, althoughthe absolute
extent of type II cell apoptosis required to induce fibrosis is not
in HPS mice, and TGF-?1 has been shown to enhance Fas-
mediated epithelial cell apoptosis and pulmonary fibrosis (44).
However, our data would suggest that TGF-? is not the sole
contributor toPearl type IIcell apoptosis and fibrosis, asapopto-
sis, which occurred as early as 5 h after bleomycin challenge,
preceded a detectable increase in TGF-? levels in vivo.
Apoptosis occurs via an extrinsic pathway in response to
activation of cell membrane receptors as well as via an intrinsic
pathway triggered by release of mitochondrial products. The
includes TNF receptor and the Fas receptor (CD95). The Fas
Young, Pasula, Gulleman, et al.: Bleomycin-Induced Pulmonary Fibrosis in HPS Mice73
receptor can be activated by either Fas ligand (FasL) on the
surface of cytotoxic lymphocytes, or by a soluble form of FasL
anti-Fas ligand antibody prevents bleomycin-induced epithelial
apoptosis and pulmonary fibrosis (46), as do caspase inhibitors
(47). Furthermore, experimental induction of apoptosis in mice
via inhalation of anti-Fas antibody has been shown to result in
pulmonary fibrosis (48).
We have previously reported that Pearl mice have increased
basal pulmonary levels of sFas (22). Based on knowledge that
nantlyinthelytic granuleofactivated Tlymphocytes andnatural
killer cells (49), and that AP-3 has previously been shown to be
critical for the movement of T-cell lytic granules to the immuno-
logical synapse (50), we hypothesized that AP-3 deficiency in
Pearl mice would lead to abnormalities in the Fas/FasL system
that would promote apoptosis via an extrinsic pathway. How-
ever, we found no baseline differences in expression of Fas
receptor or FasL by ELISA or immunohistochemistry. While
levels of FasL were elevated in Pearl mice as compared to WT
mice, the difference did not occur until 7 d after bleomycin
challenge, the time point at which fibrosis was already evident.
An intrinsic apoptotic mechanism is perhaps more likely in
HPS-affected cells. The finding that alveolar apoptosis is in-
creased in both HPS2 and HPS1 mice suggests a common,
mistrafficking-mediated mechanism of apoptotic susceptibility
ciated with marked phenotypic variability with respect to pene-
trance, age of onset, and disease severity (10, 11). Evidence of
ER stress (13) and susceptibility to bleomycin-inducedapoptosis
and fibrosis from SP-C models (17) suggests that environmental
compensated type II cells, may provide a “second hit” which
nisms of HPS type II cell apoptotic susceptibility.
bleomycin model is one of the most widely studied. Bleomycin
is associated with a myriad of injurious effects, and numerous
mechanisms have been postulated to influence the outcome of
bleomycin challenge.This studyidentifies HPSmice asa promis-
basis of pulmonary fibrosis using genetic and cellular replace-
to bleomycin-induced alveolar type II cell apoptosis, pulmonary
fibrosis, and mortality. Cellular and molecular insights into the
pathogenesis of HPS lung disease may enhance the understand-
ing and therapeutic approach to more common fibrotic lung
Conflict of Interest Statement: None of the authors has a financial relationship
with a commercial entity that has an interest in the subject of this manuscript.
Acknowledgments: The authors thank Dr. Richard Swank for providing the Pearl
and Pale Ear mice. They thank Dr. William J. Martin, II for guidance in design
of bleomycin studies, Camille Kapita for technical assistance with intratracheal
challenges, and Dr. Ping Wang for assistance with measurement of murine lung
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