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Am J Respir Crit Care Med. 2012 November 15; 186(10): 1014–1024.
Published online 2012 November 15. doi:  10.1164/rccm.201207-1206OC
PMCID: PMC3530211

The Alveolar Epithelium Determines Susceptibility to Lung Fibrosis in Hermansky-Pudlak Syndrome


Rationale: Hermansky-Pudlak syndrome (HPS) is a family of recessive disorders of intracellular trafficking defects that are associated with highly penetrant pulmonary fibrosis. Naturally occurring HPS mice reliably model important features of the human disease, including constitutive alveolar macrophage activation and susceptibility to profibrotic stimuli.

Objectives: To decipher which cell lineage(s) in the alveolar compartment is the predominant driver of fibrotic susceptibility in HPS.

Methods: We used five different HPS and Chediak-Higashi mouse models to evaluate genotype-specific fibrotic susceptibility. To determine whether intrinsic defects in HPS alveolar macrophages cause fibrotic susceptibility, we generated bone marrow chimeras in HPS and wild-type mice. To directly test the contribution of the pulmonary epithelium, we developed a transgenic model with epithelial-specific correction of the HPS2 defect in an HPS mouse model.

Measurements and Main Results: Bone marrow transplantation experiments demonstrated that both constitutive alveolar macrophage activation and increased susceptibility to bleomycin-induced fibrosis were conferred by the genotype of the lung epithelium, rather than that of the bone marrow–derived, cellular compartment. Furthermore, transgenic epithelial-specific correction of the HPS defect significantly attenuated bleomycin-induced alveolar epithelial apoptosis, fibrotic susceptibility, and macrophage activation. Type II cell apoptosis was genotype specific, caspase dependent, and correlated with the degree of fibrotic susceptibility.

Conclusions: We conclude that pulmonary fibrosis in naturally occurring HPS mice is driven by intracellular trafficking defects that lower the threshold for pulmonary epithelial apoptosis. Our findings demonstrate a pivotal role for the alveolar epithelium in the maintenance of alveolar homeostasis and regulation of alveolar macrophage activation.

Keywords: lung fibrosis, alveolar epithelial cells, alveolar macrophage, Hermansky-Pudlak syndrome, adaptor protein 3

At a Glance Commentary

Scientific Knowledge on the Subject

Hermansky-Pudlak syndrome (HPS) is a highly penetrant genetic disorder of pulmonary fibrosis, and there is strong evidence that macrophage-mediated inflammation precedes pulmonary fibrosis in patients with HPS. The spontaneous development of fibrosis in the HPS murine model recapitulates important features of the human disease, including constitutive alveolar macrophage activation and genotype-specific susceptibility to profibrotic stimuli.

What This Study Adds to the Field

Constitutive alveolar macrophage activation and increased susceptibility to bleomycin-induced fibrosis are attributable to HPS mutations in the alveolar epithelium. These findings demonstrate a pivotal role for the epithelium in the maintenance of alveolar homeostasis and regulation of alveolar macrophage activation and have implications for both for patients with Hermansky-Pudlak syndrome and for those with fibrotic lung diseases from other causes.

Pulmonary fibrosis is a final common pathway in many forms of interstitial lung diseases (ILD). Inflammation plays a prominent role in some forms of ILD and is a driver of fibrosis in other organs. However, idiopathic pulmonary fibrosis (IPF), the most common and enigmatic form of lethal pulmonary fibrosis in adults, does not respond to antiinflammatory therapies. Currently, the development of preventative and therapeutic strategies remains limited by incomplete understanding of the mechanisms underlying alveolar fibrosis (1).

Hermansky-Pudlak syndrome (HPS), the most penetrant of the genetic pulmonary fibrosis syndromes, provides a compelling paradigm for studying the cellular pathogenesis of pulmonary fibrosis. HPS gene products are ubiquitously expressed, and recessive mutations result in defects in heterooligomeric intracellular protein trafficking complexes and oculocutaneous albinism, bleeding diathesis, and sometimes granulomatous colitis (2). There are currently nine genetic loci associated with HPS in humans, and pulmonary fibrosis has been associated with some but not all genotypes, including HPS-1 and HPS-2 (26). Lung histology from patients with HPS typically reveals the usual interstitial pneumonia pattern found in IPF (7).

The alveolus is composed of and inhabited by numerous cell types, most of which have been extensively studied in efforts to understand the pathogenesis of pulmonary fibrosis. Both alveolar macrophages (813) and alveolar epithelial cells (1417) have been implicated in the pathogenesis of fibrosis in humans and experimental models, including in HPS (1823), although the causal relationships have remained unknown. Type II cell hyperplasia and endoplasmic reticulum (ER) stress have been identified in end-stage HPS lung disease (23). However, there is strong evidence that macrophage-mediated inflammation precedes pulmonary fibrosis in patients with HPS, and based on these data, bone marrow transplantation has been proposed as a potential therapy (19).

Naturally occurring HPS mouse models share many features of the human disease, but spontaneous pulmonary fibrosis does not occur (24). However, HPS-1 mice have an exaggerated fibrotic response to silica (25), and both the HPS-1 and HPS-2 models are exquisitely susceptible to bleomycin-induced fibrosis (18). Recent reports of spontaneous fibrosis in an HPS double-mutant mouse, generated by mating the HPS-1 and HPS-2 mutant mice, support the use of HPS models to study pulmonary fibrosis (21, 23). The gene product of HPS2 in mice and humans is the β3A subunit of the Adaptor Protein-3 (AP-3) complex, a heterooligomer that functions in organelle biogenesis and protein trafficking (26, 27). Mutations in individual AP-3 subunits result in instability and ubiquitin-mediated degradation of the entire AP-3 complex (26, 28), and loss of AP-3 function leads to protein mistrafficking in a variety of cell types (29). Both HPS-1 and HPS-2 mutant mice exhibit alveolar macrophage activation and fibrotic susceptibility and therefore provide a platform to experimentally model human HPS disease (18, 20). We therefore used cell-specific genetic correction and cellular replacement to decipher which cell lineage(s) in the alveolar compartment is the predominant driver of HPS fibrotic susceptibility. Some results have been previously reported in abstract form (3032).



Table 1 details the HPS mouse models studied and the corresponding disease-causing human HPS genotypes. Mice with homozygous mutations (hereafter by genotype and mutant [mt] or knock-out [ko]) on the C57BL/6J background were used in these studies (24, 27, 33, 34). The online supplement details the source of models used. Mice were housed in a barrier facility and studied using procedures approved by the Institutional Animal Care and Use Committees at the University of Cincinnati, Cincinnati Children’s Hospital Medical Center, and Vanderbilt University.


Bleomycin Challenge

Pharmaceutical grade bleomycin sulfate (0.025 units) was administered by intratracheal instillation (18).

Quantitation of Lung Collagen Content

Whole lung total soluble collagen was measured using the Sircol assay (Biocolor; Accurate Chemical and Scientific Corporation, Westburg, NY).

Lung Histology, Immunohistochemistry, and Evaluation of Apoptosis

Lung tissues were prepared and studied as described in the online supplement. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was performed using the In Situ Cell Death Detection Kit (Roche Diagnostics, Mannheim, Germany).

Isolation of Murine Alveolar Macrophages and Type II Cells

Alveolar macrophages were isolated as previously described (20). Type II cells were isolated using Dispase and negative selection on antibody-coated plates to separate leukocyte and monocyte populations (35). Detailed methods and culture conditions are provided in the online supplement. Cytokine levels were measured in the cell culture media supernatant by ELISA (R&D Systems, Minneapolis, MN). Cytotoxicity was determined using an lactate dehydrogenase (LDH) assay (Promega, Madison, WI).

Quantitative Real-Time Polymerase Chain Reaction Analysis and Western Blot Analysis

Total RNA for quantitative polymerase chain reaction was isolated and reverse transcribed by standard methods, with quantification on an Applied Biosystems Step One Plus Cycler. Primer probe sequences and Western blot protocols and antibody information are provided in the online supplement.

Bone Marrow Transplantation

Recipient mice were exposed to split dose whole body irradiation of 7 Gy then 4.75 Gy followed by tail vein injection of whole marrow, as described in the online supplement. To facilitate identification of donor versus recipient cell populations, we used C57BL/6J mice, which express green fluorescent protein under the direction of the human ubiquitin C promoter (WT-GFP), as wild-type control mice and donor mice in some experiments.

Epithelial-Specific Transgenic Correction of AP-3 in HPS Mice

The cDNA for the murine HPS2 gene product, AP-3/β3A, was fully sequenced, and a construct was generated with the following elements (in order): human SPC promoter (3.7 kb), β globin sequence, AP3 (3.4 kb), and polyadenylation signal. This construct was linearized and injected into the pronuclei of fertilized mouse C57BL/6J embryos and implanted into pseudopregnant C57BL/6J dams. Founders were crossed with HPS2 mice as detailed in the online supplement.

Statistical Analysis

Numeric data are presented as mean ± SEM. Statistical analyses were performed using GraphPad Prism. Parametric data were evaluated by analysis of variance with Tukey post hoc analysis, nonparametric data by the Mann-Whitney test, and survival analysis using the log-rank test. A two-sided probability value less than 0.05 was regarded as significant.


Susceptibility to Bleomycin-induced Fibrosis Occurs Only in Those Mouse Models with HPS Mutations Corresponding to Human Genotypes Associated with Lung Disease

ILD has been observed in patients with HPS-1, HPS-2, and HPS-4, but not HPS-3 or Chediak-Higashi syndrome (CHS), a disorder with features that overlap with HPS (4, 5). We aimed to determine whether susceptibility to bleomycin-induced fibrosis in HPS mouse models was limited to HPS mutations associated with lung fibrosis in humans with these different albinism syndromes. Bleomycin-induced 14-day mortality was 90 to 100% in HPS1mt, HPS2mt, and HPS2ko mice, all genotypes associated with human fibrosis. In marked contrast, mortality was 0 to 10% in bleomycin-challenged wild-type, HPS3mt, and CHSmt mice (Figure 1a), genotypes not known to be associated with human fibrosis. Lung histology and total collagen content were normal in unchallenged HPS and CHS mice up to 12 weeks of age (not shown). However, 7 days after bleomycin challenge, histologic evidence of diffuse fibrosis with substantial architectural distortion was evident in HPS1mt, HPS2mt, and HPS2ko mice (Figure 1b; Figure E1 in the online supplement). Only limited injury and pulmonary fibrosis was observed in bleomycin-challenged HPS3mt and CHSmt mice. Consistent with these results, total lung collagen content at Day 7 was not significantly different in bleomycin-challenged HPS3mt or CHSmt mice in comparison with wild-type mice, but was significantly elevated in bleomycin-challenged HPS1mt, HPS2mt, and HPS2ko mice (Figure 1c). Collectively, these results demonstrate that HPS mouse models faithfully replicate the fibrotic susceptibility of human HPS genotypes.

Figure 1.
Susceptibility to bleomycin-induced pulmonary fibrosis occurs only in those Hermansky-Pudlak syndrome (HPS) mouse models corresponding to genotypes linked to human lung fibrosis. HPS1mt, HPS2mt, HPS2ko, HPS3mt, CHSmt, and strain-matched C57BL/6J wild-type ...

Genotype of Bone Marrow–derived Cells Does Not Determine Fibrotic Susceptibility

Alveolar macrophage dysregulation has been reported in patients with HPS-1 before the onset of pulmonary fibrosis (19). To determine whether macrophage dysfunction contributes to the disease pathogenesis, we generated reciprocal bone marrow chimeric mice. Fluorescence-activated cell sorter analysis of spleen cell populations revealed that donor cells accounted for 99.1 ± 0.9% of cells in HPS2mt recipients of wild-type marrow and 99.0 ± 0.8% of cells in wild-type recipients of HPS2mt marrow 90 days after transplantation (Figures 2a and 2b). In whole lung leukocyte populations, donor cell percentages were 95.2 ± 2.7% and 97.8 ± 1.4% for HPS2mt and wild-type recipients, respectively (Figures 2c and 2d). Greater than 93% of bronchoalveolar lavage (BAL) leukocytes were of donor origin in all transplanted mice (Figure 2e).

Figure 2.
Generation of bone marrow chimeras. (a, b) Representative fluorescence-activated cell sorter (FACS) analysis of spleen cell populations 90 days after bone marrow transplantation. C57BL/6J mice that express green fluorescent protein (GFP) under the control ...

At more than 90 days after bone marrow transplantation, survival, lung histology, and collagen content in unchallenged mice were unchanged compared with age-matched control mice (not shown). After bleomycin exposure, excess mortality occurred in HPS2mt recipients despite transplantation with wild-type marrow (Figure 3a). Conversely, no mortality occurred, and lung histology revealed little evidence of fibrosis in wild-type mice that received HPS2mt marrow. Despite transplantation with wild-type marrow, HPS2mt mice displayed diffuse histologic evidence of pulmonary fibrosis and increased lung collagen content after bleomycin challenge without improvement compared with HPS2mt chimeric control mice (Figures 3b and 3d, Figure E2). To exclude potentially confounding influences from the small number of remaining host macrophages that are known to persist at earlier time points (36), we also studied a cohort of mice at 220 days after marrow transplantation. In this study, total lung collagen content at 7 days after bleomycin was 244 ± 13.5 μg for HPS2mt recipients of wild-type marrow and 236.1 ± 27.2 μg for HPS2mt recipients of HPS2mt marrow (not significant). To further confirm the relevance of these findings to HPS-1 disease, we also performed selected bone marrow transplantation experiments with HPS1mt mice. We found that transplantation of wild-type marrow failed to rescue the fibrotic susceptibility of recipient HPS1mt mice. Furthermore, transplantation of HPS1mt marrow into wild-type mice did not confer fibrotic susceptibility (Figures 3c and 3d). These data indicate that fibrotic susceptibility derives from genetic defects in nonhematopoietic cells.

Figure 3.
Bone marrow transplantation of wild-type (WT) whole marrow fails to rescue the fibrotic susceptibility of HPS2mt and HPS1mt mice, and transplantation of HPS2mt or HPS1mt marrow into WT mice does not confer fibrotic susceptibility. Transplanted mice, age ...

The Alveolar Macrophage Activation Phenotype in HPS Mice Is Not Corrected by Bone Marrow Transplantation

Our prior studies have demonstrated that HPS1mt and HPS2mt murine alveolar macrophages exhibit enhanced basal production of cytokines, chemokines, and nitric oxide as well as hyperresponsiveness to LPS and other toll-like receptor agonists (20). We have also shown that BAL fluid from HPS mice has macrophage-activating capability for wild-type alveolar macrophages. Here, we used bone marrow chimeric mice to determine whether macrophage dysfunction is intrinsic to HPS mononuclear cells. Alveolar macrophages were isolated from unchallenged mice 90 days after bone marrow transplantation. The genotype of the recipient, but not the bone marrow donor, determined the macrophage activation phenotype as assessed by basal levels of tumor necrosis factor (TNF)-α and macrophage inflammatory protein (MIP)-1α in the cell culture media supernatant (Figures 3e–3h). Our studies indicate that the HPS activated macrophage phenotype does not appear to derive from a cell-autonomous defect.

Generation of hSPC-AP3b1 Transgenic Mice

Although most pulmonary fibrosis occurs in patients with HPS-1, ILD also occurs in patients with HPS-2 (4, 6), and the body of biochemical information and reagents available to study HPS-2 are more robust (26, 33, 34, 37, 38). To directly test whether fibrotic susceptibility of HPS2mt mice originates from the pulmonary epithelium, we developed a transgenic model with epithelial-specific correction of the β3a subunit of AP-3 in the lungs of HPS2mt mice (Figures 4a–4c). Mutations in individual AP-3 subunits (such as β3 in HPS2mt mice) are known to result in instability and ubiquitin-mediated degradation of the entire AP-3 complex. Therefore, loss and functional restoration of the β3A protein product can be documented by the absence or presence, respectively, of other AP-3 subunits (26). Transgene (TG)+ mice, but not TGneg mice, showed functional correction of AP-3 stability in type II epithelial cells and lung but not the spleen, as revealed by the presence of the μ3 subunit or the delta subunit (Figures 4d and 4e). Faint bands detected in TGneg HPS2mt mice are consistent with prior reports that the HPS2mt is a hypomorph (34). We confirmed our findings in a second founder line and found no difference in the extent of functional correction and rescue. We found that HPS1mt (not shown) and HPS2mt type II cells secreted levels of monocyte chemotactic protein (MCP)-1 that were more than threefold greater than wild-type type II cells and that epithelial transgenic AP3b1 expression in HPS2mt mice significantly corrected the exaggerated MCP-1 secretion (Figure 4f). A similar pattern was observed for type II cell secretion of chemokine (C-X-C motif) ligand 1 (CXCL1) (Figure 4g). Type II cells in patients and mice with HPS have been reported to contain enlarged lamellar bodies. Epithelial correction of AP3b1 also resulted in significant reduction in lamellar body size in HPS2mt mice (Figure 4h, Figure E3).

Figure 4.
Transgenic correction of the defective β3 subunit stabilizes the AP-3 complex as evidenced by detection of the mu and delta subunits of the heterooligomer. (a) Construct used for generation of hSPC-AP3 transgenic mice. The cDNA for murine AP-3/β3A ...

Epithelial-Specific Correction of AP3b1 Protects HPS2mt Mice from Bleomycin-induced Mortality and Pulmonary Fibrosis

In comparison with HPS2mt TGneg littermate control mice, 6- to 8-week-old HPS2mt TG+ mice had significantly reduced mortality after bleomycin challenge (Figure 5a). Seven days after bleomycin was administered, mortality was greater than 50% in TGneg mice, whereas all TG+ mice were alive at this time point, although mortality subsequently occurred with a delayed time course. Lung histology and collagen content were analyzed 7 days after bleomycin challenge in surviving mice. Histologic evidence of fibrosis was present in TG+ mice but was mild in degree compared with the surviving TGneg mice (Figure 5b and Figure E4). Both founder lines of TG+ HPS2mt mice had significant reduction in total lung collagen content compared with their TGneg littermate control mice, although levels remained greater than those of wild-type mice (Figure 5c). One founder line was also back-crossed into HPS2ko mice to determine whether the mutant HPS2 allele interfered with transgene expression. A similar reduction in the extent of bleomycin-induced lung collagen accumulation was observed when the high expression founder line was back-crossed into HPS2ko mice (Figure 5c). Interestingly, epithelial-specific correction of AP-3 in HPS2mt mice also resulted in significant reduction in alveolar macrophage production of TNF-α and MIP-1α compared with TGneg littermate control mice (Figures 5d and 5e). Taken together with the failure of bone marrow transplantation to rescue the macrophage phenotype, these data indicate that HPS mutations in alveolar epithelial cells are responsible for alveolar macrophage activation.

Figure 5.
Epithelial-specific transgenic correction of AP-3 protects Hermansky-Pudlak syndrome (HPS) mice from bleomycin-induced mortality and pulmonary fibrosis and also dampens constitutive alveolar macrophage activation. (a) Increased survival of transgene-positive ...

Extent of Alveolar Type II Cell Apoptosis Is Dependent on HPS Genotype and Is a Key Early Event Determining Fibrotic Susceptibility in HPS Mice

Consistent with our previous findings that the early inflammatory response is similar in wild-type and HPS2mt mice (18), we also found no differences in indicators of lung injury after bleomycin challenge, including wet-to-dry lung weight ratios and BAL protein levels (Figure E5). However, 5 hours after bleomycin challenge, HPS1mt, HPS2mt, and the HPS2ko mice had prominent and accelerated type II cell apoptosis (Figure 6a), in contrast to the minimal numbers of TUNEL-positive type II cells in wild-type, HPS3mt, and CHSmt mice. Furthermore, we found that expression of AP3b1 in HPS2mt type II cells reduced the extent of type II cell apoptosis compared with HPS2mt-TGneg mice (Figure 6b and Figure E6). In vitro studies with isolated type II cells from unchallenged HPS2mt mice confirmed a dose-dependent susceptibility to bleomycin-induced cell death that was greater than that of wild-type cells (Figure 6c).

Figure 6.
Alveolar epithelial apoptosis correlates with fibrotic susceptibility in Hermansky-Pudlak syndrome (HPS) mice. (a) Evaluation of early alveolar apoptosis in HPS mouse models. Lungs were inflation-fixed 5 hours after intratracheal bleomycin challenge, ...

We tested the effect of prophylactic pan-caspase inhibition to determine whether apoptosis played a causal role in the fibrotic susceptibility of HPS mice. Starting 24 hours before intratracheal bleomycin challenge, HPS2mt or wild-type mice received quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone (Q-VD-Oph, intraperitoneal dosing 20 mg/kg, every other day) or vehicle (dimethyl sulfoxide) control. At 7 days after bleomycin challenge, mortality was 0% in wild-type mice, 10% in the HPS2mt group treated with Q-VD-Oph, and 30% in the vehicle (dimethyl sulfoxide)-treated HPS2mt group. Bleomycin-challenged HPS2mt mice treated with the pan-caspase inhibitor exhibited lung collagen content at 7 days that was no greater than that observed in wild-type mice (Figure 6d). Collectively, these data suggest that AP-3 deficiency is responsible for enhanced type II cell apoptosis in HPS2mt mice and that epithelial apoptosis plays a mechanistic role in the fibrotic susceptibility of HPS mice.

Mahavadi and colleagues have previously reported that ER stress underlies epithelial apoptosis in HPS, based in part on studies performed in HPS1/2 double-mutant mice at an advanced age when fibrosis was present (23). To determine whether ER stress could underlie the increased susceptibility to epithelial cell apoptosis in our models, we performed comprehensive ER stress–associated gene and protein expression studies in type II cells and whole lung tissue from HPS1mt and HPS2mt mice. We found no evidence of increased ER stress in HPS mutants compared with wild-type control mice at baseline or after bleomycin challenge that could explain the observed early epithelial apoptosis (Figures E7–E10).


Through murine bone marrow transplantation and generation of a transgenic model with epithelial-specific gene correction, our studies demonstrate that the pulmonary epithelium is paramount in the fibrotic susceptibility of HPS mice. Targeted reexpression of the missing subunit of the AP-3 complex in the lung epithelium of HPS2mt mice restored type II cell homeostasis, including cytokine production and lamellar body size, and reduced subsequent susceptibility to bleomycin-induced early type II cell apoptosis and fibrosis. In contrast, bone marrow transplantation did not protect HPS recipients from excess bleomycin-induced fibrosis. Similarly, dysregulated alveolar macrophage activation was determined by the recipient and not the donor, and transgenic correction in alveolar epithelial cells reduced cytokine production by alveolar macrophages, suggesting that the activated macrophage phenotype seen in HPS is regulated through a paracrine mechanism. Together, our data indicate that intracellular trafficking defects secondary to HPS mutations in alveolar epithelial cells contribute to the profibrotic phenotype in HPS through increased vulnerability to apoptosis and a persistent state of epithelial-driven macrophage activation in the lungs.

We studied a number of different mouse models corresponding to HPS genotypes that are either associated or not associated with human pulmonary fibrosis. As an additional disease control, we studied Chediak-Higashi syndrome mice, a related trafficking disorder with overlapping clinical features and giant lamellar bodies in type II cells but no reported pulmonary fibrosis in patients. Bleomycin is a chemotherapeutic agent associated with pulmonary inflammation, interstitial expansion, and irreversible pulmonary fibrosis in humans. Although widely studied, the bleomycin mouse model recapitulates only the first two of these features, and its value as a mimic of human disease is debated (39). Our findings that the fibrotic susceptibility of HPS mice to bleomycin segregates with HPS genotypes associated with pulmonary fibrosis in humans support the validity of the bleomycin model as a read-out of HPS fibrotic susceptibility.

Alveolar macrophage dysfunction has been implicated in the pulmonary fibrosis that occurs in patients with HPS (19), and our previous studies have demonstrated that HPS mice have constitutive alveolar macrophage activation that mimics the human phenotype (20). Therefore, we performed comprehensive whole marrow transplantation studies in HPS2mt and HPS1mt mice. As controls, HPS2mt mice were transplanted with HPS2mt marrow, to exclude potential confounding effects of irradiation. Furthermore, we studied mice at conservatively late time points after transplant to limit any potential contribution from the small numbers of resident host macrophages that are known to persist after transplant (36). We found that transplantation of wild-type marrow failed to rescue the fibrotic susceptibility of HPS1mt and HPS2mt recipient mice. Although bone marrow transplantation has been proposed for HPS, extrapolation of our findings would suggest that bone marrow transplantation is unlikely to protect patients with HPS from pulmonary fibrosis. Furthermore, because the bleeding complications of HPS generally respond favorably to routine platelet transfusion, bone marrow transplantation is not usually considered for the sole purpose of reducing bleeding risk. Several murine studies demonstrate that the lung recruits circulating bone marrow–derived cells to aid in repair after bleomycin-induced injury (4043). Our data demonstrate that HPS defects in bone marrow–derived cells are not sufficient for fibrotic susceptibility in HPS mice. However, we have not excluded the possibility that bone marrow–derived cells play a secondary role as effector cells in promoting or mitigating HPS pulmonary fibrosis.

Given the results of the bone marrow transplant experiments, we proceeded to directly test the role of HPS mutations in the lung epithelium of mice. In our transgenic model, epithelial-specific correction resulted in highly significant, but incomplete, protection from bleomycin injury compared with littermate control mice. The direction and degree of phenotypic correction was consistent across all outcome parameters studied, including histology, collagen content, cytokine production, apoptosis, and mortality. There was no difference in the extent of protection in a high versus lower expressing transgenic line. There are a number of known limitations of this transgenic model system that may explain incomplete correction of the pulmonary phenotype in HPS mice, including that expression driven with the human SP-C promoter is known to be variable and restricted to a subset of lung epithelial cells (44, 45). Furthermore, allergic inflammation has been reported to down-regulate SP-C promoter expression (46), and bleomycin may cause time-dependent attenuation of AP3b1 transgenic expression. Finally, we find that dominant negative interference of endogenous mutant HPS proteins is unlikely, given that the extent of transgenic rescue observed was similar in the complete knock-out (HPS2ko) and the hypomorphic (HPS2mt) backgrounds. Our data do not exclude the possibility that additional mechanisms contribute to fibrotic susceptibility and mortality in HPS mice.

A striking and consistent abnormality in bleomycin-challenged HPS mice is the accelerated apoptosis of type II cells. The extent of alveolar epithelial apoptosis correlated with extent of fibrosis in different HPS mouse models, and transgenic epithelial correction of the HPS2 defect resulted in significant reduction in type II cell apoptosis. Studies demonstrating that a pan-caspase inhibitor protected HPS mice from bleomycin-induced fibrosis provide additional evidence that apoptosis plays a causal role in HPS pulmonary fibrosis, although we cannot exclude effects the drug may have had on other cell types that could be playing a role in fibrosis. Although the albinism and bleeding phenotypes are fairly consistent across different HPS genotypes, pulmonary fibrosis is restricted to only certain HPS genotypes in humans, consistent with distinct and divergent trafficking functions of HPS proteins in the alveolar epithelium. The mechanistic relationship between HPS intracellular trafficking defects and epithelial vulnerability to apoptosis remain incompletely understood at this time. We propose that our data, in conjunction with experimental models using the Fas-Fas ligand system (14, 15) and transgenic diphtheria-toxin targeted ablation of the epithelium (16), support the concept that alveolar epithelial cell apoptosis is a key event in the pathogenesis of pulmonary fibrosis. However, although ER stress may be causal in some genetic forms of pulmonary fibrosis (4749), alveolar epithelial vulnerability in HPS appears to occur by a different mechanism. As a result, further study of HPS trafficking defects may elucidate additional mechanisms responsible for pulmonary fibrosis.

In summary, our studies demonstrate that HPS fibrotic susceptibility is due to intracellular trafficking defects in the alveolar epithelium, which may also contribute to fibrosis through paracrine activation of alveolar macrophages. HPS mouse models provide a tractable mimic of the human disease and will be valuable for further deciphering mechanisms of pulmonary fibrosis in HPS. Our hope is that insights gained from this rare genetic disease will have broader implications for understanding the pathogenesis of more common fibrotic lung diseases.

Supplementary Material

Online Supplement:


The authors thank Richard Swank for breeding pairs of HPS1mt pale ear and HPS2mt pearl mice, and Sally Mansour and Margit Burmeister for the AP3b1−/− mice (HPS2ko). We also thank Stephan Glasser and Jeffrey Whitsett for providing the human SP-C promoter, Andrew Peden for providing the original AP-3 delta subunit antibody, Jon Neumann in the Transgenic Mouse Core at the University of Cincinnati for technical assistance in the generation of the transgenic mice, and Jeff Bailey and Victoria Summey in The Comprehensive Mouse and Cancer Core at Cincinnati Children’s Hospital Medical Center for technical assistance with murine bone marrow transplantation.


Funded by the American Thoracic Society/Hermansky-Pudlak Syndrome Network Partnership Grant (L.R.Y.); Parker B. Francis Foundation Fellowship (L.R.Y.); National Institutes of Health grants K08 HL082757 (L.R.Y.), HL85317 (T.S.B.), HL92870 (T.S.B.), and HL68861 (F.X.M.); and the Department of Veterans Affairs (T.S.B. and F.X.M.).

Author Contributions: L.R.Y. was responsible for the overall project and wrote the initial manuscript. P.M.G. performed the majority of the animal and cell culture experiments. T.E.W. was responsible for generation of the transgenic construct. J.P.B. contributed to the design and conduct of the endoplasmic reticulum stress experiments. G.H.D. performed electron microscopy analysis. T.S.B. contributed to the data analysis and manuscript preparations. F.X.M. contributed to the overall project inception, experimental design, data analysis, and execution. All authors edited the manuscript.

This article has an online supplement, which is accessible from this issue’s table of contents at

Originally Published in Press as DOI: 10.1164/rccm.201207-1206OC on October 4, 2012

Author disclosures are available with the text of this article at


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