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Published ahead of print on June 12, 2003, doi:10.1165/rcmb.2003-0148OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 677-682, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2003-0148OC

Peroxisome Proliferator-Activated Receptor-{gamma} Is Deficient in Alveolar Macrophages from Patients with Alveolar Proteinosis

Tracey L. Bonfield, Carol F. Farver, Barbara P. Barna, Anagha Malur, Susamma Abraham, Baisakhi Raychaudhuri, Mani S. Kavuru and Mary Jane Thomassen

Departments of Pulmonary and Critical Care Medicine, Anatomic Pathology, and Cell Biology, The Cleveland Clinic Foundation, Cleveland, Ohio

Address correspondence to: Dr. Mary Jane Thomassen, Department of Pulmonary and Critical Care Medicine, 9500 Euclid Avenue, Cleveland Clinic Foundation, Desk A90, Cleveland, OH 44195-5038. E-mail: thomasm{at}ccf.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is a ligand-activated, nuclear transcription factor that regulates genes involved in lipid and glucose metabolism, inflammation, and other pathways. The hematopoietic growth factor, granulocyte macrophage colony-stimulating factor (GM-CSF), is essential for lung homeostasis and is thought to regulate surfactant clearance, but mechanisms involved are unknown. GM-CSF is reported to stimulate PPAR-{gamma}, but the activation status of PPAR-{gamma} in human alveolar macrophages has not been defined. In pulmonary alveolar proteinosis (PAP), a rare interstitial lung disease, surfactant accumulates in alveolar airspaces, resident macrophages become engorged with lipoproteinaceous material, and GM-CSF deficiency is strongly implicated in pathogenesis. Here we show that PPAR-{gamma} mRNA and protein are highly expressed in alveolar macrophages of healthy control subjects but severely deficient in PAP in a cell-specific manner. Further, we show that the PPAR-{gamma}–regulated lipid scavenger receptor, CD36, is also deficient in PAP. PPAR-{gamma} and CD36 deficiency are not intrinsic to PAP alveolar macrophages, but can be upregulated by GM-CSF therapy. Moreover, GM-CSF treatment of patients with PAP fully restores PPAR-{gamma} to healthy control levels. Based upon these novel findings, we hypothesize that GM-CSF regulates lung homeostasis via PPAR-{gamma}–dependent pathways.

Abbreviations: granulocyte macrophage colony-stimulating factor, GM-CSF • interleukin, IL • pulmonary alveolar proteinosis, PAP • peroxisome proliferator-activated receptor-{gamma}, PPAR-{gamma}


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is a member of a superfamily of intracellular ligand-activated receptors that function as transcription factors (1). Although weakly expressed in monocytes, PPAR-{gamma} expression intensifies with differentiation into macrophages and is predominantly nuclear in location (2). PPAR-{gamma} expression has been reported in monocytes/macrophages (3–5), although the status in human alveolar macrophages has not been established. Critical roles for PPARs have been reported in regulation of genes involved in lipid and glucose metabolism as well as in inflammation (6). Moreover, PPAR-{gamma} ligands have shown therapeutic activities in models of atherosclerosis (7) and inflammatory diseases (8).

PAP is a rare interstitial lung disease of unknown etiology in which the alveoli become filled with surfactant material (9). Surfactant, a complex mixture of phospholipids and proteins, serves to reduce surface tension within the lung (10). Surfactant accumulation in PAP is thought to be due to inefficient catabolism by alveolar macrophages and/or type II epithelial cells (11, 12). Mice deficient in either granulocyte macrophage colony-stimulating factor (GM-CSF) or its receptor fail to catabolize surfactant and develop a lung lesion histologically similar to pulmonary alveolar proteinosis (PAP) (13–15). This murine model of PAP can be reversed by site-specific application of GM-CSF, either via aerosol or pulmonary epithelial cell expression of GM-CSF (16, 17). Such results suggest that GM-CSF is essential to surfactant homeostasis by the lung.

The autoimmune nature of human PAP was initially recognized by Kitamura and coworkers and by Tanaka and colleagues, who noted circulating anti–GM-CSF autoantibodies neutralize GM-CSF biological activity, thus resulting in GM-CSF deficiency (18, 19). Further studies in patients with PAP confirmed that anti–GM-CSF antibodies have clinical utility for both diagnosis and disease severity (20–23). Furthermore, no primary defect responsible for surfactant dysregulation has yet been identified in PAP (24, 25). Treatment with recombinant GM-CSF has shown some promise in reversing the clinical symptoms of PAP (9, 26, 27), particularly in patients with low titers of anti–GM-CSF (20). Because GM-CSF also represents an upregulator of PPAR-{gamma} in cell culture studies (28, 29), we hypothesized that in PAP, a GM-CSF–deficient condition, PPAR-{gamma} expression might be deficient and that GM-CSF treatment might correct PPAR-{gamma} deficiency, thus suggesting a potential pathway by which GM-CSF could exert a beneficial effect in PAP.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study Population
This protocol was approved by the Cleveland Clinic Foundation Institutional Review Board and written informed consent was obtained from all subjects. Healthy control individuals had no history of lung disease and were not on medication. The diagnosis of idiopathic PAP was established by histopathologic examination of material from open lung or transbronchial biopsies showing the characteristic filling of the alveoli with eosinophilic amorphous material with preserved lung architecture and absence of inflammation, and exclusion of secondary etiologies by negative lung cultures or occupational history (9). All patients with PAP were symptomatic with dyspnea, were hypoxemic on room air, and had typical alveolar infiltrates on radiographs. Patients participated in a prospective clinical trial of recombinant human GM-CSF (Leukine; Berlex, Seattle, WA) as described previously (26). Treatment consisted of 250 mcg/d by subcutaneous administration, with increased dosage every 2 wk and maximum daily dose of 18 mcg/kg/d by 8 wk. Median duration of therapy was 25.5 wk (20). Patients were evaluated at baseline before initiation of GM-CSF therapy and during therapy.

Cell Collection and Culture
Alveolar macrophages were derived from bronchoalveolar lavage (BAL) obtained by fiberoptic bronchoscopy as previously described (30). Differential cell counts were obtained from cytospins stained with a modified Wright's stain. Mean viability of lavage cells was greater than 95% as determined by trypan blue dye exclusion. For culture, BAL cells were plated into 24-well plates (300,000 alveolar macrophages/well) or chamber slides (60,000 cells/well) in RPMI 1640 medium supplemented with 5% human AB serum (Gemini, Calabasas, CA), L-glutamine, and antibiotics. Bronchial epithelial cells were obtained as previously described and characterized by immunocytochemistry for cytokeratin content (31).

Immunocytochemistry and Flow Cytometry Analysis
PPAR-{gamma} protein expression was evaluated in cytospin preparations from freshly isolated alveolar macrophages and bronchial epithelial cells and in macrophages cultured 24–48 h with and without GM-CSF (100 ng/ml; Berlex) by immunocytochemistry. Cells were fixed with 4% paraformaldehyde, permeabilized with Triton x 100, and stained with rabbit polyclonal anti–PPAR-{gamma} (1:1,000; Santa Cruz, Santa Cruz, CA), followed by ALEXA-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR). Slides were also stained with propidium iodine to facilitate nuclear localization and evaluated by confocal microscopy and quantified by the BIOQUANT true color system (R&M Biometrics, Inc., Nashville, TN) as previously described (32). Alveolar macrophages were evaluated for CD36 using flow cytometry and were stained with 1:1,000 phycoerythrin-conjugated anti-CD36 (BD PharMingen, San Jose, CA) and analyzed on BD FACscan Analyzer. Results are expressed as mean percentage of positive staining alveolar macrophages for 10,000 events.

RNA Purification and Analysis
Total RNA was extracted from BAL cells by RNAeasy protocol (Qiagen, Valencia, CA). Expression of mRNA was determined by real time RT-PCR using the ABI Prism 7,000 Detection System (TaqMan; Applied Biosystems, Foster City, CA.) according to the manufacturer's instructions. RNA specimens were analyzed in duplicate using primer sets for a housekeeping gene (GAPDH) and PPAR-{gamma}, CD36, or IL-10 (ABI). Threshold cycle (CT) values for genes of interest were normalized to GAPDH and used to calculate the relative quantity of mRNA expression in PAP (or GM-CSF–treated) samples relative to untreated or healthy control values. Data are expressed as fold change in mRNA expression relative to control values.

Statistics
Data were analyzed by one-way ANOVA and Student's t test using Prism software (GraphPad, Inc., San Diego, CA.). Significance was defined as P <= 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PPAR-{gamma} Protein and mRNA Expression Are Reduced in PAP Alveolar Macrophages
Freshly isolated alveolar macrophages obtained from healthy control subjects stained intensely for PPAR-{gamma} (Figure 1A) compared with PAP alveolar macrophages (Figure 1B). We evaluated the presence of PPAR-{gamma} protein in five different patients with PAP and five healthy control subjects. Nuclear localization of PPAR-{gamma} was clearly evident in healthy control subjects (Figure 1C), with almost no nuclear staining in PAP alveolar macrophages from any of the five different patients with PAP (Figure 1D). Quantification of nuclear staining demonstrates greater staining in healthy control macrophages as compared with PAP (P = 0.0001; see Figure 1E). Autofluorescence due to surfactant phospholipids, however, was readily discernible in PAP sections. Cytospins evaluated with secondary antibody alone showed no evidence of immunostaining (data not shown).



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  		Figure 1. PPAR-{gamma} protein is expressed in healthy control but not PAP alveolar macrophages. Cytospins from BAL cells of healthy controls (HC) (A, C) and PAP (B, D) alveolar macrophages were immunostained for PPAR-{gamma} and evaluated for immunofluorescence (green) with (C, D) and without (A, B) propidium iodine (red) overlay to localize nuclei. Images were captured at x400 magnification. Prominent nuclear PPAR-{gamma} staining is visible in alveolar macrophages of HC but not PAP. Immunoquantification (E) shows the percentage of nuclear staining in PAP alveolar macrophages as compared with healthy controls (n = 5 different fields, P = 0.0001). Similar results were seen in an additional four patients with PAP and in four healthy control subjects.

 
To determine whether PPAR-{gamma} gene expression was also deficient in PAP, mRNA expression was quantified by real-time RT-PCR. PPAR-{gamma} mRNA expression was significantly reduced in PAP BAL cells (P = 0.0005) compared with those from healthy control subjects, indicating a deficiency at the transcriptional level (Figure 2).



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  		Figure 2. PPAR-{gamma} mRNA expression is deficient in PAP alveolar macrophages. PPAR-{gamma} mRNA expression was quantified in alveolar macrophages of patients with PAP (n = 6) and healthy control subjects (HC) (n = 7) by real-time RT-PCR. Relative PPAR-{gamma} mRNA expression in PAP was significantly less (P = 0.0005) than HC.

 
PPAR-{gamma} Expression Is Not Deficient in PAP Bronchial Epithelial Cells
Bronchial epithelial cells from healthy control subjects and from patients with PAP were evaluated for PPAR-{gamma} protein and mRNA expression. Because we have observed elevated interleukin (IL)-10 in PAP BAL fluid, we also investigated epithelial cell IL-10 mRNA (24). PAP bronchial epithelial cells demonstrated PPAR-{gamma} protein (Figure 3A) and mRNA levels comparable to those of control subjects (Figure 3B), whereas IL-10 mRNA, as anticipated, was elevated. These results suggest that deficiency of PPAR-{gamma} in PAP is specific to alveolar macrophages and not manifested by other cells in the lung.



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  		Figure 3. PPAR-{gamma} expression of PAP bronchial epithelial cells is not deficient. PPAR-{gamma} protein expression was evaluated by immunocytochemistry (A). PPAR-{gamma} protein levels in PAP bronchial epithelial cells were not different from controls. The image was captured at x800 magnification. PPAR-{gamma} and IL-10 mRNA expression were quantified by real-time RT-PCR in PAP (n = 3) and healthy control (HC) (n = 2) bronchial epithelial cells (B). IL-10 was elevated but PPAR-{gamma} was similar to HC.

 
GM-CSF Upregulates Alveolar Macrophage PPAR-{gamma} Expression In Vitro
We next investigated whether human alveolar macrophage PPAR-{gamma} expression was sensitive to GM-CSF modulation as reported previously for other hematopoietic cell types (33). Alveolar macrophages from healthy control subjects were cultured overnight to render them quiescent, then cultured an additional 24–48 h with or without GM-CSF (100 ng/ml). Untreated cultured cells exhibited less PPAR-{gamma} than freshly isolated cells (Figure 4A), but after exposure to GM-CSF, nuclear PPAR-{gamma} protein intensified (Figure 4A versus 4B). To ascertain whether GM-CSF upregulated PPAR-{gamma} gene expression in vitro, alveolar macrophage mRNA was analyzed by real-time RT-PCR. GM-CSF significantly (P = 0.004) enhanced PPAR-{gamma} mRNA expression in healthy control macrophages by 8-fold (Figure 4C).



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  		Figure 4. Alveolar macrophage PPAR-{gamma} expression is upregulated by GM-CSF in vitro. PPAR-{gamma} expression of healthy control (HC) (n = 3) alveolar macrophages was determined by immunocytochemistry at baseline and after 24–48 h culture with GM-CSF (100 ng/ml) (A and B). Increased nuclear staining for PPAR-{gamma} is visible after GM-CSF exposure in tissue culture (B). Images were captured at x400 magnification. GM-CSF also significantly (P = 0.004) increased PPAR-{gamma} mRNA expression in HC (C).

 
PPAR-{gamma} Deficiency of PAP Alveolar Macrophages Is Reversible by GM-CSF Treatment In Vitro and In Vivo
To investigate whether the subnormal PPAR-{gamma} expression of PAP alveolar macrophages was modifiable with appropriate stimuli, PAP macrophages were cultured with GM-CSF in vitro. PPAR-{gamma} mRNA expression rose by 20-fold in PAP alveolar macrophages after 24–48 h exposure to GM-CSF (P = 0.005; Figure 5A), suggesting that lack of stimulus rather than the presence of intrinsic cellular defects is responsible for the reduced PPAR-{gamma} expression of PAP cells. Based on this observation, we investigated whether patients with PAP enrolled in a clinical trial of GM-CSF therapy exhibited changes in alveolar macrophage PPAR-{gamma} expression. Results indicate that GM-CSF therapy dramatically upregulated PPAR-{gamma} levels in PAP alveolar macrophages to control range (Figure 5B). Expression of PPAR-{gamma} in PAP at baseline was 37-fold lower than in control subjects before therapy and rose to control levels (P = 0.009 compared with baseline) after GM-CSF treatment (Figure 5B).



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  		Figure 5. PAP alveolar macrophage PPAR-{gamma} expression is upregulated by GM-CSF in vitro and in vivo. PAP alveolar macrophages (n = 2) were evaluated for PPAR-{gamma} expression by real-time RT-PCR before and after 24–48 h culture with GM-CSF (100 ng/ml) (A). PPAR-{gamma} mRNA expression was also quantified in alveolar macrophages from untreated patients with PAP at baseline (n = 6), patients with PAP receiving GM-CSF therapy (n = 5), and healthy control subjects (HC) (n = 5) (B). GM-CSF therapy significantly increased (P = 0.009) PPAR-{gamma} mRNA expression in patients with PAP compared with baseline.

 
CD36 Receptor and Gene Expression Is Decreased in PAP
CD36 is a lipid scavenger receptor regulated by PPAR-{gamma} (34); thus, we investigated the CD36 cell surface and gene expression in PAP and healthy control alveolar macrophages. Using flow cytometry we found that PAP alveolar macrophages (n = 4) had significantly less CD36 on their surface than healthy control (n = 5) alveolar macrophages (Figure 6A, P = 0.002). We also found that PAP BAL cells from patients before GM-CSF therapy had significantly less CD36 mRNA than healthy control subjects (Figure 6B; n = 6, P = 0.044). In patients on GM-CSF therapy (n = 5), levels of CD36 mRNA were similar to those of healthy control subjects (n = 5, P = 0.129).



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  		Figure 6. CD36 receptor and message expression is decreased in PAP. We investigated the CD36 cell surface expression (A) and message (B) of PAP and healthy control alveolar macrophages. Using flow cytometry we found that PAP alveolar macrophages (n = 4) had significantly less CD36 on their surface than healthy control (n = 5) alveolar macrophages (A, P = 0.002). We also found that PAP BAL cells from patients before GM-CSF therapy have significantly less CD36 mRNA as compared with healthy control subjects (B, n = 6, P = 0.044). Patients on GM-CSF therapy (n = 5) had levels of CD36 mRNA similar to healthy control subjects (n = 5, P = 0.129).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The novel findings presented here show for the first time that: (i) PPAR-{gamma} is highly expressed in alveolar macrophages of healthy individuals but significantly deficient in PAP; (ii) deficiency of PPAR-{gamma} in PAP is cell type–specific in the lung (alveolar macrophages but not bronchial epithelial cells are affected); (iii) in vitro and in vivo GM-CSF treatment restores PPAR-{gamma} expression in PAP alveolar macrophages to healthy control levels; and (iv) the lipid scavenger receptor CD36 surface and gene expression is deficient. Results reported here for the first time implicate PPAR-{gamma} as a potential factor in GM-CSF regulation of lung homeostasis.

PPAR-{gamma} expression is ubiquitous within the lung and has been shown to be constitutive in human airway epithelium (35) and smooth muscle cells (36). The selectivity of PPAR-{gamma} deficiency for PAP alveolar macrophages and not airway epithelium may suggest that macrophages use PPAR-{gamma} pathways differently than epithelial cells and are therefore more sensitive to GM-CSF deprivation. Whether PPAR-{gamma} signaling is crucial for surfactant catabolism by alveolar macrophages is unknown. Further, there are no data regarding the status of PPAR-{gamma} expression in alveolar macrophages of the GM-CSF knockout mouse which exhibits PAP-like pathology (13, 14).

PPAR-{gamma} represents a potent transcriptional regulator of genes governing lipid and glucose metabolic pathways in adipocytes, macrophages, and other cell types (6, 37). In vitro studies have shown that PPAR-{gamma} is required for functioning of the CD36 lipid scavenger receptor, which downregulates another scavenger receptor, SR-A (34). Mice deficient in CD36 show impaired lipid transport (38). Activation of PPAR-{gamma} also induces gene expression of ABCA 1, a transporter molecule regulating apoA1-mediated cholesterol efflux in macrophages (39). PPAR-{gamma} expression itself can be upregulated by oxidized low density lipoprotein and is highly expressed in foam cells of atherosclerotic plaques (40). Interestingly, therapeutic administration of PPAR-{gamma} ligands appears to have a beneficial effect in atherosclerosis and inflammatory diseases (reviewed in Ref. 37). We have shown that PAP alveolar macrophages express less CD36 on their surface, suggesting a potential mechanism by which PPAR-{gamma} deficiency contributes to PAP pathophysiology. The apparently pleiotrophic actions of PPAR-{gamma} noted thus far in the literature are intriguing and together with the present findings suggest that in the future PAP may benefit from a combination of PPAR-{gamma} activators and GM-CSF. Further studies are required, however, to define mechanisms of surfactant catabolism and the possible involvement of PPAR-{gamma} in CD36 regulation in PAP.

Currently, GM-CSF is an approved therapy for the clinical treatment of neutropenia following chemotherapy but no studies to date have examined PPAR-{gamma} expression in treated patients (41, 42). In PAP, the conventional treatment has been whole-lung lavage administered under general anesthesia to allow removal of accumulated surfactant material (9). More recently, GM-CSF administration has been reported to be beneficial in PAP and therefore has been suggested as an alternative to whole-lung lavage (26, 27). The GM-CSF treatment studies described in the present paper are ongoing, and further analyses will be necessary regarding alveolar macrophage PPAR-{gamma} levels and PAP clinical responses. Nevertheless, the current findings clearly indicate that alveolar macrophages of patients with PAP have a severe deficiency of PPAR-{gamma} and that systemic GM-CSF therapy is effective in reversing PPAR-{gamma} deficiency. We hypothesize that the efficacy of GM-CSF therapy is achieved via PPAR-{gamma}–dependent pathways.


   Acknowledgments
 
This work was funded by NIH HL67676 and the generous support of Regina Taussig.

Received in original form April 28, 2003

Received in final form June 4, 2003


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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