Rationale: Idiopathic pulmonary fibrosis (IPF) is caused by alterations in expression of proteins involved in multiple pathways, including matrix deposition, inflammation, injury, and repair.
Objectives: To understand the pathogenic changes in lung protein expression in IPF and to evaluate apolipoprotein (Apo) A-I as a candidate therapeutic molecule.
Methods: Two-dimensional electrophoresis was adopted for differential display proteomics. Reverse-transcriptase polymerase chain reaction, Western blotting, immunohistochemical staining, and ELISA were performed for identification and quantitative measurement of Apo A-I in bronchoalveolar lavage fluids from subjects with IPF and experimental bleomycin-induced mice.
Measurements and Main Results: Sixteen protein spots showed differences in relative intensity between IPF (n = 14) and healthy control subjects (n = 8). Nano liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed increase of haptoglobulin and decrease of α1-antitrypsin, α1-antichymotrypsin, macrophage capping protein, angiotensinogen, hemoglobin chain B, Apo A-I, clusterin, protein disulfide isomerase A3, immunoglobulin, and complement C4A in IPF compared with normal control subjects (P = 0.006–0.044). Apo A-I concentrations were lower in bronchoalveolar lavage fluids from subjects with IPF (n = 28) than in normal control subjects (n = 18; P < 0.01). In bleomycin-treated mice, Apo A-I protein in BALF was lower than that in sham-treated control animals. Immunohistochemical analysis showed positive staining on intraalveolar macrophages and epithelial cells of the lungs. Intranasal treatment with Apo A-I protein reduced the bleomycin-induced increases in number of inflammatory cells and collagen deposition in sham-treated mice in a dose-dependent manner.
Conclusions: Alterations of several inflammatory and antiinflammatory proteins in the lungs may be related to the pathogenesis of IPF, and local treatment with Apo A-I is very effective against the development of experimental lung injury and fibrosis.
High-throughput and whole-proteome studies have not been performed in research to understand changes in lung protein expression in idiopathic pulmonary fibrosis (IPF).
Proteomic analysis of bronchoalveolar lavage fluid demonstrated alterations of several inflammatory and antiinflammatory proteins in bronchoalveolar lavage fluid of subjects with IPF. As a novel finding, apolipoprotein (Apo) A-I concentrations were decreased in the lungs of IPF and experimental bleomycin-induced fibrosis model. In addition, intranasal treatment with Apo A-I protein reduced bleomycin-induced increases in a number of inflammatory cells and collagen deposition. These data indicate that Apo A-I may be useful in therapeutic strategies.
Bronchoalveolar lavage (BAL) conducted with fiberoptic bronchoscopy has been widely used to collect cells and other soluble components from the epithelial lining fluids that cover the airway and the alveoli. BAL fluid thus contains proteins secreted from different cell types, including epithelial and inflammatory cells, and a wide variety of proteins from the bloodstream. Therefore, analysis of BAL fluid may reveal important pathologic mediators in the airway and alveolar wall lining fluids (11, 12). However, there has been only limited proteome analysis of BAL fluids from subjects with IPF and patients with systemic sclerosis (13, 14).
In this study, we adopted differential display proteomics for comparison between normal control subjects and subjects with IPF to obtain a better understanding of the pathogenic mechanisms and events operating in the bronchoalveolar fluid in subjects with IPF and to evaluate apolipoprotein A-I (Apo A-I) as a candidate therapeutic molecule in experimental animals. Some of the results of the present study have been previously reported in the form of an abstract (15).
The diagnosis of IPF was based on an international consensus statement by the American Thoracic Society and the European Respiratory Society (16). Histologic diagnosis of usual interstitial pneumonia (UIP) had been confirmed by surgical lung biopsy specimens in all subjects. For the proof of histology, two local pathologists reviewed the slides under the condition to be informed of the age and sex of the patients and the presence of unilateral or bilateral pulmonary disease by the chest physician's assistant (PA) and the computed tomogram. None of the patients with IPF had any evidence of underlying collagen vascular diseases clinically and by laboratory studies. The normal control subjects were recruited from hospital personnel. They had no respiratory symptoms; normal range of FVC and FEV1 (>75% of the predicted value); and normal findings on a simple chest radiogram. The clinical characteristics are demonstrated in Table 1.
|Age, yr||60 (45–72)||66 (58–75)|
|FVC, % predicted||88.5 (82.3–94)||51.5 (43.3–72.3)*|
|Dlco, % predicted||ND||49 (43.5–57)|
|Fibrosis score||3.17 (2.67–3.67)|
|GGO score||2.67 (2.67–3.25)|
|BAL cell differential counts|
|Alveolar macrophages, %||93.71 (86.32–97.81)||71.08 (37.67–80.30)*|
|Neutrophils, %||3.65 (1.69–7.29)||14.75 (7.72–38.07)*|
|Lymphocytes, %||2.75 (1.88–4.11)||4.96 (1.45–13.33)†|
| Eosinophils, %||0.90 (0.58–2.87)||2.00 (0.81–4.44)*|
BAL had been performed in the most involved lobe on computed tomography in the subjects without any immunosuppressive therapy and in the right middle lobe of normal control subjects as described previously (17). The institutional review board for human studies approved the protocol and informed written consent was obtained from all subjects. BAL fluids from 14 patients with IPF who never smoked and 8 normal control subjects who never smoked were subjected to two-dimensional gel electrophoresis (2-DE). Clinical characteristics of these patients are provided in the online supplement (see Table E1).
BAL fluids were analyzed by 2-DE, and the levels of Apo A-I in BAL fluids were measured in 28 patients with IPF and 18 normal control subjects. The demographic characteristics of the subjects are summarized in Table 1. BAL procedures and preparation were performed as described previously (17). The cytocentrifuge slides of BAL cells were air-dried, fixed in methanol, and stained with Diff-Quik. Five hundred leukocytes were enumerated and foamy macrophages were counted as described previously (18). None of the patients with IPF had any evidence of underlying collagen vascular disease either clinically or based on laboratory data. Histologic diagnosis was confirmed by surgical lung biopsy in all subjects as described previously (16). BAL fluid aliquots were desalted by overnight dialysis (relative molecular mass cut-off 3,500) against Tris-HCl buffer. A total of 1 mg of proteins of the dialyzed BAL fluid were precipitated with 10% trichloroacetic acid in acetone and resuspended in the sample solution. Immobiline DryStrips (Amersham Biosciences, Piscataway, NJ) were used for isoelectric focusing and the second dimension by 7.5–20% SDS-polyacrylamide gel electrophoresis was performed. The gels were visualized with Coomassie brilliant blue G-250. Digitized images were analyzed using the ImageMaster program to calculate the two-dimensional spot intensity by integrating the optical density over the spot area (the spot “volume”). Differentially expressed protein spots were excised from the gels, cut into smaller pieces, and digested with trypsin (Promega, Madison, WI), as described previously (19). Protein identification was performed using Agilent 1100 Series nano-LC for MS/MS and database searches. Additional detail is provided in the online supplement.
On Day 0, the mice were administered 2 U/kg of bleomycin by intratracheal instillation. On Day 4, Apo A-I purified from human plasma was administered. On Day 14, after sacrifice, the tissues were subjected to hematoxylin and eosin staining to permit morphometric analysis. The total amount of soluble collagen was determined using a Sircol collagen assay kit. For immunohistochemical analysis of Apo A-I protein, BAL cells and lung tissue on slides were incubated at 4°C overnight with goat antimouse Apo A-I polyclonal antibody. For negative control stain, lung tissues on slides were treated without the antibody. The total amount of Apo A-I protein and mRNA in BAL fluids was measured using a sandwich ELISA kit and Western blotting analysis and reverse-transcriptase polymerase chain reaction (RT-PCR) as described previously (19). Additional detail is provided in the online supplement.
The total amount of Apo A-I protein in BAL fluids was measured using a sandwich ELISA kit and Western blotting analysis as described previously (19). Apo A-I mRNA expression was measured by RT-PCR on total RNA extracted from and in situ hybridization on BAL cells as described previously (20). Total RNA of BAL cells and lung tissues were extracted using TRI REAGENT (Molecular Research Center, Toyama, Japan). Primers and probes for human Apo A-I and β-actin gene and mouse Gapdh and Apo A-I gene were designed for the selected genes using GeneFisher software (http://bibiserv.techfak.uni-bielefeld.de/genefisher). The primer sequences are provided in the online supplement. Additional detail is provided in the online supplement.
Statistical analyses were performed with SPSS 10.0 (SPSS Inc., Chicago, IL). The Mann-Whitney test (two-sample rank sum test) was used to analyze differences between two groups. Simple linear regression analyses were performed to analyze correlations between variables, and Pearson correlation coefficients (R) were determined from these analyses. All data are expressed as median values and interquartile ranges, and significance was defined as P < 0.05.
2-DE was performed to determine the differential expression of proteins in BAL fluid between the 8 normal control subjects and the 14 patients with IPF. The BAL samples were resolved by one-dimensional gel electrophoresis on IPG gel strips over a pH range of 3–10, followed by 2-DE on homogeneous 7.5–20% SDS-polyacrylamide gel electrophoresis (Figure 1). After Coomassie Brilliant Blue G-250 stain and analysis, a median number of 591 protein spots (range, 460–790 spots), groups normal control subjects; median 585 (range, 482–683), IPF; median 596 (range, 507–726) were detected in 1 mg of BAL fluid. All of the identified spots were localized in the range of pH 4–10 with a molecular mass range of 10–150 kD. The relative intensities (percentages) of the spots are expressed in Table 2 as median values of each group and the locations of identified protein spots are shown in a master image. Sixteen proteins showed significant differences in relative intensity between the two groups according to the Mann-Whitney U test (P < 0.05). These spots were excised from the gels and incubated with trypsin for in-gel digestion, and then identified by LC-MS/MS. The results of these analyses are summarized in Table 2. The relative intensity of spot 8 (haptoglobin) was four times higher in the IPF group than in the normal control group (P = 0.04). In contrast, the relative intensities of the other 15 spots were significantly lower in the former group than in the latter (Figure 1, Table 2). The differentially expressed proteins listed here represent a wide range of biologic categories. Proteins related to cellular defense mechanisms involving antiinflammatory and antioxidant activity were the most common. Among them, the levels of antiinflammatory proteins, including α1-antitrypsin (spot 1), α1-antichymotrypsin (spot 6), macrophage capping protein (spot 7), Apo A-I (spot 2), angiotensinogen (spot 4), and hemoglobin chain B (spot 5), decreased significantly in patients with IPF compared with normal control subjects (P = 0.006–0.04). The cytoskeletal proteins protein disulfide isomerase A3 (spot 10) and β-actin (spots 3 and 11) also decreased in patients with IPF compared with normal control subjects (P = 0.007–0.016). The inflammatory and immune mediators immunoglobulin heavy chain γ 2 (spot 13), immunoglobulin J chain (spot 14), myosin D (spot 12), and complement C4A (spot 15) decreased significantly in the IPF group compared with normal control subjects (P = 0.004–0.037) (Table 2). 2-DE analysis of BAL fluids was restricted to the nonsmokers.
MW (kD) PI
Relative Intensity NC IPF
|1||α1-Antitrypsin||21361198||(K)VFSNGADLSGVTEEAPLK(L)||54.93/4.95||0.281 > 0.014||0.006||Antiinflammatory|
|2||Apolipoprotein A-I||113992||(K)DSGRDYVSQFEGSALGK(Q)||30.71/5.44||0.115 > 0.038||0.007||Antiinflammatory|
|3||Beta actin||62897625||(R)LDLAGRDLTDYLMK(I)||42.08/5.37||0.106 > 0.017||0.007||Cytoskeleton|
|4||Angiotensinogen||15079348||(R)AAMVGMLANFLGFR(I)||53.36/5.87||0.029 > 0.014||0.013||Antiinflammatory|
|5||Chain B1, Hemoglobin mutant||2981647||(K)VNVDEVGGEALGR(L)||15.88/6.75||0.064 > 0.004||0.016||Antiinflammatory|
|6||α1-Antichymotrypsin||177933||(K)EQLSLLDRFTEDAK(R)||45.57/5.32||0.020 > 0.003||0.033||Antiinflammatory|
|7||Macrophage capping protein||729022||(R)EVQGNESDLFMSYFPR(G)||38.77/5.88||0.034 > 0.014||0.039||Antiinflammatory|
|8||Haptoglobin||3337390||(R)VMPICLPSKDYAEVGR(V)||38.72/6.14||0.041 < 0.245||0.04||Antiinflammatory|
|9||Clusterin||338305||(R)ASSIIDELFQDR(F)||36.99/5.74||0.141 > 0.021||0.044||Antioxidant|
|10||Protein disulfide isomerase A3||37589929||(R)LDLAGRDLTDYLMK(I)||57.04/5.88||0.106 > 0.017||0.007||Cytoskeleton|
|11||Beta actin||15277503||(K)EYDDNGEGITIFRPLHLANK(F)||42.08/5.37||0.034 > 0.001||0.016||Cytoskeleton|
|12||Myosin 1D||21040432||(K)LLNIYGRDTIEQYK(G)||50.02/6.54||0.050 > 0.009||0.011||Inflammatory|
|13||Immunoglobulin gamma 2 heavy chain||25987833||(R)VVSVLTVVHQDWLNGK(E)||36.35/7.51||0.104 > 0.009||0.017||Inflammatory|
|14||Immunoglobulin J chain||21489959||(R)SSEDPNEDIVER(N)||18.09/5.12||0.557 > 0.088||0.017||Inflammatory|
|15||Complement C4A||40737468||(R)VQQPDCREPFLSCCQFAESLR(K)||58.38/5.76||0.059 > 0.008||0.037||Inflammatory|
|16||Aminopeptidase PILS||6381989||(K)QTWTDEGSVSERMLR(S)||107.11/5.97||0.057 > 0.013||0.004||Inflammatory|
To validate the changes in Apo A-I determined on 2-DE, case control studies were conducted. The amount of Apo A-I was measured in BAL fluid from 28 patients with IPF and in 18 normal control subjects by ELISA (Figure 2). The normalized concentration of Apo A-I with total protein was twofold lower in the IPF group compared with the normal control subjects (median/range: 7.5/1–50.3 ng/μg of BAL protein vs. 14.6/1.7–88.5 ng/μg of BAL protein; P < 0.01, respectively) (Figure 2C). The log percentage of foamy macrophages among the total macrophages was significantly higher in the subjects with IPF than in normal control subjects (median/range: 13.2/4–46.4% vs. 2.4/0.6–19%; P < 0.001, respectively) (Figure 2D). The log percentages of foamy macrophages were inversely correlated with the levels of Apo A-I in BAL fluids of the patients with IPF and normal control subjects (r = −0.359; P = 0.019; n = 42) (Figure 2E). However, the correlation disappeared when the far right located two values of normal control subjects were removed (r = −0.034; P = 0.642). We investigated the association of pulmonary function tests and Apo A-I protein concentrations. There was no good association between Apo A-I concentration and lung functions (FVC and diffusing capacity of carbon monoxide) at the initial visit (data not shown). One interesting finding is the presence of inverse correlation between Apo A-I concentration and fibrosis scores (Figure 2F) measured as described by Katzerooni and colleagues (21).
Expression of Apo A-I protein in BAL cells was examined by immunocytochemical staining. In BAL cells, Apo A-I decreased markedly in the large cells from six patients with IPF compared with those of six normal control subjects (Figure 2H). Apo A-I mRNA measured by RT-PCR and by in situ hybridization was demonstrated in the BAL cells of six normal control subjects, but very weakly in those of six patients with IPF (Figures 2I and 2J).
To investigate whether Apo A-I expression was altered in the lungs in an experimental model of fibrosis (Figure 3A), we measured Apo A-I mRNA and protein in the lung tissues and lavage cells of bleomycin-treated mice. Expression of Apo A-I was analyzed by RT-PCR of lung tissues, Western blotting, and immunohistochemical staining of BAL cells and lung tissue from bleomycin-treated mice and sham-treated control animals. Apo A-I mRNA was less expressed in the lung tissues of bleomycin-treated mice than that of sham-treated control animals (Figure 3B) and at significantly higher levels in liver tissues of both groups (Figure 3B). Western blot analysis also showed a positive band with a molecular weight of 30.7 kD in BALF from sham and bleomycin-treated mice (Figure 3C). The total amount of Apo A-I was significantly reduced in the bleomycin-treated mice compared with the control animals. On immunohistochemical analysis, positive staining was observed on intraalveolar macrophages and epithelial lining cells of the lungs from sham and bleomycin-treated mice, whereas stain was not observed in the negative control animals (Figure 3E).
To investigate whether Apo A-I replacement is capable of suppressing the development of pulmonary inflammation and fibrosis in vivo, the whole molecule of human Apo A-I protein was administered into bleomycin-treated mice (Figure 3A). After bleomycin challenge, the numbers of inflammatory cells including macrophages, neutrophils, and lymphocytes in the BAL fluid increased significantly compared with sham control animals (Figure 4A). Intranasal treatment with Apo A-I protein significantly reduced the bleomycin-induced increases in numbers of inflammatory cells including neutrophils, macrophages, and lymphocytes in BAL fluids to the levels of phosphate-buffered saline–challenged controls in a dose-dependent manner (Figure 4A). Apo A-I at a dose of 7.3 pmol/kg or higher suppressed the increases in numbers of macrophages, neutrophils, and lymphocytes in BAL fluids (P < 0.01) to the same levels as in sham-treated control animals. The therapeutic effect of Apo A-I was also observed in lung tissues stained with hematoxylin and eosin (Figure 4B). Intranasal treatment with Apo A-I protein decreased the bleomycin-induced inflammatory cell infiltration in a dose-dependent manner. To analyze collagen deposition in the lung, Masson trichrome stain and Sircol collagen assay were applied to lung tissue sections and lysates, respectively. Collagen deposition was detected in the interstitium of the lungs after bleomycin treatment. Treatment with Apo A-I abrogated the bleomycin-induced deposition of collagen almost completely at Day 14 (Figure 4C). The histologic findings were confirmed by analysis of the total amount of collagen in lung tissue lysates by ELISA. The total amount of collagen was doubled on Day 14 after bleomycin treatment, and intranasal administration with Apo A-I significantly decreased the bleomycin-induced increment of collagen to the levels of sham-treated mice in a dose-dependent manner (P < 0.05)(Figure 5).
IPF is a multifactorial disease that requires global analysis, and therefore cannot be understood through analysis of individual or small numbers of genes or mediators. A metaanalysis showed that cigarette smoking and exposure to agriculture and farming, livestock, wood and metal dust, and stone and silica were associated with significantly increased risk of IPF (22). In response to environmental exposure, airway and parenchymal lung cells showed altered expression of proteins plausibly associated with the mechanisms of development of pulmonary fibrosis, including oxidant injury, immunologic responses, and fibrotic responses (23). Therefore, high-throughput, and whole-proteome studies are needed to understand the proteomic contribution to IPF. We adopted a proteomics approach as an initial approach in the present study.
Using 2-DE as a screening tool, we identified 16 proteins in BAL fluids that exhibited differential expression between patients with IPF and normal control subjects. Because the intraassay variation of 2-DE analysis may lead to false–positive results, validation using enough numbers of cases is mandatory. In the present study, 14 subjects with IPF and 8 normal control subjects underwent 2-DE to reduce the risk.
The proteins identified could be classified into four different groups based on their cellular functions and participation in biochemical pathways: (1) antiinflammatory; (2) cytoskeleton; (3) coagulation (changed to antioxidant); and (4) immune and metabolism. Among them, antiinflammatory proteins were most commonly observed (Table 2). These included Apo A-I, haptoglobin, α1-antitrypsin, α1-antichymotrypsin, macrophage capping protein, clusterin, hemoglobin B chain, and angiotensinogen.
The relative intensity of one of these proteins, α1-antitrypsin (spot 1), was 1:20 in the IPF group compared with normal control subjects (Table 2). α1-Antitrypsin is a circulating serine protease inhibitor that protects the lung parenchyma from the damaging effects of proteases, particularly neutrophil elastase (24). It is well known that qualitative and quantitative deficiencies of α1-antitrypsin are related to the development of chronic obstructive pulmonary disease (25), although the relation with the development of IPF is still controversial (26, 27). α1-Antichymotrypsin shows nucleic acid and protein sequence homology with α1-antitrypsin and was shown to be an important mediator in preventing fibrosis in an experimental model of lung fibrosis (28). Because these mediators are affected by smoking, we selected subjects with IPF and normal control subjects who had never smoked.
In the present study, the inflammatory proteins were reduced in the relative deficiency of Apo A-I in the patients with IPF compared with the normal control subjects. The antiinflammatory mechanisms of action of Apo A-I are diverse, including inhibition of tumor necrosis factor, IL-6, and IL-8 release (29). Thus, many inflammatory mediators were expected to be elevated in BAL fluid analysis on 2-DE study. One explanation for the discrepancy is that the 2-DE analysis has a limitation of poor sensitivity to detect very low abundant proteins less than 100 pg/ml (30). The clinically measured proteins seem to be divided into three major classes: (1) plasma proteins at the high abundance end, (2) tissue leakage proteins in the middle, and (3) cytokines at the low abundance end. Most inflammatory and antiinflammatory mediators reported in the previous IPF studies (17, 31), which in general act locally, are probably not detected in 2-DE analysis because they are diluted from microliter or milliliter volumes of tissue.
Of the 16 proteins identified, we selected Apo A-I for further validation, because this protein was recently shown to have an antiinflammatory effect (32, 33). Apo A-I levels were twofold lower in the IPF group compared with the normal control subjects. These observations indicated that attenuated Apo A-I concentration in the alveolar lining fluid is associated with the development of IPF. However, there is the possibility that residual serum high-density lipoprotein (HDL) could contribute to the Apo A-I content of the lungs during the surgical process of specimens.
We also demonstrated the in situ production of Apo A-I in the lung, although the major source in peripheral blood is known to be the liver (34). Human Apo A-I is synthesized as a preproapoprotein with a 24–amino acid NH2-terminal extension. The presegment, 18–amino acid residues long, is cleaved cotranslationally by signal peptidase. The remaining proprotein containing a hexapeptide prosegment (RHFWQQ) covalently linked to the NH2 terminus of mature Apo A-I is secreted into the medium. In the human liver and intestine, pro–Apo A-I is converted to Apo A-I after its secretion (35, 36). The Apo A-I observed on 2-DE of the present study may be a mixture of mature and pro form.
To date, there have been few reports regarding Apo A-I in the lung lining fluids. Analysis of the hydrophobic fraction of the inflammatory BAL using 2-DE identified increased levels of Apo A-I in the inflammatory BAL (37). Wattiez and coworkers (38) reported that the relative intensity of Apo A-I in BAL fluid of normal control subjects was 0.13 ± 0.09, which is very similar to the level of our study (0.115 ± 0.08). The relative intensity of Apo A-I measured in three subjects with IPF was 0.17 ± 0.07, which was much higher that of our study (0.038 ± 0.09). The discrepancy might be derived from the small number of subjects and different staining method (silver stain versus Coomassie blue stain). Other studies on protein profiles in BALF from sarcoidosis and patients with IPF demonstrated that sarcoidosis and patients with IPF yield different protein profiles: low-molecular-weight proteins were more abundant in IPF and plasma proteins were more abundant in sarcoidosis (38, 39). Numerous groups of plasma proteins were more abundant in BALF from sarcoidosis than patients with IPF, whereas other proteins, mostly of low relative molecular mass and local cellular origin, were more abundant in IPF than sarcoidosis (13). Rottoli and colleagues (13) also reported the decreased levels of Apo A-I fragment, α1-antitrypsin, and α1-antichymotrypsin in IPF compared with those of normal control subjects. In our study, these molecules were also lower in IPF subjects compared with those in normal control subjects. These proteins are carbonylated by oxidation in IPF (14). However, Fietta and colleagues reported that quantity of Apo A-I did not significantly vary between the subjects with systemic sclerosis with and those without pulmonary fibrosis (40).
Apo A-I is the major Apo of HDL, although other Apos (A-II, A-IV, C-I, C-II, C-III, and E) also act as lipid acceptors (34). ATP-binding cassette transporter A-I (ABCA-I) promotes transfer of cholesterol and phospholipids from cells to lipid-free serum Apo, particularly Apo A-I, initiating the formation of HDL (41). ABCA-I was found in several tissues, including cells of the small intestine, liver, brain, kidney, and lung, including pulmonary macrophages (42) and type II pneumocytes (43, 44). The localization of Apo A-I in the present study showed good agreement with that of ABCA-I. In studies of ABCA-I gene-targeted mice, Abca1−/– pups exhibited severe respiratory distress, lung congestion, and bronchopulmonary dysplasia. In addition, Abca1−/– mice showed accumulation of cholesterol in alveolar macrophages and type II pneumocytes (45).
In the present study, the proportion of foamy macrophages was inversely correlated with the level of Apo A-I in BAL fluids from subjects with IPF and normal control subjects. The correlation of foamy cells and Apo A-I concentration in normal control subjects and subjects with asthma disappeared without the far right located two values of normal control subjects (r = −0.034; P = 0.642). Thus, the association of Apo A-I with foamy cell formation needs to be reevaluated with more subjects. Foamy cells are generated because of defects in the transfer of cholesterol and phospholipids (46). Thus, the transfer of cholesterol and phospholipids from cells to Apo A-I may be essential to maintain the lung free from inflammation. To confirm the contribution of locally produced Apo A-I in the lung to inflammation and fibrosis, various amounts (0.073–73 pmol/kg) of Apo A-I were administered to mice by the nasal route. Intranasal treatment with Apo A-I substantially attenuated the bleomycin-induced inflammation and fibrosis (Figure 4A). These findings indicate that Apo A-I is an essential factor in modulating inflammation and fibrosis in bleomycin-induced experimental pulmonary fibrosis. Different animal models of pulmonary fibrosis have been developed to investigate IPF. Among them, bleomycin models in rodents have been most commonly used as an alternative in many studies, as used in the present study. Over the years, numerous agents have been shown to inhibit fibrosis in this model. However, to date none of these compounds are used in the clinical management of IPF and none has shown a comparable antifibrotic effect in humans. However, bleomycin models in rodents have been most commonly used as an alternative in many studies (47, 48).
The antiinflammatory mechanism of action of Apo A-I has recently been demonstrated. HDL was initially found to neutralize LPS activity in vitro and in vivo. Because Apo A-I is the major Apo of HDL, the antiinflammatory effect of HDL may be caused by the action of Apo A-I. Several antiinflammatory effects have been demonstrated in animal models of LPS injury. Apo A-I was shown effectively to protect against LPS-induced endotoxemia and acute lung damage (32). Clinically, a low serum level of Apo A-I is associated with the disease severity of patients in intensive care (49) and recombinant HDL reduced flulike symptoms during endotoxemia in a human study (50), although there have been no previous reports in patients with IPF. Therefore, we are currently examining the association between Apo A-I concentration in BAL fluids and long-term prognosis of patients with IPF. Apo A-I and Apo A-I mimetic peptides exert antiinflammatory effects against influenza A 2infection–induced apoptosis of Type II pneumocytes (33). When the virus-infected cells were incubated with Apo A-I mimetic peptides, production of proinflammatory oxidized phospholipids was inhibited. Because Apo A-I mimetic peptides, such as D-4F, bind lipids with high avidity (51), Apo A-I seems to have a key functional role in scavenging and removing unnecessary lipids.
Apo A-I also induced production of prostaglandin E2 and IL-10 as antiinflammatory mediators (52). Apo A-I was shown to reduce the function of activated neutrophils (53) and suppress the expression of intercellular adhesion molecule-1 and P-selectin on the endothelium (54). In addition, Apo A-I blocks T-cell and natural killer cell stimulation of dendritic cells, and thus inhibits IL-12p70 and IFN-γ production (55). Therefore, Apo A-I seems to play an important role in modulating both innate immune responses and inflammatory responses, and adaptive immune responses, perhaps through an as yet unidentified common channel on various inflammatory cell types.
Using differential display proteomics and various validation tools, we showed that attenuation of Apo A-I level in the lung is related to the development of IPF, a multifactorial disease with several contributing factors in disease pathogenesis (56–59) and experimental bleomycin-induced fibrosis. Replacement of Apo A-I prevented the bleomycin-induced inflammation and fibrosis in the experimental animal model. Modulation of Apo A-I gene or protein expression in the lung has not been examined in detail. The modulation of Apo A-I activity offers prospects for new therapeutic approaches in the treatment of IPF and other inflammatory respiratory diseases.
The authors thank the editors from textcheck.com, both native speakers of English, for their editing for grammar and typographic errors. For a certificate, see: http://www.textcheck.com/certificate/ox3Vhg.
|1.||King TE Jr, Costabel U, Cordier J-F, doPico GA, du Bois RM, Lynch D, Lynch JP III, Myers JL, Panos RJ, Raghu G, et al. Idiopathic pulmonary fibrosis: diagnosis and treatment. Am J Respir Crit Care Med 2000;161:646–664.|
|2.||Selman M, King TE, Pardo A. Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann Intern Med 2001;134:136–151.|
|3.||Lim GI, Lee KH, Jeong SW, Uh ST, Jin SY, Lee DH, Park JS, Choi DL, Kang CH, Park CS. Clinical features of interstitial lung diseases. Korean J Intern Med 1996;11:113–121.|
|4.||Wahidi MM, Schwartz DA, Raghu G. Genetics of familial pulmonary fibrosis and other variants. In: Lynch JP, editor. Idiopathic pulmonary fibrosis. New York: Marcel Dekker; 2004. pp. 31–54.|
|5.||Garantziotis S, Steele MP, Schwartz DA. Pulmonary fibrosis: thinking outside of the lungs. J Clin Invest 2004;114:319–321.|
|6.||Maher TM, Wells AU, Laurent GJ. Idiopathic pulmonary fibrosis: multiple causes and multiple mechanisms? Eur Respir J 2007;30:835–839.|
|7.||Kinnula VL, Fattman CL, Tan RJ, Oury TD. Oxidative stress in pulmonary fibrosis: a possible role for redox modulatory therapy. Am J Respir Crit Care Med 2005;172:417–422.|
|8.||Keane MP, Strieter RM, Belperio JA. Mechanisms and mediators of pulmonary fibrosis. Crit Rev Immunol 2005;25:429–463.|
|9.||Kaminski N. Microarray analysis of idiopathic pulmonary fibrosis. Am J Respir Cell Mol Biol 2003;29(Suppl. 3):S32–S36.|
|10.||Zuo F, Kaminski N, Eugui E, Allard J, Yakhini Z, Ben-Dor A, Lollini L, Morris D, Kim Y, DeLustro B, et al. Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans. Proc Natl Acad Sci U S A 2002;99:6292–6297.|
|11.||Plymoth A, Lofdahl CG, Ekberg-Jansson A, Dahlback M, Lindberg H, Fehniger TE, Marko-Varga G. Human bronchoalveolar lavage: biofluid analysis with special emphasis on sample preparation. Proteomics 2003;3:962–972.|
|12.||Lindahl M, Stahlbom B, Tagesson C. Newly identified proteins in human nasal and bronchoalveolar lavage fluids: potential biomedical and clinical applications. Electrophoresis 1999;20:3670–3676.|
|13.||Rottoli P, Magi B, Perari MG, Liberatori S, Nikiforakis N, Bargagli E, Cianti R, Bini L, Pallini V. Cytokine profile and proteome analysis in bronchoalveolar lavage of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis and idiopathic pulmonary fibrosis. Proteomics 2005;5:1423–1430.|
|14.||Rottoli P, Magi B, Cianti R, Bargagli E, Vagaggini C, Nikiforakis N, Pallini V, Bini L. Carbonylated proteins in bronchoalveolar lavage of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis and idiopathic pulmonary fibrosis. Proteomics 2005;5:2612–2618.|
|15.||Lee YH, Kim KH, Kim TH, Lee SH, Cha JY, Jung S, Jang AS, Park SW, Uh ST, Kim YH, et al. Role of lung apolipoprotein A1 in idiopathic pulmonary fibrosis: anti-inflammatory and anti-fibrotic effect [abstract]. In: Proceedings of the 18th KOGO Conference. Seoul: Korea Genome Organization; 2009. pp. 108–109.|
|16.||American Thoracic Society, European Respiratory Society. American Thoracic Society/European Respiratory Society international multidisciplinary consensus classification of the idiopathic interstitial pneumonias. Am J Respir Crit Care Med 2002;165:277–304.|
|17.||Park CS, Chung SW, Ki SY, Lim GI, Uh ST, Kim YH, Choi DI, Park JS, Lee DW, Kitaichi M. Increased levels of interleukin-6 are associated with lymphocytosis in bronchoalveolar lavage fluids of idiopathic nonspecific interstitial pneumonia. Am J Respir Crit Care Med 2000;162:1162–1168.|
|18.||Bedrossian CW, Warren CJ, Ohar J, Bhan R. Amiodarone pulmonary toxicity: cytopathology, ultrastructure, and immunocytochemistry. Ann Diagn Pathol 1997;1:47–56.|
|19.||Cha MH, Rhim T, Kim KH, Jang AS, Paik YK, Park CS. Proteomic identification of macrophage migration-inhibitory factor upon exposure to TiO2 particles. Mol Cell Proteomics 2007;6:56–63.|
|20.||Han SJ, Kim JH, Noh YJ, Chang HS, Kim CS, Kim KS, Ki SY, Park CS, Chung IY. Interleukin (IL)-5 downregulates tumor necrosis factor (TNF)-induced eotaxin messenger RNA (mRNA) expression in eosinophils. Induction of eotaxin mRNA by TNF and IL-5 in eosinophils. Am J Respir Cell Mol Biol 1999;21:303–310.|
|21.||Kazerooni EA, Martinez FJ, Flint A, Jamadar DA, Gross BH, Spizarny DL, Cascade PN, Whyte RI, Lynch JP III, Toews G. Thin-section CT obtained at 10 mm increments versus limited three-level thin-section CT for idiopathic pulmonary fibrosis: correlation with pathologic scoring. AJR Am J Roentgenol 1997;169:977–983.|
|22.||Taskar VS, Coultas DB. Is idiopathic pulmonary fibrosis an environmental disease. Proc Am Thorac Soc 2006;3:293–298.|
|23.||Nemery B, Bast A, Behr J, Borm PJ, Bourke SJ, Camus PH, De Vuyst P, Jansen HM, Kinnula VL, Lison D, et al. Interstitial lung disease induced by exogenous agents: factors governing susceptibility. Eur Respir J Suppl 2001;32:30s–42s.|
|24.||Abboud RT, Vimalanathan S. Pathogenesis of COPD. Part I. The role of protease antiprotease imbalance in emphysema. Int J Tuberc Lung Dis 2008;12:361–367.|
|25.||Dahl M, Hersh CP, Ly NP, Berkey CS, Silverman EK, Nordestgaard BG. The protease inhibitor PI*S allele and COPD: a meta-analysis. Eur Respir J 2005;26:67–76.|
|26.||Geddes DM, Webley M, Brewerton DA, Turton CW, Turner-Warwick M, Murphy AH, Ward AM. Alpha 1-antitrypsin phenotypes in fibrosing alveolitis and rheumatoid arthritis. Lancet 1977;2:1049–1051.|
|27.||Hubbard R, Baoku Y, Kalsheker N, Britton J, Johnston I. Alpha1-antitrypsin phenotypes in patients with cryptogenic fibrosing alveolitis: a case-control study. Eur Respir J 1997;10:2881–2883.|
|28.||Nagareda T, Tanaka A, Terada N, Imakon Y, Shiomi H, Matsubara H, Noro A, Nishizawa Y. Suppressive effect of anti-alpha 1-antichymotrypsin serum on pulmonary fibrosis induced by phorbol myristate acetate in vivo. Lab Invest 1995;73:541–546.|
|29.||Galbois A, Thabut D, Tazi KA, Rudler M, Mohammadi MS, Bonnefont-Rousselot D, Bennani H, Bezeaud A, Tellier Z, Guichard C, et al. Ex vivo effects of high-density lipoprotein exposure on the lipopolysaccharide-induced inflammatory response in patients with severe cirrhosis. Hepatology 2009;49:175–184.|
|30.||Anderson NL, Anderson NG. The human plasma proteome: history, character, and diagnostic prospects. Mol Cell Proteomics 2002;1:845–867.|
|31.||Ishii H, Mukae H, Kadota J, Fujii T, Abe K, Ashitani J, Kohno S. Increased levels of interleukin-18 in bronchoalveolar lavage fluid of patients with idiopathic nonspecific interstitial pneumonia. Respiration 2005;72:39–45.|
|32.||Yan YJ, Li Y, Lou B, Wu MP. Beneficial effects of ApoA-I on LPS-induced acute lung injury and endotoxemia in mice. Life Sci 2006;79:210–215.|
|33.||Van Lenten BJ, Wagner AC, Navab M, Anantharamaiah GM, Hui EK, Nayak DP, Fogelman AM. D-4F, an Apo A1 mimetic peptide, inhibits the inflammatory response induced by influenza A infection of human type II pneumocytes. Circulation 2004;110:3252–3258.|
|34.||Bolanos-Garcia VM, Miguel RN. On the structure and function of apolipoproteins: more than a family of lipid-binding proteins. Prog Biophys Mol Biol 2003;83:47–68.|
|35.||Chau P, Fielding PE, Fielding CJ. Bone morphogenetic protein-1 (BMP-1) cleaves human proapolipoprotein A1 and regulates its activation for lipid binding. Biochemistry 2007;46:8445–8450.|
|36.||Edelstein C, Gordon JI, Toscas K, Sims HF, Strauss AW, Scanu AM. In vitro conversion of proapoprotein A-I to apoprotein A-I. Partial characterization of an extracellular enzyme activity. J Biol Chem 1983;258:11430–11433.|
|37.||de Torre C, Ying SX, Munson PJ, Meduri GU, Suffredini AF. Proteomic analysis of inflammatory biomarkers in bronchoalveolar lavage. Proteomics 2006;6:3949–3957.|
|38.||Wattiez R, Hermans C, Cruyt C, Bernard A, Falmagne P. Human bronchoalveolar lavage fluid protein two-dimensional database: study of interstitial lung diseases. Electrophoresis 2000;21:2703–2712.|
|39.||Magi B, Bini L, Perari MG, Fossi A, Sanchez JC, Hochstrasser D, Paesano S, Raggiaschi R, Santucci A, Pallini V, et al. Bronchoalveolar lavage fluid protein composition in patients with sarcoidosis and idiopathic pulmonary fibrosis: a two-dimensional electrophoretic study. Electrophoresis 2002;23:3434–3444.|
|40.||Fietta AM, Bardoni AM, Salvini R, Passadore I, Morosini M, Cavagna L, Codullo V, Pozzil E, Meloni F, Montecucco C. Analysis of bronchoalveolar lavage fluid proteome from systemic sclerosis patients with or without functional, clinical and radiological signs of lung fibrosis. Arthritis Res Ther 2006;8:R160 10.1186/ar2067.|
|41.||Fitzgerald ML, Mendez AJ, Moore KJ, Andersson LP, Panjeton HA, Freeman MW. ATP-binding cassette transporter A1 contains an NH2-terminal signal anchor sequence that translocates the protein's first hydrophilic domain to the exoplasmic space. J Biol Chem 2001;276:15139–15145.|
|42.||Lawn RM, Wade DP, Couse TL, Wilcox JN. Localization of human ATP-binding cassette transporter 1 (ABC1) in normal and atherosclerotic tissues. Arterioscler Thromb Vasc Biol 2001;21:378–385.|
|43.||Bortnick AE, Favari E, Tao JQ, Francone OL, Reilly M, Zhang Y, Rothblat GH, Bates SR. Identification and characterization of rodent ABCA1 in isolated type II pneumocytes. Am J Physiol Lung Cell Mol Physiol 2003;285:L869–L878.|
|44.||Bates SR, Tao JQ, Yu KJ, Borok Z, Crandall ED, Collins HL, Rothblat GH. Expression and biological activity of ABCA1 in alveolar epithelial cells. Am J Respir Cell Mol Biol 2008;38:283–292.|
|45.||McNeish J, Aiello RJ, Guyot D, Turit T, Gabel C, Aldinger C, Hoppe KL, Roach ML, Royer LJ, de Wet J, et al. High density lipoprotein deficiency and foam cell accumulation in mice with targeted disruption of ATP-binding cassette transporter-1. Proc Natl Acad Sci U S A 2000;97:4245–4250.|
|46.||Yamashita S, Sakai N, Hirano K, Ishigami M, Maruyama T, Nakajima N, Matsuzawa Y. Roles of plasma lipid transfer proteins in reverse cholesterol transport. Front Biosci 2001;6:D366–D387.|
|47.||Moore BB, Hogaboam CM. Murine models of pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol 2008;294:L152–L160.|
|48.||Moeller A, Ask K, Warburton D, Gauldie J, Kolb M. The bleomycin animal model: a useful tool to investigate treatment options for idiopathic pulmonary fibrosis? Int J Biochem Cell Biol 2008;40:362–382.|
|49.||Chenaud C, Merlani PG, Roux-Lombard P, Burger D, Harbarth S, Luyasu S, Graf JD, Dayer JM, Ricou B. Low apolipoprotein A-I level at intensive care unit admission and systemic inflammatory response syndrome exacerbation. Crit Care Med 2004;32:632–637.|
|50.||Jiao YL, Wu MP. Apolipoprotein A-I diminishes acute lung injury and sepsis in mice induced by lipoteichoic acid. Cytokine 2008;43:83–87.|
|51.||Hsieh GR, Schnickel GT, Garcia C, Shefizadeh A, Fishbein MC, Ardehali A. Inflammation/oxidation in chronic rejection: apolipoprotein A-I mimetic peptide reduces chronic rejection of transplanted hearts. Transplantation 2007;84:238–243.|
|52.||Li Y, Dong JB, Wu MP. Human ApoA-I overexpression diminishes LPS-induced systemic inflammation and multiple organ damage in mice. Eur J Pharmacol 2008;590:417–422.|
|53.||Liao XL, Lou B, Ma J, Wu MP. Neutrophils activation can be diminished by apolipoprotein A-I. Life Sci 2005;77:325–335.|
|54.||Shi N, Wu MP. Apolipoprotein A-I attenuates renal ischemia/reperfusion injury in rats. J Biomed Sci 2008;15:577–583.|
|55.||Kim KD, Lim HY, Lee HG, Yoon DY, Choe YK, Choi I, Paik SG, Kim YS, Yang Y, Lim JS. Apolipoprotein A-I induces IL-10 and PGE2 production in human monocytes and inhibits dendritic cell differentiation and maturation. Biochem Biophys Res Commun 2005;338:1126–1136.|
|56.||Richter AG, McKeown S, Rathinam S, Harper L, Rajesh P, McAuley DF, Heljasvaara R, Thickett DR. Soluble endostatin is a novel inhibitor of epithelial repair in idiopathic pulmonary fibrosis. Thorax 2009;64:156–161.|
|57.||Bargagli E, Olivieri C, Prasse A, Bianchi N, Magi B, Cianti R, Bini L, Rottoli P, Calgranulin B. S100A9) levels in bronchoalveolar lavage fluid of patients with interstitial lung diseases. Inflammation 2008;31:351–354.|
|58.||Huh JW, Kim DS, Oh YM, Shim TS, Lim CM, Lee SD, Koh Y, Kim WS, Kim WD, Kim KR. Is metalloproteinase-7 specific for idiopathic pulmonary fibrosis? Chest 2008;133:1101–1106.|
|59.||Baran CP, Opalek JM, McMaken S, Newland CA, O'Brien JM Jr, Hunter MG, Bringardner BD, Monick MM, Brigstock DR, Stromberg PC, et al. Important roles for macrophage colony-stimulating factor, CC chemokine ligand 2, and mononuclear phagocytes in the pathogenesis of pulmonary fibrosis. Am J Respir Crit Care Med 2007;176:78–89.|