American Journal of Respiratory and Critical Care Medicine

Rationale: Injury to alveolar epithelial cells is central to the pathophysiology of idiopathic pulmonary fibrosis (IPF). An abnormal autoimmune response directed against antigens of the alveolar epithelium may contribute to the disease.

Objectives: To detect circulating autoantibodies (autoAbs) directed against epithelial structures.

Methods: We performed immunoblot by separating human placental amnion extract or alveolar epithelial cell (A549 cell line) proteins on polyacrylamide gels, blotting on nitrocellulose membranes, and incubating with serum from patients with IPF (n = 40) or healthy subjects (n = 40). Proteomic analysis and mass spectrometry characterized the target protein. Inhibition experiments performed with the correspondent recombinant protein confirmed our results.

Measurements and Main Results: We identified IgG autoAbs recognizing a 200-kD protein in the serum of patients with IPF. Proteomic analysis identified this protein as human periplakin (PPL), a component of desmosomes. Anti-PPL Abs were found by immunoblot in both serum and bronchoalveolar lavage in patients with IPF: 16/40 (40%) of them were positive versus none of the control subjects. Immunohistochemistry revealed that PPL was strongly expressed in bronchial and alveolar epithelium, but that PPL exhibited changes in intracellular localization among normal and fibrotic alveolar epithelium. In an alveolar epithelial wound repair assay, an anti-PPL IgG decreased cell migration. Recombinant PPL induced bronchoalveolar lavage T lymphocyte proliferation. Patients with IPF with anti-PPL Abs had a more severe respiratory disease, despite no difference in survival.

Conclusions: We found a new circulating autoAb directed against PPL in patients with IPF, associated with a more severe disease.

Scientific Knowledge on the Subject

The pathophysiology of idiopathic pulmonary fibrosis (IPF) remains elusive and the role of autoimmunity in the disease is debated.

What This Study Adds to the Field

This study demonstrates for the first time the presence of circulating anti-periplakin antibodies in the serum of patients with IPF. The antibodies were also detected in bronchoalveolar lavage fluid and were associated with a more severe disease. Periplakin is strongly expressed in the alveolar epithelium in normal and fibrotic lung.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease, characterized by abnormal fibroproliferation leading to chronic respiratory failure and death. The etiology of IPF is unknown, but a repeated injury to the alveolar epithelium from an unknown trigger, a persistent immuno-inflammatory phase, and a dysregulated tissue repair are generally considered as important mechanisms for IPF development (1).

An autoimmune response in the pathogenesis of IPF is strongly suggested by several data. First, B-cell aggregates are often observed in IPF lungs, organized with activated T lymphocytes and mature dendritic cells, suggesting intense antigen-presentation activity in the lung parenchyma (2, 3). Second, circulating CD4 T cells from patients with IPF exhibit typical characteristics of activation. Indeed, they produce cytokines helping autoantibody production by B cells, and also fibrogenic mediators, such as IL-10, transforming growth factor β-1, or tumor necrosis factor-α (4). Third, CD4 T cells purified from lymph nodes from patients with IPF proliferate when cultured with autologous lung tissue protein extracts (4). The responsible antigen(s) remains to be identified, but several studies point to the alveolar epithelial cell as a possible target of autoimmunity in IPF, as circulating autoantibodies directed toward epithelial cells have been detected by different groups (59).

Cell–cell adhesion is critical for maintaining the integrity of the alveolar epithelium. Cell–cell adhesion is based on different structures, such as desmosomes, hemidesmosomes, and tight junctions. Some protein components of intercellular junctions are targets for autoreactivity in autoimmune blistering cutaneous diseases (10). We hypothesized that those proteins could be a target for autoreactivity in IPF. To test this hypothesis, we developed an immunoblot to detect circulating antibodies directed against epithelial antigens using a placental amnion extract as an epithelial antigen source, as previously published (11, 12). Using proteomic analysis and mass spectrometry, we demonstrate the presence of antibodies directed against periplakin (PPL) in the serum and BAL fluid of patients with IPF. The presence of antibodies correlated with disease severity in a population of 40 patients with IPF. Moreover, we demonstrate that an anti-PPL antibody (Ab) is able to decrease epithelial reparation in vitro and that recombinant PPL induces alveolar T lymphocyte proliferation in some patients, reinforcing the potential role of PPL in the pathophysiology of IPF.

Some of the results of these studies have been previously reported in the form of an abstract (13).


Forty patients with IPF, who did not receive steroids or immunosuppressants, consecutively seen in our center between January 2004 and December 2007, were evaluated. IPF was diagnosed according to the American Thoracic Society/European Respiratory Society criteria (14). Systematic search for autoimmunity, including antinuclear antibodies (titer > 1/160) and rheumatoid factor, was negative. Antineutrophil cytoplasmic antibodies without specificity were detected in two patients. Peripheral blood was obtained by venipuncture at the time of diagnosis of pulmonary fibrosis. The local ethics committee approved the study.

High-resolution computed tomography scans of the chest were reviewed by a senior thoracic radiologist (M.-P.D.) and a senior pulmonologist (B.C.) without knowledge of biological results. A semiquantitative scoring of the extent of fibrosis and ground glass opacities was performed, as previously described by Kazerooni and colleagues (15).

A group of 40 healthy volunteers (5 males, mean age 40 yr) recruited by solicitation from the hospital personnel, a second group of 10 patients (2 males, mean age 58 yr) with connective tissue–associated lung fibrosis (dermatomyositis, n = 2; Sjögren syndrome, n = 5; rheumatoid arthritis, n = 1; systemic sclerosis, n = 1; undifferentiated connective tissue disease, n = 1), and a third group of 10 patients with chronic obstructive pulmonary disease (COPD) (all males, mean age 68 yr; 5 patients were Global Initiative for Chronic Obstructive Lung Disease [GOLD] stage 3 and 5 were stage 4; mean cumulative tobacco exposure was 55 ± 5 pack-years) were evaluated as control subjects for serum analysis. Written informed consent was obtained from all subjects.

Immunoblot Analysis

Venous whole blood was collected onto a vacuum tube without anticoagulant and allowed to clot. After coagulation, sera were obtained after centrifugation (1,700 × g for 10 min at 4°C) and immediately frozen at −80°C until use.

Circulating autoantibodies were searched by immunoblot, using human placental amnion extract as a substrate and anti-human IgG as secondary Ab, as previously described by us and others (11, 12). In some experiments, to determine whether autoantibodies could be detected in the bronchoalveolar lavage (BAL) fluid, the membranes were incubated with BAL fluid supernatant (500 μl) from three patients with IPF and three control patients without lung disease.

In some experiments, we also used cellular extracts from A549 cells as a pulmonary epithelial cell antigenic substrate, after checking in preliminary experiments that A549 cells expressed PPL.

Proteomic Analysis

The 200-kD molecular weight band corresponding to the band observed by immunoblot was analyzed using the matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, as described on the online supplement. The mass spectrometry spectra were searched in combination against the NCBInr and SwissProt databases using the Mascot search engine (see online supplement for detailed protocol).

Inhibition Experiments

We prepared a recombinant full-length PPL (rPPL) using a bacterial expression constructs of PPL from Alasdair Steven (16) (see online supplement for detailed protocol). To confirm that antibodies detected in the serum from patients with IPF recognized PPL, we performed immunoblots with purified rPPL (2 μg) as a substrate with the sera from all patients.

Inhibition experiments were done in four patients with IPF: two samples from each serum were incubated overnight, at 4°C, with an excess of purified rPPL (7 μg) or with buffer only, before performing immunoblots. We considered the experiment as successful when the reactive rPPL band disappeared after absorption.

Epithelial Cell Migration

To analyze cell migration, we used an alveolar epithelial wound repair assay with A549 cells (17). The cells were incubated with a polyclonal anti-PPL Ab (SC-16754; Santa Cruz Biotechnology, Santa Cruz, CA) or the corresponding IgG isotype (GTX27127, Gen Tex Inc., Mundolsheim, France) or with BAL fluid (25% vol/vol) or serum (5%) from patients (positive or negative for the anti-PPL Ab). The detailed protocol is available in the online supplement.

BAL and Blood Lymphocyte Proliferation

We isolated alveolar macrophages and T lymphocytes from BAL fluid as well as blood monocytes and T lymphocytes in five patients with IPF with (n = 3) or without (n = 2) anti-PPL circulating Abs (see online supplement for detailed protocol). We assessed the ability of alveolar macrophages to present rPPL to autologous alveolar T lymphocytes, according to the technique of Todd and Schlossman (18). Briefly, purified alveolar macrophages were “pulsed” with rPPL, γ-irradiated, and then cocultured with purified T lymphocytes before T-cell proliferation was measured by 3H-thymidine incorporation. Similar experiments were performed with autologous blood monocytes and T lymphocytes. Results are expressed as the ratio between T-lymphocytes proliferation induced by 0.1-μg PPL pulsed macrophages/monocytes and proliferation induced by unpulsed cells.

Immunohistochemistry and Immunofluorescence for PPL

Lung surgical biopsies from three patients with IPF without circulating anti-PPL antibodies were immediately frozen in liquid nitrogen and stored at −80°C until use for immunohistochemical studies. Normal lung parenchyma resected during surgical treatment of recurrent pneumothorax from three patients was used as control tissue. At least one section with hyperplastic alveolar epithelium was studied in the fibrotic lung from each IPF sample. Detailed procedures for immunohistochemistry and immunofluorescence analysis are available on the online supplement.

Statistical Analysis

All values were analyzed using GraphPad Prism 5.01. The Mann-Whitney U test was performed. P values less than 0.05 were considered significant. Unless specified, all data are expressed as mean ± SEM. We compared the survival of PPL-positive and PPL-negative patients with IPF with the log-rank test.

Sera of Patients with IPF React with a 200-kD Epithelial Protein

Immunoblot analysis showed that the serum of patients with IPF contained circulating antibodies directed against a main protein from the placental extract, with an apparent molecular weight around 200 kD compatible with the PPL target recognized by the serum of a patient with paraneoplastic pemphigus (Figure 1A). This 200-kD band was detected in the serum from 16 of 40 (40%) patients with IPF, whereas it was never detected in the serum from control healthy subjects or from patients with COPD. The prevalence of anti-PPL positivity in the IPF population (16/40) was significantly higher than in the control population (0/40) (P < 0.0001, Fisher exact test).

As expected, immunoblot with the paraneoplastic pemphigus serum exhibited several bands on this placental amnion extract, corresponding to other members of the plakin family (desmoplakin I/II, 250/215 kD; envoplakin, 210 kD). These bands were never detected with the serum of patients with IPF, healthy control subjects, or patients with COPD.

Immunoblot analysis was also performed with the serum of 10 patients with lung fibrosis in the context of connective tissue diseases. Only one patient with dermatomyositis was positive for the 200-kD band (data not shown).

Finally, we found that the autoantibodies from these patients targeted the same 200-kD protein when A549 cell lysate was used as a substrate (Figure 1B).

PPL Is the Target for Autoreactivity in IPF

To identify the target protein, we performed a proteomic analysis from excised gel bands. Analyzed by mass spectrometry, the significant band was matching to one protein only, with a significant MOWSE (Molecular Weight Search) score (see online supplement). The results obtained with National Center for Biotechnology Information (NCBI) and SwissProt databases characterized this protein as a mammalian protein with a calculated molecular mass of 205 kD corresponding to human PPL with 22% of sequence coverage (NCBI access number: AAD17459; SwissProt access number: O60437).

To confirm that the protein was PPL, we first incubated membranes with a commercially available polyclonal anti-human PPL Ab that exhibited a 200-kD band similar to patients with IPF in immunoblots (Figures 1A and 1B, last lane).

In a second set of experiments, immunoblots were performed using the recombinant human PPL (purified full-length C-terminal PPL) as a substrate. All the sera that were positive with the placental extract substrate also exhibited a strong reactivity with a 200-kD protein (Figure 1C), confirming that PPL was the target of autoimmunity in our patients.

Two of the sera (Figure 1C, patients 1 and 3) showed a weak reaction to a minor additional 125-kD band, that would correspond to a N-terminal truncated form of the recombinant PPL. A similar pattern was observed with the serum from a patient with paraneoplastic pemphigus. The serum of patients with IPF that did not react with placental amnion extract, and the serum from control subjects, did not react with recombinant PPL (Figure 1C, patient 8 and control).

When the serum of patients with IPF was preincubated with recombinant PPL, the reactivity against recombinant PPL disappeared (Figure 1C, patients 1–4), thus confirming the presence of antibodies specifically directed against PPL in the serum from patients with IPF.

We examined the isotype of antibodies in 13 anti-PPL–positive patients. Only six (38%) of them had IgG4 autoantibodies. We also detected anti-PPL IgA antibodies in nine patients and IgM in one (data not shown).

We asked whether the circulating Ab could reach the alveolar lumen. Immunoblot was performed with BAL fluid from patients with IPF (n = 3). BAL fluid also reacted with the 200-kD recombinant PPL, whereas the BAL fluid from control subjects was negative (Figure 1D). These results demonstrate that anti-PPL antibodies are present in the epithelial lining fluid of the alveolus as well as in the serum.

PPL Is Expressed in the Normal and Fibrotic Human Lung

We detected the expression and localization of PPL in the lung by immunohistochemistry, in three IPF lung samples and three control lung samples. In the normal human lung, PPL expression was limited to epithelial cells. PPL was strongly expressed in bronchial epithelial cells and in alveolar epithelial cells (both type I and type II pneumocytes) (Figure 2, panel 1A). In the fibrotic lung, a similar pattern of expression was observed, with a strong expression of PPL in the hyperplastic alveolar epithelium in areas of lung fibrosis (Figure 2, panel 1B). This pattern of expression was observed in all IPF samples studied.

When analyzed by immunofluorescence (Figure 2, panel 2), PPL expression was localized to the lateral and apical side of bronchial epithelial cells, in both normal (Figure 2, panel 2A) and fibrotic (Figure 2, panel 2C) lungs. By contrast, the distribution of PPL in the alveolar epithelium was very different in normal lung and fibrotic lung. Indeed, in control lungs, the protein was localized to the lateral side of the pneumocytes and colocalized with β-catenin (Figure 2, panel 2B), whereas in fibrotic lungs, PPL was diffusely detected in the cytoplasm as well at the apex of the cells in the hyperplastic alveolar epithelium (Figure 2, panel 2D). This pattern of distribution was observed in all IPF lung samples studied.

An Anti-PPL Antibody Inhibits Epithelial Cell Migration In Vitro

In an alveolar epithelial wound repair assay, incubation of the cells with 10 μg/L anti-PPL IgG reduced cell migration and wound closure, whereas the control isotype had no effect (Figure 3). This effect was not observed with 1 μg/ml anti-PPL IgG. In the same assay, we evaluated the wound repair of the cells incubated with BAL fluid or with serum from patients with IPF. We did not observe any difference in wound closure according to the anti-PPL Abs status (positive or negative) (data not shown). In the same experiments, we checked for a cytotoxic effect of the anti-PPL Ab on the A549 cell line. As compared with anti-PPL–negative serum, the anti-PPL–positive serum did not significantly alter the morphology, the proliferation rate, and the glucose consumption rate of A549 cells (data not shown).

PPL-induced Proliferation of BAL T Lymphocytes

When autologous T cells were cocultured with alveolar macrophages pulsed with 0.1-μg rPPL, we observed a marked increase in T-lymphocyte proliferation as compared with T cells cocultured with macrophages that were not exposed to rPPL, in four of five patients studied (Figure 4). By contrast, rPPL-pulsed blood monocytes obtained from the same patients at the same time did not significantly modify T-cell proliferation as compared with unpulsed blood monocytes. Although there was minimal incorporation of 3H-thymidine by T lymphocytes, similar results were obtained in the absence of CD3 coating (data not shown). However, PPL-induced BAL T-lymphocyte proliferation was not restricted to anti-PPL Ab-positive patients, as a significant proliferation was observed in one of the two anti-PPL Ab-negative patients (Figure 4, patient 3).

Antibodies Directed against PPL Are Associated with a More Severe Disease

Patients with IPF with or without antibodies directed against PPL have similar demographic characteristics and tobacco smoke habits (Table 1). As anti-PPL antibodies have been associated with paraneoplastic pemphigus, patients were specifically checked for the presence of skin or mucosal lesions. No lesion was detected and no skin disease developed at follow-up. A skin biopsy was performed in three anti-PPL–positive patients; no abnormality was detected and direct immunofluorescence was negative.


Anti-Periplakin Positive (N = 16)

Anti-Periplakin Negative (N = 24)

P Value
Age at diagnosis, yr72.5 ± 270.4 ± 20.5
Male, %81750.5
Smokers or ex-smokers, %81700.5
Pack-years27 ± 626 ± 50.7
Pulmonary function tests
 FEV1, % predicted71 ± 485 ± 30.06
 TLC, % predicted56 ± 368 ± 30.008
 FVC, % predicted66 ± 379 ± 50.02
 DlCO, % predicted32 ± 347 ± 50.03
 PaO2 room air, mm Hg62 ± 473 ± 20.05
Bronchoalveolar lavage
 Total cellularity, 103 cells/ml184 ± 40206 ± 270.2
 % Macrophages76 ± 369 ± 30.1
 % Lymphocytes7.5 ± 1.58 ± 10.7
 % Neutrophils11 ± 219 ± 30.04
 % Eosinophils7 ± 23 ± 0.80.05
Treatment, % patients
 Oral steroids81450.046

Definition of abbreviation: DlCO = diffusing capacity for carbon monoxide.

Data are expressed as mean ± SEM unless otherwise noted.

The detection of anti-PPL antibodies was associated with a more severe respiratory disease, as assessed by a more severe restrictive pattern on lung function tests, a lower diffusing capacity of carbon monoxide, and a lower PaO2 at rest (Table 1). Among anti-PPL IgG-positive patients, six of them had IgG4 autoantibodies, nine of them had IgA autoantibodies, and one had IgM. The clinical characteristics of these patients were not different from other IgG-positive patients.

BAL fluid cellularity was similar in the two groups, but the detection of anti-PPL antibodies was associated with a higher percentage of eosinophils and lower percentage of neutrophils (Table 1). Quantitative analysis of high-resolution computed tomography of the lung showed that ground glass score and fibrosis score were similar in the two groups. Survival was not different among the two groups (P = 0.22, see Figure E1 in the online supplement).

We present evidence that pulmonary autoimmunity is a component of IPF and have identified PPL as a target autoantigen. Circulating IgG autoantibodies directed against PPL, a protein expressed in bronchial and alveolar epithelium, were found in the serum of 40% of patients with IPF, in 1 of 10 of patients with connective tissue disease–associated lung fibrosis, in 0 of 10 patients with GOLD stage 3–4 COPD, and in 0 of 40 healthy control subjects. The detection of anti-PPL Ab was associated with a more severe respiratory disease in patients with IPF.

PPL is a small protein from the plakin family, closely related to envoplakin and localized to desmosomes and intermediate filaments (19). PPL has been mainly studied in keratinocytes, but its expression is wide among tissues, especially in lung, heart, kidney, pancreas, and intestine (20). Anti-PPL antibodies have been identified in patients with paraneoplastic pemphigus; however, they are always associated in this situation with antibodies directed against other members of the plakin family (especially envoplakin, and/or desmoplakin I and II and BP230), an unidentified 170-kD protein, and sometimes against two members of the cadherin family (desmoglein 1 and desmoglein 3) (21, 22), which were never detected in the patients with IPF that we studied. It must be noted that none of our patients with IPF had mucosal or cutaneous lesions. Three patients had skin biopsies, without any IgG deposition observed by direct immunofluorescence. The lack of other autoantibodies possibly explains why anti-PPL antibodies did not associate with cutaneous disease in patients with IPF. However, we can also imagine that the PPL epitopes that are recognized by paraneoplastic pemphigus sera and by IPF sera could be different.

One important question is whether the anti-PPL Ab could play a pathogenic role in lung fibrosis. Our in vitro studies did not directly demonstrate that a patient's blood or BAL anti-PPL Abs influence epithelial repair. However, we observed that a commercially available polyclonal anti-PPL Ab inhibits alveolar epithelial repair in vitro, whereas the isotypic control Ab has no effect; this provides indirect evidence that anti-PPL Ab could modulate alveolar repair. The lack of significant effect of BAL or serum from anti-PPL–positive patients with IPF as compared with anti-PPL–negative patients may be because of lower concentrations of anti-PPL Abs in these fluids or because of multiple other molecules potentially interfering with epithelial repair during IPF.

In paraneoplastic pemphigus, the circulating antibodies are also considered to have a direct pathogenic effect on squamous epithelia (2325). Furthermore, serum of patients with paraneoplastic pemphigus reacts with rat alveolar and bronchial epithelium (26, 27), suggesting that anti-plakin antibodies could be involved in the tracheobronchial injury observed during paraneoplastic pemphigus (27, 28).

In the lung, we show in this work that PPL is constitutively expressed in both bronchial and alveolar epithelia and that the intracellular distribution of PPL is modified in the hyperplastic alveolar epithelium of fibrotic lungs as it becomes strongly expressed at the apical surface of the cells. As we show that the anti-PPL Ab is present in the epithelial lining fluid of seropositive patients, one could hypothesize that a direct interaction of the Ab with its cellular target occurs in situ with possible deleterious consequences on alveolar epithelium homeostasis and repair. Indeed, PPL interacts with intermediate filaments in the cell (29) and is required for the reorganization of keratin intermediate filaments network at the wound edge of simple epithelial cell monolayers and for the correct closure of an experimental wound (30). The C-terminal domain of PPL is also linked to several intracellular proteins, such as protein kinase B ATK1/PKB (31) or FcγRI (32), that may let us hypothesize that changes in the protein structure could induce changes in cellular functions.

In the present study, all the IPF sera tested reacted with the full-length recombinant PPL, but two patients recognized also an additional minor band of about 125 kD, which could correspond to a partial N-terminal truncated form of the recombinant PPL. Previous investigations demonstrated that antibodies found in paraneoplastic pemphigus are directed against various regions of PPL, excepting the C-terminal homologous domain (26, 33). Further work is needed to identify the domains recognized by the anti-PPL antibodies in IPF.

Several autoantibodies have been described, using different substrates, in the serum from patients with IPF in stable condition (46) or during exacerbation (34), directed against different cellular components: cytokeratin-8 (8, 35), cytokeratin-19 (9), vimentin (7), DNA topoisomerase II, annexin I (34), or collagen (36). Circulating IgG antibodies binding to pulmonary epithelial cells have been observed in patients with IPF, but their target was not identified (5). It must be noted that our immunoblots performed with A549 cell lysates exhibit quite similar patterns, confirming that autoreactivity during IPF is directed against multiple targets. However, none of these antibodies has been associated with the severity of IPF, contrary to the anti-PPL antibodies that we have detected. This suggests that PPL antibodies may contribute directly to IPF pathophysiology. Assessing whether the detection of anti-PPL Abs may be a potential biomarker for disease severity and/or progression will require further longitudinal studies.

Interestingly, among anti-PPL IgG-positive patients, six of them had IgG4 autoantibodies, nine of them had IgA autoantibodies, and one had IgM. The clinical characteristics of these patients were not different from other IgG-positive patients. In autoimmune cutaneous bullous diseases, the subtypes of autoantibodies are associated with different clinical phenotypes. For instance, during active pemphigus, IgG4 represents the major IgG subset of pathogenic anti-desmoglein 3 autoantibodies (37). By contrast, although IgA autoantibodies directed against several dermoepidermal targets are commonly found in autoimmune bullous diseases (38), their role has not been yet clearly established.

Our group has recently demonstrated that a neolymphoidogenesis occurs in the lung in patients with IPF (2, 3). The ectopic localization of immune cells in the lung could promote autoimmunity toward epithelial antigens, such as PPL, as suggested by the evidence of PPL-induced BAL lymphocyte proliferation in some patients with IPF. Interestingly, one patient with lung fibrosis associated with dermatomyositis demonstrated the 200-kD band on immunoblot, demonstrating that anti-PPL autoreactivity could arise in nonidiopathic fibrotic lung diseases.

In the present study, patients were classified on the presence or the absence of autoantibodies in the 1/25 diluted serum. The titration of these autoantibodies could give additional information, in relation to severity for example. We are planning to develop an ELISA that would allow quantifying the antibodies and better understanding their evolution during the disease course.

In conclusion, this study is the first demonstration of the presence of anti-PPL antibodies in serum and BAL fluid in patients with IPF, supporting previous studies implicating an autoimmune response in the pathogenesis of IPF. Anti-PPL antibodies have the potential to interfere with alveolar repair and are associated with a more severe disease. The prognosis value of anti-PPL antibodies during IPF needs to be prospectively evaluated in a larger cohort.

The authors thank the Institut Fédératif de Recherche 02 (IFR 02, Université Paris Diderot, Paris, France, for technical assistance in expression and purification of recombinant PPL. They also thank Pr. Alasdair C. Steven and Dr. Shyh-Ing Jang (Laboratory of Skin Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institute of Health, Bethesda, MD) for providing the plasmid pET-PPL. They also thank Céline Champagnat for technical contribution in immunoblotting experiments and Dr. Gabriel Thabut for last-minute help.

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Correspondence and requests for reprints should be addressed to Bruno Crestani, M.D., Ph.D., Service de Pneumologie A, Hôpital Bichat, 46 rue Henri Huchard, 75877 Paris cedex 18, France. E-mail:


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