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Published ahead of print on August 28, 2003, doi:10.1164/rccm.200212-1514OC
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American Journal of Respiratory and Critical Care Medicine Vol 168. pp. 1156-1161, (2003)
© 2003 American Thoracic Society

Defect of Hepatocyte Growth Factor Secretion by Fibroblasts in Idiopathic Pulmonary Fibrosis

Sylvain Marchand-Adam, Joëlle Marchal, Murielle Cohen, Paul Soler, Bénédicte Gerard, Yves Castier, Guy Lesèche, Dominique Valeyre, Hervé Mal, Michel Aubier, Monique Dehoux and Bruno Crestani

INSERM unit 408, Faculté Xavier Bichat; and Service de Pneumologie, Laboratoire de Biochimie A, and Laboratoire de Biochimie Hormonale et Génétique, Hôpital Bichat, Assistance Publique-Hôpitaux de Paris, Paris; Service de Chirurgie Thoracique et Vasculaire and Service de Pneumologie, Hôpital Beaujon, Assistance Publique-Hôpitaux de Paris, Clichy; and Service de Pneumologie, Hôpital Avicenne, Bobigny, France

Correspondence and requests for reprints should be addressed to Bruno Crestani, M.D., Service de Pneumologie, Hôpital Bichat, 16 rue Henri Huchard, 75877 Paris Cedex 18, France. E-mail: bruno.crestani{at}bch.ap-hop-paris.fr


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatocyte growth factor (HGF) is a growth factor that protects alveolar epithelial cells from pulmonary fibrosis in various animal models. We compared in vitro HGF production by human lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF, n = 8) and from control subjects (n = 6). Basal HGF secretion by IPF fibroblasts was decreased by 50% when compared with control fibroblasts (p < 0.05). HGF was secreted mainly in the cleaved mature form, both in IPF and control fibroblasts. HGF messenger RNA levels were reduced in IPF fibroblasts. Prostaglandin (PG) E2 secretion by IPF fibroblasts was low when compared with control subjects (p < 0.05). After the addition of PGE2 (10-6 M) or dibutyryl cyclic AMP (10-3 M), HGF secretion by IPF fibroblasts reached the level of control subjects. Inhibition of PGE2 synthesis with indomethacin reduced HGF secretion by control fibroblasts but had no effect on IPF fibroblasts. HGF secretion by control fibroblasts was also slightly inhibited by transforming growth factor (TGF)-ß1 and stimulated by anti–TGF-ß antibody, whereas both agents had no effect on IPF fibroblasts. Our results demonstrate a defect in HGF production by IPF fibroblasts that seems secondary to a defect in PGE2 secretion.

Key Words: indomethacin • pulmonary alveoli • transforming growth factor ß • usual interstitial pneumonia

Pulmonary fibrosis is characterized by fibroblast proliferation and extracellular matrix remodeling, which result in irreversible distortion of the lung architecture. Alveolar epithelial injury and delayed repair are now considered a critical event in lung fibrogenesis (1). Epithelial–mesenchymal interactions are a central component of the lung repair process, in which the integrity of the alveolar epithelium is essential to limit activation and proliferation of fibroblasts (2), whereas pulmonary fibroblasts are important sources of cytokines, growth factors, and mediators that control Type II pneumocyte proliferation and differentiation.

Data indicate perturbations of epithelial–mesenchymal interactions in pulmonary fibrosis. Indeed, apoptotic alveolar epithelial cells are observed adjacent to the site of fibroproliferation in vivo (3), and fibroblasts from fibrotic lung induce the apoptosis of alveolar epithelial cells in vitro whereas normal fibroblasts do not (4–6). We hypothesized that the delayed epithelial repair that is central to pulmonary fibrosis might be further enhanced by the dysregulated secretion of epithelium-directed growth factors produced by fibroblasts from fibrotic lung.

Among the growth factors produced by fibroblasts, hepatocyte growth factor (HGF) plays a key role in alveolar homeostasis (7). HGF is secreted by pulmonary fibroblasts as a single-chain molecule that is cleaved proteolytically to an active heterodimer. HGF has been demonstrated to increase migration (8) and proliferation (9–11) of Type II pneumocytes in vitro and in vivo, and to limit lung fibrosis in vivo after bleomycin injury in rodents when given intravenously (12) or intratracheally (13). HGF acts through the MET receptor, a membrane-bound tyrosine kinase (14). HGF production by fibroblasts is regulated by various mediators such as prostaglandins (15), cytokines (16), and hormones (17). Despite the important functions of HGF in pulmonary homeostasis, nothing is known concerning HGF secretion by fibroblasts in pulmonary fibrosis.

Therefore, the aim of this study was (1) to compare in vitro HGF production by human lung fibroblasts from control subjects and from patients with idiopathic pulmonary fibrosis (IPF), and (2) to evaluate the regulation of HGF production by lung fibroblasts in pulmonary fibrosis. Some of the results of these studies have been previously reported in the form of an abstract (18).


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
For details, see the online supplement.

This study was approved by the ethics committee of Paris-Bichat University Hospital.

Patients with Lung Fibrosis
Fibroblasts were derived from lung tissue samples from eight patients with IPF. Lung samples were obtained by open lung biopsy (n = 3) or at the time of lung transplantation (n = 5). IPF was diagnosed according to American Thoracic Society–European Respiratory Society consensus criteria (19), including the characteristic morphology of usual interstitial pneumonia. Patients (7 men and one woman) were, on average, 58 years of age (range, 44–69 years of age). Seven were ex-smokers and one was a never-smoker. Time elapsed to biopsy after the beginning of symptoms was 55 months (range, 4–123 months). All patients showed a restrictive functional pattern (see Table E1 in the online supplement). At the time of lung biopsy, three patients were treated with oral corticosteroids, associated with azathioprin in one patient.

Control Patients
Fibroblasts were derived from lung samples from six patients (three men and three women) undergoing lung surgery for removal of a primary lung tumor. Normal lung from a noninvolved segment, remote from the solidary lesion, was obtained. The median age was 63.5 years (range, 42–70 years). Three patients were never-smokers, two were ex-smokers, and one was an active smoker.

Culture of Fibroblasts
Human lung fibroblasts were cultured from lung explants according to Akamine and colleagues (20). Fibroblasts were cultured with Dulbecco's modified Eagle's medium–10% fetal calf serum supplemented with antibiotics. The cells were used at Passage 5.

All cell cultures were evaluated immunohistochemically at Passage 5. Essentially 100% of the cells were fibroblasts as indicated by strong labeling with anti–prolyl-4-hydroxylase, anti-vimentin, and anti-CD90 monoclonal antibodies (21). Seven of the eight IPF fibroblast lines and two of the six control subject fibroblast lines contained 5 to 10% {alpha}-smooth muscle actin-positive cells, indicating a certain degree of differentiation toward a myofibroblast phenotype. Staining with anti–smooth muscle myosin heavy chain-1, anti-cytokeratin, and anti-CD31 antibodies was always negative, indicating that the cultures did not contain smooth muscle cells or epithelial or endothelial cells.

Regulation of HGF Production by Fibroblasts
Fibroblasts were grown to confluence in 12-well tissue culture plates and then washed twice in phosphate-buffered saline (PBS) and cultured in 1 ml of serum-free Dulbecco's modified Eagle's medium, either under basal conditions or exposed to various pharmacologic agents: 10-6 M prostaglandin (PG) D2, 10-6 M PGE2, 10-3 M dibutyryl cyclic AMP (dbcAMP), 2 x 10-7 M phorbol myristate acetate (PMA), 10-5 M dexamethasone, or indomethacin (2.5 µg/ml). In further experiments, cells from control subjects (n = 6) and patients with IPF (n = 5) were cultured with increasing concentrations of PGE2 (10-9 to 10-6 M). In some experiments (six IPF and five control subject cultures), the cells were cultured with human TGF-ß1 (10 ng/ml), anti–TGF-ß neutralizing antibody (1 µg/ml), or nonspecific mouse IgG (1 µg/ml).

After an 18-hour incubation period, the supernatants were saved for HGF determination. The protein content of the cell monolayer was measured by the Bio-Rad (Hercules, CA) protein assay.

Determination of HGF and PGE2 Concentrations in Fibroblast Supernatants
HGF was measured in fibroblast supernatants with an HGF Quantikine ELISA kit (R&D Systems, Madison, WI). Basal HGF concentrations were expressed as picograms of HGF per microgram of protein in the cell monolayer per milliliter of medium.

PGE2 was measured in nonstimulated fibroblast supernatants with a PGE2 high-sensitivity ELISA kit (R&D Systems). PGE2 concentrations were expressed as picomoles per microgram of protein in the cell monolayer

HGF Western Blotting
Fibroblast supernatants and recombinant human HGF (rh-HGF, a mixture of pro-HGF and mature HGF, according to the manufacturer) were examined by Western blotting as previously described (22).

Quantitative Analysis of HGF Messenger RNA
Fibroblasts were analyzed by reverse transcriptase real-time polymerase chain reaction (RT-PCR) to quantify HGF messenger RNA (mRNA). Transcripts of porphobilinogen deaminase (PBGD) served as endogenous RNA controls (see Table E2 in the online supplement). The results were expressed as HGF:PBGD mRNA ratios (six IPF and five control subjects).

Statistical Analysis
All data were expressed as median (range) values. Differences between IPF and control subject fibroblasts were determined using the Mann–Whitney U test. To compare the effects of various agents on baseline conditions, we used the Wilcoxon paired nonparametric test for group comparisons. Correlations were assessed with the Spearman rank-order test. A p value less than 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Baseline HGF Secretion by Fibroblasts
HGF was detected in all fibroblast supernatants. HGF concentrations were lower in IPF fibroblast supernatants (23.4 pg/µg protein per milliliter [range, 3.2–31.3]) than in control fibroblasts (49.4 pg/µg protein per milliliter [range, 28.7–127.1]; p < 0.05) (Figure 1) . Western blot analysis (Figure 2) demonstrated that HGF in fibroblast supernatants was mainly in the cleaved mature form (presence of the 69-kD {alpha} chain), both in IPF and control fibroblasts.



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  		Figure 1. Hepatocyte growth factor (HGF) concentrations (top) and HGF:porphobilinogen deaminase (PBGD) messenger RNA (mRNA) ratios (bottom) were lower in idiopathic pulmonary fibrosis (IPF) fibroblasts than in control subject fibroblasts. Individual values and medians (bars) are shown. *p < 0.05 compared with control subject fibroblasts.

 


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  		Figure 2. Representative Western blot analysis of HGF in fibroblast supernatants taken from one patient with IPF and one control patient and cultured under basal conditions. Recombinant human HGF (a mixture of pro-HGF and mature HGF) was loaded and blotted in parallel. The molecular masses of protein standards are indicated. Similar results were obtained in all samples studied. HGF was in the cleaved mature form as evidenced by detection of the 69-kD {alpha} chain.

 
HGF mRNA was detected in all lung fibroblasts. The HGF:PBGD mRNA ratio was lower in IPF fibroblasts (0.007 [range, 0.002–0.290]; n = 6) than in control fibroblasts (0.327 [range, 0.221–0.405], n = 5; p = 0.02) (Figure 1).

HGF concentrations did not correlate with any of the lung function test results either in patients or in control subjects, or in the whole group. Among IPF fibroblasts, in vitro HGF secretion was not different in corticosteroid-treated patients.

Defect of HGF Secretion by IPF Fibroblasts: Effect of PMA
PMA increased HGF secretion in all control subjects whereas there was no significant response in IPF fibroblasts (Figure 3) .



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  		Figure 3. HGF concentrations in fibroblast supernatants taken from patients with IPF (n = 5) and control subjects (n = 5) and cultured under basal condition or exposed to 2 x 10-7 M phorbol myristate acetate (PMA), expressed as pg/µg protein/ml. Individual values and medians (bars) are shown. *p < 0.05 compared with basal control subject fibroblasts; **p < 0.01 compared with PMA-stimulated control subject fibroblasts.

 
Defect of HGF Secretion by IPF Fibroblasts: Role of PGE2
IPF fibroblasts secrete low levels of PGE2 (23, 24), a prostaglandin known to stimulate HGF secretion in some fibroblast cell lines (15). To determine whether decreased HGF production by IPF fibroblasts was secondary to decreased PGE2 production, we cultured IPF and control fibroblasts with 10-6 M PGE2; 10-6 M PGD2; or 10-3 M dbcAMP, a stable analog of cAMP, a second messenger of PGE2; indomethacin (2.5 µg/ml), a cyclooxygenase inhibitor; or 10-5 M dexamethasone, a potent inhibitor of prostaglandin synthesis.

In control fibroblasts, inhibition of prostaglandin production with indomethacin decreased HGF production to the level observed with IPF fibroblasts (17.5 pg/µg protein per milliliter [range, 12.9–32.1]; p < 0.05 versus baseline) (Figure 4) whereas it did not modify HGF production by IPF fibroblasts (9.9 pg/µg protein per milliliter [range, 2.4–34.5]; p = 0.30) (Figure 5) . Similarly, dexamethasone inhibited HGF production by control subject fibroblasts but did not modify HGF secretion by IPF fibroblasts (Figures 4 and 5). Addition of PGE2 induced a 3-fold increase of HGF production by IPF fibroblasts (p < 0.05). PGE2-stimulated HGF secretion by IPF fibroblasts reached the level of baseline HGF secretion of control fibroblasts. Dibutyryl cyclic AMP mimicked the effect of PGE2 (42.5 pg/µg protein per milliliter [range, 3.5–90.0]; p < 0.05 compared with baseline). By contrast, PGD2 had no effect.



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  		Figure 4. HGF concentrations in control fibroblast supernatants (n = 5) cultured under basal conditions or exposed to 10-6 M prostaglandin (PG) D2, 10-6 M PGE2, 10-3 M dbcAMP, 10-5 M dexamethasone (Dexa), or indomethacin (indo, 2.5 µg/ml), expressed as pg/µg protein/ml. Individual values and medians (bars) are shown. {dagger}p < 0.05 compared with basal conditions.

 


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  		Figure 5. HGF concentrations in IPF fibroblast supernatants (n = 8) cultured under basal conditions or exposed to 10-6 M PGD2, 10-6 M PGE2, 10-3 M dbcAMP, 10-5 M dexamethasone (dexa), or indomethacin (indo, 2.5 µg/ml), expressed as pg/µg protein/ml. Individual values and medians (bars) are shown. {dagger}p < 0.05 compared with basal conditions.

 
Dose–response experiments showed that all control fibroblasts responded to 10-8 to 10-6 M PGE2. PGE2 at 10-9 M stimulated HGF secretion in four of six control cell lines (Figure 6) . All IPF fibroblasts increased HGF secretion with 10-6 M PGE2, but the response to lower concentrations of PGE2 was weak and heterogeneous.



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  		Figure 6. HGF concentrations in fibroblast supernatants cultured under basal conditions or exposed to increasing concentrations of PGE2 (10-9 to 10-6 M) and expressed as pg/µg protein/ml. Data are from six control subjects (top) and five patients with IPF (bottom). Individual values and medians (bars) are shown. {dagger}p < 0.05; {dagger}{dagger}p < 0.01 when compared with basal conditions.

 
To confirm the lack of PGE2 secretion in IPF fibroblasts, PGE2 concentration was measured in fibroblast supernatants. Baseline PGE2 concentration was profoundly reduced in IPF fibroblasts (4 pmol/µg protein per liter [range, 1–30]) when compared with control fibroblasts (96 pmol/µg protein per liter [range, 6–190]) (p < 0.05) (Figure 7) . HGF concentration did not correlate with PGE2 concentration ({rho} = 0.35, p = 0.25).



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  		Figure 7. PGE2 concentrations (pmol/µg protein/L) in fibroblast supernatants taken from patients with IPF (n = 8) and control subjects (n = 5) and cultured under basal conditions. Individual values and medians (bars) are shown. *p < 0.05 compared with control subject fibroblasts.

 
Defect of HGF Secretion by IPF Fibroblasts: Role of TGF-ß1
As TGF-ß1 has been shown to decrease HGF secretion by lung fibroblasts (17), we asked whether an autocrine loop involving TGF-ß1 could explain the inhibition of HGF secretion by IPF fibroblasts.

TGF-ß1 (10 ng/ml) slightly inhibited HGF secretion by control subject fibroblasts (16.7 pg/µg protein per milliliter [range, 12.8–29.5] versus 23.4 pg/µg protein per milliliter [range, 13.2–29.9] under basal conditions; p < 0.05) (Figure 8) . Inhibition of endogenous TGF-ß with a neutralizing anti–TGF-ß antibody slightly increased HGF secretion by control fibroblasts whereas it had no effect on IPF fibroblasts.



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  		Figure 8. HGF concentrations in IPF (n = 8) and control (n = 5) fibroblast supernatants cultured under basal condition or exposed to human TGF-ß1 (10 ng/ml) or anti–TGF-ß neutralizing antibody (1 µg/ml). HGF concentrations are expressed as pg/µg protein/ml. Individual values and medians (bars) are shown. {dagger}p < 0.05 when compared with basal conditions.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The main findings of this study are (1) that human lung fibroblasts from patients with IPF have a decreased capacity to secrete HGF in vitro when compared with control fibroblasts, and (2) that exogenous PGE2 increases HGF secretion to the level of control fibroblasts whereas PGD2, PMA, and anti–TGF-ß have no effect.

Fibroblasts plays a critical role in the complex repair process that follows lung injury and have been implicated in the pathophysiology of lung fibrosis (1). We studied HGF secretion by pulmonary fibroblasts isolated from normal and fibrotic lung by the explant technique. Fibroblast populations were heterogeneous as assessed by immunohistochemical labeling. In particular, the majority of our IPF fibroblast lines contained 5–10% myofibroblasts as assessed by the expression of {alpha}-smooth muscle actin. Myofibroblasts constitute one fibroblast subpopulation and are usually increased in human and experimental lung fibrosis (25). However, because myofibroblasts constitute a minority of the cells that we studied in vitro, it is unlikely that the reduced expression of HGF by pulmonary fibroblasts isolated from fibrotic lungs was specifically linked to the myofibroblast phenotype.

Some of the fibroblast cultures were obtained from patients treated with low doses of corticosteroids at the time of lung sampling. Steroid treatment could not explain the inhibition of HGF production observed in fibroblasts from pulmonary fibrosis because HGF secretion was not different in fibroblasts from patients treated or not treated with steroids.

To our knowledge, this is the first study of HGF production by IPF fibroblasts. We observed that the HGF concentration in IPF fibroblast supernatants was reduced by 50% when compared with control fibroblasts. Our results suggest that the difference was transcriptional because the steady state HGF mRNA level was lower in IPF fibroblasts than in control fibroblasts as assessed by quantitative RT-PCR. We also demonstrated that HGF was secreted in its cleaved biologically active form in the supernatants of control and IPF fibroblasts as assessed by Western blotting.

We observed that indomethacin, a cyclooxygenase inhibitor, strongly decreased HGF production in control fibroblasts. This suggests that endogenous prostaglandins modulate the basal level of HGF secretion by normal pulmonary fibroblasts in vitro. Several cytokines and inflammatory mediators have been shown to modulate HGF expression by fibroblasts in vitro. Specifically, prostaglandins act as inducers of gene expression of HGF. Matsumoto and colleagues showed that the addition of prostaglandins to cultures of human skin fibroblasts and Medical Research Council (MRC)-5 human embryonic lung fibroblasts led to a marked induction of the production of HGF, with a concomitant increase in HGF mRNA content (15). The effect of prostaglandins was different in the two cell types. In skin fibroblasts, PGE1 and PGE2 analogs were the most potent in stimulating HGF production, over 50-fold, whereas PGD2 and PGI2 were less potent (15). In MRC-5 lung fibroblasts, PGE1 and PGE2 analogs were the most potent in stimulating HGF production (3-fold) whereas PGD2 and PGI2 had no effect (15). In that study, PGE2 activated HGF production by a protein kinase A-dependent pathway (15). Because PGE2 is the major eicosanoid product of lung fibroblasts (26), PGE2 may play a key role in the autocrine–paracrine control of HGF expression by lung fibroblasts.

Inhibition of prostaglandin synthesis with indomethacin had no effect on HGF production by IPF fibroblasts, whereas addition of PGE2 increased HGF secretion by IPF fibroblasts to the level of control fibroblasts. Lung fibroblasts isolated from patients with IPF have been shown previously to present a defect in cyclooxygenase-2 expression and a failure in their capacity to synthesize PGE2 (23, 24). Similarly, lung fibroblasts isolated from rats with bleomycin-induced lung fibrosis have a diminished capacity to secrete PGE2 (27). In the present study, we confirmed these data because PGE2 secretion by IPF fibroblasts was 10-fold lower than that by control fibroblasts. More importantly, the exogenous addition of PGE2, but not PGD2, to IPF fibroblast cultures increased HGF production to the level of control subjects. As expected, dbcAMP mimicked the effects of PGE2, suggesting that PGE2 acted through the stimulation of a protein kinase A-dependent pathway as previously shown by Matsumoto and colleagues (15). PMA is a known inducer of COX-2 expression. The difference between control subject and IPF fibroblasts was even greater after PMA stimulation, because we observed that PMA did increase HGF secretion by control fibroblasts but had no effect on IPF fibroblasts. Altogether, our results support the existence of an autocrine–paracrine regulation of HGF production in lung fibroblasts mediated by PGE2 and its disruption in IPF fibroblasts. Because HGF has been shown to induce COX-2 in mouse fibroblasts (28), it is possible that the lack of HGF contributes to the lack of PGE2.

Our data suggest that IPF fibroblasts are not only poor producers of PGE2 but also poor responders to exogenous PGE2. Indeed, whereas all control fibroblasts increased HGF secretion when stimulated with 10-8 M PGE2, the response of IPF fibroblasts cultures to 10-8 M PGE2 was weak and heterogeneous. This could contribute to the lack of correlation between HGF and PGE2 concentrations, either in control subjects or in patients with IPF.

Several lines of evidence indicate that PGE2 is an anti-fibrotic mediator in the lung. PGE2 has potent inhibitory effects on fibroblast proliferation (29), collagen production (30), and fibroblast-to-myofibroblast transition (31). PGE2 inhibits TGF-ß-stimulated increases in {alpha}1-collagen and connective tissue growth factor mRNAs (32, 33) and downregulates the expression of the PDGF {alpha}-receptor subtype (34). Cox-2-/- mice have a reduced capacity of PGE2 secretion and are susceptible to the development of pulmonary fibrosis after exposure to intratracheal bleomycin (24) or vanadium pentoxide (35). Our data support the view that part of the protective effect of PGE2 on pulmonary fibrosis occurs through the stimulation of HGF production by pulmonary fibroblasts. HGF acts as an antifibrotic molecule. In vivo, the provision of HGF limits lung fibrosis after bleomycin injury in rodents when given intravenously (12), intratracheally (13), or after the transfer of naked plasmid DNA (36), even several days after the insult. HGF promotes repair of the alveolar epithelium. Indeed, HGF has been identified as a mitogen (9–11), motogen (8), and morphogen for alveolar epithelial cells. HGF is also able to protect cells from apoptosis in vitro (37). Reduction of HGF secretion by fibroblasts from fibrotic lung may contribute to their capacity to induce the apoptosis of alveolar epithelial cells in vitro whereas normal fibroblasts do not (4), an important point because apoptotic alveolar epithelial cells are observed adjacent to the site of fibroproliferation in vivo in the fibrotic lung (3).

We observed that a neutralizing anti–TGF-ß1 antibody slightly increased HGF concentration in control fibroblast supernatants but not in IPF fibroblast supernatants. This suggests that endogenous TGF-ß1 inhibits HGF production by control lung fibroblasts, but has no effect on IPF fibroblasts. Matsumoto and colleagues showed that TGF-ß1 inhibited HGF synthesis by MRC-5 lung fibroblasts in a dose-dependent manner (17). Maximal inhibition was 60–70%. TGF-ß1 acted at the posttranscriptional level (38). The effect of TGF-ß1 on HGF secretion by IPF fibroblasts has not been previously tested, to our knowledge. In the literature, the response of IPF fibroblasts to TGF-ß1 appears to be heterogeneous. For example, TGF-ß1 increased PGE2 secretion by some fibroblast cell lines whereas it decreased PGE2 secretion by other IPF fibroblast lines (23).

The reduction of HGF secretion by pulmonary fibroblasts in vitro apparently contrasts with the observation that in patients with IPF, bronchoalveolar lavage fluid levels of HGF are elevated (39–41). However, the nature of the HGF-producing cells in pulmonary fibrosis remains a matter of debate. In rats exposed to oxidative stress, the source of increased HGF seems to be the lung fibroblast (42). We previously showed that alveolar neutrophils were an important source of HGF in pulmonary fibrosis whereas human alveolar macrophages were not (41).

We conclude that HGF secretion by pulmonary fibroblasts obtained from patients with pulmonary fibrosis is profoundly decreased, a defect that appears to be secondary to the reduced secretion of PGE2.


   Acknowledgments
 
The authors thank Professor Eric Vicaud (Lariboisière Saint Louis Medical School, University Paris 7) for helpful advice concerning statistics, and Veronique Leçon for real-time polymerase chain reaction preparations.


   FOOTNOTES
 
Dr. Marchand-Adam is the recipient of a grant from the Fondation pour la Recherche Médicale (Prix Mariane Josso) and the Fondation Benaid.

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Conflict of Interest Statement: S.M-A. has no declared conflict of interest; J.M. has no declared conflict of interest; M.C. has no declared conflict of interest; P.S. has no declared conflict of interest; B.G. has no declared conflict of interest; Y.C. has no declared conflict of interest; G.L. has no declared conflict of interest; D.V. has no declared conflict of interest; H.M. has no declared conflict of interest; M.A. has no declared conflict of interest; M.D. has no declared conflict of interest; B.C. has no declared conflict of interest.

Received in original form December 20, 2002; accepted in final form August 19, 2003


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