Introduction

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The increase in intracellular levels of cAMP and the subsequent activation of its cAMP-dependent protein kinase results in a reduction of inflammation and slight immunosuppression (Torphy, 1998). Intracellular concentration of cyclic nucleotides is mainly governed on the one hand by the activation of adenylyl cyclase coupled with adrenoceptor and G protein and on the other hand by cyclic nucleotide breakdown by phosphodiesterases (PDEs). PDEs constitute a superfamily of enzymes that hydrolyze the 3'-ribose phosphate bond of the naturally occurring 3',5'-cyclic monophosphate second messenger nucleotide to form biologically inert 5'-nucleotide monophosphate. To date, PDEs are divided at least into 16 families, which all demonstrate distinct substrate specificities and regulatory characteristics (Torphy, 1998; Souness et al., 2000). Among these families, PDE3, PDE4, and PDE7 are responsible for cAMP hydrolysis; however, PDE4 is mainly present in the inflammatory cells. Due to their potent anti-inflammatory activity, the interest in selective PDE4 inhibitors in the treatment of pulmonary inflammatory disorders has been greatly increasing for the past few years (for reviews see Barnette, 1999; Schmidt et al., 1999).

Airway inflammation is a feature of numerous pulmonary diseases such as acute lung injury, asthma, acute respiratory distress syndrome, and chronic obstructive pulmonary disease (COPD). Acute lung injury is characterized by high microvascular permeability, low-pressure pulmonary edema, refractory hypoxemia, and respiratory failure. The onset of acute lung injury is usually an early symptom of a multiple organ failure associated with sepsis. Endotoxin, or lipopolysaccharide (LPS), is described as an important inducer of lung injury (Parsons et al., 1989). Neutrophil sequestration and activation in the pulmonary microcirculation appeared to be a key event in the development of lung injury (Worthen et al., 1987). When activated, sequestered neutrophils constitute a source of numerous inflammatory mediators, which may contribute to basement membrane destruction and airway remodeling, two processes occurring in most pulmonary diseases including acute lung injury and COPD (O'Connor and FitzGerald, 1994).

Basement membranes are thin extracellular matrices that underlie most epithelia and endothelia and play a major role in various biological processes, particularly tissue remodeling. Matrix metalloproteinases (MMPs) are grouped in a family of zinc- and calcium-dependent enzymes (O'Connor and FitzGerald, 1994) that are characterized by their substrate specificity and are involved in tissue remodeling. Among MMPs, gelatinase A (MMP-2) and gelatinase B (MMP-9) have been shown to degrade type IV collagen, the major constituent of basement membranes. MMP-2 is preferentially secreted by fibroblasts and various epithelial cells, whereas MMP-9 is specially released by inflammatory cells (for a recent review see Corbel et al., 2000).

Transforming growth factor (TGF)- is a cytokine of particular interest with wide biological functions related to cell proliferation, differentiation, and migration (Roberts and Sporn, 1990). TGF- stimulates in vitro collagen and fibronectin production by fibroblasts, and thus, TGF- is thought to play a key role in the regulation of extracellular matrix (ECM) synthesis (Ignotz and Massague, 1986; Fine and Goldstein, 1987). TGF- overproduction may lead to fibrosis via pathological accumulation of ECM proteins (Border and Ruoslahti, 1992; Border and Noble, 1994).

We previously demonstrated that corticosteroids modulated MMP activities in bronchoalveolar lavage fluid (BALF) recovered from mice exposed to LPS aerosol (Corbel et al., 1999). The present study was undertaken in a murine model of acute pulmonary inflammation to evaluate the possible effect of RP 73-401 on mediators involved in airway remodeling; to do that, we analyzed the effect of RP 73-401 pretreatment on MMP-9 activity and TGF- release in BALFs of LPS-exposed mice. Because the effect of PDE4 inhibitors appeared to be partly mediated through the release of the anti-inflammatory cytokine, IL-10 (Kambayashi et al., 1995; Eigler et al., 1998), which controls the formation of tumor necrosis factor (TNF)-, we also examined the role of the TNF-/IL-10 balance in the modulatory activities attributed to RP 73-401 in our model.

	Experimental Procedures

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Materials. LPS from Escherichia coli (0.55 B5), gelatin, EDTA, and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO). May-Grünwald and Giemsa stains were obtained from RAL (Paris, France). Sodium pentobarbital was purchased from Sanofi santé animale (Libourne, France). Acrylamide was purchased from ICN Pharmaceuticals Biochemicals Division (Aurora, OH). Coomassie blue was purchased from Bio-Rad (München, Germany). RPMI 1640 medium, penicillin, streptomycin, L-glutamine, SDS, and Tris solution were obtained from Laboratories Eurobio (Les Ulis, France). Recombinant mouse TNF-, recombinant mouse IL-10, and antimouse TGF- monoclonal antibodies were provided by R & D Systems (Minneapolis, MN). RP 73-401 (piclamilast) was a kind gift of Dr. J.J. Bourguignon (Faculté de Pharmacie, Illkirch, France) and was suspended in 1% carboxymethylcellulose (vehicle) for oral administration.

Animals and Experimental Protocols. Ten-week-old male BALB/c mice (CERJ, Le Genest Saint Isle, France) were exposed to an aerosol of LPS (100 µg/ml) in saline solution (0.9% NaCl) or to an aerosol of the saline solution alone (negative control group) for 60 min. Nonanesthetized mice were placed into a Plexiglas chamber (30 × 50 × 30 cm) directly connected to a Devilbiss ultrasonic nebulizer (Ultra-Neb 99; Sunrise Medical, Sommerset, PA) that generated particles with an aerodynamic diameter that averaged 0.5 to 3 µm. In indicated experiments, TNF- (0.1 µg) or IL-10 (0.5 µg) was administered intranasally (50 µl in 0.9% NaCl solution) under a weak and quick ether anesthesia. RP 73-401 (1, 3, 10, or 30 mg/kg) was administered orally 2 h before LPS exposure or TNF- administration. In experiments evaluating IL-10 and TGF- levels in BALF or using TNF- administration, only the highest dose of RP 73-401 (30 mg/kg) was tested.

Bronchoalveolar Lavage. Eighteen to 24 h after LPS exposure or TNF- treatment, mice were anesthetized i.p. with pentobarbital sodium (60 mg/kg). After semiexcision of the trachea, a plastic cannula was inserted, and airspaces were washed using a 1-ml syringe with 0.5 ml of 0.9% NaCl containing 2.6 mM EDTA. This operation was accomplished 10 times, and the recovery of the total lavage volume (5 ml) exceeded 95%. Bronchoalveolar lavage was centrifuged (600g for 10 min at 4°C), and the fluid phase of the first milliliter of BALF was aliquoted and frozen at 80°C until the mediators were assessed.

TNF- and IL-10 Measurements. Amounts of TNF- and IL-10 were quantified in the BALFs by enzyme-linked immunosorbent assay (R & D Systems).

Zymography Analysis. Aliquots of BALFs were subjected to electrophoresis on a 4.5% acrylamide stacking gel/7% acrylamide separating gel containing 1 mg/ml gelatin in the presence of SDS under nonreducing conditions, as previously described (Corbel et al., 1999). After electrophoresis, gels were washed twice with 2.5% Triton X-100, rinsed with water, and incubated at 37°C overnight in 50 mM Tris, 5 mM CaCl₂, and 2 nM ZnCl₂, pH 8. The gels were stained with Coomassie Brilliant Blue and destained in a solution of 25% ethanol and 10% acetic acid. Gelatinase activities appeared as clear bands against a blue background. Molecular weights of gelatinolytic bands were estimated using recombinant protein molecular weight markers (10,000-225,000) (Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK). Enzyme amount was quantified by measuring the intensity of the negative bands using a densitometric analyzer with Densylab (Bioprobe Systems, Les Ulis, France). Results are expressed as a percentage of a band of migration of an internal standard loaded onto each gel to allow comparison between gels.

Western Blot for TGF- Determination. Standardized protein quantities (2 µg) of BALF were loaded to perform SDS-polyacrylamide gel electrophoresis under reducing conditions and were transferred electrophoretically onto nitrocellulose membranes (Hybond-ECL; Amersham). After blocking the filters with 3% bovine serum albumin in Tris-buffered saline (TBS), they were incubated with an antimouse TGF- monoclonal antibody diluted in TBS containing 0.1% bovine serum albumin and 0.3% Nonidet P-40. The filters were then washed three times for 10 min in TBS and incubated with peroxidase-conjugated IgG. All incubations were performed at room temperature for 2 h. Antibody binding was detected by an enhanced chemiluminescence system (Amersham), and blots were exposed to X-ray films. Results are expressed as a percentage of sample loaded onto filter. This sample was used as an internal standard of intensity to allow comparison between filters. Under reducing conditions, this antibody detects the active TGF- homodimer (25 kDa).

Expression of Results and Statistical Analysis. The results are expressed as mean ± S.E.M. Statistical analysis was performed with Statview software from Abacus Concepts, Inc. (Berkeley, CA) on an Apple computer. Analysis of treatment effects between the different groups was performed with analysis of variance. Comparison of multiple treatment interactions was accomplished by Newman-Keuls tests. Other comparisons were performed with Fisher's t test. For each analysis, p < 0.05 was considered statistically significant.

	Results

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Effect of RP 73-401 Pretreatment on LPS-Induced Inflammatory Cell Recruitment in BAL. Exposure of mice to LPS aerosol led to a significant increase in the total number of BAL cells compared with saline-exposed mice (control negative group) (Table 1). The most significant increase in a particular cell type was noted for neutrophils and, to a lesser extent, for lymphocytes. RP 73-401 pretreatment led to a dose-dependent inhibition of the LPS-induced increase in the total BAL cells and neutrophils. However, only RP 73-401 doses equal to 10 and 30 mg/kg elicited significant reductions (P < 0.05 and P < 0.01, respectively). The increase in lymphocyte numbers was significantly reduced only if RP 73-401 was used at a 30 mg/kg concentration. No significant changes in BAL macrophage and eosinophil numbers were observed.

Effect of RP 73-401 Pretreatment on LPS-Induced Cytokine Release in BAL. TNF- and IL-10 were quantified in BALFs (Figs. 1 and 2, respectively). The TNF- level was markedly increased after LPS challenge compared with saline-exposed mice (P < 0.05). This increase was significantly and dose-dependently reduced when LPS-exposed mice were pretreated by RP 73-401 (1-30 mg/kg). The TNF- level in BALF of saline-exposed (control) mice was not modified by RP 73-401 pretreatment.

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Effect of RP 73-401 pretreatment (1-30 mg/kg) on the level of TNF- in BALFs from LPS-exposed (filled bars; n = 6) or saline-exposed (open bars; n = 3) mice. Results are presented as mean ± S.E.M. $, P < 0.05 compared with nontreated control mice exposed to saline solution alone; *, P < 0.05 compared with nontreated control mice exposed to LPS (n = 4-17).

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Effect of RP 73-401 pretreatment (30 mg/kg) on the level of IL-10 in BALFs from LPS- or saline-exposed mice. Treatment with RP 73-401 (30 mg/kg) did not modify the level of IL-10 in control mice (data not shown). Results are presented as mean ± S.E.M. *, P < 0.05 (n = 4-6).

Effect of RP 73-401 Pretreatment on LPS-Induced MMP-9 Activity and TGF- Production in BALF. Gelatinase B (MMP-9) activity in BALFs of LPS-exposed mice was markedly induced compared with control mice (Fig. 3, top; lanes D and E in comparison with lane A; saline-exposed mice), and Fig. 3, lanes B and C (30 mg/kg RP 73-401 pretreated- and saline-exposed mice) (P < 0.05). RP 73-401 pretreatment markedly reduced LPS-induced MMP-9 activity in BALFs [Fig. 3, top; lanes F and G (30 mg/kg RP 73-401 pretreated- and LPS-exposed mice) and Fig. 3, bottom]. RP 73-401 pretreatment was significant at all concentrations tested except 1 mg/kg, a dose that was not efficient in reducing LPS-induced effects. Similarly, LPS exposure induced a significant enhancement in the TGF- level in BALFs compared with saline-exposed mice (Fig. 4). This increase was significantly attenuated by a 30 mg/kg RP 73-401 pretreatment.

View larger version (41K): [in this window] [in a new window]   Fig. 3.   Effect of RP 73-401 (1-30 mg/kg) pretreatment on MMP-9 activity in BALFs from LPS-exposed mice. Top, negative image of gelatin zymogram performed with BALFs samples. Lane A, BALFs from control mice (nontreated mice exposed to saline solution); lanes B and C, BALFs from mice pretreated with 30 mg/kg RP 73-401 and aerosolized with saline solution; lanes D and E, BALFs from mice pretreated with vehicle and exposed to LPS; lanes F and G, BAL fluids from mice pretreated with 30 mg/kg RP 73-401 and exposed to LPS; and lane H, molecular weight marker. Bottom, effect of RP 73-401 pretreatment on MMP-9 activity in BALFs from LPS-exposed mice. Enzyme activity was quantified by measuring the intensity of the bands using image analysis. Results are expressed as mean ± S.E.M. of relative percentages of a band of migration of one sample loaded onto each gel. $, P < 0.05 compared with nontreated control mice exposed to saline solution alone; *, P < 0.05 compared with nontreated control mice exposed to LPS (n = 5-16).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Top, Western blot analysis of TGF- in BALF from mice. Lane A, control saline mice; lane B, mice treated with 30 mg/kg RP 73-401 and exposed to saline; lane C, mice treated with 30 mg/kg RP 73-401 and exposed to LPS; and lane D, mice treated with vehicle and exposed to LPS. Bottom, relative intensity of the effect of RP 73-401 pretreatment (30 mg/kg) on the level of TGF- in BALF from LPS (filled bars; n = 6)- or saline (open bars; n = 4)-treated mice. Results are presented as mean ± S.E.M. of percentage of the intensity of an internal standard to allow comparison between filters. *, P < 0.05.

Effect of IL-10 Pretreatment on the LPS-Induced Effects in BAL. Intranasal administration of IL-10 elicited a significant reduction in the enhanced number of total cells and neutrophils induced by LPS exposure (Table 2). The numbers of macrophages, eosinophils and lymphocytes were not modified by either LPS exposure or IL-10 pretreatment (Table 2). IL-10 pretreatment led to a significant decrease in TNF- release (Fig. 5, top) and MMP-9 activity (Fig. 5, middle) in the BALF of LPS-exposed mice. In contrast, although there was a clear trend of IL-10 to reduced LPS-induced release of TGF- in BALFs, we did not reach any significance (Fig. 5, bottom).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Effect of IL-10 (0.5 µg) on the levels of TNF- (top), MMP-9 activity (middle), and TGF- (bottom) in BALFs from LPS (filled bars; n = 8)- or saline (open bars; n = 5)-treated mice. Results are presented as mean ± S.E.M. *, P < 0.05.

Effect of RP 73-401 Pretreatment on the TNF--Induced Effects in BAL. We wondered whether TNF- might be responsible for some of the LPS-induced effects in BALFs. Intranasal administration of TNF- induced a significant increase in the total cell and neutrophil numbers in BALs, which was partially but significantly inhibited by a 30 mg/kg RP 73-401 pretreatment (Table 3). TNF- induced a decrease in BALF IL-10, which was restored by RP 73-401 pretreatment (30 mg/kg) (Fig. 6, top). TNF- also elicited enhanced MMP-9 activity (Fig. 6, middle) and TGF- release (Fig. 6, bottom) in BALFs. However, a 30 mg/kg RP 73-401 pretreatment was ineffective in reducing both parameters (Fig. 6, middle and bottom).

View this table: [in this window] [in a new window]   TABLE 3 Influence of RP 73-401 pretreatment on TNF--induced changes in BAL cell composition RP 73-401 was administered orally at the dose of 30 mg/kg, 2 h before intranasal injection of TNF- (0.1 µg/mice); BAL was performed 24 h after TNF- administration.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Effect of RP 73-401 (30 mg/kg) on the levels of IL-10 release (top), MMP-9 activity (middle), and TGF- secretion (bottom) in BALFs from TNF-- (0.1 µg; n = 10) or saline-treated (control; n = 8) mice. RP 73-401 pretreatment (30 mg/kg) did not modify the level of MMP-9 activity or TGF- and IL-10 release in BALFs from control mice (data not shown). Results are presented as mean ± S.E.M. *, P < 0.05.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

In the present study, we showed that the selective PDE4 inhibitor, RP 73-401, inhibited neutrophil recruitment, MMP-9 activity, and TGF- release in BAL fluids from mice exposed to LPS aerosol. Furthermore, RP 73-401 pretreatment led to a decrease in TNF- but an increase in IL-10 release in BALF of LPS-exposed mice, suggesting that the overall inhibitory effect of RP 73-401 is associated with a modulation of the proinflammatory (TNF-)/anti-inflammatory (IL-10) cytokine balance.

As expected, LPS exposure of BALB/c mice was characterized by a massive recruitment of inflammatory cells in the airways, namely neutrophils, that was accompanied by an increase in TNF- level and a reduction of IL-10 level in BALF. Furthermore, LPS exposure induced a marked enhancement in MMP-9 activity in BALF compared with saline-exposed mice, as previously described in other species (D'Ortho et al., 1994; Delclaux et al., 1997; Corbel et al., 1999). These observations suggested that the model used in our study reproduced some features of acute lung injury and COPD (Shapiro, 1994).

In agreement with previous animal studies (Kips et al., 1993; Turner et al., 1993; Goncalves de Moraes et al., 1998; Miotla et al., 1998; Escofier et al., 1999; Boichot et al., 2000), oral treatment with selective PDE4 inhibitors significantly inhibited pulmonary inflammation induced by LPS exposure. The selective PDE4 inhibitors were described to be active in reducing neutrophil influx and TNF- release in BAL fluids. In addition, the present study clearly reports that the reduction of the inflammatory process was associated with a decrease in MMP-9 activity in BAL. Although alveolar macrophages spontaneously release MMP-9 (Welgus et al., 1990; Shapiro, 1994), it also seems that neutrophils are mainly involved in MMP-9 secretion (Hibbs et al., 1985; Yao et al., 1997). In addition, activated neutrophils produce other proteases and reactive oxygen species that activate latent metalloproteases to their active forms (Palmgren et al., 1992). Although 3 mg/kg RP 73-401 nonsignificantly reduced airway neutrophilia by about 40% but significantly inhibited MMP-9 activity, it is conceivable that the reduction of MMP-9 observed in our experiments might be due to reduced airway neutrophilia caused by RP 73-401 pretreatment. Indeed, MMP-9 is secreted from preformed granules of neutrophils under the influence of LPS and chemotactic factors, a mechanism that does not require direct de novo synthesis of MMP-9 (Masure et al., 1991). Alternatively, the involvement of alveolar macrophages is not excluded because these cells are an important source of MMP-9 (Welgus et al., 1990; Shapiro, 1994) and PDE4 inhibitors may reduce their activation (Torphy, 1998, Barnette 1999). The investigation of the effect of RP 73-401 on neutrophil activation may further provide a final statement.

TNF- is mainly secreted by mononuclear cells such as alveolar macrophages (Vassali, 1992) and plays an important role in the recruitment of neutrophils to sites of injury. TNF- activates neutrophils and mononuclear cells (Vassali, 1992) and also stimulates the secretion of MMPs (Yao et al., 1997). PDE4 inhibitors are particularly effective in reducing LPS-induced TNF- synthesis by human (Gantner et al., 1997) or murine alveolar macrophages (Schade and Schudt, 1993; Goncalves de Moraes et al., 1998). TNF- production by LPS-activated macrophages is inhibited by IL-10 (Fiorentino et al., 1991), which also decreases MMP biosynthesis in human mononuclear phagocytes (Lacraz et al., 1995). Thus, it is likely that during the development of acute lung injury and inflammation, inflammatory cell recruitment and the balance between TNF- and IL-10 may modulate MMP activities.

We wondered whether some of the LPS-induced effects might be due to TNF- itself. Thus, we directly administered TNF- by the intranasal route, and we demonstrated that, similarly to LPS exposure, TNF- administration induced airway neutrophilia, enhanced MMP-9 activity, and reduced the IL-10 level in BALF. These results are consistent with data demonstrating that TNF- favored neutrophil sequestration and migration (Ulich et al., 1993; Denis et al., 1994; Goncalves de Moraes et al., 1996) and that the difference in the levels of TNF- and IL-10 modulates cell recruitment. Interestingly, a 30 mg/kg RP 73-401 pretreatment partially but significantly reduced airway neutrophilia and enhanced significantly IL-10 release but did not reduce MMP-9 activity in BAL of TNF--exposed mice. These results are consistent with previous studies showing that PDE4 inhibitors alone are moderately effective toward the in vitro activity of TNF- on cell adhesion molecule expression (Morandini et al., 1996; Blease et al., 1998). A combination of PDE4 inhibitors and ₂-agonists is necessary to obtain a significant inhibition. It is possible that inhibition of PDE4 is insufficient to elevate intracellular cyclic AMP to levels that have functional consequences (Blease et al., 1998). Therefore, the combined effect of the activation of adenylate cyclase with elevated cyclic AMP and inhibition of its metabolism may be useful to provide a significant effect. Our results suggest that selective PDE4 inhibitor may interfere with the release of TNF- from competent cells rather than their own activity and that this effect may be mediated in part by IL-10 production.

IL-10 is an important negative modulator of the immune response and is secreted by several cell types, including Th₁ and Th₂ T lymphocytes, macrophages, monocytes, B cells, and keratinocytes (Moore et al., 1993). It has been reported that IL-10 inhibited proinflammatory cytokine synthesis and release at transcriptional and posttranscriptional levels. IL-10 seems to be involved in PDE4 inhibitor mechanisms of action either directly (Kambayashi et al., 1995) or via an increase in cAMP (Eigler et al., 1998). Kambayashi and colleagues (1995) showed that the selective PDE4 inhibitor rolipram increased IL-10 secretion and decreased TNF- release by LPS-stimulated mouse peritoneal macrophages. Moreover, anti-IL-10 antibodies suppressed the inhibitory effect of rolipram on TNF- release. Kambayashi et al. (1995) hypothesized that, besides the intracellular cAMP increase, IL-10 may participate in the anti-inflammatory activity of PDE4 inhibitors. More recently, Salez et al. (2000) have reported that rolipram also elicited an enhancement in IL-10 release from mouse peritoneal macrophages treated with LPS but not from mouse alveolar macrophages. In the present study, we demonstrated that in contrast to the study of Salez et al. (2000), LPS induced a decrease in IL-10 release and RP 73-401 pretreatment restored the IL-10 level in BALF. We also confirmed that IL-10 pretreatment was able to reduce cell recruitment, TNF- levels, and MMP-9 activity in BAL fluids from LPS-exposed mice.

TGF- plays a key role in the regulation of ECM production. In vitro, TGF- stimulates fibroblast production of ECM proteins, including collagen and fibronectin (Ignotz and Massague, 1986; Fine and Goldstein, 1987). TGF- overproduction leads to fibrosis via a pathological accumulation of ECM components (Border and Ruoslahti, 1992; Border and Noble, 1994). Aberrant TGF- expression has been reported through in vivo studies of numerous diseases in which inflammatory and fibrotic lesions occur (Border and Noble, 1994). Thus, by inhibiting the effect of TGF-, the net amount of collagen accumulated during the fibrotic process may be reduced. Previous studies have demonstrated that inhibition of TGF- by antibodies raised against TGF- or by TGF--soluble receptor may lead to the reduction of bleomycin-induced pulmonary fibrosis (Giri et al., 1993; Wang et al., 1999). Because TGF- has been shown to play a key role in airway remodeling, we investigated the effect of RP 73-401 on TGF- release in BALFs of mice. We presently report that RP 73-401 pretreatment of LPS-exposed mice elicited a marked inhibition of TGF- release in BAL. Because we showed that RP 73-401 modulated the TNF-/IL-10 balance, we further investigated the influence of these two cytokines on TGF- production. TGF- release in BALF was inhibited, although nonsignificantly, following the administration of IL-10 in mice. In contrast, TGF- release in BAL was significantly enhanced following administration of TNF-, but this effect was not reduced by RP 73-401 pretreatment, indicating that RP 73-401 did not act directly on the TNF- pathway. However, the marked reduction elicited by RP 73-401 on TGF- production and MMP-9 activity in BALF of LPS-exposed mice suggested that selective PDE4 may modulate airway remodeling occurring in several pathologies such as acute lung injury, acute respiratory distress syndrome, and COPD. In this context, selective PDE4 inhibitors may be proposed as therapeutical candidates for the treatment of pathologies with intense airway remodeling.

In conclusion, we assessed the effect of RP 73-401 on pulmonary inflammation and airway remodeling. We also showed that the efficacy of RP 73-401 is partially mediated by its effect on the balance between TNF- and IL-10 release. Our results also suggested that selective PDE4 inhibitors such as RP 73-401 might modulate tissue remodeling associated with acute lung injury and might provide new data for the therapeutic development of selective PDE4 inhibitors in inflammatory lung diseases.