Prostaglandin (PG) D2 exerts contrasting activities in the inflamed lung via two receptors, the D prostanoid receptor (DP) and the chemoattractant receptor-homologous molecule expressed on T helper 2 lymphocytes. DP activation is known mainly to inhibit proinflammatory cell functions. We tested the effect of a DP-specific agonist, (4S)-(3-[(3R,S)-3-cyclohexyl-3-hydroxypropyl]-2,5-dioxo)-4-imidazolidineheptanoic acid (BW245C), on pulmonary fibroblast functions in vitro and in a mouse model of lung fibrosis induced by bleomycin. DP mRNA expression was detected in cultured mouse lung primary fibroblasts and human fetal lung fibroblasts and found to be up- and down-regulated by interleukin-13 and transforming growth factor (TGF)-β, respectively. Although micromolar concentrations of BW245C and PGD2 did not affect mouse fibroblast collagen synthesis or differentiation in myofibroblasts, they both inhibited fibroblast basal and TGF-β-induced proliferation in vitro. The repeated administration of BW245C (500 nmol/kg body weight instilled transorally in the lungs 2 days before and three times per week for 3 weeks) in bleomycin-treated mice significantly decreased both inflammatory cell recruitment and collagen accumulation in the lung (21 days). Our results indicate that BW245C can reduce lung fibrosis in part via its activity on fibroblast proliferation and suggest that DP activation should be considered as a new therapeutic target in fibroproliferative lung diseases.
Eicosanoids play multiple roles in inflammatory processes, and contrasting activities have often been reported depending on the organ and pathological process considered. In the inflamed lung, prostaglandin (PG) D2 is generated mainly by mast cells and, to a lesser extent, by alveolar macrophages and T helper 2 (Th2) cells and acts differently on target cells depending on the receptor that is bound: the D prostanoid receptor (DP or DP1) or the chemoattractant receptor-homologous molecule expressed on Th2 lymphocytes (CRTH2 or DP2) (Kostenis and Ulven, 2006). In the lung, most data on these receptors come from models of allergic responses. In asthma and atopic dermatitis, activation of CRTH2 potentiated eosinophil recruitment on the sites of inflammation (Spik et al., 2005). The chemotaxis of Th2 lymphocytes was also increased by selective agonists of CRTH2 (Hirai et al., 2001). Blocking CRTH2 with an antagonist decreased eosinophilia and mucus deposition in an ovalbumin-induced allergic model (Uller et al., 2007). Although these data clearly point to a proinflammatory activity of CRTH2, opposite effects of CRTH2 deficiency have been reported on inflammation in asthma and allergic rhinitis models (Chevalier et al., 2005; Nomiya et al., 2008).
In contrast, DP mediates mostly anti-inflammatory processes that counteract the activity of CRTH2, and its activation regulates several functions during the early phase of the immune response, notably in dendritic cells (DCs). PGD2 or the selective DP agonist (4S)-(3-[(3R,S)-3-cyclohexyl-3-hydroxypropyl]-2,5-dioxo)-4-imidazolidineheptanoic acid (BW245C) inhibits DC chemotaxis and T cell activation (Hammad et al., 2003; Gosset et al., 2005). In experimental models of allergy in the lung or skin, DP activation reduced DC migration from the site of inflammation to the draining lymph nodes, T lymphocyte proliferation and secretion of cytokines, and eosinophil recruitment. Several authors have suggested that DP is crucial in the Th2/Th1 balance because DP deficiency or its specific activation oriented the immune response, respectively, to a Th2 or Th1 phenotype (Angeli et al., 2004; Hammad et al., 2007). However, the overall anti-inflammatory activity of DP remains controversial because DP-deficient mice displayed a decreased asthmatic reaction in an ovalbumin-induced model of lung inflammation (Matsuoka et al., 2000). This suggests that unknown activities of PGD2/DP remain to be discovered and, possibly, new DP-bearing cells remain to be identified. In the lung, DP was identified mainly on leukocytes such as DCs and eosinophils (Gervais et al., 2001; Faveeuw et al., 2003). Matsuoka et al., (2000) also detected the receptor on alveolar and bronchial epithelial cells by immunohistochemistry.
In addition to leukocytes that maintain a protracted inflammatory status in the lung, mesenchymal cells play a central role in the establishment of lung fibrotic diseases not only by producing excessive amounts of extracellular matrix proteins that accumulate in the pulmonary interstitium but also by acting as inflammatory cells secreting cytokines and chemokines (Smith et al., 1997; Phan, 2002). PGE2 has long been known to repress fibroblast proliferation, differentiation, and collagen synthesis in vitro (Huang et al., 2007; Thomas et al., 2007). In the bleomycin (bleo) model of experimental lung fibrosis, results, however, have been inconclusive in PGE2 receptor-deficient mice (Moore et al., 2005; Lovgren et al., 2006). Another prostaglandin, PGF2α, was identified as a profibrotic factor because mice deficient for its receptor showed reduced lung fibrosis after administration of bleomycin, independently of the prototypic profibrotic cytokine transforming growth factor (TGF)-β. PGF2α was shown to act directly on pulmonary fibroblasts by stimulating their proliferation and collagen synthesis (Oga et al., 2009).
In contrast, PGD2 has been shown to decrease basal, TGF-β-induced, and TNF-α-induced proliferation of fibroblast cell lines (Hori et al., 1989; Okuda-Ashitaka et al., 1990). First evidence of a beneficial effect of PGD2 on pulmonary fibrosis was obtained in bleomycin-treated mice intravenously injected with human PGD synthase-transfected fibroblasts (Ando et al., 2003). However, specifically targeting DP, and not CRTH2 known to mediate proinflammatory responses, would provide a more effective approach for the treatment of lung fibrosis. We therefore hypothesized that an agonist of DP could exert inhibitory activity on lung fibroblasts.
In this article, we identify the expression of DP in primary lung fibroblasts and show an in vitro inhibitory activity of its selective agonist BW245C on fibroblast proliferation. We also record a significant beneficial effect of BW245C administration in bleomycin-induced lung inflammation and fibrosis in mice.
Materials and Methods
Animals and Treatments.
Female C57BL/6 mice were obtained from the local breeding facility (Animalerie Centrale, Université Catholique de Louvain, Brussels, Belgium). Ten- to 12-week-old animals were housed in positive pressure air-conditioned units (25°C, 50% relative humidity) on a 12-h light/dark cycle with free access to water and laboratory animal food. For instillation, animals were anesthetized with a mix of Ketalar (Warner-Lambert, Zaventem, Belgium) and Rompun (Bayer, Leverkusen, Germany) (1 and 0.2 mg/mouse i.p., respectively). Bleomycin (Aventis, Brussels, Belgium) was suspended in sterile 0.9% NaCl, and 1 U/kg body weight (b.wt.) (50 μl/20 g b.wt.) was instilled transorally into the lungs via the trachea. Control mice were instilled with a corresponding volume of 0.9% NaCl. To activate DP in vivo, 10 nmol of BW245C/20 g b.wt. (Cayman Chemical, Ann Arbor, MI) (Orchard et al., 1983) was transorally instilled in the lung of mice 2 days before bleomycin treatment and then three times per week. Control mice received the same volume of vehicle [phosphate-buffered saline (PBS) plus 0.74% EtOH]. Mice were sacrificed 21 days after instillation with an overdose of sodium pentobarbital (15 mg/animal i.p.) to estimate the amplitude of lung inflammation and fibrosis (see below). To assess DP mRNA expression, whole lungs from mice treated with NaCl or bleo (1 U/kg b.wt.) were collected 7, 14, or 21 days after instillation, snap-frozen in liquid nitrogen, and homogenized in TRIzol (Invitrogen, Carlsbad, CA) on ice with an Ultra-Turrax T25 homogenizer (Janke and Kunkel, Brussels, Belgium). In a separate experiment, mice were instilled with NaCl or bleo (1 U/kg b.wt.) 21 days before CD45+ and CD45− cell isolation from the lungs.
Isolation of CD45+ and CD45− Lung Cells.
Perfused whole lungs of C57BL/6 mice were infused with 1 ml of Hank's buffered salt solution (Invitrogen) containing 2 mg of pronase (Sigma-Aldrich, St. Louis, MO) and 0.1 mg of DNase (Worthington Biochemicals, Freehold, NJ). After 20-min incubation, the lungs were transferred in 3 ml of fetal bovine serum (FBS; Invitrogen) and disrupted with a sterile syringe. The resulting cell suspension was filtered (70 μm) and resuspended in magnetic-activated cell sorting buffer (IMag; BD Biosciences, Erembodegem, Belgium). CD45+ cells were separated from CD45− cells by using mouse CD45 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. Resulting positive (CD45+) and negative (CD45−) cell fractions were centrifuged and resuspended in TRIzol for RNA extraction.
Culture of Mouse Lung Primary Fibroblasts.
The whole lung was disrupted as described above. After centrifugation, cells were resuspended in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% FBS and 1% antibiotic-antimycotic (AA; Invitrogen) and transferred into a flat tissue culture flask (two lungs/75 cm2). After 1 week of proliferation at 37°C under 5% CO2, cells were detached with 0.25% trypsin (Invitrogen), centrifuged, and resuspended in DMEM/10% FBS/1% AA. Cell number and viability were determined with Trypan blue (Sigma-Aldrich). The cells were seeded into 96-well culture plates and incubated at 37°C for 24 h before applying the treatments. All experiments were performed with first-passage fibroblasts.
For RNA extraction, 5 × 104 fibroblasts were cultured for 24 h. Medium was then replaced with fresh DMEM/0.1% FBS/1% AA containing recombinant mouse IL-13 (R&D Systems, Minneapolis, MN) and/or human TGF-β1 (R&D Systems). Fibroblasts were collected in TRIzol after 24 h.
For proliferation tests, 3 × 104 fibroblasts were cultured for 24 h. Medium was then replaced by 50 μl of fresh DMEM/0.1% FBS/1% AA containing human TGF-β1, PGD2 (Cayman Chemical), and/or BW245C. After 1 h, 0.1 μCi of [3H]thymidine (GE Healthcare, Little Chalfont, Buckinghamshire, UK) in 50 μl of DMEM/0.1% FBS/1% AA was added for an additional 48 h. Cells were then collected on microfilter plates, and thymidine incorporation was measured, after addition of scintillating liquid, with a Top Count microplate scintillation counter (Canberra-Packard, Schwadorf, Austria).
Culture of Human Fetal Lung Fibroblast Cell Line.
Human fetal lung (HFL) fibroblasts (HFL-1; obtained from American Type Culture Collection, Manassas, VA) were cultured in F12K medium (Invitrogen) containing 10% FBS and 1% AA at 37°C under 5% CO2. At preconfluence, cells were detached with 0.25% trypsin (Invitrogen), centrifuged, and resuspended in F12K/10% FBS/1% AA. Cell number and viability were determined with Trypan blue (Sigma Aldrich). A total of 2 × 105 cells were seeded into 48-well culture plates and incubated at 37°C for 24 h before applying the treatments. Medium was then replaced with fresh F12K/0.1% FBS/1% AA containing recombinant mouse IL-13 (R&D Systems) and/or human TGF-β1 (R&D Systems). Fibroblasts were collected in TRIzol after 24 h.
RNA Extraction and Quantification.
RNA extraction was performed according to the TRIzol manufacturer's instructions. Total RNA was reverse-transcribed with Moloney murine leukemia virus reverse transcriptase (Invitrogen) and 350 pmol of random hexamers (Eurogentec, Seraing, Belgium) in a final volume of 25 μl. The resulting cDNA was then diluted 10× and used as template in subsequent polymerase chain reaction (PCR). Sequences of interest were amplified by using the following forward primers: 5′-CCTGCCTTTAATTTATCGTGCG (mouse DP), 5′-GGAGTAATGGTTGGAATGGGC [α-smooth muscle actin (SMA)], 5′-CCGGCTCCTGCTCCTCTT (procollagen I α1), 5′-CACTATGTGTTCTCTGCCCGTAATT (human DP), 5′-CGGCTACCACATCCAAGGAA (18S RNA) and reverse primers: 5′-CCACTATGGAAATCACAGACAGGA (mouse DP), 5′-GCCTTAGGGTTCAGTGGTGC (α-SMA), 5′-GGGTTGGGACAGTCCAGTTCT (procollagen I α1), 5′-TCTTCAGAGGTCCTGTTTTTCTCC (human DP), 5′-ATACGCTATTGGAGCTGGAATTACC (18S RNA). mRNA expression of these genes was quantified by real-time PCR using SYBR Green technology on an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) according to the following program: 10 min 95°C, (15 s 95°C, 1 min 60°C) × 40. Five microliters of diluted cDNA or standards was amplified with 300 nM of the described primers using Power SYBR Green PCR Master Mix (Applied Biosystems) in a total volume of 25 μl. PCR product specificity was verified by taking a dissociation curve and agarose gel electrophoresis. Results are presented as a ratio of gene expression to the expression of the reference gene 18S RNA.
Bronchoalveolar Lavages and Lung Homogenates.
Bronchoalveolar lavage (BAL) was performed by cannulating the trachea and infusing the lungs with 1 ml of PBS. Whole lungs were perfused with PBS, excised, and then placed in 3 ml of cold PBS for biochemical analyses. Lungs were homogenized on ice with an Ultra-Turrax T25 and stored at −80°C. Lavages were centrifuged for 10 min at 400g (4°C). Cell-free supernatants were used for biochemical measurements, and cell pellets were resuspended in PBS. Total BAL cells were counted and cytocentrifuged onto glass slides for differentiation by light microscopy after Diff-Quick staining (200 cells counted; Dade Behring AG, Düdingen, Switzerland).
Lung Collagen Content.
Collagen deposition was estimated by measuring hydroxyproline content in lung homogenates and soluble total collagen in lung homogenate supernatants. Hydroxyproline was assessed by high-pressure liquid chromatography analysis on hydrolyzed lung homogenates (6 N HCl at 108°C during 24 h) as described previously (Biondi et al., 1997). Soluble collagen was measured with the Sircol Collagen Assay Kit (Biocolor, Newtownabbey, UK) according to the manufacturer's instructions.
The following analyses all were performed on the BAL fluid (BALF). Lactate dehydrogenase (LDH) activity was assayed as described previously (Arras et al., 2001). Fibronectin was determined by enzyme-linked immunosorbent assay (ELISA) as detailed previously (Huaux et al., 2002), and hyaluronic acid was determined by Duoset ELISA (R&D Systems). Total TGF-β1 was quantified by DuoSet ELISA (R&D Systems), interferon-γ was quantified with the OptEIA kit (BD Biosciences, Erembodegem, Belgium), and IL-13 was quantified with the mouse Quantikine kit (R&D Systems), according to the manufacturers' instructions.
Differences were evaluated by using t tests or one-way analysis of variance, followed by Dunnett's test, as appropriate. Statistical significance was considered at p < 0.05. Data analysis was performed with InStat version 3.05 for Windows 95/NT (GraphPad Software Inc., San Diego, CA).
DP Is Expressed by Lung Fibroblasts and Modulated by IL-13 and TGF-β.
To address the hypothesis of a potential activity of DP in pulmonary fibroblasts, we examined the expression of this receptor by those cells. We first showed that DP mRNA expression was significantly stronger (6-fold) in mouse structural lung cells (CD45− fraction) compared with leukocytes (CD45+ fraction; Fig. 1A). We then cultured lung primary fibroblasts from naive C57BL/6 mice and showed DP transcript expression in this isolated cell type (Fig. 1B). Because profibrotic cytokines such as TGF-β and IL-13 are known to alter fibroblast gene expression and phenotype in vitro and in vivo (Wynn, 2008), we tested their effect on DP expression. We were surprised to find that TGF-β1 down-regulated DP expression, whereas IL-13 had the opposite effect (Fig. 1B). Simultaneous addition of both cytokines also reduced DP expression, indicating that the inhibitory effect of TGF-β1 overcomes that of IL-13. Because adherent cell types other than fibroblasts could still be present in our primary cultures, although probably in a low amount, we verified that DP is expressed by well characterized lung fibroblasts. Therefore, we assessed DP expression in the fibroblast cell line HFL-1 treated or not with TGF-β1 or/and IL-13. Figure 1C indicates that HFL-1 cells produce DP transcripts, although in a lower amount than mouse lung primary fibroblasts. Moreover, its expression was very similarly regulated by TGF-β1 and IL-13 compared with mouse primary fibroblasts: TGF-β1 down-regulated DP expression (not significant), and IL-13 strongly and significantly up-regulated DP. Finally, the simultaneous addition of TGF-β1 and IL-13 to HFL-1 increased DP mRNA in contrast with the effect observed in mouse primary fibroblasts.
BW245C and PGD2 Do Not Affect Lung Fibroblast Differentiation or Collagen Synthesis.
In the fibrotic lung, fibroblasts are characterized by a differentiation in myofibroblasts and an increased proliferation and collagen production (Phan, 2002). These processes are induced in vivo and in vitro by TGF-β. Because DP is expressed in mouse lung primary fibroblasts, we tested the effect of BW245C on collagen synthesis, differentiation, and proliferation of these cells. These endpoints were also assessed with the endogenous DP ligand PGD2. α-SMA and procollagen I α1 transcripts were measured as markers of differentiation to myofibroblasts and collagen production, respectively. TGF-β1 activated both processes but neither BW245C nor PGD2 consistently modified the effects of TGF-β1 (Fig. 2, A and B).
BW245C and PGD2 Inhibit Lung Fibroblast Proliferation.
A recent technology based on the measurement of the impedance of cultured cells allowing us to compare the attachment, spreading, and proliferation of adherent cells showed that PGD2 and BW245C were able to alter lung fibroblast phenotype (data not shown). Because fibroblast proliferation is crucial for the establishment of fibrosis, lung fibroblast proliferation was assessed by [3H]thymidine incorporation 48 h after adding TGF-β1 and/or BW245C/PGD2. Both BW245C and PGD2 inhibited the basal proliferation of fibroblasts (Fig. 2C). TGF-β1 doubled primary fibroblast proliferation and, again, BW245C and PGD2 significantly counteracted this growth factor-induced proliferation.
BW245C Reduces Lung Inflammation and Fibrosis.
We then tested the effect of BW245C on the development of experimental pulmonary fibrosis induced by bleomycin in mice. To assess the relevance of testing the activity of this DP agonist in this model, we first quantified the mRNA expression of DP in the lungs after bleomycin administration. DP transcripts were detected in the lung and rapidly increased after bleomycin treatment (Fig. 3). This up-regulation was at least maintained 21 days after the treatment. At this time point, i.e., when fibrosis is established (Huaux et al., 2003), DP was overexpressed mainly by structural cells in the fibrotic lung because DP mRNA was almost exclusively increased in pulmonary CD45− isolated cells and not in CD45+ cells (Fig. 1A).
Then, we treated C57BL/6 mice transorally with a single dose of 1 U bleomycin/kg b.wt. and during all of the experimental procedures with the vehicle or 500 nmol BW245C/kg b.wt. starting 2 days before bleomycin treatment and then three times a week. Parameters of inflammation and fibrosis were monitored 21 days after bleomycin administration. LDH activity measured in cell-free BALF, as a marker of cell damage, was increased by bleomycin and significantly reduced by the administration of BW245C (Fig. 4A). Similar results were obtained for alveolar cell influx (105 ± 2.104 total BAL cells in NaCl/vehicle mice and 5.105 ± 2.105 for bleo/vehicle mice versus 2.105 ± 2.104 for bleo/BW245C mice; not statistically significant; n = 6–7). BAL cell differentials showed that the proportion of BAL lymphocytes was already elevated in the NaCl group, probably because of the experimental procedure (repeated transoral instillations) and/or the low EtOH content in the vehicle of BW245C (Fig. 4B). As expected, bleomycin induced the recruitment of lymphocytes in the alveolar space 21 days after treatment, as reflected by the increased percentage of lymphocytes and the corresponding simultaneous decrease of macrophage percentage (Fig. 4B). On the contrary, we observed very similar macrophage and lymphocyte proportions in the NaCl and bleo/BW245C groups, and BW245C almost completely abrogated the bleo-induced recruitment of lymphocytes (154.103 ± 73.103 lymphocytes for bleo/vehicle mice versus 57.103 ± 9.103 for bleo/BW245C).
Because fibrosis is associated with an accumulation of fibronectin and hyaluronic acid (Hernnäs et al., 1992), we measured these markers in BALF. Both extracellular matrix proteins were increased in the BALF of fibrotic lungs compared with naive lungs and reduced by the treatment with BW245C, although not significantly (Fig. 5, A and B). Collagen accumulation assessed by measuring lung total OH-proline and soluble collagen was increased by the administration of bleomycin. Treatment with BW245C significantly reduced the accumulation of lung collagen induced by bleomycin (Fig. 5, C and D).
Because BW245C reduced lung inflammation and fibrosis induced by bleomycin, important cytokines for these processes in the lung were measured in the BALF, i.e., the profibrotic TGF-β1 and IL-13 and the antifibrotic interferon-γ. None of these mediators were found to be differentially accumulated in the BALF of bleo/BW245C mice compared with bleo/vehicle (data not shown).
Fibroblasts that proliferate and produce excessive amounts of extracellular matrix proteins during the pulmonary fibrotic process originate from the lung and derive from lung epithelial cells or circulating progenitors (Kim et al., 2006; Lama and Phan, 2006). TGF-β and IL-13 are known to induce and potentiate lung fibrosis and stimulate fibroblast profibrotic metabolism (Wynn, 2008).
We report here, for the first time, the expression of DP by primary lung fibroblasts and the inhibitory effect of a DP agonist (BW245C) on their in vitro proliferation. Administration of BW245C also significantly reduced lung inflammation and fibrosis in vivo in an experimental model of pulmonary fibrosis.
The expression of DP by lung fibroblasts was already suggested by the presence of DP transcripts in a mouse embryonic fibroblast cell line (Mandal et al., 2004). We found that the expression of DP in mouse lung primary fibroblasts is differently regulated by IL-13 and TGF-β. Considering that a DP agonist was found to repress fibroblast proliferation and IL-13 increased DP expression in fibroblasts, our data suggest that, in addition to its profibrotic activities, IL-13 may exert some control on these functions, e.g., by inhibiting fibroblast proliferation. The inhibitory effect of TGF-β on DP expression prevailed, however, on that of IL-13 in mouse fibroblasts. We also showed that DP mRNA is produced by HFL-1 and its expression in this cell line is similarly regulated by IL-13 and TGF-β compared with mouse primary fibroblasts. These data strongly support that DP is expressed by mouse and human lung fibroblasts and suggest that it is similarly regulated under fibrotic conditions in both species.
In the fibrotic lung, both cytokines are present in increased amounts (Broekelmann et al., 1991; Hancock et al., 1998) but whether they are produced simultaneously and at identical sites is not clear. However, after bleomycin treatment, DP transcripts were strongly up-regulated in the lung, mainly in CD45− cells at day 21. This might indicate that in vivo 1) other structural cells than fibroblasts (epithelial, smooth muscle, or endothelial cells) may significantly overexpress DP in the fibrotic lung, 2) among CD45− cells, the proportion of fibroblasts over other cells was dramatically increased, and/or 3) another unidentified mechanism/mediator than TGF-β/IL-13 triggered in response to bleomycin increases DP expression by fibroblasts.
We also showed that BW245C and PGD2 decreased both basal and TGF-β1-induced fibroblast proliferation, prompting us to evaluate the effect of the DP agonist on the establishment of fibrosis. Administration of BW245C to bleomycin-treated mice decreased collagen deposition in the lung. BW245C treatment also had an inhibitory effect on inflammatory parameters, notably alveolar recruitment of lymphocytes, which are key players in bleomycin-induced lung inflammation and fibrosis (Sharma et al., 1996). Although no direct effect of PGD2/DP on lymphocytes has been reported yet, an indirect activity on T lymphocyte proliferation and differentiation mediated by dendritic cells is well documented in allergic models of inflammation (Hammad et al., 2003; Angeli et al., 2004). Thus, we propose that the overall antifibrotic effect of BW245C can be attributed both to its anti-inflammatory, as described here and in the literature (Kostenis and Ulven, 2006), and its fibroblast antiproliferative activities.
PGD2 is nonenzymatically converted to 15-deoxy-δ-12,14-prostaglandin J2 (15d-PGJ2) that interacts with DP and CRTH2 on the cell surface but also intracellularly with other targets, such as the peroxisome proliferator-activated receptor-γ, and exerts anti-inflammatory activities (Scher and Pillinger, 2005). In vitro, 15d-PGJ2 inhibits TGF-β-induced differentiation and collagen production of fibroblasts (Burgess et al., 2005). In our experimental setting, PGD2, most probably partially converted to 15d-PGJ2, did not affect these fibroblast phenotypes. By contrast, PGD2, like BW245C, affected fibroblast proliferation.
A weak, but significant, interaction of BW245C with PGE2 receptors EP2 and EP4 has been reported (Kabashima and Narumiya, 2003). Because EP4 mediates PGE2 inhibitory effect on fibroblast proliferation (Moore et al., 2005), we cannot rule out the possibility that part of BW245C activity in the present study is EP4-dependent. Contrary to PGD2 or even PGE2, both of which can interact with several receptors, including DP, EP2, and EP4, associated with inhibition of effector cell functions but also CRTH2, EP1, and EP3, which tend to promote cellular activation (Vancheri et al., 2004; Kostenis and Ulven, 2006), BW245C specifically binds to inhibitory receptors and seems like a more selective option for the treatment of lung fibrotic conditions.
In conclusion, our results indicate that the DP agonist BW245C should be considered as a potent, selective, and locally active drug in fibroproliferative lung diseases.
We thank Yousof Yakoub for excellent technical assistance and Virginie Rabolli for help with cell culture.
This work was supported by Action de Recherche Concertée, Communauté Française de Belgique, and the European Commission under FP7-HEALTH-F4–2008 Resolve [Contract 202047]. F.H. is a Research Associate with the Fonds National de la Recherche Scientifique of Belgium.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- D prostanoid receptor
- (4S)-(3-[(3R,S)-3-cyclohexyl-3-hydroxypropyl]-2,5-dioxo)-4-imidazolidineheptanoic acid
- chemoattractant receptor-homologous molecule expressed on T helper 2 lymphocytes
- T helper 2
- transforming growth factor
- dendritic cell
- body weight
- human fetal lung
- 15-deoxy-δ-12,14-prostaglandin J2
- phosphate-buffered saline
- fetal bovine serum
- Dulbecco's modified Eagle's medium
- polymerase chain reaction
- smooth muscle actin
- bronchoalveolar lavage
- BAL fluid
- lactate dehydrogenase
- enzyme-linked immunosorbent assay
- Received April 23, 2010.
- Accepted August 17, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics