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. 2002 May;136(2):287–295. doi: 10.1038/sj.bjp.0704705

Regulation by PGE2 of the production of interleukin-6, macrophage colony stimulating factor, and vascular endothelial growth factor in human synovial fibroblasts

Hideo Inoue 1,*, Makiko Takamori 1, Yoshihito Shimoyama 1, Hideaki Ishibashi 2, Seizo Yamamoto 2, Yasuko Koshihara 3
PMCID: PMC1573344  PMID: 12010778

Abstract

  1. We examined the effects of endogenous prostaglandin E2 (PGE2) on the production of interleukin-6 (IL-6), macrophage colony stimulating factor (M-CSF), and vascular endothelial growth factor (VEGF) by interleukin-1β (IL-1β)-stimulated human synovial fibroblasts.

  2. NS-398 (1 μM), a cyclo-oxygenase-2 (COX-2) inhibitor, inhibited IL-6 and VEGF production (35±4% and 26±2%, respectively) but enhanced M-CSF production (38±4%) by IL-1β (1 ng ml−1) in synovial fibroblasts isolated from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Exogenous PGE2 completely abolished the effects of NS-398 on the production of each mediator by OA fibroblasts stimulated with IL-1β.

  3. 8-Bromo cyclic AMP and dibutyryl cyclic AMP, cyclic AMP analogues, mimicked the effects of PGE2 on IL-6, M-CSF, and VEGF production by OA fibroblasts.

  4. The EP2 selective receptor agonist ONO-AE1-259 (2 nM) and the EP4 selective receptor agonist ONO-AE1-329 (2 or 20 nM), but not the EP1 selective receptor agonist ONO-DI-004 (1 μM) and the EP3 selective receptor agonist ONO-AE-248 (1 μM), replaced the effects of PGE2 on IL-6, M-CSF, and VEGF production by OA and RA fibroblasts stimulated with IL-1β in the presence of NS-398.

  5. Both OA and RA fibroblasts expressed mRNA encoding EP2 and EP4 but not EP1 receptors. In addition, up-regulation of EP2 and EP4 receptor mRNAs was observed at 3 h after IL-1β treatment.

  6. These results suggest that endogenous PGE2 regulates the production of IL-6, M-CSF, and VEGF by IL-1β-stimulated human synovial fibroblasts through the activation of EP2 and EP4 receptors with increase in cyclic AMP.

Keywords: Synovial fibroblasts, PGE2, EP receptor subtypes, EP receptor agonists, IL-6, M-CSF, VEGF, rheumatoid arthritis, osteoarthritis

Introduction

The inflammatory process is a complex mechanism, orchestrated by both infiltrating cells and mesenchymally derived cells. Pro-inflammatory cytokines are considered to play important roles in the initiation and development of joint diseases, including RA and OA (Feldmann et al., 1996; Westacott & Sharif, 1996; Isomaki & Punnonen, 1997). IL-1, as well as tumour necrosis factor-α (TNF-α), is the most prominent cytokine for developing synovial inflammation and plays a predominant role in the etiopathology of joint disease. It has been established that IL-1 enhances synovial fibroblast DNA synthesis (Butler et al., 1988) and activates synoviocytes to secret soluble mediators such as collagenase (Dayer et al., 1986), PGE2 (Dayer et al., 1986), IL-6 (Guerne et al., 1989), and VEGF (Jackson et al., 1997). These mediators are involved in inflammatory response, joint destruction, and angiogenesis. PGs and related eicosanoids also contribute to inflammatory responses in joint diseases (Robinson et al., 1975). PG production is mediated by two isoforms of cyclo-oxygenase (COX) (DeWitt, 1991; Xie et al., 1991), a constitutive form (COX-1), and an inducible form (COX-2). The induction of COX-2 by IL-1β in synovial cells is associated with an increase in PGE2 production (Crofford et al., 1994; Hulkower et al., 1994). In addition, the expression of COX-2 is elevated in a disease-related pattern of synovial tissue from patients with arthritis (Siegle et al., 1998).

PGE2 is able to control the production of diverse chemical mediators in various cells. While PGE2 induces VEGF expression in RA synovial fibroblasts (Ben-Av et al., 1995) and human prostate cancer cells (Liu et al., 1999), it reduces TNF-α production by rodent macrophages (Kunkel et al., 1988) and M-CSF production by human monocytes (Lee et al., 1990). We previously reported that PGE2 induces the production of parathyroid hormone-related peptide in OA synovial fibroblasts (Yoshida et al., 1998). Furthermore, PGE2 is suggested to regulate the production of cytokines such as IL-6, IL-8, IL-11, and granulocyte macrophage colony stimulating factor (GM-CSF) by IL-1-stimulated synovial fibroblasts (Agro et al., 1996; Mino et al., 1998). However, it is not well understood whether endogenous PGE2 participates in the production of soluble factors by human synovial fibroblasts through PGE2 receptors.

The physiological and pharmacological actions of PGE2 on cell growth and function are mediated by a specific group of seven transmembrane receptors. PGE2 receptors have been classified into four subtypes, designated the EP1, EP2, EP3, and EP4 receptors (Coleman et al., 1994; Negishi et al., 1995). EP1 receptors associate phospholipase C and phosphoinositol turnover, and stimulate the release of intracellular calcium. EP2 and EP4 receptors increase cyclic AMP levels via activation of adenylate cyclase, whereas EP3 receptor variants mediate multiple signal pathways such as inhibition or stimulation of cyclic AMP levels, activation of phospholipase C, and mobilization of intracellular calcium. Recently, the prostanoid derivative EP receptor agonists such as ONO-DI-004 for EP1, ONO-AE1-259 for EP2, ONO-AE-248 for EP3, and ONO-AE1-329 for EP4 (Figure 1) have been developed (Suzawa et al., 2000) and used to examine the role and function of PGE2 receptor subtypes (Zacharowski et al., 1999; Suzawa et al., 2000; Yamane et al., 2000). We report here, for the first time, that endogenous PGE2 regulates the production of IL-6, VEGF, and M-CSF by IL-1β in synovial fibroblasts obtained from patients with OA and RA through the activation of EP2 and EP4 receptors. Furthermore, the results from this study favour the hypothesis that PGE2 plays an important role as a regulator in the production of chemical mediators driven inflammation.

Figure 1.

Figure 1

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Chemical structures of ONO-DI-004, ONO-AE1-259, ONO-AE-248, and ONO-AE1-329.

Methods

Reagents

Recombinant human IL-1β was purchased from Becton Dickinson Labware (Bedford, MA, U.S.A.). PGE2 and pentoxifylline were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). 8-Bromo cyclicAMP and dibutyryl cyclic AMP were purchased from Biomol Research Laboratory (Plymouth Meeting, PA, U.S.A.), and NS-398 (N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulphonamide) was purchased from Cayman Chemical Co. (Ann Arbor, MI, U.S.A.). ONO-DI-004 ((17S)-2,5-ethano-6-oxo-17,20-dimethylPGE1), ONO-AE1-259((16S)-9-deoxy-9β-chloro-15-deoxy-16-hydroxy-17, 17-trimethylene-19, 20-didehydroPGE2 sodium salt), ONO-AE-248 (11, 15-O-dimethylPGE2), and ONO-AE1-329 (16-(3-methoxymethyl)phenyl-ω-tetranor-3, 7-dithiaPGE1) were generous gifts from Ono Pharmaceutical Co. (Osaka, Japan).

Synovial fibroblast culture

Primary cultures of human synovial fibroblasts were obtained by enzymatic digestion of synovial tissue obtained from patients with OA and RA (Takayanagi et al., 1997). Fibroblasts were cultured in α-minimum essential medium (α-MEM; Gibco BRL, Gaithersbung, MD, U.S.A.) containing heat-inactivated 10% foetal calf serum (FCS) (Biowhittaker, Walkersville, MD, U.S.A.) and 60 μg ml−1 kanamycin sulphate (Gibco BRL) at 37°C in a humidified atmosphere of 95% air and 5% CO2. The culture medium was replaced twice each week. The confluent cells were dispersed with trypsinization and then transferred to new plastic dishes in a split ratio of 1 : 2 or 1 : 4. The cells at more than six passages (eight population doubling levels, PDL) were used for subsequent experiments. These cells consisted of fibroblasts alone, with no dendritic or monocytic cells.

Synovial fibroblasts were plated onto 24-well plates (Becton Dickinson Labware) at 2×104 cells ml−1 to perform the experiments. When cells grew up to confluence, cells were kept in 0.5 ml α-MEM containing 0.5% FCS for 48 h and then exposed to a concentration of 1 ng ml−1 IL-1β (Inoue et al., 2001) in the presence or absence of different agents dissolved in DMSO (0.1%) for 24 h. Each study was repeated another one or two times.

Measurement of IL-6, IL-8, M-CSF, PGE2, and VEGF levels in the culture medium

The culture media were collected at 24 h after IL-1β stimulation and kept at −80°C until used for assay. Concentrations of IL-6 (detection limit: 3 pg ml−1), M-CSF (detection limit: 9 pg ml−1), PGE2 (detection limit roughly 12 pg ml−1), and VEGF (detection limit: 5 pg ml−1) in the culture supernatant were measured directly by the enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's directions. ELISA kits used were purchased from PerSeptive Biosystems (Framingham, MA, U.S.A.) for IL-6, R & D systems (Minneapolis, MN, U.S.A.) for M-CSF and VEGF, and Cayman Chemical for PGE2. Sample and standard dilutions were made with experimental medium, and results were expressed as the mean±s.e.m. The statistical significance was determined with the Tukey–Kramer multiple comparison test after one-way analysis of variance. Appropriate groups were compared by Student's t-test.

RT–PCR analysis of EP receptor subtypes

Total RNA was extracted from synovial fibroblasts (106 cells) by acid guanidine-phenol-chloroform extraction using ISOGEN® (Nippon Gene, Toyama, Japan) and treated with a DNA-free kit (Ambion, Austin, TX, U.S.A.) for elimination of contaminating DNA. cDNA was synthesized from isolated RNA with oligo-dT primer and AMV reverse transcriptase XL (TaKaRa RNA PCR kit (AMV) Ver. 2.1, Takara, Osaka, Japan), and used as templates for PCR. PCR for EP2 and EP4 receptors was carried out for 30–32 cycles of 95°C for 1 min, 58°C for 1 min, and 72°C for 1 min, followed by a final of 72°C for 6 min. That for EP1 and EP3 receptors was performed for 42 cycles of 95°C for 1 min, 65°C for 1 min, and 72°C for 1 min, followed by a final of 72°C for 6 min. The synthetic primers for the human PGE2 receptors were designed to according to Zeng et al. (1998) (Table 1). The constitutively expressed gene encoding GAPDH was used as internal control in RT–PCR to normalize the amounts of mRNA in each sample. The cDNA of the GAPDH gene was amplified from the same volume of cDNAs as for EP receptors, but only 10–12 cycles of PCR. The PCR products were analysed by electrophoresis in a 2% agarose gel visualized using ethidium bromide. No PCR product was amplified without reverse-transcription reaction.

Table 1.

Gene-specific primer sequences used in PCR amplification

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Results

Effects of exogenous PGE2 on the spontaneous production of IL-6, M-CSF, and VEGF by OA fibroblasts

We first examined whether PGE2 alone affects the spontaneous release of IL-6, M-CSF, and VEGF from OA fibroblasts (Table 2). OA fibroblasts themselves released immunoreactive IL-6, M-CSF, and VEGF for 24 h culture without any stimulants. Among them, spontaneous level of M-CSF was higher than that of IL-6 and VEGF. PGE2 dose-dependently increased the secretion of IL-6 and VEGF from OA fibroblasts. When cells were treated with 1 ng ml−1 PGE2, significant (P<0.05) increases in the release of IL-6 and VEGF were observed. Also, levels of IL-6 and VEGF were further enhanced a 12 and 2 fold by 20 ng ml−1 PGE2, respectively, compared with PGE2-untreated cells. In contrast, PGE2 at a concentration of 20 ng ml−1 significantly (P<0.05) decreased M-CSF level.

Table 2.

Effects of exogenous PGE2 on the spontaneous production of IL-6, M-CSF, and VEGF by OA synovial fibroblasts

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Effects of exogenous PGE2 on IL-6, M-CSF, and VEGF production by OA fibroblasts stimulated with IL-1β in the presence of NS-398

We had recently reported that IL-1β induced the production of IL-6, M-CSF, PGE2, and VEGF in synovial fibroblasts of patients with OA and RA (Inoue et al., 2001). In fact, IL-1β (1 ng ml−1) markedly increased IL-6, M-CSF, and VEGF production by OA synovial fibroblasts (Figure 2). Basal levels of IL-6, M-CSF, and VEGF were 119±4, 540±7, and 120±5 pg ml−1 (n=5–6), respectively. Increase in IL-6 production by IL-1β was greater than that in M-CSF and VEGF production. The release of PGE2 was also enhanced significantly (P<0.001, Student's t-test) by IL-1β from the basal value 0.030±0.003 to 8.1±0.5 ng ml−1 (n=6). NS-398, a COX-2 inhibitor (Futaki et al., 1994), at a dose of 1 μM which completely blocks the production of PGE2 in this model (Inoue et al., 2001), significantly (P<0.01 or P<0.001) inhibited IL-6 and VEGF production. In contrast, treatment with NS-398 resulted in approximately 30% increase in M-CSF production compared with IL-1β alone value. Furthermore, treatment of OA fibroblasts with PGE2 completely abolished the effects of NS-398 on IL-1β-induced IL-6, M-CSF, and VEGF production. Simultaneous addition of 10 or 20 ng ml−1 PGE2 to IL-1β resulted in further enhancement of IL-6 and VEGF production in the presence of NS-398. Contrary, PGE2 dose-dependently inhibited the enhancement by NS-398 of IL-1β-induced M-CSF production. M-CSF level increased in response to IL-1β was significantly (P<0.01) reduced up to 64% by 20 ng ml−1 PGE2.

Figure 2.

Figure 2

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Effects of exogenous PGE2 on IL-6, M-CSF, and VEGF production by OA synovial fibroblasts stimulated with IL-1β in the presence of NS-398. Confluent fibroblasts were treated with IL-1β (1 ng ml−1) in the presence or absence of NS-398 (1 μM) and PGE2 at various concentrations for 24 h.+: with and −: without. Values are the mean±s.e.m. of 5–6 samples. *P<0.05 or **P<0.01 vs IL-1β (Student's t-test). † P<0.01 vs IL-1β+NS-398 (Tukey–Kramer multiple test).

Involvement of cyclic AMP in the production of IL-6, M-CSF, and VEGF by OA fibroblasts stimulated with IL-1β

Effects of cyclic AMP analogues were examined on IL-6, M-CSF, and VEGF production by IL-1β in OA fibroblasts. 8-Bromo-cyclic AMP and dibutyryl-cyclic AMP, cyclic AMP analogues, at 1 mM significantly (P<0.01, Student's t-test) increased the basal level of IL-6 from 84±3 to 2490±260 and 2364±54 pg ml−1 (n=5), and of VEGF from 115±3 to 289±14 and 340±5 pg ml−1 (n=5), respectively. In contrast, 8-bromo-cyclic AMP and dibutyryl-cyclic AMP markedly (P<0.01, Student's t-test) reduced the basal level of M-CSF from 509±8 to 288±11 and 344±12 pg ml−1 (n=5), respectively. Both compounds dose-dependently enhanced IL-1β-induced IL-6 and VEGF production in the presence of NS-398 (Figure 3). However, dibutyryl-cyclic AMP at a high dose (1 mM) failed to enhance IL-6 production. 8-Bromo-cyclic AMP and dibutyryl-cyclic AMP significantly (P<0.01) inhibited M-CSF production by IL-1β in a dose-dependent manner. Furthermore, pentoxifylline (0.5 mM), a non-selective phosphodiesterase inhibitor (Ward & Clissold, 1987), significantly (P<0.05, Student's t-test) enhanced VEGF production in response to IL-1β (IL-1β: 583±31 vs pentoxifylline: 695±29 pg ml−1, n=5) whereas it markedly (P<0.01, Student's t-test) reduced IL-1β-induced M-CSF production (IL-1β: 1992±47 vs pentoxyfylline: 1647±36 pg ml−1, n=5).

Figure 3.

Figure 3

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Effects of cyclic AMP analogues on IL-6, M-CSF, and VEGF production by OA synovial fibroblasts stimulated with IL-1β in the presence of NS-398. Confluent fibroblasts were treated with IL-1β (1 ng ml−1) in the presence or absence of NS-398 (1 μM) and dibutyryl-cyclic AMP (dbcAMP) or 8-bromo-cyclic AMP (8brcAMP) at various concentrations for 24 h.+: with and −: without. Values are the mean±s.e.m. of 5–6 samples. *P<0.05 or **P<0.01 vs IL-1β (Student's t-test). †P<0.01 vs IL-1β+NS-398 (Tukey–Kramer multiple test).

Effects of EP receptor agonists on IL-6, M-CSF, and VEGF production by IL-1β in OA and RA fibroblasts

We next determined whether PGE2 regulates IL-1β-stimulated IL-6, M-CSF, and VEGF production via PGE2 receptor subtypes by employing selective EP receptor agonists such as ONO-DI-004, ONO-AE1-259, ONO-AE-248, and ONO-AE1-329. The specificities of the agonists have been confirmed by the binding assay for the respective receptor subtypes expressed in CHO cells (Suzawa et al., 2000). In addition, ONO-AE1-259 and ONO-AE1-329 have no difference in binding affinity and agonist activity between mouse and human EP receptors (personal communications). Basal levels of IL-6, M-CSF, and VEGF in OA fibroblasts were 107±12, 527±13, and 120±7 pg ml−1 (n=5–6), respectively. Similar to the exogenous PGE2, the EP2 receptor agonist ONO-AE1-259 and the EP4 receptor agonist ONO-AE1-329 at a dose of 2 or 20 nM not only enhanced the production of IL-6 and VEGF but also reduced M-CSF production by OA fibroblasts treated with IL-1β in the presence of NS-398 (Table 3). However, the EP1 receptor agonist ONO-DI-004 and the EP3 receptor agonist ONO-AE-248 had no effect on the production of each mediator until 1 μM. Both agonists for EP2 and EP4 receptors, as well as PGE2, enhanced spontaneous levels of IL-6 and VEGF, and attenuated spontaneous M-CSF level in non-stimulated OA fibroblasts (data not shown).

Table 3.

Effects of EP receptor agonists on IL-1β-induced IL-6, M-CSF, and VEGF production in OA synovial fibroblasts

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Furthermore, we examined the effects of EP receptor agonists on IL-1β-induced IL-6, M-CSF, and VEGF production in RA synovial fibroblasts (Table 4). Basal levels of IL-6, M-CSF, and VEGF were 202±7, 357±9, and 69±4 pg ml−1 (n=5–6), respectively. Similar to OA fibroblasts, basal level of M-CSF was higher than that of IL-6 and VEGF in RA fibroblasts. In addition, IL-1β strongly induced IL-6 production compared with M-CSF and VEGF production. IL-6 and VEGF production in response to IL-1β were inhibited, but M-CSF production was enhanced by treatment with NS-398. In OA and RA fibroblasts, inhibition by NS-398 of IL-1β-induced IL-6 and VEGF production was 35±4% and 26±2% (n=4–5 experiments), respectively, whereas enhancement of M-CSF was 38±4% (n=5 experiments). As expected, the EP2 receptor agonist ONO-AE1-259 and the EP4 receptor agonist ONO-AE1-329 at a dose of 2 nM were effective in enhancing IL-6 and VEGF production, and in inhibiting M-CSF production by IL-1β-stimulated RA fibroblasts. In contrast to this, EP1 and EP3 receptor agonists at 1 μM did not change the production of IL-6, M-CSF, and VEGF by IL-1β in the presence of NS-398. EP2 receptor agonist effectively attenuated M-CSF production compared with EP4 receptor agonist in both OA and RA fibroblasts.

Table 4.

Effects of EP receptor agonists on IL-1β-induced IL-6, M-CSF, and VEGF production in RA synovial fibroblasts

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Enhancement by IL-1β of EP2 and EP4 receptor mRNAs in OA and RA fibroblasts

As EP2 and EP4 receptors were suggested to be involved in the production of IL-6, M-CSF, and VEGF by IL-1β, we examined expression of mRNA encoding EP receptor subtypes in OA and RA synovial fibroblasts by RT–PCR. Co-expression of EP2, EP3, and EP4 receptor mRNAs was confirmed in total RNA isolated from OA fibroblasts (Figure 4a, lane 1). Expression of EP2, EP3, and EP4 receptor mRNAs was up-regulated at up to 3 h after IL-1β stimulation (Figure 4a, lane 3). As shown in Figure 4b, mRNA encoding EP2 and EP4 receptors was confirmed in RA fibroblasts (Figure 4b, lane 1) and up regulated at 3 h after IL-1β stimulation (Figure 4b, lane 2). EP3 receptor mRNA was not observed in either IL-1β-treated or -untreated RA fibroblasts. Nevertheless, RA fibroblasts derived from other donors expressed EP3 receptor mRNA as well as EP2 and EP4 receptor mRNAs (data not shown). Expression of EP1 receptor mRNA was not observed in both OA and RA fibroblasts, and induced even by treatment with IL-1β (data not shown).

Figure 4.

Figure 4

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Time-course of mRNA encoding PGE2 receptor subtypes in OA (a) and RA (b) synovial fibroblasts treated with IL-1β. Total RNA was isolated from confluent fibroblasts at 1, 3, 6, 12 or 24 h after IL-1β (1 ng ml−1) treatment and subjected to RT–PCR with a specific primer for each EP receptor or GAPDH. After RT–PCR, amplified products were analysed by agarose gel electrophoresis with ethidium bromide staining. The cultures without IL-1β treatment served as the control. (a) Lane 1: control; lane 2 : 1 h; lane 3 : 3 h; lane 4 : 6 h; lane 5 : 12 h; lane 6 : 24 h. (b) Lane 1: control; lane 2 : 3 h. The figure was one representative of three independent experiments.

Discussion

In this study, we have presented evidence showing that endogenous PGE2 regulates the production of IL-6, M-CSF, and VEGF in response to IL-1β through the activation of EP2 and EP4 receptors in human synivial fibroblasts. Synovial fluid of patients with RA and OA contains many soluble mediators, such as cytokines and growth factors, which contribute to the development of joint diseases (Feldmann et al., 1996; Westacott & Sharif, 1996). IL-6 increases B-cell proliferation, immunoglobulin production, and acute phase protein production, and may stimulate rheumatoid factor secretion in the synovium (Isomaki & Punnonen, 1997). M-CSF, as well as granulocyte macrophage-colony stimulating factor (GM-CSF), activates monocytes/macrophages to produce cytokines and increases the number of mature macrophages in the inflamed synovium (Moss & Hamilton, 2000). VEGF plays an important role of angiogenesis and endothelial migration during development of RA synovitis (Folkman, 1995; Nagashima et al., 1995). In addition, inflammatory mediators including cytokines and PGs are also involved in the induction of angiogenensis (Fidler & Ellis, 1994). In our study, the COX-2 inhibitor NS-398 inhibited IL-6 and VEGF production as well as PGE2 production in response to IL-1β whereas it enhanced M-CSF production in synovial fibroblasts, suggesting that autocrine stimulation by PGs is involved in the production of them. In fact, exogenous PGE2 abolished the effect of NS-398 in response to IL-1β. PGE2 also potentiated the spontaneous production of IL-6 and VEGF, and attenuated M-CSF production by non-stimulated synovial fibroblasts. However, the ability of PGE2 to produce IL-6 and VEGF was low compared with IL-1β. Thus, endogenous PGE2 seems to play as a promoter in the production of cytokine and growth factor by synovial fibroblasts stimulated with IL-1β. Others have found that PGE2 enhances IL-6 production but inhibits GM-CSF production by IL-1α-stimulated human synovial fibroblasts (Agro et al., 1996). IL-11, a functionally pleiotropic cytokine classified as an IL-6-type cytokine, produced by RA fibroblasts treated with IL-1α and TNF-α is inhibited by indomethacin, and its inhibition is prevented by PGE2 treatment (Mino et al., 1998). Furthermore, PGE2 participates in the upregulation of VEGF by cobalt chloride-stimulated hypoxia in human prostate cancer cells (Liu et al., 1999). Taken together, our results indicate that endogenous PGE2 regulates the production of IL-6, M-CSF, and VEGF by IL-1β in synovial fibroblasts derived from arthritis. In addition to PGE2, other prostanoids also might modulate the production of cytokine and growth factor in response to IL-1β, as suggested by others that prostacyclin regulates the release of GM-CSF and granulocyte-colony stimulating factor from human vascular smooth muscle cells stimulated with IL-1β (Stanford et al., 2000).

It has been reported that human synovial fibroblasts express mRNA encoding the EP1 and EP2 subtypes of PGE2 receptors (Ben-Av et al., 1995). We confirmed that both OA and RA fibroblasts express EP2, EP3, and EP4, but not EP1, receptor mRNAs, although there was difference in expression of EP3 receptor mRNA between RA donors. A recent report has shown that RA fibroblasts express EP3 receptor mRNA as well as EP2 and EP4 receptor mRNAs (Yoshida et al., 2001). Thus, it is conceivable that the expression of EP receptor mRNAs in synovial fibroblasts may be affected by the condition of diseases or the medical application for donors. We also found that the expression of mRNA encoding EP2 and EP4 mRNAs was up-regulated by IL-1β treatment in both OA and RA fibroblasts. IL-1β is known to augment EP1 receptor level, as well as PGE2 production, in amnion cells (Spaziani et al., 1997). However, in the present study, the induction of mRNA encoding EP1 receptor by IL-1β was not observed in synovial fibroblasts. In addition, specific agonists for EP2 and EP4 receptors excluding EP1 and EP3 receptors enhanced IL-6 and VEGF production, and attenuated M-CSF production by fibroblasts stimulated with IL-1β in the presence of NS-398. Moreover, there was no difference in the effects of EP receptor agonists between OA and RA fibroblasts. These findings suggest that both EP2 and EP4 receptors may participate in the regulation of IL-1β-induced IL-6, M-CSF, and VEGF production in human synovial fibroblasts. Additionally, it seems likely that an increase in expression of mRNA encoding EP2 and EP4 receptors contributes to the PGE2 effects in this model. Recently, PGE2 has been found to induce parathyroid hormone-related peptide via EP2 and EP4 receptors in RA fibroblasts treated with IL-1α (Yoshida et al., 2001). Others have reported that while EP3 receptors mediate the production of matrix metalloproteinase-9 in response to PGE2 (Zeng et al., 1996), EP2 and EP4 receptors regulate the production of IL-6 by PGE2 in human leukemic T cells (Zeng et al., 1998) and mediate the modulation of TNF-α-induced M-CSF synthesis by PGE2 in human bone marrow stromal cells (Besse et al., 1999). Furthermore, PGE2 is shown to down-regulate adhesion molecule-1 expression by interferon-γ via EP2 receptor in human gingival fibroblasts (Noguchi et al., 1999). Expression of VEGF mRNA by PGE2 in synovial fibroblasts has been suggested to be related to the activation of EP2 receptor (Ben-Av et al., 1995). It has also been shown that the augmentation of IL-6 production is mediated mainly by EP2 receptor whereas the suppression of TNF-α production is predominantly regulated by EP4 receptor in lipopolysaccharide-stimulated mouse neutrophils (Yamane et al., 2000). These reports indicate that EP2 and EP4 receptors mediate the production of soluble factors in cells through cyclic AMP dependent-mechanism. Accordingly, we examined whether cyclic AMP contributes to the regulation by PGE2 of the production of IL-6, M-CSF, and VEGF in synovial fibroblasts.

PGE2 is known to stimulate cyclic AMP accumulation in Swiss 3T3 fibroblasts (Burch et al., 1989). In the present study, the cyclic AMP analogues, 8-bromo-cyclic AMP and dibutyryl-cyclic AMP, themselves increased spontaneous levels of IL-6 and VEGF but reduced M-CSF level in OA fibroblasts. Furthermore, the cyclic AMP analogues mimicked the effects of PGE2 on the production of soluble factors by IL-1β, indicating that cyclic AMP is involved as a second messenger in the PGE2 effects. This is further supported by the finding that pentoxifylline, a non-selective phosphodiesterase inhibitor, also enhanced VEGF production and attenuated M-CSF production by fibroblasts stimulated with IL-1β. EP2 and EP4 receptors generate cyclic AMP production through stimulation of adenyl cyclase (Coleman et al., 1994; Negishi et al., 1995). In fact, it has been reported that the EP2 receptor agonist ONO-AE1-259 and the EP4 receptor agonist ONO-AE1-329, as well as PGE2, increase cyclic AMP formation (Yamane et al., 2000). It is therefore, well possible that cyclic AMP accumulation facilitates the process of IL-6 and VEGF production and interferes with that of M-CSF production in synovial fibroblasts, although the potentiation by PGE2 of IgE-induced IL-6 and GM-CSF production in rodent mast cells is mediated via EP1 and/or EP3 receptors with cyclic AMP-independent mechanisms (Gomi et al., 2000). Furthermore, the activation of cyclic AMP-dependent protein kinase A by EP2 and EP4 receptors may mediate the signals for the PGE2 effects (Ben-Av et al., 1995; Zeng et al., 1998; Suzawa et al., 2000). However, the signalling pathways after cyclic AMP accumulation leading to the PGE2 regulation on the production of IL-6, M-CSF, and VEGF in response to IL-1β remain to be examined.

In conclusion, we have demonstrated for the first time that endogenous PGE2 regulates, at least in part, the production of IL-6, M-CSF, and VEGF by IL-1β through the activation of EP2 and EP4 receptors in synovial fibroblasts from patients with OA and RA. This implies that in addition to the function of inflammatory mediator, PGE2 plays an important role as a regulator in the production of various soluble factors at inflammatory sites.

Acknowledgments

The authors thank Ono Pharmaceutical Co. Ltd. (Osaka, Japan) for providing us with PGE2 receptor subtype agonists such as ONO-DI-004, ONO-AE1-259, ONO-AE-248, and ONO-AE1-329. We would also like to acknowledge Dr. T. Sato, Minophagen Research Laboratory, for professional help.

Abbreviations

COX

cyclo-oxygenase

DMSO

dimethyl sulfoxide

ELISA

enzyme-linked immunosorbent assay

FCS

foetal calf serum

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

GM-CSF

granulocyte macrophage-colony stimulating factor

IL

interleukin

M-CSF

macrophage colony stimulating factor

OA

osteoarthritis

PGE2

prostaglandin E2

RA

rheumatoid arthritis

RT–PCR

reverse transcription–polymerase chain reaction

TNF-α

tumour necrosis factor-α

VEGF

vascular endothelial growth factor

α-MEM

α-minimum essential medium

References

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