Skip to main content
NCBI home page
Annals of the Rheumatic Diseases logoLink to Annals of the Rheumatic Diseases
. 2004 Oct;63(10):1197–1204. doi: 10.1136/ard.2003.011163

EP2/EP4 signalling inhibits monocyte chemoattractant protein-1 production induced by interleukin 1ß in synovial fibroblasts

R Largo 1, I Diez-Ortego 1, O Sanchez-Pernaute 1, M Lopez-Armada 1, M Alvarez-Soria 1, J Egido 1, G Herrero-Beaumont 1
PMCID: PMC1754778  PMID: 15361371

Abstract

Background: Besides its proinflammatory properties, prostaglandin E2 (PGE2) acts as a regulator of the expression of inducible genes. Inhibition of PGE2 synthesis might thus result in a paradoxical deleterious effect on inflammation.

Objective: To examine the effect of PGE2 on monocyte chemoattractant protein-1 (MCP-1) expression in cultured synovial fibroblasts (SF) stimulated with interleukin (IL)1ß.

Methods: MCP-1 expression was assessed in SF stimulated with IL1ß in the presence of PGE2 or different NSAIDs by RT-PCR or northern blot and immunocytochemistry. Expression of cyclo-oxygenase (COX) isoforms was studied by western blot techniques. The role of PGE2 receptors (EP) in PGE2 action was assessed employing EP receptor subtype-specific agonists.

Results: PGE2 significantly inhibited IL1ß induced MCP-1 expression in SF in a dose dependent manner. IL1ß increased COX-2 and did not alter COX-1 synthesis in SF. 11-Deoxy-PGE1, an EP2/EP4 agonist, reproduced PGE2 action on MCP-1 expression. Butaprost, a selective EP2 agonist, was less potent than PGE2. Sulprostone, an EP1/EP3 agonist, had no effect on IL1ß induced MCP-1 expression. Inhibition of endogenous PGE2 synthesis by NSAIDs further enhanced MCP-1 mRNA expression in IL1ß stimulated SF, an effect prevented by addition of exogenous PGE2.

Conclusion: Activation of EP2/EP4 receptors down regulates the expression of MCP-1 in IL1ß stimulated SF, while PGE2 pharmacological inhibition cuts off this signalling pathway and results in a superinduction of MCP-1 expression. The data suggest that NSAIDs may intercept a natural regulatory circuit controlling the magnitude of inflammation, which questions their continuous administration in inflammatory joint diseases.

Full Text

The Full Text of this article is available as a PDF (287.5 KB).

Figure 1.

Figure 1

Open in a new tab

 Effect of PGE2 on MCP-1 expression induced by IL1ß in human and rabbit SF. Cells were incubated for 4 hours with 10 U/ml IL1ß and increasing concentrations of PGE2. (A) A representative northern blot autoradiograph of four different experiments carried out in human SF. Panels (B) and (C) show the average increase of MCP-1 mRNA levels in human and rabbit SF, respectively, compared with non-stimulated cells (n = 4). Data are expressed in arbitrary densitometric units (AU) after correction by mRNA levels of GAPDH, which was employed as housekeeping gene. 10–6 M PGE2 did not alter MCP-1 gene expression v vehicle. *p<0.05 v vehicle; †p<0.05 v IL1ß alone.

Figure 2.

Figure 2

Open in a new tab

 MCP-1 protein detection by immunofluorescence. In untreated human SF, a slight cytoplasmic immunoreactivity to anti-MCP-1 was found (A). Cells treated with 10 U/ml IL1ß for 4 hours showed an intense cytoplasmic fluorescence with the antibody (B). Co-incubation with 10 U/ml IL1ß and 10–6 M PGE2 markedly diminished the MCP-1 signal, in comparison with cells stimulated with IL1ß alone (C). Exposure of SF to 10–6 M PGE2 did not alter the MCP-1 immunostaining pattern in comparison with vehicle (D).

Figure 3.

Figure 3

Open in a new tab

 PGE2 release (A) and COX isoforms (B) in SF incubated with IL1ß. (A) PGE2 release was measured in rabbit SF culture supernatants in triplicate at 4 hours of incubation with 10 U/ml IL1ß and/or 10–6 M MXC or 10–6 M DCF (pg/ml; n = 2). Untreated cells: 248 (21); 10 U/ml IL1ß: 1643 (453); 10–6 M MXC: 110 (25); 10–6 M DCF: 39 (0); MXC+IL1ß: 104.5 (18); DCF+IL1ß: 57.5 (32). (B) Human SF were stimulated with 10 U/ml IL1ß and/or 10–6 M PGE2 for 4 hours. Upper panel: representative western blots of COX-2, COX-1, and α-tubulin are shown. Lower panel: a densitometric analysis of COX-2 levels expressed in arbitrary units (AU) after correction by α-tubulin (n = 4). *p<0.05 v vehicle; †p<0.05 v IL1ß alone.

Figure 4.

Figure 4

Open in a new tab

 Effect of PGE2 synthesis inhibition mediated by NSAIDs on MCP-1 expression. Rabbit SF were pre-incubated for 1 hour with 10–6 M MXC or 10–6 M DCF, and then 10 U/ml IL1ß and/or 10–6 M PGE2 were added for 4 hours. Upper panel shows a representative RT-PCR autoradiograph of MCP-1 and GAPDH. Lower panel shows the analysis of MCP-1 gene expression expressed in densitometric arbitrary units (AU) after normalisation by GAPDH levels (n = 4). *p<0.05 v vehicle; †p<0.05 v IL1ß alone; ‡p<0.05 v IL1ß+PGE2; **p<0.05 v the NSAID+IL1ß.

Figure 5.

Figure 5

Open in a new tab

 Effect of specific EP receptor-subtype agonists on MCP-1 expression induced by IL1ß. The role of EP receptors in MCP-1 regulation was studied in rabbit SF by RT-PCR, at 4 hours of incubation with 10 U/ml IL1ß and/or the following molecules: the EP2/EP4 agonist 11-deoxy-PGE1 (11-D; 10–6 M), the EP2 agonist butaprost (BT; 10–6 M), the EP1/EP3 agonist sulprostone (SP; 10–6 M), PGE2 (10–6 M), and the AC inhibitor MDL-12,330A (MDL, 10–5 M, 30 minutes preincubation). Upper panel shows a representative autoradiograph of MCP-1 and the housekeeping gene GAPDH. Lower panel shows the analysis for MCP-1 expression in densitometric arbitrary units (AU) relative to that of GAPDH (n = 4). *p<0.05 v vehicle; †p<0.05 v IL1ß alone.

Figure 6.

Figure 6

Open in a new tab

 Effect of PGE2 or the EP2/EP4 specific agonist 11-deoxy-PGE1 (11-D) on NF-κB DNA binding induced by IL1ß. Human SF were incubated with 10 U/ml IL1ß and/or 10–6 M PGE2 or 10–6 M 11-deoxy-PGE1 for 60 minutes, and NF-κB activity was measured by EMSA. A representative autoradiograph of four different experiments with similar results is shown.

Figure 7.

Figure 7

Open in a new tab

 Signalling pathways controlling MCP-1 expression in SF, in which IL1ß and PGE2 are involved. Mechanisms triggered by IL1ß (pink arrows); PGE2 dependent signals (blue track); effects of NSAIDs (orange); action of MDL-12,330A, an AC inhibitor (green). Crossed bars: blockade of pathways; continuous lines: mechanisms explored in our study; broken lines: data from published reports. PGE2 receptors (EP1 to EP4) are shown in connection with each specific agonist employed in this study and with their second messengers. IL1ß is one of the strongest activators of SF in inflammation. Many of its actions are mediated by PGE2 prompt synthesis. This is warranted by (a) activation of phospholipase A2 (PLA2) with generation of arachidonic acid (AA), which is the substrate for the action of COX and (b) increased availability of COX-2, whose synthesis is activated by the cytokine. IL1ß promotes recruitment of mononuclear cells to the inflamed joint by the induction of MCP-1 gene expression in SF. IL1ß is an activator of NF-κB, which controls transcription of MCP-1. At the same time, levels of PGE2 increase in response to IL1ß. When targeting EP4, PGE2 could act by tempering inflammation. Addition of PGE2 or EP4 agonists leads to a down regulation of MCP-1 expression induced by IL1ß in SF. Thus, IL1ß could initiate lesion producing mechanism as well as restoring mechanisms in the target cell. It may even increase cell sensitivity to PGE2 by up regulating the expression of EP4 receptors. EP4 is coupled with cAMP generation, being the second messenger able to block translocation to the nucleus of NF-κB active components, where they trigger transcription of inducible genes, such as MCP-1. Thus, addition of MDL-12,330A, an inhibitor of AC, to IL1ß plus PGE2, impairs PGE2 regulation of MCP-1 gene transcription. Finally, the regulating circuit is intercepted by the employment of NSAIDs owing to the dramatic reduction of PGE2 levels that these molecules provoke. Furthermore, NSAID blockade of COX may account for deviation of AA metabolism to the production of proinflammatory mediators generated by lipoxygenases (LO), some of which can activate NF-κB.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Accomazzo Maria Rosa, Cattaneo Sarah, Nicosia Simonetta, Rovati G. Enrico. Bell-shaped curves for prostaglandin-induced modulation of adenylate cyclase: two mutually opposing effects. Eur J Pharmacol. 2002 Nov 15;454(2-3):107–114. doi: 10.1016/s0014-2999(02)02486-x. [DOI] [PubMed] [Google Scholar]
  2. Akahoshi T., Wada C., Endo H., Hirota K., Hosaka S., Takagishi K., Kondo H., Kashiwazaki S., Matsushima K. Expression of monocyte chemotactic and activating factor in rheumatoid arthritis. Regulation of its production in synovial cells by interleukin-1 and tumor necrosis factor. Arthritis Rheum. 1993 Jun;36(6):762–771. doi: 10.1002/art.1780360605. [DOI] [PubMed] [Google Scholar]
  3. Badolato R., Oppenheim J. J. Role of cytokines, acute-phase proteins, and chemokines in the progression of rheumatoid arthritis. Semin Arthritis Rheum. 1996 Oct;26(2):526–538. doi: 10.1016/s0049-0172(96)80041-2. [DOI] [PubMed] [Google Scholar]
  4. Baggiolini M., Dewald B., Moser B. Interleukin-8 and related chemotactic cytokines--CXC and CC chemokines. Adv Immunol. 1994;55:97–179. [PubMed] [Google Scholar]
  5. Bertolini A., Ottani A., Sandrini M. Dual acting anti-inflammatory drugs: a reappraisal. Pharmacol Res. 2001 Dec;44(6):437–450. doi: 10.1006/phrs.2001.0872. [DOI] [PubMed] [Google Scholar]
  6. Bonizzi G., Piette J., Schoonbroodt S., Greimers R., Havard L., Merville M. P., Bours V. Reactive oxygen intermediate-dependent NF-kappaB activation by interleukin-1beta requires 5-lipoxygenase or NADPH oxidase activity. Mol Cell Biol. 1999 Mar;19(3):1950–1960. doi: 10.1128/mcb.19.3.1950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  8. De Benedetti F., Pignatti P., Bernasconi S., Gerloni V., Matsushima K., Caporali R., Montecucco C. M., Sozzani S., Fantini F., Martini A. Interleukin 8 and monocyte chemoattractant protein-1 in patients with juvenile rheumatoid arthritis. Relation to onset types, disease activity, and synovial fluid leukocytes. J Rheumatol. 1999 Feb;26(2):425–431. [PubMed] [Google Scholar]
  9. DeWitt D. L. Prostaglandin endoperoxide synthase: regulation of enzyme expression. Biochim Biophys Acta. 1991 May 8;1083(2):121–134. doi: 10.1016/0005-2760(91)90032-d. [DOI] [PubMed] [Google Scholar]
  10. Ellingsen T., Buus A., Stengaard-Pedersen K. Plasma monocyte chemoattractant protein 1 is a marker for joint inflammation in rheumatoid arthritis. J Rheumatol. 2001 Jan;28(1):41–46. [PubMed] [Google Scholar]
  11. Goetzl E. J., An S., Smith W. L. Specificity of expression and effects of eicosanoid mediators in normal physiology and human diseases. FASEB J. 1995 Aug;9(11):1051–1058. doi: 10.1096/fasebj.9.11.7649404. [DOI] [PubMed] [Google Scholar]
  12. Gutierrez S., Palacios I., Sánchez-Pernaute O., Hernández P., Moreno J., Egido J., Herrero-Beaumont G. SAMe restores the changes in the proliferation and in the synthesis of fibronectin and proteoglycans induced by tumour necrosis factor alpha on cultured rabbit synovial cells. Br J Rheumatol. 1997 Jan;36(1):27–31. doi: 10.1093/rheumatology/36.1.27. [DOI] [PubMed] [Google Scholar]
  13. Gómez-Garre D., Largo R., Tejera N., Fortes J., Manzarbeitia F., Egido J. Activation of NF-kappaB in tubular epithelial cells of rats with intense proteinuria: role of angiotensin II and endothelin-1. Hypertension. 2001 Apr;37(4):1171–1178. doi: 10.1161/01.hyp.37.4.1171. [DOI] [PubMed] [Google Scholar]
  14. Hachicha M., Rathanaswami P., Schall T. J., McColl S. R. Production of monocyte chemotactic protein-1 in human type B synoviocytes. Synergistic effect of tumor necrosis factor alpha and interferon-gamma. Arthritis Rheum. 1993 Jan;36(1):26–34. doi: 10.1002/art.1780360106. [DOI] [PubMed] [Google Scholar]
  15. Harigai M., Hara M., Yoshimura T., Leonard E. J., Inoue K., Kashiwazaki S. Monocyte chemoattractant protein-1 (MCP-1) in inflammatory joint diseases and its involvement in the cytokine network of rheumatoid synovium. Clin Immunol Immunopathol. 1993 Oct;69(1):83–91. doi: 10.1006/clin.1993.1153. [DOI] [PubMed] [Google Scholar]
  16. Haringman J. J., Kraan M. C., Smeets T. J. M., Zwinderman K. H., Tak P. P. Chemokine blockade and chronic inflammatory disease: proof of concept in patients with rheumatoid arthritis. Ann Rheum Dis. 2003 Aug;62(8):715–721. doi: 10.1136/ard.62.8.715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ishibashi R., Tanaka I., Kotani M., Muro S., Goto M., Sugawara A., Mukoyama M., Sugimoto Y., Ichikawa A., Narumiya S. Roles of prostaglandin E receptors in mesangial cells under high-glucose conditions. Kidney Int. 1999 Aug;56(2):589–600. doi: 10.1046/j.1523-1755.1999.00566.x. [DOI] [PubMed] [Google Scholar]
  18. Kabashima Kenji, Saji Tomomi, Murata Takahiko, Nagamachi Miyako, Matsuoka Toshiyuki, Segi Eri, Tsuboi Kazuhito, Sugimoto Yukihiko, Kobayashi Takuya, Miyachi Yoshiki. The prostaglandin receptor EP4 suppresses colitis, mucosal damage and CD4 cell activation in the gut. J Clin Invest. 2002 Apr;109(7):883–893. doi: 10.1172/JCI14459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kasama T., Strieter R. M., Lukacs N. W., Lincoln P. M., Burdick M. D., Kunkel S. L. Interleukin-10 expression and chemokine regulation during the evolution of murine type II collagen-induced arthritis. J Clin Invest. 1995 Jun;95(6):2868–2876. doi: 10.1172/JCI117993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Katrib Amel, Smith Malcolm D., Ahern Michael J., Slavotinek John, Stafford Leanne, Cuello Carolyn, Bertouch James V., McNeil H. Patrick, Youssef Peter P. Reduced chemokine and matrix metalloproteinase expression in patients with rheumatoid arthritis achieving remission. J Rheumatol. 2003 Jan;30(1):10–21. [PubMed] [Google Scholar]
  21. López-Armada M. J., Sánchez-Pernaute O., Largo R., Diez-Ortego I., Palacios I., Egido J., Herrero-Beaumont G. Modulation of cell recruitment by anti-inflammatory agents in antigen-induced arthritis. Ann Rheum Dis. 2002 Nov;61(11):1027–1030. doi: 10.1136/ard.61.11.1027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nanki T., Nagasaka K., Hayashida K., Saita Y., Miyasaka N. Chemokines regulate IL-6 and IL-8 production by fibroblast-like synoviocytes from patients with rheumatoid arthritis. J Immunol. 2001 Nov 1;167(9):5381–5385. doi: 10.4049/jimmunol.167.9.5381. [DOI] [PubMed] [Google Scholar]
  23. Narumiya S., Sugimoto Y., Ushikubi F. Prostanoid receptors: structures, properties, and functions. Physiol Rev. 1999 Oct;79(4):1193–1226. doi: 10.1152/physrev.1999.79.4.1193. [DOI] [PubMed] [Google Scholar]
  24. Noguchi K., Iwasaki K., Shitashige M., Ishikawa I. Prostaglandin E2 receptors of the EP2 and EP4 subtypes downregulate tumor necrosis factor alpha-induced intercellular adhesion molecule-1 expression in human gingival fibroblasts. J Periodontal Res. 1999 Nov;34(8):478–485. doi: 10.1111/j.1600-0765.1999.tb02284.x. [DOI] [PubMed] [Google Scholar]
  25. Noguchi Kazuyuki, Shitashige Miki, Endo Hirahito, Kondo Hirobumi, Ishikawa Isao. Binary regulation of interleukin (IL)-6 production by EP1 and EP2/EP4 subtypes of PGE2 receptors in IL-1beta-stimulated human gingival fibroblasts. J Periodontal Res. 2002 Feb;37(1):29–36. doi: 10.1034/j.1600-0765.2002.00641.x. [DOI] [PubMed] [Google Scholar]
  26. Ollivier V., Parry G. C., Cobb R. R., de Prost D., Mackman N. Elevated cyclic AMP inhibits NF-kappaB-mediated transcription in human monocytic cells and endothelial cells. J Biol Chem. 1996 Aug 23;271(34):20828–20835. doi: 10.1074/jbc.271.34.20828. [DOI] [PubMed] [Google Scholar]
  27. Palacios I., Lopez-Armada M. J., Hernandez P., Sanchez-Pernaute O., Gutierrez S., Miguelez R., Martinez J., Egido J., Herrero-Beaumont G. Tenidap decreases IL-8 and monocyte chemotactic peptide-1 (MCP-1) mRNA expression in the synovial tissue of rabbits with antigen arthritis and in cultured synovial cells. Clin Exp Immunol. 1998 Mar;111(3):588–596. doi: 10.1046/j.1365-2249.1998.00530.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pulsatelli L., Dolzani P., Piacentini A., Silvestri T., Ruggeri R., Gualtieri G., Meliconi R., Facchini A. Chemokine production by human chondrocytes. J Rheumatol. 1999 Sep;26(9):1992–2001. [PubMed] [Google Scholar]
  29. Schmitz T., Leroy M. J., Dallot E., Breuiller-Fouche M., Ferre F., Cabrol D. Interleukin-1beta induces glycosaminoglycan synthesis via the prostaglandin E2 pathway in cultured human cervical fibroblasts. Mol Hum Reprod. 2003 Jan;9(1):1–8. doi: 10.1093/molehr/gag007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Seitz M., Loetscher P., Dewald B., Towbin H., Ceska M., Baggiolini M. Production of interleukin-1 receptor antagonist, inflammatory chemotactic proteins, and prostaglandin E by rheumatoid and osteoarthritic synoviocytes--regulation by IFN-gamma and IL-4. J Immunol. 1994 Feb 15;152(4):2060–2065. [PubMed] [Google Scholar]
  31. Tak P. P., Smeets T. J., Daha M. R., Kluin P. M., Meijers K. A., Brand R., Meinders A. E., Breedveld F. C. Analysis of the synovial cell infiltrate in early rheumatoid synovial tissue in relation to local disease activity. Arthritis Rheum. 1997 Feb;40(2):217–225. doi: 10.1002/art.1780400206. [DOI] [PubMed] [Google Scholar]
  32. Takahashi Hideo K., Iwagaki Hiromi, Yoshino Tadashi, Mori Shuji, Morichika Toshihiko, Itoh Hideyuki, Yokoyama Minori, Kubo Shinichiro, Kondo Eisaku, Akagi Tadaatsu. Prostaglandin E(2) inhibits IL-18-induced ICAM-1 and B7.2 expression through EP2/EP4 receptors in human peripheral blood mononuclear cells. J Immunol. 2002 May 1;168(9):4446–4454. doi: 10.4049/jimmunol.168.9.4446. [DOI] [PubMed] [Google Scholar]
  33. Takayama Kiyoshi, García-Cardena Guillermo, Sukhova Galina K., Comander Jason, Gimbrone Michael A., Jr, Libby Peter. Prostaglandin E2 suppresses chemokine production in human macrophages through the EP4 receptor. J Biol Chem. 2002 Sep 4;277(46):44147–44154. doi: 10.1074/jbc.M204810200. [DOI] [PubMed] [Google Scholar]
  34. Tang L., Loutzenhiser K., Loutzenhiser R. Biphasic actions of prostaglandin E(2) on the renal afferent arteriole : role of EP(3) and EP(4) receptors. Circ Res. 2000 Mar 31;86(6):663–670. doi: 10.1161/01.res.86.6.663. [DOI] [PubMed] [Google Scholar]
  35. Ueda A., Okuda K., Ohno S., Shirai A., Igarashi T., Matsunaga K., Fukushima J., Kawamoto S., Ishigatsubo Y., Okubo T. NF-kappa B and Sp1 regulate transcription of the human monocyte chemoattractant protein-1 gene. J Immunol. 1994 Sep 1;153(5):2052–2063. [PubMed] [Google Scholar]
  36. Villiger P. M., Terkeltaub R., Lotz M. Production of monocyte chemoattractant protein-1 by inflamed synovial tissue and cultured synoviocytes. J Immunol. 1992 Jul 15;149(2):722–727. [PubMed] [Google Scholar]
  37. Yanni G., Whelan A., Feighery C., Bresnihan B. Synovial tissue macrophages and joint erosion in rheumatoid arthritis. Ann Rheum Dis. 1994 Jan;53(1):39–44. doi: 10.1136/ard.53.1.39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yoshida T., Sakamoto H., Horiuchi T., Yamamoto S., Suematsu A., Oda H., Koshihara Y. Involvement of prostaglandin E(2) in interleukin-1alpha-induced parathyroid hormone-related peptide production in synovial fibroblasts of patients with rheumatoid arthritis. J Clin Endocrinol Metab. 2001 Jul;86(7):3272–3278. doi: 10.1210/jcem.86.7.7687. [DOI] [PubMed] [Google Scholar]

Articles from Annals of the Rheumatic Diseases are provided here courtesy of BMJ Publishing Group

RESOURCES