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Role of the endogenous prostaglandin E2 in human lung fibroblast interleukin-11 production
RESPIRATORY
MEDICINE
(1999) 93, 637-642
Role of the endogenous prostaglandin E2 In human lung fibroblast interleukin-11 production S. MATSUI, N. YAMASHITA, T. MINO, H. TAKI, E. SUGIYAMA, R. HAYASHI, M. MARUYAMA AND M. KOBAYASHI First Department of Internal Medicine, Toyama Medical and Pharmaceutical University, Sugitani, Toyama 930-0194, Japan Interleukin-11 (IL-l 1) is known to be a member of the interleukin-6 (IL-6)-type cytokine family. IL-l 1 is likely to be a major determinant of immune regulation in acute and chronic inflammatory lung diseases,although it is not directly linked with specific diseaseprocesses. It has already been shown that although unstimulated human lung fibroblasts did not produce significant amounts of IL- 11, the addition of interleukin- lcr (IL- ICC)and/or transforming growth factor-p (TGF-/?) stimulated fibroblasts dose-dependently to produce IL-l 1. Northern blot analysis showed that these stimulators also upregulated IL-l 1 mRNA expression. As it has been previously reported that IL-l and TGF-fi stimulate prostaglandin E2 (PGE2) release from lung fibroblasts, we investigate here the role of endogenous PGEz and the direct effects of the two inhibitors of prostaglandin synthesis, indomethacin and dexamethasone, on IL-l 1 production by human lung fibroblasts. The addition of indomethacin, a cycle-oxygenaseinhibitor, resulted in significant suppression of IL-11 production and mRNA expression in lung fibroblasts. There was no detectable effect of PGE, alone on IL-11 levels; however, the suppression of IL-11 production by indomethacin was almost completely reversed by addition of PGE2. In contrast, suppression of IL-1 1 production by indomenthacin was not reversed by addition of thromboxane B2 and carbocyclic thromboxane AZ. In addition, dexamethasone completely suppressed IL-l 1 production and downregulated IL-l 1 mRNA. These results suggest that endogenous PGEz acts as an autocrine stimulus for IL-11 production by human lung fibroblasts activated by IL-la and TGF-fi. I&FIR.
MED. (1999) 93, 637-642
Introduction Macrophage/monotype-derived cytokines, including interleukin-1 (IL-l), tumour necrosis factor (TNF) and transforming growth factor-p (TGF-I), have been shown to be important contributors to the inflammatory and fibrotic processesof the lung (1,2). Recent data have suggested that the local stromal cell population, such as lung fibroblasts, is also a significant contributor to the local cytokine pool which causesinflammatory and fibrotic responsesin airway disorders (3). Interleukin-6 (IL-6), interleukin-8 (IL-8), granulocyte-macrophage colony stimulating factor (GMCSF) and granulocyte-CSF (G-CSF) are all major products of lung fibrobalsts and are found in various inflamed tissues (3-5). Interleukin-1 1 (IL-l 1) was originally identified from the primate bone marrow-derived stromal cell line, PU-34, based on its ability to stimulate the proliferation of IL-6-
Received25 September1998and acceptedin revisedform 17May 1999. Correspondenceshouldbe addressedto: S.Matsui. Fax: + 81-76434-5025.
0954-6111/99/090637+06 $12.00/O
0
1999 HARCOLIRT
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dependent cells (6). In accordance with this finding, most studies on IL-1 1 have focused on its roles in haematopoiesis (7-9). However, IL-11 has other bioactivities such as the induction of acute phase protein production (lo), B-cell activation (11) and augmentation of the production of metalloproteinase inhibitor (12). Recent studies have also demonstrated that IL-l 1 has protective effects in a number of animal models of acute and chronic inflammation or injury (13-15). This protection is due, at least in part, to the ability of IL-1 1 to regulate the production of proinflammatory cytokines and nitric oxide by macrophages (13, 15). Since lung stromal cellshave the ability to produce IL-11 in response to various stimuli (16, 17) IL-11 may have an important role in the modulation of inflammatory and fibrotic responses in human lung disorders. The products of the cycle-oxygenase pathway, such as prostaglandin Ez (PGE& also participate in inflammatory responses(18). An inhibitory effect of PGE2 on immune cell function has been documented. PGEz can inhibit the release of IL-l and TNF from myeloid cells (19). In addition, Hamilton et al. showed that fibroblast-derived colony stimulating factors were modulated by endogenous and exogenous prostanoids (20). Therefore, a link between PGEz and stromal cell cytokine production appears to be 0
1999 HARCO~RT
PUBLISHERS
LTD
638 S. MATSLJIETAL evident. We have previously demonstrated that freshly prepared rheumatoid synovial cells produced large amounts of IL-11 and the production of IL-11 by fresh rheumatoid synovial cells was regulated by PGE2 (21). However, the regulation of IL-11 expression in freshly isolated human lung fibroblasts has not been clarified. Thus, in this study, we investigated the role of endogenous PGE2 and the direct effects of the two inhibitors of prostaglandin synthesis, indomethacin and dexamethasone, on IL-l 1 production by human lung fibroblasts.
with various concentrations of IL-la and/or TGF-P for the time indicated. After incubation, the cell-free culture supernatants were stored at -20°C until assay for IL-11 or PGE2. To examine the role of endogenous PGEz on IL-la and TGF-P-induced IL-1 1 production, lung fibroblasts were incubated with IL-lx and TGF-/I in the presence and absence of 1O-5 M indomethacin or 10e6 M dexamethasone for 24 h. After incubation, the cell-free culture supernatants were stored at -20°C until assay for IL-I 1.
Materials and Methods
MEASUREMENT PROSTAGLANDIN
MATERIALS Human recombinant IL-la was kindly provided by Dainippon Pharmaceutical Co. (Osaka, Japan). Recombinant TGF-Pi was purchased from R&D Systems (Minneapolis, MN, U.S.A.). PGE2, thromboxane B2 and carbocyclic thromboxane A2 were purchased from Cayman Chemical Co. (Ann Arbor, MI, U.S.A.). Indomethacin and dexamethasone were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Dulbecco’s modified Eagle’s medium (DMEM) and phosphate-buffered saline (PBS(-)) were purchased from Nissui Pharmaceutical Co. (Tokyo, Japan).
CELL
PREPARATION
Human lung tissues were obtained from lungs of patients undergoing lobectomy because of lung cancer. The normal part of the lobe was washed with PBS and the pleural tissues and bronchi were removed after resection. Human lung fibroblasts were prepared as previously described (22). Briefly, specimens were minced and enzymatically digested with 0.5-l .O mg ml-’ clostridium collagenase (Wako Pure Chemical Industries) and 5-10 mg ml-’ deoxyribonuclease 1 (Sigma Chemical Co. St. Louis, MO, U.S.A.) for 2-3 h. After digestion, the single cells were filtrated through gauze, washed three times with DMEM and filtered through nylon mesh. The cells were resuspended in complete medium [DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U ml-’ penicillin G, 50 mg ml-’ streptomycin, 2 mM L-glutamine and non-essential amino acids (GIBCO BRL, Gland Island, NY, U.S.A.)]. The cells were cultured overnight to adhere to a plastic dish. The dish was washed to remove non-adherent cells. After three to four passages, fibroblasts were used as freshly isolated human lung fibroblasts and grown to confluence in 5% Co2/95% air in complete medium.
LUNG
FIBROBLAST
CULTURE
Lung fibroblasts were treated with typsin and plated at a density of 2 x lo5 cellsper well in 24-well flat-bottom plates. To examine whether treatment of freshly isolated human lung fibroblasts with IL-la and/or TGF-P might modulate their production of IL-l 1 or PGEs, the cells were incubated
OF IL-11 PROTEIN E2
AND
IL-11 protein was measured with an ELISA assay kit (Quantikine TM human IL-l 1 ELISA kit; R&D Systems). PGEz was measured with ELISA assay kit purchased from Neogen TM corporation (Lexington, KY, U.S.A.).
mRNA
ISOLATION
AND
ANALYSIS
Lung fibroblasts were incubated with IL-la and/or TGF-P for 24 h in the presence and absence of 1O-5 M indomethatin or 10e6 M dexamethasone. Total celluiar RNA in the treated cells was extracted by acid guanadinium thiocyanate-phenol-chloroform extraction method using ISOGEN (Nippon Gene, Tokyo, Japan), and Northern blot analysis was carried out as previously described (22). Fifteen micrograms of total RNA from each experimental condition was size fractionated by electrophoresis through 1% agarose /17% formaldehyde gels, transferred to nylon membranes (Amersham, Tokyo, Japan) and hybridized with digoxigenin-labelled cDNA probes (Boehringer Mannheim, Germany). IL-11 cDNA was a gift of Dr Paul Schendel (Genetics Institute, Cambridge, MA, U.S.A.). The intensity of the band was analysed by National Institute of Health (NIH) Image software.
STATISTICAL
ANALYSIS
Values are presented as the mean +(SEM). Data were analysed by a Student’s t-test. P-values less than 0.05 were considered as statistically significant.
Results As shown in Fig. 1, lung fibroblasts produced a trace amount of IL-11 without stimuli. IL-la or TGF-j stimulated lung fibroblasts to produce IL-11 in a dosedependent manner [Fig. l(a,b)]. Incubation with both cytokines resulted in higher IL-11 production than with either cytokine alone [Fig. l(c)]. As shown in Fig. 2, the amounts of IL-11 production in response to IL-la and TGF-P increased in a timedependent manner, and reached a maximum at 24 h. As shown in Fig. 3, unstimulated lung fibroblasts produced a trace amount of PGE2. A low level of PGEz production was detectable under TGF-P stimulation, while
PROSTAGLANDINE2 ANDINTERLEUKIN-11 PRODUCTION
639
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FIG. 1. Effect of IL-lx and TGF-1 on IL-l 1 production by freshly isolated human lung tibroblasts. Cells were incubated with various concentrations of IL-lol (a), TGF-/3 (b) or 1.0 ng ml-‘IL-la plus 5.0 ng ml-‘TGF-P (c) for 24 h. IL-11 was measured by ELISA in the supernatant. Data are expressed as mean +SEM of at least three independent experiments. *P
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FIG. 2. Time course of IL-la and TGF-P-induced IL-l 1 production by freshly isolated human lung fibroblasts. Cells were incubated with 1.0 ng ml-’ IL-lc( plus 5.0 ng m - ’ TGF-fi for the time indicated, and IL-l 1 was measured by ELISA in the supernatants. Data are expressed as mean + SEM of three independent experiments.
a significant increase in PGEz level was detectable after IL-la stimulation. Incubation with both cytokines resulted in higher PGEz production than with either cytokine alone (Fig. 3). As shown in Fig. 4, the production of IL-11 was significantly inhibited by indomethacin. In addition, dexamethasone completely inhibited IL-I 1 production (Fig. 4). The levels of IL-11 mRNA expressed by unstimulated cells were extremely low, while a significant increase in IL11 mRNA levels was observed upon stimulation with 1.0 ng ml-’ IL-la or 5.0 ngml-’ TGF-p (Fig. 5). Lung fibroblasts incubated with IL-la and TGF-P in combination contained significantly higher levels of IL-11 mRNA than fibroblasts incubated with the cytokines individually. Furthermore, indomethacin inhibited IL- 1I mRNA in
Medium
IL-la
TGF-b
IL-la + TGF-j?
FIG. 3. Effect of IL- 1cIand TGF-/3 on PGEz production by freshly isolated human lung fibroblasts. Cells were incubated with 1.0 ng ml-’ IL-la and/or 5.0 ng ml-‘TGF-P for 24 h. PGE2 was measured by ELISA in the supernatants. Data are expressed as mean ~SEM of four independent experiments. *P
640
S. MATSUI
ET AL
GAPDH
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TGF-P IL-lx IL-la IL-la +TGF-/3 ++‘FB-$ ;TzD-B
FIG. 4. Inhibitory effects of indomethacin or dexamethasone on IL-lcr and TGF-P induced IL-1 1 production by freshly isolated human lung tibroblasts. Cells were incubated with I.0 ng ml-’ IL-ICC and 5.0 ng ml-’ TGF-b in the presence and absence of lO-‘h/r indomethacin (IND) or 10M6~ dexamethasone (DEX) for 24 h. IL- 11 was measured by ELISA in the supernatants. Data are expressed as mean f SEM of three independent experiments. #P
Discussion IL-l 1 was initially reported as a haematopoietic cytokine with thrombopoietic activity (7). In addition to this haematopoietic effects, however, it has been demonstrated that IL-11 protects a variety of models of tissue from inflammation and injury (13-15). This protection is due to the ability of IL-11 to regulate macrophage production of pro-inflammatory cytokines such as TNF and IL-l. Recently, Leng and Elias reported that IL-11 can also inhibit interleukin-12 (IL-12) production by macrophages (23). Excessive IL-12 production has been implicated in the pathogenesis of a number of diseases including sarcoidosis, sepsis and Sjogren’s syndrome (24-26). Lung stromal cells have the ability to produce IL- 11 in response to cytokines, such as IL-la and TGF-fl (16) and infection with some respiratory viruses (17). Thus IL-1 1 may have an important role in the modulation of inflammatory responses in lung disorders. In agreement with previous results on lung fibroblast and epithelial cell lines (16,17), we demonstrated that freshly isolated human lung fibroblasts produce immunoreactive IL-11 in response to IL-la and TGF-P. The stimulatory effect of IL-la and TGF-8 on IL-I 1 production was detected at the concentrations of 1 .O ng ml-’ and 5.0 ng ml-‘, respectively.
MediumIL-lclTGF-P
IZ-la IL-la a-la +TGF$ +TGF-P +TGF-P +DEX +IND
FIG. 5. Inhibition of IL-lee and/or TGF-j3 induced IL-l 1 mRNA expression in freshly isolated human lung fibroblasts by indomenthacin or dexamethasone. Cells were incubated with 1.0 ng ml-i IL-la and or 5.0 ng ml-” TGF-P for 24 h in the presence and absence of lo-’ M indomethacin (IND) or 10e6 M dexamethasone (DEX). Total cellular RNA was extracted, and the levels of IL-1 1 mRNA were evaluated by Northern blot analysis. Densitometric ratios of IL-1 l/GAPDH are shown in lower panel. The data shown are the means of three independent experiments.
Prostaglandin Ez is a product of the arachidonic acid metabolism cascade and can be synthesized by a wide range of cell types. Lung fibroblasts are also a source of PGE2 during inflammatory process (27). It has been reported that IL-la and TGF-P stimulate PGEz synthesis in lung fibroblasts (28). Furthermore, PGE2 is reported to regulate IL-1 1 production positively in osteoblasts and synovial cells (21, 29). The present study showed that indomethacin blocked IL-la and TGF-fl induction of PGEz by human lung fibroblasts. It also suppressed IL-1 1 production and its gene expression in human lung fibroblasts. The suppression of IL-11 production by indomethacin was almost completely reversed by addition of exogenous PGE2. In contrast, the suppression of IL-1 1 production by indomethacin was not reversed by the addition of thromboxane B2 and carbocyclic thromboxane AZ. Therefore, endogenous PGE2 appeared to act as an autocrine stimulus for IL-1 1 production. Our data also showed that dexamethasone inhibited IL-I 1 production and mRNA expression almost completely in IL-ICC and TGF-b stimulated lung fibroblasts. However, the suppression of IL-11 production by dexamethasone was not reversed by the addition of PGE2. The
PROSTAGLANDIN
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1500
Acknowledgments
T
1000
We wish to thank Dr Sigeki Sugiyama for supplying lung tissues for fibroblasts isolation and Dr Tomohito Hamazaki for a critical review of the manuscript.
T
3 d 2
PRODUCTION
human lung tissue in order to clarify the pathogenesis of the inflamation process.
2000
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References
+
100
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6. Recovery of inhibitory effect of indomenthacin on IL-I 1 production by addition of PGE2. IL-la (I.0 ng ml-‘) and TGF-fi (5.0 ng ml-‘)-treated lung fibroblasts were incubated with 10e5 M indomethacin (IND) in the presence and absence of various concentration of PGE2. IL- 11 was measured by ELISA in the supernatants. Data are expressed as mean f SEM of three independent experiments.
FIG.
mechanisms of inhibition by dexamethasone remain to be clarified. It has been shown that glucocorticoids inhibit the induction of the gene coding for inducible cycle-oxygenase (COX-2) in monocytes and epithelial cells (30). However, glucocorticoids may not only block COX-2, but also block the synthesis of cytokines in several ways. Glucocorticoids may inhibit the synthesis of cytokine receptors, such as the IL-2 receptor (31). Several cytokines produce their cellular effects by activating transcription factors, such as AP-1 and NF-KB, which activate or repress target genes that are regulated in an opposing manner by glucocorticoid receptor (32). This activation is strongly inhibited by glucocorticoids. As the IL-l 1 gene contains the AP-I site, which is known to be important for IL-11 transcription, in the 5’ flanking lesion (33,34), dexamethasone may inhibit the production of IL-11 via the inhibition of AP-1 activity. In summary, we have demonstrated that IL-la and TGF-/I stimulated freshly isolated human lung fibroblasts to produce IL-11 in a dose- and time-dependent manner along with increased IL-11 mRNA, and that endogenous PGE:! appeared to act as an autocrine stimulus for IL-la and TGF-P-induced IL-1 1 production by lung fibroblasts. The lung is constantly exposed to various environmental stimuli, including micro-organisms and chemical compounds. Alveolar macrophages, mast cells and eosinophils stimulated with external and endogenous stimuli release many cytokines and arachidonic acid metabolites including IL- 1, TGF and PGE2. IL- 11 production by lung ‘ribroblasts is regulated by these molecules. It is important to examine the IL-1 1 production and regulation mechanisms in normal
1. Zang Y, Lee TC, Guillemin B, Yu M-C, Rom WN. Enhanced IL-l/? and tumor necrosis factor-a release and messenger RNA expression in macrophages from idiopathic pulmonary fibrosis or after asbestos exposure. J Inwnunol 1993; 150: 41884196. 2. Fine A, Goldstein RH. The effect of transforming growth factor-p on cell proliferation and collagen formation by lung fibroblasts. J Biol Chem 1987; 262: 3879-3902. 3. Rolfe MW, Kunkel SL, Standiford TJ, et al. Pulmonary fibroblast expression of interleukin-8: a model for alveolar macrophage-derived cytokine networking. Am J Respir Cell Mol Biol 1991; 5: 4933501. 4. Leizer T, Cebon J, Layton JE, Hamilton JA. Cytokine regulation of colony-stimulating factor production in cultured human synovial fibroblasts: I. induction of GM-CSF and G-CSF production by interleukin-1 and tumor necrosis factor. Blood 1990; 76: 1989-1995. 5. Elias JA, Lents V. IL-l and tumor necrosis factor synergistically stimulate fibroblast IL-6 production and stabilize IL-6 messenger RNA. J Immunol 1990; 145: 161-166. 6. Paul SR, Bennett F, Clavetti JA et al. Molecular cloning of a cDNA encoding interleukin- 11, a stromal cell-derived lymphopoietic cytokine. Proc Nat1 Acad Sci USA 1990; 87: 7512-7516. 7. Neben TY, Loebelenz J, Hayes L, et al. Recombinant human interleukin-1 1 stimulates megakaryocytopoiesis and increases peripheral platelets in normal and splenectomized mice. Blood 1993; 81: 901-908. 8. Quesniaux VFJ, Clark SC, Turner K, Fagg B. Interleukin-1 1 stimulates multiple phases of erythropoiesis in vitro. Blood 1992; 80: 1218-1223. 9. Keller DC, Du XX, Srour EF, Hoffman R, Williams DA. Interleukin-1 1 inhibits adipogenesis and stimulates myelopoiesis in human long-term marrow cultures. Blood 1993; 82: 1428-1435. 10. Baumann H, Schendel P. Interleukin-11 regulates the hepatic expression of the same plasma protein genes as interleukin-6. J Biol Chem 1991; 226: 2042420427. 11. Anderson KC, Morimoto C, Paul SR, et al. Interleukin-1 1 promotes accessory cell-dependent B-cell differentation in humans. Blood 1992; 80: 2797-2804. 12. Maier R, Ganu V, Lots M. Interleukin-1 1, an inducible cytokine in human articular chondrocytes and synoviocytes, stimulates the production of the tissue
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inhibitor of metalloproteinases. J Biol Chem 1993; 268: 21527-21532. 13. Redlich AA, Gao X, Rockwell S, Kelly M; Elias JA. IL- 11 enhances survival and decreasesTNF production after radiation-induced thoracic injury. J Immunol 1996; 157: 1705-1710. 14. Orazi A; Du X, Yang Z, Kashai M, Williams DA. Interleukin-1 1 prevents apotosis and accelerates recovery of small intestinal mucosa in mice treated with combined chemotherapy and radiation. Lab Invest 1996; 75: 33-42. 15. Trepicchio WL, Bozza M, Pedneault G, Dorner AJ. Recombinant human IL-l 1 attenuates the inflammatory response through down-regulation of proinflammatory cytokine release and nitric oxide production. J Immunol1996;
157: 3627-3634.
16. Elias JA, Zheng T, Whiting NL, et al. Interleukin-1 and transforming growth factor-p regulation of fibroblastderived IL- 11. J Immunol 1994; 152: 2421-2429. 17. Einarsson 0, Geba GP, Zhu 2, Landry M, Elias JA. Interleukin-I 1: stimulation in vivo and in vitro by respiratory viruses and induction of airways hyperresponsiveness.J Clin Invest 1996; 97: 915-924. 18. Portanova JP, Zhang Y, Anderson GD et al. Selective neutralization of prostaglandin E2 blocks inflammation, hyperalgesia, and interleukin 6 production in vivo. J Exp Med 1996; 184: 883-891. 19. Bachwich PR, Chensue SW, Larrick JW, Kunkel SL. Tumor necrosis factor stimulates interleukin-1 and prostaglandin E2 production in resting macrophages. Biochem Biophys Res Commun 1986; 136: 94-101. 20. Hamilton JA, Piccoli DS, Cebon J, et al. Cytokine regulation of colony-stimulating factor (CSF) production in cultured human synovial fibroblasts. II. Similarities and differences in the control of interleukin-l induction of granulocyte-macrophage CSF and granulocyte-CSFproduction. Blood 1992;79: 1413-1419. 21. Taki H, Sugiyama E, Mino T, Kuroda A, Kobayashi M. Differential inhibitory effects of indomethacin, dexamethasone, and interferon-gamma (IFN-y) on IL-11 production by rheumatoid synovial cells. Clin Exp Immunol 1998;112: 133-138. 22. Hayashi R, Yamashita N, Matsui S: et al. Bradykinin
stimulates interleukin-8 production by human lung fibroblasts Immunology 1998; 95: 507-511. 23. Leng SX, Elias JA. Interleukin-I 1 inhibits macrophage interleukin-12 production. J Immunol 1997; 159: 2161-2168.
24. Moller DR, Forman JD, Liu MC, ef al. Enhanced expression of IL-12 associated with Thl cytokine profiles in active pulmonary sarcoidosis. J Immounol 1996; 1564952-4960. 25. Ozmen L, Pericin M, Hakimi J, et al. Interleukin-12,
interferon-y, and tumor necrosis factor CIare the key cytokines of the generalized Shwartsman reaction. J Exp Med 1994; 180: 907-915. 26. Yanagi K, Haneji N, Hamano H, Takahashi M, Higashiyama H, Hayashi Y. In vivo role of IL-10 and IL-12 during development of Sjiigren’s syndrome in MRL/lpr mice. Cell Immunol 1996; 168: 243-250. 27. Elias JA, Gustilo K, Baeder W, Freundlich B. Synergistic stimulation of fibroblast. Prostaglandin production by recombinant interleukin 1 and turnor necrosis factor. J Immunol 1987; 138: 3812-3816. 28. Jackson BA, Goldstein RH, Roy R, Cozzani M, Taylor L, Polgar P. Effects of transforming growth factor p and interleukin-l/3 on expression of cyclooxygenase 1 and 2 and phospholipase Az mRNA in lung fibroblasts and endothelial cells in culture. Biochem Biophys Res Commun 1993; 197: 1465-1474. 29. Romas E; Udagawa N, Zhou H, et al. The role of gp 130-mediated signals in osteoclast development: regulation of interleukin 11 production by osteoblasts and distribution of its receptor in bone marrow cultures. J Exp Med 1996; 183: 258 l-2591. 30. Mitchell JA, Belvisi MG, Akarasereenont P, et al. Induction of cycle-oxygenase-2 by cytokines in human pulmonary epithelial cells: regulation by dexamethasone. Bu J Pharmacol 1994; 113: 1008-1014. 31. Grabstein K, Dower S, Gillis S, Urdal V, Larsen A. Expression of interleukin 2, interferon-r and the IL-2 receptor by human peripheral blood lymphocytes. J Inlmunol 1986; 136: 45034508. 32. Yang-Yen H-F, Chambard J-C, Sun Y-L, et al. Transcriptional interference between c-jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction. Cell 1990; 62: 1205-1215. 33. Yang L, Yang Y-C. Regulation of interleukin (IL)-11 gene expression in IL-l induced primate bone marrow stromal cells. J Biol Chem 1994; 269: 32732-32739. 34. Tang W, Yang L, Yang Y-C, Leng SX; Elias JA. Transforming growth factor-p stimulates interleukin-1 1 transcription via complex activating protein-l-dependent pathways. J Biol Chem 1998; 273: 5506-5513.
MEDICINE
(1999) 93, 637-642
Role of the endogenous prostaglandin E2 In human lung fibroblast interleukin-11 production S. MATSUI, N. YAMASHITA, T. MINO, H. TAKI, E. SUGIYAMA, R. HAYASHI, M. MARUYAMA AND M. KOBAYASHI First Department of Internal Medicine, Toyama Medical and Pharmaceutical University, Sugitani, Toyama 930-0194, Japan Interleukin-11 (IL-l 1) is known to be a member of the interleukin-6 (IL-6)-type cytokine family. IL-l 1 is likely to be a major determinant of immune regulation in acute and chronic inflammatory lung diseases,although it is not directly linked with specific diseaseprocesses. It has already been shown that although unstimulated human lung fibroblasts did not produce significant amounts of IL- 11, the addition of interleukin- lcr (IL- ICC)and/or transforming growth factor-p (TGF-/?) stimulated fibroblasts dose-dependently to produce IL-l 1. Northern blot analysis showed that these stimulators also upregulated IL-l 1 mRNA expression. As it has been previously reported that IL-l and TGF-fi stimulate prostaglandin E2 (PGE2) release from lung fibroblasts, we investigate here the role of endogenous PGEz and the direct effects of the two inhibitors of prostaglandin synthesis, indomethacin and dexamethasone, on IL-l 1 production by human lung fibroblasts. The addition of indomethacin, a cycle-oxygenaseinhibitor, resulted in significant suppression of IL-11 production and mRNA expression in lung fibroblasts. There was no detectable effect of PGE, alone on IL-11 levels; however, the suppression of IL-11 production by indomethacin was almost completely reversed by addition of PGE2. In contrast, suppression of IL-1 1 production by indomenthacin was not reversed by addition of thromboxane B2 and carbocyclic thromboxane AZ. In addition, dexamethasone completely suppressed IL-l 1 production and downregulated IL-l 1 mRNA. These results suggest that endogenous PGEz acts as an autocrine stimulus for IL-11 production by human lung fibroblasts activated by IL-la and TGF-fi. I&FIR.
MED. (1999) 93, 637-642
Introduction Macrophage/monotype-derived cytokines, including interleukin-1 (IL-l), tumour necrosis factor (TNF) and transforming growth factor-p (TGF-I), have been shown to be important contributors to the inflammatory and fibrotic processesof the lung (1,2). Recent data have suggested that the local stromal cell population, such as lung fibroblasts, is also a significant contributor to the local cytokine pool which causesinflammatory and fibrotic responsesin airway disorders (3). Interleukin-6 (IL-6), interleukin-8 (IL-8), granulocyte-macrophage colony stimulating factor (GMCSF) and granulocyte-CSF (G-CSF) are all major products of lung fibrobalsts and are found in various inflamed tissues (3-5). Interleukin-1 1 (IL-l 1) was originally identified from the primate bone marrow-derived stromal cell line, PU-34, based on its ability to stimulate the proliferation of IL-6-
Received25 September1998and acceptedin revisedform 17May 1999. Correspondenceshouldbe addressedto: S.Matsui. Fax: + 81-76434-5025.
0954-6111/99/090637+06 $12.00/O
0
1999 HARCOLIRT
PUBLISHERS
LTD
dependent cells (6). In accordance with this finding, most studies on IL-1 1 have focused on its roles in haematopoiesis (7-9). However, IL-11 has other bioactivities such as the induction of acute phase protein production (lo), B-cell activation (11) and augmentation of the production of metalloproteinase inhibitor (12). Recent studies have also demonstrated that IL-l 1 has protective effects in a number of animal models of acute and chronic inflammation or injury (13-15). This protection is due, at least in part, to the ability of IL-1 1 to regulate the production of proinflammatory cytokines and nitric oxide by macrophages (13, 15). Since lung stromal cellshave the ability to produce IL-11 in response to various stimuli (16, 17) IL-11 may have an important role in the modulation of inflammatory and fibrotic responses in human lung disorders. The products of the cycle-oxygenase pathway, such as prostaglandin Ez (PGE& also participate in inflammatory responses(18). An inhibitory effect of PGE2 on immune cell function has been documented. PGEz can inhibit the release of IL-l and TNF from myeloid cells (19). In addition, Hamilton et al. showed that fibroblast-derived colony stimulating factors were modulated by endogenous and exogenous prostanoids (20). Therefore, a link between PGEz and stromal cell cytokine production appears to be 0
1999 HARCO~RT
PUBLISHERS
LTD
638 S. MATSLJIETAL evident. We have previously demonstrated that freshly prepared rheumatoid synovial cells produced large amounts of IL-11 and the production of IL-11 by fresh rheumatoid synovial cells was regulated by PGE2 (21). However, the regulation of IL-11 expression in freshly isolated human lung fibroblasts has not been clarified. Thus, in this study, we investigated the role of endogenous PGE2 and the direct effects of the two inhibitors of prostaglandin synthesis, indomethacin and dexamethasone, on IL-l 1 production by human lung fibroblasts.
with various concentrations of IL-la and/or TGF-P for the time indicated. After incubation, the cell-free culture supernatants were stored at -20°C until assay for IL-11 or PGE2. To examine the role of endogenous PGEz on IL-la and TGF-P-induced IL-1 1 production, lung fibroblasts were incubated with IL-lx and TGF-/I in the presence and absence of 1O-5 M indomethacin or 10e6 M dexamethasone for 24 h. After incubation, the cell-free culture supernatants were stored at -20°C until assay for IL-I 1.
Materials and Methods
MEASUREMENT PROSTAGLANDIN
MATERIALS Human recombinant IL-la was kindly provided by Dainippon Pharmaceutical Co. (Osaka, Japan). Recombinant TGF-Pi was purchased from R&D Systems (Minneapolis, MN, U.S.A.). PGE2, thromboxane B2 and carbocyclic thromboxane A2 were purchased from Cayman Chemical Co. (Ann Arbor, MI, U.S.A.). Indomethacin and dexamethasone were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Dulbecco’s modified Eagle’s medium (DMEM) and phosphate-buffered saline (PBS(-)) were purchased from Nissui Pharmaceutical Co. (Tokyo, Japan).
CELL
PREPARATION
Human lung tissues were obtained from lungs of patients undergoing lobectomy because of lung cancer. The normal part of the lobe was washed with PBS and the pleural tissues and bronchi were removed after resection. Human lung fibroblasts were prepared as previously described (22). Briefly, specimens were minced and enzymatically digested with 0.5-l .O mg ml-’ clostridium collagenase (Wako Pure Chemical Industries) and 5-10 mg ml-’ deoxyribonuclease 1 (Sigma Chemical Co. St. Louis, MO, U.S.A.) for 2-3 h. After digestion, the single cells were filtrated through gauze, washed three times with DMEM and filtered through nylon mesh. The cells were resuspended in complete medium [DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U ml-’ penicillin G, 50 mg ml-’ streptomycin, 2 mM L-glutamine and non-essential amino acids (GIBCO BRL, Gland Island, NY, U.S.A.)]. The cells were cultured overnight to adhere to a plastic dish. The dish was washed to remove non-adherent cells. After three to four passages, fibroblasts were used as freshly isolated human lung fibroblasts and grown to confluence in 5% Co2/95% air in complete medium.
LUNG
FIBROBLAST
CULTURE
Lung fibroblasts were treated with typsin and plated at a density of 2 x lo5 cellsper well in 24-well flat-bottom plates. To examine whether treatment of freshly isolated human lung fibroblasts with IL-la and/or TGF-P might modulate their production of IL-l 1 or PGEs, the cells were incubated
OF IL-11 PROTEIN E2
AND
IL-11 protein was measured with an ELISA assay kit (Quantikine TM human IL-l 1 ELISA kit; R&D Systems). PGEz was measured with ELISA assay kit purchased from Neogen TM corporation (Lexington, KY, U.S.A.).
mRNA
ISOLATION
AND
ANALYSIS
Lung fibroblasts were incubated with IL-la and/or TGF-P for 24 h in the presence and absence of 1O-5 M indomethatin or 10e6 M dexamethasone. Total celluiar RNA in the treated cells was extracted by acid guanadinium thiocyanate-phenol-chloroform extraction method using ISOGEN (Nippon Gene, Tokyo, Japan), and Northern blot analysis was carried out as previously described (22). Fifteen micrograms of total RNA from each experimental condition was size fractionated by electrophoresis through 1% agarose /17% formaldehyde gels, transferred to nylon membranes (Amersham, Tokyo, Japan) and hybridized with digoxigenin-labelled cDNA probes (Boehringer Mannheim, Germany). IL-11 cDNA was a gift of Dr Paul Schendel (Genetics Institute, Cambridge, MA, U.S.A.). The intensity of the band was analysed by National Institute of Health (NIH) Image software.
STATISTICAL
ANALYSIS
Values are presented as the mean +(SEM). Data were analysed by a Student’s t-test. P-values less than 0.05 were considered as statistically significant.
Results As shown in Fig. 1, lung fibroblasts produced a trace amount of IL-11 without stimuli. IL-la or TGF-j stimulated lung fibroblasts to produce IL-11 in a dosedependent manner [Fig. l(a,b)]. Incubation with both cytokines resulted in higher IL-11 production than with either cytokine alone [Fig. l(c)]. As shown in Fig. 2, the amounts of IL-11 production in response to IL-la and TGF-P increased in a timedependent manner, and reached a maximum at 24 h. As shown in Fig. 3, unstimulated lung fibroblasts produced a trace amount of PGE2. A low level of PGEz production was detectable under TGF-P stimulation, while
PROSTAGLANDINE2 ANDINTERLEUKIN-11 PRODUCTION
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FIG. 1. Effect of IL-lx and TGF-1 on IL-l 1 production by freshly isolated human lung tibroblasts. Cells were incubated with various concentrations of IL-lol (a), TGF-/3 (b) or 1.0 ng ml-‘IL-la plus 5.0 ng ml-‘TGF-P (c) for 24 h. IL-11 was measured by ELISA in the supernatant. Data are expressed as mean +SEM of at least three independent experiments. *P
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FIG. 2. Time course of IL-la and TGF-P-induced IL-l 1 production by freshly isolated human lung fibroblasts. Cells were incubated with 1.0 ng ml-’ IL-lc( plus 5.0 ng m - ’ TGF-fi for the time indicated, and IL-l 1 was measured by ELISA in the supernatants. Data are expressed as mean + SEM of three independent experiments.
a significant increase in PGEz level was detectable after IL-la stimulation. Incubation with both cytokines resulted in higher PGEz production than with either cytokine alone (Fig. 3). As shown in Fig. 4, the production of IL-11 was significantly inhibited by indomethacin. In addition, dexamethasone completely inhibited IL-I 1 production (Fig. 4). The levels of IL-11 mRNA expressed by unstimulated cells were extremely low, while a significant increase in IL11 mRNA levels was observed upon stimulation with 1.0 ng ml-’ IL-la or 5.0 ngml-’ TGF-p (Fig. 5). Lung fibroblasts incubated with IL-la and TGF-P in combination contained significantly higher levels of IL-11 mRNA than fibroblasts incubated with the cytokines individually. Furthermore, indomethacin inhibited IL- 1I mRNA in
Medium
IL-la
TGF-b
IL-la + TGF-j?
FIG. 3. Effect of IL- 1cIand TGF-/3 on PGEz production by freshly isolated human lung fibroblasts. Cells were incubated with 1.0 ng ml-’ IL-la and/or 5.0 ng ml-‘TGF-P for 24 h. PGE2 was measured by ELISA in the supernatants. Data are expressed as mean ~SEM of four independent experiments. *P
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FIG. 4. Inhibitory effects of indomethacin or dexamethasone on IL-lcr and TGF-P induced IL-1 1 production by freshly isolated human lung tibroblasts. Cells were incubated with I.0 ng ml-’ IL-ICC and 5.0 ng ml-’ TGF-b in the presence and absence of lO-‘h/r indomethacin (IND) or 10M6~ dexamethasone (DEX) for 24 h. IL- 11 was measured by ELISA in the supernatants. Data are expressed as mean f SEM of three independent experiments. #P
Discussion IL-l 1 was initially reported as a haematopoietic cytokine with thrombopoietic activity (7). In addition to this haematopoietic effects, however, it has been demonstrated that IL-11 protects a variety of models of tissue from inflammation and injury (13-15). This protection is due to the ability of IL-11 to regulate macrophage production of pro-inflammatory cytokines such as TNF and IL-l. Recently, Leng and Elias reported that IL-11 can also inhibit interleukin-12 (IL-12) production by macrophages (23). Excessive IL-12 production has been implicated in the pathogenesis of a number of diseases including sarcoidosis, sepsis and Sjogren’s syndrome (24-26). Lung stromal cells have the ability to produce IL- 11 in response to cytokines, such as IL-la and TGF-fl (16) and infection with some respiratory viruses (17). Thus IL-1 1 may have an important role in the modulation of inflammatory responses in lung disorders. In agreement with previous results on lung fibroblast and epithelial cell lines (16,17), we demonstrated that freshly isolated human lung fibroblasts produce immunoreactive IL-11 in response to IL-la and TGF-P. The stimulatory effect of IL-la and TGF-8 on IL-I 1 production was detected at the concentrations of 1 .O ng ml-’ and 5.0 ng ml-‘, respectively.
MediumIL-lclTGF-P
IZ-la IL-la a-la +TGF$ +TGF-P +TGF-P +DEX +IND
FIG. 5. Inhibition of IL-lee and/or TGF-j3 induced IL-l 1 mRNA expression in freshly isolated human lung fibroblasts by indomenthacin or dexamethasone. Cells were incubated with 1.0 ng ml-i IL-la and or 5.0 ng ml-” TGF-P for 24 h in the presence and absence of lo-’ M indomethacin (IND) or 10e6 M dexamethasone (DEX). Total cellular RNA was extracted, and the levels of IL-1 1 mRNA were evaluated by Northern blot analysis. Densitometric ratios of IL-1 l/GAPDH are shown in lower panel. The data shown are the means of three independent experiments.
Prostaglandin Ez is a product of the arachidonic acid metabolism cascade and can be synthesized by a wide range of cell types. Lung fibroblasts are also a source of PGE2 during inflammatory process (27). It has been reported that IL-la and TGF-P stimulate PGEz synthesis in lung fibroblasts (28). Furthermore, PGE2 is reported to regulate IL-1 1 production positively in osteoblasts and synovial cells (21, 29). The present study showed that indomethacin blocked IL-la and TGF-fl induction of PGEz by human lung fibroblasts. It also suppressed IL-1 1 production and its gene expression in human lung fibroblasts. The suppression of IL-11 production by indomethacin was almost completely reversed by addition of exogenous PGE2. In contrast, the suppression of IL-1 1 production by indomethacin was not reversed by the addition of thromboxane B2 and carbocyclic thromboxane AZ. Therefore, endogenous PGE2 appeared to act as an autocrine stimulus for IL-1 1 production. Our data also showed that dexamethasone inhibited IL-I 1 production and mRNA expression almost completely in IL-ICC and TGF-b stimulated lung fibroblasts. However, the suppression of IL-11 production by dexamethasone was not reversed by the addition of PGE2. The
PROSTAGLANDIN
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Acknowledgments
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1000
We wish to thank Dr Sigeki Sugiyama for supplying lung tissues for fibroblasts isolation and Dr Tomohito Hamazaki for a critical review of the manuscript.
T
3 d 2
PRODUCTION
human lung tissue in order to clarify the pathogenesis of the inflamation process.
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References
+
100
1000 (ngru-‘)
6. Recovery of inhibitory effect of indomenthacin on IL-I 1 production by addition of PGE2. IL-la (I.0 ng ml-‘) and TGF-fi (5.0 ng ml-‘)-treated lung fibroblasts were incubated with 10e5 M indomethacin (IND) in the presence and absence of various concentration of PGE2. IL- 11 was measured by ELISA in the supernatants. Data are expressed as mean f SEM of three independent experiments.
FIG.
mechanisms of inhibition by dexamethasone remain to be clarified. It has been shown that glucocorticoids inhibit the induction of the gene coding for inducible cycle-oxygenase (COX-2) in monocytes and epithelial cells (30). However, glucocorticoids may not only block COX-2, but also block the synthesis of cytokines in several ways. Glucocorticoids may inhibit the synthesis of cytokine receptors, such as the IL-2 receptor (31). Several cytokines produce their cellular effects by activating transcription factors, such as AP-1 and NF-KB, which activate or repress target genes that are regulated in an opposing manner by glucocorticoid receptor (32). This activation is strongly inhibited by glucocorticoids. As the IL-l 1 gene contains the AP-I site, which is known to be important for IL-11 transcription, in the 5’ flanking lesion (33,34), dexamethasone may inhibit the production of IL-11 via the inhibition of AP-1 activity. In summary, we have demonstrated that IL-la and TGF-/I stimulated freshly isolated human lung fibroblasts to produce IL-11 in a dose- and time-dependent manner along with increased IL-11 mRNA, and that endogenous PGE:! appeared to act as an autocrine stimulus for IL-la and TGF-P-induced IL-1 1 production by lung fibroblasts. The lung is constantly exposed to various environmental stimuli, including micro-organisms and chemical compounds. Alveolar macrophages, mast cells and eosinophils stimulated with external and endogenous stimuli release many cytokines and arachidonic acid metabolites including IL- 1, TGF and PGE2. IL- 11 production by lung ‘ribroblasts is regulated by these molecules. It is important to examine the IL-1 1 production and regulation mechanisms in normal
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S. MATSUI
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