- Email: [email protected]
REGULATION OF INTERLEUKIN 6 PRODUCTION IN A HUMAN GASTRIC EPITHELIAL CELL LINE MKN-28
doi:10.1006/cyto.1999.0604, available online at http://www.idealibrary.com on
SHORT COMMUNICATION
REGULATION OF INTERLEUKIN 6 PRODUCTION IN A HUMAN GASTRIC EPITHELIAL CELL LINE MKN-28 Song-Ze Ding,1 Chi-Hin Cho,2 Shiu-Kum Lam1 Interleukin (IL-) 6 is closely related to gastrointestinal diseases. The question of whether gastric epithelial cell contributes to IL-6 production remains undefined. We aim to evaluate the regulatory pathway of IL-6 expression in gastric epithelial cells, by using different inflammatory cytokines, endotoxin, or protein kinase modulators. IL-6 was measured by ELISA. Phorbol-12myristate-13-acetate (PMA), calcium ionophore A23187, TNF-, IL-1, oncostatin M (OSM) but not lipopolysaccharide stimulated IL-6 production from gastric epithelial cell line MKN-28. Blocking protein tyrosine kinase (PTK) activation by herbimycin A or genistein, or blocking NF-B activation by pyrrolidinedithiocarbamate, reduced the IL-6 expression induced by TNF-, IL-1 and OSM. Dexamethasone mimicked this effect. Protein kinase (PK) C inhibitor only reduced the PMA and OSM induced IL-6 production. Both inhibitors and activators for PKA and G-protein as well as IL-10 had no effects on IL-6 expression. These results indicate that inflammatory cytokines are crucial for IL-6 regulation in gastric epithelial cells. The IL-6 signal pathway is mediated through PTK, NF-B, and also involve PKC, intracellular calcium and sensitive to dexamethasone, but is not related to PKA, G-protein and IL-10. 2000 Academic Press
Interleukin (IL-) 6 is involved in the pathogenesis of gastrointestinal diseases, including inflammatory bowel disease and Helicobacter pylori infection-related disease.1,2 In H. pylori-infected gastritis patients, the mucosal IL-6 levels are significantly increased. This increase, together with mucosal TNF-, correlate with mucosal polymorphonuclear or monocyte infiltration and H. pylori colonization. More interestingly, mucosal IL-6 is found to be closely related to the mucosal TNF- level,2 suggesting that in addition to TNF-, IL-1 and IL-8, IL-6 is also involved in gastric mucosal inflammatory processes. IL-6 is a pleiotropic cytokine. Its major functions include regulation of From the Departments of 1Medicine and 2Pharmacology, The University of Hong Kong, Hong Kong, China Correspondence to: Prof. Shiu-Kum Lam, Head, Department of Medicine, Queen Mary Hospital, The University of Hong Kong, 102 Pokfulam Road, Hong Kong, China; E-mail: [email protected] Received 15 September 1998; received in revised form 20 August 1999; accepted for publication 25 August 1999 2000 Academic Press 1043–4666/00/071129+07 $35.00/0 KEY WORDS: tumour necrosis factor-/interleukin 1/oncostatin M/MKN-28/protein kinase/dexamethasone/signal transduction/ gastric epithelial CYTOKINE, Vol. 12, No. 7 (July), 2000: pp 1129–1135
growth and differentiation of various cell types, haematopoiesis and inflammation. IL-6 induces final maturation of B cells into antibody producing cells and stimulates T cell growth and cytotoxic T cell differentiation. It also induces acute phase reactions in response to inflammation or tissue injury.3,4 IL-6 has also been indicated to play an essential role in the development of B cell lymphoproliferation or malignancy.5,6 This may have particular significance during H. pylori infection, since this has recently been demonstrated closely related to the development of B cell lymphoma.7 Our unpublished data indeed indicate that both HP and its toxin stimulate IL-6 expression from gastric epithelial cell line MKN-28. Except direct stimulation by these pathogenic substances, we hypothesize that IL-6 may also be induced by inflammatory cytokines such as TNF- or IL-1. This hypothesis is also supported by the fact that gastric epithelial cells are able to express IL-6 mRNA. Some gastric cancer cell lines are also able to produce IL-6 when cultured in large quantity in vitro.8 However, it is still unknown regarding the source of IL-6 in gastric mucosa. Also if the gastric epithelial cell can express IL-6 after inflammatory cytokine stimulation, it would be interesting to 1129
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study the signal pathways that mediate such a response. In the present study, we examined the production of IL-6 from gastric epithelial cell line MKN-28 when stimulated by inflammatory cytokines TNF-, IL-1, oncostatin M (OSM) and lipopolysaccharide (LPS). We also investigated the possible signal pathways which might involve in the IL-6 expression, including protein kinase (PK), intracellular calcium, G-protein and nuclear transcription factor NF-B.
CYTOKINE, Vol. 12, No. 7 (July, 2000: 1129–1135)
Effects of protein tyrosine kinase and NF-B inhibitors on IL-6 expression Herbimycin A (Fig. 3A), genistein (Fig. 3B) and NF-B inhibitor pyrrolidinedithiocarbamate (PDTC, Fig. 3C) showed dose-dependently reduced TNF--, IL-1- and OSM-induced IL-6 expression from MKN-28 cells. Significant difference was shown over the control (P<0.05–0.01).
Effects of dexamethasone and IL-10 on IL-6 expression RESULTS Effects of TNF-, IL-1, OSM and LPS on the release of IL-6 from MKN-28 cells TNF-, IL-1 and OSM but not LPS timedependently increased IL-6 production in MKN-28 cells over the 36-h incubation compared to the control. MKN-28 cells spontaneously secreted very small amount of IL-6 in cell culture media, ranging from 0 to 4.85 ng/ml (n=8). TNF-, IL-1 and OSM induced a 16-, 14- and 9-fold increase in IL-6 production, respectively, during the 24-h incubation period over the control, and continuously increased to the end of 36 h. LPS even at a very high dose (100 g/ml) induced only a slight but insignificant increase in IL-6 production (Fig. 1A). OSM also induced a dose-dependent increase in IL-6 production from MKN-28 cells (Fig. 1B). Synergistic effects on IL-6 expression were observed when the cytokines were combined together (Fig. 1C).
Effects of protein kinase A on IL-6 expression The role of PKA on IL-6 expression could be excluded by two lines of observations. PKA inhibitor H8, at the doses of 0.5, 5 or 25 M showed no significant influence on IL-6 expression induced by TNF-, IL-1 and OSM over the control. A specific PKA activator, forskolin, at concentrations of 0.1, 1, 10 and 25 M showed no significant increase in IL-6 production over the control (data not shown).
Effects of protein kinase C on IL-6 expression Phorbol-12-myristate-13-acetate (PMA, 100 ng/ ml) significantly increased IL-6 expression 10-fold over the control during the 24-h incubation. This increase could be abolished by GF109203X (10 M) by 96.8%. Depletion of PKC by using PMA (2000 ng/ml) preincubation for 24 h, abolished IL-6 production by 83.8%. Whereas the TNF--, IL-1- or OSM-induced IL-6 expression was not affected under this experimental condition (Fig. 2A). Although GF109203X showed no effect on TNF-- and IL-1-induced IL-6 expression, it dose-dependently reduced the IL-6 production induced by OSM from MKN-28 cells (Fig. 2B).
Incubation of dexamethasone from 10 8–10 5 M reduced TNF--, IL-1- and OSM-induced IL-6 expression by 34.76–48.58%, 41.3–43.4% and 25.96– 32% respectively. Significant difference was shown over the control groups (P<0.05). Recombinant IL-10 at the doses of 1, 10 and 100 ng/ml showed no significant effects on the above cytokine induced IL-6 expression (data not shown).
Effects of intracellular calcium on IL-6 expression A23187, a calcium ionophore, induced a significant increase in IL-6 production from MKN-28 cells at 50 M doses or above (Fig. 4A). PKC depletion by PMA preincubation did not significantly affect the A23187-induced IL-6 production (Fig. 4B). These data suggested that Ca2+ might also play a role in inducing IL-6 expression.
Effects of G-protein inhibitor and activator on IL-6 expression Cholera toxin (0.5–5 g/ml) was unable to increase IL-6 production. Pertusis toxin either alone or in combination with TNF-, IL-1 and OSM showed no effect on IL-6 production induced by the above cytokines (data not shown).
Cell viability The absorption value in all test groups showed no significant difference when compared with the control group. Optical density (OD) in the control group was 0.6070.051, while the OD values in other groups ranged from 0.4760.012 to 0.7290.023, and no significant difference was demonstrated.
DISCUSSION Production of IL-6 by mucosal epithelial cell may have crucial effect in the mediation of inflammatory and immune responses initiated by infection or injury.3,4,9 IL-6 has been shown to be produced by several cell types including epithelial cell, endothelial cells, fibroblasts, hepatocytes, osteoblasts and leukocytes,4,10,11 the present study indicates that gastric
Cytokine-induced IL-6 signal in MKN-28 cells / 1131
epithelial cell line MKN-28 secretes IL-6 in response to inflammatory cytokine TNF-, IL-1 and OSM
A 40
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stimulation, while the detailed study of IL-6 production in gastric epithelial cells has not been previously reported. The role of PKA and PKC in IL-6 production induced by TNF- and IL-1 can be excluded by the fact that their inhibitor cannot affects IL-6 production. The role of PKC on OSM-induced IL-6 expression is not yet clear. On the one hand PMA depletion showed no effect on the OSM-induced IL-6 expression, indicating that at least one of the pathways for OSM stimulation was not PKC-dependent. On the other hand, PKC inhibitor GF 109203X could dose-dependently reduce the IL-6 expression induced by OSM. Two possibilities could account for these contradictory results. It is noted that OSM use two different cellular receptors, type I and type II, which mediate functionally different but overlapping cellular signalling, including tyrosine phosphorylation, mitogen activated protein (MAP) kinase induction, growth inhibition and IL-6 production.10–13 It is possible that when PKC is depleted by PMA, another receptor or pathway can still transduce cellular signals to produce IL-6. It is also possible that an unknown GF109203X-sensitive kinase is involved in the signal pathway. This is also in line with the previous observations that there is an unknown H7-sensitive pathway. H7 is a broad PKC or serine/threonine kinase inhibitor. It can inhibit PP90rsk kinase activation and regulate some primary response gene as well as IL-6 production.10,11,13 Further investigations are warranted to clarify the exact mechanism of this PKC-sensitive mechanism. OSM is originally a member of IL-6 subfamily, and has recently been defined as an inflammatory mediator.14 Its biological functions include stimulating adhesion molecule expression, promoting polymorphonuclear leukocyte migration and adhesion on endothelial cells, and induction of IL-6 and IL-8 expression from endothelial cells. It has been shown that OSM utilizes the tyrosine phosphorylation signal pathway that leads to IL-6 production and involves JAK and STAT pathway in the epithelial and endothelial cells.9,15 However, its effect on gastric epithelial cell has not been investigated. In this report we demonstrate that OSM has similar effects to TNF- and IL-1 in inducing IL-6 expression from MKN-28 cells; the
30 Figure 1. Effects of TNF-, IL-1, OSM and LPS on MKN-28 cell IL-6 expression.
20 10 0
TNF-α IL-1β OSM
TNF-α TNF-α IL-1β IL-1β OSM
OSM
Gastric epithelial cells (MKN-28) 2104 in 10% FCS RPMI-1640 were seeded in 96-well culture plates overnight. They were stimulated with TNF- ( ), IL-1 ( ), OSM () and LPS ( ) to study the time-course response of IL-6 (A), dose response (B), and the synergistic effects of TNF-, IL-1 and OSM on IL-6 production from MKN-28 cells (C). The supernatant was collected and subjected to ELISA assay for IL-6. The experiment was performed in triplicate. Values are the meansSE of three or four separate experiments.
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CYTOKINE, Vol. 12, No. 7 (July, 2000: 1129–1135)
A 35
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signal pathway requires PTK, NF-B as well as PKCindependent and GF109203X-sensitive mechanisms. Activation of MAP kinase and protein tyrosine phosphorylation are important signal transduction pathways that involve IL-6 expression. In the present observation, herbimycin A and genistein have been shown to attenuate IL-6 production induced by TNF-, IL-1 and OSM. These results are in line with previous observations that herbimycin A or genistein inhibit tyrosine kinase activation and subsequent IL-6 production in intestinal epithelial cells and monocytes.16 We therefore suggest that in addition to the above-mentioned cell systems, PTK is also involved in IL-6 production in gastric epithelial cells. NF-B has been shown to mediate IL-8 and IL-6 signal transduction. This process requires another transcription
25
0
Figure 2. Effects of PKC on MKN-28 cell IL-6 expression. MKN-28 cells 2104 in 10% FCS RPMI-1640 were seeded in 96-well culture plates overnight. Phorbol-12-myristate-13-acetate (PMA) at 100 ng/ml was incubated to stimulate MKN-28 cell IL-6 expression. The PKC depletion was carried out using PMA at the dose of 2000 ng/ml incubated for 24 h with MKN-28 cells. They were then subjected to PMA (100 ng/ml), TNF-, IL-1 and OSM (10 ng/ ml) stimulation for another 24 h (A). The role of PKC was further studied by the specific PKC inhibitor GF109203X incubated with stimulators for 24 h to evaluate its role in TNF--, IL-1- and OSM-induced IL-6 production (B). The experiment was performed in triplicate. Values are the meansSE of three or four separate experiments. *P<0.05 when compared with the control groups.
30
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Figure 3. Effects of PTK and NF-B inhibitors on MKN-28 cell IL-6 expression. MKN-28 cells (2104) in 10% FCS RPMI-1640 were seeded in 96-well culture plates overnight. Herbimycin A at 0.1–5 M (A), genistein at 1–50 M (B) and PDTC at 50 and 100 M (C) were incubated for 30 min before TNF-, IL-1 and OSM (10 ng/ml) stimulation for 24 h. The supernatant was then collected and subjected to ELISA assay for IL-6. The experiment was performed in triplicate. Values are the meansSE of three or four separate experiments. *P<0.05, **P<0.01 when compared with the control.
factor, NF-IL6, for co-operation. PDTC is a specific inhibitor for NF-B activation and PDTC by itself, or
Cytokine-induced IL-6 signal in MKN-28 cells / 1133
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Figure 4.
A23187 (50 µM) No depletion of PKC
A23187 (50 µM)
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Effects of A23187 on MKN-28 cell IL-6 expression.
MKN-28 cells (2104) in 10% FCS RPMI-1640 were seeded in 96-well culture plates overnight. Calcium ionophore A23187 at different doses was incubated alone (A), or incubated at 50 M after PKC depletion (B) to study its effect on IL-6 production. The experiment was performed in triplicate. Values are the meansSE of three separate experiments.
in co-operation with TNF-, leads to the inhibition of IL-8, IL-6 and GM-CSF gene and protein expression in epithelium, endothelium and monocytes.16,17 In the present study, TNF--, IL-1- and OSM-induced IL-6 production from MKN-28 cell can be dosedependently reduced by PDTC without affecting the cell viability, indicating that NF-B mediates cytokine induced IL-6 signal in MKN-28 cells. It has been shown that dexamethasone can reduce IL-6 production induced by LPS, TNF- and IL-1 as well as its mRNA expression from different cell systems; the mechanisms have been suggested to inhibit IL-6 mRNA accumulation and transcription.18 The present results indicate that dexamethasone not only reduces the TNF-- and IL-1-induced IL-6 production, but also downregulates the OSM-induced IL-6
expression induced by the same type of cells. IL-10 either alone or in combination with the above stimulus fails to show any effect on IL-6 expression, indicating that IL-10 cannot regulate the IL-6 signal pathway in MKN-28 cells. Cytoplasmic-free calcium is one of the most important secondary messengers. This intracellular mediator binds to calmodulin and subsequently activates varies target protein and enzymes and initiates different cell biological changes.19 A23187, a calcium ionophore which has been shown to increase intracellular calcium and Ca2+ mobilization, induces IL-6 production from monocytes, mast cells, astrocytes and IL-8 production from gastric epithelial cell line (AGS). The signal pathway is suggested through PKC or Ca2+ activation. In the present study, A23187 induces IL-6 production only at a 50-M dose or higher. As A23187 moderately stimulates PKC activation, we used PKC depletion to abolish the effect of PKC and to see the effect of calcium ionophore on IL-6 expression. The results indicated that A23187-induced IL-6 production was not affected by the PKC depletion, suggesting that PKC activation may not play an important role in A23187-induced IL-6 production, and that intracellular calcium is probably directly involved in the IL-6 production. The fact that G-protein sub-family Gs protein activator cholera toxin and Gi-protein subfamily inhibitor pertusis toxin failed to affect the IL-6 production induced by TNF-, IL-1 and OSM indicated that G-protein do not participate in the production of the IL-6. In summary, we confirm that in addition to the previously described cell systems, gastric epithelial cells can also express IL-6. This expression may contribute to the increased IL-6 mucosal level during inflammation and the development of gastric B cell lymphoma. The present study also demonstrates that TNF-, IL-1 and OSM are important regulators of IL-6 protein production in gastric epithelial cell line MKN-28. The signal pathway is mediated through PTK and NF-B activation, and also involves intracellular calcium, PKC and dexamethasone-sensitive mechanisms.
MATERIALS AND METHODS Chemical and reagents The routinely used chemicals and reagents were purchased from Sigma (Sigma Chemical Company, St. Louis, MO, USA); herbimycin A, genistein, GF109203X, H8; calcium ionophere A23187, cholera toxin and pertusis toxin were ordered from Calbiochem (Calbiochem, San Diego, CA, USA); forskolin was ordered from Gibco-BRL (Life Technology, Grand Island, NY, USA); human recombinant TNF-, IL-1 and IL-10, were purchased from Genzyme (Genzyme Diagnostics, Cambridge, MA, USA); human
1134 / Ding et al.
recombinant oncostatin M was ordered from R&D systems (R&D systems, Minneapolis, MN, USA).
Cell culture Human gastric epithelial cell line MKN-28, a welldifferentiated gastric adenocarcinoma cell line was obtained from Japanese Cancer Research Resources Bank (Tokyo, Japan). The cell was cultured in RPMI-1640 medium with 10% fetal calf serum (FCS), 100 U/ml penicillin G and 100 g/ml streptomycin in an incubator at 37C, 100% humidity and 5% CO2. The cells (2104) in 100 l volume in 10% FCS RPMI-1640 were seeded into 96-well culture plates in triplicate overnight. They were stimulated by TNF-, IL-1, OSM, LPS, forskolin and PMA, respectively. The supernatant was collected 2, 8, 24 and 36 h later, respectively, for IL-6 assay. MKN-28 cells were incubated with 2000 ng/ ml of PMA for 24 h to deplete PKC. They were then stimulated with PMA (100 ng/ml), TNF- (10 ng/ml), IL-1 (10 ng/ml), OSM (10 ng/ml) and A23187 (50 M) again for 24 h to determine if PKC depletion could affect IL-6 expression. MKN-28 cells spontaneously secreted very small amounts of IL-6 in the culture medium; when stimulated with TNF-, IL-1 and OSM at the 10 ng/ml dose, they were able to produce large amount of IL-6 in 24 h. This dose was subsequently used to determine which kinase was involved in the expression of IL-6. GF109203X, H8, herbimycin A and genistein, PDTC, dexamethasone or recombinant human IL-10 were incubated 30 min before the stimulus challenge. In the experiment to study the role of NF-B in the IL-6 regulation, Cytokine was incubated with MKN-28 cells for 6 h to study the effects of blocking NF-B activation on IL-6 production induced by the above cytokines.
Determination IL-6 concentration The cell culture supernatant IL-6 concentration was determined by enzyme-linked immunosorbent assay (ELISA), with a commercial ELISA kit from BioSource Europe SA (Fleurus, Belgium). The IL-6 value was expressed as ng/ml. The minimal detectable concentration of IL-6 by this method was 2 pg/ml, and had no cross-reaction with other interleukins, GM-CSF or OSM.
Cell viability Cell viability was assayed with routine 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) reduction method as described previously.20
Statistics Student’s t-test was used to analysis the difference in all groups. A P-value less than 0.05 was taken as significant.
Acknowledgements This project was partially supported by Hong Kong Research Grant Council and Simon KY Lee Gastroenterology and Hepatology Funds, The University of Hong Kong. Parts of the contents were
CYTOKINE, Vol. 12, No. 7 (July, 2000: 1129–1135)
presented in an abstract form at the American Gastroenterology Association Annual Meeting of Digestive Disease Week, 17–20 May 1998, in New Orleans, Louisiana, USA.
REFERENCES 1. Crabtree JE, Shallcross TM, Heatley RV, Wyatt JI (1991) Mucosal tumour necrosis factor alpha and interleukin-6 in patients with Helicobacter pylori associated gastritis. Gut 32:1473–1477. 2. Yamaoka Y, Kita M, Kodama T, Sawai N, Kashima K, Imanishi J (1997) Induction of various cytokines and development of severe mucosal inflammation by CagA gene positive Helicobacter pylori strains. Gut 41:442–451. 3. Hibi M, Nakajima K, Hirano T (1996) IL-6 cytokine family and signal transduction: a model of the cytokine system. J Mol Med 74:1–12. 4. Simpson RJ, Hammacher A, Smith DK, Matthews JM, Ward LD (1997) Interleukin-6: structure-function relationships. Protein Sci 6:929–955. 5. Dolcetti R, Boiocchi M (1996) Cellular and molecular bases of B-cell clonal expansions. Clin Exp Rheumatol 14 (suppl 14):S3– 13. 6. Hilbert DM, Kopf M, Mock BA, Kohler G, Rudikoff S (1995) Interleukin 6 is essential for in vivo development of B lineage neoplasms. J Exp Med 182:243–248. 7. Hussell T, Isaacson PG, Crabtree JE, Spencer J (1993) The response of cells from low-grade B-cell gastric lymphomas of mucosa-associated lymphoid tissue to Helicobacter pylori. Lancet 342:571–574. 8. Wu CW, Wang SR, Chao MF, Wu TC, Lui WY, P’eng KF, Chi CW (1996) Serum interleukin-6 levels reflect disease status of gastric cancer. Am J Gastroenterol 91:1417–1422. 9. Hartner A, Coppelt-Struebe M, Hocke GM, Sterzel RB (1997) Differential regulation of chemokines by leukemia inhibitory factor, interleukin-6 and oncostatin M. Kidney Int 51:1754–1760. 10. Hirano T, Matsuda T, Nakajima K (1994) Signal transduction through gp130 that is shared among the receptors for the interleukin 6 related cytokine subfamily. Stem Cells Dayt 12:262– 277. 11. Nakajima K, Matsuda T, Fujitani Y, Kojima H, Yamanaka Y, Nakae K, Takeda T, Hirano T (1995) Signal transduction through IL-6 receptor: involvement of multiple protein kinases, stat factors, and a novel H-7-sensitive pathway. Ann NY Acad Sci 762:55–70. 12. Thoma B, Bird TA, Friend DJ, Gearing DP, Dower SK (1994) Oncostatin M and leukemia inhibitory factor trigger overlapping and different signals through partially shared receptor complexes. J Biol Chem 269:6215–6222. 13. Yin T, Yang YC (1994) Mitogen-activated protein kinases and ribosomal S6 protein kinases are involved in signaling pathways shared by interleukin-11, interleukin-6, leukemia inhibitory factor, and oncostatin M in mouse 3T3-L1 cells. J Biol Chem 269:3731– 3738. 14. Modur V, Feldhaus MJ, Weyrich AS, Jicha DL, Prescott SM, Zimmerman GA, McIntyre TM (1997) Oncostatin M is a proinflammatory mediator, in vivo effects correlate with endothelial cell expression of inflammatory cytokines and adhesion molecules. J Clin Invest 100:158–168. 15. Giordano V, Falco GD, Chiari R, Quinto I, Pelicci PG, Bartholomew L, Delmastro P, Gadina M, Scala G (1997) Shc mediates IL-6 signaling by interacting with gp130 and Jak2 kinase. J Immunol 158:4097–4103. 16. Parikh AA, Salzman AL, Kane CD, Fischer JE, Hasselgren PO (1997) IL-6 production in human intestinal epithelial cells following stimulation with IL-1 is associated with activation of the transcription factor NF-B. J Surg Res 69:139–144. 17. Munoz C, Pascual-Salcedo D, Castellanos MC, Alfranca A, Aragones J, Vara A, Redondo JM, de Landazuri MO (1996) Pyrrolidine dithiocarbamate inhibits the production of interleukin-6,
Cytokine-induced IL-6 signal in MKN-28 cells / 1135 interleukin-8, and granulocyte-macrophage colony-stimulating factor by human endothelial cells in response to inflammatory mediators: modulation of NF-B and AP1 transcription factors activity. Blood 88:3482–3490. 18. Tobler A, Meier R, Seitz M, Dewald B, Baggiolini M, Fey MF (1992) Glucocorticoids downregulate gene expression of GM-CSF, NAP-1/IL-8, and IL-6, but not of M-CSF in human fibroblasts. Blood 79:45–51.
19. Beales ILP, Calam J (1997) Stimulation of IL-8 production in human gastric epithelial cells by Helicobacter pylori, IL-1 and TNF- requires tyrosine kinase activity, but not protein kinase C. Cytokine 9:514–520. 20. Ding SZ, Cho CH, Lam SK (1997) Helicobacter pylori induces interleukin-8 expression in endothelial cells and the signal pathway is protein tyrosine kinase dependent. Biochem Biophys Res Commun 240:561–565.
SHORT COMMUNICATION
REGULATION OF INTERLEUKIN 6 PRODUCTION IN A HUMAN GASTRIC EPITHELIAL CELL LINE MKN-28 Song-Ze Ding,1 Chi-Hin Cho,2 Shiu-Kum Lam1 Interleukin (IL-) 6 is closely related to gastrointestinal diseases. The question of whether gastric epithelial cell contributes to IL-6 production remains undefined. We aim to evaluate the regulatory pathway of IL-6 expression in gastric epithelial cells, by using different inflammatory cytokines, endotoxin, or protein kinase modulators. IL-6 was measured by ELISA. Phorbol-12myristate-13-acetate (PMA), calcium ionophore A23187, TNF-, IL-1, oncostatin M (OSM) but not lipopolysaccharide stimulated IL-6 production from gastric epithelial cell line MKN-28. Blocking protein tyrosine kinase (PTK) activation by herbimycin A or genistein, or blocking NF-B activation by pyrrolidinedithiocarbamate, reduced the IL-6 expression induced by TNF-, IL-1 and OSM. Dexamethasone mimicked this effect. Protein kinase (PK) C inhibitor only reduced the PMA and OSM induced IL-6 production. Both inhibitors and activators for PKA and G-protein as well as IL-10 had no effects on IL-6 expression. These results indicate that inflammatory cytokines are crucial for IL-6 regulation in gastric epithelial cells. The IL-6 signal pathway is mediated through PTK, NF-B, and also involve PKC, intracellular calcium and sensitive to dexamethasone, but is not related to PKA, G-protein and IL-10. 2000 Academic Press
Interleukin (IL-) 6 is involved in the pathogenesis of gastrointestinal diseases, including inflammatory bowel disease and Helicobacter pylori infection-related disease.1,2 In H. pylori-infected gastritis patients, the mucosal IL-6 levels are significantly increased. This increase, together with mucosal TNF-, correlate with mucosal polymorphonuclear or monocyte infiltration and H. pylori colonization. More interestingly, mucosal IL-6 is found to be closely related to the mucosal TNF- level,2 suggesting that in addition to TNF-, IL-1 and IL-8, IL-6 is also involved in gastric mucosal inflammatory processes. IL-6 is a pleiotropic cytokine. Its major functions include regulation of From the Departments of 1Medicine and 2Pharmacology, The University of Hong Kong, Hong Kong, China Correspondence to: Prof. Shiu-Kum Lam, Head, Department of Medicine, Queen Mary Hospital, The University of Hong Kong, 102 Pokfulam Road, Hong Kong, China; E-mail: [email protected] Received 15 September 1998; received in revised form 20 August 1999; accepted for publication 25 August 1999 2000 Academic Press 1043–4666/00/071129+07 $35.00/0 KEY WORDS: tumour necrosis factor-/interleukin 1/oncostatin M/MKN-28/protein kinase/dexamethasone/signal transduction/ gastric epithelial CYTOKINE, Vol. 12, No. 7 (July), 2000: pp 1129–1135
growth and differentiation of various cell types, haematopoiesis and inflammation. IL-6 induces final maturation of B cells into antibody producing cells and stimulates T cell growth and cytotoxic T cell differentiation. It also induces acute phase reactions in response to inflammation or tissue injury.3,4 IL-6 has also been indicated to play an essential role in the development of B cell lymphoproliferation or malignancy.5,6 This may have particular significance during H. pylori infection, since this has recently been demonstrated closely related to the development of B cell lymphoma.7 Our unpublished data indeed indicate that both HP and its toxin stimulate IL-6 expression from gastric epithelial cell line MKN-28. Except direct stimulation by these pathogenic substances, we hypothesize that IL-6 may also be induced by inflammatory cytokines such as TNF- or IL-1. This hypothesis is also supported by the fact that gastric epithelial cells are able to express IL-6 mRNA. Some gastric cancer cell lines are also able to produce IL-6 when cultured in large quantity in vitro.8 However, it is still unknown regarding the source of IL-6 in gastric mucosa. Also if the gastric epithelial cell can express IL-6 after inflammatory cytokine stimulation, it would be interesting to 1129
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study the signal pathways that mediate such a response. In the present study, we examined the production of IL-6 from gastric epithelial cell line MKN-28 when stimulated by inflammatory cytokines TNF-, IL-1, oncostatin M (OSM) and lipopolysaccharide (LPS). We also investigated the possible signal pathways which might involve in the IL-6 expression, including protein kinase (PK), intracellular calcium, G-protein and nuclear transcription factor NF-B.
CYTOKINE, Vol. 12, No. 7 (July, 2000: 1129–1135)
Effects of protein tyrosine kinase and NF-B inhibitors on IL-6 expression Herbimycin A (Fig. 3A), genistein (Fig. 3B) and NF-B inhibitor pyrrolidinedithiocarbamate (PDTC, Fig. 3C) showed dose-dependently reduced TNF--, IL-1- and OSM-induced IL-6 expression from MKN-28 cells. Significant difference was shown over the control (P<0.05–0.01).
Effects of dexamethasone and IL-10 on IL-6 expression RESULTS Effects of TNF-, IL-1, OSM and LPS on the release of IL-6 from MKN-28 cells TNF-, IL-1 and OSM but not LPS timedependently increased IL-6 production in MKN-28 cells over the 36-h incubation compared to the control. MKN-28 cells spontaneously secreted very small amount of IL-6 in cell culture media, ranging from 0 to 4.85 ng/ml (n=8). TNF-, IL-1 and OSM induced a 16-, 14- and 9-fold increase in IL-6 production, respectively, during the 24-h incubation period over the control, and continuously increased to the end of 36 h. LPS even at a very high dose (100 g/ml) induced only a slight but insignificant increase in IL-6 production (Fig. 1A). OSM also induced a dose-dependent increase in IL-6 production from MKN-28 cells (Fig. 1B). Synergistic effects on IL-6 expression were observed when the cytokines were combined together (Fig. 1C).
Effects of protein kinase A on IL-6 expression The role of PKA on IL-6 expression could be excluded by two lines of observations. PKA inhibitor H8, at the doses of 0.5, 5 or 25 M showed no significant influence on IL-6 expression induced by TNF-, IL-1 and OSM over the control. A specific PKA activator, forskolin, at concentrations of 0.1, 1, 10 and 25 M showed no significant increase in IL-6 production over the control (data not shown).
Effects of protein kinase C on IL-6 expression Phorbol-12-myristate-13-acetate (PMA, 100 ng/ ml) significantly increased IL-6 expression 10-fold over the control during the 24-h incubation. This increase could be abolished by GF109203X (10 M) by 96.8%. Depletion of PKC by using PMA (2000 ng/ml) preincubation for 24 h, abolished IL-6 production by 83.8%. Whereas the TNF--, IL-1- or OSM-induced IL-6 expression was not affected under this experimental condition (Fig. 2A). Although GF109203X showed no effect on TNF-- and IL-1-induced IL-6 expression, it dose-dependently reduced the IL-6 production induced by OSM from MKN-28 cells (Fig. 2B).
Incubation of dexamethasone from 10 8–10 5 M reduced TNF--, IL-1- and OSM-induced IL-6 expression by 34.76–48.58%, 41.3–43.4% and 25.96– 32% respectively. Significant difference was shown over the control groups (P<0.05). Recombinant IL-10 at the doses of 1, 10 and 100 ng/ml showed no significant effects on the above cytokine induced IL-6 expression (data not shown).
Effects of intracellular calcium on IL-6 expression A23187, a calcium ionophore, induced a significant increase in IL-6 production from MKN-28 cells at 50 M doses or above (Fig. 4A). PKC depletion by PMA preincubation did not significantly affect the A23187-induced IL-6 production (Fig. 4B). These data suggested that Ca2+ might also play a role in inducing IL-6 expression.
Effects of G-protein inhibitor and activator on IL-6 expression Cholera toxin (0.5–5 g/ml) was unable to increase IL-6 production. Pertusis toxin either alone or in combination with TNF-, IL-1 and OSM showed no effect on IL-6 production induced by the above cytokines (data not shown).
Cell viability The absorption value in all test groups showed no significant difference when compared with the control group. Optical density (OD) in the control group was 0.6070.051, while the OD values in other groups ranged from 0.4760.012 to 0.7290.023, and no significant difference was demonstrated.
DISCUSSION Production of IL-6 by mucosal epithelial cell may have crucial effect in the mediation of inflammatory and immune responses initiated by infection or injury.3,4,9 IL-6 has been shown to be produced by several cell types including epithelial cell, endothelial cells, fibroblasts, hepatocytes, osteoblasts and leukocytes,4,10,11 the present study indicates that gastric
Cytokine-induced IL-6 signal in MKN-28 cells / 1131
epithelial cell line MKN-28 secretes IL-6 in response to inflammatory cytokine TNF-, IL-1 and OSM
A 40
IL-6 expression (ng/ml)
35 30 25 20 15 10 5 0 2
8
24
36
10 Dose (ng/ml)
100
Time (h) B 35
IL-6 expression (ng/ml)
30 25 20 15 10 5 0
0.1
1
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IL-6 concentration (ng/ml)
50 40
stimulation, while the detailed study of IL-6 production in gastric epithelial cells has not been previously reported. The role of PKA and PKC in IL-6 production induced by TNF- and IL-1 can be excluded by the fact that their inhibitor cannot affects IL-6 production. The role of PKC on OSM-induced IL-6 expression is not yet clear. On the one hand PMA depletion showed no effect on the OSM-induced IL-6 expression, indicating that at least one of the pathways for OSM stimulation was not PKC-dependent. On the other hand, PKC inhibitor GF 109203X could dose-dependently reduce the IL-6 expression induced by OSM. Two possibilities could account for these contradictory results. It is noted that OSM use two different cellular receptors, type I and type II, which mediate functionally different but overlapping cellular signalling, including tyrosine phosphorylation, mitogen activated protein (MAP) kinase induction, growth inhibition and IL-6 production.10–13 It is possible that when PKC is depleted by PMA, another receptor or pathway can still transduce cellular signals to produce IL-6. It is also possible that an unknown GF109203X-sensitive kinase is involved in the signal pathway. This is also in line with the previous observations that there is an unknown H7-sensitive pathway. H7 is a broad PKC or serine/threonine kinase inhibitor. It can inhibit PP90rsk kinase activation and regulate some primary response gene as well as IL-6 production.10,11,13 Further investigations are warranted to clarify the exact mechanism of this PKC-sensitive mechanism. OSM is originally a member of IL-6 subfamily, and has recently been defined as an inflammatory mediator.14 Its biological functions include stimulating adhesion molecule expression, promoting polymorphonuclear leukocyte migration and adhesion on endothelial cells, and induction of IL-6 and IL-8 expression from endothelial cells. It has been shown that OSM utilizes the tyrosine phosphorylation signal pathway that leads to IL-6 production and involves JAK and STAT pathway in the epithelial and endothelial cells.9,15 However, its effect on gastric epithelial cell has not been investigated. In this report we demonstrate that OSM has similar effects to TNF- and IL-1 in inducing IL-6 expression from MKN-28 cells; the
30 Figure 1. Effects of TNF-, IL-1, OSM and LPS on MKN-28 cell IL-6 expression.
20 10 0
TNF-α IL-1β OSM
TNF-α TNF-α IL-1β IL-1β OSM
OSM
Gastric epithelial cells (MKN-28) 2104 in 10% FCS RPMI-1640 were seeded in 96-well culture plates overnight. They were stimulated with TNF- ( ), IL-1 ( ), OSM () and LPS ( ) to study the time-course response of IL-6 (A), dose response (B), and the synergistic effects of TNF-, IL-1 and OSM on IL-6 production from MKN-28 cells (C). The supernatant was collected and subjected to ELISA assay for IL-6. The experiment was performed in triplicate. Values are the meansSE of three or four separate experiments.
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CYTOKINE, Vol. 12, No. 7 (July, 2000: 1129–1135)
A 35
30
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30 25 20 15 10
*
*
*
5 0
0 10 25 50 0 1 10 25 50 0 10 25 50 TNF-α OSM IL-1β GF109203X concentration (µM)
IL-6 concentration (ng/ml)
IL-6 concentration (ng/ml)
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PMA TNF-α IL-1β OSM
GF109203X
Before depletion of PKC
25
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0 1 10 25 50 0 1 10 25 50 0 1 10 25 50 TNF-α OSM IL-1β Genistein concentration (µM)
25 IL-6 concentration (ng/ml)
signal pathway requires PTK, NF-B as well as PKCindependent and GF109203X-sensitive mechanisms. Activation of MAP kinase and protein tyrosine phosphorylation are important signal transduction pathways that involve IL-6 expression. In the present observation, herbimycin A and genistein have been shown to attenuate IL-6 production induced by TNF-, IL-1 and OSM. These results are in line with previous observations that herbimycin A or genistein inhibit tyrosine kinase activation and subsequent IL-6 production in intestinal epithelial cells and monocytes.16 We therefore suggest that in addition to the above-mentioned cell systems, PTK is also involved in IL-6 production in gastric epithelial cells. NF-B has been shown to mediate IL-8 and IL-6 signal transduction. This process requires another transcription
25
0
Figure 2. Effects of PKC on MKN-28 cell IL-6 expression. MKN-28 cells 2104 in 10% FCS RPMI-1640 were seeded in 96-well culture plates overnight. Phorbol-12-myristate-13-acetate (PMA) at 100 ng/ml was incubated to stimulate MKN-28 cell IL-6 expression. The PKC depletion was carried out using PMA at the dose of 2000 ng/ml incubated for 24 h with MKN-28 cells. They were then subjected to PMA (100 ng/ml), TNF-, IL-1 and OSM (10 ng/ ml) stimulation for another 24 h (A). The role of PKC was further studied by the specific PKC inhibitor GF109203X incubated with stimulators for 24 h to evaluate its role in TNF--, IL-1- and OSM-induced IL-6 production (B). The experiment was performed in triplicate. Values are the meansSE of three or four separate experiments. *P<0.05 when compared with the control groups.
30
20 **
*
15
** 10 *
5 0
0
*
0 50 100 0 50 100 50 100 TNF-α OSM IL-1β PDTC concentration (µM)
Figure 3. Effects of PTK and NF-B inhibitors on MKN-28 cell IL-6 expression. MKN-28 cells (2104) in 10% FCS RPMI-1640 were seeded in 96-well culture plates overnight. Herbimycin A at 0.1–5 M (A), genistein at 1–50 M (B) and PDTC at 50 and 100 M (C) were incubated for 30 min before TNF-, IL-1 and OSM (10 ng/ml) stimulation for 24 h. The supernatant was then collected and subjected to ELISA assay for IL-6. The experiment was performed in triplicate. Values are the meansSE of three or four separate experiments. *P<0.05, **P<0.01 when compared with the control.
factor, NF-IL6, for co-operation. PDTC is a specific inhibitor for NF-B activation and PDTC by itself, or
Cytokine-induced IL-6 signal in MKN-28 cells / 1133
A
IL-6 concentration (ng/ml)
20
15
10
5
0
0
0.1 1 10 25 A23187 concentration (µM)
50
B
IL-6 concentration (ng/ml)
20
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5
0
Figure 4.
A23187 (50 µM) No depletion of PKC
A23187 (50 µM)
PMA (100 ng/ml) With depletion of PKC
Effects of A23187 on MKN-28 cell IL-6 expression.
MKN-28 cells (2104) in 10% FCS RPMI-1640 were seeded in 96-well culture plates overnight. Calcium ionophore A23187 at different doses was incubated alone (A), or incubated at 50 M after PKC depletion (B) to study its effect on IL-6 production. The experiment was performed in triplicate. Values are the meansSE of three separate experiments.
in co-operation with TNF-, leads to the inhibition of IL-8, IL-6 and GM-CSF gene and protein expression in epithelium, endothelium and monocytes.16,17 In the present study, TNF--, IL-1- and OSM-induced IL-6 production from MKN-28 cell can be dosedependently reduced by PDTC without affecting the cell viability, indicating that NF-B mediates cytokine induced IL-6 signal in MKN-28 cells. It has been shown that dexamethasone can reduce IL-6 production induced by LPS, TNF- and IL-1 as well as its mRNA expression from different cell systems; the mechanisms have been suggested to inhibit IL-6 mRNA accumulation and transcription.18 The present results indicate that dexamethasone not only reduces the TNF-- and IL-1-induced IL-6 production, but also downregulates the OSM-induced IL-6
expression induced by the same type of cells. IL-10 either alone or in combination with the above stimulus fails to show any effect on IL-6 expression, indicating that IL-10 cannot regulate the IL-6 signal pathway in MKN-28 cells. Cytoplasmic-free calcium is one of the most important secondary messengers. This intracellular mediator binds to calmodulin and subsequently activates varies target protein and enzymes and initiates different cell biological changes.19 A23187, a calcium ionophore which has been shown to increase intracellular calcium and Ca2+ mobilization, induces IL-6 production from monocytes, mast cells, astrocytes and IL-8 production from gastric epithelial cell line (AGS). The signal pathway is suggested through PKC or Ca2+ activation. In the present study, A23187 induces IL-6 production only at a 50-M dose or higher. As A23187 moderately stimulates PKC activation, we used PKC depletion to abolish the effect of PKC and to see the effect of calcium ionophore on IL-6 expression. The results indicated that A23187-induced IL-6 production was not affected by the PKC depletion, suggesting that PKC activation may not play an important role in A23187-induced IL-6 production, and that intracellular calcium is probably directly involved in the IL-6 production. The fact that G-protein sub-family Gs protein activator cholera toxin and Gi-protein subfamily inhibitor pertusis toxin failed to affect the IL-6 production induced by TNF-, IL-1 and OSM indicated that G-protein do not participate in the production of the IL-6. In summary, we confirm that in addition to the previously described cell systems, gastric epithelial cells can also express IL-6. This expression may contribute to the increased IL-6 mucosal level during inflammation and the development of gastric B cell lymphoma. The present study also demonstrates that TNF-, IL-1 and OSM are important regulators of IL-6 protein production in gastric epithelial cell line MKN-28. The signal pathway is mediated through PTK and NF-B activation, and also involves intracellular calcium, PKC and dexamethasone-sensitive mechanisms.
MATERIALS AND METHODS Chemical and reagents The routinely used chemicals and reagents were purchased from Sigma (Sigma Chemical Company, St. Louis, MO, USA); herbimycin A, genistein, GF109203X, H8; calcium ionophere A23187, cholera toxin and pertusis toxin were ordered from Calbiochem (Calbiochem, San Diego, CA, USA); forskolin was ordered from Gibco-BRL (Life Technology, Grand Island, NY, USA); human recombinant TNF-, IL-1 and IL-10, were purchased from Genzyme (Genzyme Diagnostics, Cambridge, MA, USA); human
1134 / Ding et al.
recombinant oncostatin M was ordered from R&D systems (R&D systems, Minneapolis, MN, USA).
Cell culture Human gastric epithelial cell line MKN-28, a welldifferentiated gastric adenocarcinoma cell line was obtained from Japanese Cancer Research Resources Bank (Tokyo, Japan). The cell was cultured in RPMI-1640 medium with 10% fetal calf serum (FCS), 100 U/ml penicillin G and 100 g/ml streptomycin in an incubator at 37C, 100% humidity and 5% CO2. The cells (2104) in 100 l volume in 10% FCS RPMI-1640 were seeded into 96-well culture plates in triplicate overnight. They were stimulated by TNF-, IL-1, OSM, LPS, forskolin and PMA, respectively. The supernatant was collected 2, 8, 24 and 36 h later, respectively, for IL-6 assay. MKN-28 cells were incubated with 2000 ng/ ml of PMA for 24 h to deplete PKC. They were then stimulated with PMA (100 ng/ml), TNF- (10 ng/ml), IL-1 (10 ng/ml), OSM (10 ng/ml) and A23187 (50 M) again for 24 h to determine if PKC depletion could affect IL-6 expression. MKN-28 cells spontaneously secreted very small amounts of IL-6 in the culture medium; when stimulated with TNF-, IL-1 and OSM at the 10 ng/ml dose, they were able to produce large amount of IL-6 in 24 h. This dose was subsequently used to determine which kinase was involved in the expression of IL-6. GF109203X, H8, herbimycin A and genistein, PDTC, dexamethasone or recombinant human IL-10 were incubated 30 min before the stimulus challenge. In the experiment to study the role of NF-B in the IL-6 regulation, Cytokine was incubated with MKN-28 cells for 6 h to study the effects of blocking NF-B activation on IL-6 production induced by the above cytokines.
Determination IL-6 concentration The cell culture supernatant IL-6 concentration was determined by enzyme-linked immunosorbent assay (ELISA), with a commercial ELISA kit from BioSource Europe SA (Fleurus, Belgium). The IL-6 value was expressed as ng/ml. The minimal detectable concentration of IL-6 by this method was 2 pg/ml, and had no cross-reaction with other interleukins, GM-CSF or OSM.
Cell viability Cell viability was assayed with routine 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) reduction method as described previously.20
Statistics Student’s t-test was used to analysis the difference in all groups. A P-value less than 0.05 was taken as significant.
Acknowledgements This project was partially supported by Hong Kong Research Grant Council and Simon KY Lee Gastroenterology and Hepatology Funds, The University of Hong Kong. Parts of the contents were
CYTOKINE, Vol. 12, No. 7 (July, 2000: 1129–1135)
presented in an abstract form at the American Gastroenterology Association Annual Meeting of Digestive Disease Week, 17–20 May 1998, in New Orleans, Louisiana, USA.
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