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Nutritional Immunology

Fatty Acids Enhance GRO/CINC-1 and Interleukin-6 Production in Rat Intestinal Epithelial Cells¹

Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan and Second Department of Internal Medicine, National Defense Medical College, Saitama, Japan ^*

²To whom correspondence should be addressed. E-mail: miura{at}me.ndmc.ac.jp.

ABSTRACT

Intestinal mucosal immunity is modulated by cytokine release from intestinal cells, but little is known about the relation between nutrient absorption and cytokine release. In this study, we examined how exposure to fatty acids affects the production of growth-regulated oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1) and interleukin (IL)-6 in rat intestinal epithelial cells (IEC). The long-chain fatty acids, oleic, linoleic and arachidonic acids, and the middle-chain fatty acid octanoic acid were administered to subconfluent cultures of IEC-6 cells alone, or in combination with IL-1ß and transforming growth factor (TGF)-ß. The GRO/CINC-1 and IL-6 concentrations in culture media were determined by sandwich enzyme immunoassay. In epithelial cells, GRO/CINC-1 and IL-6 mRNA expression were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and mitogen-activated protein kinase (MAPK) activities determined by immunoblotting. Administration of long-chain fatty acids significantly increased the GRO/CINC-1 and IL-6 secretion into culture media, and this secretion was markedly increased (P < 0.05) in the presence of IL-1ß or TGF-ß. Octanoic acid had no effect on GRO/CINC-1 or IL-6 production. Furthermore, treatment with long-chain fatty acids significantly enhanced the GRO/CINC-1 and IL-6 expression that was induced by IL-1ß or TGF-ß. MAPK activity was significantly enhanced by treatment with long-chain fatty acids. Inhibitors of phospholipase C, protein kinase C or MAPK significantly reduced the fatty acid–induced increase in GRO/CINC-1 secretion, whereas a calcium/calmodulin inhibitor did not attenuate the secretion. These results suggest that long-chain fatty acids enhance cytokine release under conditions of inflammatory stimulation in the intestinal mucosa.

KEY WORDS: • long-chain fatty acids • GRO/CINC-1 • interleukin-6 • mitogen-activated protein kinase • proinflammatory cytokines • intestinal epithelial cells

Intestinal epithelial cells (IEC)⁴ form the first line of defense against the microorganisms and food-derived antigens contained in the intestinal lumen. These cells not only form a continuous barrier to prevent the entry of foreign intestinal antigens, but also actively participate in intestinal immune networks by the transport of protective immunoglobulin (Ig)A by processing and presenting antigen as antigen-presenting cells and by cytokine production. Isolated IEC cells and IEC cell lines may secrete several proinflammatory cytokines such as interleukin (IL)-8 (1 ) and IL-6 (2 –4 ). The C-X-C chemokines, such as IL-8 and growth-regulated oncogene (GRO), play an important role in the chemoattraction of neutrophils to sites of inflammation and in the activation of those cells (5 ). Several groups have previously reported rapid upregulation of expression of members of the C-X-C chemokine family in IEC after invasion with gram-negative and gram-positive bacteria or Cryptosporidium (4 , 6 –8 ).

McGee et al. (2 , 3 ) also found that IEC can produce significant levels of IL-6, and cholera toxin (CT) has been shown to induce these cells to secrete IL-6 (9 –10 ). IL-6 not only has a role in the inflammatory response, but has also been shown to preferentially enhance IgA secretion (11 ). The inflammatory cytokine IL-1ß (3 ) and, to a lesser degree, transforming growth factor (TGF)-ß1 (2 ), could also induce IEC-6 cells to secrete significantly elevated levels of IL-6, and a combination of these two cytokines induced an even greater level of IL-6 secretion by these cells (3 ), suggesting that IEC are an important source of IL-6 in the mucosal immune response of the gut. There is considerable evidence to show that dietary fat can modulate different immune functions (12 –14 ). This contention is supported by several reports that demonstrate specific in vitro and in vivo actions of saturated and unsaturated fatty acids on lymphocyte function, although the effects of specific forms of dietary fat on the immune system remain controversial (14 –17 ). A dependence of lymphocyte function in intestinal lymphatics on lipid absorption has been proposed (18 ). Recently we demonstrated that the administration of olive oil significantly increases the migration of T lymphocytes into postcapillary venules of Peyer’s patches (19 ). Although fat absorption may affect the movement of immune effector cells into gut-associated lymphoid tissues, little attention has been devoted to discerning how fat exposure might affect the immunological function of intestinal epithelial cells.

In the present report we explore the possibility that exposure to long chain fatty acids affects the expression and release of growth-regulated oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1) (rat IL-8) and IL-6, using an in vitro model of monolayers of nontransformed rat intestinal epithelial IEC-6 cells. We also examined the effect of fatty acid administration on cytokine-induced production of GRO/CINC-1 and IL-6 in IEC cells stimulated with IL-1ß and TGF-ß. These findings suggest that fatty acids can facilitate a coordinated response by epithelial cells and cellular constituents of the intestinal mucosal immune system.

MATERIALS AND METHODS

Cell line and cell culture.

The IEC-6 cell line was purchased from the American Type Culture Collection (Rockville, MD). IEC-6 cells were established in vitro from rat small intestine epithelial cells. These cells exhibit a number of features characteristic of normal small intestinal crypt cells (20 ).

Monolayer cultures were grown in plastic dishes at 37°C in an atmosphere of 95% air and 5% CO₂. The complete medium routinely consisted of Dulbecco’s modified Eagle medium (DMEM), containing 5% fetal bovine serum (FBS), 10 mg/L insulin, 50 kU/L penicillin, 50 mg/L streptomycin and 4 mmol/L glutamine. The cells were suspended into trypsin by gentle scraping with a rubber-tipped spatula. The cell suspension was centrifuged at 100 x g for 5 min at 4°C and resuspended. Cells (1 x 10⁴) were transferred to new 2-cm diameter plastic dishes, and routinely subcultured with 3 mL of fresh medium. At subconfluence, fatty acid micelles were administered to the culture medium. Cell numbers were determined before and 48 h after each treatment as follows. Upon termination of incubation, reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul-fophenyl)-2H-tetrazolium, inner salts to formazan was assessed by using a CellTiter 96 AQ nonradioactive cell proliferation assay kit (Promega Madison, WI). The absorbance of the formazan at 490 nm was measured.

The number of cells with membrane barrier dysfunction was also determined by using a cationic fluorescence dye, propidium iodide (PI, Molecular Probes, Eugene OR). The PI-positive cells was designated as damaged cells.

Administration of fatty acids.

Mixed micellar solutions were prepared according to the method of Johnston and Borgström (21 ). Long-chain fatty acids (oleic, linoleic arachidonic acids) (Sigma Chemical, St. Louis, MO) and monoolein were mixed and dissolved in DMEM containing 5% FBS with taurocholate. The final concentration in micellar solution was 20 mmol/L for sodium taurocholate, 19.2 mmol/L for long-chain fatty acids and 9.6 mmol/L for monoolein. Each fatty acid micelle was administered to the culture medium at a final concentration of from 0.01 to 0.2 mmol/L.

Octanoic acid emulsion was prepared by dissolving 19.2 mmol/L octanoic acid (Sigma) in phosphate buffer with 20 mmol/L sodium taurocholate. The pH was adjusted to 7.4 after sonication.

Determination of GRO/CINC-1 and IL-6 release.

After treatment with fatty acids, culture medium was collected after 24 or 48 h and the concentration of GRO/CINC-1 was determined by a sandwich enzyme immunoassay (EIA) (Immuno Biological Laboratories, Gunma, Japan). The concentration of IL-6 was also determined by EIA using a rat IL-6 EIA kit (Immuno Biological Laboratories). Samples of medium were stored at -70°C in polypropylene tube. A 50 µL-aliquot of sample was combined with 50 µL of assay buffer (0.01 mol/L PBS, pH 7.2, containing 0.05% Tween 20 and 10 g/L bovine serum albumin), added to 98-well microplates coated with monoclonal antibody against GRO/CINC-1 or IL-6 and incubated at 37°C for 1 h. After samples were discarded and washed with PBS, 100 µL of anti-rat GRO/CINC-1 or anti-rat IL-6 (N-terminal), rabbit IgG, Fab'-horse radish peroxidase (HRP) was added to each well, and reincubated at 37°C for 30 min. After five more washings with PBS, o-phenylendiamine with buffer containing H₂O₂ as the chromogen was added, and the microplate was set in a dark box for 15 min. H₂SO₄ (1 mol/L) was added to stop the reaction. Bound enzyme activity was measured at 490 nm with an ELISA reader (NJ-2000, Inter Med.,Tokyo,Japan). A standard curve was plotted and used to determine the quantity of GRO/CINC-1 or IL-6.

Administration of IL-1ß, TGF-ß and various inhibitors.

Human recombinant IL-1ß (Sigma) was added to the culture media of IEC-6 cells at final concentrations of 0.05–2.0 µg/L, and GRO/CINC-1 or IL-6 release was determined 48 h later. Similarly, porcine TGF-ß1 (Sigma) at concentrations of 0.05–2.0 µg/L was administered to culture media. In some experiments, both IL-1ß and TGF-ß1 were administered. Furthermore, in another set of experiments, these reagents were administered together with fatty acids and the effect of fatty acids on cytokine-induced GRO/CINC-1 release was investigated.

The effects of various inhibitors of intracellular signaling on fatty acid–induced GRO/CINC-1 release were assessed by addition at appropriate concentrations to the culture media. Inhibitors included the phospholipase C (PLC) inhibitor 2-nitro-4-carboxyphenyl-N,N'-diphenylcarbamate (NCDC; Sigma), the protein kinase C (PKC) inhibitors, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and staurosporine (Sigma), and the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide hydrochloride (W-7)(Sigma). The effects of MAPK inhibitors, PD98059; 2-[2-amino-3-methoxyphenyl]-4H-1-benzopyran-4-one (Sigma) and U0126, bis-[amino-[(2-aminophenil)thio]methylene butanedinitrite](BIOMOL Research Laboratory, Plymouth, PA), were also determined. These inhibitors of intracellular signaling were added to the culture media just before the addition of fatty acids to cells.

RNA extraction and PCR amplification of GRO/CINC-1 and IL-6.

Total RNA was isolated from IEC-6 cells using RNAzol (Biotcx, Houston, TX). Cells were lysed with 1.0 mL RNAzol/dish. Isolation and extraction were performed according to the manufacturer’s suggested protocol. Briefly, RNA was extracted with chloroform, precipitated with isopropanol and washed with 70% ethanol. The concentration of the extracted RNA was calculated by measuring the optical density at 260 nm. The ratio of the optical density at 260 nm to that at 280 nm was always >1.9. The quality of RNA was assessed by the intactness of 28S and 18S bands and the lack of degradation on agarose-gel electrophoresis.

Aliquots of RNA (5 µg) were reverse-transcribed using an RT-PCR kit from Stratagene (La Jolla, CA). Briefly, 5 µg of RNA in 38 µL of diethyl pyrocarbonate–treated water was mixed with 0.3 µg of oligo (dT), heated at 65°C for 5 min and then cooled slowly at room temperature. The following reagents were added to the tubes: 5 µL of 10X concentrated synthesis buffer (final concentration, 10 mmol/L Tris HCl, pH 8.3, 50 mmol/L KCl, 1.5 mmol/L MgCl₂), 1 µL of RNase inhibitor (40 U/µL), 2 µL of 100 mmol/L dNTPs, and 1 µL of Moloney murine leukemia virus reverse transcriptase (50 U/µL). The reaction mixture was incubated for 1 h at 37°C before being terminated by incubating the tube at 90°C for 5 min and on ice for 10 min. The tube was stored at -80°C until PCR was performed using the Takara Taq kit (recombinant Taq DNA polymerase; Takara Biochemicals, Tokyo, Japan), with rat-specific primers prepared on a DNA synthesizer (Sawady Technology, Tokyo, Japan). The primers were designed according to cDNA sequences of rat GRO/CINC-1 (sense, 5'-CTGTGCTGGCCACCAGCCGC-3'; antisense, 5'-ACAGTCCTTGGAACTTCTCTG-3').

The cDNA amplification products were predicted to be 907 bp in length. To initiate the PCR, 1 µL of RT product and 50 pmol of primers were added to the PCR master mix, consisting of 10X PCR reaction buffer (final concentrations: 10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 1.5 mmol/L MgCl₂) and 200 µmol/L dNTPs in a total of 100 µL in each tube. Tubes were placed in a Programmed temperature control system (Applied Biosystems, Tokyo, Japan) that was programmed as follows: 1) incubation at 94°C for 3 min (initial denaturation); 2) 30 cycles of the following sequential steps: 94°C for 1 min (denaturation), 60°C for 1 min (annealing), and 72°C for 3 min (extension); and 3) incubation at 72°C for 7 min (final extension). Reaction products were separated by agarose gel electrophoresis and stained with ethidium bromide. DNA bands were visualized with an ultraviolet transilluminator.

The primers for IL-6 were sense; 5'-TGGAGTCACAGAAGGAGTGGCTAAG-3', antisense; 5'-TCTGACCACAGTGAGGAATGTCCAC-3'.

The cDNA amplification products were shown at 508 bp in length. PCR was performed under the same conditions as described above except for the conditions of annealing (58°C for 1 min) and extension (72°C for 90 s). GRO/CINC-1 or IL-6 mRNA abundance was measured densitometrically, normalized to control GADPH mRNA and expressed as relative increase to controls.

MAPK activity assay.

Mitogen-activated protein kinase (MAPK) was determined in IEC-6 cells before and after exposure to fatty acids by using a p44/42 MAPK assay kit (New England Biolabs, Beverly, MA). The process was performed according to the manufacturer’s suggested protocol. Briefly, to harvest cells, culture medium was removed and cells were rinsed once with ice-cold PBS. PBS was removed and 1X ice-cold cell lysis buffer plus 1 mmol/L phenylmethylsulfonyl fluouride was added to each plate. The plate was incubated on ice for 5 min. Cells were scraped, transferred to tubes and sonicated 4 times for 5 s. Lysate (200 µL) containing 150 µg of protein was incubated with phospho-specific p44/42 MAPK monoclonal antibody (1:100 dilution) for 4 h at 4°C, protein A sepharose beads (20 µL) (Sigma) were added, and the lysate reincubated for 3 h at 4°C. The lysate was centrifuged at 15,000 x g for 30 s at 4°C, and the pellet was washed with 500 µL of 1X lysis buffer and kinase buffer. The pellet was suspended in 50 µL of 1X kinase buffer and incubated with 200 µmol/L of ATP and 2 µg of Elk1 fusion protein (2 g/L) for 30 min at 30°C. This allows immunoprecipitated active MAPK to phosphorylate Elk1. To terminate the reaction, lysate was mixed with 25 µL of 3X SDS sample buffer, boiled for 5 min and loaded on an SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane and probed with the appropriate antibody. Western blotting was performed as follows: initial blocking with blocking buffer, incubation with primary antibody (phospho-specific Elk1 antibody, 1:1000) overnight, followed by incubation with HRP-conjugated anti-rabbit secondary antibody (1:2000) and HRP-conjugated anti-biotin antibody (1:2000) for 1 h at room temperature. After the membrane was washed 3 times with 15 mL of Tris-buffered saline with Tween 20 and incubated with 10 mmol/L 1X LumiGLO for 1 min at room temperature, the bands were measured by exposure to X-ray film. Activation of MAPK was estimated by densitometric scanning and expressed as a percentage of controls.

Statistical analysis.

All results were expressed as means ± SEM. Differences among groups were evaluated by one-way ANOVA and Fisher’s post-hoc test. Statistical significance of difference was set at P < 0.05.

RESULTS

The percentage of damaged IEC-6 cells (PI-positive cells) 48 h after incubation increased with various concentrations of fatty acids (Fig. 1 ). The damaged cell number started to increase at 0.2 mmol/L of oleic acid or linoleic acid and had increased significantly at >0.5 mmol/L. Therefore, fatty acid concentrations < 0.2 mmol/L were used for the following experiments. The effect of fatty acid administration on IEC-6 cell number was determined at 48 h. Either oleic acid or linoleic acid at 0.1 mmol/L did not significantly affect cell growth compared with controls during the experiments (oleic acid-treated, 91.9 ± 5.9% of control; linoleic acid-treated, 96.3 ± 4.0% of control).

View larger version (13K): [in this window] [in a new window]   Figure 1. Effect of fatty acids on intestinal epithelial cell (IEC)-6 viability. Values are mean (n = 4) percentages of damaged cells (propidium iodide–positive cells) 48 h after incubation with various concentrations of fatty acids. Oleic or linoleic acid micelles were administered to the culture medium at a final concentration of from 0.01 to 0.2 mmol/L.

View larger version (54K): [in this window] [in a new window]   Figure 2. Effects of fatty acids on growth-regulated oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1) release from intestinal epithelial cells (IEC)-6. The concentration of GRO/CINC-1 was determined in culture media by EIA 48 h after treatment with various fatty acids. The long-chain fatty acids, oleic, linoleic acid and arachidonic acids, were administered at final concentrations of 0.01–0.1 mmol/L. The middle-chain fatty acid, octanoic acid, was administered at a concentration of 0.05–0.1 mmol/L. Taurocholate treatment (0.1 mmol/L) alone was used as a control. Values are means ± SEM, n = 6. *P < 0.05 vs. taurocholate alone (control).

View larger version (51K): [in this window] [in a new window]   Figure 3. The combined effect of long-chain fatty acids and interleukin (IL)-1ß or transforming growth factor (TGF)-ß on growth-regulated oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1) release in intestinal epithelial cells (IEC)-6. The concentration of GRO/CINC-1 was determined in culture media 48 h after administration of fatty acids and cytokines by EIA. Taurocholate treatment (0.1 mmol/L) alone was used as a control. IL-1ß or TGF-ß was administered at a concentration of 2 µg/L. In some experiments, IL-1ß and TGF-ß were administered simultaneously. Oleic or linoleic acid (0.1 mmol/L) was administered at the same time as IL-1ß or TGF-ß. Values are means ± SEM, n = 6. *P < 0.05 vs. controls. #P < 0.05 vs. IL-1ß or TGF-ß alone. P < 0.05 vs. IL-1ß + TGF-ß.

View larger version (34K): [in this window] [in a new window]   Figure 4. Effect of various inhibitors of intracellular signals on growth-regulated oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1) release from intestinal epithelial cells (IEC)-6 in the absence and presence of fatty acids (oleic acid and linoleic acid). 2-Nitro-4-carboxyphenyl-N, N'-diphenylcarbamate (NCDC; 200 µmol/L), 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7; 30 µmol/L), staurosporine (3 nmol/L) or N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide hydrochloride (W-7; 30 µmol/L) was added to the culture media with oleic or linoleic acid (0.1 mmol/L). Values are means ± SEM, n = 6. #P < 0.05 vs. control or fatty acid alone.

View larger version (33K): [in this window] [in a new window]   Figure 5. Effect of various inhibitors of mitogen-activated protein kinase (MAPK) on growth-regulated oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1) release from intestinal epithelial cells (IEC)-6 in the absence and presence of fatty acids (oleic and linoleic acids). PD98059 (50 µmol/L) or U0126 (20 µmol/L) was added to the culture media with oleic or linoleic acid (0.1 mmol/L). Values are means ± SEM, n = 6. #P < 0.05 vs. control or fatty acid alone.

View larger version (52K): [in this window] [in a new window]   Figure 6. Effects of fatty acids and the combined effect with interleukin (IL)-1ß or transforming growth factor (TGF)-ß on growth-regulated oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1) mRNA expression in intestinal epithelial cells (IEC)-6. Expression of GRO/CINC-1 mRNA 6 h after the administration of oleic acid (A) or linoleic acid (B) was determined by reverse transcriptase-polymerase chain reaction. Fatty acids were administered at a final concentration of 0.1 mmol/L. The effect of IL-1ß and TGF-ß administration (2 µg/L), and the combined effect of these cytokines with long-chain fatty acids on GRO/CINC-1 mRNA expression were also examined. Taurocholate treatment (0.1 mmol/L) alone was a control. IEC-6 cells expressed a 907-bp GRO/CINC-1 mRNA. This shows a representative picture from four experiments with similar results. GRO/CINC-1 mRNA abundance was normalized to control GADPH mRNA and expressed as relative increase to control at the bottom. Values are means (SEM), n = 4. *P < 0.05 vs. controls. #P < 0.05 vs. IL-1ß or TGF-ß alone.

View larger version (39K): [in this window] [in a new window]   Figure 7. Effects of fatty acids and the combined effect with interleukin (IL)-1ß or transforming growth factor (TGF)-ß on IL-6 release in intestinal epithelial cells (IEC)-6. The concentration of IL-6 was determined by EIA in culture media 48 h after treatment with various fatty acids. The long-chain fatty acids, oleic or linoleic acid, were administered at the final concentration of 0.1 mmol/L. Taurocholate treatment (0.1 mmol/L) alone was used as a control. The effect of IL-1ß and TGF-ß administration (2 µg/L), and the combined effect of these cytokines with long-chain fatty acids on IL-6 release was also examined. Values are means ± SEM, n = 6. *P < 0.05 vs. taurocholate alone (control). #P < 0.05 vs. IL-1ß or TGF-ß alone.

View larger version (52K): [in this window] [in a new window]   Figure 8. Effects of fatty acids and the combined effect with interleukin (IL)-1ß or transforming growth factor (TGF)-ß on IL-6 mRNA expression in intestinal epithelial cells (IEC)-6. Expression of IL-6 mRNA 6 h after the administration of oleic acid (A) or linoleic acid (B) was determined by reverse transcriptase-polymerase chain reaction. Fatty acids were administered at a final concentration of 0.1 mmol/L. The effect of IL-1ß and TGF-ß administration (2 µg/L), and the combined effect of these cytokines with long-chain fatty acids on IL-6 mRNA expression were also examined. Taurocholate treatment (0.1 mmol/L) was a control. IEC-6 cells expressed IL-6 mRNA at 508 bp. This shows a representative picture from four experiments with similar results. IL-6 mRNA abundance was normalized to control GADPH mRNA and expressed as relative increase to control at the bottom. Values are means (SEM), n = 4. *P < 0.05 vs. controls. #P < 0.05 vs. IL-1ß or TGF-ß alone.

View larger version (59K): [in this window] [in a new window]   Figure 9. Effect of oleic acid treatment on mitogen-activated protein kinase (MAPK) activation in intestinal epithelial cells (IEC)-6. MAPK-associated phosphoryltation was determined by Western blot analysis 1 and 3 h after the exposure to oleic acid (0.1 mmol/L). Immunoprecipitated active MAPK was incubated with phosphospecific fusion protein (phospho-Elk) and the fatty acid–induced increase by phosphorylation of Elk 1 fusion protein corresponding to the activation of MAPK was determined. This shows a representative picture from four experiments with similar results. Activation of MAPK was estimated by densitometric scanning and expressed as a percentage of control at the bottom. Values are means (SEM), n = 4. *P < 0.05 vs. controls.

DISCUSSION

The intestinal epithelium is continuously exposed at the epithelial cell’s apical surface to luminal factors, including nutrients. The ability of epithelial cells to secrete cytokines in response to inflammatory or pathogenic stimuli is well established (1 , 22 ); however, the possibility that epithelial cells respond to variations in the normal environment of the intestinal lumen is less well documented. This study shows for the first time that long-chain fatty acids can stimulate small intestinal epithelial cells to secrete inflammatory cytokines, such as GRO/CINC-1 and IL-6. The unsaturated fatty acids, linoleic and oleic acids, are both major fatty acids in all subclasses of lipoproteins and the major fatty acids in food; therefore we chose to use these fatty acids in this study. Various fatty acids have been reported to modulate the immunological role and function of lymphocytes and phagocytes (18 , 23 , 24 ). We demonstrated (18 ) that absorption of oleic acid stimulates the mitogen-induced blast transformation of lymphocytes in intestinal lymphatics and that the enhanced lymphocyte function may be closely related to the formation and secretion of lipoproteins into intestinal lymph. The results of the present study strongly suggest that long-chain unsaturated fatty acids in the intestinal milieu regulate epithelial participation in the inflammatory process of the intestinal mucosa by stimulating cytokine secretion. It was interesting that administration of a medium-chain fatty acid, most of which is thought to be transported directly to portal blood without being metabolized in the epithelial cells (25 ), did not significantly affect cytokine production in IEC cells.

Rat GRO/CINC-1 is a 72-amino acid peptide that is a member of the IL-8 superfamily (26 ). We showed that GRO/CINC-1 is a potent stimulator that evokes not only locomotive but also secretagogue activation of neutrophils via a CD18-dependent mechanism in vivo (27 ). IL-8 is secreted by granulocytes, endothelial cells, fibroblasts, T cells, keratinocytes, monocytes/macrophages (28 ) and by intestinal epithelial cells (1 , 6 ). It has been reported that one of the factors in the lumen, butyrate, a short-chain fatty acid, may alter the production of IL-8 by intestinal epithelial cells (29 ). Furthermore, butyrate significantly enhanced IL-8 secretion by cells stimulated with IL-1ß or lipopolysaccharide (29 ).

In this study, enhanced expression of IL-6 was also demonstrated after exposure to long-chain fatty acids in IEC cells. IL-6 is involved in both inflammatory and normal immune responses and has a wide range of effects including the induction of the acute phase protein response, macrophage differentiation and the proliferation of T cells (30 ). IL-6, either alone (11 ) or in combination with IL-5, has also been shown to induce Peyer’s patch B cells to produce high levels of IgA. These findings that long-chain fatty acids can facilitate secretion of proinflammatory cytokines such as GRO/CINC-1 and IL-6 by epithelial cells suggest an important mechanism by which nutrients alert the intestinal immune system to possible infections through secretion of these two cytokines.

The cytokines IL-1ß, IL-6, TGF-ß and C-X-C chemokine (IL-8 and GRO/CINC-1) are present in inflamed intestinal mucosa and other sites of inflammation in the body. However, little is known about how different cell types at the intestinal mucosa, including IEC, interact in this cytokine network. McGee et al. (3 ) showed that TGF-ß and IL-1ß can enhance IL-6 secretion by IEC-6 cells, and a combination of TGF-ß and IL-1ß synergistically enhanced IL-6 secretion by these cells. In the present study, we found that administration of long-chain fatty acids, but not octanoic acid, produced a synergistic enhancement of GRO/CINC-1 and IL-6 secretion by IEC-6 cells when these fatty acids were administered in combination with IL-1ß and TGF-ß. Our results also suggest the possibility that fatty acid stimulation and cytokine stimulation may use different signal pathways in the secretion of C-X-C chemokine. Enhanced levels of IL-1 have been found in mucosal tissues infected with enteropathogenic Escherichia coli or experimentally induced colitis, as well as in mucosal biopsies with active inflammatory bowel disease (31 ).

Like IL-1 and IL-6, TGF-ß has been shown to have a role in the inflammatory response, can be found in platelets and is secreted by macrophages, lymphocytes and intestinal epithelial cells (32 , 33 ). Taken together, our present results suggest that exposure of intestinal epithelial cells to long-chain fatty acids significantly activates secretion of C-X-C chemokine or IL-6 from these cells under conditions of cytokine stimulation, and suggests a possible mechanism by which fatty acid exposure may play an active role in mucosal inflammatory response. Recently we found a significant immunologic reversal effect of dietary supplementation with oleic acid on the intestinal immune system, including intestinal lymphatics altered by elemental diet (34 ). The dietary concentration of fat may be closely related to immunologic function of gut-associated lymphoid tissues, and may be an important factor in the management of inflammatory bowel diseases including Crohn’s disease.

To examine further the intracellular mechanisms involved in fatty acid–induced cytokine production, IEC-6 cells were pretreated with various inhibitors of signal transduction. Even in the absence of fatty acids, GRO/CINC-1 production was partially inhibited by NCDC, an inhibitor of phosphoinositide-specific PLC (35 ), as well as by the potent PKC inhibitors, H7 and staurosporine (36 ). These results suggest that GRO/CINC-1 production under unstimulated conditions is partially dependent on PLC and PKC activities. On the other hand fatty acid-induced GRO/CINC-1 synthesis was attenuated by NCDC or PKC inhibitors, suggesting that activation of both PLC and PKC are important in fatty acid–induced cytokine synthesis in intestinal epithelial cells. We speculate that the enhanced GRO/CINC-1 production may be largely dependent on these enzymes. PKC is physiologically activated by diacylglycerol, which is released by the hydrolysis of phosphatidylinositol 4,5-bisphosphate by PLC, and 1,4,5-inositoltriphosphate (IP3) is a co-product. The primary function of IP3 is to mobilize calcium from internal stores. Interestingly, fatty acid–induced GRO/CINC-1 synthesis was not significantly inhibited by the calmodulin inhibitor, W7. The exact signaling pathways by which PLC and PKC mediate fatty acid–induced cytokine production must be further clarified.

Two members of the MAPK family, extracellular signal-regulated kinases, (ERK)1 and ERK2, have been shown to modulate cell physiology through phosphorylation of proteins and to regulate gene expression in many different ways. For instance, ERK 1/2 phosphorylates both p90rsk, an S6 protein kinase that stimulates protein synthesis, and p62YCF, a transcription factor that increases c-fos transcription (37 ). In the gastrointestinal tract, activation of the MAPK signal transduction pathway seems to be especially important in the healing of wounded mucosa. Recently, Hobbie et al. (38 ) demonstrated that stimulation of the MAPK pathways by Salmonella typhimurium may be directly responsible for the bacteria-induced activation of the AP-1 and nuclear factor-B transcription factors and subsequent synthesis of proinflammatory cytokines including IL-8. In another study, inhibition of p38 MAPK has been shown to result in inhibition of IL-6 expression as a consequence of tumor necrosis- stimulation (39 ). In the present study, we demonstrated that potent MAPK inhibitors PD98059 (40 ) and U0126 (41 ) both attenuated fatty acid–induced GRO/CINC-1 release from IEC-6 cells. Although in this study we have demonstrated the possible link between ERK1/2 stimulation and cytokine production in intestinal epithelial cells after exposure to long-chain fatty acids, the detailed pathways leading to activation of ERK1/2 by fatty acid exposure remain to be clarified.

FOOTNOTES

¹ Supported in part by Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture of Japan and by grants from Keio University, School of Medicine and National Defense Medical College.

³ To whom reprint requests should be addressed. E-mail: hishii{at}med.keio.ac.jp.

⁴ Abbreviations used: CT, cholera toxin; DMEM, Dulbecco’s modified Eagle medium; EIA, enzyme immunoassay; ERK, extracellular signal-regulated kinases; FBS, fetal bovine serum; GRO/CINC-1, growth-regulated oncogene/cytokine-induced neutrophil chemoattractant-1; H-7, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine; HRP, horse radish peroxidase; IEC, intestinal epithelial cells; Ig, immunoglobulin; IL, interleukin; IP3, 1,4,5-inositoltriphosphate; MAPK, mitogen-activated protein kinase; NCDC, 2-nitro-4-carboxyphenyl-N, N'-diphenylcarbamate; PKC, protein kinase C; PLC, phospholipase C; RT-PCR, reverse transcriptase-polymerase chain reaction; TGF, transforming growth factor; W-7, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide hydrochloride.

Manuscript received 2 January 2001. Initial review completed 19 March 2001. Revision accepted 21 August 2001.

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