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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Tumor Necrosis Factor--Induced Cytokine-Induced Neutrophil Chemoattractant-1 (CINC-1) Production by Rat Gastric Epithelial Cells: Role of Reactive Oxygen Species and Nuclear Factor-B

Vascular Cell Biology/Inflammation Program, Lawson Health Research Institute, London, Ontario, Canada (O.H., G.C., P.R.K.); Department of Inflammation and Immunology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto, Japan (O.H., T.T., M.S. T.Y.); Department of Molecular Gastroenterology and Hepatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto, Japan (Y.N., S.K., N.Y.); and Division of Gastroenterology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan (H.M.)

Received October 31, 2003; accepted January 23, 2004.

	Abstract

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Rat cytokine-induced neutrophil chemoattractant-1 (CINC-1), a counterpart of the human growth-regulated oncogene product (GRO), has been suggested to participate in neutrophil recruitment in an experimental model of gastritis in rat. However, the mechanism(s) involved in regulation of CINC-1 production by the gastric mucosa remains unclear. The aim of this study was to investigate the mechanism(s) of CINC-1 production by rat gastric mucosa in vitro. All experiments were performed using rat normal gastric mucosal cell line (RGM-1). RGM-1s were stimulated with tumor necrosis factor (TNF)-, and CINC-1 mRNA levels (reverse transcription-polymerase chain reaction) and protein secretion (enzyme-linked immunosorbent assay) were assessed. The production of reactive oxygen species (ROS) and nuclear factor (NF)-B activation (translocation to the nuclei) in response to TNF- stimulation was evaluated using fluorescence microscopy in the presence or absence of the inhibitors of mitochondrial electron flow and NF-B activation. Stimulation of RGM-1 cells with TNF- resulted in an increase in intracellular oxidative stress, NF-B translocation to the nuclei, and up-regulation of CINC-1 mRNA and protein, which was prevented by interfering with mitochondria-dependent ROS production and NF-B activation. Taken together, these findings indicate that CINC-1, a counterpart of the human GRO, production by rat gastric epithelial cells in response to TNF- stimulation is an oxidant stress-mediated and NF-B-dependent event.

TNF- is a proinflammatory cytokine and has been shown recently to be a key mediator in aspirin- and H. pylori-induced gastric mucosal injuries. The following evidence supports this hypothesis: aspirin administration in rats results in an early increase in plasma TNF- levels (Fiorucci et al., 1999), aspirin causes a time- and concentration-dependent increase in macrophage TNF- mRNA expression and cytokine release (Fiorucci et al., 1998), gastric mucosal injury induced by aspirin administration or H. pylori infection results in TNF--dependent induction of the apoptosis signaling cascade in gastric epithelial cells (Fiorucci et al., 1998), TNF- exerts direct cytotoxic effects on gastric epithelium (Naito et al., 2001), and TNF- gene polymorphisms are related to the development of peptic ulcers (Lanas et al., 2001) and infection with the H. pylori cagA subtype (Yea et al., 2001). However, the mechanism(s) involved in TNF--induced CINC-1 production by gastric mucosa remains unclear. In the present study, we investigated the intracellular regulatory mechanism(s) of CINC-1 production by the rat gastric mucosal cells (RGM-1) in response to TNF- stimulation in vitro.

	Materials and Methods

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Gastric Epithelial Cell Line. The rat gastric mucosal cell line RGM-1 (RCB-0876 at Riken Cell Bank, Tsukuba, Japan), established by Matsui and Ohno, was used (Kobayashi et al., 1996). RGM-1 cells have characteristics of gastric mucous-producing cells and carry prostaglandin EP₄ receptors (Hassan et al., 1996; Kobayashi et al., 1996). RGM-1 does not express transcripts of the gastrin receptor, the H₂ receptor, histidine decarboxylase, somatostatin, or pepsinogen 1 (Hassan et al., 1996). RGM-1 cells were grown in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin. The cells were incubated at 37°C in a humidified atmosphere with 5% CO₂. For the experiments, RGM-1 cells were trypsinized and seeded into 96-well cell culture plates or eight-well chamber glass slides. Experiments were performed when the cells were confluent.

Inhibitors. The following inhibitors were used: proteasome inhibitor (PSI; Calbiochem, San Diego, CA), nuclear factor B (NF-B) inhibitor, pyrrolidine dithiocarbamate (PDTC; Sigma, St. Louis, MO), mitochondrial complex I electron flow inhibitors, rotenone, and diphenyleneiodonium (DPI; Sigma).

Enzyme-Linked Immunosorbent Assay (ELISA). RGM-1 cells were cultured in 96-well plates until confluence and stimulated with pro-inflammatory cytokines: rat TNF- (Genzyme-Techne, Cambridge, MA), rat IL-1 (Genzyme-Techne), or bacterial lipopolysaccharide (LPS; from Escherichia coli 055:B5) (Difco, Detroit, MI). CINC-1 production (release in culture supernatants) was assessed using an ELISA kit (Immuno-Biological Laboratories Co., Ltd., Gunma, Japan) according to the manufacturer's instructions. Briefly, 100 µl of cell supernatants were placed into 96-well plates coated with rabbit anti-rat GRO/CINC-1 IgG and incubated for 1 h at room temperature. Following this, the supernatants were removed, and the wells were washed with 1% bovine serum albumin and 0.05% Tween-20 in phosphate-buffered saline (PBS). Subsequently, horseradish peroxidase-conjugated rabbit-anti rat GRO/CINC-1 antibody (Fab' fragments) was added, and the amount of CINC-1 was determined colorimetrically 30 min later (490-nm wavelength; MPR-A4i microplate reader; Tosoh, Tokyo, Japan) using tetra methyl benzidine as a substrate.

RNA Extraction and Reverse Transcription (RT)-Polymerase Chain Reaction (PCR) Amplification of CINC-1. RGM-1 cells were cultured in 75-cm² cell culture flasks and treated with TNF- (10 ng/ml) in the presence or absence of NF-B inhibitors. After 2 h of stimulation, total RNA from 2 x 10⁷ RGM-1 cells was extracted by the single-step guanidium thiocyanate-phenol-chloroform method using ISOGEN reagent (Nippon Gene, Toyama, Japan) according to the manufacturer's suggested protocol, and the RNA concentration was determined spectrophotometrically at 260/280 nm. The RNA was used for RT-PCR amplification. The amplification was carried out in a 50-µl mixture containing 2 µl of RT product, 0.6 µM of both the sense and antisense primers, and 0.4 mM dNTP mixture with 0.5 µl of TaqDNA polymerase (Takara Biochemicals, Shiga, Japan). The settings for the thermal profile were as follows: initial denaturation (3 min/94°C) followed by 30 amplification cycles: 1 min/94°C, 1 min/60°C, and 3 min/72°C followed by final extension of 7 min/72°C). Primers were designed according to the cDNA sequences of rat CINC-1 (sense, 5'-CTGTGCTGGCCACCAGCCGC-3'; and antisense, 5'-ACAGTCCTTGGAACTTCTCTG-3'). The PCR products were separated electrophoretically in a 2.5% agarose gel and stained with ethidium bromide. DNA bands were visualized with an ultraviolet transilluminator.

Immunocytochemical Staining for the NF-B Subunit RelA (p65). RGM-1 cells were grown on eight-well chamber glass slides (Lab-Tek Chamber Slide; Nunc, Rochester, NY) until confluence. Following stimulation with TNF- (10 ng/ml) for 2 h, the cells were fixed and permeabilized with a mixture of acetone and 100% methanol (1:1 acetone:methanol) for 15 min at room temperature. After washing with PBS containing 1% bovine serum albumin, cells were incubated with a rabbit polyclonal antibody directed against NF-B subunit, RelA (p65) (Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 2 h at 37°C. Subsequently, the cells were incubated with fluorescein isothiocyanate-conjugated goat-anti-rabbit IgG (Biochemical Technologies Inc., Stoughton, MA) for 2 h at room temperature, and NF-B staining (fluorescence intensity) was observed using the IX70–23FL/DIC-SP inverted fluorescence microscope (Olympus, Tokyo, Japan). Photographic images (MPEG format) were taken from four random fields.

Production of Reactive Oxygen Species (ROS). RGM-1 cells were grown on eight-well chamber glass slides (Nunc) until confluence. Cells were stimulated with TNF- (10 ng/ml) for 1 h or H₂O₂ (100 µM) for 20 min as a positive control. Subsequently, the cells were washed twice with PBS and incubated with 5-(and 6)- carboxy-2',7'- dichlorodihydrofluorescein diacetate (CH₂DCF-DA; Molecular Probes, Eugene, OR) at a final concentration of 50 µM for 30 min or with dihydrorhodamine-123 (DHR-123; Molecular Probes) at a final concentration of 20 µM for 15 min. Cell fluorescence was observed using an IX70–23FL/DIC-SP inverted fluorescence microscope (Olympus). The excitation/emission wavelengths for CH₂DCF-DA and DHR-123 were 504/530 nm and 505/529 nm, respectively. Photographic images (MPEG format) were taken from four random fields.

Statistical Analysis. The results are presented as the mean ± S.E.M. Data were compared by two-way analysis of variance. Differences were considered significant if the P value was less than 0.05 based on Fisher's protected least significant difference tests. Statistical analysis was performed using the Stat View 5.0-J program (Abacus Concepts, Berkeley, CA).

	Results

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Production of CINC-1 by RGM-1 in Response to Various Proinflammatory Cytokines. Our initial experiments were carried out to establish whether rat gastric epithelial cell can produce CINC-1 in response to proinflammatory cytokines, such as rat TNF- (0.1–100 ng/ml), rat IL-1 (0.001–1 ng/ml), and bacterial LPS (0.1–100 ng/ml). As shown in Fig. 1, proinflammatory cytokines and LPS significantly increased production of CINC-1 by RGM-1 cells after 6 h of stimulation. Control (unstimulated) RGM-1 cells produced a small amount of CINC-1 (774.6 ± 19.8 pg/ml) during the same period of time.

View larger version (18K): [in this window] [in a new window]   Fig. 1. The effects of proinflammatory cytokines on CINC-1 production by RGM-1 cells. RGM-1 cells were cultured in 96-well plates until confluence and stimulated with different proinflammatory cytokines for 6 h. CINC-1 concentrations in the cell culture supernatants were assessed using ELISA after stimulation with rat TNF- (0–100 ng/ml) (A), rat IL-1 (0 - 1 ng/ml) (B), and bacterial LPS (0–100 ng/ml) (C). Data presented are means ± S.E.M. of three experiments. *, P < 0.05 compared with unstimulated cells.

Time Course of CINC-1 Production by RGM-1 Cells in Response to TNF- Stimulation. In these experiments, we characterized the effects of TNF--induced CINC-1 production by RGM-1. To this end, RGM-1 cells were stimulated with rat TNF- (10 and 100 ng/ml) for various periods of time (0–12 h), and CINC-1 concentration in cell culture supernatants was measured by ELISA. As shown in Fig. 2, TNF- enhanced CINC-1 production in a dose- and time-dependent manner.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Time course of CINC-1 production by RGM-1 cells after stimulation with TNF-. RGM-1 cells were cultured in 96-well plates until confluence and stimulated with TNF- (1 and 10 ng/ml) for 12 h. GRO/CINC-1 concentrations in cell culture supernatants were assessed using ELISA. Data are means ± S.E.M. of three experiments.

CINC-1 mRNA Expression in RGM-1 Cells following TNF- Stimulation. In this series of experiments, CINC-1 mRNA expression in RGM-1 cells was assessed. In parallel, the effect of PSI, a proteasome inhibitor, on CINC-1 mRNA expression also was examined. RT-PCR analysis (Fig. 3) indicates that TNF- up-regulates CINC-1 mRNA in RGM-1 cells in comparison with unstimulated cells (compare lanes 1 and 2). PSI, which prevents NF-B activation, markedly attenuated TNF--induced CINC-1 mRNA expression in RGM-1 cells (compare lanes 2 and 3).

View larger version (49K): [in this window] [in a new window]   Fig. 3. The effects of TNF- on GRO/CINC-1 mRNA expression in RGM-1 cells. RGM-1 cells were cultured in 10-cm2 cell culture dishes until confluence. Subsequently, the cells were stimulated with TNF- (10 ng/ml) for 3 h in the presence or absence of proteasome inhibitor, PSI (12.5 µM). PSI was added to the cell monolayers 1 h prior to TNF-. GRO/CINC-1 mRNA expression was assessed by RT-PCR analysis. Image is representative of three independent experiments. M, molecular weight marker.

TNF--Induced Activation of NF-B in RGM-1 Cells. Activation of NF-B [RelA (p65)] (translocation from the cytoplasm to the nuclei) was determined by immunofluorescence using an antibody specific for RelA (p65). In the unstimulated cells, RelA (p65) was localized exclusively to the cell cytoplasm (Fig. 4A, arrow). Stimulation of RGM-1 with TNF- for 2 h resulted in a nuclear staining of p65, indicating activation of NF-B (Fig. 4B, arrows). In addition, TNF--induced activation of NF-B was prevented by interfering with the activity of mitochondrial electron flow complex I by both rotenone (5 µM) and DPI (5 µM) (data not shown).

View larger version (82K): [in this window] [in a new window]   Fig. 4. Immunofluorescence analysis of NF-B in RGM-1 cells. RGM-1 cells were grown on gelatin-coated eight-well chamber glass slides and stimulated with TNF- (10 ng/ml) for 2 h. Subsequently, the cells were fixed and immunostained for NF-B subunit, RelA (p65), as described under Materials and Methods. In unstimulated cells, NF-B p65 subunit is predominantly localized to the cytoplasm (arrows, A), whereas stimulation with TNF- results in translocation of p65 to the cell nuclei (arrows, B). Images are representatives of three independent experiments and are presented in black and white. Bar = 10 µm.

Induction of Oxidative Stress in RGM-1 Cells by TNF-. Oxidative stress in RGM-1 cells in response to TNF- or H₂O₂ was assessed using oxidant-sensitive fluorescence probes CH₂DCF-DA and DHR-123. CH₂DCF-DA and DHR-123 exhibit no fluorescence in unstimulated cells and become fluorescent if interacted with ROS. Stimulation of RGM-1 cells with H₂O₂ (as a positive control) resulted in a strong oxidation of both CH₂DCF-DA (Fig. 5B) and DHR-123 (Fig. 6B). It is important to note that stimulation of RGM-1 cells with TNF- resulted in a moderate in comparison with H₂O₂-induced increase in CH₂DCF-DA and DHR-123 oxidation, indicating that TNF- alone can also induce production of ROS in RGM-1 cells (Figs. 5C and 6C). Moreover, interfering with mitochondrial electron flow complex I activity either by rotenone or DPI (5 µM) inhibited TNF--induced oxidant production by RGM-1 cells (data not shown).

View larger version (49K): [in this window] [in a new window]   Fig. 5. Cytoplasmic ROS production in RGM-1 cells stimulated with TNF-. RGM-1 cells were grown on gelatin-coated eight-well chamber glass slides. Cells were stimulated with TNF- (10 ng/ml) for 1 h or H2O2 (100 µM) (as a positive control) for 20 min. Subsequently, the cells were washed and incubated with CH2DCF-DA. Cell fluorescence was observed by inverted fluorescence microscope at excitation/emission wavelength of 504/530 nm, respectively. A, unstimulated RGM-1; B, H2O2-stimulated RGM-1 (positive control); and C, TNF--stimulated RGM-1. Images are representatives of three independent experiments and presented in black and white. Bar = 20 µm.

View larger version (46K): [in this window] [in a new window]   Fig. 6. Mitochondria-associated ROS production in RGM-1 cells stimulated with TNF-. Experimental conditions were the same as described in Fig. 5. After stimulation the cells were washed and incubated for 15 min with dihydrorhodamine-123. DHR-123 fluorescence was observed using inverted fluorescence microscope at excitation/emission wavelength of 505/529 nm, respectively. A, unstimulated RGM-1; B, H2O2-stimulated RGM-1 (positive control); and C, TNF--stimulated RGM-1. Images are representatives of three independent experiments and presented in black and white. Bar = 20 µm.

Effects of NF-B Inhibitors and Inhibitors of ROS Production on CINC-1 Release by RGM-1. In this series of experiments, we assessed the effects of NF-B inhibitors and inhibitors interfering with mitochondria-dependent oxidant production (rotenone and DPI) on TNF--induced CINC-1 production by RGM-1 cells. To this end, RGM-1 cells were stimulated with different concentrations of TNF- (0–10 ng/ml) in the presence or absence of selected inhibitors. As shown in Fig. 7, TNF- induced a significant increase in CINC-1 production in a dose-dependent manner. This TNF--induced effect was completely abolished by NF-B inhibitors, PDTC (Fig. 7A) and PSI (Fig. 7B). Similarly, TNF--induced production of CINC-1 was prevented by the inhibitors of mitochondrial electron flow rotenone (5–10 µM) (Fig. 8A) and DPI (5–10 µM) (Fig. 8B) in a dose-dependent manner. In these experiments, all inhibitors at the concentrations used did not affect the viability of RGM-1 cells during the course of 7 h, as measured by the water-soluble tetrazolium-1 assay (Dojin Laboratory, Kumamoto, Japan) performed according to the manufacturer's instructions (data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 7. Effects of NF-B inhibitors on GRO/CINC-1 production by RGM-1 cells. RGM-1 cells were cultured in 96-well plates until confluence and stimulated with TNF- (0, 1, 5, and 10 ng/ml) for 6 h in the presence or absence of proteasome inhibitor, PSI (0, 6.25, and 12.5 µM) (A) or PDTC (0, 6.25, and 12.5 µM) (B). GRO/CINC-1 concentrations in cell culture supernatants were assessed using ELISA. Data are means ± S.E.M. of three experiments. *, P < 0.05 compared with unstimulated control. **/#, P < 0.05 compared with TNF- stimulation.

View larger version (20K): [in this window] [in a new window]   Fig. 8. Effect of rotenone and DPI on GRO/CINC-1 production by RGM-1 cells. RGM-1 cells were cultured in 96-well plates until confluence and stimulated with TNF- (10 ng/ml) for 6 h in the presence or absence of mitochondrial electron flow inhibitors, rotenone (0, 1, and 5 µM) (A), or DPI (0, 1, and 5 µM) (B). GRO/CINC-1 concentrations in cell culture supernatants were assessed using ELISA. Data are means ± S.E.M. of three experiments. *, P < 0.05 compared with unstimulated control. **/#, P < 0.05 compared with TNF- stimulation.

	Discussion

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In the present study, we describe the mechanism of TNF--induced CINC-1 production by rat gastric epithelial cells. Although a rat counterpart of human IL-8 has not been identified yet, CINC-1, a counterpart of the human GRO (a member of IL-8 family), has been suggested to play a critical role as a mediator of neutrophil infiltration in rats during inflammation. Our results demonstrate that rat gastric epithelial cells, upon stimulation with TNF-, IL-1, or LPS, produce and secrete high levels of CINC-1 into cell culture supernatants. Various cells (e.g., renal epithelial cells, cardiac myocytes, and intestinal epithelial cells) produce CINC-1 in response to inflammatory stimuli such as IL-1, TNF-, or LPS (Watanabe et al., 1989; Seino et al., 1995; Yoshida et al., 2001). It also has been shown that gastric ulcer tissue (Takahashi et al., 1999) and gastric fibroblasts (Takahashi et al., 2001) produce CINC-1 in response to inflammatory stimuli. The present study shows for the first time that various proinflammatory cytokines can induce CINC-1 production by rat gastric epithelial cells. In addition, we focused on the mechanism of TNF--induced CINC-1 production by RGM-1 cells.

TNF- is a proinflammatory cytokine and has been shown recently to be a key mediator in aspirin- and H. pylori-induced gastric mucosal injuries (Santucci et al., 1995). Therefore, investigation of the mechanism(s) involved in TNF--induced CINC-1 production is important in light of the growing interest in TNF- as a therapeutic target for treatment of H. pylori- or nonsteroid anti-inflammatory drug-induced gastric mucosal inflammation.

Our results have demonstrated that unstimulated RGM-1 cells produce small amounts of CINC-1 (approximately 700 pg/ml). In accordance with previous studies, this concentration of CINC-1 is capable of inducing weak migratory effects on polymorphonuclear neutrophils (Watanabe et al., 1989; Watanabe et al., 1991). In the present study, stimulation of RGM-1 cells with TNF- resulted in a dose- and time-dependent production of CINC-1 (maximum concentration 1500 pg/ml). This concentration of CINC-1 can promote a severe polymorphonuclear neutrophil infiltration into affected areas in vivo (Watanabe et al., 1991). Our study also indicates that TNF--induced release of CINC-1 is regulated at the transcriptional level, i.e., TNF- up-regulates CINC-1 mRNA levels in RGM-1 cells.

One of the potential candidates involved in the control of CINC-1 transcription is NF-B. It has been shown that the CINC-1 promoter contains an NF-B-binding domain, thus supporting the role of NF-B in regulating the transcription of CINC-1 (Shibata et al., 1998). Generally, NF-B exists in the cytoplasm predominantly as a heterodimer consisting of subunits designated as p50 and p65. It is prevented from entering the nuclei by virtue of its association with inhibitory proteins, IB. Various cytokines (e.g., TNF- and IL-1) activate NF-B by inducing phosphorylation, ubiquitination, and subsequent degradation of IB by the proteasome pathway. The loss of IB allows the NF-B dimers to translocate to the nuclei and initiate the transcription of the target genes (Cepinskas et al., 2003).

To address the role of NF-B in this phenomenon, we used fluorescence microscopy to assess activation of NF-B [translocation of NF-B subunit RelA (p65) from the cytoplasm to the nuclei] in the absence or presence of the inhibitors of mitochondrial electron flow (rotenone and DPI) and NF-B activation (PSI and PDTC). PSI is a potent and selective peptide aldehyde inhibitor of the proteasome that inhibits NF-B in a dose-dependent manner (Adams, 1996). The antioxidant PDTC is also a potent and specific inhibitor of NF-B activation (Schreck et al., 1992). The present study indicates that Rel A (p65) is translocated to the nuclei of RGM-1 cells upon TNF- stimulation and that interfering with NF-B activation results in an inhibition of CINC-1 production by RGM-1 cells in response to TNF- stimulation. In addition, interfering with mitochondrial electron flow at complex I (rotenone and DPI) also effectively prevents TNF--induced activation of NF-B in RGM-1 cells (data not shown), suggesting the potential role of oxidants in this phenomenon. These results strongly support the role of NF-Bin TNF--induced CINC-1 production by gastric epithelial cells in vitro.

A recent study has found that distinct types of non-phagocytic cells can produce small amounts of O₂^- (Teshima et al., 2000) and that these ROS regulate signal transduction (Schoonbroodt and Piette, 2000). To determine the intracellular production of ROS, we used two different fluorogenic probes, CH₂DCF-DA and DHR-123. CH₂DCF-DA can be deacetylated in cells, where it can react quantitatively with intracellular radicals, mainly H₂O₂, to be converted to its fluorescent product, 2,7-dichlorofluorescein, which is retained within the cells and, thus, provides an index of cell cytosolic oxidation (Royall and Ischiropoulos, 1993). Fluorescent product of DHR-123 oxidation, rhodamine123, is a positively charged lipophilic compound that accumulates within mitochondria to a marked degree, with little loss to the extracellular space, thus being an excellent marker for measuring intracellular oxidative stress (Goossens et al., 1995; Cepinskas et al., 1999; Ischiropoulos et al., 1999; Lievre et al., 2001; Qu et al., 2001; Yuyama et al., 2003).

In the present study, stimulation of RGM-1 with TNF- resulted in a moderate production of ROS, which was attenuated by interfering with mitochondrial complex I activity (rotenone and DPI; data not shown). As can be seen in the Figs. 5 and 6, ROS production by TNF--stimulated RGM-1 cells was less than that from H₂O₂-stimulated RGM-1. In addition, because the concentration of TNF- used in this study did not affect the viability of RGM-1 cells (data not shown), it is obvious that TNF--induced ROS production in RGM-1 cells is not associated with the cytotoxic effects of TNF- but rather is involved in a signaling cascade, similarly as observed in gastric pit cells (Teshima et al., 2000) and endothelial cells (Jones et al., 1996).

Production of ROS including superoxide, H₂O₂, singlet oxygen, and hydroxyl radicals is necessary for normal cellular metabolism. Xanthine oxidase, NAD(P)H oxidase, peroxisomes, the endoplasmic reticulum, and the mitochondrial electron transport systems are cellular sources of ROS production (Li et al., 1997). Within the intact stomach, possible sources of ROS are gastric mucosal cells, endothelial cells, or neutrophils. Within gastric epithelial cells, ROS (superoxide) can be produced by NAD(P)H or other oxidases, such as cytochrome P450 and the mitochondrial electron transport chain. Mitochondrial respiration consumes approximately 90% of the oxygen used by the cells and as such is generally considered as the major source of cellular ROS production under the basal (physiological) conditions (Li et al., 1997). Two sites of the mitochondrial respiratory chain have been identified as sources of ROS: complex 1 (NADH ubiquinone oxidoreductase) and complex 3 (the ubiquinone reductase site) (Liu et al., 2002). In the mitochondrial membrane, the ubiquinone reductase activity of complex 1 is sensitive to rotenone (Ko et al., 2001; Gyulkhandanyan et al., 2003). DPI inhibits the mitochondrial complex 1, especially between NADH and ferritin-sulfur clusters (Majander et al., 1994). Several other flavoproteins are also sensitive to DPI. DPI has been used frequently to inhibit ROS production mediated by various flavoenzymes, including NAD(P)H oxidase of phagocytes (Hancock and Jones, 1987), nitric oxide synthase (Stuehr et al., 1991), xanthine oxidase, and NADPH-cytochrome P-450 reductase (Li and Trush, 1998). In the present study, rotenone, a mitochondrial electron flow inhibitor, and DPI, an inhibitor of NADPH oxidase, even at low, non-cytotoxic concentrations, completely prevented TNF--induced oxidant stress and CINC-1 production. These findings indicate that ROS generated from the mitochondrial respiratory chain and NADPH oxidase are involved in TNF--mediated signaling associated to the up-regulation of CINC-1 transcription/synthesis.

In summary, our data indicate that various proinflammatory cytokines can up-regulate CINC-1 release from rat gastric epithelial cells. It appears that TNF--induced CINC-1 production by RGM-1 cells is mediated by the intracellular oxidants and requires activation of NF-B. Further studies are warranted to unravel the mechanisms involved in this phenomenon in more detail. In addition, rat gastric mucosal epithelial cells may provide an excellent model to study the role of the proinflammatory cytokines and CXC chemokines during inflammation.

	Footnotes

 
This work was supported by Grant-in-Aid for Scientific Research 14570943 (to Y.N.) and 15390178 (to T.Y.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

DOI: 10.1124/jpet.103.062216.

ABBREVIATIONS. IL-8, interleukin-8; GRO, growth-regulated oncogene product; CINC-1, cytokine-induced neutrophil chemoattractant-1; TNF-, tumor necrosis factor-; IL-1, interleukin-1; LPS, lipopolysaccharide; RGM-1, rat gastric mucosal cells; PSI, proteasome inhibitor; NF-B, nuclear factor-B; PDTC, pyrrolidine dithiocarbamate; DPI, diphenyleneiodonium; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; RT, reverse transcription; PCR, polymerase chain reaction ROS, reactive oxygen species; CH₂DCF-DA, 5-(and 6-)carboxy-2',7'-dichlorodihydrofluorescein diacetate; DHR-123, dihydrorhodamine-123.

Address correspondence to: Dr. Yuji Naito, Department of Molecular Gastroenterology and Hepatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kajiicho, Kawaramachidori Hirokouji Agaru, Kamigyou-ku, Kyoto 602-8566, Japan. E-mail: ynaito{at}koto.kpu-m.ac.jp

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