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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Objective

Rebamipide, a gastroprotective agent, has the ability to scavenge reactive oxygen radicals. Increased oxidative stress is implicated in the pathogenesis of rheumatoid arthritis (RA). We undertook this study to investigate the impact of rebamipide on the development of arthritis and the pathophysiologic mechanisms by which rebamipide attenuates arthritis severity in a murine model of RA.

Methods

Collagen-induced arthritis (CIA) was induced in DBA/1J mice. Anti–type II collagen antibody titers and interleukin-17 (IL-17) levels were determined using enzyme-linked immunosorbent assay. The expression of transcription factors was analyzed by immunostaining and Western blotting. Frequencies of IL-17–producing CD4+ T cells (Th17 cells) and CD4+CD25+FoxP3+ Treg cells were analyzed by flow cytometry.

Results

Rebamipide reduced the clinical arthritis score and severity of histologic inflammation and cartilage destruction in a dose-dependent manner. The joints isolated from rebamipide-treated mice with CIA showed decreased expression of nitrotyrosine, an oxidative stress marker. Rebamipide-treated mice showed lower circulating levels of type II collagen–specific IgG, IgG1, and IgG2a. Whereas the number of Th17 cells in spleens was decreased in rebamipide-treated mice with CIA, a significant increase in the number of Treg cells in spleens was observed. In vitro, rebamipide inhibited Th17 cell differentiation through STAT-3/retinoic acid receptor–related orphan nuclear receptor γt and reciprocally induced Treg cell differentiation through FoxP3. Rebamipide increased Nrf2 nuclear activities in murine CD4+ T cells and LBRM-33 murine T lymphoma cells. Heme oxygenase 1 (HO-1) expression in the spleens was markedly increased in rebamipide-treated mice.

Conclusion

The inhibitory effects of rebamipide on joint inflammation are associated with recovery from an imbalance between Th17 cells and Treg cells and with activation of an Nrf2/HO-1 antioxidant pathway.

Rheumatoid arthritis (RA) is an autoimmune polyarthritis that causes the deterioration of joint function. Although the pathogenesis of RA is not fully understood, genetic and immunologic factors clearly contribute. Adaptive immunity, a Th1/Th2 cell imbalance, is at the center of RA pathophysiology, especially in the early phases of the disease ([1-4]). In addition, repeated innate immune reactions are also involved ([5]). The key paradigm concerning Th1/Th2 cell imbalance in RA was changed by the discovery of interleukin-17 (IL-17)–expressing CD4+ T cells, known as Th17 cells ([6, 7]). Based on data from animal models of autoimmune arthritis, Th17 cells are believed to play pivotal roles in disease pathogenesis ([7, 8]). In addition to Th17 cells, the other major subset of CD4+ T cells is CD4+CD25+ Treg cells ([9]). The function of Treg cells has been revealed to be impaired in several autoimmune diseases ([10, 11]). While the exact roles of Treg cells in human RA remain elusive, their potential to control disease progression and their association with remission following successful treatment in RA patients are becoming increasingly clear ([12, 13]).

Reactive oxygen species (ROS) are normally present in all aerobic cells and are required for normal cellular metabolism. Oxidative stress implies a disturbed cell state in which ROS production exceeds the neutralization capacity. In humans, oxidative stress has received attention as a potential causative factor for several diseases with an inflammatory component, such as cardiovascular disease and diabetes mellitus ([14-16]). Also, several lines of evidence suggest that oxidative stress may be a risk factor for the development of RA ([17, 18]). Approximately 2 decades ago, Blake et al showed that exercise in inflamed joints, but not in normal joints, produced hypoxia-reperfusion injury through increased synovial capillary perfusion pressure that occurred immediately after cessation of exercise ([19]). This may contribute to oxidative stress and suggests a potential mechanism of persistent synovial inflammation in RA joints.

Owing to the potential of commonly used drugs to induce gastrointestinal (GI) side effects, including gastritis and peptic ulceration ([20-22]), mucosal-protective agents are prescribed for RA patients with a high risk of such side effects ([23]). Rebamipide is a gastroprotective agent used for the treatment of gastritis and gastric ulcers. It is also prescribed for RA patients to attenuate or prevent GI symptoms. Interestingly, substantial research has demonstrated that rebamipide acts as an oxygen radical scavenger and also exhibits antiinflammatory activity ([24, 25]). Given that rebamipide has antioxidant and antiinflammatory properties, we aimed to investigate its potential to prevent the development of autoimmune arthritis.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Animals

Male DBA/1J mice ages 4–6 weeks were purchased from Orient Bio. The animals were maintained under specific pathogen–free conditions at the Catholic Research Institute of Medical Science of the Catholic University of Korea and were fed standard mouse chow and water. All experimental procedures were examined and approved by the Animal Research Ethics Committee of the Catholic University of Korea; the procedures conformed to all guidelines of the National Institutes of Health.

Collagen-induced arthritis (CIA) induction and injection of rebamipide

Type II collagen (CII) was dissolved overnight at 4°C in 0.1N acetic acid (4 mg/ml), with gentle rotation. Mice were injected intradermally at the base of the tail with 100 μg CII emulsified in Freund's complete adjuvant (1:1 weight/volume; Chondrex). Two weeks later, mice were boosted intradermally with 100 μg CII in Freund's incomplete adjuvant. To assess the influence of rebamipide on symptom severity in the CIA model, mice were treated with rebamipide (0.6 or 6 mg/kg) in 10% carboxymethylcellulose (CMC) solution (vehicle) or with vehicle alone by oral feeding every day after booster immunization for 4 weeks. The arthritis index in these mice was scored twice weekly and expressed as the sum of the scores of 4 limbs.

Mixed lymphocyte reaction

Single-cell suspensions prepared from the spleens were cultured at 2 × 105 cells/well in 96-well plates with CII (100 μg/ml). During the last 16–18 hours of the 3-day assay, cells were pulsed with 1 μCi of 3H-thymidine (GE Healthcare) per well. The incorporation of 3H-thymidine was determined using a Betaplate scintillation counter (Perkin-Elmer).

Immunohistochemistry

Mouse joint tissues were fixed in 4% paraformaldehyde, decalcified in a histologic decalcifying agent (Calci-Clear Rapid; National Diagnostics), embedded in paraffin, and sectioned. The sections were stained with hematoxylin and eosin, Safranin O, and toluidine blue to detect proteoglycans. Tissues were first incubated with primary antibodies against tumor necrosis factor α (TNFα), IL- 1β, IL-6, and IL-17 (Abcam) as well as with an antibody against nitrotyrosine and an isotype control (Santa Cruz Biotechnology) overnight at 4°C. Then, the tissues were incubated for 1 hour with a biotinylated secondary antibody and a streptavidin–peroxidase complex. The final colored product was developed using diaminobenzidine chromogen (Thermo Scientific). Finally, the sections were counterstained with hematoxylin and photographed using a photomicroscope (Olympus). Tartrate-resistant acid phosphatase (TRAP) staining was performed with a commercial kit (catalog no. 387-A; Sigma) according to the manufacturer's instructions, omitting counterstaining with hematoxylin.

Confocal microscopy of immunostaining

Spleen tissues were snap-frozen in liquid nitrogen and stored at −70°C. Spleen tissue sections (7 μm) were fixed in acetone and stained for Treg cells using fluorescein isothiocyanate (FITC)–labeled anti-FoxP3, phycoerythrin (PE)–labeled anti-CD4 (both from eBioscience), and allophycocyanin (APC)–labeled anti-CD25 (BioLegend) antibodies. To stain Th17 cells, PE-labeled anti–IL-17 (eBioscience), FITC-labeled anti-CD4 (eBioscience), and PE-labeled anti–STAT-3 (pTyr705 or pSer727; BD Biosciences) antibodies were used. To detect STAT-3 phosphorylation, FITC-labeled anti-CD4, APC-labeled anti–STAT-3 (BD Biosciences), and PE-labeled anti–STAT-3 (pTyr705 and pSer727) antibodies were used. After incubation overnight at 4°C and staining, sections were analyzed using an LSM 510 Meta confocal microscopy system (Carl Zeiss). Positive cells were counted visually at higher magnification by 4 individuals. LBRM-33 cells were cultured at a density of 1 × 106/ml in 6-well plates, exposed to rebamipide or vehicle (DMSO) for 1 hour, harvested, and fixed at room temperature with 2% paraformaldehyde in phosphate buffered saline (PBS). They were then washed 3 times with PBS for 5 minutes and permeabilized with 0.1% Triton X-100 in PBS. Nonspecific antibody binding was blocked through incubation with 5% normal goat serum in PBS for 1 hour at room temperature. Then, cells were incubated overnight at 4°C with a rabbit polyclonal anti-Nrf2 antibody (1:100 dilution; Abcam) and washed 3 times with PBS.

Next, a secondary antibody (FITC-labeled goat anti-rabbit IgG, 1:200 dilution; Santa Cruz Biotechnology) was applied, and the cells were incubated in a dark chamber for 45 minutes. They were then counterstained with DAPI for 5 minutes. After washing with PBS, antifade mounting medium (Gel Mount; Biomeda) and a coverslip were applied. Staining was evaluated using a fluorescence microscope (BX50; Olympus).

Murine T cell isolation and differentiation

The DBA/1J mouse spleens were collected for cell preparation and washed twice with PBS. The spleens were minced, and the red blood cells were lysed with 0.83% ammonium chloride. The cells were filtered through a cell strainer and centrifuged at 1,300 revolutions per minute for 5 minutes at 4°C. To purify splenic CD4+ T cells, the splenocytes were incubated with CD4-coated magnetic beads and isolated using MACS separation columns (Miltenyi Biotec). Among CD4+ T cells, Th0 cells were stimulated only with plate-bound anti-CD3 (0.5 μg/ml; BD PharMingen) and soluble anti-CD28 (1 μg/ml; eBioscience), with no added cytokines. Th17 cells were polarized with plate-bound anti-CD3 (0.5 μg/ml), soluble anti-CD28 (1 μg/ml), anti–interferon-γ (anti-IFNγ; 2 μg/ml), anti–IL-4 (2 μg/ml), IL-6 (20 ng/ml), and transforming growth factor β (TGFβ; 2 ng/ml) for 72 hours. Th1 cells were polarized with plate-bound anti-CD3 (0.5 μg/ml), soluble anti-CD28 (1 μg/ml), anti–IL-4 (2 μg/ml), and IL-12 (10 ng/ml) for 72 hours. Cells were pretreated with rebamipide for 2 hours and then stimulated under the required polarizing conditions. To examine the production of IL-1β and IL-12 in non–T cells, CD4+ T cell–depleted cells were stimulated with lipopolysaccharide (LPS; 100 ng/ml) in the presence of rebamipide (1,000 μM) for 72 hours. The production of IL-1β and IL-12 was measured by sandwich enzyme-linked immunosorbent assay (ELISA).

Human CD4+ T cell isolation and differentiation

CD4+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) using a CD4+ T cell isolation kit according to the instructions of the manufacturer (Miltenyi Biotec). Th17 cells were stimulated with plate-bound anti-CD3 (0.5 μg/ml) and soluble anti-CD28 (0.5 μg/ml), anti-IFNγ (2 μg/ml), anti–IL-4 (2 μg/ml), IL-1β (20 ng/ml), and IL-6 (20 ng/ml) for 72 hours. Cells were pretreated with rebamipide for 2 hours and then stimulated under the required polarizing conditions.

Intracellular staining and flow cytometry

The following antibodies were used for intracellular staining of mouse cells: PerCP–Cy5.5–conjugated anti-CD4, APC-conjugated anti-CD25, FITC-conjugated anti–IL-17A, and PE-conjugated anti-FoxP3 (all from eBioscience). The following antibodies were used for intracellular staining of human cells: PE–Cy7–conjugated anti-CD4, APC-conjugated anti-CD25, FITC-conjugated anti-p53 (all from BD PharMingen), and FITC-conjugated anti-FoxP3 and PE-conjugated anti–IL-17A (both from eBioscience). Intracellular staining was performed as previously described ([26]). To stain STAT-3 phosphorylated at Tyr705, we pretreated splenocytes from DBA/1J mice for 2 hours with rebamipide. The cells were cultured for 72 hours under conditions for Th17 cell differentiation and then stained with PE-conjugated anti–STAT-3 (pTyr705) using Fixation Buffer and Perm Buffer III, according to the manufacturer's instructions. All data were analyzed using FlowJo software (Tree Star).

Measurement of IL-17A, IL-21, and IL-6 in culture supernatants

The amounts of IL-17A, IL-21, IL-22, IFNγ, IL-1β, and IL-12 were measured using a sandwich ELISA (R&D Systems). The amounts of antibody to CII IgG1 and IgG2a in blood from the orbital sinus and in serum were measured using a mouse IgG1 and IgG2a ELISA quantitation kit (Bethyl Laboratories). Absorbance was measured at 405 nm on an ELISA microplate reader (Molecular Devices).

Real-time polymerase chain reaction (PCR).

Messenger RNA (mRNA) was extracted using TRI Reagent (Molecular Research Center) according to the manufacturer's instructions. Complementary DNA was synthesized using a SuperScript Reverse Transcription system (Takara). A LightCycler 2.0 instrument (software version 4.0; Roche Diagnostics) was used for PCR amplification. All reactions were performed with LightCycler FastStart DNA Master SYBR Green I (Takara) according to the manufacturer's instructions. The following primers were used for mouse samples: for IL-17, 5′-CCT-CAA-AGC-TCA-GCG-TGT-CC-3′ (sense) and 5′-GAG-CTC-ACT-TTT-GCG-CCA-AG-3′ (antisense); for FoxP3, 5′-GGC-CCT-TCT-CCA-GGA-CAG-A-3′ (sense) and 5′-GCT-GAT-CAT-GGC-TGG-GTT-GT-3′ (antisense); for STAT-3, 5′-GAC-CCG-CCA-ACA-AAT-TAA-GA-3′ (sense) and 5′-TCG-TGG-TAA-ACT-GGA-CAC-CA-3′ (antisense); for CCR6, 5′-CCA-TGA-CTG-ACG-TCT-ACC-TGT-TGA-ACA-3′ (sense) and 5′-GAA-CAG-CTC-CAG-TCC-CAT-ACC-CAG-CAG-3′ (antisense); for retinoic acid receptor–related orphan nuclear receptor γt (RORγt), 5′-TGT-CCT-GGG-CTA-CCC-TAC-TG-3′ (sense) and 5′-GTG-CAG-GAG-TAG-GCC-ACA-TT-3′ (antisense); and, for β-actin, 5′-GTA-CGA-CCA-GAG-GCA-TAC-AGG-3′ (sense) and 5′-GAT-GAC-GAT-ATC-GCT-GCG-CTG-3′ (antisense).

The following primers were used for human samples: for IL-17, 5′-CAA-CCG-ATC-CAC-CTC-ACC-TT-3′ (sense) and 5′-GGC-ACT-TTG-CCT-CCC-AGA-T-3′ (antisense); for IL-21, 5′-GAG-TGG-TCA-GCT-TTT-TCC-TGT-T-3′ (sense) and 5′-AGG-AAT-TCT-TTG-GGT-GGT-TTT-T-3′ (antisense); for IL-22, 5′-CTC-CTT-CTC-TTG-GCC-CTC-TT-3′ (sense) and 5′-GTT-CAG-CAC-CTG-CTT-CAT-CA-3′ (antisense); and, for β-actin, 5′-GGA-CTT-CGA-GCA-AGA-GAT-GG-3′ (sense) and 5′-TGT-GTT-GGG-GTA-CAG-GTC-TTT-G-3′ (antisense). Relative levels of gene expression were normalized against β-actin.

Western blot analysis

Cell lysates were prepared from ∼1 × 107 cells by homogenization in lysis buffer. To detect Nrf2 and heme oxygenase 1 (HO-1), nuclear and cytoplasmic protein extracts of cells were obtained using NE-PER nuclear extraction reagents (Pierce). Protein concentration was determined using the Bradford method (Bio-Rad). Protein samples were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech). The membrane was preincubated with 5% skim milk in Tris buffered saline (TBS) for 2 hours at room temperature. Primary antibodies against STAT-3, pSTAT-3, lamin B, Nrf2, and β-actin (all from Cell Signaling Technology) and against histone deacetylase 2 (HDAC-2) and tubulin (both from Abcam), diluted 1:1,000 in 5% bovine serum albumin/TBS containing 0.1% Tween 20 (TBST), were added, and the samples were incubated overnight at 4°C. The samples were washed 4 times with TBST, horseradish peroxidase–conjugated secondary antibodies were added, and the samples were incubated for 1 hour at room temperature. The samples were then washed with TBST, and the hybridized bands were detected with an ECL detection kit (Pierce) and Hyperfilm (Agfa). HDAC-2, tubulin, and β-actin were used as loading controls.

Statistical analysis

Statistical analyses were performed using GraphPad Prism software, version 4 for Windows. P values were calculated by 2-tailed t-test and two-way analysis of variance (grouped). P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Rebamipide attenuates the development of inflammatory arthritis in a dose-dependent manner

We investigated whether rebamipide would suppress inflammation and joint destruction in an experimental murine model of RA (CIA). The results showed that rebamipide administered orally from day 14 after primary immunization with CII emulsified in Freund's complete adjuvant reduced the clinical arthritis score in a dose-dependent manner as compared with mice with CIA treated with vehicle (10% CMC solution) (Figure 1A). Serum levels of CII-specific IgG, IgG1, and IgG2a antibodies were significantly lower in rebamipide-treated mice than in vehicle-treated mice (Figure 1B). Assessment of cartilage destruction by Safranin O and toluidine blue staining showed that the hind paw joints of mice with CIA treated with rebamipide exhibited a significant reduction in cartilage loss compared with the joints of vehicle-treated mice. TRAP staining was used to examine the inhibition of osteoclastogenic activity by rebamipide. TRAP-positive cells were rarely observed in joints isolated from rebamipide-treated mice (Figure 1C). Rebamipide also reduced inflammation and cartilage damage in a dose-dependent manner (Figure 1C). To ascertain whether CII-specific T cell differentiation was inhibited by rebamipide treatment, splenocytes isolated from each group of mice were cultured with CII. T cell proliferative responses to CII were significantly inhibited in rebamipide-treated mice compared with those in controls (Figure 1D).

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Figure 1. Treatment with rebamipide suppresses inflammatory arthritis in mice with collagen-induced arthritis (CIA). CIA was induced in DBA/1J mice by immunization with type II collagen (CII) in adjuvant. A, Changes in arthritis scores in rebamipide-treated mice compared with vehicle-treated mice. Rebamipide dissolved in 10% carboxymethylcellulose solution (vehicle) was given orally to 2 different groups (each receiving 0.6 or 6 mg/kg; n = 5 mice per group) daily for 4 weeks, starting after booster immunization. A third group (n = 5) received vehicle alone. B, Concentrations of CII-specific IgG, IgG1, and IgG2a, determined by enzyme-linked immunosorbent assay of serum obtained from 5 mice per group. C, Histologic features of the joints of mice with CIA treated with rebamipide or vehicle. Mice were killed on day 70 after secondary immunization. Left, Tissue sections from the joints of each mouse were stained with hematoxylin and eosin (H&E), toluidine blue, Safranin O, and tartrate-resistant acid phosphatase (TRAP). Joints from mice treated with rebamipide showed mild erosive arthritis, while joints from mice treated with vehicle showed markedly erosive and destructive arthritis. Decreased numbers of TRAP-positive osteoclasts were found in joints from rebamipide-treated mice. Right, Histologic scores for inflammation and cartilage damage in mice treated with rebamipide (n = 5 per group) or vehicle (n = 5) are shown. D, Decreased T cell responses to CII in mice with CIA treated with rebamipide. Splenocytes collected from each group of mice (n = 6 per group) were cultured with CII for 72 hours, and T cell proliferative responses were determined by 3H-thymidine incorporation assay. Values are the mean ± SD. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 versus vehicle-treated mice.

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The antiinflammatory effects of rebamipide are associated with attenuated oxidative stress in the joints of mice with CIA

TNFα, IL-1β, IL-6, and IL-17 are considered to be proinflammatory cytokines that are implicated in the pathogenesis of RA ([27]). Compared with those of vehicle-treated mice with CIA, the joints of rebamipide-treated mice with CIA demonstrated profoundly decreased cell populations expressing TNFα, IL-1β, IL-6, and IL-17 (Figure 2). To determine the degree of oxidative damage to the joints, immunohistochemistry was used to assess the expression of nitrotyrosine on day 70 after secondary immunization. Interestingly, the expression of nitrotyrosine was significantly decreased in the joints of rebamipide-treated mice with CIA, and the decrease tended to be greater in mice treated with the higher dose (6 mg/kg) than in mice treated with the lower dose (0.6 mg/kg) (Figure 2).

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Figure 2. Effects of rebamipide on inflammatory molecules and oxidative stress in the joints of mice with collagen-induced arthritis (CIA). A, Tissue sections from joints of mice with CIA treated with rebamipide or vehicle were stained with anti–tumor necrosis factor α (anti-TNFα), anti–interleukin-1β (anti–IL-1β), anti–IL-6, anti–IL-17, and antinitrotyrosine antibodies, or with an isotype control. The cells stained with each antibody are shown in brown. B, Expression of proinflammatory molecules and nitrotyrosine (an oxidative stress marker) was significantly attenuated in the joints of rebamipide-treated mice with CIA compared with expression in the joints of vehicle-treated animals. Values are the mean ± SD. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 versus vehicle-treated mice.

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Rebamipide represses Th17 cell differentiation and reciprocally induces FoxP3+ Treg cell differentiation in vivo and in vitro

We counted CD4+CD25+FoxP3+ Treg cells and CD4+IL-17+ Th17 cells in spleen tissues from mice treated with rebamipide or vehicle. The results demonstrated that spleen tissues from mice treated with rebamipide showed dose-dependent increases in the number of FoxP3+ Treg cells and reciprocal decreases in the number of Th17 cells compared with spleen tissues from mice treated with vehicle (Figure 3A). CD4+ T cells isolated from spleens of each group of mice were stimulated with anti-CD3 (0.5 μg/ml) plus anti-CD28 (1 μg/ml) for 3 days. IL-17 production in culture supernatants of cells from rebamipide-treated mice was significantly inhibited compared with that in culture supernatants of cells from vehicle-treated mice (Figure 3B). Next, we attempted to ascertain whether treatment with rebamipide modulates Th17 cell and Treg cell differentiation in vitro. To investigate the effects of rebamipide under Th17 cell–polarizing conditions, isolated murine CD4+ T cells were cultured in the presence of anti-CD3, anti-CD28, TGFβ, IFNγ, IL-4, and IL-6 with or without rebamipide for 72 hours. We found that the number of IL-17–expressing CD4+ T cells was substantially decreased by the addition of rebamipide, whereas the number of CD4+CD25+ cells expressing FoxP3 was augmented in a dose-dependent manner (Figure 3C). Also, rebamipide tended to reduce the level of IL-17 in the culture supernatant (Figure 3D).

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Figure 3. Reciprocal effects of rebamipide on Th17 cell and Treg cell differentiation in vivo and in vitro.A, Left, Spleen tissues from each mouse were stained for CD4+CD25+FoxP3+ Treg cells and CD4+IL-17+ Th17 cells using monoclonal antibodies against CD4 (green) and IL-17 (red) (top) or against CD4 (red), CD25 (blue), and FoxP3 (green) (bottom). Original magnification × 400. Right, CD4+CD25+FoxP3+ T cells and CD4+IL-17+ T cells were counted visually at higher magnification (projected on a screen) by 4 individuals, and mean ± SD values are presented in the form of a histogram. B, Murine CD4+ T cells isolated from spleens from each group of mice were cultured with anti-CD3 (0.5 μg/ml) plus anti-CD28 (1 μg/ml) for 72 hours. IL-17 levels in culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Values are the mean ± SD. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 versus vehicle-treated mice. C, Splenic CD4+ T cells from DBA/1J mice were cultured under Th17 cell–inducing conditions in the presence or absence of rebamipide. Three days later, the cells were stained with antibodies against CD4, IL-17, CD25, and FoxP3. Left, A plot from 1 representative experiment shows the frequencies of CD25+FoxP3+ and IL-17+ cells among CD4+ T cells. Right, Mean ± SD values are presented in the form of a histogram. Data are representative of 4 independent experiments with similar results. D, IL-17 levels in culture supernatants from C were measured by ELISA. E, Expression of IL-17, FoxP3, retinoic acid receptor–related orphan nuclear receptor γt (RORγt), STAT-3, and CCR6 mRNA was determined by real-time polymerase chain reaction. Values are the mean ± SD. ∗ = P < 0.05; ∗∗∗ = P < 0.001 versus vehicle-treated cells. F, Interferon-γ (IFNγ) levels were measured in culture supernatants from murine CD4+ T cells cultured for 72 hours under Th1 cell–polarizing conditions in the presence or absence of rebamipide. G, CD4− T cells were cultured with lipopolysaccharide (LPS) stimulation (100 ng/ml) in the presence or absence of rebamipide for 72 hours. The concentrations of IL-12 and IL-1β were measured in the culture supernatants. Values are the mean ± SD. ∗ = P < 0.05 versus vehicle-treated cells. See Figure 2 for other definitions.

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The mRNA levels of molecules involved in Th17 cell differentiation (RORγt, STAT-3, and CCR6) were decreased in rebamipide-treated splenocytes compared with vehicle-treated cells. Conversely, the mRNA level of FoxP3, a key transcription factor for Treg cell differentiation, was increased by treatment with 300 μM rebamipide (Figure 3E). To elucidate the antiarthritic mechanisms of rebamipide, murine CD4+ T cells were cultured under Th1 cell–polarizing conditions. The concentrations of IFNγ (a Th1 cell–associated cytokine) produced by these cells were not affected by rebamipide (Figure 3F). Next, murine CD4− T cells were stimulated with LPS in the presence or absence of rebamipide for 72 hours. The levels of IL-1β and IL-12 were measured in the culture supernatants. Rebamipide did not affect the level of IL-12 (a Th1 cell–polarizing cytokine) produced by these cells, whereas it significantly inhibited IL-1β production (Figure 3G). Taken together, the immunoregulatory property of rebamipide may be specific for Th17/Treg cell differentiation, rather than for Th1 cell differentiation.

Inhibition of Th17 cell differentiation by rebamipide is mediated by modulation of pSTAT-3 expression

To investigate the signaling pathway that mediates the rebamipide-induced regulation of Th17 cell differentiation shown in vivo and in vitro, CD4+ T cells isolated from normal DBA/1J mice were pretreated with rebamipide or vehicle and then cultured under Th17 cell–polarizing conditions. Then, the expression levels of STAT-3 and its phosphorylated forms in murine CD4+ T cells were evaluated by Western blotting. The expression levels of STAT-3 (pTyr705) and STAT-3 (pSer727) were decreased by treatment with rebamipide (Figure 4A). In addition, splenocytes isolated from normal DBA/1J mice were cultured under Th17 cell–polarizing conditions for 72 hours with rebamipide or vehicle (DMSO). Flow cytometric analysis showed that levels of STAT-3 (pTyr705) were attenuated by treatment with rebamipide (Figure 4B).

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Figure 4. Effects of rebamipide on phosphorylation of STAT-3. A, Mouse splenic CD4+ T cells were pretreated with rebamipide (10 mM) or vehicle for 2 hours and then incubated under Th17 cell–inducing conditions for 12 hours. Lysates were prepared and analyzed by Western blotting to detect phosphorylated and total forms of STAT-3. Treatment of murine splenic CD4+ T cells with rebamipide specifically reduced levels of STAT-3 phosphorylated at Tyr705 (pSTAT3 [Y705] or pSTAT3Tyr705) and of STAT-3 phosphorylated at Ser727 (pSTAT3 [S727] or pSTAT3Ser727). B, Splenocytes isolated from DBA/1J mice were cultured under Th17 cell–inducing conditions in the presence of rebamipide or vehicle. Three days later, CD4+ T cells expressing pSTAT3Tyr705 were analyzed by flow cytometry. The results showed that treatment of murine splenic CD4+ T cells with rebamipide reduced pSTAT3Tyr705 levels. C, Left, Spleens from each group of mice were examined by immunofluorescence staining with monoclonal antibodies against CD4 (green) and STAT-3 (blue) (top), CD4 (green) and pSTAT3Tyr705 (red) (middle), or CD4 (green) and pSTAT3Ser727 (red) (bottom). Original magnification × 400. Right, Graphs show the number of positive cells. Values are the mean ± SD. ∗ = P < 0.05; ∗∗ = P < 0.01 versus vehicle-treated mice. CIA = collagen-induced arthritis.

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To ascertain whether levels of the phosphorylated forms of STAT-3 differ in vivo between rebamipide-treated mice with CIA and vehicle-treated mice, confocal microscopic studies of spleens isolated from each group were performed. As shown in Figure 4C, the number of STAT-3 (pTyr705)–expressing cells was significantly decreased in the spleens of rebamipide-treated mice. Although the number of STAT-3 (pSer727)–expressing cells in spleens isolated from rebamipide-treated mice with CIA tended to be decreased, the difference was not statistically significant. Therefore, our findings suggest that rebamipide inhibits Th17 cell differentiation through STAT-3 phosphorylation.

Rebamipide significantly increases HO-1 expression in the spleens of rebamipide-treated mice with CIA in a dose-dependent manner and induces nuclear translocation of Nrf2 in murine CD4+ T cells and LBRM-33 cells

Nrf2 is a key transcription factor that plays a central role in the protection of cells against oxidative stresses. Nrf2 up-regulates several genes encoding antioxidant proteins such as HO-1 ([28]). We examined whether HO-1 expression was influenced by the oral administration of rebamipide to mice with CIA. The results showed that total Nrf2 expression in the spleens of mice with CIA did not differ between rebamipide-treated animals and vehicle-treated animals. However, HO-1 expression in the spleens of rebamipide-treated mice with CIA was profoundly increased compared with that in vehicle-treated animals (Figure 5A). Nrf2 exerts its protective effect when it accumulates and translocates to the nucleus. Therefore, we determined to ascertain the effect of rebamipide on nuclear Nrf2 expression. We confirmed the effects of rebamipide in murine CD4+ T cells isolated from the spleens of normal DBA/1J mice. Compared with treatment with vehicle, treatment with rebamipide for 5 hours dramatically augmented the nuclear expression of Nrf2 (Figure 5B).

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Figure 5. Activation of Nrf2/heme oxygenase 1 (HO-1) upon exposure to rebamipide (Reba). A, The expression of Nrf2 and HO-1 in spleen tissues from mice with collagen-induced arthritis (CIA) treated with rebamipide was determined by immunohistochemical staining. The results showed markedly increased expression of HO-1 in the spleens of rebamipide-treated mice with CIA. B, Splenic CD4+ T cells isolated from DBA/1J mice were treated with 1 mM rebamipide. After 5 hours, nuclear and cytoplasmic protein extracts were prepared from the stimulated murine CD4+ T cells and were analyzed by Western blotting with antibodies against Nrf2, histone deacetylase 2 (HDAC-2), and β-actin. C, Murine CD4− T cells generated by depleting CD4+ T cells in normal DBA/1J mice were cultured with lipopolysaccharide (LPS; 100 ng/ml) in the presence or absence of rebamipide (1 mM). Left, After 24 hours, nuclear and cytoplasmic protein extracts were prepared from the stimulated murine CD4− T cells and were analyzed by Western blotting. Rebamipide induced the nuclear translocation of Nrf2 in murine CD4− T cells. HDAC-2 was examined as a loading control for nuclear extracts. Right, Graph shows the Nrf2:HDAC-2 ratio. D, Nuclear extracts from LBRM-33 cells were prepared after treatment with rebamipide, vehicle (DMSO), culture media, or sulforaphane (SFN) for 1 hour. Rebamipide increased nuclear expression of Nrf2 in a dose-dependent manner. E, Nuclei (DAPI; blue) of LBRM-33 cells exposed to rebamipide or DMSO for 1 hour were costained with Nrf2 (green). Nuclear accumulation of Nrf2 was visualized by immunofluorescence microscopy.

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To investigate whether rebamipide could activate Nrf2 signal not only in CD4+ T cells but also in other immune cells, isolated CD4− T cells from the spleens of normal DBA/1J mice were stimulated with LPS in the presence or absence of rebamipide. Rebamipide significantly enhanced the nuclear translocation of Nrf2 in murine CD4− T cells (Figure 5C). Nuclear expression of Nrf2 in LBRM-33 murine T lymphoma cells was also up-regulated by rebamipide treatment as compared with the Nrf2 agonist sulforaphane (Figures 5D and E). Cell viability was not affected by rebamipide treatment (data not shown). These findings suggest that rebamipide activates Nrf2 by stimulating its nuclear translocation, resulting in enhanced HO-1 production in mice with CIA.

Rebamipide inhibits Th17 cell differentiation and Th17 cell–associated gene expression and reciprocally promotes CD4+CD25+FoxP3+ Treg cell differentiation in human PBMCs

We further investigated the effects of rebamipide on human CD4+ T cells isolated from PBMCs from normal healthy volunteers. Purified CD4+ T cells were cultured with plate-bound anti-CD3 and soluble anti-CD28 under Th17 cell–polarizing conditions in the presence or absence of rebamipide for 72 hours. The doses of rebamipide used ranged from 1 μM to 250 μM. As shown in Figure 6A, treatment with 250 μM rebamipide significantly inhibited Th17 cell differentiation in human CD4+ T cells. The IL-17 concentration in the culture supernatant was also significantly decreased by treatment with 250 μM rebamipide (Figure 6B).

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Figure 6. Regulatory effects of rebamipide on human Th17 cell and Treg cell differentiation. Human CD4+ T cells from peripheral blood mononuclear cells were treated for 2 hours with rebamipide or vehicle and were then stimulated under Th17 cell–polarizing conditions for 3 days. A, The population of CD4+ interleukin-17–positive (IL-17+) cells was analyzed by flow cytometry. Th17 cell differentiation was inhibited by 250 μM rebamipide. B, IL-17 levels in culture supernatants were measured by enzyme-linked immunosorbent assay. C, Expression of IL-17, IL-21, and IL-22 mRNA was determined by real-time polymerase chain reaction. D, The population of CD4+CD25+FoxP3+ cells was analyzed by flow cytometry. Treg cell differentiation was increased by 10 μM rebamipide. Values are the mean ± SD. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 versus DMSO-treated cells.

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We next assessed the mRNA levels of IL-17 and the Th17 cell–related cytokines IL-21 and IL-22. The mRNA levels of these cytokines were significantly decreased by treatment with rebamipide compared with those in DMSO-treated cells (Figure 6C). We examined whether rebamipide also enhances Treg cell differentiation in human CD4+ T cells. Differentiation of CD4+CD25+FoxP3+ Treg cells under Th17 cell–polarizing conditions was increased by rebamipide treatment (Figure 6D). Thus, human CD4+ T cell reactions to rebamipide correspond to those observed in murine CD4+ T cells. We conclude that rebamipide diverted the differentiation of human and murine CD4+ T cells toward a Treg cell phenotype even under Th17 cell–polarizing conditions and reciprocally suppressed Th17 cell differentiation through specific inhibition of STAT-3 (pTyr705). Moreover, the antiinflammatory and antioxidant effects of rebamipide appear to be related to Nrf2/HO-1 signaling pathway activation.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

In this study, rebamipide effectively reduced the clinical and histologic scores in mice with CIA, an animal model of RA. The main mechanism by which rebamipide exerts its antiarthritic efficacy is the reciprocal regulation of Th17 cell and Treg cell differentiation. The inhibitory effect of rebamipide on Th17 cell differentiation depends primarily on inhibition of STAT-3 phosphorylation. The opposing effect on Treg cell differentiation was achieved through FoxP3 induction. Interestingly, rebamipide treatment in arthritic mice resulted in profound HO-1 induction. Consistent with this finding, treatment of murine CD4+ T cells with rebamipide significantly activated Nrf2, a key transcription factor promoting expression of antioxidant genes including HO-1.

Th17 cells and Treg cells display discrete and opposite functions. However, the recent notion of developmental plasticity of Treg cells and Th17 cells led to a further twist in the relationship between these 2 distinct T cell subsets. Human Treg cells are able to convert to Th17 cells in the context of the inflammatory milieu ([29]). The transcription factor RORγt, which is critical for Th17 cell generation in mice, was also found to be expressed in a proportion of IL-10–secreting FoxP3+ T cells ([30]). FoxP3 binds directly to RORγt, and the presence of inflammatory cytokines relieves FoxP3-mediated inhibition of RORγt. Hence, FoxP3+ Treg cells could be converted to Th17 cells in the presence of inflammatory signals. Therefore, attempts to reset the differentiation of naive cells from Th17 cells toward Treg cells would be an attractive treatment strategy for human diseases in which the imbalance between pro- and antiinflammatory mediators has been suggested to be a pathophysiologic mechanism. In our study, oral administration of rebamipide successfully reduced inflammatory arthritis severity through reciprocal regulation of the differentiation of Th17 cells and FoxP3-expressing Treg cells.

Our data demonstrated that levels of IFNγ (a Th1 cell–associated cytokine) and IL-12 (a Th1 cell–polarizing cytokine) were not affected by rebamipide, whereas IL-1β production was significantly inhibited. These results suggested that Th1 cell immunity may not be implicated in rebamipide-induced immune alterations. Animal studies suggested potential cross-talk between Nrf2 and the NF-κB pathway ([31]). It could be postulated that Nrf2-induced inhibition of NF-κB/IL-1β activity regulates the differentiation of effector T cells. Actually, the Th17 cell–specific transcription factor RORγt is an NF-κB target gene ([32]).

Rebamipide promotes the production of endogenous prostaglandins and has cytoprotective antiulcer effects ([33]). A recent meta-analysis showed that rebamipide is effective and safe for defending against nonsteroidal antiinflammatory drug (NSAID)–induced gastroduodenal and lower GI injuries ([34]). Although cyclooxygenase 2 (COX-2)–selective inhibitors were developed to prevent nonselective NSAID–induced GI complications, gastroduodenal ulcers were identified in 3–10% of patients taking a COX-2–selective inhibitor ([35-37]). Interestingly, a recent study showed that rebamipide add-on therapy is effective for preventing the occurrence of peptic ulcers in arthritis patients taking a COX-2–selective inhibitor ([38]). Therefore, it may be fortuitous that rebamipide, which has been prescribed in clinical practice, exerted antiarthritic effects and also activated Nrf2/HO-1 signaling.

In conclusion, oral administration of rebamipide in mice with CIA reduces clinical arthritis severity, histologic inflammation, and cartilage destruction in a dose-dependent manner. Rebamipide treatment of mice with CIA markedly inhibited oxidative damage and reduced proinflammatory cytokine expression in inflamed joints. The antiarthritic effect of rebamipide may be associated with significant expansion of FoxP3-expressing Treg cells and reciprocal suppression of Th17 cell differentiation. Of great interest, rebamipide treatment induced the nuclear translocation of Nrf2 in vitro, suggesting activation of its antioxidant response pathway. Moreover, HO-1 expression was dramatically increased in the spleens of rebamipide-treated mice with CIA compared with those of vehicle-treated mice. These data suggest that rebamipide may be beneficial for treatment of patients with RA.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Drs. Cho and Min had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Moon, J.-S. Park, Sung-Hwan Park, Cho, Min.

Acquisition of data. Moon, J.-S. Park, Woo, Lim, S.-M. Kim, S.-Y. Lee, E.-K. Kim, H. J. Lee, W. S. Lee, Sang-Hi Park, Jeong, Cho, Min.

Analysis and interpretation of data. Moon, J.-S. Park, Woo, S.-Y. Lee, H. J. Lee, W. S. Lee, Sang-Hi Park, Sung-Hwan Park, H.-Y. Kim, Cho, Min.

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  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES
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