International Immunopharmacology 20 (2014) 269–275
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Gallium nitrate ameliorates type II collagen-induced arthritis in mice Jae-Hyeog Choi a,1, Jong-Hwan Lee b,1, Kug-Hwan Roh a, Su-Kil Seo a, Il-Whan Choi a, Sae-Gwang Park a, Jun-Goo Lim c, Won-Jin Lee d, Myoung-Hun Kim d, Kwang-rae Cho d, Young- Jae Kim d,⁎ a
Department of Microbiology and Immunology, College of Medicine, Inje University, Busan, Republic of Korea Department of Anesthesiology and Pain Medicine, College of Medicine, Dong-A University, Busan, Republic of Korea Shin Tong Pain Clinic, Busan, Republic of Korea d Department of Anesthesiology and Pain Medicine, Busan Paik Hospital, College of Medicine, Inje University, Busan, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 23 July 2013 Received in revised form 5 March 2014 Accepted 6 March 2014 Available online 19 March 2014 Keywords: Collagen type II-induced arthritis Gallium nitrate IgG Pro-inflammatory cytokines Matrix metalloproteinases Nuclear factor κB
a b s t r a c t Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease. Gallium nitrate has been reported to reserve immunosuppressive activities. Therefore, we assessed the therapeutic effects of gallium nitrate in the mouse model of developed type II collagen-induced arthritis (CIA). CIA was induced by bovine type II collagen with Complete Freund's adjuvant. CIA mice were intraperitoneally treated from day 36 to day 49 after immunization with 3.5 mg/kg/day, 7 mg/kg/day gallium nitrate or vehicle. Gallium nitrate ameliorated the progression of mice with CIA. The clinical symptoms of collagen-induced arthritis did not progress after treatment with gallium nitrate. Gallium nitrate inhibited the increase of CD4+ T cell populations (p b 0.05) and also inhibited the type II collagen-specific IgG2a-isotype autoantibodies (p b 0.05). Gallium nitrate reduced the serum levels of TNF-α, IL-6 and IFN-γ (p b 0.05) and the mRNA expression levels of these cytokine and MMPs (MMP2 and MMP9) in joint tissues. Western blotting of members of the NF-κB signaling pathway revealed that gallium nitrate inhibits the activation of NF-κB by blocking IκB degradation. These data suggest that gallium nitrate is a potential therapeutic agent for autoimmune inflammatory arthritis through its inhibition of the NF-κB pathway, and these results may help to elucidate gallium nitrate-mediated mechanisms of immunosuppression in patients with RA. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is a chronic, systemic and inflammatory disorder caused by a loss of immune tolerance that compromises immune homeostasis and results in the onset of autoimmune disorders [1–3]. A variety of approaches have been suggested for treating RA. The goals are to relieve pain, reduce inflammation, slow or stop joint damage, and improve the quality of life. A number of medications have been demonstrated to promote and suppress immunity in different ways. However, the drug's effectiveness and potential side effects are important considerations for the treatment of RA [4]. Gallium nitrate is the gallium salt of nitric acid with the chemical formula Ga(NO3)2 and is used to treat hypercalcemia secondary to cancer because gallium nitrate inhibits osteoclast activity, thereby lowering the amount of free calcium in blood [5]. This inhibition of osteoclasts can mediate the therapeutic effect on RA. It also has been demonstrated that gallium nitrate has potent anti-inflammatory, anti-microbial, and immune modulatory activity in several animal and clinical studies [6–8]. In addition, gallium nitrate has been demonstrated to prevent ⁎ Corresponding author at: Department of Anesthesiology and Pain Medicine, Busan Paik Hospital, College of Medicine, Inje University, Busan 614-735, Republic of Korea. Tel.: +82 51 890 6520; fax: +82 51 898 4216. E-mail address: [email protected] (Y.-J. Kim). 1 Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.intimp.2014.03.005 1567-5769/© 2014 Elsevier B.V. All rights reserved.
adjuvant inflammatory arthritis [8]. However, the immune modulatory mechanism of gallium nitrate has remained unknown. This study was conducted to evaluate whether gallium nitrate can ameliorate the progression of RA via focused anti-inflammatory and immune modulatory activity. Gallium nitrate was administered when arthritic symptoms developed after immunization with type II collagen. After treatment with gallium nitrate, clinical symptoms of collageninduced arthritis (CIA) including clinical scores, hind paw thickness and joint structure did not progress. Gallium nitrate also inhibited the increase of the CD4+ T cell population and type II collagen-specific IgG1 and IgG2a autoantibodies. In addition, gallium nitrate-treated CIA mice exhibited reduced levels of inflammatory cytokines (TNF-α, IL-6 and INF-γ) and MMP2 and MMP9. The activation of NF-κB in joint tissues was also decreased. This study reveals that gallium nitrate can inhibit the progression of CIA through the modulation of inflammation and the immune response. 2. Materials and methods 2.1. Animals Five- to six-week-old male DBA/1J mice were purchased from Orient Bio, Inc. (Sungnam, Korea) and maintained in the Animal Care Facility at the Inje University School of Medicine. All experimental procedures
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were examined and approved by the Institutional Animal Care and Use Committee of Inje University.
from eBiosciences, Inc. (San Diego, CA): anti-CD3-phycoerythrin (PE), anti-CD4-fluoresceinisothiocyanate (FITC) and anti-CD8-PECy5.
2.2. Induction of CIA and treatment with gallium nitrate
2.8. Reverse transcription-polymerase chain reaction
CIA was induced in six- to seven-week-old male DBA/1 mice by intradermal tail-base injection of 100 μg bovine type II collagen (bCII) (Chondrex, Inc., Redmond, WA) emulsified with Complete Freund's adjuvant (CFA) containing 4.0 mg/ml Mycobacterium tuberculosis H37RA (Chondrex). Mice were boosted three weeks later with bCII emulsified in Incomplete Freund's adjuvant (IFA) and were observed up to 70 days after immunization. Mice were divided randomly into three groups (n = 6 per group), in which the mice with CIA were intraperitoneally injected from day 36 to day 49 after immunization (14 days) with 3.5 mg/kg/day or 7 mg/kg/day gallium nitrate (Navicular Research Division, EbyPharma LLC, TX) or TDW (triple distilled water) as a vehicle. Gallium nitrate was diluted with distilled water because the original stock of gallium nitrate contains distilled water. Dexamethasone was used as a reference drug and was given at 5 mg/kg by oral gavage, three times a week for two weeks on day 36 after immunization.
Joint tissues were collected after seven days (PI 43 days) and 14 days (PI 50 days) of gallium nitrate injection. Joint tissues were homogenized, and total RNA was purified by using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA) and quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). The cDNA was synthesized with 2 μg RNA. The amount of mRNA was determined using Accupower® premix (Bioneer, Korea). The specific primers used for PCR were as follows: for TNF-α, 5′-CCACACCGTCAG CCGATTTG-3′ and 5′-CACCCATTCCCTTCACAGAGC-3′; for IL-6, 5′-TGGG AAATCGTGGAAATGAG-3′ and 5′-GAAGGACTCTGGCTTTGTCTT-3′; for IFN-γ, 5′-GCCACGGCACAGTCATTGAAA-3′ and 5′-TTTCGCCTTGCTGTTG CTGA-3′; for MMP2, 5′-CCCCGATGCTGATACTGA-3′ and 5′-CTGTCCGC CAAATAAACC-3′; for MMP9, 5′-CTGGACAGCCAGACACTAAA-3′ and 5′CTCGCGGCAAGTCTTCAGAG-3′; and for GAPDH, 5′-TTCACCACCATGGA GAAGGC-3′ and 5′-GGCATGGACTGTGGTCATGA-3′. 2.9. Western blot analysis
2.3. Assessment of arthritis Clinically, arthritic severity was characterized by observations of joint properties and the inflammation of the surrounding tissue. The clinical arthritis score of joint inflammation was assessed from 0 to 4 as previously described. The maximal arthritis score per paw is 4, and the maximal disease score per mouse is 16 [9]. Paw swelling was determined by measuring the thickness of the hind paw using a thickness gauge (dial type) (Mitutoyo, Tokyo, Japan). 2.4. Histopathology of the ankle joint On day 70 after immunization, the mice were sacrificed and the hind paws were fixed in 10% buffered formalin, decalcified, and embedded in paraffin. Joint sections (5- to 7-μm) were prepared and stained with hematoxylin and eosin. The histological findings (original magnification, 40 ×) were examined for synovial hyperplasia and mononuclear cell infiltration.
Joint tissues were collected after 14 days (PI 50 days) of injection with gallium nitrate. Joint tissues were homogenized with protease and phosphatase inhibitor in RIPA lysis buffer (Thermo Scientific, Rockford, IL). The homogenates, which contained 20 μg of protein, were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. The blot was probed with 1 μg/ml of primary antibody against NF-κBp65, IκBα, pIκBα and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA). Horseradish peroxidase-conjugated anti-rabbit (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) or anti-mouse IgG (Santa Cruz Biotechnology) was used as a secondary antibody. 2.10. Statistical analysis Statistical analysis was performed using GraphPad Prism (version 5 for Windows; GraphPad Software, San Diego, CA). p values were calculated by t-test. p b 0.05 was considered to be statistically significant. 3. Results
2.5. Measurement of collagen-specific antibodies 3.1. Effect of gallium nitrate on the progression of CIA Serum concentrations of anti-bCII IgG1, IgG2a and IgG2b isotypes were measured by enzyme-linked immunosorbent assay (ELISA). In brief, collect 100 μl of blood was collected using a heparin-coated capillary tube from the retro-orbital vascular plexus at days 36, 43, 50, and 56 after immunization. Microtiter plates were coated with bCII (10 μg/ml), blocked, and incubated with serially diluted test sera. Bound IgG was detected by incubation with horseradish peroxidase (HRP)-conjugated rat anti-mouse IgG1, IgG2a or IgG2b, and tetramethylbenzidine (TMB) substrate. Absorbance (450 nm) was measured with an ELISA plate reader, and the values were represented in arbitrary OD units. 2.6. Determination of serum cytokine levels of TNF-α, IL-6 and IFN-γ TNF-α, IL-6 and IFN-γ were measured using the BD Cytometric Bead Array (CBA) Mouse Inflammation kit (BD Biosciences, San Jose, CA) according to the manufacturer's instructions. 2.7. Flow cytometry Spleen cells of six mice from each treated group were prepared a single cell suspension. The cells were washed, incubated with antibodies for FACS analysis and acquired on a FACS Canto II flow cytometer (BD Biosciences, San Jose, CA). The following antibodies were obtained
To test whether gallium nitrate can be effective after the onset of arthritis, treatment was begun when arthritis had developed in more than two ankles (Fig. 1). Arthritis appeared at approximately 28 to 30 days after the initial immunization, or approximately one week following the booster immunization. Mice were randomly divided into four groups (vehicle, 3.5 mg/kg gallium nitrate, 7 mg/kg gallium nitrate and dexamethasone) on day 33 and treated with gallium nitrate (3.5 mg/kg or 7 mg/kg) from day 36 to day 49. Dexamethasone (5 mg/kg) was added three times a week for two weeks by oral gavage on day 36 after immunization. During the administration of gallium nitrate, the mice appeared healthy, and no indications of toxicity were found. On day 36, the clinical scores and hind paw thickness of each group were, respectively, 3.0 ± 1.0 and 1.99 ± 0.32 mm (vehicle), 3.2 ± 0.8 and 1.93 ± 0.38 mm (3.5 mg/kg gallium nitrate), 3.2 ± 0.6 and 1.97 ± 0.39 mm (7 mg/kg gallium nitrate) and 3.2 ± 0.8 and 1.98 ± 0.08 (dexamethasone). Arthritis symptoms were continuously progressive in the vehicle group, with hind paw thickness peaking at 2.94 ± 0.38 mm on day 50, and clinical scores continuously increased until the end of the experiments (12.8 ± 2.7 at day 70) (Fig. 1). The clinical scores of mice treated with 3.5 mg/kg and 7 mg/kg gallium nitrate were significantly different from the vehicle group (p b 0.05) at 46 and 44 days, respectively (Fig. 1A). Mice treated with 3.5 mg/kg gallium nitrate exhibited a progressive increase of
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Fig. 1. Gallium nitrate mitigates the clinical arthritis score and the hind paw thickness in mice with CIA. DBA/1 mice were immunized with bCII in CFA, boosted with bCII in IFA and monitored for the development of arthritis. Mice were injected intraperitoneally with 0.2 ml of 3.5 mg/kg or 7 mg/kg gallium nitrate or vehicle from days 36 to 49 (14 injections). As a control, some mice received 5 mg/kg dexamethasone by oral gavage 3 times a week for 14 days, beginning on day 36. (A) Clinical arthritis score. Mice were monitored every other day using a standard scoring system. (B) Hind paw thickness. The thickness of each paw was evaluated with a thickness gauge. (C) Histopathology of ankle joints in CIA mice. Sections of hind paw joint were stained with hematoxylin and eosin on day 70 after bCII immunization. Joints of CIA mice treated with vehicle (a), 3.5 mg/kg gallium nitrate (b) and 7 mg/kg gallium nitrate (c). (Original magnification, 40×) The data are represented as the mean ± SD with n = 6 mice per group.*, p b 0.05; **, p b 0.01 and ***, p b 0.001 compared with the vehicle treatment.
hind paw thickness until day 48 (2.29 ± 0.43 mm), after which hind paw thickness stabilized. The hind paw thickness of mice treated with 7 mg/kg gallium nitrate progressively increased until day 42 (2.27 ± 0.50 mm) but exhibited a progressive decrease after day 42 (Fig. 1B). Mice treated with dexamethasone showed a significant decrease in both clinical score and hind paw thickness compared to the vehicle group. However, dexamethasone-treated mice were less effective than both of the gallium nitrate-treated groups in terms of clinical score and hind paw thickness. Therefore, the dexamethasone treatment group was excluded from the subsequent
experiment. The results suggest that the treatment of CIA mice with gallium nitrate resulted in a significant dose-dependent amelioration of the clinical arthritis score and the thickness of the hind paw compared to the vehicle (p b 0.05). 3.2. Effect of gallium nitrate on histopathology of CIA mice To evaluate the histological changes in the ankle joints, mice were sacrificed at the end of the experiments (day 70), and joints were fixed with formalin, decalcified, and stained with H&E (Fig. 1C). Ankle
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joints of the vehicle-treated mice showed marked infiltration of inflammatory cells, narrowing joint space, synovial hyperplasia, and destruction of bone and cartilage (Fig. 1C, microphotograph a). However, the joints of mice treated with 3.5 mg/kg gallium nitrate showed less inflammatory cell infiltration, well-preserved joint spaces and minimal synovial hyperplasia (Fig. 1C, microphotograph b). The joints of mice treated with 7 mg/kg gallium nitrate were nearly histologically normal (Fig. 1C, microphotograph c). Therefore, gallium nitrate-treated mice exhibited ameliorated clinical symptoms of CIA, and the histological changes to the joints were prevented from progressing further. 3.3. Characterization of the cell populations in gallium nitrate-treated CIA mice To determine whether there was any change in the T cell population in gallium nitrate-treated CIA mice, we analyzed the T cell population in the spleens of mice treated with 3.5 mg/kg and 7 mg/kg gallium nitrate and the vehicle group by flow cytometry. Splenocytes were harvested two different times, early (gallium nitrate treatment for seven days, PI 43 days) and late (gallium nitrate treatment for 14 days, PI 50 days)
time points. CD4+ T cells were markedly reduced by gallium nitrate treatment for 7 days and 14 days (Fig. 2). Based on these results, we concluded that type II collagen-specific CD4+ T cells were reduced by gallium nitrate treatment. 3.4. Gallium nitrate treatment inhibits the collagen type II-specific humoral immune response To test whether gallium nitrate reduces the production of autoantibody, sera were collected at days 36, 43, 50 and 56. Type II collagen (CII)-specific antibodies were detected by isotype-specific ELISA (Fig. 2C). Serum concentrations of IgG1 and IgG2b anti-CII antibodies were slightly decreased by disease progression, but the concentration of IgG2a was stable in untreated CIA mice. Gallium nitrate treatments reduced serum levels of anti-CII IgG2a autoantibody and induced rapid decreases in anti-CII autoantibody serum levels below vehicle levels in a dose-dependent manner (p b 0.05). However, gallium nitrate treatments did not change the CII-specific IgG2b antibody serum levels. These findings indicate that gallium nitrate influences type II collagenspecific autoantibody production by B cells during CIA.
Fig. 2. Gallium nitrate inhibits the increase of CII-specific CD4+ T cells in the spleen and collagen type II-specific autoantibody levels in mice with CIA. Splenic cells were labeled with conjugated Abs specific for CD3, CD4 and CD8 and analyzed by flow cytometry. (A) For spleen cells, CD3+ cells were gated and CD4+ and CD8+ cells were analyzed. The results shown are from one of three representative experiments. (B) CD4+ T helper cells. (C) Serum levels of CII-specific autoantibody in CIA mice treated with 3.5 mg/kg and 7 mg/kg gallium nitrate or vehicle. Sera were collected at the indicated dates. The levels of IgG1, IgG2a and IgG2b were measured by ELISA. Data are the mean ± SD. *, p b 0.05 and **, p b 0.01 compared with the vehicle treatment.
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To investigate the effect of gallium nitrate on CIA progression in mice, RT-PCR and Western blotting were performed on joint tissue. The MMP2 mRNA level was decreased at day 7 by treatment with 7 mg/kg gallium nitrate and was slightly increased by treatment with both 3.5 and 7 mg/kg at day 14, but not significantly. The MMP9 mRNA level was decreased at day 14 by treatment with 7 mg/kg gallium nitrate and was slightly increased by 7 mg/kg gallium nitrate at day 7, but not significantly (Fig. 4A). Previous studies showed that MMPs implicated in the pathogenesis of RA are regulated at the level of gene transcription by NF-κB DNA binding protein and NF-κB was markedly increased and the levels of IκB protein was decreased in mice with CIA [11,12]. Therefore, to test the mechanism of gallium nitrate-mediated inhibition of MMP2 and MMP9 expression associated with the NF-κB signaling pathway, this pathway was evaluated by Western blotting. As shown in Fig. 4B, NF-κB activity was inhibited in gallium nitratetreated mice and the decrease of IκB resulting from degradation was suppressed by gallium nitrate treatment. These data suggest that gallium nitrate reduced local inflammation and tissue destruction in the CIA mice through the suppression of NF-κB activation. 4. Discussion Our results demonstrated the effects of gallium nitrate in a CIA model, on both the gross and microscopic levels. Gallium nitrate effectively alleviated CIA in mice, as indicated by the notable reductions in the clinical arthritis score, hind paw thickness and the histological lesions of CIA. The therapeutic effects of gallium nitrate might be caused by immune modulatory and anti-inflammatory activities. Immune modulatory activity was revealed through the suppression of the Th-1 response, including the increased population of CD4+ T cells, autoantibodies, and serum IFN-γ. Anti-inflammatory activity was demonstrated through the reduction of local and systemic levels of pro-inflammatory cytokines and the suppression of MMP gene expression. These effects of gallium nitrate might be caused by decreased NF-κB activity. To evaluate the immunological and anti-inflammatory features of gallium nitrate, the CIA model using the arthritis-susceptible DBA/1 mouse was used. This model is one of the most commonly used models and has many of the pathological features of human RA [13]. In the CIA model, gallium nitrate was administered from day 36, when the clinical score of three per mouse, to day 49. Even seven days of gallium nitrate treatment resulted in the maintenance of the clinical score. In addition, the clinical score did not increase after treatment was stopped. These findings suggested that the therapeutic effects of gallium nitrate might be caused by the modulation of the immune response.
TNF-α α
IL-6
150
30
100
20
**
50 0
Pg/ml
3.6. Gallium nitrate reduces MMP expression and inhibits NF-κB activation in the joints of CIA mice
B
Pg/ml
The pro-inflammatory cytokines TNF-α and IL-6 seem to be related to the development of CIA [10]. The mRNA and serum levels of TNF-α were significantly reduced in the group treated with 7 mg/kg gallium nitrate. The mRNA and serum levels of IL-6 were significantly reduced in the group treated with 7 mg/kg gallium nitrate, but the mRNA level of IL-6 was not significantly reduced in the group treated with 3.5 mg/kg gallium nitrate (Fig. 3A, B). Thus, gallium nitrate was significantly effective at reducing the pro-inflammatory cytokine levels in CIA. To evaluate the effect of gallium nitrate on the T cell response, the IFN-γ mRNA levels in joint tissues were detected with RT-PCR, and serum levels of IFN-γ were measured with CBA. The mRNA and serum levels of IFN-γ were significantly reduced in the gallium nitratetreated groups compared to the vehicle-treated group (Fig. 3A, B). These results suggested that gallium nitrate may inhibit the local and systemic T cell responses in CIA mouse.
A
10
*
*
0
IFN-γ 4 3
Pg/ml
3.5. Gallium nitrate treatment modulates cytokine production in CIA mice
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2 1 0
*
***
Fig. 3. Gallium nitrate treatment decreases the production of cytokines in mice with CIA. (A) mRNA and serum levels of cytokines were measured in collagen type II-induced arthritic mice treated with 3.5 mg/kg and 7 mg/kg gallium nitrate or vehicle. Joint tissues were collected after 7 or 14 days of injection with gallium nitrate. The mRNA levels of TNF-α and IL-6 were measured by reverse transcription-polymerase chain reaction. GAPDH was used as the loading control. (B) Serum cytokine levels in collagen type IIinduced arthritic mice treated with 3.5 mg/kg and 7 mg/kg gallium nitrate or vehicle. Sera were collected on day 56. The levels of mouse inflammatory cytokines are measured using a CBA Mouse Inflammation kit and flow cytometry. Data are the mean ± SD. *, p b 0.05; **, p b 0.01; and ***, p b 0.001 compared with vehicle treatment.
Although the etiology of RA remains unknown and controversy persists concerning the role of humoral and cellular autoimmunity in the pathogenesis of RA, CD4+ T cells and pro-inflammatory cytokines play an important role in CIA [10,14,15]. In addition, the severity of CIA is associated with a Th1-type immune response with the production of IgG2a autoantibodies and a high level of IFN-γ [16,17]. Our data showed that CIA mice have increased the number of CD4+ T cells in the spleen and gallium nitrate reduced that increase. However, CD3, CD8, early activated (CD3+CD69+) and regulatory T cells (CD4+CD25+) were not changed by the treatment (data not shown). CII specific IgG2a autoantibody was significantly reduced by treatment with gallium nitrate, but IgG2b was not. Gallium is known as an irreducible mimic of Fe3 +, which is readily reducible to Fe2+ [18]. Th-1 cells are much more sensitive to the inactivation by iron deprivation than are Th-2 cells [19]. However, it also possible that gallium nitrate suppresses the gene
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signaling pathway is involved in the development and activation of Th-1 responses [26]. We investigated whether the activation of NF-κB signaling pathway was inhibited by gallium nitrate. Our data showed that gallium nitrate reduced p65 and p-IκB and increased IκB, indicating that gallium nitrate inhibits the NF-κB activation by blocking IκB degradation. These data reveal that the transcription factor NF-κB is a critical regulator in gallium nitrate-treated CIA. It has also been reported that NF-κB can mediate abnormal apoptosis and the proliferation of RA fibroblast-like synovial cells, and it may also affect the differentiation and activation of bone resorbing activity of osteoclasts [26]. In further research efforts, the effect of gallium nitrate on RA fibroblast-like synovial cells and osteoclasts should be examined. 5. Conclusions Gallium nitrate significantly improved the severity of arthritis in the CIA model, attenuated inflammatory and immune responses and regulated the production of cytokines and MMPs by suppressing the NF-κB signaling pathway. These data may provide a basis for understanding gallium nitrate-mediated mechanisms of immunosuppressive and anti-inflammatory effects in patients with RA. Taken together, these data indicate that gallium nitrate may be useful as a therapeutic agent for RA, as well as other inflammatory autoimmune disorders. References
Fig. 4. Gallium nitrate treatment alters gene expression and suppresses NF-κB activation in mice with CIA. (A) The mRNA levels of MMPs were measured in collagen type II-induced arthritic mice treated with 3.5 mg/kg and 7 mg/kg gallium nitrate or vehicle. Joint tissues were collected after 7 or 14 days of gallium nitrate injection. The mRNA levels of MMP2 and MMP9 were measured by reverse transcription-polymerase chain reaction. GAPDH was used as the loading control. (B) NF-κbp65, IκBα and phosphor-IκBα protein levels were measured in collagen type II-induced arthritic mice treated with 3.5 mg/kg and 7 mg/kg gallium nitrate or vehicle. Joint tissue was collected after 7 or 14 days of gallium nitrate injection. NF-κbp65, IκBα and phosphor-IκBα protein levels were measured by Western blotting. GAPDH was used as the loading control.
expression of IgG2a specifically. When we tested the effect of gallium nitrate on the levels of pro-inflammatory and Th1-type cytokines, we determined that gallium nitrate significantly reduced the mRNA and serum levels of TNF-α, IL-6 and IFN-γ in CIA mice. However, our results showed that mRNA and protein expression did not correlate with one another in the gallium nitrate-treated CIA model. It is possible that the lack of correlation is caused by complicated biological processes, such as translational modification, regulatory mechanisms and different experimental mRNA and protein degradation rates [20]. The results indicated that the inactivation of Th1 and the anti-inflammatory action of gallium nitrate may be associated with inhibiting the release of proinflammatory and Th1 type cytokines and Th1 type autoantibodies. Matrix metalloproteinases (MMPs) play a central role in articular cartilage degradation and synovial inflammation [21]. MMP2 (gelatinase A) and MMP9 (gelatinase B) are enhanced in the joints of patients with RA [22] and have been known to play an important role in progression and cartilage degradation via the digestion of collagen [23]. In addition, MMP9 is thought to be a main effector in the migration of macrophages and neutrophils during inflammatory disease such as RA [24]. Our data showed that gallium nitrate treatment reduced the expression of MMP2 and MMP9 in the joint tissues of CIA mice. NF-κB plays a key role in inflammatory process responsible for the pathogenesis of RA [25]. It has also been reported that the NF-κB
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Gallium nitrate ameliorates type II collagen-induced arthritis in mice Jae-Hyeog Choi a,1, Jong-Hwan Lee b,1, Kug-Hwan Roh a, Su-Kil Seo a, Il-Whan Choi a, Sae-Gwang Park a, Jun-Goo Lim c, Won-Jin Lee d, Myoung-Hun Kim d, Kwang-rae Cho d, Young- Jae Kim d,⁎ a
Department of Microbiology and Immunology, College of Medicine, Inje University, Busan, Republic of Korea Department of Anesthesiology and Pain Medicine, College of Medicine, Dong-A University, Busan, Republic of Korea Shin Tong Pain Clinic, Busan, Republic of Korea d Department of Anesthesiology and Pain Medicine, Busan Paik Hospital, College of Medicine, Inje University, Busan, Republic of Korea b c
a r t i c l e
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Article history: Received 23 July 2013 Received in revised form 5 March 2014 Accepted 6 March 2014 Available online 19 March 2014 Keywords: Collagen type II-induced arthritis Gallium nitrate IgG Pro-inflammatory cytokines Matrix metalloproteinases Nuclear factor κB
a b s t r a c t Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease. Gallium nitrate has been reported to reserve immunosuppressive activities. Therefore, we assessed the therapeutic effects of gallium nitrate in the mouse model of developed type II collagen-induced arthritis (CIA). CIA was induced by bovine type II collagen with Complete Freund's adjuvant. CIA mice were intraperitoneally treated from day 36 to day 49 after immunization with 3.5 mg/kg/day, 7 mg/kg/day gallium nitrate or vehicle. Gallium nitrate ameliorated the progression of mice with CIA. The clinical symptoms of collagen-induced arthritis did not progress after treatment with gallium nitrate. Gallium nitrate inhibited the increase of CD4+ T cell populations (p b 0.05) and also inhibited the type II collagen-specific IgG2a-isotype autoantibodies (p b 0.05). Gallium nitrate reduced the serum levels of TNF-α, IL-6 and IFN-γ (p b 0.05) and the mRNA expression levels of these cytokine and MMPs (MMP2 and MMP9) in joint tissues. Western blotting of members of the NF-κB signaling pathway revealed that gallium nitrate inhibits the activation of NF-κB by blocking IκB degradation. These data suggest that gallium nitrate is a potential therapeutic agent for autoimmune inflammatory arthritis through its inhibition of the NF-κB pathway, and these results may help to elucidate gallium nitrate-mediated mechanisms of immunosuppression in patients with RA. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is a chronic, systemic and inflammatory disorder caused by a loss of immune tolerance that compromises immune homeostasis and results in the onset of autoimmune disorders [1–3]. A variety of approaches have been suggested for treating RA. The goals are to relieve pain, reduce inflammation, slow or stop joint damage, and improve the quality of life. A number of medications have been demonstrated to promote and suppress immunity in different ways. However, the drug's effectiveness and potential side effects are important considerations for the treatment of RA [4]. Gallium nitrate is the gallium salt of nitric acid with the chemical formula Ga(NO3)2 and is used to treat hypercalcemia secondary to cancer because gallium nitrate inhibits osteoclast activity, thereby lowering the amount of free calcium in blood [5]. This inhibition of osteoclasts can mediate the therapeutic effect on RA. It also has been demonstrated that gallium nitrate has potent anti-inflammatory, anti-microbial, and immune modulatory activity in several animal and clinical studies [6–8]. In addition, gallium nitrate has been demonstrated to prevent ⁎ Corresponding author at: Department of Anesthesiology and Pain Medicine, Busan Paik Hospital, College of Medicine, Inje University, Busan 614-735, Republic of Korea. Tel.: +82 51 890 6520; fax: +82 51 898 4216. E-mail address: [email protected] (Y.-J. Kim). 1 Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.intimp.2014.03.005 1567-5769/© 2014 Elsevier B.V. All rights reserved.
adjuvant inflammatory arthritis [8]. However, the immune modulatory mechanism of gallium nitrate has remained unknown. This study was conducted to evaluate whether gallium nitrate can ameliorate the progression of RA via focused anti-inflammatory and immune modulatory activity. Gallium nitrate was administered when arthritic symptoms developed after immunization with type II collagen. After treatment with gallium nitrate, clinical symptoms of collageninduced arthritis (CIA) including clinical scores, hind paw thickness and joint structure did not progress. Gallium nitrate also inhibited the increase of the CD4+ T cell population and type II collagen-specific IgG1 and IgG2a autoantibodies. In addition, gallium nitrate-treated CIA mice exhibited reduced levels of inflammatory cytokines (TNF-α, IL-6 and INF-γ) and MMP2 and MMP9. The activation of NF-κB in joint tissues was also decreased. This study reveals that gallium nitrate can inhibit the progression of CIA through the modulation of inflammation and the immune response. 2. Materials and methods 2.1. Animals Five- to six-week-old male DBA/1J mice were purchased from Orient Bio, Inc. (Sungnam, Korea) and maintained in the Animal Care Facility at the Inje University School of Medicine. All experimental procedures
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were examined and approved by the Institutional Animal Care and Use Committee of Inje University.
from eBiosciences, Inc. (San Diego, CA): anti-CD3-phycoerythrin (PE), anti-CD4-fluoresceinisothiocyanate (FITC) and anti-CD8-PECy5.
2.2. Induction of CIA and treatment with gallium nitrate
2.8. Reverse transcription-polymerase chain reaction
CIA was induced in six- to seven-week-old male DBA/1 mice by intradermal tail-base injection of 100 μg bovine type II collagen (bCII) (Chondrex, Inc., Redmond, WA) emulsified with Complete Freund's adjuvant (CFA) containing 4.0 mg/ml Mycobacterium tuberculosis H37RA (Chondrex). Mice were boosted three weeks later with bCII emulsified in Incomplete Freund's adjuvant (IFA) and were observed up to 70 days after immunization. Mice were divided randomly into three groups (n = 6 per group), in which the mice with CIA were intraperitoneally injected from day 36 to day 49 after immunization (14 days) with 3.5 mg/kg/day or 7 mg/kg/day gallium nitrate (Navicular Research Division, EbyPharma LLC, TX) or TDW (triple distilled water) as a vehicle. Gallium nitrate was diluted with distilled water because the original stock of gallium nitrate contains distilled water. Dexamethasone was used as a reference drug and was given at 5 mg/kg by oral gavage, three times a week for two weeks on day 36 after immunization.
Joint tissues were collected after seven days (PI 43 days) and 14 days (PI 50 days) of gallium nitrate injection. Joint tissues were homogenized, and total RNA was purified by using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA) and quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). The cDNA was synthesized with 2 μg RNA. The amount of mRNA was determined using Accupower® premix (Bioneer, Korea). The specific primers used for PCR were as follows: for TNF-α, 5′-CCACACCGTCAG CCGATTTG-3′ and 5′-CACCCATTCCCTTCACAGAGC-3′; for IL-6, 5′-TGGG AAATCGTGGAAATGAG-3′ and 5′-GAAGGACTCTGGCTTTGTCTT-3′; for IFN-γ, 5′-GCCACGGCACAGTCATTGAAA-3′ and 5′-TTTCGCCTTGCTGTTG CTGA-3′; for MMP2, 5′-CCCCGATGCTGATACTGA-3′ and 5′-CTGTCCGC CAAATAAACC-3′; for MMP9, 5′-CTGGACAGCCAGACACTAAA-3′ and 5′CTCGCGGCAAGTCTTCAGAG-3′; and for GAPDH, 5′-TTCACCACCATGGA GAAGGC-3′ and 5′-GGCATGGACTGTGGTCATGA-3′. 2.9. Western blot analysis
2.3. Assessment of arthritis Clinically, arthritic severity was characterized by observations of joint properties and the inflammation of the surrounding tissue. The clinical arthritis score of joint inflammation was assessed from 0 to 4 as previously described. The maximal arthritis score per paw is 4, and the maximal disease score per mouse is 16 [9]. Paw swelling was determined by measuring the thickness of the hind paw using a thickness gauge (dial type) (Mitutoyo, Tokyo, Japan). 2.4. Histopathology of the ankle joint On day 70 after immunization, the mice were sacrificed and the hind paws were fixed in 10% buffered formalin, decalcified, and embedded in paraffin. Joint sections (5- to 7-μm) were prepared and stained with hematoxylin and eosin. The histological findings (original magnification, 40 ×) were examined for synovial hyperplasia and mononuclear cell infiltration.
Joint tissues were collected after 14 days (PI 50 days) of injection with gallium nitrate. Joint tissues were homogenized with protease and phosphatase inhibitor in RIPA lysis buffer (Thermo Scientific, Rockford, IL). The homogenates, which contained 20 μg of protein, were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. The blot was probed with 1 μg/ml of primary antibody against NF-κBp65, IκBα, pIκBα and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA). Horseradish peroxidase-conjugated anti-rabbit (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) or anti-mouse IgG (Santa Cruz Biotechnology) was used as a secondary antibody. 2.10. Statistical analysis Statistical analysis was performed using GraphPad Prism (version 5 for Windows; GraphPad Software, San Diego, CA). p values were calculated by t-test. p b 0.05 was considered to be statistically significant. 3. Results
2.5. Measurement of collagen-specific antibodies 3.1. Effect of gallium nitrate on the progression of CIA Serum concentrations of anti-bCII IgG1, IgG2a and IgG2b isotypes were measured by enzyme-linked immunosorbent assay (ELISA). In brief, collect 100 μl of blood was collected using a heparin-coated capillary tube from the retro-orbital vascular plexus at days 36, 43, 50, and 56 after immunization. Microtiter plates were coated with bCII (10 μg/ml), blocked, and incubated with serially diluted test sera. Bound IgG was detected by incubation with horseradish peroxidase (HRP)-conjugated rat anti-mouse IgG1, IgG2a or IgG2b, and tetramethylbenzidine (TMB) substrate. Absorbance (450 nm) was measured with an ELISA plate reader, and the values were represented in arbitrary OD units. 2.6. Determination of serum cytokine levels of TNF-α, IL-6 and IFN-γ TNF-α, IL-6 and IFN-γ were measured using the BD Cytometric Bead Array (CBA) Mouse Inflammation kit (BD Biosciences, San Jose, CA) according to the manufacturer's instructions. 2.7. Flow cytometry Spleen cells of six mice from each treated group were prepared a single cell suspension. The cells were washed, incubated with antibodies for FACS analysis and acquired on a FACS Canto II flow cytometer (BD Biosciences, San Jose, CA). The following antibodies were obtained
To test whether gallium nitrate can be effective after the onset of arthritis, treatment was begun when arthritis had developed in more than two ankles (Fig. 1). Arthritis appeared at approximately 28 to 30 days after the initial immunization, or approximately one week following the booster immunization. Mice were randomly divided into four groups (vehicle, 3.5 mg/kg gallium nitrate, 7 mg/kg gallium nitrate and dexamethasone) on day 33 and treated with gallium nitrate (3.5 mg/kg or 7 mg/kg) from day 36 to day 49. Dexamethasone (5 mg/kg) was added three times a week for two weeks by oral gavage on day 36 after immunization. During the administration of gallium nitrate, the mice appeared healthy, and no indications of toxicity were found. On day 36, the clinical scores and hind paw thickness of each group were, respectively, 3.0 ± 1.0 and 1.99 ± 0.32 mm (vehicle), 3.2 ± 0.8 and 1.93 ± 0.38 mm (3.5 mg/kg gallium nitrate), 3.2 ± 0.6 and 1.97 ± 0.39 mm (7 mg/kg gallium nitrate) and 3.2 ± 0.8 and 1.98 ± 0.08 (dexamethasone). Arthritis symptoms were continuously progressive in the vehicle group, with hind paw thickness peaking at 2.94 ± 0.38 mm on day 50, and clinical scores continuously increased until the end of the experiments (12.8 ± 2.7 at day 70) (Fig. 1). The clinical scores of mice treated with 3.5 mg/kg and 7 mg/kg gallium nitrate were significantly different from the vehicle group (p b 0.05) at 46 and 44 days, respectively (Fig. 1A). Mice treated with 3.5 mg/kg gallium nitrate exhibited a progressive increase of
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Fig. 1. Gallium nitrate mitigates the clinical arthritis score and the hind paw thickness in mice with CIA. DBA/1 mice were immunized with bCII in CFA, boosted with bCII in IFA and monitored for the development of arthritis. Mice were injected intraperitoneally with 0.2 ml of 3.5 mg/kg or 7 mg/kg gallium nitrate or vehicle from days 36 to 49 (14 injections). As a control, some mice received 5 mg/kg dexamethasone by oral gavage 3 times a week for 14 days, beginning on day 36. (A) Clinical arthritis score. Mice were monitored every other day using a standard scoring system. (B) Hind paw thickness. The thickness of each paw was evaluated with a thickness gauge. (C) Histopathology of ankle joints in CIA mice. Sections of hind paw joint were stained with hematoxylin and eosin on day 70 after bCII immunization. Joints of CIA mice treated with vehicle (a), 3.5 mg/kg gallium nitrate (b) and 7 mg/kg gallium nitrate (c). (Original magnification, 40×) The data are represented as the mean ± SD with n = 6 mice per group.*, p b 0.05; **, p b 0.01 and ***, p b 0.001 compared with the vehicle treatment.
hind paw thickness until day 48 (2.29 ± 0.43 mm), after which hind paw thickness stabilized. The hind paw thickness of mice treated with 7 mg/kg gallium nitrate progressively increased until day 42 (2.27 ± 0.50 mm) but exhibited a progressive decrease after day 42 (Fig. 1B). Mice treated with dexamethasone showed a significant decrease in both clinical score and hind paw thickness compared to the vehicle group. However, dexamethasone-treated mice were less effective than both of the gallium nitrate-treated groups in terms of clinical score and hind paw thickness. Therefore, the dexamethasone treatment group was excluded from the subsequent
experiment. The results suggest that the treatment of CIA mice with gallium nitrate resulted in a significant dose-dependent amelioration of the clinical arthritis score and the thickness of the hind paw compared to the vehicle (p b 0.05). 3.2. Effect of gallium nitrate on histopathology of CIA mice To evaluate the histological changes in the ankle joints, mice were sacrificed at the end of the experiments (day 70), and joints were fixed with formalin, decalcified, and stained with H&E (Fig. 1C). Ankle
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joints of the vehicle-treated mice showed marked infiltration of inflammatory cells, narrowing joint space, synovial hyperplasia, and destruction of bone and cartilage (Fig. 1C, microphotograph a). However, the joints of mice treated with 3.5 mg/kg gallium nitrate showed less inflammatory cell infiltration, well-preserved joint spaces and minimal synovial hyperplasia (Fig. 1C, microphotograph b). The joints of mice treated with 7 mg/kg gallium nitrate were nearly histologically normal (Fig. 1C, microphotograph c). Therefore, gallium nitrate-treated mice exhibited ameliorated clinical symptoms of CIA, and the histological changes to the joints were prevented from progressing further. 3.3. Characterization of the cell populations in gallium nitrate-treated CIA mice To determine whether there was any change in the T cell population in gallium nitrate-treated CIA mice, we analyzed the T cell population in the spleens of mice treated with 3.5 mg/kg and 7 mg/kg gallium nitrate and the vehicle group by flow cytometry. Splenocytes were harvested two different times, early (gallium nitrate treatment for seven days, PI 43 days) and late (gallium nitrate treatment for 14 days, PI 50 days)
time points. CD4+ T cells were markedly reduced by gallium nitrate treatment for 7 days and 14 days (Fig. 2). Based on these results, we concluded that type II collagen-specific CD4+ T cells were reduced by gallium nitrate treatment. 3.4. Gallium nitrate treatment inhibits the collagen type II-specific humoral immune response To test whether gallium nitrate reduces the production of autoantibody, sera were collected at days 36, 43, 50 and 56. Type II collagen (CII)-specific antibodies were detected by isotype-specific ELISA (Fig. 2C). Serum concentrations of IgG1 and IgG2b anti-CII antibodies were slightly decreased by disease progression, but the concentration of IgG2a was stable in untreated CIA mice. Gallium nitrate treatments reduced serum levels of anti-CII IgG2a autoantibody and induced rapid decreases in anti-CII autoantibody serum levels below vehicle levels in a dose-dependent manner (p b 0.05). However, gallium nitrate treatments did not change the CII-specific IgG2b antibody serum levels. These findings indicate that gallium nitrate influences type II collagenspecific autoantibody production by B cells during CIA.
Fig. 2. Gallium nitrate inhibits the increase of CII-specific CD4+ T cells in the spleen and collagen type II-specific autoantibody levels in mice with CIA. Splenic cells were labeled with conjugated Abs specific for CD3, CD4 and CD8 and analyzed by flow cytometry. (A) For spleen cells, CD3+ cells were gated and CD4+ and CD8+ cells were analyzed. The results shown are from one of three representative experiments. (B) CD4+ T helper cells. (C) Serum levels of CII-specific autoantibody in CIA mice treated with 3.5 mg/kg and 7 mg/kg gallium nitrate or vehicle. Sera were collected at the indicated dates. The levels of IgG1, IgG2a and IgG2b were measured by ELISA. Data are the mean ± SD. *, p b 0.05 and **, p b 0.01 compared with the vehicle treatment.
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To investigate the effect of gallium nitrate on CIA progression in mice, RT-PCR and Western blotting were performed on joint tissue. The MMP2 mRNA level was decreased at day 7 by treatment with 7 mg/kg gallium nitrate and was slightly increased by treatment with both 3.5 and 7 mg/kg at day 14, but not significantly. The MMP9 mRNA level was decreased at day 14 by treatment with 7 mg/kg gallium nitrate and was slightly increased by 7 mg/kg gallium nitrate at day 7, but not significantly (Fig. 4A). Previous studies showed that MMPs implicated in the pathogenesis of RA are regulated at the level of gene transcription by NF-κB DNA binding protein and NF-κB was markedly increased and the levels of IκB protein was decreased in mice with CIA [11,12]. Therefore, to test the mechanism of gallium nitrate-mediated inhibition of MMP2 and MMP9 expression associated with the NF-κB signaling pathway, this pathway was evaluated by Western blotting. As shown in Fig. 4B, NF-κB activity was inhibited in gallium nitratetreated mice and the decrease of IκB resulting from degradation was suppressed by gallium nitrate treatment. These data suggest that gallium nitrate reduced local inflammation and tissue destruction in the CIA mice through the suppression of NF-κB activation. 4. Discussion Our results demonstrated the effects of gallium nitrate in a CIA model, on both the gross and microscopic levels. Gallium nitrate effectively alleviated CIA in mice, as indicated by the notable reductions in the clinical arthritis score, hind paw thickness and the histological lesions of CIA. The therapeutic effects of gallium nitrate might be caused by immune modulatory and anti-inflammatory activities. Immune modulatory activity was revealed through the suppression of the Th-1 response, including the increased population of CD4+ T cells, autoantibodies, and serum IFN-γ. Anti-inflammatory activity was demonstrated through the reduction of local and systemic levels of pro-inflammatory cytokines and the suppression of MMP gene expression. These effects of gallium nitrate might be caused by decreased NF-κB activity. To evaluate the immunological and anti-inflammatory features of gallium nitrate, the CIA model using the arthritis-susceptible DBA/1 mouse was used. This model is one of the most commonly used models and has many of the pathological features of human RA [13]. In the CIA model, gallium nitrate was administered from day 36, when the clinical score of three per mouse, to day 49. Even seven days of gallium nitrate treatment resulted in the maintenance of the clinical score. In addition, the clinical score did not increase after treatment was stopped. These findings suggested that the therapeutic effects of gallium nitrate might be caused by the modulation of the immune response.
TNF-α α
IL-6
150
30
100
20
**
50 0
Pg/ml
3.6. Gallium nitrate reduces MMP expression and inhibits NF-κB activation in the joints of CIA mice
B
Pg/ml
The pro-inflammatory cytokines TNF-α and IL-6 seem to be related to the development of CIA [10]. The mRNA and serum levels of TNF-α were significantly reduced in the group treated with 7 mg/kg gallium nitrate. The mRNA and serum levels of IL-6 were significantly reduced in the group treated with 7 mg/kg gallium nitrate, but the mRNA level of IL-6 was not significantly reduced in the group treated with 3.5 mg/kg gallium nitrate (Fig. 3A, B). Thus, gallium nitrate was significantly effective at reducing the pro-inflammatory cytokine levels in CIA. To evaluate the effect of gallium nitrate on the T cell response, the IFN-γ mRNA levels in joint tissues were detected with RT-PCR, and serum levels of IFN-γ were measured with CBA. The mRNA and serum levels of IFN-γ were significantly reduced in the gallium nitratetreated groups compared to the vehicle-treated group (Fig. 3A, B). These results suggested that gallium nitrate may inhibit the local and systemic T cell responses in CIA mouse.
A
10
*
*
0
IFN-γ 4 3
Pg/ml
3.5. Gallium nitrate treatment modulates cytokine production in CIA mice
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2 1 0
*
***
Fig. 3. Gallium nitrate treatment decreases the production of cytokines in mice with CIA. (A) mRNA and serum levels of cytokines were measured in collagen type II-induced arthritic mice treated with 3.5 mg/kg and 7 mg/kg gallium nitrate or vehicle. Joint tissues were collected after 7 or 14 days of injection with gallium nitrate. The mRNA levels of TNF-α and IL-6 were measured by reverse transcription-polymerase chain reaction. GAPDH was used as the loading control. (B) Serum cytokine levels in collagen type IIinduced arthritic mice treated with 3.5 mg/kg and 7 mg/kg gallium nitrate or vehicle. Sera were collected on day 56. The levels of mouse inflammatory cytokines are measured using a CBA Mouse Inflammation kit and flow cytometry. Data are the mean ± SD. *, p b 0.05; **, p b 0.01; and ***, p b 0.001 compared with vehicle treatment.
Although the etiology of RA remains unknown and controversy persists concerning the role of humoral and cellular autoimmunity in the pathogenesis of RA, CD4+ T cells and pro-inflammatory cytokines play an important role in CIA [10,14,15]. In addition, the severity of CIA is associated with a Th1-type immune response with the production of IgG2a autoantibodies and a high level of IFN-γ [16,17]. Our data showed that CIA mice have increased the number of CD4+ T cells in the spleen and gallium nitrate reduced that increase. However, CD3, CD8, early activated (CD3+CD69+) and regulatory T cells (CD4+CD25+) were not changed by the treatment (data not shown). CII specific IgG2a autoantibody was significantly reduced by treatment with gallium nitrate, but IgG2b was not. Gallium is known as an irreducible mimic of Fe3 +, which is readily reducible to Fe2+ [18]. Th-1 cells are much more sensitive to the inactivation by iron deprivation than are Th-2 cells [19]. However, it also possible that gallium nitrate suppresses the gene
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signaling pathway is involved in the development and activation of Th-1 responses [26]. We investigated whether the activation of NF-κB signaling pathway was inhibited by gallium nitrate. Our data showed that gallium nitrate reduced p65 and p-IκB and increased IκB, indicating that gallium nitrate inhibits the NF-κB activation by blocking IκB degradation. These data reveal that the transcription factor NF-κB is a critical regulator in gallium nitrate-treated CIA. It has also been reported that NF-κB can mediate abnormal apoptosis and the proliferation of RA fibroblast-like synovial cells, and it may also affect the differentiation and activation of bone resorbing activity of osteoclasts [26]. In further research efforts, the effect of gallium nitrate on RA fibroblast-like synovial cells and osteoclasts should be examined. 5. Conclusions Gallium nitrate significantly improved the severity of arthritis in the CIA model, attenuated inflammatory and immune responses and regulated the production of cytokines and MMPs by suppressing the NF-κB signaling pathway. These data may provide a basis for understanding gallium nitrate-mediated mechanisms of immunosuppressive and anti-inflammatory effects in patients with RA. Taken together, these data indicate that gallium nitrate may be useful as a therapeutic agent for RA, as well as other inflammatory autoimmune disorders. References
Fig. 4. Gallium nitrate treatment alters gene expression and suppresses NF-κB activation in mice with CIA. (A) The mRNA levels of MMPs were measured in collagen type II-induced arthritic mice treated with 3.5 mg/kg and 7 mg/kg gallium nitrate or vehicle. Joint tissues were collected after 7 or 14 days of gallium nitrate injection. The mRNA levels of MMP2 and MMP9 were measured by reverse transcription-polymerase chain reaction. GAPDH was used as the loading control. (B) NF-κbp65, IκBα and phosphor-IκBα protein levels were measured in collagen type II-induced arthritic mice treated with 3.5 mg/kg and 7 mg/kg gallium nitrate or vehicle. Joint tissue was collected after 7 or 14 days of gallium nitrate injection. NF-κbp65, IκBα and phosphor-IκBα protein levels were measured by Western blotting. GAPDH was used as the loading control.
expression of IgG2a specifically. When we tested the effect of gallium nitrate on the levels of pro-inflammatory and Th1-type cytokines, we determined that gallium nitrate significantly reduced the mRNA and serum levels of TNF-α, IL-6 and IFN-γ in CIA mice. However, our results showed that mRNA and protein expression did not correlate with one another in the gallium nitrate-treated CIA model. It is possible that the lack of correlation is caused by complicated biological processes, such as translational modification, regulatory mechanisms and different experimental mRNA and protein degradation rates [20]. The results indicated that the inactivation of Th1 and the anti-inflammatory action of gallium nitrate may be associated with inhibiting the release of proinflammatory and Th1 type cytokines and Th1 type autoantibodies. Matrix metalloproteinases (MMPs) play a central role in articular cartilage degradation and synovial inflammation [21]. MMP2 (gelatinase A) and MMP9 (gelatinase B) are enhanced in the joints of patients with RA [22] and have been known to play an important role in progression and cartilage degradation via the digestion of collagen [23]. In addition, MMP9 is thought to be a main effector in the migration of macrophages and neutrophils during inflammatory disease such as RA [24]. Our data showed that gallium nitrate treatment reduced the expression of MMP2 and MMP9 in the joint tissues of CIA mice. NF-κB plays a key role in inflammatory process responsible for the pathogenesis of RA [25]. It has also been reported that the NF-κB
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