Male DBA/1J mice were purchased from Charles River Japan (Yokohama, Japan). The mice were specific pathogen-free and were kept in cages in a room maintained at 20 to 26°C at a relative humidity of 35 to 75%. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Chugai Pharmaceutical Co., Ltd.

GPI-induced arthritis was induced as previously described, with modifications 21. In brief, male DBA/1J mice (6 weeks old) were immunized intradermally at the base of the tail with 300 μg of recombinant GPI-glutathione S-transferase fusion protein (GPI-GST) emulsified with an equal volume of complete adjuvant H37Ra (DIFCO, Detroit, MI, USA). Pertussis toxin (200 ng/200 μL) was injected on the day of immunization and 2 days after immunization. Clinical symptoms of arthritis were evaluated visually and assigned a scale of 0 to 3 for each limb (maximum score/mouse = 12).

MTX (Sigma Aldrich, St Louis, MO, USA) dissolved in 7% sodium bicarbonate solution was given orally 3 times a week from the first day of immunization. Ten days after immunization, mice were intraperitoneally injected once with 4 mg of rat anti-mouse IL-6R monoclonal antibody, MR16-1 22. The vehicle group mice were administered 7% sodium bicarbonate solution orally, and were intraperitoneally injected with phosphate buffer saline (PBS). The MTX group mice were administered MTX orally, and were intraperitoneally injected with PBS. The MR16-1 group mice were administered 7% sodium bicarbonate solution orally and were intraperitoneally injected with MR16-1. The MTX plus MR16-1 group mice were administered MTX orally and were intraperitoneally injected with MR16-1. Each group consisted of 8 or 9 animals.

Immediately after the animals were killed, whole hind limbs were immersed in RNAlater RNA Stabilization Reagent (Qiagen, Valencia, CA, USA) and stored at -80°C until total RNA extraction. The whole hind limbs were excised, immediately soaked in TRIzol (Invitrogen, Carlsbad, CA, USA), and crushed in a bead mill (TissueLyser II; Qiagen, Valencia, CA, USA). Total RNA was extracted using an RNeasy kit (Qiagen) according to the kit manufacturer's protocol. Amounts of total RNA obtained from a single whole hind limb were 4.5 to 18 μg (intact mice), 15to 60 μg (non-treated immunized mice), 9 to 60 μg (MTX-treated immunized mice), 12 to 30 μg (MR16-1-treated immunized mice), and 4.2 and 30 μg (MTX- and MR16-1-treated immunized mice). cDNA was synthesized with an Omniscript RT kit (Qiagen) using random 9-mer primers (TaKaRa, Shiga, Japan) according to the kit manufacturer's protocol. Quantitative real-time polymerase chain reaction (PCR) was performed by running a TaqMan gene expression assay (Applied Biosystems, Foster City, CA, USA), targeting mouse SLC19A1, IL-6, TNF-α, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), on an ABI PRISM 7500 system (Applied Biosystems) according to the manufacturer's protocol.

For CD4 T cell and B cell subset sorting, splenocytes were labeled with antibodies to CD4 and B220 and sorted to 97% purity by using a fluorescence-activated cell sorter (FACSAria III; BD Biosciences, Franklin Lakes, NJ, USA). Total RNA was extracted using an RNeasy kit (Qiagen) according to the kit manufacturer's protocol. Synthesis of cDNA and measurement of mRNA levels by quantitative real-time PCR were performed by the same methods as described above.

Arthritic mice were killed and the synovial tissues removed from their hind limbs. Synovial tissues were incubated at 37°C for 180 min in α-MEM supplemented with 10% fetal bovine serum (FBS) and containing 0.5 mg/mL of Liberase Blendzyme2 (Roche Diagnostics, Basel, Switzerland). After incubation of the synovial tissues with the Liberase Blendzyme2, the resulting cells were cultured in a culture flask in α-MEM supplemented with 10% FBS and the non-adherent cells were removed and discarded. Synovial cells were then subcultured in α-MEM supplemented with 10% FBS to a density of 2 × 10⁵ cells/35 mL in a T175 flask. In this study, synovial cells from passages two to five were used.

Synovial cells (5 × 10⁴ cells/2 mL/well) were cultured in α-MEM supplemented with 10% FBS for 24 h. After this pre-culture, cells were cultured with mouse IL-6 (Peprotech, Rocky Hill, NJ, USA) and soluble mouse IL-6R (R&D Systems, Minneapolis, MN, USA), TNF-α, or MTX for 24 h. Total RNA was extracted using an RNeasy kit (Qiagen) according to the kit manufacturer's protocol. cDNA was synthesized with an Omniscript RT kit (Qiagen) using random 9-mer primers (Takara) according to the kit manufacturer's protocol. Quantitative real-time PCR was performed by running a TaqMan gene expression assay, targeting mouse SLC19A1, multidrug resistance protein-1 (MRP-1), breast cancer resistance protein (BCRP), and GAPDH, on an ABI PRISM 7500 system (Applied Biosystems) according to the manufacturer's protocol.

Synovial cells (5 × 10³ cells/0.2 mL/well) were cultured either with medium alone or with mouse IL-6, mouse sIL-6R, and MTX in a 96-well flat bottom plate for 3 days. Synovial cell proliferation was assessed using the Cell Proliferation ELISA system (GE Healthcare UK, Buckinghamshire, UK) according to the manufacturer's instructions. Briefly, bromodeoxyuridine (BrdU) was added for the last 3 h, and its uptake was detected with anti-BrdU antibody. Substrate was added to elicit a colorimetric reaction, and absorbance at 450 nm was measured using a microplate reader (SLT Inc., Salzburg, Austria). The data are expressed in terms of optical density (OD) values.

Synovial cells (5 × 10⁴ cells/2 mL/well) were cultured for 24 h in α-MEM supplemented with 10% FBS. After this pre-culture, cells were cultured with mouse IL-6 and mouse sIL-6R for an additional 24 h. After washing cells with PBS, cells were incubated in the presence 100 nM of Alexa Fluor 488 conjugated MTX (Invitrogen) for 5 h. After incubation, cells were treated with trypsin-EDTA (Gibco, Grand Island, NY, USA) to obtain a single cell suspension. Intracellular accumulation of MTX was measured with a fluorescence-activated cell sorter (FACSCanto II; BD Biosciences) and calculated mean fluorescence intensities (MFIs) were analyzed using FACSDiva software (BD Bioscience).

Serum samples were collected 15 days after immunization, and the concentrations of serum amyloid A (SAA) and IL-6 were measured by SAA mouse ELISA kit (Invitrogen) and Mouse IL-6 Quantikine ELISA Kit (R&D systems), respectively.

Statistical significances were estimated by Wilcoxon's test, unpaired t-test, and Dunnett's multiple comparison test using a statistical software package (SAS Institute Japan, Tokyo, Japan), with the significance level set to 5%.

MTX was administered orally 3 times a week for 4 weeks. MTX (10 mg/kg) suppressed the progression of arthritis, but from day 20 its efficacy gradually diminished (Figure 1A).

The mRNA expressions of SLC19A1 in whole hind limbs and immune cells were examined 15 days after immunization (peak of arthritis). The SLC19A1 expression in immunized mice was lower than that in intact mice (Figure 1B-D). Moreover, the expression level of SLC19A1 in whole hind limbs was further decreased by treatment with MTX (Figure 1B). TNF-α and IL-6 mRNA expressions in whole hind limbs were up-regulated in immunized mice with arthritis compared with intact mice, and MTX treatment did not reduce TNF-α or IL-6 mRNA expressions (Figure 1E, F). IL-1 mRNA expression was not detectable (data not shown).

We examined the effects of MTX, TNF-α and IL-6 on the expression of SLC19A1 in synovial cells. IL-6 or sIL-6R alone did not affect SLC19A1 mRNA expression (data not shown), but IL-6 + sIL-6R significantly decreased SLC19A1 expression in synovial cells (Figure 2A). MTX, but not TNF-α, significantly decreased the expression of SLC19A1 in synovial cells (Figure 2B, C). Moreover, in the presence of IL-6 + sIL-6R, MTX further reduced SLC19A1 expression (Figure 2D).

To investigate if MTX and IL-6 + sIL-6R affected the accumulation of MTX in synovial cells, the synovial cells were pre-treated with MTX and IL-6 + sIL-6R, and then MTX uptake into cells was examined. As shown in Figure 2E, pre-treatment with IL-6 + sIL-6R, or with MTX, reduced the accumulation of fluorescent conjugated MTX in synovial cells, and pre-treatment with IL-6 + sIL-6R + MTX further reduced the accumulation of fluorescent conjugated MTX in synovial cells.

Because we previously reported that MTX suppressed the proliferation of synovial cells from RA patients 23, we examined the effect of IL-6 + sIL-6R on MTX-induced suppression of proliferation of mouse synovial cells in vitro. MTX clearly inhibited the proliferation of mouse synovial cells in a dose-dependent manner (Figure 3). Interestingly, the inhibitory effect of MTX was weakened by the co-addition of IL-6 + sIL-6R.

Iwanami et al. reported that the development of GPI-induced arthritis was almost completely blocked by the injection of MR16-1 on days 0, 3, or 8 after immunization, whereas injection of MR16-1 on day 14, at the peak of arthritis, did not ameliorate arthritis, because injection of MR16-1 on day 14 did not inhibit T_h17 induction 17. A similar result was obtained in our study; namely, injection of MR16-1 on days 0 or 5 completely blocked the onset of arthritis, but the injection on day 10 did not ameliorate arthritis (data not shown). From these results, we decided to administer MR16-1 on day 10, because this regimen can exclude the direct effect of MR16-1 on the progression of arthritis.

Next, we examined whether the combination use of MTX and MR16-1 would affect the inhibitory effect of MTX on GPI-induced arthritis. Surprisingly, although MR16-1 monotherapy did not reduce arthritis score, concomitant use of MTX and MR16-1 significantly reduced the progression of arthritis compared with the vehicle group or the MTX group (Figure 4A).

To examine whether this phenomenon was induced by blocking IL-6, we measured concentrations of IL-6 and SAA in serum on day 15. Serum concentration of IL-6 was significantly elevated in vehicle-treated arthritic mice compared with intact mice (Figure 4B). Although serum IL-6 concentration did not significantly change in the MTX group compared with the vehicle-treated group, dramatic elevation of serum IL-6 level was observed in the MR16-1 group and the MTX plus MR16-1 group. It has been shown that IL-6 induces SAA 24, and because SAA is a beneficial marker of IL-6 activity, we also measured the serum level of SAA. Levels of SAA in the vehicle group were increased to 100 times the levels in intact mice, and were only slightly reduced in MTX-treated immunized mice. On the other hand, SAA induction was completely inhibited in the MR16-1 group and the MTX plus MR16-1 group (Figure 4C). We also noted that the body weights in all groups were unchanged throughout the experiments (data not shown).

The expressions of SLC19A1 mRNA in whole hind limbs, CD4 T cells and B cells were increased in the MR16-1 group and in the MTX plus MR16-1 group compared with those in the vehicle group (Figure 5A-C). Moreover, the levels of SLC19A1 mRNA expression significantly increased in the MTX plus MR16-1 group compared with those in the MTX group.