DHMEQ was synthesized as described previously 22. It was dissolved in 100% dimethyl sulfoxide (DMSO) at 20 mg/ml and kept in aliquots at -30°C. Before use in cell culture, it was diluted with the medium described below to a final DMSO concentration of 0.05% or less, at which no effect of DMSO per se on NF-κB activity was observed.

Animal experiments were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University. Male 8-week-old DBA/1J mice were purchased from Oriental Yeast (Tokyo, Japan). Bovine collagen type II (Collagen Research Center, Tokyo, Japan) was dissolved in 50 mmol/l acetic acid at 4 mg/ml and emulsified in an equal volume of Freund's complete adjuvant (Difco Laboratories, Detroit, MI, USA). Mice were immunized intradermally with 100 μl of the emulsion at the base of the tail. After 21 days (day 0) the same amount of the antigen emulsified in the same adjuvant was intradermally injected at the base of the tail as a booster immunization. By day 5, 20 out of the 25 mice developed signs of arthritis and were randomly allocated to two groups of 10 mice each: an experimental group and a control group. From days 5 to 18, 100 μg DHMEQ (5 mg/kg body weight) dissolved in 50 μl of 100% DMSO was injected subcutaneously every day into the inguinal region of the mice in the experimental group. Mice in the control group received 50 μl DMSO, injected similarly.

The thickness of each hind paw was measured using a pair of digital slide calipers by an investigator who was blinded to the treatment groups. Out of 16 joints in each hind paw (i.e. the ankle, midfoot, first to fifth metatarsophalangeal [MTP] joints, interphalangeal joint of the first toe, and second to fifth proximal and distal interphalangeal joints), the swollen joints were identified using magnified pictures taken with a digital camera and counted. Radiographic assessment of bilateral second to fourth MTP joints was carried out using the following scoring systems: for soft tissue swelling 0 = not obvious, 1 = mild, 2 = marked; and for bone erosion 0 = not obvious, 1 = erosion < 0.3 mm in diameter, 2 = erosion > 0.3 mm in diameter. Use of this system yields a possible score between 0 and 12 per animal for each item. The left hind paw of each mouse was dissected, formalin-fixed, decalcified, embedded in paraffin, and stained with hematoxylin and eosin. Synovitis and bone destruction around the MTP joints of the sections were scored as follows: synovitis 0 = not obvious, 1 = synovitis < 0.2 mm at maximum thickness, 2 = synovitis > 0.2 mm at maximum thickness; bone destruction 0 = not obvious, 1 = obvious bone erosion, 2 = marked bone erosion associated with penetration of pannus into the marrow space. Radiographic and histopathologic assessments were performed by five investigators who were blinded to the assignment of mouse groups.

RA FLS lines were established, as described previously 23, from the synovial tissues of RA patients obtained at surgery. RA patients fulfilled the American College of Rheumatology criteria. All procedures involving human tissues were approved by the Ethics Committee of Tokyo Medical and Dental University, and consent forms were obtained from the patients involved in the study. The cells were cultured in 100 mm dishes with Dulbecco's modified Eagle's medium (high glucose) containing 10% heat-inactivated fetal calf serum (FCS; Givco, Rockville, MD, USA) and antibiotics. Cells were passaged between four and eight times and were used when the cultures had reached about 80% cell layer confluence.

RA FLS in 100 mm dishes were starved for 16 hours in medium without FCS, and then 10 μg/ml DHMEQ or vehicle was added. Twenty minutes later they were stimulated with 5 ng/ml TNF-α (PeproTech, London, UK) for 30 minutes, washed with phosphate-buffered saline (PBS), and detached from the dishes by treatment with trypsin-EDTA. Total RNA was isolated using RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and treated with DNase I (Takara, Ohtsu, Japan), and RT-PCR was carried out using OneStep RT-PCR Kit (Qiagen) and the following primers: β-actin (5'-GTCCTCTCCCAAGTCCACACA, 3'-CTGGTCTCAAGTCAGTGTACAGGTAA), CC chemokine ligand (CCL)5 (formerly called RANTES; 5'-CGCTGTCATCCTCATTGCTA, 3'-GCTGTCTCGAACTCCTGACC), CCL2 (formerly called MCP-1; 5'-GCCTCCAGCATGAAAGTCTC, 3'-TAAAACAGGGTGTCTGGGGA), IL-1β (5'-TGCACGATGCACCTGTACGA, 3'-AGGCCCAAGGCCACAGGTAT), IL-6 (5'-GTTCCTGCAGAAAAAGGCAAAG, 3'-CTGAGGTGCCCATGCTACATTT), matrix metalloproteinase (MMP)-3 (5'-ATGGAGCTGCAAGGGGTGAG, 3'-CCCGTCACCTCCAATCCAAG), and vascular endothelial cell growth factor (VEGF) (5'-ATTGGAGCCTTGCCTTGCTG, 3'-CCAGGGTCTCGATTGGATGG). The cycling program was 94°C for 1 minute, 62°C for 1 minute, and 72°C for 1 minute for 25 or 30 cycles, followed by a final extension for 1 minute. The PCR products were electrophoresed on 1.5% agarose gel and stained with ethidium bromide. The relative intensities of the bands were quantified using image analysis software (NIH Image version 1.63; National Institute of Health, Bethesda, MD, USA).

RA-FLS were cultured and starved similarly as described above except 24-well culture plates were used. Twenty minutes after addition of DHMEQ (10 μg/ml) or vehicle, TNF-α (5 ng/ml) was added and 24 hours later the supernatant was harvested and kept at -20°C until use. ELISA kits for CCL2, CCL5, IL-1β and IL-6 were purchased from BioSource (Camacillo, CA, USA), and that for MMP-3 was from MBL (Nagoya, Japan).

RA-FLS were cultured and starved as described above except 60 mm culture dishes were used. Twenty minutes after addition of DHMEQ (10 μg/ml) or vehicle, TNF-α (5 ng/ml) was added and 14 hours later the cells were washed with PBS, detached by trypsin-EDTA, and suspended in PBS. The prepared cells were incubated with monoclonal antibody to intercellular adhesion molecule (ICAM)-1 (BD Sciences, San Jose, CA, USA), vascular cell adhesion molecule (VCAM)-1 (BD Sciences), or isotype-matched mouse IgG for 20 minutes, followed by a detection with phycoerythrin-conjugated goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL, USA), and analyzed with an Epics XL flow cytometer (Beckman Coulter, Miami, FL, USA).

For detection of apoptotic cells, cells were prepared as above, DHMEQ (0, 5, or 10 μg/ml) or 1 μmol/l staurosporin (Wako, Osaka, Japan) was added, the cells were stimulated with TNF-α (5 ng/ml) 20 minutes later, further incubated for 14 hours, and stained with Cy3-labeled annexin V (MBL).

RA FLS were cultured at a density of 10⁴/well in 96-well culture plates for 18 hours and then starved for 24 hours in Dulbecco's modified Eagle's medium with 2 μmol/l 2-mercaptoethanol, without FCS. After serum starvation, 0–10 μg/ml DHMEQ was added followed 20 minutes later by addition of 5 ng/ml TNF-α and 5 μCi/ml [³H]thymidine. The cells were further cultured for 48 hours and incorporation of ³H during the last 24 hours was measured in a scintillation counter.

RA FLS were cultured, starved, and stimulated as described above for RT-PCR, and nuclear extracts were prepared using NucBaster Protein Extraction Kit (Novagen, Darmstadt, Germany). The protein concentrations of the extracts were estimated by BCA Protein Assay Kit (Pierce, Rockford, IL, USA), and the extracts were kept at -80°C until use. ³²P-labeled oligonucleotide containing the NF-κB binding sequence (5'-AGTTGAGGGGACTTTCCCAGGC-3') was used as a probe. Ten micrograms of the nuclear extract was incubated with 2 μg poly(dI-dC) for 30 minutes at room temperature in 20 μl reaction buffer containing 20 mmol/l HEPES, 20% glycerol, 100 mmol/l KCl, and 0.2 mmol/l EDTA, at pH 7.9. Following incubation, ³²P-labeled probe was added to the mixture with or without 100-fold excess unlabeled oligonucleotide as a competitor and incubated for a further 30 minutes at room temperature. The protein-DNA complexes were separated from the free probe by 4% PAGE. For supershift assays, 1 μg of antibody to p50, p52 (Santa Cruz Biotech, Santa Cruz, CA, USA), or p65 (Chemicon, Temecula, CA, USA) was added to the sample and incubated for 30 minutes at 4°C before electrophoresis.

Results were compared using two-sided, unpaired Student's t-tests.

The in vivo anti-inflammatory effect of DHMEQ was first demonstrated in a type of CIA model described by Matsumoto and coworkers 21, in which they showed a prophylactic effect of this compound when administered at 2–4 mg/kg, three times a week, from the day of booster immunization, although the CIA protocol was different from that in the present study and they did not use adjuvant. To test the therapeutic effect on a standard CIA model, we included only those mice that had apparently begun to develop arthritis by day 5 after the booster immunization with collagen and complete adjuvant, and started therapy with DHMEQ at 100 μg (5 mg/kg) daily.

After 14 days of therapy (from days 5 to 18), the thickness of the hind paws in the DHMEQ treated group (mean ± SD; 5.97 ± 0.66 mm) was significantly lower than that in the control group (6.95 ± 0.88 mm) that received vehicle alone (Fig. 1a). The number of swollen joints was also significantly lower in the DHMEQ-treated group (9.20 ± 4.64 versus 14.20 ± 3.68; Fig. 1b). During the first week of treatment, these young animals exhibited growth retardation, as estimated from their body weights, likely resulting from severe inflammation. However, this slow-down was significantly less in the DHMEQ group (weight gain from days 5 to 12; 1.29 ± 1.25 g) than in the control group (0.14 ± 1.01 g; Fig. 1c), suggesting that DHMEQ alleviated inflammation and was tolerable at the dose tested. Radiographs showed various degrees of soft tissue swelling and destructive changes in bone (Fig. 2a, b). The scores of these findings were both significantly lower in the group treated with DHMEQ (Fig. 2c, d). At the histologic level, various degrees of synovitis and bone destruction were observed (Fig. 3a, b). In severe cases, marked infiltration of mononuclear cells were present within the synovium, and pannus was frequently observed to penetrate into the bone marrow space. Again, DHMEQ reduced these findings significantly (Fig. 3c, d).

To examine the mechanisms underlying the antiarthritic effect of DHMEQ, as well as its effect on human cells, RA FLS lines were established from several patients with RA and used for the present experiments. We should like to note that it was previously confirmed that these cells expressed neither CD14 nor HLA class II 23, which means that they did not contain either macrophages or dendritic cells.

The effect of DHMEQ on NF-κB activation in RA FLS was examined using electrophoretic mobility shift assay (Fig. 4). Unstimulated RA FLS in serum-free medium exhibited only a faint band corresponding to NF-κB, but stimulation with 5 ng/ml TNF-α increased the intensity of the band dramatically. In supershift assays, anti-p50 antibody virtually abrogated the band of NF-κB, and anti-p65 antibody also remarkably diminished the intensity of the band. Anti-p52 antibody was much less effective, suggesting that the major components of the activated NF-κB in TNF-α-stimulated RA FLS were p65 and p50. An excess amount of unlabeled NF-κB probe abolished the band, confirming the specificity of this assay. When DHMEQ was added 20 minutes before the stimulation with TNF-α, the band representing NF-κB was abrogated almost completely at 10 μg/ml but not significantly at 1 μg/ml. Based on these findings, the following experiments were carried out using 10 μg/ml DHMEQ unless otherwise indicated.

Among the many molecules that are involved in inflammatory responses, a set of representative chemokines, ILs, MMP-3, and VEGF were selected to test the effect of DHMEQ. IL-6 is known to be an NF-κB-dependent cytokine 2425 and is one of the key cytokines in RA pathogenesis, as evidenced by the fact that an anti-IL-6 receptor monoclonal antibody has been shown to reduce significantly RA disease activity in clinical trials 26. We previously showed that chemokines CCL2 and CCL5 play a role not only in inflammatory cell migration but also in activation of RA FLS in an autocrine or paracrine manner 23. Other investigators have shown that an antagonist to CCL2 suppressed arthritis in a murine model 27. MMP-3 is among the cartilage-degrading enzymes and is known to be regulated by the NF-κB pathway in RA synovium 28. VEGF is one of the angiogenic factors that are involved in the neovascularization in RA joints 29.

The effect of DHMEQ on mRNA expression of these key molecules was first examined by RT-PCR. As shown in Fig. 5, there was some heterogeneity in the mRNA expression levels depending on the cell line used. However, mRNA levels of CCL2, IL-6, and MMP-3 tended to be consistently increased by TNF-α stimulation and suppressed by DHMEQ. CCL5 mRNA was not detected in one of the cell lines (#5), but in other cell lines it was enhanced by TNF-α and suppressed by DHMEQ. IL-1β mRNA was barely detectable in some of the cell lines tested, at least under these assay conditions. However, after TNF-α stimulation IL-1β mRNA was clearly expressed in cell line #2 and faintly in lines #1 and #5; in all cases the expression was diminished by treatment with DHMEQ. In contrast, the level of VEGF mRNA was neither significantly increased by TNF-α nor suppressed by DHMEQ. We applied a constant amount of RNA to each tube, and observed virtually constant intensity of β-actin mRNA, irrespective of treatment with DHMEQ; this suggested that this compound did not affect the housekeeping activity of the cells.

The suppressive effect of DHMEQ on the TNF-α-induced expression of CCL2, CCL5, IL-1β, IL-6 and MMP-3 was further tested at the protein level by ELISA (Fig. 6). CCL2, CCL5 and IL-6 were barely detected in the serum-free culture supernatant of the cells unless the cells were stimulated, but were produced abundantly after TNF-α stimulation. This production was significantly suppressed by DHMEQ. Similarly, production of MMP-3 tended to be suppressed by DHMEQ, although this was not statistically significant. Because the level of IL-1β was lower than the detection limit (10 pg/ml), even after stimulation with TNF-α, we could not confirm the effect of DHMEQ on IL-1β expression by ELISA.

Adhesion molecules ICAM-1 (CD54) and VCAM-1 (CD106) are expressed at higher levels in the synovial tissue of RA than in osteoarthritis 3031 and are implicated in the interaction between leukocytes and RA FLS that contributes to the synovitis. Flow cytometric analysis showed that 2.7 ± 0.5% (mean ± standard deviation [SD] of four independent experiments) of RA FLS expressed ICAM-1 in serum-free medium, and the ratio of cells expressing ICAM-1 markedly increased up to 44.5 ± 11.7% after stimulation with TNF-α (Fig. 7). In the presence of DHMEQ, however, this ratio significantly decreased to 5.3 ± 3.4%. On the other hand, VCAM-1 was expressed on only 0.9 ± 1.0% (mean ± SD of three independent experiments) of the unstimulated cells, but 29.0 ± 11.2% of the cells expressed VCAM-1 after TNF-α stimulation (Fig. 8); this ratio significantly decreased to 3.1 ± 2.5% by the effect of DHMEQ.

One of the prominent characteristics of RA FLS is their proliferative activity, which leads to pannus formation as well as swelling of the joints, and pathways controlling the proliferation of RA FLS include an NF-κB-dependent pathway 32. Representative data shown in Fig. 9 indicate that RA FLS incorporate a certain amount of [³H]thymidine even without stimulation (mean ± SD; 207 ± 36 counts/minute), which was significantly higher than the background level (29 ± 1.2 counts/minute; P < 0.01). This moderate activity increased to 582 ± 53 counts/minute with stimulation with TNF-α, and DHMEQ significantly suppressed this proliferative activity in a dose-dependent manner. At 5.0 μg/ml or higher concentration of DHMEQ, proliferative activity of the cells was lower than that of unstimulated cells, suggesting that DHMEQ suppressed spontaneous proliferation as well as TNF-α-induced proliferation.

To test whether DHMEQ exhibits cytotoxicity at the concentration that suppressed inflammatory mediators, serum-starved RA FLS were further incubated for 14 hours after stimulation with TNF-α in serum-free medium with DHMEQ or the apoptosis inducer staurosporin. Thereafter, expression of annexin V binding phospholipid on the cell surface – an indicator of early phase of apoptosis – was measured using Cy3-labeled annexin V. With staurosporin, 10.1% of the cells were annexin V positive and the mean fluorescence intensity was 1.1 (Fig, 10). In contrast, in the presence of 5 and 10 μg/ml DHMEQ, the ratio and mean fluorescence intensity of annexin V positive cells remained less than 1% and 0.5%, respectively. Trypan blue dye exclusion test also showed virtually 100% viability of the cells incubated with DHMEQ (not shown).