Recombinant mouse il 6

MOG peptide 35–55 (MOG₃₅₋₅₅; MEVGWYRSPFSRVVHLYRNGK) was synthesized and purified by Operon Biotechnologies (Tokyo, Japan). Incomplete Freund's adjuvant was obtained from Sigma-Aldrich (St. Louis, MO, USA). Heat-killed Mycobacterium tuberculosis H37Ra was obtained from Difco (Detroit, MI, USA), and pertussis toxin was obtained from List Biological Laboratories (Campbell, CA, USA). Recombinant mouse IL-6, IL-19, and TGF-β1 were obtained from R&D Systems (Minneapolis, MN, USA).

Splenic CD4⁺ T cells were isolated from 6-week-old NOD/ShiLtJ mice. For Th17 cell differentiation, the cells were stimulated with anti-CD3 (0.5 µg/mL), anti-CD28 (1 µg/mL), anti-IFN-γ (10 µg/mL) and anti-IL-4 (10 µg/mL) antibodies, IL-6 (20 ng/mL), and transforming growth factor-β (TGF-β) (2 ng/mL) for 3 days. Recombinant mouse IL-6 and antibodies to IFN-γ and IL-4 were purchased from R&D Systems (Minneapolis, MN, USA), and TGF-β was purchased from PeproTech. Cells were pretreated with RA (Sigma-Aldrich, St. Louis, MO, USA) at concentrations of 0.2–1 μM for 2 h and then stimulated under the required conditions.

Supernatants were collected from stimulated DCs (16 hrs), and cytokine production was measured by ELISA according to standard protocol. Capture and detection antibody pairs for IL-6 (MP5-20F3 and MP5-32C11), IL-10 (JES5-2A5 and SXC-1), and IL-12 (C15.6 and C17.8) were purchased from BD Biosciences. Recombinant mouse IL-6 (R&D), mouse IL-10 (R&D), and mouse IL-12 (Peprotech) were used as standards. Cytokine-antibody complexes were visualized by the addition of Tetramethyl Benzidine (TMB) solution (Life Technologies), and color development was stopped by the addition of TMB stop solution (Life Technologies). Absorbance at 492 nm was measured on a microplate reader (Biotek).

A mouse model for “hyperalgesic priming” originally developed by Levine and colleagues^{65 (link)} was used for the study. Animals were placed in acrylic boxes with wire mesh floors, and baseline plantar mechanical sensitivity was measured after habituation for 1 h using the up-down method^{66 (link)}. Briefly, Von Frey monofilaments (Stoelting, Wood Dale, IL, USA) were firmly applied to the plantar surface of left hindpaw for 5 s and the up-down method was used to estimate the withdrawal threshold in grams (g). To establish hyperalgesic priming, we co-administered the SPOT-ONs (0.3, 1 μg) with recombinant mouse IL-6 (1.25 ng; R&D Systems) or mouse 2.5S NGF (50 ng; Millipore) in 25 μL sterile PBS into the left hindpaw with an intraplantar (i.pl.) injection and measured their mechanical withdrawal thresholds at various time points after administration. Following complete resolution of the initial mechanical hypersensitivity (day 9), mice were again assessed for their mechanical withdrawal threshold and subsequently injected into the left hindpaw with PGE₂ (100 ng; Cayman Chemical) in 25 μL sterile 0.9% NaCl. Afterwards, mechanical withdrawal thresholds were measured at 3 and 24 h post PGE₂.

CD4⁺CD25⁻ T cells were isolated from spleens of 8–12-week-old C57BL/6 mice as follows: single-cell suspensions were made by crushing the spleen through a cell strainer, and red blood cells (RBCs) were lysed with RBC lysis buffer. CD4⁺ T cells were then purified using magnetic-activated cell sorting (MACS) column a CD4⁺ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol.
Spleen cells (3 × 10⁶) were stimulated in 6-well plates with 5 μg/mL plate-bound anti-CD3e antibody (eBioscience, San Diego, USA) and 2 μg/mL soluble anti-CD28 antibody (eBioscience) for 24 h. The activated cells were then collected, and seeded into 96-well round-bottom plates (2 × 10⁴) for Th17 differentiation in the presence of 5 ng/mL recombinant human TGF-β (R&D Systems, Minneapolis, MN), 30 ng/mL recombinant mouse IL-6 (R&D Systems, Minneapolis, MN), and either the screening compounds (5 μM final concentration) or DMSO as control. After culturing for 2 days, fresh medium (100 uL/well) containing recombinant human TGF-β, recombinant mouse IL-6, and the previously-introduced screening compounds was added to the cells, which were then cultured for additional 2 days [26 (link)].

Recombinant mouse IL-6 was purchased from R&D Systems (No. 406-ML-025, USA) and dissolved IL-6 in 0.9% NaCl solution. In the IL-6 group, 10 μg/kg Recombinant mouse IL-6 was intravenously administered 15 min prior to the onset of myocardial ischemia, whereas the control group received the same volume of 0.9% NaCl over the same period as described previously [34 (link)]. There was no difference in mortality between the groups. When the injection was complete, we began the in situ myocardial ischemia induction.