KRN TCR transgenic mice on the C57BL/6 (B6) background and B6 mice congenic for H-2g7 (B6.g7) were gifts from Diane Mathis and Christophe Benoist (Harvard Medical School, Boston, Massachusetts and Institute de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France). Ccr5-deficient (B6.129P2-Ccr5tm1Kuz/J)  and wild-type B6 mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). The mice were intercrossed as needed to create the animals used for this study.
Animals were maintained in our specific pathogen-free animal facility at the University of Minnesota (Minneapolis, MN) under protocols approved by the Institutional Animal Care and Use Committee of the University.
Genotypes of mice were determined by polymerase chain reaction (PCR) amplification of genomic DNA extracted from mouse ear punched tissues.
Determination of serum CCL5 levels
Serum concentrations of CCL5/RANTES were measured using a commercially available enzyme-linked immunosorbent assay, according to the manufacturer’s instructions (R&D Systems).
Tissue sections were prepared as previously described [17, 18]. Briefly, paraffin sections of 10% buffered formalin-fixed and decalcified ankles were deparaffinized and placed in acidic antigen retrieval solution (R&D Systems) at 95 °C for 10 min. Sections were cooled at room temperature for 20 min and rinsed under cold tap water for 5 min. Bloxall (Vector Laboratories) was applied to sections to block endogenous peroxidases for 10 min. Fc receptors were blocked with anti-CD16/32 (clone 70–0161-M001, Tonbo Biosciences) and anti-CD64 (clone 139,302, BioLegend) in 2% bovine serum albumin (BSA). Sections were incubated overnight with anti-CCR5-Biotin (clone 107,003, BioLegend) or control Armenian Hamster IgG-Biotin at 4 °C at 1:100 dilution. Sections were incubated with Vectastain ABC reagent (Vector Laboratories) for 30 min followed by ImmPACT DAB peroxidase substrate (Vector Laboratories) for 10 min. Sections were cleared with Xylene and mounted with Vectamount (Vector Laboratories).
Compound synthesis and administration
MCC22 and MCC14 were synthesized as described previously . Morphine was purchased from Mallinckrodt Pharmaceuticals Company (Mallinckrodt Chemical, St. Louis, Missouri). All the compounds were dissolved in 10% (wt/vol) dimethyl sulfoxide (DMSO) and then diluted to less than 1% DMSO in the final solutions. Vehicle control was 1% DMSO. All the compounds were administrated via intraperitoneal injection.
Mechanical hypersensitivity test
The mechanical pain threshold was quantified by measuring the hind paw withdrawal response using an electronic Von Frey anesthesiometer (IITC Life Science, Woodland Hills, CA). Briefly, animals were placed into enclosures in a test chamber with a metal mesh floor through which the von Frey rigid tip monofilaments were applied, and mechanical pain thresholds (in grams) were measured. The mechanical threshold (in grams) of both hind paws was averaged for each animal. Baseline measurements were recorded before each dose of drug or vehicle. Animals were tested again for their hind paw withdrawal 2 h after drug administration. Drugs were administered once daily for up to 15 consecutive days. Recordings were taken on days indicated in each Figure. After a maximum of 15 days, drug administration was stopped and the animals were monitored to record the accumulative analgesic effect of the administered drug.
A drug tolerance test was performed by administering the ED90 dose (defined as the dose at which 90% of animals experience an analgesic effect) of drug twice daily for 9 days. Electronic Von Frey stimulation was performed on days 1, 3, and 9, and the mechanical thresholds were recorded.
Grip strength measurement
Forelimb grip strength was recorded using a Chatillon Force Gauge DFE II (Ametek) according to the manufacturer’s instructions.
Scoring of mouse nest building was performed as described previously . Briefly, nest scores (scale of 1–5) are determined by the wall height of nest/dome made by mice around them in the cage using the same amount of nesting material (1 = nesting material scattered in the cage, 2 = flat nest, 3 = less than half height of nesting dome, 4 = half height of nesting dome, 5 = greater than half height of nesting dome). Double-blinded scoring was performed to measure the height of the nesting dome.
Assessment of ankle thickness and arthritis severity scores were performed as per standard protocols . In brief, ankle thickness measurements were determined by measuring across the malleoli using a Kafer dial thickness gauge with flat anvils (Long Island Indicator Service, Hauppauge, NY). Arthritis severity scores are determined using a scale of 0–3 for each paw, where 0 is normal and 3 is maximum arthritis severity; the maximum total score for a given mouse is 12.
Histopathology of ankles
Paraffin sections of 10% buffered formalin-fixed and decalcified ankles were stained with hematoxylin and eosin. Sections were scored from 0 for normal to 5 for inflammation, fibroplasia, and cartilage injury per the scoring system of Caplazi and Diehl .
Prism 5.01 (GraphPad Software, Inc., La Jolla, CA) was used for all analysis and graphical representation of data. For tests involving multiple comparisons, one-way or repeated measures two-way analysis of variance (ANOVA) were used followed by post-hoc Tukey’s or Sidak’s multiple-comparison tests, as appropriate. For comparisons of two groups, the paired t test or Mann-Whitney U test was used as appropriate. The particular statistical tests used for each experiment are indicated in the Figure legends. ED50 values with 95% confidence intervals (CIs) were computed with Prism using nonlinear regression methods. For all tests, P values < 0.05 were considered significant.