Autoantibodies to citrullinated proteins induce joint pain independent of inflammation via a chemokine-dependent mechanism

Abstract

Objective: An interesting and so far unexplained feature of chronic pain in autoimmune disease is the frequent disconnect between pain and inflammation. This is illustrated well in rheumatoid arthritis (RA) where pain in joints (arthralgia) may precede joint inflammation and persist even after successful anti-inflammatory treatment. In the present study, we have addressed the possibility that autoantibodies against citrullinated proteins (ACPA), present in RA, may be directly responsible for the induction of pain, independent of inflammation.

Methods: Antibodies purified from human patients with RA, healthy donors and murinised monoclonal ACPA were injected into mice. Pain-like behaviour was monitored for up to 28 days, and tissues were analysed for signs of pathology. Mouse osteoclasts were cultured and stimulated with antibodies, and supernatants analysed for release of factors. Mice were treated with CXCR1/2 (interleukin (IL) 8 receptor) antagonist reparixin.

Results: Mice injected with either human or murinised ACPA developed long-lasting pronounced pain-like behaviour in the absence of inflammation, while non-ACPA IgG from patients with RA or control monoclonal IgG were without pronociceptive effect. This effect was coupled to ACPA-mediated activation of osteoclasts and release of the nociceptive chemokine CXCL1 (analogue to human IL-8). ACPA-induced pain-like behaviour was reversed with reparixin.

Conclusions: The data suggest that CXCL1/IL-8, released from osteoclasts in an autoantibody-dependent manner, produces pain by activating sensory neurons. The identification of this new pain pathway may open new avenues for pain treatment in RA and also in other painful diseases associated with autoantibody production and/or osteoclast activation.

Keywords: Ant-CCP; Autoantibodies; Fibromyalgis/Pain Syndromes; Rheumatoid Arthritis.

Figure 1

Mechanical and thermal sensitivity and locomotor activity in mice following injection of human antibodies. Mechanical sensitivity in mice injected intravenously with saline (sal), IgG from healthy donors, IgG from patients with ACPA⁻ RA or IgG from patients with ACPA⁺ RA (4 mg, n=9/group) (A) and purified human (h) ACPA IgG (batch 1, 1 mg, n=4), non-ACPA IgG from the same patients (FT, 1 mg, n=6) and IgG from healthy donors (1 mg, n=6) (B). ACPA and FT from batch 1 were injected into a different strain of mice (n=7/group) and mechanical sensitivity assessed over time (C), cold sensitivity days 7 and 28 (E and F) and heat sensitivity day 25 (G). Total movement (H), ambulatory (directional) movement (I) and rearing (J) were monitored 12 h the third night (same mice as in C). Arthritis scores (0–60) (D). Mechanical sensitivity days 5 and 7 (K) and total movement (L), ambulatory movement (M) and rearing (N) during third night after injection with 1 mg ACPA batch 1–3 (n=3 each) or 0.5 mg (n=7), 0.125 mg (n=6) ACPA batch 2 or corresponding FT (n=6/group) or saline. Data are presented as mean±SEM. *p<0.05, **p<0.01, and ***p<0.001 are compared with saline. ACPA, anti-citrullinated protein antibodies; FT, flow through; RA, rheumatoid arthritis.

Figure 2

Mechanical sensitivity following injection of murinised monoclonal ACPA in mice. Specificities of the monoclonal antibodies derived from B cells of human patients with RA measured with ELISA, using CEP-1, fib36–52, vim60–75 and CCP peptides. Control antibody E2 binds human tetanus (A). Visual inflammation score (0–60) for all monoclonal antibodies (B). Two milligrams of D10 (n=12) (C), C7 (n=7) (C), B2 (n=7) (D), control antibody E2 (n=7) (D) or saline (sal, n=18) were injected and mechanical sensitivity was measured over 20 days. Data are presented as mean±SEM. *p<0.05; **p<0.01 and ***p<0.001 are compared with saline. ACPA, anti-citrullinated protein antibodies; RA, rheumatoid arthritis.

Figure 3

Location of antibodies, histology and gene expression in mice after injection of human ACPA. Mice were perfused with saline (sal) to remove blood, and the presence of human IgG in different tissues 7 days after intravenous injection of 1 mg of ACPA₃, FT₃ or IgG from healthy control (HC) was assessed by western blot. Plasma was used as the positive control (A). Representative ankle joint and tibial bone sections stained with H&E 7 days after injection of human ACPA₃ (n=3), saline (n=4) or 15 days after induction of collagen antibody-induced arthritis (CAIA, positive control) (n=3) (B) were scored for bone erosion (C), loss of cartilage (D) and synovitis (E). Ankle joint extracts were analysed by qPCR for changes in mRNA levels 7 days after injection of human ACPA_2–3 (n=6) or saline (n=6) and data expressed as relative expression unit (F). Fluorescence image of paws (G), presented as a heat map after intravenous injection with MMPsense680, which becomes fluorescent in the presence of active MMPs in mice injected with saline, human ACPA₃ or anticollagen antibodies as the positive control (n=3/group). Data are presented as mean±SEM; *p<0.05 and **p<0.01 are compared with saline. ACPA, anti-citrullinated protein antibodies; FT, flow through.

Figure 4

Effect of ACPA on primary peripheral neurons. Mouse dorsal root ganglions were cultured and stimulated with ACPA or FT (both 1 µg/mL). A representative trace showing Ca²⁺ during stimulation with antibodies and KCl (50 mM) (A). Calcium signal were recorded from 243 cells, where few cells showed a minor response to stimulation (2.5% for ACPA and 1.7% for FT) (B). A total of 24 cells were patched and ionic currents were recorded in whole-cell voltage clamp mode (C). None (0/24) of the recorded cells gave inward current response to ACPA, while 33% (8/24) gave response to capsaicin (1 μM) (D). ACPA, anti-citrullinated protein antibodies; FT, flow through.

Figure 5

Binding of ACPA in tibial bone marrow and effect of ACPA on cultured osteoclasts. Colocalisation of ACPA: marker for macrophage/osteoclasts (CD68) in subchondral bone (A) and synovia (B), and marker for sensory nerve fibres (CGRP) in tibial bone marrow (C). ACPA and CD68 binding in cultured mouse bone marrow without permeabilisation of the plasma membrane (D). CXCL1 (E) and CXCL2 (F) levels in the supernatant of cultured mouse osteoclasts after stimulation with human ACPA (1 μg/mL), FT (1 μg/mL) or saline (n=6 mice/group). Three different cohorts of littermates were used (E–F). Number of osteoclasts per well at the end of experiment day 14 (G). Data are presented as mean±SEM. **p<0.01 and ***p<0.001 are compared with saline. Scale bar is 25 μm. ACPA, anti-citrullinated protein antibodies; FT, flow through.

Figure 6

Effect of reparixin on ACPA-induced hypersensitivity. Mechanical hypersensitivity after injection of CXCL1 (30 ng, n=7), CXCL2 (30 ng, n=7) or mixed CXCL1/2 (15 ng each, n=10) or saline (n=20) into the ankle joint (A). Mechanical sensitivity after intravenous injection of mouse monoclonal ACPA D10 and B2 (1 mg each, n=18) or saline (n=9) and treatment with reparixin (30 mg/kg/day, s.c., n=9) or saline (n=9) for 6 days, starting on day 6 (B). Hyperalgesic index comparing area under the curve for reparixin-treated or saline-treated mice from day 6 (C). Cold (D) and heat (E) sensitivity were tested on days 19 and 26, respectively. Results are from two separate experiments. Statistical significance (two-way analysis of variance (ANOVA)) between mACPA/saline and saline is marked by # and difference between mACPA/saline and mACPA/reparixin is marked with * (A). Mechanical sensitivity of mice injected with either saline (n=5) or reparixin (30 mg/kg/day, s.c., n=5) on day 6–12 (E). Data are presented as mean±SEM. * or ^#p<0.05, **p<0.01 and ***p<0.001 are compared with saline. ACPA, anti-citrullinated protein antibodies.