Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 216 (8), 1904-1924

Cartilage-binding Antibodies Induce Pain Through Immune Complex-Mediated Activation of Neurons

Affiliations

Cartilage-binding Antibodies Induce Pain Through Immune Complex-Mediated Activation of Neurons

Alex Bersellini Farinotti et al. J Exp Med.

Abstract

Rheumatoid arthritis-associated joint pain is frequently observed independent of disease activity, suggesting unidentified pain mechanisms. We demonstrate that antibodies binding to cartilage, specific for collagen type II (CII) or cartilage oligomeric matrix protein (COMP), elicit mechanical hypersensitivity in mice, uncoupled from visual, histological and molecular indications of inflammation. Cartilage antibody-induced pain-like behavior does not depend on complement activation or joint inflammation, but instead on tissue antigen recognition and local immune complex (IC) formation. smFISH and IHC suggest that neuronal Fcgr1 and Fcgr2b mRNA are transported to peripheral ends of primary afferents. CII-ICs directly activate cultured WT but not FcRγ chain-deficient DRG neurons. In line with this observation, CII-IC does not induce mechanical hypersensitivity in FcRγ chain-deficient mice. Furthermore, injection of CII antibodies does not generate pain-like behavior in FcRγ chain-deficient mice or mice lacking activating FcγRs in neurons. In summary, this study defines functional coupling between autoantibodies and pain transmission that may facilitate the development of new disease-relevant pain therapeutics.

Figures

Figure 1.
Figure 1.
Injection of anti-CII antibodies induces pain-like behavior before visual, histological, and molecular signs of arthritis. (A–C) B10.RIII mice injected with anti-CII mAbs (n = 19; saline controls n = 17) started developing joint inflammation around day 6 (A). On day 9, all animals displayed signs of arthritis (B). Mechanical hypersensitivity (C) was observed already on days 3 and 5, before onset of arthritis, and persisted throughout day 21. (D) Representative H&E histology of B10.RIII mouse ankle joints collected 5 and 15 d after injection of anti-CII mAbs. While an inflammatory infiltrate, bone erosion, and cartilage serration were visible on day 15, no signs of joint pathology was detectable on day 5 or in saline controls. Scale bar represents 100 µm. *, ▼, and V point to signs of synovitis, bone erosion, and cartilage destruction, respectively. (E–G) Scores for inflammatory hallmarks as synovitis (E), bone erosion (F), and loss of cartilage (G) revealed mild ankle joint pathology in two of eight mice day 5 and prominent signs in all mice day 15 (n = 5). Control mice represent pooled time-matched saline-injected mice (n = 4+4). (H and I) Quantitative PCR analysis of joint extracts showed a significant increase in mRNA levels of most of the inflammatory factors investigated at day 15 (n = 7), while none of them were elevated at day 5 of CAIA (n = 6), compared with saline controls (n = 5; H and I). (J and K) Activation of MMPs was significantly increased only after 15 d of CAIA, while no changes were detected at day 5 (n = 3/group, B10.RIII mice). Data are presented as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with saline controls. REU, relative expression unit.
Figure 2.
Figure 2.
Anti-CII antibodies injected either as a cocktail or as individual antibodies induce mechanical hypersensitivity and reduce locomotion before inflammation. (A–C) Anti-CII mAbs (n = 10) induced mechanical hypersensitivity as early as 2 d after injection (A) compared with saline controls (n = 9) in B10.RIII mice. Arthritis scores (B) and incidence (C) were not detectable until day 4 and remained very low also on day 5. (D) Total movement (left) and rearing (right) significantly decreased in B10.RIII mice injected with the anti-CII mAb cocktail (n = 15), compared with controls (n = 19). (E–G) When injected individually, the four mAbs (M2139, UL1, CIIC1, and CIIC2) induced mechanical hypersensitivity similarly to the cocktail (E; n = 5–9/group, B10.RIII mice). No considerable signs of inflammation (F and G) were detected. (H) Total movement (left) and rearing (right) were reduced in M2139 mAb–injected B10.RIII mice (n = 5), compared with saline (n = 5) or isotype control (n = 5). (I–K) Injection of M2139 mAb–induced mechanical hypersensitivity for 21 d (I) even at doses that did not induce visual signs of inflammation (J and K; n = 5, B10.RIII mice). Axes in Fig. 2 (A and E) are interrupted to make the difference between groups clearer to visualize. Data are presented as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with saline controls. For Fig. 2 E, significance is shown with symbols: * for M2139 and CIIC2 mAbs, # for CIIC1, and † for UL1 compared with saline controls. For Fig. 2 I, significance is shown with symbols: * for 4 mg M2139, # for 2 mg M2139, † for 1 mg M2139, and ‡ for 0.5 mg M2139 compared with each respective baseline.
Figure 3.
Figure 3.
Anti-CII antibody–induced pain-like behavior is not mediated by complement activation or cartilage destruction. (A) Injection of the C5a-receptor antagonist PMX53 (C5aR ant; n = 5) did not prevent anti-CII mAb–induced mechanical hypersensitivity (n = 4, B10.RIII mice) compared with vehicle (saline)-injected controls (n = 7, B10.RIII mice). (B and C) Antagonizing the C5a-receptor (n = 5, B10.RIII mice) did not prevent anti-CII mAb–induced reduction in total movement (B) and rearing (C) compared with saline controls (n = 19, B10.RIII mice). (D–F) Complement 5–deficient (C5−/−) mice developed mechanical hypersensitivity (D; n = 5) and displayed a reduction in total movement (E) and rearing (F; n = 4) comparable to WT B10Q mice (n = 6–8) after injection of anti-CII mAbs. (G) B10.RIII mice injected with the nonarthritogenic CIIF4 antibody (n = 8) developed mechanical hypersensitivity from day 3 after injection compared with saline controls (n = 7). Data are presented as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with saline controls.
Figure 4.
Figure 4.
FcγRs are expressed in mouse DRG neurons. (A) Microarray data showed mRNA for Fcgr1–4 in CBA mouse DRG (n = 3). (B) Quantitative PCR showed mRNA for Fcgr1–4 in B10.RIII mouse DRG (n = 10). (C) Publicly available RNA sequencing of C57BL/6 mouse DRGs show the presence for Fcgr1–4 (n = 3). (D) smFISH showed mRNA molecules for Fcgr1, Fcgr2b, and Fcgr3 in BALB/c mouse DRG colocalizing with NeuN. Scatter graph shows number of mRNA molecules in neuronal soma. Scale bars represent 100 µm and 10 µm in close-up images. (E) FcγRI protein expression was detected by Western blotting in DRGs from WT BALB/c and B10.RIII mice, but not from FcRγ-chain−/− mice. (F) Proteomic analysis identified peptides specific for FcγRIIb in BALB/c mouse DRG. See also Figs. S1 and S4.
Figure 5.
Figure 5.
FcγRI and FcγRIIb are expressed in the DRG and in nerve fibers in the skin. (A and B) FcγRI immunoreactivity was detected in WT BALB/c DRGs, but not in FcRγ-chain−/− mice (A), colocalizing with Iba1-positive resident macrophages (B). Scale bars represent 100 µm and 50 µm, respectively. (C and D) FcγRIIb immunoreactivity was detected in BALB/c mouse DRG and retained in FcRγ-chain−/− mice (C), colocalizing with TrkA-positive neurons (D). Scale bars represent 100 µm and 50 µm, respectively. (E and F) FcγRI (E) and FcγRIIb (F) immunoreactivity was detected in PGP9.5-positive nerve fibers in BALB/c mouse glabrous skin. Scale bars represent 5 µm. (G–I) smFISH on BALB/c mouse sciatic nerves after ligation (G) revealed accumulation of mRNA molecules for Fcgr1 (H) and Fcgr2b (I) proximal to the site of ligation (ipsilateral), while barely any signal was found in the contralateral intact nerve. Scale bars represent 10 µm. See also Figs. S2, S3, and S4.
Figure 6.
Figure 6.
CII-IC stimulation of DRG cell cultures leads to increased neuronal excitability. (A) FcγRI and FcγRIIb are expressed in BALB/c mouse DRG neurons in culture as shown by colocalization with βIII-tubulin. Scale bars represent 10 µm. (B and C) CII-IC stimulation of BALB/c mouse DRG cell cultures resulted in increased intracellular [Ca2+] signal (B) and also evoked positive inward currents (C). (D and E) CII-IC stimulation evoked CGRP release in DRG cell cultures from WT BALB/c mice (D) but not from FcRγ-chain−/− mice (E). Capsaicin (CAP) was used as positive control. Data are presented as mean ± SEM. ***, P < 0.001.
Figure 7.
Figure 7.
Different ICs promote pain-like behavior in vivo, and FcγRIV−/− mice develop mechanical hypersensitivity despite lack of CAIA. (A and B) I.a. injection of CII-IC–induced mechanical hypersensitivity in WT BALB/c mice (n = 14–21/group; A) but not in FcRγ-chain−/− mice (n = 8–10/group; B). (C and D) Systemic administration of anti-CII mAbs evoked mechanical hypersensitivity in WT BALB/c mice (n = 8/group; C) but not FcRγ-chain−/− mice (n = 8/group; D). (E) I.a. injection of IgG-IC induced mechanical hypersensitivity in WT BALB/c mice (n = 6/group). (F) I.a. injection of COMP-IC induced mechanical hypersensitivity in WT C57BL/6 mice (n = 7/group). (G and H) Systemic administration of anti-COMP mAb evoked mechanical hypersensitivity in WT BALB/c mice (n = 5; G), in the absence of any signs of inflammation (H). (I–K) Systemic injection of anti-CII mAbs induced pain-like behavior in both WT C57BL/6 mice (n = 4; I) and FcγRIV−/− mice (n = 6; J), even if no signs of inflammation were observed in the latter (K). Axes in Fig. 7 (A–D) are interrupted to make the difference between groups clearer to visualize. Data are presented as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with saline/PBS controls.
Figure 8.
Figure 8.
The pronociceptive properties of anti-CII antibodies are dependent on the Fc region, glycosylation, and interaction with FcγRI in the joint. (A–C) B10.RIII mice injected with anti-CII mAb Fab fragments (n = 8) did not develop mechanical hypersensitivity (day 5; A) compared with CAIA (n = 13) and control (n = 7) mice. They also did not show reduction in total movement (B) or rearing (C; night 3, n = 8–19/group). (D) B10.RIII mice injected with EndoS-treated anti-CII mAbs did not develop mechanical hypersensitivity (n = 3/group). BL, baseline. (E–G) B10.RIII mice injected with EndoS-treated anti-CII mAb M2139 did not develop mechanical hypersensitivity (day 5; E) or display a reduction in locomotor activity (F and G; night 3, n = 6–7/group). (H–J) BALB/c mice lacking activating FcγRs in myeloid cells (KO-WT; I) developed mechanical hypersensitivity after injection of anti-CII mAbs compared with controls (WT-WT; J). In contrast, mice lacking activating FcγRs in nonmyeloid cells (WT-KO), including neurons, were protected (H; n = 8–9/group). Data are presented as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with controls.
Figure 9.
Figure 9.
FcγRI and FcγRIII are expressed in human DRG. (A) Publicly available data show the presence of Fcgr mRNA in human DRGs (n = 6). Fcgr3a is the most highly expressed. (B and C) FcγRI immunoreactivity was detected in human DRGs (n = 4). The lack of colocalization with NeuN and the morphology of the FcγRI-positive cells suggest FcγRI expression in resident macrophages, similarly to mice (scale bars represent 100 µm and 10 µm in close-up images). White arrows indicate FcγRI-positive cells, which are negative for NeuN. (D and E) Immunoreactivity for the activating FcγRIII in human DRGs (n = 4) colocalized with the neuronal marker NeuN (scale bars represent 100 µm and 10 µm, respectively). White arrows point to double-positive neurons (FcγRIII and NeuN).

Comment in

Similar articles

See all similar articles

Cited by 5 PubMed Central articles

References

    1. Agalave N.M., Larsson M., Abdelmoaty S., Su J., Baharpoor A., Lundbäck P., Palmblad K., Andersson U., Harris H., and Svensson C.I. 2014. Spinal HMGB1 induces TLR4-mediated long-lasting hypersensitivity and glial activation and regulates pain-like behavior in experimental arthritis. Pain. 155:1802–1813. 10.1016/j.pain.2014.06.007 - DOI - PubMed
    1. Amirahmadi S.F., Whittingham S., Crombie D.E., Nandakumar K.S., Holmdahl R., Mackay I.R., van Damme M.P., and Rowley M.J. 2005. Arthritogenic anti-type II collagen antibodies are pathogenic for cartilage-derived chondrocytes independent of inflammatory cells. Arthritis Rheum. 52:1897–1906. 10.1002/art.21097 - DOI - PubMed
    1. Andoh T., and Kuraishi Y. 2004. Direct action of immunoglobulin G on primary sensory neurons through Fc gamma receptor I. FASEB J. 18:182–184. 10.1096/fj.02-1169fje - DOI - PubMed
    1. Bas D.B., Su J., Sandor K., Agalave N.M., Lundberg J., Codeluppi S., Baharpoor A., Nandakumar K.S., Holmdahl R., and Svensson C.I. 2012. Collagen antibody-induced arthritis evokes persistent pain with spinal glial involvement and transient prostaglandin dependency. Arthritis Rheum. 64:3886–3896. 10.1002/art.37686 - DOI - PubMed
    1. Boyle D.L., Rosengren S., Bugbee W., Kavanaugh A., and Firestein G.S. 2003. Quantitative biomarker analysis of synovial gene expression by real-time PCR. Arthritis Res. Ther. 5:R352–R360. 10.1186/ar1004 - DOI - PMC - PubMed
Feedback