Transfer of complex regional pain syndrome to mice via human autoantibodies is mediated by interleukin-1-induced mechanisms

Abstract

Neuroimmune interactions may contribute to severe pain and regional inflammatory and autonomic signs in complex regional pain syndrome (CRPS), a posttraumatic pain disorder. Here, we investigated peripheral and central immune mechanisms in a translational passive transfer trauma mouse model of CRPS. Small plantar skin-muscle incision was performed in female C57BL/6 mice treated daily with purified serum immunoglobulin G (IgG) from patients with longstanding CRPS or healthy volunteers followed by assessment of paw edema, hyperalgesia, inflammation, and central glial activation. CRPS IgG significantly increased and prolonged swelling and induced stable hyperalgesia of the incised paw compared with IgG from healthy controls. After a short-lasting paw inflammatory response in all groups, CRPS IgG-injected mice displayed sustained, profound microglia and astrocyte activation in the dorsal horn of the spinal cord and pain-related brain regions, indicating central sensitization. Genetic deletion of interleukin-1 (IL-1) using IL-1αβ knockout (KO) mice and perioperative IL-1 receptor type 1 (IL-1R1) blockade with the drug anakinra, but not treatment with the glucocorticoid prednisolone, prevented these changes. Anakinra treatment also reversed the established sensitization phenotype when initiated 8 days after incision. Furthermore, with the generation of an IL-1β floxed^(fl/fl) mouse line, we demonstrated that CRPS IgG-induced changes are in part mediated by microglia-derived IL-1β, suggesting that both peripheral and central inflammatory mechanisms contribute to the transferred disease phenotype. These results indicate that persistent CRPS is often contributed to by autoantibodies and highlight a potential therapeutic use for clinically licensed antagonists, such as anakinra, to prevent or treat CRPS via blocking IL-1 actions.

Keywords: CRPS; anakinra; autoantibody; complex regional pain syndrome; interleukin-1.

Fig. 1.

Effect of intraperitoneal injection of serum IgG derived from CRPS patients or healthy controls on plantar incision-induced mechanical hyperalgesia (A) and swelling (B) of the injured mouse hind paw. IgG was administered daily starting on day 0. The right hind paws were incised on day 0 about 6 h after Ig injection. Shown are pooled results from all three long-term experiments to either day 10 or 13 with three different IgG preparations (2–4) (individual results are in Fig. 2, and patients details are in SI Appendix, Table S1). Data are means ± SEM. Two-way ANOVA was followed by Bonferroni’s multiple comparison test. Healthy indicates the healthy control IgG-injected group, and CRPS indicates the CRPS IgG-injected group. *P < 0.05 (vs. saline-treated control mice); **P < 0.01 (vs. saline-treated control mice); ***P < 0.001 (vs. saline-treated control mice); ^#P < 0.05 (vs. healthy IgG-treated mice); ^##P < 0.01 (vs. healthy IgG-treated mice); ^###P < 0.001 (vs. healthy IgG-treated mice).

Fig. 2.

Imaging ROS demonstrates the development of inflammation in the injured hind paws of mice. In vivo images of L-012–derived bioluminescence were obtained during general anesthesia on days 2, 6, and 13 after paw incision. Typical images, with red color indicating strong bioluminescence, are shown in A, and quantification of the bioluminescence intensity is in B. Data at each time point represent the pooled results from experiments conducted with separate CRPS/healthy control IgG preparations (numbers of preparations per time point are in brackets) (details are in SI Appendix, Fig. S4 and Table S2) and are shown as means ± SEM of n = 6–18 mice per group. One-way ANOVA was followed by Bonferroni’s multiple comparison test. Healthy indicates the healthy control IgG-injected group, and CRPS indicates the CRPS IgG-injected group. *P < 0.05 vs. respective control groups; **P < 0.01 vs. respective control groups; ^#P < 0.05 vs. respective intact side; ^##P < 0.01 vs. respective intact side.

Fig. 3.

Effects of human IgG transfer on sensory neuropeptide and inflammatory cytokine concentrations in the hind paws. Concentrations of (A) SP and (B) CGRP were measured by RIA in hind paw homogenates excised after they were killed. Concentrations of (C) IL-6, (D) TNF-α, (E) MCP-1, and (F) IL-1β were measured by cytometric bead array from the same samples. Data are from one to three experiments per time point (brackets below x axes) each with different patient preparations. Shown are means ± SEM. One-way ANOVA was followed by Bonferroni’s multiple comparison test. Healthy indicates the healthy control IgG-injected group, and CRPS indicates the CRPS IgG-injected group. **P < 0.01 vs. respective control groups; ^#P < 0.05 vs. respective intact side; ^##P < 0.01 vs. respective intact side; ^###P < 0.001 vs. respective intact side;

Fig. 4.

Glial activation in the L5 spinal cord dorsal horn ipsilateral to the paw injury. A–C show GFAP immunopositivity marking astrocytes, and G–I show Iba1 immunopositivity marking microglia cells, with (A and G) saline, (B and H) healthy control IgG, and (C and I) CRPS IgG injections. The GFAP-immunopositive sections shown are from day 6, and Iba1 sections are from day 13 after paw incision. Quantification of astrocyte reactivity (D–F) and microglia staining (J–L) in lamina I–II dorsal horn of the L4–L6 spinal cord and deeper laminae at 3, 6, and 13 d after hind paw incision. Each panel represents the pooled results from two experiments with two different samples (3 and 4). Shown are means ± SEM of six to seven mice per group. One-way ANOVA was followed by Bonferroni’s modified post hoc test. Healthy indicates the healthy control IgG-injected group, and CRPS indicates the CRPS IgG-injected group. *P < 0.05 vs. respective control groups; **P < 0.01 vs. respective control groups; ***P < 0.001 vs. respective control groups; ^#P < 0.05 vs. respective contralateral side; ^##P < 0.01 vs. respective contralateral side; ^###P < 0.001 vs. respective contralateral side.

Fig. 5.

Effects of prophylactic steroid or anakinra treatment (days 0–6) and delayed (therapeutic) anakinra treatment on CRPS IgG-induced mechanical hyperalgesia and glial activation in the spinal cord. A and B show mechanical hyperalgesia in groups of animals injected intraperitoneally first with human IgG or saline and 3 h later with 4 mg/kg prednisolone, 10 mg/kg anakinra, or saline vehicle on each day between days 0 and 6. D and E show dorsal horn glia cell activation in these mice on day 6: (D) GFAP (astrocyte) and (E) Iba-1 (microglia). Results represent the average values derived from two independent experiments with different preparations for each treatment, with four experiments in total; saline, healthy control IgG, and CRPS IgG outcomes are pooled from these experiments. (C and F) Late anakinra treatment starting on day 8: (C) behavioral outcome and (F) dorsal horn microglia cell count on day 13. Data are shown as means ± SEM. Two-way ANOVA was followed by Bonferroni’s multiple comparison test. One-way ANOVA was followed by Bonferroni’s modified post hoc test. Healthy indicates the healthy control IgG-injected group, and CRPS indicates the CRPS IgG-injected group. Significance symbols for the behavioral data as follows: *P < 0.05 (CRPS IgG vs. saline-treated control mice); **P < 0.01 (CRPS IgG vs. saline-treated control mice); ***P < 0.001 (CRPS IgG vs. saline-treated control mice); ^#P < 0.05 (CRPS IgG vs. healthy control IgG-treated mice); ^###P < 0.001 (CRPS IgG vs. healthy control IgG-treated mice); ⁺⁺⁺P < 0.001 (anakinra plus CRPS IgG vs. CRPS IgG-injected mice). Significance immunohistochemistry data: *P < 0.05 vs. respective control groups; **P < 0.01 vs. respective control groups; ***P < 0.001 vs. respective control groups; ^#P < 0.05 vs. respective contralateral side; ^##P < 0.01 vs. respective contralateral side; ^###P < 0.001 vs. respective contralateral side.

Fig. 6.

Deletion of IL-1αβ or microglia-derived IL-1β fully or partially prevents development of the CRPS IgG-induced phenotype in mice. (A) A population of microglia identified by immunostaining against P2Y12, a specific microglial marker in the brain (59), displays a morphologically activated phenotype and shows immunopositivity for IL-1β at day 7 in the deep laminae of the L4–L5 spinal cord near the central canal. (Scale bar: 50 μm.) (B and C) IL-1αβ KO mice are fully protected and M-IL-1βKO mice are partially protected from the development of the CRPS IgG-induced phenotype: (B) paw hyperalgesia and (C) paw edema. Two-way ANOVA was followed by Bonferroni’s multiple comparison test. Significance in B and C: **P < 0.01 (CRPS IgG WT vs. healthy IgG WT); ***P < 0.001 (CRPS IgG WT vs. healthy IgG WT); ^#P < 0.05 (CRPS IgG M-IL-1β KO vs. CRPS IgG WT); ^##P < 0.01 (CRPS IgG M-IL-1β KO vs. CRPS IgG WT); ^###P < 0.001 (CRPS IgG IL-1αβ KO vs. CRPS IgG WT); ⁺⁺P < 0.01 (CRPS IgG IL-1αβ KO vs. CRPS IgG M-IL-1β KO); ⁺⁺⁺P < 0.001 (CRPS IgG IL-1αβ KO vs. CRPS IgG M-IL-1β KO). (D) CRPS IgG-induced microglia activation is abrogated in IL-1αβ KO but not in M-IL-1β KO mice. Data are pooled from two experiments with different CRPS IgG preparations for each mouse type and are shown as means ± SEM. Healthy indicates the healthy control IgG-injected group, and CRPS indicates the CRPS IgG-injected group. One-way ANOVA was followed by Bonferroni’s modified post hoc test. Significance values in D: **P < 0.01 vs. respective control groups; ***P < 0.001 vs. respective control groups; ^#P < 0.05 vs. respective contralateral side; ^###P < 0.001 vs. respective contralateral side.