Proceedings of The Physiological Society

Physiology 2016 (Dublin, Ireland) (2016) Proc Physiol Soc 37, PCA292

Poster Communications

Involvement of macrophage-derived high mobility group box 1 in paclitaxel-induced neuropathic pain in mice

R. Domoto1, D. Yamasoba1, H. Yamanishi1, F. Sekiguchi1, M. Tsubota1, M. Nishibori2, A. Kawabata1

1. Laboratory of Pharmacology and Pathophysiology, Faculty of Pharmacy, Kindai University, Higashi-Osaka, Japan. 2. Department of Pharmacology, Okayama University Graduate School of Medicine, Okayama, Japan.


High mobility group box 1 (HMGB1), a nuclear protein, is released from various cells including macrophages (Mφs), and aggravates inflammation through multiple targets including the receptor for advanced glycation end products (RAGE) and Toll-like receptor 4 (TLR4). NF-κB participates in HMGB1 secretion and also in downstream signals of RAGE and TLR4. Given our recent evidence for the involvement of peripheral HMGB1 in pain processing [1, 2], we investigated the role of Mφ-derived HMGB1 in the neuropathic pain induced by paclitaxel (PTX), an anticancer drug, in mice and analyzed the effect of PTX on HMGB1 secretion in Mφ-like RAW264.7 cells, particularly in relation to NF-κB signals. Male ddY mice (20-25 g) received i.p. administration of PTX at 4 mg kg-1 on day 0, 2, 4 and 6, 4 times in total. Neuropathic hyperalgesia was evaluated by determining mechanical nociceptive threshold in the right hindpaw with von Frey test. After decapitation, the sciatic nerve was excised for immunostaining of F4/80, a Mφ marker. An anti-HMGB1 neutralizing antibody (anti-HMGB1) at 1 mg kg-1 and pyrrolidine dithiocarbamate (PDTC), an NF-κB inhibitor, at 50 mg kg-1 were injected i.p. once a day, 7 times in total, from day 0 of PTX treatment. Liposomal clodronate (Cld), known to deplete Mφ, at 1.05 mg per mouse was injected i.p. on day 6 of PTX treatment. RAW264.7 cells were stimulated with PTX at 1 µM for 24 hours in the absence and presence of PDTC at 100 μM. HMGB1 levels in the supernatant were determined by Western blotting. Data are shown as the mean±S.E.M. Statistical significance was performed by Kruskal-Wallis H-test followed by a least significant difference-type test for behavioral data and by ANOVA followed by Tukey's test for all other data. PTX treatment caused delayed decrease in nociceptive threshold 9-10 days after the onset of PTX administration, which was inhibited by repeated administration of anti-HMGB1 [vehicle (V)+V 0.59±0.03 g, V+PTX 0.21±0.07 g (p<0.01 vs. V+V), anti-HMGB1+PTX 0.64±0.05 g (p<0.05 vs. V+PTX), n=5], or PDTC [V+V 0.59±0.08 g, V+PTX 0.12±0.02 g (p<0.01 vs. V+V), PDTC+PTX 0.58±0.07 g (p<0.01 vs. V+PTX), n=5]. Depletion of Mφ by Cld also significantly eliminated the PTX-induced hyperalgesia [V+V 0.51±0.04 g, V+PTX 0.15±0.04 g (p<0.01 vs. V+V), Cld+PTX 0.41±0.06 g (p<0.05 vs. V+PTX), n=5]. The F4/80-positive cells increased in the sciatic nerve after PTX treatment [V 6.5±0.7 vs PTX 13.1±1.0 cells per field, p<0.001, n=16]. In RAW264.7 cells, stimulation with PTX for 24 hours markedly increased HMGB1 secretion, which was inhibited by PDTC [V+V 0.02±0.002 (arbitrary unit), V+PTX 0.88±0.18 (p<0.01 vs. V+V), PDTC+PTX 0.35±0.10 (p<0.05 vs. V+PTX), n=5]. Our data suggest that PTX stimulates Mφs, which in turn secrete HMGB1 in an NF-kB-dependent manner, leading to neuropathic pain in mice.

Where applicable, experiments conform with Society ethical requirements