Nuclear high mobility group box 1 (HMGB1) is released from various cells including macrophages (Mφ), promoting inflammation via the receptor for advanced glycation end products (RAGE), Toll-like receptor 4 (TLR4) and CXC chemokine receptor 4 (CXCR4). Given evidence for the role of HMGB1 in inflammatory pain [1-3], we tested if HMGB1 mediates acute pancreatitis-related pain. Male ddY mice (4 weeks old) received cerulein (Cer) (i.p., 50 μg kg-1) every 1 hour, 6 times in total. Referred hyperalgesia in the upper abdomen was evaluated by assessing nociceptive scores (0, 1 or 2) in response to stimulation with von Frey hairs, as reported previously [4]. Under urethane (i.p., 1.5-1.8 g kg-1) anesthesia, blood was withdrawn from the abdominal aorta for plasma amylase assay, and the pancreas was excised and weighed after decapitation. Mφ infiltration was examined by immunostaining of F4/80. Results are shown as means±SEM. Behavioral data were analyzed by Kruskal-Wallis H test followed by a least significant difference-type test, and all other data were by ANOVA followed by Tukey’s test. Cer treatment increased pancreatic weight (PW) and plasma amylase activity (AA), and caused referred hyperalgesia (increased nociceptive score), and the anti-HMGB1-neutralizing antibody (Ab) (i.p., 1 mg kg-1) abolished the referred hyperalgesia, but not increased PW or AA [PW (mg g-1 body weight): vehicle (V)+V 9.0±0.92, control IgG (IgG)+Cer 13.8±0.52 (p<0.01 vs. V+V), Ab+Cer 12.1±0.08 (n.s. vs. IgG+Cer); AA: V+V 100±16.4, IgG+Cer 388±87.8 (p<0.01 vs. V+V), Ab+Cer 280±33.6% (n.s. vs. IgG+Cer); total nociceptive score (TNS) from 10 challenges with 0.16 g hair: V+V 2.7±0.67, IgG+Cer 9.2±0.39 (p<0.05 vs. V+V), Ab+Cer 1.2±0.20 (p<0.01 vs. IgG+Cer) (n=5-6)]. The Cer-induced referred hyperalgesia was prevented by i.p. ethyl pyruvate (EP) (80 mg kg-1), known to inhibit HMGB1 release from Mφ, or liposomal clodronate (LClo) (1.05 mg per mouse) that depletes Mφ [TNS: V+V 4.5±0.64, V+Cer 8.0±0.31 (p<0.05 vs. V+V), EP+Cer 4.2±0.48 (p<0.01 vs. V+Cer); control liposome (cL)+V 5.3±0.21, cL+Cer 9.5±0.50 (p<0.05 vs. V+V), LClo+Cer 6.0±0.36 (p<0.05 vs. cL+Cer) (n=4-6)]. The F4/80-positive cells in the pancreas increased after Cer treatment [V 2.6±0.43 vs Cer 9.0±0.82 cells per field, p<0.001, n=13-16]. FPS-ZM1 (FPS) (1 mg kg-1), lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS) (0.5 mg kg-1) and AMD3100 (AMD) (8 mg kg-1), antagonists of RAGE, TLR4 and CXCR4, respectively, when administered i.p., reduced the referred hyperalgesia [TNS: V+V 5.3±0.33, V+Cer 10.1±0.40 (p<0.01 vs. V+V), FPS+Cer 4.8±0.40 (p<0.001 vs. V+Cer); V+V 3.8±0.65, V+Cer 7.8±0.74 (p<0.01 vs. V+V), LPS-RS+Cer 5.0±0.81 (p<0.05 vs. V+Cer); V+V 2.2±0.85, V+Cer 9.5±0.86 (p<0.05 vs. V+V), AMD+Cer 2.2±0.62 (p<0.05 vs. V+Cer) (n=4-6)]. Our data thus suggest that Mφ-derived HMGB1 mediates pancreatitis-related pain by activating RAGE, TLR4 and CXCR4 in mice.