Macrophage (Mφ)-derived high mobility group box 1 (HMGB1), a nuclear protein, promotes inflammation via Toll-like receptor 4 (TLR4), receptor for advanced glycation end products (RAGE), CXC chemokine receptor 4 (CXCR4), etc. We have shown that HMGB1 mediates bladder pain accompanying cyclophosphamide-induced cystitis in mice, a model for interstitial cystitis (IC)/bladder pain syndrome (BPS) [1]. To study the pathophysiology of BPS without apparent bladder injury, we examined the role of HMGB1 in the bladder pain induced by intravesical (i.ves.) administration of substance P (SP) in mice. Under inhalation anesthesia with isoflurane, female ddY mice (17-30 g) received i.ves. SP at 6 nmol per mouse (200 μL of 30 μM solution, for 30 min) twice. To evaluate referred hyperalgesia (RH), 24 hours after i.ves. SP, nociceptive responses following stimulation of the skin region between the anus and urethral opening with von Frey hairs were scored, as reported previously [1], and the bladder was isolated, weighed and subjected to hematoxylin/eosin staining after cervical dislocation. Data show the mean ± SEM. Statistical significance was analyzed 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. SP treatment caused RH [total nociceptive score (TNS) from 10 challenges with 0.4 g hair: vehicle (V) 3.17±1.08 vs. SP 10.17±0.4, p<0.01, n=6], whereas it produced no change in bladder histology and only slight increase in bladder weight, an indicator of swelling [V 0.96±0.05 vs. SP 1.23±0.09 mg g-1 body weight, p<0.05, n=6]. The SP-induced RH was prevented by i.p. administration of an anti-HMGB1-neutralizing antibody (Ab) at 1 mg kg-1 [V+V 6.6±0.72, control IgG (IgG)+SP 10.63±0.46 (p<0.001 vs. V+V), Ab+SP 3.5±0.69 (p<0.01 vs. IgG+SP), n=8-10] and also by i.p. liposomal clodronate (LClo) (1.05 mg per mouse) that depletes Mφ or ethyl pyruvate (EP), known to inhibit HMGB1 release from Mφ, at 80 mg kg-1 [TNS: control liposome (cL)+V 5.4±0.51, cL+SP 9.6±0.24 (p<0.01 vs. cL+V), LClo+SP 6.2±0.37 (p<0.05 vs. cL+SP), n=5; V+V 6.5±0.76, V+SP 9.83±0.4 (p<0.05 vs. V+V), EP+SP 5.8±0.20 (p<0.01 vs. V+SP), n=5-6]. The RH following i.ves. SP was also reduced by i.p. low molecular weight heparin (LMWH), known to inhibit RAGE, at 2.5 mg kg-1 or AMD3100 (AMD), a CXCR4 antagonist, at 8 mg kg-1, but not LPS-RS, a TLR4 antagonist, at 0.5 mg kg-1 [TNS: V+V 8.13±0.44, V+SP 11.71±0.52 (p<0.01 vs. V+V), LMWH+SP 8.0±0.45 (p<0.01 vs. V+SP), n=5-6; V+V 7.75±0.25, V+SP 10.75±0.25 (p<0.05 vs. V+V), AMD+SP 8.5±0.50 (p<0.01 vs. V+SP), n=4; V+V 8.17±0.60, V+SP 12.0±0.52 (p<0.001 vs. V+V), LPS-RS+SP 10.8±0.37 (n.s. vs. V+SP), n=5-6]. Thus, Mφ-derived HMGB1 appears to mediate SP-induced bladder pain in mice, a model for BPS without apparent bladder injury, by activating RAGE and CXCR4.