{"paper_id":"388932ec-39dc-4264-b4c2-3d1c9a62d546","body_text":"Overt nociception/overt pain-like behavior models are widely used to assess the\nactivity of novel candidate analgesic drugs and their mechanisms of action. These\ntests involve the injection of stimuli with irritating characteristics, which\nrapidly promotes behaviors such as abdominal writhing, flinching, or licking of the\ninjected paw ( 1 - 6 ). The stimuli are generally chemical [e.g.,\nphenyl- p -benzoquinone (PBQ), acetic acid, formalin] ( 1 - 3 ),\nbut may also be biological (e.g., zymosan) ( 5 ) or even an antigen ( 4 ).\nAbdominal writhing induced by acetic acid or PBQ is dependent on the release of\ninflammatory mediators, such as cytokines and prostanoids ( 2 , 5 ). In the PBQ model,\nwrithing depends on the cytokines interleukin (IL)-18, interferon gamma (IFN-γ), and\nendothelin-1 (ET-1) ( 2 ). The mechanism for\nmediating the acetic acid-induced writhing response ( 2  depends on activation of peritoneal macrophages and mast cells that\nthen release cytokines, such as tumor necrosis factor-α (TNF-α), IL-1β, and IL-8, as\nwell as eicosanoids and sympathomimetic amines ( 5 ). Nevertheless, despite differences in the peripheral mediators\ninvolved in PBQ- and acetic acid-induced writhing responses, both depend on spinal\nactivation of mitogen-activated protein kinases, phosphatidylinositol 3-kinase and\nmicroglia ( 1 ).\nIn the formalin test, the subcutaneous injection of formalin into the mouse hind-paw\ninduces a nociceptive response that consists of two phases. Phase 1 (0-5 min after\nformalin injection) is the neurogenic phase and is generally attributed to a direct\neffect of the stimulus on nociceptors, whereas phase 2 (10-30 min after formalin\ninjection) involves the subsequent development of inflammation, which is mediated by\ncytokines, such as TNF-α, IL-1β, IL-6, and IL-8, and prostaglandins (PGs) ( 6 - 9 ).\nOvert pain-like behavior can also be induced by antigen challenge in immunized\nanimals ( 4 ). For instance, intraplantar\ninjection of ovalbumin (OVA) induces significantly more paw licking in immunized\nmice than in non-immunized mice, by an ET-1-dependent mechanism ( 4 ).\nIL-33 is the most recent addition to the IL-1 cytokine family that includes IL-1β and\nIL-18. IL-33 exerts its biological activity by interacting with a heteromeric\nreceptor composed of ST2, the IL-33 specific subunit of the receptor, and the IL-1\nreceptor accessory protein, which is shared with IL-18 ( 10 , 11 ). IL-33 is a\npleiotropic cytokine implicated in various inflammatory conditions and diseases\n( 11 ). IL-33 plays a role in Th1, Th2, and\nTh17 adaptive responses, innate inflammation, and as an endogenous danger signal\n( 11 ).\nIL-33/ST2 signaling is involved in pain ( 12 ).\nIL-33 mediates methylated bovine serum albumin (mBSA)-induced cutaneous and\narticular mechanical hyperalgesia in immunized mice via activation of the TNF-α →\nIL-1β → IFN-γ → ET-1 → PGE 2  signaling cascade ( 12 ). Furthermore, IL-33/ST2 signaling contributes to\ncarrageenan-induced innate inflammatory pain, triggering the production of TNF-α,\nCXCL1, IL-1β, ET-1, and PGE 2  ( 13 ).\nThese data suggest that the role of IL-33 in pain is wider than just its involvement\nin innate and Th1/Th17-dependent mechanical hyperalgesia ( 12 , 13 ) because the\nmolecules produced in response to IL-33 also mediate nociceptive responses in other\npain models, as well as promoting overt pain behaviors ( 2 , 4 , 5 , 7 ).\nThus, in this study, we used ST2-deficient mice to investigate the role of IL-33/ST2\nsignaling in abdominal writhing induced by acetic acid and PBQ, and in formalin- and\nOVA challenge-induced paw flinch and licking responses in naive and immunized\nanimals, respectively.\n\nAcetic acid and formalin were obtained from Mallinckrodt Baker S.A. (Mexico), PBQ\nand OVA were from Sigma-Aldrich (USA), and DMSO was from Merck (Germany). The\ndoses of these stimuli were chosen based on pilot studies and previous data from\nour laboratory ( 1 , 2 , 1,2,6 ).\nAll experiments were performed on sex-matched BALB/c wild-type\n(ST2 +/+ ) and BALB/c background ST2-deficient (ST2 -/- ) mice\n( 14 ), weighing 20-25 g. The\nexperiments were conducted between 9:00 am and 5:00 pm. The mice were bred at\nthe Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil.\nAnimal care and handling procedures were in accordance with the International\nAssociation for the Study of Pain guidelines and with the approval of the Ethics\nCommittee of the Faculdade de Medicina de Ribeirão Preto, Universidade de São\nPaulo, Brazil. All experiments were double-blinded.\nThe PBQ- and acetic acid-induced writhing model experiments were performed as\ndescribed previously, using the same doses ( 1 , 2 ). PBQ (630 µg diluted in\n10 mL 2% DMSO in saline), acetic acid [0.6% (v/v) diluted in saline], or vehicle\nwas injected (10 mL/kg) into the peritoneal cavity of each mouse. Next, each\nmouse was placed in a large glass cylinder, and the intensity of nociceptive\nbehavior was quantified by counting the total number of writhing responses\n(contraction of the abdominal muscles together with stretching of hind limbs or\nrotation of the trunk) occurring between 0 and 20 min after stimulus injection.\nThe intensity of the writhing response was expressed as the cumulative number of\nmovements occurring in 2-min bins over 20 min.\nThe number of paw flinches and time spent licking the paw were counted between 0\nand 30 min after intraplantar injection of formalin [2.5% (v/v) diluted in 25 µL\nsaline] or vehicle as described previously ( 6 ). The period was divided into 5-min bins, which clearly\ndemonstrated the presence of the first (0-5 min) and second (10-30 min) phases\nthat are characteristic of the model ( 6 - 9 ).\nMouse motor coordination/function was evaluated using the rota-rod test. The\napparatus consisted of a 2.5-cm diameter bar that was subdivided into 6\ncompartments by 25-cm diameter disks (model 7600; Ugo Basile, Italy). Mice were\nplaced on the bar while it was rotating at constant speeds of 10 or 15 rotations\nper min (rpm), and the duration that they were able to remain on it was\ndetermined. The cutoff time used was 180 s.\nMice were placed in a 10-cm diameter glass cylinder on a hot plate (Hot Plate\nHP-2002, Insight Equipamentos, Brazil) maintained at 55°C. The reaction time was\nscored when the animal jumped, flinched or licked its paws. A maximum latency\n(cutoff) was set at 30 s to avoid tissue damage ( 6 ).\nMice were immunized with a single subcutaneous dose of 50 µg OVA plus 5 mg\nAl(OH) 3 , diluted in 200 µL sterile saline. After 14 days, mice\nwere challenged with 1 µg/paw OVA or vehicle (saline). The number of paw\nflinches and time spent licking the paw was determined over 30 min ( 4 ). The dose of the antigen challenge was\ndetermined by preliminary studies in our laboratory.\nResults are reported as means ±SE of 7 mice per group per experiment, and\nrepresent two separate experiments. Statistical differences between groups were\ndetermined by the two-tailed Student  t -test for unpaired\nsamples ( Figures 1  and  3 ) or one-way ANOVA followed by the\nBonferroni  t -test ( Figure\n2 ). All statistical analyses were performed using the GraphPad Prism\n5 software (USA). The significance level was set at P<0.05.\n\nThe role of IL-33/ST2 was evaluated in PBQ- and acetic acid-induced overt\npain-like behavior. ST2 -/-  mice showed reduced PBQ-induced writhing\nresponses compared with ST2 +/+  mice ( Figure 1A ). The differences were significant at each interval\nbetween 10 and 20 min. Acetic acid-induced writhing responses were also\ndiminished in ST2 -/-  mice, compared with ST2 +/+  mice\n( Figure 1B ), and the differences were\nsignificant between 4 and 20 min. The vehicles, saline and 2% DMSO in saline,\ndid not induce writhing responses in any of the mice (data not shown).\nST2 +/+  and ST2 -/-  mice challenged with 1 µg/paw OVA 14 days\nafter immunization with the antigen displayed paw flinches and licking behavior.\nBoth the paw flinches ( Figure 2A ) and time\nspent licking the paw ( Figure 2B ) induced\nby OVA challenge were reduced significantly in ST2 -/-  mice, compared\nwith ST2 +/+  mice. Vehicle (saline), in immunized mice, and OVA\nchallenge in the sham-immunized group did not induce significant flinching or\nlicking behaviors. ST2 -/-  mice showed the same responses as\nST2 +/+  mice in the sham-immunized group challenged with OVA and\nthe immunized group challenged with saline (data not shown).\nThe paw flinch responses of ST2 -/-  mice were reduced at all time\npoints compared with those of ST2 +/+  mice ( Figure 3A ), and the time spent licking the injected paw was\nreduced in ST2 -/-  compared with ST2 +/+  mice during the\n15-20-min and 25-30-min intervals ( Figure\n3B ). Injection of vehicle (saline) did not induce flinch or licking\nresponses in either ST2 +/+  or ST2 -/-  mice (data not\nshown).\nRota-rod and hot plate tests were used to evaluate the role of IL-33/ST2\nsignaling on motor coordination/function and thermal nociceptive threshold. No\nsignificant differences (P>0.05) were observed in the time that\nST2 -/-  mice (179.80±0.20 or 162.40±11.03 s) and ST2 +/+ \nmice (178.70±1.30 or 175.70±4.30 s) remained on the rota-rod at 10 or 15 rpm,\nrespectively. In the hot plate test, no significant difference (P>0.05) was\nobserved in the reaction time of ST2 -/-  mice (10.45±0.50 s) and\nST2 +/+  mice (11.45±1.04 s; data not shown).\n\nIL-33 is a pleiotropic cytokine involved in adaptive and innate immune responses\n( 11 ). The role of IL-33/ST2 signaling in\npain was first demonstrated using a Th1/Th17 immunization protocol. Treatment with\nsoluble ST2 reduced antigen challenge-induced mechanical cutaneous and articular\nhyperalgesia in mice by preventing the production of inflammatory molecules,\nincluding the cytokines TNF-α, IL-1β and IFN-γ, ET-1, and PGE 2  ( 12 ). It is interesting to note that IL-18 also\nmediates hyperalgesia induced by antigen challenge in immunized mice by triggering\nthe production of IFN-γ, ET-1, and PGE 2  ( 15 ) and is an important cytokine in the PBQ-induced writhing response\n( 2 ). We reason that IL-33/ST2 signaling\ncould also contribute to overt pain-like responses, as already observed in relation\nto IL-18.\nFurthermore, since IL-33 and IL-18 receptors share a common beta chain, the IL-1\nreceptor accessory protein ( 11 ), and both\nreceptors mediate hyperalgesia in antigen-induced inflammation via IFN-γ, ET-1 and\nPGE 2 , a similar profile of responses to their activation might be\nexpected. However, IL-18 mediates the PBQ- but not the acetic acid-induced writhing\nresponse ( 2 ), whereas IL-33/ST2 mediates the\nnociceptive response in both models. IL-33/ST2 triggers production of TNF-α and\nIL-1β, which are important cytokines in the acetic acid-induced writhing response\n( 5 ). This mechanism of IL-33, compared\nwith that of IL-18, explains why IL-33/ST2 mediates both the acetic acid- and\nPBQ-induced writhing responses. Thus, it seems likely that the role of IL-33/ST2 in\npain may be broader than that of IL-18. Furthermore, consistent with a role of IL-33\nin abdominal pain, IL-33 peritoneal fluid and serum levels are elevated in 75 and\n23% of patients with endometriosis, respectively, primarily in deeply infiltrating\nendometriosis presenting painful symptoms, such as dysmenorrhea ( 16 ).\nIn the OVA challenge in immunized mice, the results indicate a role for IL-33/ST2 in\nTh2 inflammation-induced overt pain-like behavior, increasing the possible role of\nIL-33/ST2 in nociception beyond Th1/Th17 and innate responses, as demonstrated\npreviously ( 12 , 13 ). In the OVA model, mast cell degranulation induces\nET-1/ET A  receptor-dependent overt pain-like behavior ( 4 ). Likewise, in the model of Th1/Th17\ninflammation in immunized mice challenged with mBSA, and in innate inflammation\ninduced by carrageenin, IL-33/ST2 also induces ET-1-dependent mechanical\nhyperalgesia ( 12 , 13 ). Thus, it seems reasonable to expect that IL-33/ST2 could\ntrigger an ET-1-dependent nociceptive response in Th2 inflammation models, but this\nphenomenon remains to be explored further.\nIn the formalin test, ST2 deficiency resulted in a decreased response in both phases,\nmainly in the flinch response assay, but with significant inhibition seen also in\nthe second phase of the licking response. In the first phase of the formalin test,\nthere is participation by mast cell-derived mediators, such as histamine ( 8 ), and, considering that IL-33 activates the\nconstitutively expressed ST2 receptors in mast cells ( 10 , 11 ), it is possible\nthat involvement of IL-33/ST2 in the first phase of the formalin test might be\nrelated to the activation of mast cell-derived mediators. The reduction in\nformalin-induced nociception was more evident in the second phase, with inhibition\nof both flinching and licking responses. In that phase, there is production of\ncytokines, such as TNF-α and IL-1β, and inhibition of the activity of those\ncytokines reduces nociceptive behavior ( 7 ).\nIn antigen- and carrageenin-induced hyperalgesia IL-33/ST2, signaling mediates the\nproduction of hyperalgesic TNF-α and IL-1β ( 12 , 13 ). Thus, it seems reasonable\nto suggest that IL-33/ST2 could induce overt pain-like behavior in the second phase\nof the formalin test by triggering the production of nociceptive cytokines, such as\nTNF-α and IL-1β.\nDuring the review process of this manuscript, it was reported that the intraplantar\nor intrathecal administration of IL-33 induces overt pain-like behavior. Moreover,\nintraplantar or intrathecal administration of IL-33 increases, and treatment with\nsoluble ST2 (a decoy receptor for IL-33) reduces, the overt pain-like behavior\ninduced by formalin ( 17 ). Those results are\nconsistent with the present data and the rationale that IL-33/ST2 signaling itself\ncan trigger overt pain-like behavior.\nFurthermore, IL-33 is constitutively expressed in normal human tissues, and its level\nis abundant in endothelial and epithelial cells  in vivo , indicating\nthat IL-33 can be released promptly. IL-33 has a role as a nuclear factor and is\nconsidered to be a “danger” signal, similar to HMGB1 and IL-1α, functioning as an\nalarm to the immune system when endothelial and/or epithelial cells are subject to\ndamage ( 18 ). This concept is in line with the\ninvolvement of IL-33/ST2 in acute nociceptive events such as the writhing response,\nin which the stimulus is injected into the highly vascularized peritoneal cavity,\nand the paw flinch and licking responses, with the possible release of IL-33 by\nepithelial cells and keratinocytes and activation of mast cells, fibroblasts, and\nmacrophages ( 11 , 12 , 15 , 16 , 18 , 19 ). Furthermore, ST2 is\nexpressed by neurons, and IL-33 is expressed by neurons and astrocytes in the spinal\ncord of mice in a model of encephalomyelitis ( 20 ), suggesting that IL-33 could be produced by astrocytes and neurons\nand act on ST2 receptors expressed by neurons in the spinal cord, which is\nconsistent with the overt pain-like behavior induced by the intrathecal injection of\nIL-33 ( 17 ). Nevertheless, the cellular\nsources and targets of IL-33 remain to be determined in pain models to establish\nwhether it has direct and/or indirect effects on nociceptive neurons.\nNotably, differences were not observed between naive ST2 -/-  and\nST2 +/+  mice in the rota-rod, hot plate (data not shown), or\nelectronic pressure meter tests ( 12 , 13 ). These data indicate that the motor\ncoordination/function in ST2 -/-  mice is preserved, and that there is no\nalteration of basal nociceptive responses to thermal or mechanical stimuli.\nIn conclusion, the present data indicate that, in addition to promoting mechanical\nhyperalgesia ( 12 , 13 ), IL-33/ST2 signaling is important in overt pain-like\nbehavior triggered by a variety of phlogistic agents including acetic acid, PBQ,\nformalin, and OVA challenge in immunized mice. These results increase the relevance\nof IL-33/ST2 signaling in nociception and suggest that the potential of IL-33\ntargeting therapies to control inflammatory pain deserves to be investigated\nfurther.","source_license":"CC-BY-4.0","license_restricted":false}