Saburo Saito

Morphine-induced antinociception is partially reduced in interleukin-31 (IL-31) receptor A (IL-31RA)-deficient mice, indicating that IL-31RA is crucial for morphine-induced peripheral antinociception. Herein, we examined the combined effects of IL-31 and morphine on the antinociceptive activity and itch-associated scratching behavior (LLS) in mice and elucidated the regulatory mechanisms. A hot-plate test was used to assess antinociception. LLS was automatically detected and recorded via a computer. IL-31RA mRNA expression was assessed using real-time polymerase chain reaction. Repeated pre-treatment with IL-31 resulted in significant antinociceptive activity. Repeated administration of morphine decreased the morphine-induced antinociceptive activity, LLS counts, and regular dose and inhibited IL-31-induced LLS. These results suggested that the repeated administration of morphine depleted inter-neuronal IL-31RA levels, preventing morphine-induced antinociception. Therefore, IL-31 may be helpful as an adjunct analgesic to morphine. To explore the benefits of IL-31, its influence on morphine-induced antinociceptive tolerance in mice was examined. An IL-31 and morphine combination increased the analgesic action, which increased the expression of DRG neuronal IL-31RA, elucidating the site of peripheral antinociception of morphine. This site may induce exocytosis of IL-31RA in the sensory nervous system. Collectively, the suppressive effect of IL-31 on morphine-induced antinociceptive tolerance may result from IL-31RA supplementation in sensory nerves.

Morphine-induced antinociception is partially reduced in interleukin-31 (IL-31) receptor A (IL-31RA)-deficient mice, indicating that IL-31RA is crucial for morphine-induced peripheral antinociception. Herein, we examined the combined effects of IL-31 and morphine on antinociceptive activity and itch-associated scratching behavior (LLS) in mice, and elucidated the regulatory mechanisms. A hot-plate test was used to assess antinociception. LLS was automatically detected and recorded via computer. IL-31RA mRNA expression was assessed using real-time polymerase chain reaction. Repeated pre-treatment with IL-31 resulted in significant antinociceptive activity. Repeated administration of morphine decreased the morphine-induced antinociceptive activity, LLS counts, and regular dose, and inhibited IL-31-induced LLS. These results suggested that repeated administration of morphine depleted inter-neuronal IL-31RA levels, preventing morphine-induced antinociception. Therefore, IL-31 may be helpful as an adjunct analgesic to morphine. To explore the benefits of IL-31, its influence on morphine-induced antinociceptive tolerance in mice was examined. IL-31 and morphine combination increased analgesic action which increased the expression of DRG neuronal IL-31RA, elucidating the site of peripheral antinociception of morphine. This site may induce exocytosis of IL-31RA in the sensory nervous system. Collectively, the suppressive effect of IL-31 on morphine-induced antinociceptive tolerance may result from IL-31RA supplementation in sensory nerves.

The theory that an itch inhibits pain has been refuted; however, previous research did not investigate this theory for an interleukin-31 (IL-31)-induced itch. Previously, we have found that morphine-induced antinociception was partially reduced in IL-31 receptor A (IL-31RA)-deficient (IL-31RAKI) mice, indicating that IL-31RA may play an important role in morphine-induced peripheral antinociception. In the present study, we evaluated the effect of IL-31-induced analgesia on a 2,4,6-trinitrochlorobenzene (TNCB)-sensitized mice using a hot-plate test. This test evaluated the antinociceptive activity of morphine and non-steroidal anti-inflammatory drugs (NSAIDs). Repeated pretreatment with IL-31 showed significant antinociceptive action. Furthermore, its combination with morphine, but not with NSAIDs, increased the analgesic action. In contrast, treatment with TNCB and capsaicin decreased antinociception. Moreover, TNCB increased IL-31RA expression in the dorsal root ganglia at 24 h, whereas capsaicin inhibited it. The comparative action of several analgesics on TNCB or capsaicin was evaluated using a hot-plate test, which revealed that the antinociceptive activity was decreased or disappeared in response to capsaicin-induced pain in IL-31RAKI mice. These results indicate that the analgesic action of IL-31 involves the peripheral nervous system, which affects sensory nerves. These results provide a basis for developing novel analgesics using this mechanism.

The theory that an itch inhibits pain has been refuted; however, previous research did not investigate this theory for interleukin-31 (IL-31)-induced itch. Previously, we have found that morphine-induced antinociception was partially reduced in IL-31 receptor A (IL-31RA)-deficient (IL-31RAKI) mice, indicating that IL-31RA may play an important role in morphine-induced peripheral antinociception. In the present study, we evaluated the effect of IL-31-induced analgesia on a 2,4,6-trinitrochlorobenzene (TNCB)-sensitized mice using a hot-plate test. This test evaluated the antinociceptive activity of morphine, and non-steroidal anti-inflammatory drugs (NSAIDs). Repeated pretreatment with IL-31 showed significant antinociceptive action. Furthermore, its combination with morphine, but not with NSAIDs, increased the analgesic action. In contrast, treatment with TNCB and capsaicin decreased antinociception. Moreover, TNCB increased IL-31RA expression in the dorsal root ganglia at 24 h, whereas capsaicin inhibited it. The comparative action of several analgesics on TNCB or capsaicin was evaluated using a hot-plate test, which revealed that the antinociceptive activity was decreased or disappeared in response to capsaicin-induced pain and in IL-31RAKI mice. These results indicate that the analgesic action of IL-31 involves the peripheral nervous system, which affects sensory nerve. These results provide a basis for developing novel analgesics using this mechanism.

Atopic dermatitis (AD) is a common skin disease caused by genetic and environmental factors. However, the mechanisms underlying AD development remain unclear. In this study, we examined the genetic factors contributing to the onset of itch-associated scratching in different strains of mice. Interleukin-31 (IL-31) induces severe scratching and dermatitis in mice. However, the site of action of IL-31 remains unclear. Cutaneous IL-31 and IL-31 receptor A (IL-31RA) mRNAs in the dorsal root ganglion (DRG) are expressed exclusively in the AD model, i.e., NC/Nga mice. Here we evaluated the effects of repeated administration of IL-31 on the scratching behavior in NC/Nga, BALB/c, and C57BL/6 mice. The results showed that repeated administration of IL-31 significantly increased itch-associated scratching (LLS) behavior in the three strains of mice. One hour after an intravenous IL-31 injection, BALB/c mice showed alloknesis-like behavior. Mite infestation and IL-31 administration triggered itchy skin, increased LLS counts and DRG neuronal IL-31RA expression, and eventually caused dermatitis. The dermatitis severity and LLS counts induced by mite infestation and IL-31 administration were in the order NC/Nga > BALB/c > C57BL/6. In conclusion, neuronal IL-31RA expression in the DRG was the most important genetic factor affecting the severity of LLS and dermatitis in mice.

Retinal inflammation accelerates photoreceptor cell death (PCD) caused by retinal degeneration. Minocycline, a semisynthetic broad-spectrum tetracycline antibiotic, has previously been reported to show PCD rescue effect in retinal degeneration. The purpose of this study was to assess the effect of minocycline on Cx3cr1 and Ccr2 expression in retinal degeneration. Mertk ^-/- Cx3cr1 ^GFP/+ Ccr2 ^RFP/+ mice, which enabled observation of Cx3cr1 - and Ccr2 -expression pattern in inherited retinal degeneration, were used to test the effect of minocycline. Minocycline was systemically administered to Mertk ^-/- Cx3cr1 ^GFP/+ Ccr2 ^RFP/+ mice. For observing the effect of minocycline on Cx3cr1 and Ccr2 expression, administration was started on 4-week-old mice and continued for 2 weeks. To assess the PCD rescue effect, minocycline was administered to 6-week-old mice for 2 weeks. The expression pattern of Cx3cr1-GFP and Ccr2-RFP were observed on retinal and retinal pigment epithelium (RPE) flat-mounts. The severity of retinal degeneration was assessed on retinal sections. Minocycline administration suppressed Ccr2 expression in Mertk ^-/- Cx3cr1 ^GFP/+ Ccr2 ^RFP/+ mice as observed in retinal and RPE flat-mounts. On the contrary, Cx3cr1 expression was not affected by minocycline administration. Retinal degeneration is ameliorated in minocycline administered Mertk ^-/- Cx3cr1 ^GFP/+ Ccr2 ^RFP/+ mice. In conclusions, Minocycline suppression of Ccr2 expression correlates to amelioration of retinal degeneration.

Summary: Transgenic rice seeds that contain genetically modified Cry j 1 and Cry j 2, the two major allergens of Cryptomeria japonica (Japanese cedar; JC), have been developed as immunotherapeutic candidates for JC pollinosis. Because the transgenic rice (TG‐rice) seeds express allergens containing whole amino acid sequences of Cry j 1 and Cry j 2 in the endosperm tissue (edible part of rice grain), they can potentially target all Cry j 1‐ and Cry j 2‐specific T‐cells. However, it was unknown whether antigenicity of Cry j 1 and Cry j 2 could be completely preserved in TG‐rice seeds. We verified the antigenicity of TG‐rice seeds to T‐cells through the analysis of the proliferative responses of T‐cells in Cry j 1‐ or Cry j 2‐immunized mice or T‐cell lines to TG‐rice seed extract. First, four mouse strains were immunized with Cry j 1 or Cry j 2. T‐cells in the immunized mice proliferated on treatment with TG‐rice seed extract, but not non‐transgenic wild‐type rice (WT‐rice) seed extract. Furthermore, T‐cell lines were established from the spleen cells of the immunized mice. Each T‐cell line resulted in a proliferative response to TG‐rice seed extract, but not to WT‐rice seed extract, suggesting that TG‐rice seeds certainly express T‐cell epitopes corresponding to T‐cell lines. Considering the modified amino acid sequences of Cry j 1 and Cry j 2 in TG‐rice seeds, the expression of specific T‐cell epitopes suggested that TG‐rice seeds express all possible T‐cell epitope repertoires of Cry j 1 and Cry j 2.

Japanese cedar (JC) pollinosis is a serious type I allergic disease in Japan. Although subcutaneous immunotherapy and sublingual immunotherapy have been applied to treat JC pollinosis, high doses of allergens may cause IgE-mediated allergic reactions. The transgenic rice seeds that contain genetically modified Cry j 1 and Cry j 2, the two major allergens of JC pollen, have been developed as candidates for oral immunotherapy. Although the antigens in the transgenic rice seeds (Tg-rice seeds) were engineered such that they decrease binding ability with IgE and they are of insufficient length to cross-link IgE on the surface of mast cells or basophils, the safety of Tg-rice seeds for patients with JC pollinosis was unclear. To verify the safety of Tg-rice seeds in terms of allergies, we investigated the percentage of activated basophils induced by Tg-rice seed extract in the basophil activation test. Blood samples from 29 patients with JC pollinosis were collected. Tg-rice seed extract, non-transgenic wild-type rice seed extract, and Cry j 1 and Cry j 2 were mixed with the blood with reagents. The percentage of activated basophils was assessed by CD203c expression, a basophil activation marker. The percentage of activated basophils after the stimulation with Tg-rice seed extract was 4.5 ± 1.6% (mean ± SD) compared with 62.9 ± 20.2% after Cry j 1- and Cry j 2-stimulation (difference 58.4%, P < 0.001, 95% confidence interval 51.0–65.9%). The results will contribute to the safety of Tg-rice seeds in terms of allergies.

Background: Morphine is an effective analgesic for the treatment of severe pain, but it can cause itching in patients. In the present study, we examined the possible involvement of interleukin‐31 (IL‐31) receptor A (IL‐31RA) on the morphine‐induced itching and antinociception in mice.Methods: Long‐lasting scratching (LLS) and short‐lasting scratching (SLS) were estimated as an indicator of itching and nonspecific behaviour, respectively, and antinociception was evaluated using a hot‐plate test in mice.Results: Morphine (5 mg/kg, s.c.) induced multiple episodes of SLS, few episodes of LLS, and antinociception in naive mice, with a close correlation observed between SLS or LLS counts and antinociception. In IL‐31RA‐deficient (IL‐31RA^−/−) mice, morphine (5 mg/kg, s.c.)‐induced LLS but not SLS was completely abolished, while antinociception was partially suppressed with 2.5 and 5 mg/kg but not 10 mg/kg, s.c. of morphine administration. Interestingly, intracerebroventricular (i.c.v.) administration of morphine (10 μg/mouse) significantly increased SLS but not LLS, and this effect was higher in IL‐31RA^−/− mice than that in wild‐type mice. Furthermore, following i.c.v. administration of morphine (10 μg/mouse), the antinociceptive effect was also significantly higher in IL‐31RA^−/− mice than that in wild‐type mice.Conclusion: Taken together, the present findings suggest that IL‐31RA may play a significant role, perhaps in the sensory neurons and/or spinal cord rather than in the brain, in the modulation of morphine‐induced itching and antinociception.Significance: Here, we demonstrate a possible common mediator of itching and antinociception of morphine, interleukin‐31 (IL‐31) receptor A (IL‐31RA). IL‐31RA may be a noteworthy target for considering the novel mechanism of itch and pain signalling affected by morphine.

Interleukin-31 (IL-31) is a recently identified cytokine produced by Th2 cells that is involved in the development of atopic dermatitis-induced skin inflammation and pruritus. Its receptor, IL-31RA, is expressed by a number of cell types, including epithelial cells, eosinophils, and activated monocytes and macrophages. To date, however, the regulation of Th2 responses by distinct cell types and tissues expressing IL-31RA has not been well studied. In this study, Cry j 2, one of the major allergens of Japanese cedar pollen, was administered to IL-31RA-deficient or wild-type (WT) mice via nasal or intraperitoneal injection for induction of specific Th2 responses. After nasal administration of Cry j 2, IL-31RA-deficient mice showed lower Cry j 2-specific CD4+ T cell proliferation, Th2 cytokine (IL-5 and IL-13) production, and Th2-mediated (IgE, IgG1, and IgG2b) antibody responses than WT mice. In contrast, IL-31RA-deficient mice administered Cry j 2 intraperitoneally showed stronger Th2 immune responses than WT mice. These results indicate that IL-31R signaling positively regulates Th2 responses induced by nasal administration of Cry j 2, but negatively regulates these responses when Cry j 2 is administered intraperitoneally. Collectively, these data indicate that the induction of antigen-specific Th2 immune responses might depend on tissue-specific cell types expressing IL-31RA.