Experimental Study: Interleukin-31 Augments Morphine-Induced Antinociceptive Activity and Suppress Tolerance Development in Mice.

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.

Analyzing the Antinociceptive Effect of Interleukin-31 in Mice.

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.

Interleukin-31 Receptor A Expression in the Dorsal Root Ganglion of Mice with Atopic Dermatitis.

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.

Capsaicin suppresses interleukin-31-induced itching partially involved in inhibiting the expression of dorsal root ganglion interleukin-31 receptor A in male mice.

To elucidate the mechanisms underlying the antipruritic effect of capsaicin, we investigated how topical application of capsaicin (0.01, 0.1 and 1.0% w/v) affects spontaneous scratching in NC/Nga mice, inerleukin-31 (IL-31) induced in BALB/c mice, and IL-31 receptor A (IL-31RA) and transient receptor potential vanilloid member 1 (TRPV1) mRNA expression in dorsal root ganglia (DRG). Capsaicin concentration-dependently suppressed long-lasting scratching (over 1.0 s, itch-associated scratching) and short-lasting scratching (0.3-1.0 s, locomotor activity) immediately after the application. Total long-lasting scratching and short-lasting scratching counts for 24 h and IL-31RA mRNA expression in the DRG significantly decreased with increasing concentration of capsaicin. Furthermore, 1.0% capsaicin suppressed long-lasting scratching and short-lasting scratching for more than 72 h. At this point, DRG IL-31RAmRNA was significantly decreased, but there was no change in cutaneous IL-31RA and TRPV1 mRNA. Thus capsaicin suppresses long-lasting scratching by inhibiting IL-31RA mRNA expression in the DRG. Next, we examined the effect of capsaicin on IL-31-induced long-lasting scratching in BALB/c mice. Repeated administration of IL-31 (50 µg/kg, subcutaneous) every 12 h for 3 days apparently increased long-lasting scratching counts and IL-31RA mRNA in the DRG. These increases were significantly suppressed by pretreatment with 1.0% capsaicin. TRPV1 mRNA in the DRG was also decreased within 1-24 h after capsaicin application. These results suggest that the strong and prolonged antipruritic action for IL-31-induced itching of capsaicin was caused by desensitization of C-fibers, and, in addition, the long-lasting inhibition of IL-31RA mRNA expression in the DRG.

Involvement of interleukin-31 receptor A in morphine-induced itching and antinociception in mice.

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.

Regulation of Th2 responses by different cell types expressing the interleukin-31 receptor.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Repeated administration of IL-31 upregulates IL-31 receptor A (IL-31RA) in dorsal root ganglia and causes severe itch-associated scratching behaviour in mice.

We investigated the effects of repeated administration of interleukin-31 (IL-31) on itch-associated scratching counts (long-lasting scratching, LLS) and IL-31-related receptor mRNA expression in mice. Intra-dermal (i.d.) injection of IL-31 (100 and 300 ng/site) every 12 h for 3 days significantly increased LLS. Repeated administration of IL-31 also increased the expression of IL-31 receptor A (IL-31RA) and oncostatin M receptor beta (OSMRß) in dorsal root ganglia (DRG). After the repeated administration of IL-31 was discontinued, IL-31RA expression decreased and reached the baseline level 2 days after the last dose of IL-31. LLS changed along with DRG IL-31RA expression. Moreover, IL-31-induced IL-31RA protein expression was confirmed by Western blotting analysis. These data suggest that IL-31 upregulates IL-31RA expression in DRG neuron cell bodies, and cutaneous-injected IL-31-induced itching is enhanced by DRG IL-31RA expression in mice.

A single dose of interleukin-31 (IL-31) causes continuous itch-associated scratching behaviour in mice.

We investigated the effects of a single dose of mouse interleukin-31 (IL-31) on scratching behaviour in comparison with spontaneous skin-lesion- or serotonin (5-HT)- induced scratching behaviour in NC/Nga and BALB/c mice. Intradermal (i.d.) injection of IL-31 caused a gradual increase in long-lasting scratching (LLS, over 1.5 s) about 3 h after administration followed by a gradual decrease for over 24 h after administration. I.d. injection of IL-31 significantly increased the total LLS counts/24 h but not short-lasting scratching (SLS, 0.3-1.5 s). In skin-lesioned NC/Nga mice, the LLS but not SLS counts were significantly higher than those in non-skin-lesioned NC/Nga mice. We also investigated 5-HT-induced scratching in BALB/c mice, SLS but not LLS increased immediately after the injection and then decreased to baseline after at 20 min. These results suggest that IL-31 may participate in the sensation of itching and promote scratching behaviour in skin-lesioned NC/Nga mice, an animal model of atopic dermatitis (AD).

Itch-associated scratching contributes to the development of dermatitis and hyperimmunoglobulinaemia E in NC/Nga mice.

Atopic dermatitis (AD) is related to immunoglobulin E (IgE) production, and a type-1 and type-2 helper T cell (Th1/Th2) imbalance has been hypothesized as the aetiology. While itching and scratching are important factors in the development of dermatitis, the mechanisms underlying these phenomena are poorly understood. We investigated the relationship between scratching, transepidermal water loss (TEWL), signs of dermatitis and serum Ig levels in NC/Nga mice, a model of AD. We also sensitized specific pathogen-free (SPF)-NC/Nga mice and BALB/c mice to mite antigen to determine the effects of IgE overproduction on scratching and investigated the involvement of mast cells and T/B cells in the induction of scratching using WBB6F1-W/W(v) mice and C.B.17/Icr-scid mice. Under conventional conditions, the scratch counts increased, followed by increases in TEWL and the inflammation score in NC/Nga mice that were not kept under SPF conditions. However, no change was observed in scratching, TEWL, or signs of dermatitis in mite antigen-sensitized SPF-NC/Nga and BALB/c mice, although the serum total IgE, IgG(1) and IgG(2a) levels increased. The scratch count increased significantly in both the WBB6F1-W/W(v) mice and C.B.17/Icr-scid mice when they were co-housed with skin-lesioned NC/Nga mice, raised under conventional conditions. These results show that IgE overproduction results from itch-associated scratching-induced dermatitis in NC/Nga mice.

The importance of brain PGE2 inhibition versus paw PGE2 inhibition as a mechanism for the separation of analgesic and antipyretic effects of lornoxicam in rats with paw inflammation.

OBJECTIVES: Lornoxicam is a non-selective cyclooxygenase inhibitor that exhibits strong analgesic and anti-inflammatory effects but a weak antipyretic effect in rat models. Our aim was to investigate the mechanism of separation of potencies or analgesic and antipyretic effects of lornoxicam in relation to its effect on prostaglandin E2 (PGE2) production in the inflammatory paw and the brain. METHODS: A model of acute or chronic paw inflammation was induced by Freund's complete adjuvant injection into the rat paw. Lornoxicam (0.01-1 mg/kg), celecoxib (0.3-30 mg/kg) or loxoprofen (0.3-30 mg/kg) was administered orally to the rats and the analgesic and antipyretic effects were compared. The paw hyperalgesia was assessed using the Randall-Selitto test or the flexion test. Dorsal subcutaneous body temperature was measured as indicator of pyresis. After the measurement of activities, the rats were sacrificed and the PGE2 content in the paw exudate, cerebrospinal fluid or brain hypothalamus was measured by enzyme-immunoassay. KEY FINDINGS: In a chronic model of arthritis, lornoxicam, celecoxib and loxoprofen reduced hyperalgesia with an effective dose that provides 50% inhibition (ED50) of 0.083, 3.9 and 4.3 mg/kg respectively, whereas the effective dose of these drugs in pyresis was 0.58, 0.31 and 0.71 mg/kg respectively. These drugs significantly reduced the PGE2 level in paw exudate and the cerebrospinal fluid. In acute oedematous rats, lornoxicam 0.16 mg/kg, celecoxib 4 mg/kg and loxoprofen 2.4 mg/kg significantly reduced hyperalgesia to a similar extent. On the other hand, lornoxicam did not affect the elevated body temperature, whereas celecoxib and loxoprofen significantly reduced the pyrexia to almost the normal level. These drugs significantly reduced the PGE2 level in inflamed paw exudate lo almost the normal level. On the other hand, lornoxicam did not change PGE2 level in the brain hypothalamus, whereas celecoxib and loxoprofen strongly decreased it. CONCLUSIONS: Lornoxicam exhibits strong analgesic but weak antipyretic effects in rats with paw inflammation. Such a separation of effects is related to its efficacy in the reduction of PGE2 levels in the paw and brain hypothalamus.

Modulation of scratching behavior by silencing an endogenous cyclooxygenase-1 gene in the skin through the administration of siRNA.

BACKGROUND: RNA interference (RNAi) is rapidly becoming a major tool that is revolutionizing research in the bioscience and biomedical fields. To apply the RNAi technique in vivo, it is crucial to develop appropriate methods of guiding the short interfering RNA (siRNA) molecules to the right tissues and cells. Here, we demonstrate an efficient method for performing gene knockdown in the body skin using the in vivo electro-transduction of siRNA. Using this method, we examined whether the targeted silencing of the cyclooxygenase (COX) gene in the skin could modulate the scratching behavior of an atopic dermatitis mouse model. METHODS: NC/Nga mice were used as the atopic dermatitis model. Using our optimized in vivo electroporation conditions, siRNAs were introduced into the skin; the silencing efficiency was then analyzed by Western blotting, measuring the levels of prostaglandins, and immunohistochemistry. The scratching behaviors of the mice were measured using an automatic system. RESULTS: Targeted silencing of the COX-1 gene using our in vivo siRNA technique significantly accelerated the scratching behavior of NC/Nga mice, whereas the COX-2 siRNA showed no effect. In addition, the effect of COX-1 siRNA was mimicked by treatment with a COX-1-selective inhibitor (SC-560). CONCLUSIONS: We have demonstrated the successful silencing of endogenous gene expression in the skin using the intradermal transfection of unmodified siRNA via electroporation. Using this method, we revealed that COX- 1-mediated prostaglandins may act as endogenous inhibitors of scratching behavior.

Inductions of histidine decarboxylase in mouse tissues following systemic antigen challenge: contributions made by mast cells, non-mast cells and IL-1.

BACKGROUND: Previous findings suggest that antigen challenge (AC) may induce histidine decarboxylase (HDC) in cells other than mast cells (MCs) via MC-derived IL-1. We examined this hypothesis. METHODS: Mice were sensitized to ovalbumin. After the sensitization, an AC was delivered intravenously. RESULTS: In control mice, AC markedly induced HDC at a postanaphylactic time in the liver, lung, spleen, and ears. In MC-deficient W/W(v) mice, AC also induced HDC, although the effect was weaker than in control mice. AC increased IL-1 in the tissues, the pattern being similar in W/W(v) and control mice. AC induced HDC similarly in IL-1-deficient and control mice. In control mice, AC decreased histamine in the tissues (except the liver) for several hours. CONCLUSION: (1) AC induces HDC in both MC-dependent and MC-independent ways. (2) AC induces IL-1 mostly in non-MCs, but this IL-1 is not a prerequisite for the induction of HDC by AC. (3) HDC induction may contribute to the replenishment of the reduced pool of MC histamine in the anaphylactic period. (4) In the case of MC-dependent HDC induction, AC may stimulate MCs in such a way as to induce HDC within the MCs themselves, and/or AC-stimulated MCs may stimulate HDC induction in other cells, which will need to be directly identified in future studies.

The anti-pruritic efficacy of TS-022, a prostanoid DP1 receptor agonist, is dependent on the endogenous prostaglandin D2 level in the skin of NC/Nga mice.

TS-022 is a prostanoid DP(1) receptor agonist, originally developed as a novel anti-pruritic drug for atopic dermatitis. The drug has been shown to suppress scratching and improve the skin inflammation in the NC/Nga (NC) mouse, a model of atopic dermatitis. Corticosteroids are commonly used as effective agents for the treatment of atopic dermatitis. We examined the anti-pruritic efficacy of TS-022 in NC mice cohabited with skin-lesioned NC mice, which showed spontaneous scratching without skin lesions in the early phase and chronic itching with severe dermatitis in the late phase, in comparison with that of dexamethasone. We have previously reported that prostaglandin D(2) might have a physiological role in the inhibition of pruritus. While after 2 weeks of cohabitation with skin-lesioned NC mice (early phase of dermatitis, characterized by the appearance of spontaneous scratching), topically applied TS-022 exhibited a weak anti-pruritic effect in the NC mice, after 6 weeks of cohabitation (late phase, characterized by both chronic scratching and dermatitis), the drug exerted potent anti-pruritic activity. In contrast, dexamethasone exerted potent anti-pruritic effect in both the early and late phases. Indomethacin aggravated the scratching in the early phase, but had no effect in the late phase. The skin prostaglandin D(2) level was significantly increased in the early phase, to subsequently declined and return to the basal level in the late phase. The cutaneous ability for prostaglandin D(2) production following topical application of arachidonic acid or mechanical scratching was decreased in the late phase. Moreover, the expression level of the prostanoid DP(1) receptor in the skin was increased in the late phase. These findings suggest that the potent anti-pruritic activity of TS-022 in the late phase might be attributable to the decrease of endogenous prostaglandin D(2) production and increase of prostanoid DP(1) receptor expression.

Role of scratch-induced cutaneous prostaglandin D production on atopic-like scratching behaviour in mice.

NC/Nga mice are known to develop scratching dermatitis akin to atopic dermatitis, under conventional (Conv), but not under the specific-pathogen-free (SPF) condition. In this study, we examined the effects of mechanical-scratching on the spontaneous scratching counts (sign of itching), in relation to the cutaneous prostaglandin D2 (PGD2) levels in NC/Nga or BALB/c mice. Mechanical-scratching increased the cutaneous barrier damage and PGD2 levels in both strain mice under the SPF condition. By 4 weeks of cohabitation with the skin-lesioned NC/Nga mice, both the increase in the spontaneous scratching and development of dermatitis score were higher in the Conv-NC/Nga than in the Conv-BALB/c mice. At this time-point, the cutaneous PGD2 level induced by mechanical-scratching was significantly lower in the Conv-NC/Nga when compared with that in the SPF-NC/Nga mice, and that in the Conv-BALB/c was almost equal to that in the SPF-BALB/c mice. With mechanical scratches, the cohabitation-induced scratching was suppressed in the Conv-BALB/c, but not in the Conv-NC/Nga mice. These results suggest that the scratch-induced cutaneous PGD2 inhibits scratching and the subsequent development of dermatitis in BALB/c, while the impaired scratch-induced cutaneous PGD2 production in the NC/Nga mice resulted in no suppression of scratching, and aggravated the dermatitis.

Putative mechanism of the itch-scratch circle: repeated scratching decreases the cutaneous level of prostaglandin D2, a mediator that inhibits itching.

In atopic dermatitis, scratching of the skin as a reaction to itching causes injury to the skin, which, in turn, further increases the itching resulting in the establishment of the so-called itch-scratch circle. We have shown that prostaglandin (PG) D2 plays an inhibitory role against pruritus in mice with atopic-like dermatitis; therefore, we examined the relationship between scratching and the cutaneous PGD2 level using an artificial scratching model with a wire brush. Mechanical scratching induced a temporary increase of the skin PGs levels (PGE2, PGD2, 6-ketoPGF1alpha, PGF2alpha). The skin PGD2 level and the ability of PGD2 production decreased at 48 h after repeated scratch, compared to that of normal skin, not so after single scratch. Immunohistochemical analysis and Western blotting revealed a decrease in the levels of cyclooxygenase-1 (COX-1) and hematopoietic PGD synthase in mechanically scratched skin. The reduced ability of the skin for PGD2 production following mechanical scratching could be caused by this decrease in the expression levels of COX-1 and PGD2 synthase. The results suggest that repeated scratching in mice decreases the ability of the skin to produce PGD2, which is an endogenous mediator that inhibits pruritus, resulting in the establishment of the itch-scratch circle.

Effects of TS-022, a newly developed prostanoid DP1 receptor agonist, on experimental pruritus, cutaneous barrier disruptions and atopic dermatitis in mice.

TS-022, {4-[(1R, 2S, 3R, 5R)-5-Chloro-2-((S)-3-cyclohexyl-3-hydroxyprop-1-ynyl)-3-hydroxycyclopentyl] butylthio} acetic acid monohydrate, inhibits ADP-induced platelet aggregation, an effect significantly antagonized, as in the case of prostaglandin D(2) by the prostanoid DP(1) receptor antagonist (BW A868C). TS-022 is a prostanoid DP(1) receptor agonist, originally developed as a novel anti-pruritic drug for patients with atopic dermatitis. We examined the effects of TS-022 on experimental pruritus, cutaneous barrier disruption, and atopic dermatitis and in in vitro immune function tests. Topically applied TS-022 significantly suppressed scratching in skin-lesioned NC/Nga mice from a concentration of 2.5 nM, and this scratch-suppressive activity was significantly antagonized by BW A868C. Tacrolimus (FK-506) and dexamethasone, used as reference drugs for atopic dermatitis, also exhibited suppressive effects against scratching, but only at concentrations of 125 and 25,000 microM. TS-022 applied topically, once a day for 2 days, significantly accelerated repair of the cutaneous barrier disruption caused by mechanical scratching, from concentrations of 2.5 nM. This acceleration of repair of the disrupted cutaneous barrier by this drug was also significantly antagonized by BW A868C. FK-506 and dexamethasone showed no beneficial effects on the repair of the disrupted cutaneous barrier. Repeated topical application of 2.5 microM of TS-022 and 12.5 microM of FK-506 once a day for 6 weeks significantly improved the skin inflammation scores in the NC/Nga mice. In regard to the effects of TS-022 in vitro, the inhibitory activity of TS-022 against concanavalin A-induced cytokine production by splenocytes was marginal as compared with that of FK-506 or dexamethasone. These results suggest that the beneficial therapeutic effects of TS-022 in NC/Nga mice with atopic dermatitis are mediated by its suppressive effect on scratching and its effect of accelerating repair of the disrupted cutaneous barrier, both effects being attributable to its prostanoid DP(1) receptor agonistic activity.

Time course changes of scratching counts, dermatitis symptoms, and levels of cutaneous prostaglandins in NC/Nga mice.

NC/Nga (NC) mice are known to develop dermatitis resembling atopic dermatitis (AD) in conventional (Conv) conditions, but not in specific pathogen-free (SPF) conditions. We reported that the ability of skin prostaglandin D(2) (PGD(2)) production, which might be the endogenous inhibitor of itching, was attenuated in skin-lesioned Conv-NC mice. We examined the age-related change in scratching, dermatitis symptoms, and skin PGs of SPF- and Conv-NC mice. In Conv-NC, PGD(2) increased at 7 weeks, at which scratching counts increased, but dermatitis did not develop. PGE(2), PGI(2) and PGF(2alpha) increased at 10 and 13 weeks, at which dermatitis developed. The ability to produce skin PGs was examined by measuring PGs after application of arachidonic acid or after mechanical scratching using a wire brush. In Conv-NC, PGD(2) production at 13 weeks was lower than at 7 weeks. In Conv-NC, hematopoietic PGD synthase (hPGDS) expression in the skin at 13 weeks was lower than at 7 weeks by Western blotting and immunohistochemical analysis. The increase of skin PGD(2) level in the early phase of the development of dermatitis is due to the stress of extensive scratching, but did not increase in spite of the stress of extensive scratching in the late phase, due to decreasing capacity of PGD(2) production attributable to decreasing hPGDS expression in Conv-NC mice. These results suggest that a decreased ability to produce skin PGD(2) production could enhance scratching and aggravate dermatitis in Conv-NC mice.

COX-1 inhibition enhances scratching behaviour in NC/Nga mice with atopic dermatitis.

NC/Nga (NC) mice, spontaneously develop an eczematous atopic dermatitis (AD)-like skin lesion when kept under conventional condition (Conv), but not under specific pathogen-free (SPF) conditions, have been thought to be an animal model of AD. We have previously shown that PGD(2) and arachidonic acid inhibited the scratching behaviour of NC mice, while indomethacin enhanced it. This study was designed to assess the role of cyclooxygenase (COX)-1 and COX-2 in the itch-related scratching behaviour of NC mice. We examined the expression of COX in the skin using real-time PCR and Western blotting and the effects of SC-560 (a COX-1 selective inhibitor) or NS-398 (a COX-2 selective inhibitor) on scratching behaviour in relation to skin prostaglandin (PG) levels in NC mice. COX-1 mRNA expression was unchanged and protein expression decreased in Conv NC mice compared with that of SPF mice. By contrast, COX-2 mRNA and protein expression increased in Conv NC mice. SC-560 increased scratching behaviour and significantly reduced skin PGD(2), PGE(2) and PGF(2alpha) levels, but NS-398 did not have effects on scratching and skin PG level. Moreover, the topical application of PGD(2), which might be the endogenous inhibitor of itching, suppressed the SC-560-induced enhancement of scratching behaviour by NC mice. These results suggest COX-1-coupled skin PGD(2) biosynthesis plays a physiological role in inhibiting regulation of pruritus in NC mice with AD.

Effects of indomethacin and dexamethasone on mechanical scratching-induced cutaneous barrier disruption in mice.

Effects of indomethacin and dexamethasone on recovery of cutaneous barrier disruption induced by mechanical scratching were examined. Cutaneous barrier was disrupted by scratching using a stainless-steel wire brush (mechanical scratching) and compared to cutaneous application of acetone/ether (1:1) mixture (AE) and tape-stripping. Increase of transepidermal water loss (TEWL), as an indicator of a broken skin barrier, and recovery period for mechanical scratching were higher and longer than those for AE treatment and tape-stripping and we also confirmed the severity of skin damage in a histological study. Topical application of moisturizers showed a temporal effect, rapidly decreased TEWL on mechanical scratching- or AE treatment-induced cutaneous barrier disruption, and gradually increased base levels from 4 to 12 h after treatment. Topical application of indomethacin or dexamethasone prolonged the recovery period for the cutaneous barrier, and concomitant use further worsened the status of the barrier. Additionally, we examined the effects of prostaglandins (PGs) and inflammatory cytokine on mechanical scratching-induced cutaneous barrier disruption pretreated with indomethacin and dexamethasone. As a results, PGD2 and interleukin (IL)-1beta significantly accelerated the recovery of cutaneous barrier disruption by mechanical scratching but such was not the case with PGE2, IL-1alpha, and tumor necrosis factor-alpha treatment. These results suggest that indomethacin and dexamethasone prolonged the recovery period caused by inhibition of PGD2 and IL-1beta. Mechanical scratching-induced cutaneous barrier disruption may be a useful method for evaluating means of recovery from skin damage.