Akiko Takaoka

Involvement of IL‐31 on scratching behavior in NC/Nga mice with atopic‐like dermatitis

Abstract:  Pruritus is an important symptom in atopic dermatitis (AD), but the major pruritogen have not been identified. NC/Nga mice, spontaneously develop an eczematous AD‐like skin lesion when kept under conventional conditions, but not under specific pathogen‐free (SPF) conditions, have been thought to be an animal model for AD. In this study, to determine whether newly identified cytokine, IL‐31, may be involved in pruritus of AD, we examined the IL‐31 expression in spontaneous dermatitis model which showed itch‐associated long‐lasting (over 1.5 s duration) scratching behavior and compared with that of hapten‐induced contact dermatitis model without itch‐associated long‐lasting scratching behavior, using NC/Nga mice. In NC/Nga mice cohabited with NC/Nga mice which developed severe dermatitis for 2 weeks (conventional NC/Nga mice), the numbers of long‐lasting scratching counts were significantly increased. Yet in 2,4,6‐trinitrochlorobenzene (TNCB)‐sensitized and challenged mice (TNCB‐applied NC/Nga mice), no significant increase in long‐lasting scratching counts was observed. In conventional NC/Nga mice with long‐lasting scratching behavior, expression of IL‐31 mRNA was increased, while in TNCB‐applied NC/Nga mice without long‐lasting scratching behavior, the expression of IL‐31 mRNA were unchanged. There was a good correlation between the scratching counts and expression of IL‐31 mRNA in conventional NC/Nga mice, but not so in TNCB‐applied NC/Nga mice. These results suggest that IL‐31 causes the itch‐associated scratching behavior in conventional NC/Nga mice, an experimental animal model for AD.

Critical role for OX40 ligand in the development of pathogenic Th2 cells in a murine model of asthma

Bronchial asthma is characterized by massive infiltration of eosinophils and airway hyperreactivity (AHR), which are caused by overproduction of Th2 cytokines (IL‐4, IL‐5, and IL‐13) by allergen‐specific T cells. We recently demonstrated a critical contribution of OX40 ligand (OX40L) to the development of Th2‐mediated experimental leishmaniasis. In this study, we have examined the role of OX40L in the development of Th2‐mediated pulmonary inflammation by utilizing OX40L‐deficient mice and a neutralizing anti‐OX40L mAb in a murine model of asthma. Sensitization and airway challenge with ovalbumin in wild‐type BALB/c mice induced a typical allergic asthma characterized by AHR, accumulation of eosinophils, increased mucus production, and high levels of Th2 cytokines in the lung.All these asthmatic responses were not induced in OX40L‐deficient BALB/c mice. Administration of neutralizing anti‐OX40L mAb in wild‐type BALB/c mice during the sensitization period also abolished the induction of asthmatic responses. In contrast, administration of anti‐OX40L mAb during the challenge period did not inhibit the asthmatic responses. These results indicate a critical role for OX40L in the induction phase, which leads to the development of pathogenic Th2 cells, but not in the effector phase, which includes migration and activation of pathogenic Th2 cells in the lung.

A Critical Role for Mouse CXC Chemokine(s) in Pulmonary Neutrophilia During Th Type 1-Dependent Airway Inflammation

Ag-specific Th1 and Th2 cells have been demonstrated to play a critical role in the induction of allergic diseases. Here we have investigated the precise mechanisms of Th1-induced airway inflammation. Airway inflammation was induced in BALB/c mice by transfer of freshly induced OVA-specific Th1 or Th2 cells followed by OVA inhalation. In this model, both Th1 and Th2 cells induced airway inflammation. The former induced neutrophilia in airways, whereas the latter induced eosinophilia. Moreover, we found that Th1 cells induced more severe airway hyperresponsiveness (AHR) than Th2 cells. The eosinophilia induced by Th2 cell infusion was almost completely blocked by administration of anti-IL-5 mAb, but not anti-IL-4 mAb. In contrast, Th1-induced AHR and pulmonary neutrophilia were inhibited by the administration of anti-human IL-8R Ab, which blocks the function of mouse CXC chemokine(s). These findings reveal a critical role of mouse CXC chemokine(s) in Th1-dependent pulmonary neutrophilia and AHR.

Role of scratch-induced cutaneous prostaglandin D2 production on atopic-like scratching behaviour in mice

Abstract:  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.

Pyrrole derivatives as potent inhibitors of lymphocyte-specific kinase: Structure, synthesis, and SAR

We have described the synthesis, enzyme inhibitory activity, structure-activity relationships, and proposed binding mode of a novel series of pyrrole derivatives as lymphocyte-specific kinase (Lck) inhibitors. The most potent analogs exhibited good enzyme inhibitory activity (IC(50)s <10nM) for Lck kinase inhibition.

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

Abstract:  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/Wv 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, IgG1 and IgG2a levels increased. The scratch count increased significantly in both the WBB6F1‐W/Wv 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.

Ring-fused pyrazole derivatives as potent inhibitors of lymphocyte-specific kinase (Lck): Structure, synthesis, and SAR.

We have identified a novel series of ring-fused pyrazole derivatives as lymphocyte-specific kinase (Lck) inhibitors. The most potent analogs exhibited good enzyme inhibitory activity (IC(50)s <1nM) as well as excellent cellular activity against mixed lymphocyte reaction (MLR) (IC(50)s <1nM).

Effects of the novel and potent lymphocyte-specific protein tyrosine kinase inhibitor TKM0150 on mixed lymphocyte reaction and contact hypersensitivity in mice.

Lymphocyte-specific protein tyrosine kinase (Lck) plays a critical role in T cell activation. In the present study, the effect of a newly synthesized small molecule compound, 7-[2-(dimethylamino)ethoxy]-2-(4-phenoxyphenyl)-9,10-dihydro-4H- pyrazolo[5,1-b] [1,3]benzodiazepine-3-carboxamide (TKM0150) on Lck activity was investigated. TKM0150 inhibited Lck with an 1C50 value of 0.7 nM. To evaluate if TKM0150 is a specific inhibitor of Lck, the activity against several Src (Proto-oncogene tyrosine-protein kinase Src) and non-Src family kinases were assayed. TKM150 inhibited Src family kinases, Src and Csk (c-Src kinase) (with IC50 values of 0.6 nM and 1.7 nM, respectively) as well as Fyn (p59-Fyn) and Lyn (tyrosine-protein kinase Lyn) at a dose of 1 microM; however, it did not inhibit kinase which is a non-Src family kinase in the tyrosine kinase (TK) group, nor kinases in other groups. Then, the anti-inflammtory potential of TKM0150 was evaluated by known experimental models. TKM0150 inhibited the murine mixed lymphocyte reaction (MLR) in vitro with an IC50 value of 0.7 nM, and 2,4,6-trinitro-1-chlorobenzene-induced contact hypersensitivity in vivo at a dose of 0.3 and 1% w/v administered topically. These results indicate that TKM0150 is a specific inhibitor of Lck/Src kinase and can block T cell-mediated responses in vitro and in vivo. Accordingly, TKM0150 would be expected as a drug candidate for treating T cell-mediated disorders including atopic dermatitis.