Development of animal models for cutaneous eosinophilic inflammation and analysis of its regulatory mechanisms

近年来的研究表明，肥大细胞衍生的tnf - α在半抗原诱导的接触性超敏反应中起重要作用。最近，我们发现Fc epsilon RI β链通过其ITAM的三个酪氨酸残基（Y219/Y229/Y225）的功能，控制肥大细胞产生包括tnf - α在内的促炎细胞因子。在本研究中，我们利用肥大细胞敲入小鼠研究了表达野生型或突变β链ITAM的肥大细胞在接触性超敏反应发生中的生物学功能。我们采用逆转录病毒介导的基因转移方法，将携带野生型（YYY）或Fc epsilon RI β链ITAM突变体（FFF）的肥大细胞转移到来自Fc epsilon RI β链-/-小鼠的bmmc中。将这些肥大细胞皮内移植到肥大细胞缺陷小鼠耳中（W/Wv）。将2%恶唑酮应用于剃光的腹部，使小鼠预致敏。第5天，1%恶唑酮和对照剂分别应用于右耳和左耳两侧。耳部厚度评价接触性过敏。Real-Time PCR检测组织中细胞因子的表达。结果表明，恶唑酮未引起W/Wv和Fc epsilon RI β -链-/-小鼠耳肿胀。此外，我们发现与YYY敲入小鼠相比，fff敲入小鼠的耳部肿胀明显减少。与此结果一致，在fff敲入小鼠中，CD3^<+> T细胞和嗜酸性粒细胞在炎症部位的浸润严重减少。此外，实时PCR实验表明，fff敲入小鼠耳组织中tnf - α mRNA的表达也降低。综上所述，这些结果表明，通过Fc epsilon RI β链调节肥大细胞的激活对于半抗原皮肤暴露后接触性超敏反应的发展至关重要。

Recent studies suggest that mast cell derived-TNF-alpha plays important roles in the development of contact hypersensitivity induced by hapten. Recently, we revealed that Fc epsilon RI beta-chain controls production of proinflammatory cytokines including TNF-alpha from mast cells through function of three tyrosine residues (Y219/Y229/Y225) of its ITAM. In the present study, we investigated the biological functions of mast cells expressing wild-type or mutated β-chain ITAM in development of the contact hypersensitivity employing mast cell "knock-in" mice.We prepared mast cells harboring wild-type (YYY) or Fc epsilon RI beta-chain ITAM mutant (FFF) by employing a retrovirus-mediated gene transfer into BMMCs derived from Fc epsilon RI beta-chain -/- mice. Those mast cells were intradermally transferred into ears of mast cell deficient mice (W/Wv). The mice were pre-sensitized by application of 2% oxazolone to the shaved abdomen. On day 5, 1% oxazolone and vehicle were applied to both sides of right ear and left ear, respectively. Contact hypersensitivity was evaluated by ear thickness.Cytokine expression in the tissue was analyzed by Real-Time PCR.As a result, oxazolone failed to cause ear swelling in W/Wv and Fc epsilon RI beta-chain -/- mice. In addition, we showed that ear swelling is significantly reduced in the FFF-knock-in mice as compared with that of YYY- knock-in mice. Consistent with this result, infiltration of CD3^<+> T cells and eosinophils into the inflammation sites was severely reduced in FFF-knock-in mice. Furthermore, an experiment using real-time PCR demonstrated that expression of TNF-alpha mRNA is also decreased in the ear tissue of FFF-knock-in mice. Taken together, these results suggest that regulation of mast cell activation through Fc epsilon RI beta-chain is critical for development of contact hypersensitivity following cutaneous exposure of hapten.