Elucidation of Pathoptysiological Roles of Immune Chemokines

该项目的目的是阐明免疫趋化因子的病理生理作用，这些免疫趋化因子主要针对淋巴细胞和树突状细胞。今年的主要成绩是：(1)我们发现促炎细胞因子IL-1和肿瘤坏死因子α能有效地诱导正常人表皮角质形成细胞表达LARC/CCL20，这种趋化因子可以选择性地吸引未成熟树突状细胞和α4β7阳性的记忆/效应T细胞VUACCR6。我们还发现，特应性皮炎(AD)的皮损皮肤通常含有产生LARC的表皮角质形成细胞和表皮下广泛聚集的CCR6细胞。此外，我们还发现AD患者的血浆样本中LARC水平经常升高。综上所述，这些结果支持LARC在AD中的重要病理作用。(2)IL-1、肿瘤坏死因子α等促炎细胞因子可有效诱导粘膜上皮细胞LARC的表达。毛发…此外，我们还分析了这些细胞因子对LARC启动子的激活机制，发现转录起始点-96~-87bp之间的一个核因子-kB位点对于LARC启动子的诱导是必不可少的。(3)我们发现，在AD患者血浆中，TARC/CCL17和MDC/CCL22均可通过CCR4吸引Th2型记忆/效应T细胞和寻皮型CLA记忆/效应T细胞。此外，我们还发现AD患者的血小板中含有TARC，并且其含量显著升高。我们进一步证明了干扰素-γ诱导正常人表皮角质形成细胞表达TARC和MDC。综上所述，TARC和MDC可能在AD的发病机制中发挥重要作用。此外，干扰素-γ也被认为是Th1型趋化因子的启动子，通过诱导一系列同时吸引Th1型和Th2型细胞的趋化因子，可能是AD发病的关键因素。(4)在白色念珠菌感染的慢性炎症皮肤和淋巴结中，大量成熟树突状细胞产生MDC，并与CCR4表达的T细胞聚集。因此，MDC可能在DC-T细胞相互作用中发挥重要作用。(5)在卵蛋白诱导的哮喘小鼠模型中，抗mTARC单抗可有效地阻断淋巴细胞和嗜酸性粒细胞的肺内渗入，并使乙酰甲胆碱诱导的气道阻力增加。因此，TARC很可能在哮喘中发挥重要作用。(6)多发性硬化(MS)患者急性期脑脊液中表达CCR5或CXCR3的淋巴细胞频率升高，缓解期表达CCR5的淋巴细胞频率下降。这些结果支持MS发病机制中以Th1为主的免疫反应。(7)我们在亚美尼亚仓鼠体内产生了抗MDC、LARC和SLC/CCL21等小鼠趋化因子的单抗，但未能获得具有中和活性的抗体。(8)将TARC基因敲除小鼠和αE整合素基因敲除小鼠多次回交到BALB/c和C57BL/6小鼠中，并培育出同源品系。较少

The aim of the project has been to elucidate the pathophysiological roles of the immune chemokines, which are mainly directed to lymphocytes and dendritic cells. The main accomplishments of this year are as follows. (1) We showed that the proinflammatory cytokines such as IL-1 and TNFα potently induced normal human epidermal keratinocytes to express LARC/CCL20, the chemokine known to selectively attracts immature dendritic cells and α4β7-positive memory/effector T cells vua CCR6. We also showed that the lesional skin of atopic dermatitis (AD) often contained epidermal keratinocytes producing LARC and extensive accumulation of CCR6+ cells beneath the epidermal layer. Furthermore, we showed that plasma samples from AD patients often contained elevated levels of LARC. Collectively, these results support an important pathologenic role of LARC in AD. (2) We showed that expression of LARC was potently induced in mucosal epithelial cells by proinflammatory cytokines such as IL-1 and TNFα. Fur … More thermore, we analysed the activation mechanism of the LARC promoter by these cytokines and showed that an NF-kB site from -96 to -87bp from the transcriptional initiation site was essential for the induction of the LARC promoter. (3) We showed that TARC/CCL17 and MDC/CCL22, both known to attract Th2-type memory/effector T cells and skin-seeking CLA+ memory/effector T cells via CCR4, were significantly evevated in the plasma of AD patients. Furthermore, we found that platelets contained TARC and its contents were dramatically elevated in platelets from AD patients. We further showed that IFN-γinduced normal human epidermal keratinocytes to express TARC and MDC. Collectively, TARC and MDC may play important roles in the pathogenesis of AD. Furthermore, IFN-γ, which is also known to be apotent inducer of Th1-attracting chemokines, may be a critical factor in AD pathogenesis by inducing a set of chemokines attracting both Th1 and Th2 cells. (4) We showed that a large fraction of mature dendritic cells produced MDC and were clustered with CCR4-expressing T cesss in chronically inflamed skin infected with Candida albicans as well as in lymph node. Thus, MDC may play an important role in DC-T cell interactions. (5) We showed that in an asthmatic model in mice induced by priming with and subsequent inhalating of ovalbumin, the treatment with anti-mTARC monoclonal antibody efficiently blocked lung infiltrating of lymphocytes and eosinophils and elevation of methacholine-induced airway resistance. Thus, TARC is likely to play an important role in asthma. (6) We showed that in patients with multiple sclerosis (MS), the frequencies of lymphocytes expressing CCR5 or CXCR3 was increased in the cerebrospinal fluid during exacerbation, while the frequencies of CCR5-expressinglymphocytes were decreased after remission. These results support the Th1-biased immune responses in the pathogenesis of MS. (7) We generated monoclonal antibodies to mouse chemokines such as MDC, LARC and SLC/CCL21 in Armenisan hamsters but could not get antibodies with neutralizing activities. (8) We have repeatedly backcrossed TARC-knockout mice and αE integrin-knockout mice into BALB/c and C57BL/6 mice and developed the syngeneic strains. Less