Mucosal Immunity in Integrin Alpha-E Deficient Mice

整合素αEbeta7在T细胞、肥大细胞和树突状细胞(DC)上表达，并与E-钙粘素结合，介导T细胞/上皮细胞的特异性黏附。此外，与AlphaE+/+小鼠相比，AlphaE-/-小鼠的肠道上皮内T细胞数量减少，这表明这种相互作用在体内的重要性。此外，肠源性内皮细胞上还有另一种α-Ebeta7配体，其分布尚不清楚。为了评价AlphaEbeta7在体内的作用，将CD4+CD45RBhi T细胞转移到SCID/SCID小鼠体内，产生偏向Th1的肠炎。使用抗α-Ebeta7单抗可以消除这种综合征。相比之下，当AlphaE+/+受体小鼠与AlphaE-/-与AlphaE+/+供者T细胞重组时，限制了AlphaE缺乏对T细胞的影响，疾病严重程度仅略有改善。最后，在IL-10-/-小鼠出现的偏向Th1的肠道炎症中，与αE-/-小鼠相比，AlphaE-/-小鼠肠道炎症的临床症状加速，在这种情况下，所有细胞都缺乏AlphaE。总体而言，这些研究表明，除了T细胞外，还有另一种细胞类型可以调节肠道炎症，当这些非T细胞缺乏αEbeta7时，基于Th1细胞因子的肠道炎症会恶化。重要的是，在体外研究中，与来自AlphaE+/+小鼠的DC相比，在AlphaE-/-小鼠来源的DC刺激下，幼稚的CD4+T细胞产生更多的Th1和更少的Th2细胞因子。这一发现表明，当DC缺乏αE时，偏向Th1的肠道炎症更严重，这可能是通过改变DC的定位、功能或与T细胞的相互作用而实现的。在AIM 1中，将通过流式细胞术、ELISA法和免疫组织化学方法检测αEbeta7+DC的表型、细胞因子的表达和分布。在AIM 2中，当αEbeta7缺乏仅限于受体小鼠的非T细胞时，将通过临床和显微镜评估来确定αEbeta7缺乏对肠道炎症的CD4+CD45RBhi T细胞转移模型过程的影响。在AIM 3中，将使用ELISA法和流式细胞术，在直接刺激、有或没有αEbeta7交联的情况下，或在T细胞/DC共培养中刺激后，评估αEbeta7在调节DC细胞因子表达或表面表型中的作用。在AIM 4中，将通过FACS或Northern印迹来评估E-钙粘素或其他α-Ebeta7配体在初始或最近激活的T细胞上的可能表达。此外，将使用黏附和共刺激试验来确定DC/T细胞相互作用中的αEbeta7的滚动。这些实验将深入了解整合素αEbeta7在DC定位和与Th淋巴细胞分化相关的功能中的影响，这与细胞因子失调导致炎症的肠道具有特别的相关性。

The integrin alphaEbeta7 is expressed on T cells, mast cells and dendritic cells (DCs) and binds to the E-cadherin to mediate T cell/epithelial cell specific adhesion. Furthermore, alphaE-/- mice have reduced numbers of intestinal intraepithelial T cells versus alphaE+/+ mice, indicating the importance of this interaction in vivo. In addition, there is another alphaEbeta7 ligand on intestine derived endothelial cells, whose distribution is not known. To evaluate the role of alphaEbeta7 in vivo, CD4+CD45RBhi T cells were transferred into scid/scid mice to generate Th1 biased intestinal inflammation. This syndrome was abrogated by administration of an anti-alphaEbeta7 monoclonal antibody. In contrast, when the alphaE+/+ recipient mice were reconstituted with alphaE-/- versus alphaE+/+ donor T cells, limiting the impact of alphaE deficiency to the T cell, disease severity was only modestly ameliorated. Finally, in the Th1 biased intestinal inflammation that develops in IL-10-/- mice, clinical signs of intestinal inflammation were accelerated in alphaE-/- versus alphaE-/- mice, a condition in which all cells are alphaE deficient. Overall, these studies suggest that there is another cell type, in addition to T cells, that modulates intestinal inflammation, and that when these non-T cells are alphaEbeta7 deficient, Th1 cytokine based intestinal inflammation is worsened. Importantly, in in vitro studies, naive CD4+ T cells produced more Th1 and less Th2 cytokines when stimulated in the presence of DCs derived from alphaE-/- versus alphaE+/+ mice. This finding suggests that when DCs are alphaE deficient, Th1 biased intestinal inflammation is worse, possibly through altered DC localization, function or interactions with T cells. In AIM 1, the phenotype, cytokine expression and distribution of alphaEbeta7+ DCs will be determined by flow cytometry, ELISA and immunohistochemistry. In AIM 2, the impact of alphaEbeta7 deficiency upon the course of the CD4+CD45RBhi T cell transfer model of intestinal inflammation will be determined when alphaEbeta7 deficiency is restricted to non-T cells in recipient mice, by clinical and microscopic evaluation. In AIM 3, the role of alphaEbeta7 in regulating DC cytokine expression or surface phenotype will be evaluated following direct stimulation, with or without alphaEbeta7 crosslinking, or after stimulation in T cell/DC co-cultures, using ELISA and flow cytometry. In AIM 4, the possible expression of E-cadherin or of other alphaEbeta7 ligands on naive or recently activated T cells will be assessed, by FACS or Northern blot. In addition, the roll of alphaEbeta7 in DC/T cell interactions will be determined using adhesion and co-stimulation assays. These experiments will provide insight into the impact of integrin alphaEbeta7 in DC localization and function relevant to Th lymphocyte differentiation, which have particular relevance to the intestine where cytokine dysregulation leads to inflammation.