MECHANISMS OF IGE REGULATION OF HUMAN LEUKOCYTES

IGE对人类白细胞的调节机制

• 批准号: 6349752
• 负责人: SARBJIT Singh SAINI
• 金额: $ 12.57万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6349752
• 关键词: antibody receptor; atopy; basophils; cell line; cytokine; enzyme linked immunosorbent assay; flow cytometry; genetic translation; human subject; immunogenetics; immunoglobulin E; immunoregulation; interleukin 4; monocyte; phenotype; polymerase chain reaction; protein structure; receptor expression; transfection; western blottings

Allergen crosslinking of IgE on cells bearing high affinity IgE receptors (FcepsilonRI, alphabetagamma2), such as mast cells and basophils, triggers the release of mediators that initiate allergic inflammation. Recent studies have defined a role for IgE in the regulation of FcepsilonRI expression and function on basophils. Approaches that remove IgE, or prevent its attachment to the alpha subunit of FcepsilonRI, dramatically reduce levels of basophil FcepsilonRIalpha and result in reductions of antigen-specific histamine release. Other studies show that re-exposure to IgE in vitro or in vivo reverses these effects. In contrast, preliminary studies fail to show similar IgE-dependent FcepsilonRIalpha modulation on monocytes, which express a different form of FcepsilonRI lacking the beta subunit (alphagamma2). Further, preliminary data suggest that expression of monocyte FcepsilonRI only occurs in allergic donors, but unlike basophils, appears to be independent of serum IgE levels. The goal of this proposal is to enhance our understanding of the mechanisms of IgE- dependent and IgE-independent regulation of FcepsilonRI on human basophils and monocytes, with an emphasis on the role played by the beta chain of FcepsilonRI We hypothesize that IgE-dependent enhancement of the basophil beta subunit occurs with the alpha subunit. We further hypothesize that basophils, after IgE-induced receptor enhancement, will secrete increased amounts of IL-4. We also hypothesize that cytokines present in allergic disease are needed for monocyte FcepsilonRIalpha expression and that the lack of the beta subunit in monocytes reduces IgE-dependent receptor modulation. To test these hypotheses, specific aims will 1) determine whether IgE-dependent alteration of FcepsilonRIalpha expression on basophils alters transcription and translation of the beta subunit of FcepsilonRI; 2) test whether observed FcepsilonRIalpha receptor modulation alters IL-4 release from basophils; and 3) explore mechanisms regulating FcepsilonRI expression on monocytes. Experiments will use RT-PCR and Western blotting to monitor changes in beta subunit mRNA and protein expression in lysates from basophils cultured with or without IgE. Similar basophil cultures will be examined for changes in the magnitude or releasability of IL-4 protein secretion under various stimulation conditions. Finally, regulation of monocyte FcepsilonRI expression will be examined in cultures supplemented with or without IgE and selected cytokines. Using transfection, the introduction of the beta subunit into human monocytes or monocytic cell lines bearing alphagamma2 will be tested for its' effects on IgE or cytokine mediated expression of FcepsilonRI. The results of the planned studies will further define mechanisms of FcepsilonRI regulation on basophils and monocytes, and may lead to novel FcepsilonRI-based strategies for the treatment of allergic diseases.

IgE在携带高亲和力IgE的细胞上的变应原交联 受体（Fc ε RI，α γ 2），如肥大细胞，和 嗜碱性粒细胞，触发释放介质，启动过敏 炎症 最近的研究已经确定了IgE的作用， FcepsilonRI在嗜碱性粒细胞上的表达和功能的调节。 去除IgE或防止其附着在α上的方法 Fc ε RI亚基，显著降低嗜碱性粒细胞水平 FcepsilonRI α并导致抗原特异性组胺减少 release. 其他研究表明，在体外或体内重新暴露于IgE， 逆转这些影响。 相比之下，初步研究未能表明， 对单核细胞类似IgE依赖性FcepsilonRI α调节， 表达缺乏β亚基的不同形式的FcepsilonRI （Escherichiagamma2）. 此外，初步数据表明， 单核细胞FcepsilonRI仅发生在过敏性供体中，但与 嗜碱性粒细胞，似乎是独立的血清IgE水平。的目标 这个建议是为了提高我们对IgE机制的理解- FcepsilonRI对人免疫球蛋白依赖性和非IgE依赖性调节 嗜碱性粒细胞和单核细胞，重点是β 我们假设IgE依赖性增强FcepsilonRI链， 嗜碱性粒细胞β亚基与α亚基一起出现。 我们进一步 假设嗜碱性粒细胞在IgE诱导的受体增强后， 分泌更多的IL-4。 我们还假设细胞因子 单核细胞Fc受体1 α 表达，并且单核细胞中β亚基的缺乏降低了 IgE依赖性受体调节。 为了验证这些假设， 目的将1）确定是否IgE依赖性改变， FcepsilonRI α在嗜碱性粒细胞上的表达改变了转录， Fc ε RI的β亚基的翻译; 2）测试是否观察到 Fc ε RI α受体调节改变嗜碱性粒细胞IL-4的释放 3）探讨FcepsilonRI表达的调控机制。 单核细胞实验将使用RT-PCR和Western印迹来监测 β亚基mRNA和蛋白质表达的变化， 有或没有IgE培养的嗜碱性粒细胞。类似的嗜碱性细胞培养 检查IL-4的大小或释放能力的变化 在各种刺激条件下的蛋白质分泌。 最后， 单核细胞Fc受体RI表达的调节将在 补充有或没有IgE和选择的细胞因子的培养物。 使用 转染，将β亚基引入人单核细胞 或携带γ-内酰胺酶2的单核细胞系将被测试其" 对IgE或细胞因子介导的Fc ε RI表达的影响。的 计划中的研究结果将进一步确定 FcepsilonRI调节嗜碱性粒细胞和单核细胞，并可能导致新的 基于FcepsilonRI的过敏性疾病治疗策略。