MAST CELL-SPECIFIC REGULATION OF IL-4 GENE EXPRESSION

白细胞介素4（IL-4）是一种有效的免疫调节细胞因子， 对许多靶细胞产生极其不同的影响。 IL-4发挥着 在造血细胞生长中重要生理作用， 分化、肿瘤监测和局部保护性炎症 应答 然而，IL-4产生失调已被证明具有 极其有害的影响。 病理状况，如特应性 疾病和无法解决某些寄生虫感染， 与IL-4的过度产生明显相关。 IL-4最初是 定义为T细胞细胞因子，但现在很清楚，肥大细胞是 也是重要的IL-4产生细胞。 肥大细胞分布广泛 在许多IL-4靶细胞附近的血管化组织中。 该位置为肥大细胞来源的IL-4提供了机会， 容易地访问这样的细胞，并有效地调节广泛的局部 炎症反应。 由于其独特的组织分布， 肥大细胞和因此潜在的不同功能的肥大细胞衍生的 IL-4对局部炎症反应的影响，推测其作用机制可能是 调节肥大细胞中IL-4基因表达的蛋白质 那些在T细胞中。 最近的研究证实了这一预测。 证明了 IL-4基因存在独特的顺式作用序列， 在T细胞和肥大细胞中的转录。 组织特异性的主要成分 肥大细胞中的转录活性似乎包含在一个 IL-4基因的第二内含子中的序列。 这个序列显示 肥大细胞系中的原型增强子活性， 依赖于积极和消极调节的相互作用 元素 该提案的具体目标是：1.以识别 包含IL-4的必需的正性和负性作用序列 内含子增强子 2.为了鉴定DNA结合蛋白， 特异性控制IL-4内含子增强子的功能， 确定它们在肥大细胞和细胞中的表达和结合动力学 其他血统。 3.检查新鲜分离的肥大细胞群， 细胞在刺激后表达IL-4的能力，并分析其表达的IL-4。 肥大细胞特异性转录因子在这些细胞中的表达。 4.确定IL-4内含子增强子如何与其他IL-4相互作用 调节序列以调节肥大细胞中的IL-4表达。 DNA- 蛋白结合测定和应答元件-报告基因构建体 与瞬时转染测定的配合将用于完成 这些目标。 了解组织特异性的分子基础 肥大细胞的调节是最终 设计治疗策略以选择性地限制或增强 过敏性疾病中肥大细胞或T细胞衍生的IL-4活性，或 免疫治疗肿瘤，如黑色素瘤。

Interleukin 4 (IL-4) is a potent immunoregulatory cytokine with extremely diverse effects on a number of target cells. IL-4 plays an important physiologic role in hematopoietic cell growth and differentiation, tumor surveillance and local protective inflammatory responses. However, dysregulated IL-4 production has been shown to have extremely detrimental effects. Pathological conditions such as atopic disease and inability to resolve certain parasitic infections are clearly associated with overproduction of IL-4. IL-4 was originally defined as a T cell cytokine, but it is now clear that mast cells are also important IL-4 producing cells. Mast cells are widely distributed in vascularized tissues in the proximity of many IL-4 target cells. This location provides the opportunity for mast cell-derived IL-4 to easily access such cells and effectively modulate a wide range of local inflammatory responses. Because of the unique tissue distribution of mast cells and thus potentially distinct functions of mast cell-derived IL-4 on local inflammatory responses, it was predicted that mechanisms which regulate IL-4 gene expression in mast cells would differ from those in T cells. Recent studies have confirmed this prediction. It was demonstrated that there are unique cis acting sequences which regulate IL-4 gene transcription in T and mast cells. A major component of tissue-specific transcriptional activity in mast cells appears to be contained in a sequence in the second intron of the IL-4 gene. This sequence exhibits prototypic enhancer activity in mast cell lines and its activity is dependent on the interaction of both positive and negative regulatory elements. The specific aims of this proposal are: 1. To identify essential positive and negative acting-sequences that comprise the IL-4 intronic enhancer. 2. To identify DNA binding proteins which specifically govern the function of the IL-4 intronic enhancer and determine their expression and binding dynamics in mast cells and cells of other lineages. 3. To examine populations of freshly isolated mast cells for their ability to express IL-4 upon stimulation and analyze the expression of mast cell specific transcription factors in these cells. 4. To determine how the IL-4 intronic enhancer interacts with other IL-4 regulatory sequences to modulate IL-4 expression in mast cells. DNA- protein binding assays and response element-reporter gene constructs in concert with transient transfection assays will be used to accomplish these aims. Understanding the molecular basis for tissue specific regulation in mast cells is an important first step in ultimately devising therapeutic strategies for selectively limiting or augmenting mast cell or T cell-derived IL-4 activity in allergic disease or immunotherapy of tumors such as melanoma.