MONONUCLEAR CELL FACTORS AFFECTING BASOPHILS/MAST CELLS

本建议的目的是表征可溶性因子 由调节嗜碱性粒细胞功能的单核细胞释放， 肥大细胞 这些细胞在速发型超敏反应中起主要作用 IgE抗体引起的反应（哮喘、鼻炎、过敏反应）。 嗜碱性粒细胞也在许多其他迟发性反应中局部蓄积： 接触性皮炎;排斥肿瘤，微生物，移植;和 IgE依赖性反应的慢性阶段。 据推测 单核细胞释放细胞因子，引导嗜碱性粒细胞迁移 以及嗜碱性粒细胞和肥大细胞的分泌。 我们已经确定， 表征了两种作用于嗜碱性粒细胞的人类细胞因子， 趋化活性和组胺释放活性。 这些因子将通过培养白细胞大量制备 来自正常献血者和人T细胞系的浓缩物。 因素将 通过离子交换、凝胶过滤、亲和和高效纯化 液相色谱法 然后将细胞因子用于产生 特异性抗体和测定它们对嗜碱性粒细胞和肥大细胞的作用 细胞 将通过用以下物质免疫家兔来制备多克隆抗体： 纯化的HRA，以便提供单克隆抗体的测定， 在小鼠中制备。 这些抗体将用于亲和 色谱法，细胞因子的表征，和HRA的测量， 生物液体 将比较未分级单个核细胞产生的细胞因子 用纯化的T细胞、T细胞亚群和 单核-巨噬细胞 要进行的比较是物理化学的 特性、抗原交叉反应性和功能反应 嗜碱性粒细胞 一个有待验证的假设是， 调节嗜碱性粒细胞的迁移和分泌。 我们推测，在 低浓度时，细胞因子诱导细胞运动，高浓度时， 浓度，胞吐作用。 这将通过结合不同的 浓度的细胞因子与固相或液相抗体，然后 确定功能（组胺的趋化性和释放）， 纯化的嗜碱性粒细胞的形态学（电子显微镜）反应。 通过寻找这种细胞因子，探讨HRA的生物学意义 接触性皮炎患者的水泡液中，以及 支气管肺泡灌洗液 自发和 抗原诱导哮喘患者细胞合成HRA的体外研究 将被跟踪，水平与临床病程相关， 对免疫疗法的反应。

The objective of this proposal is to characterize the soluble factors released by mononuclear cells that regulate the function of basophils and mast cells. These cells have a major role in immediate hypersensitivity reactions (asthma, rhinitis, anaphylaxis) caused by IgE antibodies. Basophils also accumulate locally in many other late onset reactions: contact dermatitis; rejection of tumors, microbes, transplants; and the chronic stage of IgE dependent reactions. It is postulated that mononuclear cells release cytokines that direct the migration of basophils and the secretion of basophils and mast cells. We have identified and characterized two human cytokines acting on basophils, a basophil chemotactic activity and a histamine-releasing activity. These factors will be prepared in large quantities by culture of leukocyte concentrates from normal blood donors and human T-cell lines. Factors will be purified by ion exchange, gel filtration, affinity and high performance liquid chromatographies. The cytokines will then be used for production of specific antibodies and for determining their effects on basophils and mast cells. Polyclonal antibodies will be prepared by immunizing rabbits with purified HRA in order to provide an assay for monoclonal antibodies to be prepared in mice. These antibodies will be used for affinity chromatography, characterization of cytokines, and measurements of HRA in biological fluids. The cytokines produced by unfractionated mononuclear cells will be compared with the products of purified T-cells, T-cell subsets and monocyte-macrophages. Comparisons to be made are physicochemical properties, antigenic cross-reactivity and functional response of basophils. One hypothesis to be tested is whether the same molecules can regulate both migration and secretion of basophils. We speculate that at low concentrations, cytokines induce cell movement, and at high concentrations, exocytosis. This will be tested by combining different concentrations of cytokines with solid or fluid phase antibodies, and then determining the function (chemotaxis and release of histamine) and morphologic (electron microscopy) responses of purified basophils. The biological significance of HRA will be studied by seeking this cytokine in blister fluid from patients with contact dermatitis, and in bronchoalveolar lavage fluid from asthmatics. The spontaneous and antigen-induced synthesis of HRA by cells from asthmatics cultured in vitro will be followed and levels correlated with the clinical course and response to immunotherapy.