DIFFERENTIAL CYTOKINE SECRETION BY BASOPHILS

描述(改编自调查者摘要)：本申请 检测人类嗜碱性粒细胞合成IL-4和IL-13的能力 利用各种激动剂和对比条件调节 (增加或抑制)每一种的生产。最初的目标，还有一个 最详细的检查，通过检查对每种细胞因子的影响 刺激c-AMP、类固醇、环孢素、FK506和酪氨酸的激动剂 这是一种激酶抑制剂。将检测IL-13蛋白和信使核糖核酸的产生， 前者利用超纯嗜碱性细胞制剂和IL-3等刺激物， 离子霉素和PMA。将使用流式细胞术来确定细胞内 细胞因子的表达。通过PKC作用的引物，如IL-1b和TNFa将 以及非IgE依赖的激动剂，如C5a或FMLP，以及 IL-3样生长因子，如GMCSF和IL-5。CD40L的微扰或 将测试CD32(免疫球蛋白G受体)对IL-4/IL-13分泌的影响。这个 特应性和非特应性受试者的细胞将被比较和关联 有特应性症状的每种细胞因子的产生水平。这个 我们将比较信使核糖核酸生产和蛋白质积累的动力学， 由于初步数据表明，IL-4的合成先于 但IL-13在mRNA水平上的上调或下调速率尚不清楚。 将寻找分泌每种细胞因子的嗜碱性粒细胞亚群 单细胞同时或连续产生IL-4和/或IL-13 演示了。他们将利用三色流式细胞术 荧光，和莫能菌素抑制蛋白质分泌。实验在 在刺激前添加哪种IL-4或IL-13将测试每一种 一方可以监管另一方。通过以下途径抑制B细胞凋亡的药物 将测试阻止NFkB激活对IL-4或IL-13的影响 生产以及精选的酪氨酸激酶抑制剂。目标2尝试 蛋白激酶C同工酶在IL-4和IL-13调节中的鉴别 到目前为止，研究的佛波酯抑制IL-4的合成，但似乎可以 刺激IL-13的产生。延长对PKC耗竭的影响 PMA对IL-3和IgE刺激的IL-13分泌的刺激作用将被检测。 将使用钙依赖型与非钙依赖型PKC抑制剂 寻求差异化效果。RT-PCR将用于定性和定量检测 半定量检测IL-4、IL-13的mRNA水平。最后，一个 将测试重组人HRF刺激IL-13的能力 IgE(+)细胞供者与IgE(-)细胞供者的分泌。直接刺激和 还将检查用HRF引爆其他分泌物的情况。各种各样的 细胞因子引物将以HRF为激动剂，IL-4和IL-13为测试对象 分泌物比较。引种预孵化的时间会有所不同 任何寻求的分泌物抑制物。

DESCRIPTION (Adapted from Investigator's abstract): This application examines the ability of human basophils to synthesize IL-4 and IL-13 utilizing a variety of agonists and contrasts conditions that modulate (augment or inhibit) production of each. The initial aim, and the one examined in greatest detail, examines the effects upon each cytokine by agonists that stimulate c-AMP, steroids, cyclosporin, FK506, and tyrosine kinase inhibitors. IL-13 protein and mRNA production will be examined, the former utilizing ultrapure basophil preparations and stimuli such as IL-3, ionomycin, and PMA. Flow cytometry will be used to determine intracellular cytokine expression. Primers acting through PKC such as IL-1b and TNFa will be tested as well as non-IgE dependent agonists such as C5a or FMLP, and IL-3-like growth factors such as GMCSF and IL-5. Perturbation of CD40L or CD32 (IgG receptor) will be tested for effects on IL-4/IL-13 secretion. The cells of atopic versus non-atopic subjects will be compared and correlation of levels of production of each cytokine with atopic symptoms sought. The kinetics of mRNA production versus protein accumulation will be compared, since preliminary data indicate that the synthesis of IL-4 precedes that of IL-13 but rates of up or down regulation at the mRNA level are unknown. Subpopulations of basophils secreting each cytokine will be sought or simultaneous or sequential production of IL-4 and/or IL-13 by single cells demonstrated. They will utilize flow cytometry with three color fluorescence, and monensin to inhibit protein secretion. Experiments in which IL-4 or IL-13 are added prior to stimulation will test whether each one can regulate the other. Drugs that inhibit apoptosis in B-cells by preventing NFkB activation will be tested for effects on IL-4 or IL-13 production as well as selected tyrosine kinase inhibitors. Aim 2 attempts to differentiate PKC isozymes in the regulation of IL-4 and IL-13 since phorbol esters examined thus far inhibit IL-4 synthesis, but seem to stimulate production of IL-13. The effect of depletion of PKC by prolonged PMA stimulation on IL-3 and IgE-stimulated IL-13 secretion will be examined. Calcium dependent versus calcium independent PKC inhibitors will be employed seeking differential effects. RT-PCR will be used for qualitative and semi-quantitative assessment of IL-4 and IL-13 mRNA levels. Finally, a recombinant human HRF will be tested for its ability to stimulate IL-13 secretion in IgE(+) versus IgE(-) cell donors. Direct stimulation and priming of other secretogogues by HRF will be examined. A variety of cytokine primers will be tested with HRF as agonist and IL-4 versus IL-13 secretion compared. The time of priming preincubation will be varied and any inhibitors of secretion sought.