DIFFERENTIAL CYTOKINE SECRETION BY BASOPHILS

描述（改编自研究者摘要）：本申请 检测人嗜碱性粒细胞合成IL-4和IL-13的能力 利用多种激动剂和对比条件， （增加或抑制）每一种的生产。 最初的目标， 最详细的检查，检查了每种细胞因子的影响， 刺激c-AMP、类固醇、环孢菌素、FK 506和酪氨酸的激动剂 激酶抑制剂。 将检查IL-13蛋白和mRNA的产生， 前者利用超纯嗜碱性粒细胞制剂和刺激物如IL-3， 离子霉素和PMA。 流式细胞术将用于确定细胞内 细胞因子表达 通过PKC如IL-1b和TNF α起作用的引物将 以及非IgE依赖性激动剂如C5 a或FMLP，和 IL-3样生长因子，如GMCSF和IL-5。 CD 40 L的扰动或 将检测CD 32（IgG受体）对IL-4/IL-13分泌的影响。 的 将比较特应性与非特应性受试者的细胞 每种细胞因子的生产水平与特应性症状寻求。 的 比较mRNA产生相对于蛋白质积累的动力学， 由于初步数据表明IL-4的合成先于 IL-13，但在mRNA水平的上调或下调率是未知的。 将寻找分泌每种细胞因子的嗜碱性粒细胞亚群，或 通过单细胞同时或顺序产生IL-4和/或IL-13 演示。 他们将利用流式细胞术， 荧光和莫能菌素以抑制蛋白质分泌。 实验 在刺激之前加入IL-4或IL-13将测试是否每种 一个可以控制另一个。 抑制B细胞凋亡的药物， 将测试预防NFkB活化对IL-4或IL-13的影响 生产以及选择的酪氨酸激酶抑制剂。 目标2次尝试 为了区分PKC同工酶在IL-4和IL-13调节中的作用， 迄今为止检测的佛波醇酯抑制IL-4的合成，但似乎 刺激IL-13产生。 PKC耗竭的影响， 将检查PMA对IL-3和IgE刺激的IL-13分泌的刺激。 将使用钙依赖性与钙非依赖性PKC抑制剂 寻求不同的效果。 RT-PCR将用于定性和 IL-4和IL-13 mRNA水平的半定量评估。 最后 将测试重组人HRF刺激IL-13的能力 在IgE（+）与IgE（-）细胞供体中的分泌。 直接刺激和 将检查HRF对其它促分泌素的引发。 各种 细胞因子引物将用HRF作为激动剂和IL-4与IL-13进行测试 分泌比较 预充预孵育的时间将有所不同， 任何寻求的分泌抑制剂。

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.