SYNERGISTIC STIMULATION AND PRIMING OF NEUTROPHILS

这项建议的长远目标是阐明 引发中涉及的生物化学事件的序列， 刺激嗜中性粒细胞产生超氧化物（O2-）。 超氧化物是 氧依赖性抗菌机制的一个关键组成部分， 对感染性生物体提供主要防御的细胞。 某些 细胞因子使嗜中性粒细胞释放增加量的O2-。 这些因素的治疗价值目前正在临床上进行测试， 对各种癌症和艾滋病患者有良好的效果。 因此，在本发明中， 本文概述的实验与理解宿主相关， 防御机制和免疫缺陷疾病。 具体而言，该项目涉及两个基本未知的领域。 这些 （1）羟基化二十碳四烯酸的作用机制 酸（HETE）在调节O2-产生中的作用，和（2） 蛋白磷酸化和O2生成。 中性粒细胞产生5-和 15-HETE在各种 情节 5-戏剧性地 增强02-释放，而15-HETE抑制它。我们建议5-HETE可以抑制02-释放。 HETE通过增加Ca 2+跨膜的流量来引发中性粒细胞 质膜，也许是通过为这种阳离子打开通道。 这将是 测量这些细胞对45 Ca 2+的摄取率， 存在 不同浓度的5-HETE。 各种各样的影响 将评价钙通道拮抗剂对该过程的影响。 的 45 Ca 2+吸收速率与O2-生成速率之间的关系 将被定义，15-HETE可能的拮抗作用将被定义。 寻求。 关于蛋白质磷酸化，我们已经观察到刺激 的 02-中性粒细胞的释放总是伴随着强烈的 两种分子量为ca. 47和49 kDa。 从数量上讲，这是两种主要的蛋白质， 在刺激这些细胞时磷酸化。 而47 kDa蛋白质 已经被广泛地描述过了，实际上对49个 kDa种类。 本文详细描述了实验以鉴定、表征 并纯化这种蛋白质。 技术 生物化学与细胞 生物学将被使用。 我们将确定49 kDa蛋白是否是 O2生成系统的组件，或者涉及一些其他组件， 刺激这些细胞的机制的方式（例如， 磷脂酶D）。 49 kDa蛋白质的活性变化， 磷酸化将寻求建立这种修饰之间的联系， 反应和细胞刺激。

The long term objectives of this proposal are to elucidate the exact sequence of biochemical events that are involved in the priming and stimulation of superoxide (02-) production by neutrophils. Superoxide is a key component of the oxygen-dependent antimicrobial mechanisms of these cells which provide a major defense against infectious organisms. Certain cytokines prime neutrophils to release increased amounts of 02-. Therapeutic value of these factors is now being tested clinically in a variety of cancer and AIDS patients with promising results. Thus, experiments outlined herein are relevant to an understanding of host- defense mechanisms and immune deficiency diseases. Specifically, this project addresses two largely unknown areas. These are: (1) the mechanism(s) of action of the hydroxylated eicosatetraenoic acids (HETE) in modulating 02- production, and (2) the link between protein phosphorylation and 02- generation. Neutrophils produce 5- and 15-HETE under various circumstances. 5-HETE dramatically potentiates 02- release, whereas 15-HETE inhibits it. We propose that 5- HETE primes neutrophils by increasing the flux of Ca2+ across the plasmalemma, perhaps by opening a channel for this cation. This will be measuring the rates of 45Ca2+ uptake by these cells in the presence of different concentrations of 5-HETE. The effects of a variety of calcium channel antagonists on this process will be evaluated. The relationships between the rates of 45Ca2+ uptake and 02- generation will be defined, and possible antagonistic effects of 15-HETE will be sought. With regard to protein phosphorylation, we have observed that stimulation of 02- release by neutrophils is always accompanied by an intense phosphorylation of two proteins with molecular weights of ca. 47 and 49 kDa. Quantitatively, these are the two major proteins which are phosphorylated upon stimulation of these cells. While the 47 kDa protein has been extensively characterized, virtually nothing is known of the 49 kDa species. Experiments are detailed herein to identify, characterize and purify this protein. Techniques of biochemistry and cell biology will be employed. We will determine whether the 49 kDa protein is a component of the 02- generating system, or is involved in some other fashion in the mechanism of stimulation of these cells (e.g., phospholipose D). Changes in the activity of the 49 kDa protein after phosphorylation will be sought to forge a link between this modification reaction and cell stimulation.