LUNG INFLAMMATION

肺部炎症

• 批准号: 2215287
• 负责人: GERALD WEISSMANN
• 金额: $ 31.51万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-06-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2215287
• 关键词: Arthus phenomenon; Shwartzman phenomenon; arachidonate; cell free system; cyclic AMP; cyclic GMP; endotoxins; flow cytometry; gel mobility shift assay; human subject; inflammation; interleukin 1; leukocyte activation /transformation; liposomes; lung injury; neutrophil; nitric oxide; northern blottings; phosphatidate; phospholipids; phosphorylation; prostaglandin receptor; prostaglandins; tumor necrosis factor alpha; vascular endothelium

We will determine how regulatory molecules released from resting and stimulated endothelial cells (+/-TNFa, Il- 1, endotoxin) modify neutrophil functions in Arthus and Shwartzman-like models of tissue injury. Using intact human neutrophils, granule-free cytoplasts, cell free systems, liposomes of varying physical structure, and human umbilical vein endothelial cells, we will test four novel hypotheses. Specific Aim I. To test the hypothesis that the Yin/Yang control of neutrophil function by cAMP/cGMP is exerted at the level of raf phosphorylation in the MAP-kinase cascade. Neutrophils, their cytoplasts and cell-free extracts exposed to the three classes of activators will be studied kinetically (5 sec to 5 min) for delta-psi, [Ca]i phospholipid turnover, lysosomal enzyme release, translocation of 47phox and 67phox components of the NADPH oxidase and ras-related proteins, carboxy methylation, cAMP, cGMP and phosphorylation/ dephosphorylation patterns studied of raf, CD11b/CD 18, MAP kinases +/- inhibitors. (calyculin, genestein, okadaiate, etc.). Specific Aim II. To test the hypothesis that endothelial cells release autocoids (adenosine, PGI2 and NO) that inhibit neutrophil-mediated endothelial activation (judged by translocation of NFkB, induction of COX II, NO synthases, expression of E-selectin, VCAM-1, ICAM-1). Neutrophils or cytoplasts will be incubated with endothelial cells that do or do not form the autocoid (+/- adenosine deaminase, +/- COX II, NO synthase inhibitors). Endothelial cell activation will be determined by electrophoretic mobility shift, Northern Blot and FACS. Specific Aim III. To test the hypothesis that lipid metabolites formed in the course of neutrophil/endothelial interactions, e.g. arachidonate (20:4) and phosphatidate (PA), regulate the association of ras-related proteins with their inhibitor/chaperones (GDI's) and with the plasmalemma. In intact cells, cytoplasts and cell-free, oxidase-competent systems we will study membrane/cytosol distribution, GDI association and phospholipid-dependent isoprenyl-directed carboxy methyltransferase (piCMT) activity with respect to O2- and/or aggregation, phosphorylation patterns in response to added 20:4 (+/- other eicosanoids, HODEs) or PA (+/- other phospholipids). Specific Aim IV. To test the hypothesis that the physical state of phospholipids (lamellar vs Hex(II)) as regulated by phospholipases A, C and D regulates a) the association of prenylated proteins with the plasmalemma and b) the effect of autocoids (PGE1, 14,15 di-HETEs, 13-HODE, 22:6, lipoxins) on the activity of piCMT and the oxidase system in natural and artificial bilayers. Lamellar (phosphatidyl choline, palmitoyl-oleyl ethanolamine) liposomes will be compared with Hex(II) forming liposomes (PA +/- Ca, dioleyl ethanolamine) for presentation to PGI2 receptors and reconstitution of the O2- system.

我们将确定监管分子如何从静止和 刺激的内皮细胞（+/- TNFA，IL-1，内毒素）修饰中性粒细胞 在Arthus和Shwartzman类模型的组织损伤模型中的功能。使用 完整的人类嗜中性粒细胞，无颗粒细胞体，无细胞系统， 不同物理结构的脂质体和人脐静脉 内皮细胞，我们将检验四个新的假设。 特定目的I.用于检验阴阳控制的假设 CAMP/CGMP的中性粒细胞功能在RAF的水平上施加 地图激酶级联反应中的磷酸化。中性粒细胞，其细胞质 暴露于三类激活剂的无细胞提取物将是 对Delta-PSI进行动力学研究（5秒至5分钟），[Ca] i磷脂 营业额，溶酶体酶释放，47phox的易位和67phox NADPH氧化酶和与RAS相关蛋白的成分，羧基 甲基化，CAMP，CGMP和磷酸化/去磷酸化模式 研究RAF，CD11b/cd 18，MAP激酶+/-抑制剂。 （钙钙蛋白酶， Genestein，Okadaiate等）。具体目标II。检验以下假设 内皮细胞释放抑制的异型型（腺苷，PGI2和NO） 中性粒细胞介导的内皮激活（通过易位 NFKB，Cox II的诱导，无合酶，E-选择素的表达，VCAM-1，，， ICAM-1）。中性粒细胞或细胞质将与内皮孵育 做或不形成异型体的细胞（+/-腺苷脱氨酶，+/- COX II，无合酶抑制剂）。内皮细胞激活将是 由电泳移动性转移，北印迹和FACS确定。 特定目标III。为了检验以下假设：脂质代谢产物形成 中性粒细胞/内皮相互作用的过程，例如蛛丝 （20：4）和磷酸（PA），调节RAS相关的关联 及其抑制剂/伴侣蛋白（GDI）的蛋白质和浆膜。 在完整的细胞中，细胞体和无细胞，氧化酶竞争的系统 将研究膜/细胞质分布，GDI关联和 磷脂依赖性异on导向的羧基转移酶 （PICMT）有关O2和/或聚集的活性，磷酸化 响应于添加的20：4（+/-其他类花生素，霍德斯）或PA的模式 （+/-其他磷脂）。特定目标IV。检验以下假设 由 磷脂酶A，C和D调节a）prenylated的缔合 蛋白质具有浆膜和b）型型型蛋白质的作用（PGE1，14,15 Di-Hetes，13-hode，22：6，脂毒素）在PICMT和 天然和人工双层中的氧化酶系统。层层（磷脂酰基 将将胆碱，棕榈酰 - 奥碱乙醇胺）与脂质体进行比较 十六进制（II）形成脂质体（PA +/- Ca，二烯基乙醇胺） 呈现向PGI2受体和O2系统的重构。