ADHESION MOLECULE INDUCT ACTIVATION IN INFLAMMATORY INJURY

白细胞移行进入缺血和再灌注心肌 再灌注后迅速恢复。 在动物模型中，心肌组织 通过实验性治疗， 嗜中性粒细胞的积聚和/或活化。 建议的实验 本申请将使用具有犬和鼠细胞的体外模型 确定中性粒细胞转化所需的粘附机制 从循环中的静止细胞到能够粘附的细胞- 对心肌细胞的依赖性损伤。 这种转变发生在体内， 中性粒细胞通过血管壁迁移到心肌组织中。 的 具体目标将在实验中解决不同的阶段， 转换定义，1）允许中性粒细胞的分子机制， 在具有剪切应力的流动条件下， 小静脉范围，2）允许中性粒细胞粘附的粘附机制， 通过血管壁结构迁移并附着在心肌细胞上， 和3）中性粒细胞产生反应性 氧、细胞因子（IL-1 β、TNF α和IL-6）和趋化因子（IL-8和 MCP-1）。 将使用两种一般的实验策略-i） 隔离已知的粘合机构（例如，通过使用重组粘附 分子），以确定其是否可以支持或影响白细胞 在这三个阶段中发挥作用，以及ii）确定相对 每个粘合剂机制对这些功能的贡献， 白细胞与精氨酸刺激的内皮细胞相互作用， 细胞外表面，和精氨酸刺激的心肌细胞。 的 后面的研究将使用单克隆抗体来阻断特异性粘合剂 功能，或来自基因工程小鼠的细胞， 特异性粘附分子。 这些研究的重点将放在CD上 18种整合素和刺激物（IL-8和血小板活化因子）， 上调该家族的两个成员（CD11b/CD18和 CD11a/CD18）、内皮细胞和肌细胞上的ICAM-1（CD54）， 选择素家族（CD62L和CD62P），以及一种不确定的粘附机制 这解释了中性粒细胞在24小时后的原发性粘附， 用IL-1刺激内皮细胞。 我们还将评估 使犬单核细胞停留在内皮细胞上的粘附步骤 在流动的条件下，并影响上述生产 细胞因子和趋化因子。 这些研究将集中在两个粘附 除了上述分子外，β 1整联蛋白、VLA 4 （CD49d/CD29）和VCAM-1（CD106）。 犬活体显微镜检查， 小鼠模型也将被用来评估一些概念， 从体外获得的数据。 这些研究将提高 了解关键的连续粘合剂步骤， 中性粒细胞和单核细胞的迁移和激活 心肌再灌注损伤

Emigration of leukocytes into ischemic and reperfused myocardium occurs rapidly following reperfusion. In animal models, myocardial tissue injury is reduced significantly by experimental therapies that limit the accumulation and/or activation of neutrophils. Experiments proposed in this application will use in vitro models with canine and murine cells to define adhesive mechanisms necessary for the transition of neutrophils from resting cells in the circulation to cells capable of adherence- dependent damage to cardiac myocytes. This transition occurs in vivo as neutrophils migrate through vessel walls into myocardial tissue. The Specific Aims will address experimentally distinct stages in this transition define, 1) the molecular mechanisms that allow neutrophils to stop on endothelium under conditions of flow with shear stresses in the venular range, 2) the adhesive mechanisms that allow neutrophils to migrate through vessel wall structures and attach to cardiac myocytes, and 3) the adhesive determinants for neutrophil production of reactive oxygen, cytokines (IL-1beta, TNFalpha, and IL-6) and chemokines (IL-8 and MCP-1). Two general experimental strategies will be used -- i) to isolate a known adhesive mechanism (e.g., by using recombinant adhesion molecules) in order to determine if it can support or influence leukocyte functions in these 3 stages, and ii) to determine the relative contributions of each adhesive mechanisms to these functions as leukocytes interact with cytokine-stimulated endothelial cells, extracellular surfaces, and cytokine-stimulated cardiac myocytes. The latter studies will use monoclonal antibodies to block specific adhesive functions, or cells from mice genetically engineered to be deficient in specific adhesion molecules. The focus of these studies will be on CD 18 integrins and stimuli (IL-8 and platelet activating factor) that upregulate the function of two members of this family (CD11b/CD18 and CD11a/CD18), ICAM-1 (CD54) on endothelium and myocytes, members of the selectin family (CD62L and CD62P), and an undefined adhesive mechanism that accounts for primary adhesion of neutrophils 24 hours after stimulation of endothelial cells with IL-1. We will also evaluate the adhesive steps that allow canine monocyte to stop on endothelial cells under conditions of flow, and that influence production of the above cytokines and chemokines. These studies will focus on two adhesion molecules in addition to those cited above, the beta1 integrin, VLA4 (CD49d/CD29), and VCAM-1 (CD106). Intravital microscopy in canine and murine models will also be used to assess some of the concepts resulting from the data obtained in vitro. These studies will enhance understanding of the critical sequential adhesive steps necessary for the emigration and activation of neutrophils and monocytes in the context of myocardial reperfusion injury.