力调节的白细胞上整合素LFA-1与ICAM-1的相互作用

Cytokine-induced activation of Integrin LFA-1 expressed on leukocyte membrane is a key event in the immune response of flowing leukocyte targeting to inflammatory sites in blood vessel wall. The activated LFA-1 at intermediate affinity state binds with its ligand expressed on endothelial cells, then as a conductor, transmits the mechanical stimulation of blood flow shear stress upon leukocytes to cell inside, lastly initiate ensuing outside-in signaling pathway to its completely activation process or to its high affinity state. However, there still is less knowledge on the mechano-chemical process. .In this project, with combination of theoretial modeling and expermental observations, we will perform systematically experiments of the flow chamber, the atomic force microscope and the contact-area fluorescence recovery after photobleaching to investigate the behaviors of the two dimensional reaction kinetics in interaction between ICAM-1 and LFA-1, which is at chemokine (IL-8)-induced intermediate affinity state, under blood flow shear stress, by measuring the involved kinetic parameters, the on- and off- rates, and the activation rate of bound LFA-1 as well as the inactivated rate of unliganded activated LFA-1. Here, we will focus at how fast the bound LFA-1 under stretching is activated fully and the unliganded LFA-1 at high affinity return to its initial intermediate affinity states. The aim of this project is reveal not only the mechanism of mechanical activation of bound LFA-1 from intermediate to high affinity state and inactivation of unliganded LFA-1 at high affinity state but also the involved molecular structural basis, not only for better understanding on physiological and pathological processes such as the inflammatory immune response of leukocyte, the formation of thrombus and metastasis of tumor, but also for novel approach to design of innovative drugs, some useful informations and new ideas to the intervention therapy of the corresponding diseases.

在循环白细胞奔赴血管壁炎症部位的过程中，细胞因子诱导的白细胞膜上整合素LFA-1的激活是白细胞免疫应答响应的一个关键事件。这些部分活化了的处于中间亲和态的LFA-1，只有和表达在内皮细胞上的配体结合，才能感受经由白细胞传递的血流剪应力刺激，开启随后发生的经由Outside-in信号通路的完全激活过程（LFA-1由中等到高亲和力态的转换）。针对这一所知甚少的力化学耦合过程，本项目拟系统开展流动腔、原子力显微镜和荧光淬灭后的恢复实验，通过理论建模与实验观测相结合，研究（剪应）力环境中，当被趋化因子（如IL-8）诱导到中间亲和力状态后，LFA-1和ICAM-1 相互作用的二维反应动力学行为、活化水平，活化与失活速率及其力学调控途径。力图通过测定这些动力学参数，揭示LFA-1机械激活的力化学协同调控机制和分子结构基础，以深化对白细胞在炎症免疫应答、血栓形成与肿瘤转移等生理病理过程中的行为的理解。

在选择素、趋化因子和流体剪应力共存的生理环境中，循环白细胞膜上整合素的激活是白细胞炎症反应及免疫应答中的关键事件。β2整合素被认为在白细胞的稳定黏附和跨膜迁移中发挥着至关重要的作用，可由选择素、趋化因子经过由内而外的信号通路激活到中等亲和力状态，然后这些部分活化的β2整合素才能与内皮细胞上的细胞间黏附分子ICAM结合，开启“由外而内”的信号通路达到高亲和力状态。整个激活过程中自始至终存在着流体剪应力的作用，但力如何参与并调控β2整合素的构象和亲和力变化依然知之甚少。本项目中，我们采用流动腔实验系统、荧光显示技术和分子动力学模拟等手段，结合理论建模和实验观测，试图揭示整合素活化过程的力化学偶联调控机制及其分子结构基础。取得了如下主要进展：.1) P-选择素介导的滚动白细胞上整合素的激活是从低到中、再到高的逐步过程，局部β2整合素的活化非常迅速发生在10秒内，而大范围或全局的活化过程则相对较长（40～300秒），活化速率随剪应力的增加呈现先增加后减小趋势，且转折拐点与P-selectin/PSGL-1逆锁键拐点基本一致，揭示P-选择素诱导的整合素激活可能受到逆锁键的调控。2) 可溶性与固定在血管壁面上的趋化因子可将整合素激活到不同的中等亲和力状态，只有固定的趋化因子才能在极短的接触时间内进一步将整合素激活到高亲和力状态。中、高两种亲和态LFA-1/ICAM-1的二维反应动力学参数均受力的调控，且转化速率与剪应力呈正相关。3) 流场下白细胞钙响应的研究发现一条快速激活β2整合素的信号通路，该快速通路涉及细胞骨架、moesin蛋白，而与细胞膜上脂筏及Syk关系不大，随后激发膜上钙离子通道使外钙内流。4) 分子动力学模拟结果发现无论选择素还是趋化因子介导，其胞质区与胞内分子的复合物只有在力作用下才能使磷酸化位点有效暴露，从而启动下游信号以激活整合素。5) 无论在白血病细胞还是血小板上，均发现了类似的力对整合素活化的调控。可为心脑血管病、白血病人的诱导综合症的治疗和相关药物开发提供参考。