VISCOELASTICITY OF WHITE BLOOD CELLS

我们过去一年的研究目标是描述尼古丁刺激的粘附受体动力学，使内皮细胞能够捕获并引导中性粒细胞到达炎症部位。从自由流动的白细胞与内皮细胞的随机相互作用到特异性粘附和渗出的转变是一种有序的机制，细胞间粘附由信号传导协调。例如，E-选择素不是在微血管内皮细胞（MVEC）上组成型表达，而是响应于细胞因子或内毒素刺激而被诱导。E-选择素支持中性粒细胞滚动，这是白细胞迁移的初步步骤。虽然大血管内皮细胞中粘附分子表达的时间模式已得到很好的表征，但MVEC的研究较少，MVEC与嗜中性粒细胞迁移的毛细血管后小静脉更密切相关。我们发现，E-选择素表达的结构特征与E-选择素和抗E-选择素单克隆抗体包被的珠之间的功能性粘附（FA）之间存在相关性。MVEC用IL-1 α刺激，同时使用流式细胞术或微量移液管FA测定进行E-选择素表达动力学的两个单独测定4-6小时。在流式细胞术测量中，在刺激6小时后，HMVEC上表达的E-选择素受体的数量增加了20倍。在HMVEC和抗E-选择素mAb包被珠之间的平行微量移液管FA测定中，FA增加10倍。虽然FA取决于受体和配体密度，使用流式细胞术测量的参数，但它也由粘附试验固有的表面接触的物理特性和有效的受体/配体正向反应速率确定。最终，我们计划用中性粒细胞取代抗体包被的珠探针，并研究特异性EC受体-中性粒细胞配体相互作用的动力学。我们完成了中性粒细胞微绒毛静态和动态长度的研究，并发表在《美国科学院院刊》上。微绒毛的顶端含有大部分的L-选择素和PSGL-1（P-选择素糖蛋白配体-1），被认为可以促进内皮细胞上中性粒细胞的初始停滞。在停止后的滚动阶段，所涉及的分子键的寿命取决于血流的剪切应力施加的拉力。用两种不同的方法，电子显微镜（与细胞固定的非收缩程序）和微量吸管操作，我们已经获得了两个可比的中性粒细胞微绒毛长度，均约0.3 - 5米的平均值。我们还发现，在拉力下，微绒毛可以延伸（微绒毛延伸）或由其形成长的薄膜圆柱体（系链形成）。如果力小于或等于34 pN（1 - 3 pN），微绒毛的长度将被延长;如果力大于61 pN（1 - 5 pN），则微绒毛将以恒定速度形成系链，该速度与力成线性关系。当力在34 pN和61 pN之间（过渡区）时，单个微绒毛中膜和细胞骨架之间的结合程度将决定微绒毛延伸或系链形成是否发生。当微绒毛伸展时，它就像弹簧一样，弹簧常数为43 pN/5 m。与刚性或不可延伸的微绒毛相反，微绒毛延伸和系链形成都可以降低施加在粘合剂结合上的拉力，从而延长在高生理剪切应力下的结合的持久性。意义：这项研究将促进对循环中性粒细胞如何影响吞噬作用的理解。

The objective of our research for the past year is to characterize cytokine-stimulated adhesion receptor kinetics that enable endothelial cells to capture and guide neutrophils to the site of inflammation. The transition from random interactions of freely-flowing leukocytes with the endothelium to specific adhesion and diapedesis is a well-ordered mechanism with intercellular adhesion coordinated by signaling. E-selectin, for example, is not constitutively expressed on microvascular endothelial cells (MVEC), but is induced in response to cytokine or endotoxin stimulation. E-selectin supports neutrophil rolling, a preliminary step in leukocyte transmigration. While the temporal patterns of adhesion molecule expression in large vessel endothelium have been well characterized, there are fewer studies of MVEC, which is more closely related to the post-capillary venules where neutrophils transmigrate. We show that there is correlation between structural characteristics of E-selectin expression and functional adhesion (FA) between E-selectin and anti-E-selectin mAb-coated beads. MVEC were stimulated with IL-1a while two separate assays of E-selectin expression kinetics were performed for 4-6 hours using either flow cytometry or the micropipette FA assay. In flow cytometry measurements, the number of E-selectin receptors expressed on HMVEC increased 20-fold following 6 hours of stimulation. In parallel micropipette FA assays between HMVEC and anti-E-selectin mAb-coated beads, FA increased 10-fold. While FA depends on receptor and ligand density, parameters measured using flow cytometry, it is also determined by physical characteristics of surface contact intrinsic to the adhesion assay and effective receptor/ligand forward reaction rates. Ultimately, we plan to replace the antibody-coated bead probe with a neutrophil and study the kinetics of specific EC receptor- neutrophil ligand interactions. We have completed the studies on the static and dynamic lengths of neutrophil microvilli and this work has been published in the Proceeding of National Academy of Science. Microvilli, with most of the L-selectin and PSGL-1 (P-selectin glycoprotein ligand-1) on their tips, are believed to promote the initial arrest of neutrophils on endothelium. At the rolling stage following arrest, the lifetimes of the involved molecular bonds depend upon the pulling force imposed by the shear stress of blood flow. With two different methods, electron microscopy (with the cells fixed by a non-shrinking procedure) and micropipette manipulation, we have obtained two comparable neutrophil microvillus lengths, both about 0.3 5m in average. We have found also that, under a pulling force, a microvillus can be extended (microvillus extension) or a long thin membrane cylinder can be formed from it (tether formation). If the force is less than or equal to 34 pN (1 3 pN), the length of the microvillus will be extended; if the force is larger than 61 pN (1 5 pN), a tether will be formed from the microvillus at a constant velocity, which depends linearly upon the force. When the force is between 34 pN and 61 pN (transition zone), the degree of association between membrane and cytoskeleton in individual microvilli will dictate whether microvillus extension or tether formation occurs. When a microvillus is extended, it acts like a spring with a spring constant of 43 pN/5m. In contrast to a rigid or non-extendible microvillus, both microvillus extension and tether formation can decrease the pulling force imposed on the adhesive bonds, hence prolonging the persistence of the bonds at high physiological shear stresses. SIGNIFICANCE: This research will advance understanding of how circulating neutrophils effect phagocytosis.