Impact of real-time shedding on binding kinetics of membrane-remaining L-selectin to PSGL-1.
Impact of real-time shedding on binding kinetics of membrane-remaining L-selectin to PSGL-1.
复制标题
DOI：
10.1152/ajpcell.00212.2018
复制
发表时间：
2019-02
期刊：
影响因子：
--
通讯作者：
Shuang Peng;Shenbao Chen;Linda Li;Chunfang Tong;Ning Li;Shouqin Lü;M. Long
中科院分区：
文献类型：
--
作者：
Shuang Peng;Shenbao Chen;Linda Li;Chunfang Tong;Ning Li;Shouqin Lü;M. Long
在炎症级联反应中，多种细胞因子诱导的L-选择素脱落在激活中性粒细胞(PMN)中起着关键作用。虽然在体内的实时脱落在中性粒细胞激活后持续约10min，但在瞬时或稳态下，依赖于时间的脱落对膜剩余L-选择素与其配体结合动力学的影响尚不清楚。在这里，我们发展了体外L-选择素脱落动力学方法，结合细胞黏附的竞争性分析，提出了一个定量实时脱落对膜残留L-选择素与P-选择素糖蛋白配体-1结合动力学影响的理论模型。结果表明，与人PMN相比，Jurkat细胞中L-选择素脱落对PMA活性的影响程度更大，但终止时间更长。同时，FMLF或IL-8刺激的PMN的终止时间比PMA刺激的更长，但其脱落程度相似。L-选择素的脱落通过三种途径减少L-选择素-PSGL-1介导的细胞黏附：减少膜锚定的L-选择素，增加可溶性L-选择素与配体的竞争性结合，以及膜剩余的L-选择素自身的构象变化。与完整细胞相比，即使在不同的过渡时间点，保留膜的L-选择素-PSGL-1对的结合亲和力在中性粒细胞和Jurkat细胞的早期均增强，在后期均降低。在切变流作用下，机械或生物化学诱导L-选择素脱落后，中性粒细胞和Jurkat细胞的滚动速度均增加。这些发现有助于我们进一步理解时间依赖性L-选择素在炎症级联反应中的作用。
L-selectin shedding induced by various cytokines is crucial in activating neutrophils (PMNs) in inflammatory cascade. While the real-time shedding in vivo lasts ~10 min after PMN activation, the impact of time-dependent shedding on binding kinetics of membrane-remaining L-selectins to its ligands is poorly understood at transient or steady state. Here, we developed an in vitro L-selectin shedding dynamics approach, together with competitive assays of cell adhesion, and proposed a theoretical model for quantifying the impact of real-time shedding on the binding kinetics of membrane-remaining L-selectins to P-selectin glycoprotein ligand-1 (PSGL-1). Our data indicated that the extent of L-selectin shedding on PMA activation is higher, but the terminating time is longer for Jurkat cells than those for human PMNs. Meanwhile, fMLF or IL-8 stimulation yields the longer terminating time than that on PMA stimulation but results in a similar shedding extent for PMNs. L-selectin shedding reduces L-selectin-PSGL-1-mediated cell adhesion in three ways: decreasing membrane-anchored L-selectins, increasing soluble L-selectins competitively binding to ligands, and presenting conformational alteration of membrane-remaining L-selectins themselves. Compared with those on intact cells, the binding affinities of membrane-remaining L-selectin-PSGL-1 pairs were all enhanced at initial and lowered at the late shedding phase for both PMN and Jurkat cells even with varied transition time points. The rolling velocities of both PMNs and Jurkat cells were increased following mechanically or biochemically induced shedding of L-selectin under shear flow. These findings help to further our understanding of the function of time-dependent L-selectin shedding during the inflammation cascade.