Key Points Synergic effects of LPS and histamine induce jagged junction morphology. Junction morphology drives TEM separately from adhesion molecules or WPBs. S1P and Rap1 signaling pathways reverse jagged junction morphology. During inflammation, endothelial cells are bombarded with cytokines and other stimuli from surrounding cells. Leukocyte extravasation and vascular leakage are both prominent but believed to be uncoupled as they occur in separate spatiotemporal patterns. In this study, we investigated a “double-hit” approach on primary human endothelial cells primed with LPS followed by histamine. Using neutrophil transendothelial migration (TEM) under physiological flow assays, we found that an LPS-primed endothelium synergistically enhanced neutrophil TEM when additionally treated with histamine, whereas the effects on neutrophil TEM of the individual stimuli were moderate to undetectable. Interestingly, the double-hit–induced TEM increase was not due to decreased endothelial barrier, increased adhesion molecule expression, or Weibel–Palade body release. Instead, we found that it was directly correlated with junctional remodeling. Compounds that increased junctional “linearity” (i.e., stability) counteracted the double-hit effect on neutrophil TEM. We conclude that a compound, in this case histamine (which has a short primary effect on vascular permeability), can have severe secondary effects on neutrophil TEM in combination with an inflammatory stimulus. This effect is due to synergic modifications of the endothelial cytoskeleton and junctional remodeling. Therefore, we hypothesize that junctional linearity is a better and more predictive readout than endothelial resistance for compounds aiming to attenuate inflammation.

中文翻译：

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由内皮粘附连接不稳定驱动的双重打击诱导的白细胞外渗

关键点 LPS 和组胺的协同作用诱导锯齿状连接形态。结形态驱动 TEM 与粘附分子或 WPB 分开。S1P 和 Rap1 信号通路逆转锯齿状连接形态。在炎症期间，内皮细胞受到来自周围细胞的细胞因子和其他刺激物的轰击。白细胞外渗和血管渗漏都很突出，但被认为是不相关的，因为它们以不同的时空模式发生。在这项研究中，我们研究了用 LPS 和组胺引发的原代人内皮细胞的“双重打击”方法。在生理流动测定下使用中性粒细胞跨内皮迁移 (TEM)，我们发现 LPS 引发的内皮在额外用组胺处理时协同增强中性粒细胞 TEM，而单个刺激对中性粒细胞 TEM 的影响是中度到检测不到的。有趣的是，双击诱导的 TEM 增加不是由于内皮屏障降低、粘附分子表达增加或 Weibel-Palade 体释放所致。相反，我们发现它与连接重塑直接相关。增加连接“线性”（即稳定性）的化合物抵消了对中性粒细胞 TEM 的双重打击效应。我们得出结论，在这种情况下，一种化合物，即组胺（对血管通透性的主要影响很短），与炎症刺激相结合，可以对中性粒细胞 TEM 产生严重的次要影响。这种效果是由于内皮细胞骨架和连接重塑的协同修饰。所以，