Estevao Carolina, Bowers Chantelle E, Luo Ding, Sarker Mosharraf, Hoeh Alexandra Eva, Frudd Karen, Turowski Patric, Greenwood John
Institute of Ophthalmology, University College London, 11-43 Bath Street, London, EC1V 9EL, UK.
School of Health and Life Sciences, Teesside University, Stephenson Street, Middlesbrough, TS1 3BA, UK.
Brain Behav Immun Health. 2021 Oct 22;18:100370. doi: 10.1016/j.bbih.2021.100370. eCollection 2021 Dec.
During neuroinflammation many chemokines alter the function of the blood-brain barrier (BBB) that regulates the entry of macromolecules and immune cells into the brain. As the milieu of the brain is altered, biochemical and structural changes contribute to the pathogenesis of neuroinflammation and may impact on neurogenesis. The chemokine CCL4, previously known as MIP-1β, is upregulated in a wide variety of central nervous system disorders, including multiple sclerosis, where it is thought to play a key role in the neuroinflammatory process. However, the effect of CCL4 on BBB endothelial cells (ECs) is unknown.
Expression and distribution of CCR5, phosphorylated p38, F-actin, zonula occludens-1 (ZO-1) and vascular endothelial cadherin (VE-cadherin) were analysed in the human BBB EC line hCMEC/D3 by Western blot and/or immunofluorescence in the presence and absence of CCL4. Barrier modulation in response to CCL4 using hCMEC/D3 monolayers was assessed by measuring molecular flux of 70 kDa RITC-dextran and transendothelial lymphocyte migration. Permeability changes in response to CCL4 were measured by an occlusion technique in pial microvessels of Wistar rats and by fluorescein angiography in mouse retinae.
CCR5, the receptor for CCL4, was expressed in hCMEC/D3 cells. CCL4 stimulation led to phosphorylation of p38 and the formation of actin stress fibres, both indicative of intracellular chemokine signalling. The distribution of junctional proteins was also altered in response to CCL4: junctional ZO-1 was reduced by 60% within 60 min. In addition, surface VE-cadherin was redistributed through internalisation. Consistent with these changes, CCL4 induced hyperpermeability and and increased transmigration of lymphocytes across monolayers of hCMEC/D3 cells
These results show that CCL4 can modify BBB function and may contribute to disease pathogenesis.
在神经炎症过程中，许多趋化因子会改变血脑屏障（BBB）的功能，血脑屏障可调节大分子和免疫细胞进入大脑。随着脑内环境的改变，生化和结构变化会促使神经炎症的发病机制形成，并可能影响神经发生。趋化因子CCL4，以前称为MIP-1β，在多种中枢神经系统疾病中上调，包括多发性硬化症，据认为它在神经炎症过程中起关键作用。然而，CCL4对血脑屏障内皮细胞（ECs）的影响尚不清楚。
在存在和不存在CCL4的情况下，通过蛋白质免疫印迹和/或免疫荧光分析人血脑屏障内皮细胞系hCMEC/D3中CCR5、磷酸化p38、F-肌动蛋白、紧密连接蛋白1（ZO-1）和血管内皮钙黏蛋白（VE-钙黏蛋白）的表达和分布。使用hCMEC/D3单层细胞评估对CCL4的屏障调节作用，通过测量70 kDa RITC-葡聚糖的分子通量和跨内皮淋巴细胞迁移来进行。通过闭塞技术在Wistar大鼠软脑膜微血管中以及通过荧光素血管造影术在小鼠视网膜中测量对CCL4的通透性变化。
CCL4的受体CCR5在hCMEC/D3细胞中表达。CCL4刺激导致p38磷酸化和肌动蛋白应力纤维形成，两者均表明细胞内趋化因子信号传导。连接蛋白的分布也因CCL4而改变：紧密连接蛋白ZO-1在60分钟内减少了60%。此外，表面VE-钙黏蛋白通过内化重新分布。与这些变化一致，CCL4诱导hCMEC/D3细胞单层的通透性增加和淋巴细胞跨膜迁移增加。
这些结果表明CCL4可改变血脑屏障功能，并可能有助于疾病发病机制的形成。
