Department of General Pediatrics, University Children’s Hospital, Heinrich-Heine-University, Düsseldorf, Germany.
J Neuroinflammation. 2011 May 18;8:51. doi: 10.1186/1742-2094-8-51.
A critical point during the course of bacterial meningitis is the excessive influx of polymorphnuclear neutrophils (PMNs) from the blood into the brain. Both paracellular and transcellular routes of leukocyte transmigration through the blood-brain barrier have been described in CNS diseases so far. Thus, we investigated the mechanism of PMN transmigration through the blood-CSF barrier under inflammatory conditions.
In an "inverted" Transwell culture model of the blood-CSF barrier, the zoonotic agent Streptococcus suis (S. suis) was used to stimulate porcine choroid plexus epithelial cells (PCPECs) specifically from the physiologically relevant basolateral side. Barrier function was analyzed by measuring TEER and TR-dextran-flux, and tight junction morphology was investigated by immunofluorescence. Route and mechanism of PMN transmigration were determined by immunofluorescence, electron microscopy and FACS analysis. Quantitative real time-PCR was used to determine expression levels of ICAM-1 and VCAM-1.
Here, we show that the transmigration of PMNs through PCPECs was significantly higher after stimulation with TNFα or infection with S. suis strain 10 compared to its non-encapsulated mutant. Barrier function was not significantly affected by PMN migration alone, but in combination with S. suis infection. Tight junction and cytoskeletal actin reorganisation were also observed after stimulation with S. suis or TNFα. Most strikingly, PMNs preferentially migrated across PCPECs via the transcellular route. Extensive sequential analyses of the PMN transmigration process with Apotome(®)-imaging and electron microscopy revealed that paracellular migrating PMNs stop just before tight junctions. Interestingly, PMNs subsequently appeared to proceed by transcellular migration via funnel-like structures developing from the apical membrane. It is noteworthy that some PMNs contained bacteria during the transmigration process. Flow cytometric and transmigration inhibition studies with integrin-specific antibodies showed that PMN traversal is dependent on CD11b/CD18. Analysis of cell adhesion molecules in PCPECs revealed a significant increase of ICAM-1 and VCAM-1 expression after TNFα and S. suis stimulation.
Our data underline the relevance of the blood-CSF barrier as a gate for leukocyte entry into the CNS and suggest a novel transcellular migration step during the pathogenesis of bacterial meningitis.
细菌性脑膜炎病程中的一个关键点是过多的多形核中性粒细胞（PMN）从血液涌入大脑。迄今为止，中枢神经系统疾病中已经描述了白细胞通过血脑屏障的细胞旁和细胞内途径的迁移。因此，我们研究了在炎症条件下PMN 通过血脑脊液屏障迁移的机制。
在血脑脊液屏障的“倒置”Transwell 培养模型中，使用人畜共患病原体猪链球菌（S. suis）特异性地从生理相关的基底外侧刺激猪脉络丛上皮细胞（PCPECs）。通过测量 TEER 和 TR-葡聚糖通量来分析屏障功能，并通过免疫荧光法研究紧密连接形态。通过免疫荧光、电子显微镜和 FACS 分析确定 PMN 迁移的途径和机制。定量实时 PCR 用于测定 ICAM-1 和 VCAM-1 的表达水平。
在这里，我们表明，与非囊泡突变株相比，TNFα 刺激或 S. suis 10 株感染后，PMN 通过 PCPECs 的迁移明显增加。PMN 单独迁移不会显著影响屏障功能，但与 S. suis 感染相结合时会影响。刺激 S. suis 或 TNFα 后也观察到紧密连接和细胞骨架肌动蛋白的重排。最引人注目的是，PMN 优先通过细胞间途径穿过 PCPECs。使用 Apotome（®）成像和电子显微镜对 PMN 迁移过程进行广泛的顺序分析表明，穿过细胞旁的 PMN 在紧密连接前停止。有趣的是，PMN 随后似乎通过从顶膜发育而来的漏斗状结构通过细胞内迁移进行。值得注意的是，在迁移过程中一些 PMN 含有细菌。使用整合素特异性抗体的流式细胞术和迁移抑制研究表明，PMN 的穿透依赖于 CD11b/CD18。PCPECs 中细胞粘附分子的分析表明，TNFα 和 S. suis 刺激后 ICAM-1 和 VCAM-1 的表达显著增加。
我们的数据强调了血脑脊液屏障作为白细胞进入中枢神经系统的门户的相关性，并提出了细菌性脑膜炎发病机制中一种新的细胞内迁移步骤。
