Chen Xiaodi, Sadowska Grazyna B, Zhang Jiyong, Kim Jeong-Eun, Cummings Erin E, Bodge Courtney A, Lim Yow-Pin, Makeyev Oleksandr, Besio Walter G, Gaitanis John, Threlkeld Steven W, Banks William A, Stonestreet Barbara S
Department of Pediatrics, The Alpert Medical School of Brown University, Women & Infants Hospital of Rhode Island, Providence, RI, USA.
ProThera Biologics, Inc., Providence, RI, USA.
Neurobiol Dis. 2015 Jan;73:118-29. doi: 10.1016/j.nbd.2014.09.007. Epub 2014 Sep 26.
We have previously shown that increases in blood-brain barrier permeability represent an important component of ischemia-reperfusion related brain injury in the fetus. Pro-inflammatory cytokines could contribute to these abnormalities in blood-brain barrier function. We have generated pharmacological quantities of mouse anti-ovine interleukin-1β monoclonal antibody and shown that this antibody has very high sensitivity and specificity for interleukin-1β protein. This antibody also neutralizes the effects of interleukin-1β protein in vitro. In the current study, we hypothesized that the neutralizing anti-interleukin-1β monoclonal antibody attenuates ischemia-reperfusion related fetal blood-brain barrier dysfunction. Instrumented ovine fetuses at 127 days of gestation were studied after 30 min of carotid occlusion and 24h of reperfusion. Groups were sham operated placebo-control- (n=5), ischemia-placebo- (n=6), ischemia-anti-IL-1β antibody- (n=7), and sham-control antibody- (n=2) treated animals. Systemic infusions of placebo (0.154M NaCl) or anti-interleukin-1β monoclonal antibody (5.1±0.6 mg/kg) were given intravenously to the same sham or ischemic group of fetuses at 15 min and 4h after ischemia. Concentrations of interleukin-1β protein and anti-interleukin-1β monoclonal antibody were measured by ELISA in fetal plasma, cerebrospinal fluid, and parietal cerebral cortex. Blood-brain barrier permeability was quantified using the blood-to-brain transfer constant (Ki) with α-aminoisobutyric acid in multiple brain regions. Interleukin-1β protein was also measured in parietal cerebral cortices and tight junction proteins in multiple brain regions by Western immunoblot. Cerebral cortical interleukin-1β protein increased (P<0.001) after ischemia-reperfusion. After anti-interleukin-1β monoclonal antibody infusions, plasma anti-interleukin-1β monoclonal antibody was elevated (P<0.001), brain anti-interleukin-1β monoclonal antibody levels were higher (P<0.03), and interleukin-1β protein concentrations (P<0.03) and protein expressions (P<0.001) were lower in the monoclonal antibody-treated group than in placebo-treated-ischemia-reperfusion group. Monoclonal antibody infusions attenuated ischemia-reperfusion-related increases in Ki across the brain regions (P<0.04), and Ki showed an inverse linear correlation (r= -0.65, P<0.02) with anti-interleukin-1β monoclonal antibody concentrations in the parietal cortex, but had little effect on tight junction protein expression. We conclude that systemic anti-interleukin-1β monoclonal antibody infusions after ischemia result in brain anti-interleukin-1β antibody uptake, and attenuate ischemia-reperfusion-related interleukin-1β protein up-regulation and increases in blood-brain barrier permeability across brain regions in the fetus. The pro-inflammatory cytokine, interleukin-1β, contributes to impaired blood-brain barrier function after ischemia in the fetus.
我们之前已经表明，血脑屏障通透性增加是胎儿缺血再灌注相关脑损伤的一个重要组成部分。促炎细胞因子可能导致血脑屏障功能出现这些异常。我们已经制备出药理剂量的小鼠抗羊白细胞介素-1β单克隆抗体，并表明该抗体对白细胞介素-1β蛋白具有非常高的敏感性和特异性。这种抗体在体外也能中和白细胞介素-1β蛋白的作用。在本研究中，我们假设中和性抗白细胞介素-1β单克隆抗体可减轻缺血再灌注相关的胎儿血脑屏障功能障碍。对妊娠127天的有仪器监测的羊胎儿进行研究，在颈动脉闭塞30分钟和再灌注24小时后进行观察。分组包括假手术安慰剂对照组（n = 5）、缺血安慰剂组（n = 6）、缺血抗IL-1β抗体组（n = 7）和假手术对照抗体组（n = 2）的动物。在缺血后15分钟和4小时，对同一假手术或缺血组的胎儿静脉内给予安慰剂（0.154M氯化钠）或抗白细胞介素-1β单克隆抗体（5.1±0.6mg/kg）进行全身输注。通过酶联免疫吸附测定法（ELISA）测量胎儿血浆、脑脊液和顶叶大脑皮质中白细胞介素-1β蛋白和抗白细胞介素-1β单克隆抗体的浓度。使用α-氨基异丁酸在多个脑区的血脑转运常数（Ki）来量化血脑屏障通透性。还通过蛋白质免疫印迹法在顶叶大脑皮质中测量白细胞介素-1β蛋白，并在多个脑区测量紧密连接蛋白。缺血再灌注后大脑皮质白细胞介素-1β蛋白增加（P<0.001）。在输注抗白细胞介素-1β单克隆抗体后，血浆抗白细胞介素-1β单克隆抗体升高（P<0.001），脑内抗白细胞介素-1β单克隆抗体水平更高（P<0.03），并且单克隆抗体治疗组中的白细胞介素-1β蛋白浓度（P<0.03）和蛋白表达（P<0.001）低于安慰剂治疗的缺血再灌注组。单克隆抗体输注减轻了全脑缺血再灌注相关的Ki增加（P<0.04），并且Ki与顶叶皮质中的抗白细胞介素-1β单克隆抗体浓度呈负线性相关（r = -0.65，P<0.02），但对紧密连接蛋白表达影响很小。我们得出结论，缺血后全身输注抗白细胞介素-1β单克隆抗体可导致脑内摄取抗白细胞介素-1β抗体，并减轻缺血再灌注相关的白细胞介素-1β蛋白上调以及胎儿全脑血脑屏障通透性增加。促炎细胞因子白细胞介素-1β导致胎儿缺血后血脑屏障功能受损。
