Jakovljevic Marija, Lavrnja Irena, Bozic Iva, Savic Danijela, Bjelobaba Ivana, Pekovic Sanja, Sévigny Jean, Nedeljkovic Nadezda, Laketa Danijela
Institute for Biological Research Sinisa Stankovic, University of Belgrade, Belgrade, Serbia.
Département de Microbiologie-Infectiologie et d'Immunologie, Faculté de Médecine, Université Laval, Québec, QC, Canada.
Front Cell Neurosci. 2017 Oct 30;11:333. doi: 10.3389/fncel.2017.00333. eCollection 2017.
The present study explores tissue and cellular distribution of ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) and the gene and protein expression in rat spinal cord during the course of experimental autoimmune encephalomyelitis (EAE). Given that NTPDase2 hydrolyzes ATP with a transient accumulation of ADP, the expression of ADP-sensitive P2 purinoceptors was analyzed as well. The autoimmune disease was actively induced in Dark Agouti female rats and the changes were analyzed 10, 15 and 29 days after the induction. These selected time points correspond to the onset ( ), peak ( ) and recovery ( ) from EAE. In control animals, NTPDase2 was confined in the white matter, in most of the glial fibrillary acidic protein (GFAP)-immunoreactive () astrocytes and in a considerable number of nestin- cells, while the other cell types were immunonegative. Immunoreactivity corresponding to NTPDase2 decreased significantly at and and then returned to the baseline levels at . The preservation of the proportion of GFAP single-labeled and GFAP/NTPDase2 double-labeled elements along the course of EAE indicated that changes in NTPDase2- occurred at fibrous astrocytes that typically express NTPDase2 in normal conditions. Significant downregulation of P2Y and P2Y receptor proteins at and several-fold induction of P2Y and P2Y receptor proteins at and/or were observed implying that the pathophysiological process in EAE may be linked to ADP signaling. Cell-surface expression of NTPDase2, NTPDase1/CD39 and ecto-5'-nucleotidase (eN/CD73) was analyzed in CD4 T cells of a draining lymph node by fluorescence-activated cell sorting. The induction of EAE was associated with a transient decrease in a number of CD4 NTPDase2 T cells in a draining lymph node, whereas the recovery was characterized by an increase in NTPDase2 cells in both CD4 and CD4 cell populations. The opposite was found for NTPDase1/CD39 and eN/CD73 cells, which slightly increased in number with progression of the disease, particularly in CD4 cells, and then decreased in the recovery. Finally, CD4 NTPDase2 cells were never observed in the spinal cord parenchyma. Taken together, our results suggest that the process of neuroinflammation in EAE may be associated with altered ADP signaling.
本研究探讨了外切核苷酸三磷酸二磷酸水解酶2（NTPDase2）在实验性自身免疫性脑脊髓炎（EAE）大鼠脊髓中的组织和细胞分布以及基因和蛋白表达。鉴于NTPDase2可水解ATP并使ADP短暂积累，因此还分析了ADP敏感的P2嘌呤受体的表达。在暗褐鼠雌性大鼠中主动诱导自身免疫性疾病，并在诱导后10、15和29天分析变化情况。这些选定的时间点分别对应EAE的发病期（ ）、高峰期（ ）和恢复期（ ）。在对照动物中，NTPDase2局限于白质，存在于大多数胶质纤维酸性蛋白（GFAP）免疫反应阳性（ ）的星形胶质细胞以及相当数量的巢蛋白细胞中，而其他细胞类型免疫阴性。与NTPDase2对应的免疫反应性在 和 时显著降低，然后在 时恢复到基线水平。在EAE病程中，GFAP单标记和GFAP/NTPDase2双标记元件的比例保持不变，这表明NTPDase2的变化发生在正常情况下通常表达NTPDase2的纤维星形胶质细胞中。观察到在 时P2Y和P2Y受体蛋白显著下调，在 和/或 时P2Y和P2Y受体蛋白有几倍的诱导，这意味着EAE中的病理生理过程可能与ADP信号传导有关。通过荧光激活细胞分选分析了引流淋巴结中CD4 T细胞表面NTPDase2、NTPDase1/CD39和胞外5'-核苷酸酶（eN/CD73）的表达。EAE的诱导与引流淋巴结中CD4 NTPDase2 T细胞数量的短暂减少有关，而恢复期的特征是CD4和CD4细胞群体中NTPDase2细胞数量增加。对于NTPDase1/CD39和eN/CD73细胞则发现相反的情况，它们的数量随着疾病进展略有增加，尤其是在CD4细胞中，然后在恢复期减少。最后，在脊髓实质中从未观察到CD4 NTPDase2细胞。综上所述，我们的结果表明EAE中的神经炎症过程可能与ADP信号传导改变有关。
