Coughlan C M, McManus C M, Sharron M, Gao Z, Murphy D, Jaffer S, Choe W, Chen W, Hesselgesser J, Gaylord H, Kalyuzhny A, Lee V M, Wolf B, Doms R W, Kolson D L
Department of Pathology and Laboratory Medicine University of Pennsylvania Medical Center, Philadelphia, PA 19104, USA.
Neuroscience. 2000;97(3):591-600. doi: 10.1016/s0306-4522(00)00024-5.
Functional chemokine receptors and chemokines are expressed by glial cells within the CNS, though relatively little is known about the patterns of neuronal chemokine receptor expression and function. We developed monoclonal antibodies to the CCR1, CCR2, CCR3, CCR6, CXCR2, CXCR3 and CXCR4 chemokine receptors to study their expression in human fetal neurons cultured from brain tissue as well as the clonally derived NT2.N human neuronal cell line (NTera 2/cl.D1). Specific monoclonal antibody labeling demonstrated expression of CCR2, CXCR2, CXCR3 and CXCR4 on neurons from both sources. Co-labeling studies revealed strong expression of CXCR3 and CXCR4 on both dendritic and axonal processes, with a weaker expression of CXCR2 and CCR2. Reverse transcriptase-polymerase chain reaction analysis of pure NT2.N neurons confirmed RNA expression for CCR2, CXCR2, CXCR3 and CXCR4. No changes in the neuronal labeling pattern of chemokine receptor expression were noted when NT2.N neurons were grown on a supporting layer of astrocytes, again consistent with similar patterns seen in primary human fetal brain cultures. Analysis of single-cell calcium transients revealed a robust response to stromal derived factor-1alpha (CXCR4) and melanocyte growth-stimulating activity (CXCR2), and variable response to monocyte chemoattractant protein-1 (CCR2) or interferon-gamma inducible protein-10 (CXCR3). Finally, we detected the release of monocyte chemoattractant protein-1 from pure cultures of NT2.N neurons, but not undifferentiated NT2 cells. These data indicate that individual neurons may not only co-express multiple functional chemokine receptors, but also that neurons themselves produce chemokines which may influence cellular function within the central nervous system.
功能性趋化因子受体和趋化因子由中枢神经系统内的神经胶质细胞表达，不过对于神经元趋化因子受体的表达模式和功能了解相对较少。我们研发了针对CCR1、CCR2、CCR3、CCR6、CXCR2、CXCR3和CXCR4趋化因子受体的单克隆抗体，以研究它们在从脑组织培养的人类胎儿神经元以及克隆衍生的NT2.N人类神经元细胞系（NTera 2/cl.D1）中的表达情况。特异性单克隆抗体标记显示，CCR2、CXCR2、CXCR3和CXCR4在这两种来源的神经元上均有表达。共标记研究揭示，CXCR3和CXCR4在树突和轴突上均有强烈表达，而CXCR2和CCR2的表达较弱。对纯NT2.N神经元进行的逆转录聚合酶链反应分析证实了CCR2、CXCR2、CXCR3和CXCR4的RNA表达。当NT2.N神经元生长在星形胶质细胞支持层上时，未观察到趋化因子受体表达的神经元标记模式有变化，这再次与人类胎儿脑原代培养中观察到的类似模式一致。单细胞钙瞬变分析显示，对基质衍生因子-1α（CXCR4）和黑素细胞生长刺激活性（CXCR2）有强烈反应，对单核细胞趋化蛋白-1（CCR2）或干扰素-γ诱导蛋白-10（CXCR3）有可变反应。最后，我们检测到NT2.N神经元纯培养物中释放单核细胞趋化蛋白-1，但未分化的NT2细胞则未释放。这些数据表明，单个神经元不仅可能共表达多种功能性趋化因子受体，而且神经元自身还产生趋化因子，这可能会影响中枢神经系统内的细胞功能。
