Immunocytochemical characterization of the expression of inducible and constitutive isoforms of nitric oxide synthase in demyelinating multiple sclerosis lesions
Immunocytochemical characterization of the expression of inducible and constitutive isoforms of nitric oxide synthase in demyelinating multiple sclerosis lesions
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DOI：
10.1097/00005072-199701000-00002
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发表时间：
1997-01-01
影响因子：
3.2
通讯作者：
VanderValk, P
中科院分区：
文献类型：
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作者：
DeGroot, CJA;Ruuls, SR;VanderValk, P
在多发性硬化症 (MS) 和对照脑和脊髓的组织切片中确定诱导型和组成型一氧化氮合酶 (iNOS/cNOS) 的细胞定位和分布。使用特定的 iNOS 和 cNOS 导向抗体应用免疫细胞化学技术。此外，还进行了 NADPH-心肌黄酶组织化学分析。为了确定 iNOS、eNOS 和 NADPH 心肌黄酶阳性细胞的身份，使用巨噬细胞标记物 KP1 (CD68) 和星形胶质细胞标记物胶质纤维酸性蛋白 (GFAP) 对组织切片进行单染色和双染色。使用中性脂质油红 O (ORO) 染色来确定髓磷脂分解和脱髓鞘的面积。此外，从活动性脱髓鞘 MS 病变中分离出的巨噬细胞进行 iNOS、cNOS、KP1 和 ORO 染色。在活动性多发性硬化症病变中，仅在活动性脱髓鞘区域内分布的血管周围和实质巨噬细胞中发现了强烈的 iNOS 免疫反应性。在这些活动性多发性硬化症病变中，在巨噬细胞中也发现了 cNOS 免疫反应性。从活动性多发性硬化症病变中分离出的巨噬细胞也显示出 iNOS 和 eNOS 的免疫反应性。此外，这些分离的巨噬细胞在体外产生一氧化氮（NO；>30μM）。在活动性 MS 病变中的 KP1 阳性血管周围和实质巨噬细胞以及 GFAP 阳性反应性星形胶质细胞以及位于慢性活动性 MS 病变超细胞边缘的反应性星形胶质细胞中检测到 NADPH 心肌黄酶活性。在活动性和慢性活动性 MS 病变中均检测到 cNOS 阳性反应性星形胶质细胞。在慢性活动性病变内部，一些残留的巨噬细胞呈弱 iNOS 阳性。在对照脑和脊髓中未检测到 iNOS 免疫反应性。这些结果表明，人类巨噬细胞能够产生自由基一氧化氮（NO），这可能有助于少突胶质细胞的细胞毒性以及多发性硬化症大脑和脊髓中髓磷脂的破坏。
The cellular localization and distribution of inducible and constitutive nitric oxide synthase (iNOS/cNOS) was determined in tissue sections from multiple sclerosis (MS) and control brain and spinal cord. Immunocytochemical techniques were applied using specific iNOS- and cNOS-directed antibodies. In addition, NADPH-diaphorase histochemistry was performed. To establish the identity of iNOS-, eNOS- and NADPH-diaphorase-positive cells single and double staining was performed on tissue sections with the macrophage marker KP1 (CD68) and with the astrocyte marker glial fibrillary acidic protein (GFAP). Areas of myelin breakdown and demyelination were determined using a staining for neutral lipids, Oil Red O (ORO). Furthermore, macrophages isolated from active demyelinating MS lesions were stained for iNOS, cNOS, KP1 and ORO. In active MS lesions strong iNOS immunoreactivity was found exclusively in perivascular and parenchymal macrophages distributed within regions of active demyelination.In these active MS lesions immunoreactivity for cNOS was also found in macrophages. Macrophages isolated from active MS lesions also showed immunoreactivity for iNOS and eNOS. Moreover, these isolated macrophages produced nitric oxide (NO; >30 mu M) in vitro. NADPH-diaphorase activity was detected in KP1-positive perivascular and parenchymal macrophages and in GFAP-positive reactive astrocytes in active MS lesions and in reactive astrocytes located in the hypercellular rims of chronic active MS lesions. cNOS-positive reactive astrocytes were detected in both active and chronic active MS lesions. Inside chronic active lesions some residual macrophages were weakly iNOS-positive. In control brain and spinal cord no iNOS immunoreactivity could be detected. These results suggests an important role for human macrophages capable of producing the free radical nitric oxide (NO), which may contribute to the cytoxicity of oligodendrocytes and destruction of myelin in MS brain and spinal cord.