IMMUNOCYTOCHEMICAL CHARACTERIZATION OF THE EXPRESSION OF INDUCIBLE AND CONSTITUTIVE ISOFORMS OF NITRIC-OXIDE SYNTHASE IN DEMYELINATING MULTIPLE-SCLEROSIS LESIONS

The cellular localization and distribution of inducible and constituti ve nitric oxide synthase (iNOS/cNOS) was determined in tissue sections from multiple sclerosis (MS) and control brain and spinal cord. Immun ocytochemical techniques were applied using specific iNOS- and cNOS-di rected antibodies. In addition, NADPH-diaphorase histochemistry was pe rformed. To establish the identity of iNOS-, eNOS- and NADPH-diaphoras e-positive cells single and double staining was performed on tissue se ctions with the macrophage marker KP1 (CD68) and with the astrocyte ma rker 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 demye linating MS lesions were stained for iNOS, cNOS, KP1 and ORO. In activ e MS lesions strong iNOS immunoreactivity was found exclusively in per ivascular and parenchymal macrophages distributed within regions of ac tive demyelination. In these active MS lesions immunoreactivity for cN OS was also found in macrophages. Macrophages isolated from active MS lesions also showed immunoreactivity for iNOS and eNOS. Moreover, thes e isolated macrophages produced nitric oxide (NO; >30 mu M) in vitro. NADPH-diaphorase activity was detected in KP1-positive perivascular an d parenchymal macrophages and in GFAP-positive reactive astrocytes in active MS lesions and in reactive astrocytes located in the hypercellu lar rims of chronic active MS lesions. cNOS-positive reactive astrocyt es were detected in both active and chronic active MS lesions. Inside chronic active lesions some residual macrophages were weakly iNOS-posi tive. In control brain and spinal cord no iNOS immunoreactivity could be detected. These results suggests an important role for human macrop hages capable of producing the free radical nitric oxide (NO), which m ay contribute to the cytoxicity of oligodendrocytes and destruction of myelin in MS brain and spinal cord.