Mice resistant to experimental autoimmune encephalomyelitis have increased thymic expression of myelin basic protein and increased MBP specific T cell tolerance.

The relationship between expression of the autoantigens in thymi and susceptibility to autoimmune disease was determined in the experimental autoimmune encephalomyelitis (EAE) model. In two different sets of MHC congenic strains of mice characterized by differential susceptibility to EAE, levels of expression of MBP were shown to be higher in the more resistant strain. These data raised the possibility that more central tolerance to MBP may occur in more resistant strains. Differential tolerance was then evidenced by a decrease in T cell responses to MBP 83-102 in the more resistant strains. Together, these data indicate that the list of non-MHC genes involved in susceptibility to autoimmune disease should include genes which regulate expression of autoantigens in thymi.

"Classic" myelin basic proteins are expressed in lymphoid tissue macrophages.

"Classic" myelin basic proteins (MBPs) are demonstrated in lymph nodes of SJL mice by western blot and RT-PCR. Interestingly, expression of these "classic" MBPs was increased during the late relapsing phase of adoptive experimental autoimmune encephalomyelitis (EAE). When splenocytes from SJL mice were separated into macrophage versus B lymphocyte-enriched populations, intact MBP isoforms were demonstrated in the macrophage-enriched population while undetectable in the B lymphocyte-enriched population. RT-PCR demonstrated "classic" MBP transcripts in splenic macrophages, as well as in a macrophage cell line (RAW). The expression of "classic" MBPs in lymphoid tissue macrophages raises the possibility that MBP-specific T cells may be exposed to autoantigen outside the central nervous system (CNS).

Temporal kinetics and cellular phenotype of TNF p55/p75 receptors in experimental allergic encephalomyelitis.

TNF-alpha and LT-alpha are thought to be involved in the immunopathology of CNS demyelinating diseases. Both cytokines induce cellular effects through 55-kDa type-1 receptors (R1) and 75-kDa type-2 receptors (R2). To date, no study has specifically identified the various cell populations that express TNF receptors (TNFR) in the inflammatory and demyelinating mouse model, EAE. Phenotyping the TNFR positive cells is important in determining when and where the ligands may be acting and playing a role in disease pathology. We observed an upregulation of TNF R1 and R2 mRNA in high endothelial venules (HEVs) in the lymph node and CNS before the onset of EAE (preclinical phase). This upregulation of TNFR expression in HEVs was followed by a rapid increase in leukocytes within the CNS after the onset of clinical disease. The temporal kinetics of these data suggest that HEVs become activated early, probably through the release of pro-inflammatory cytokines originating from circulating leukocytes. An increase in TNFR on HEVs would make these cells more susceptible to TNF-induced changes, such as increasing cellular adhesion molecules, thereby further facilitating the trafficking of leukocytes into the CNS parenchyma.

Myelin protein expression is increased in lymph nodes of mice with relapsing experimental autoimmune encephalomyelitis.

Myelin proteins had been thought to be sequestered behind the blood-brain barrier. Recently, however, myelin proteins have been found to be expressed in lymphoid tissues. The myelin basic protein (MBP) gene is embedded within a larger transcription unit called the golli-MBP gene. This larger gene encodes both the "classic" MBPs as well as the structurally related golli-MBPs. In this study, golli-MBP expression in lymph nodes was examined in four different models of relapsing experimental autoimmune encephalomyelitis (rEAE). Disease in these rEAE models was induced by the adoptive transfer of T lymphocytes specific for 18.5-kDa MBP, MBP peptide 83-102, or PLP peptide 139-151 in the SJL/J mouse and the adoptive transfer of T lymphocytes specific for MBP peptide Ac1-9 in the (SJL/J x PL/J)F1 mouse. In all four models, expression of golli-MBP BG21 mRNA was increased two- to fivefold in lymph nodes of mice 45 to 60 days post-transfer. Immunohistochemical analysis indicated that expression occurred principally in macrophages within lymph nodes. Endogenous golli-MBP epitopes within lymph node cells stimulated "classic" MBP 1-44-specific T lymphocytes, and this stimulatory ability resided within the adherent lymph node cell population. An increase in myelin protein expression within lymph nodes during rEAE has implications with regard to intra- and intermolecular epitope spreading. This is the first report describing an increase in target autoantigen expression within lymphoid tissue during an autoimmune disease.

Inducible nitric oxide synthase and nitric oxide production by oligodendrocytes.

It has been previously demonstrated that microglia and astrocytes produce micromolar amounts of nitric oxide in vitro. In this study, we demonstrate that primary rat oligodendrocytes can be stimulated to produce iNOS mRNA as detected by Northern blot and in situ hybridization analysis and a 131-kDa iNOS protein by Western blot analysis; protein was also detected in cells by single- and double-label immunohistochemistry for iNOS and the oligodendrocyte-specific marker CNPase. NO/NOS are produced as a consequence of activation of the gene encoding the inducible nitric oxide synthase as determined by inhibition with actinomycin D and cyclohexamide. The iNOS is functional, leading to calcium/calmodulin-independent NO production in these in vitro cultures.

Differential expression, cytokine modulation, and specific functions of type-1 and type-2 tumor necrosis factor receptors in rat glia.

Tumor necrosis factor alpha (TNF alpha) and lymphotoxin alpha (LT alpha) induce pleiotropic cellular effects through low-affinity 55 kDa type-1 receptors (TNFR1, CD120a) and high-affinity 75 kDa type-2 receptors (TNFR2, CD120b). Both cytokines have potent biological effects on glial cells and are strongly implicated in the pathology of central nervous system (CNS) demyelinating diseases. However, to date, neither constitutive nor cytokine-induced TNFR expression by glial cells have been definitively characterized. We therefore characterized TNF receptors at the molecular, protein, and functional levels in rat astrocytes, microglia, and oligodendrocytes. Northern blotting demonstrated that all three types of glia constitutively transcribed a single TNFR1 mRNA. IFN gamma increased transcript levels in all three types of glia, but TNF alpha increased levels only in oligodendrocytes Microglia constitutively transcribed three distinct TNFR2 mRNAs, levels of which were increased by either IFN gamma or TNF alpha. In contrast, astrocytes and oligodendrocytes constitutively transcribed nearly undetectable levels of TNFR2 mRNAs, and levels were not affected by IFN gamma, TNF alpha, or oligodendrocyte maturation. Immunocytochemical staining of glial cells corroborated Northern data by demonstrating that glia express a parallel pattern of TNFR proteins on their cell surfaces. In co-cultures of microglia plated atop irradiated astrocytes, human TNF alpha (which, on mouse cells, binds TNFR1 exclusively) induced microglial cell proliferation, whereas murine TNF alpha (which binds both TNFRs) did not. Collectively, the data show that microglia, a primary source of TNF alpha at CNS inflammatory sites, express both TNFR1 and TNFR2, whereas astrocytes and oligodendrocytes, whose embryological origin differs from that of microglia, predominantly express TNFR1. TNF alpha increases expression of TNFR1 by oligodendrocytes whereas it increases expression of TNFR2 by microglia. Microglia proliferation data suggest that signals transduced through TNFR2 directly or indirectly inhibit signals transduced through TNFR1. Different patterns of TNFR expression by glia at sites of CNS inflammation may be critical in determining whether TNF has activational, proliferative, or cytotoxic effects on these cells.

Gender differences in autoimmune demyelination in the mouse: implications for multiple sclerosis.

Gender-related differences in experimental allergic encephalomyelitis (EAE) were examined in the SJL mouse with the purpose of characterizing an animal model ideal for the study of gender-related differences in multiple sclerosis (MS). For the model to allow for study of the induction and the effector phase of disease, the adoptive EAE model was characterized. First, the SJL strain was shown to be nonresponsive with regard to the development of antisyngeneic HY-specific responses in females, thereby permitting intergender adoptive transfers of T lymphocytes during EAE induction. Then, when myelin basic protein (MBP)-specific T cells derived from females were adoptively transferred into female and male recipients, female recipients demonstrated a more rapid onset of disease (p = 0.01), greater maximal acute-phase clinical scores (p < 0.0001) and greater mean clinical scores (p < 0.0001) compared with male recipients. When MBP-specific T cells derived from males were adoptively transferred, female recipients again tended to be more severely affected. Histopathologic analysis revealed quantitative differences between genders that paralleled clinical expression. These results document a clear gender-related difference in adoptive EAE in the SJL, with clinical and histopathologic disease greater in females compared with males. This model will be a useful tool for addressing autoimmune mechanisms underlying gender-related differences in MS.

Differential sensitivity to nitric oxide in immortalized, cloned murine oligodendrocyte cell lines.

Using five different immortalized, cloned murine oligodendrocyte cells lines, we have assessed their sensitivity to the nitric oxide donor chemical S-nitroso, N-acetyl-DL-penicillamine (SNAP). The five lines were quite different in their sensitivity to SNAP as determined by assessment of viability, mitochondrial function, single-stranded DNA breaks, DNA/RNA/protein synthesis, morphology, and myelin gene expression. Single-stranded DNA breaks as well as mitochondrial and DNA damage occurred in viable cells. Thus, cell damage was independent of cell death. In the most mature oligodendrocyte cell line, cell morphology was altered and the expression of myelin basic protein mRNA was inhibited by nitric oxide in a time- and dose-dependent fashion. These findings suggest two things. First, oligodendrocytes at different states of differentiation are differentially sensitive to nitric oxide. Second, while not all oligodendrocytes may be destroyed in multiple sclerosis plaques, many could be significantly damaged and thereby nonfunctional in repair processes triggered during the pathology of this disease.