Glial reactions and degeneration of myelinated processes in spinal cord gray matter in chronic experimental autoimmune encephalomyelitis.

Multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) result in inflammatory white matter lesions in the CNS. However, information is sparse with regard to the effects of autoimmune demyelinating disease on gray matter regions. Therefore, we studied the late effects of chronic EAE in C57BL/6 mice on the spinal cord gray matter using immunohistochemistry. Here, EAE induced marked astrocytic, microglial, and macrophage activation in the ventral horn gray matter, without any motoneuron loss. Activated caspase-3 was also increased in the ventral horn gray matter. Furthermore, activated poly (ADP-ribose) polymerase (PARP), another apoptotic marker, co-localized with myelin basic protein (MBP) of oligodendrocyte processes, but not with the oligodendroglial cell body marker, adenomatous polyposis coli gene clone CC1 (APC-CC1), or with neurofilament marker (RT-97) or synaptophysin of axonal arbors. However, there was no associated increase in the number of terminal deoxynucleotidyl transferase (TdT) mediated-dUTP nick end labeling positive nuclei in the spinal cord gray matter of EAE mice. In addition, co-localization of MBP and the low-affinity neurotrophin receptor, p75, was demonstrated, further supporting the notion of apoptotic oligodendrocyte process degeneration in the gray matter of EAE mice.

Hormone-based therapies in MS.

It is well established in multiple sclerosis (MS) that the frequency of relapses decreases in late pregnancy, but increases in the post-partum period. This protective effect is probably due to hormonal and immune response changes induced by pregnancy. Oestrogens may be responsible, at least in part, for this protective effect. The potential use of oestrogens as therapy for MS is discussed.

Onset of multiple sclerosis associated with anti-TNF therapy.

Therapies aimed at inhibiting tumor necrosis factor (TNF), a proinflammatory cytokine implicated in autoimmune disease are effective, especially for rheumatoid arthritis. We report a patient with new onset MS closely associated with the initiation of anti-TNF therapy for juvenile rheumatoid arthritis. It is possible that the inhibition of TNF triggered MS in this individual.

Sex hormones in experimental autoimmune encephalomyelitis: implications for multiple sclerosis.

For decades, it has been known that females are more susceptible than males to multiple sclerosis (MS). It has also long been appreciated that during late pregnancy there is a decrease in MS disease activity. Interestingly, these two observations have also been made in an extensively used animal model for MS, experimental autoimmune encephalomyelitis (EAE) in SJL mice. Female mice are more susceptible to disease than male mice, and there is an improvement in disease during late pregnancy. In this review, the role of sex hormones in each of these two observations is characterized in this EAE model using castration and exogenous hormone treatment strategies. The gender difference in EAE susceptibility is due primarily to a protective effect of testosterone in male mice. The decrease in disease severity during late pregnancy appears to be due at least in part to high levels of estriol, which characterize this time period.

Testosterone acts directly on CD4+ T lymphocytes to increase IL-10 production.

Males are less susceptible than females to experimental autoimmune encephalomyelitis and many other autoimmune diseases. Gender differences in cytokine production have been observed in splenocytes of experimental autoimmune encephalomyelitis mice stimulated with myelin proteins and may underlie gender differences in susceptibility. As these differences should not be limited to responses specific for myelin proteins, gender differences in cytokine production upon stimulation with Ab to CD3 were examined, and the mechanisms were delineated. Splenocytes from male mice stimulated with Ab to CD3 produced more IL-10 and IL-4 and less IL-12 than those from female mice. Furthermore, splenocytes from dihydrotestosterone (DHT)-treated female mice produced more IL-10 and less IL-12 than those from placebo-treated female mice, whereas there was no difference in IL-4. IL-12 knockout mice were then used to determine whether changes in IL-10 production were mediated directly by testosterone vs indirectly by changes in IL-12. The results of these experiments favored the first hypothesis, because DHT treatment of female IL-12 knockout mice increased IL-10 production. To begin to delineate the mechanism by which DHT may be acting, the cellular source of IL-10 was determined. At both the RNA and protein levels, IL-10 was produced primarily by CD4+ T lymphocytes. CD4+ T lymphocytes were then shown to express the androgen receptor, raising the possibility that testosterone acts directly on CD4+ T lymphocytes to increase IL-10 production. In vitro experiments demonstrated increased IL-10 production following treatment of CD4+ T lymphocytes with DHT. Thus, testosterone can act directly via androgen receptors on CD4+ T lymphocytes to increase IL-10 gene expression.

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).

Decreased IL-12 production underlies the decreased ability of male lymph node cells to induce experimental autoimmune encephalomyelitis.

Myelin basic protein (MBP)-specific T lymphocytes from male SJL mice were shown to be less encephalitogenic than MBP-specific T lymphocytes from females. Mechanisms underlying this gender difference in the induction phase of EAE were examined. Following immunization with MBP, draining lymph nodes contained fewer cells, and Ag-specific proliferative responses were decreased in males as compared with females. These gender differences in the proliferative response were not unique to MBP-specific responses since they were also observed after immunization with hen eggwhite lysozyme. Short-term MBP-specific T cell lines derived from females and males mapped with identical specificity, indicating no defect in the ability of male APCs to process Ag. Interestingly, IL-12 and IFN-gamma production was decreased following Ag-specific stimulation of draining lymph node cells (LNC) from males as compared with females, but IL-10 and IL-4 were no different. While male-derived LNCs were less encephalitogenic than female derived LNCs, cotransfer and coculture of male LNCs with female LNCs demonstrated that male LNCs were not immunosuppressive. Administration of IL-12 to LNCs from male mice enhanced encephalitogenicity. These data indicate that deficient endogenous IL-12 production within draining LNCs of male SJL mice is central to gender differences in the induction phase of experimental autoimmune encephalomyelitis.

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.

Estriol ameliorates autoimmune demyelinating disease: implications for multiple sclerosis.

OBJECTIVE: To evaluate the use of estriol in the treatment of experimental autoimmune encephalomyelitis (EAE) and other cell mediated autoimmune diseases. BACKGROUND: Experimental autoimmune encephalomyelitis is a T helper 1 (Th1)-mediated autoimmune demyelinating disease that is a useful model for the study of immune responses in MS. Interestingly, both EAE and MS have been shown to be ameliorated during late pregnancy. METHODS: Estriol, progesterone, and placebo pellets were implanted in mice during the effector phase of adoptive EAE. Disease scores were compared between treatment groups, and autoantigen-specific humoral and cellular responses were examined. RESULTS: Estriol treatment reduced the severity of EAE significantly compared with placebo treatment whereas progesterone treatment had no effect. Estriol doses that induced serum estriol levels that approximated estriol levels during late pregnancy were capable of ameliorating disease. Estriol-treated EAE mice had significantly higher levels of serum antibodies of the immunoglobulin (Ig) G1 isotype specific for the autoantigen myelin basic protein (MBP). Further, MBP-specific T-lymphocyte responses from estriol-treated EAE mice were characterized by significantly increased production of the Th2 cytokine interleukin 10 (IL-10). T lymphocytes were shown to be the primary source of IL-10 within antigen-stimulated splenocyte populations. CONCLUSIONS: Estriol as a hormone involved in immune changes during pregnancy may provide a basis for the novel therapeutic use of estriol for MS and other putative Th1-mediated autoimmune diseases that improve during late pregnancy.

Myelin protein expression in lymphoid tissues: implications for peripheral tolerance.

During chronic relapsing experimental autoimmune encephalomyelitis (EAE), T lymphocytes specific for myelin protein epitopes are stimulated in vivo. When epitopes are unique from the disease-initiating myelin protein epitope, this phenomenon has been termed "epitope spreading". These T-lymphocyte responses have been detected primarily in lymph node and spleen during the relapsing phase of disease. If myelin proteins are sequestered behind the blood brain barrier, a fundamental question arises: where does the in vivo stimulation of T lymphocytes occur during relapsing EAE? While it has been thought that epitope spreading may occur within the central nervous system (CNS), here we present data supporting a novel hypothesis. Epitope spreading during EAE may not occur within the CNS, but rather within lymphoid tissues. Both myelin basic protein (MBP) and proteolipid protein (PLP) are expressed at the RNA and protein level in lymph node, thymus and spleen of SJL mice with relapsing EAE. This myelin protein expression occurs within T lymphocytes, B lymphocytes and macrophages. Further, T-lymphocyte lines from SJL mice specific for the immunodominant and subdominant epitopes of MBP and PLP can recognize endogenous protein within cells derived from lymphoid tissues. Thus, immunologically relevant myelin proteins are endogenously produced and presented within lymphoid tissues. The hypothesis that epitope spreading occurs within lymphoid tissues would explain how myelin protein-specific T lymphocytes become activated outside the CNS to allow their passage through the blood brain barrier to form new CNS lesions during relapses.

Antisense knockdown of inducible nitric oxide synthase inhibits induction of experimental autoimmune encephalomyelitis in SJL/J mice.

We used an antisense oligodeoxynucleotide (ODN) complementary to inducible nitric oxide synthase (iNOS) to inhibit experimental autoimmune encephalomyelitis (EAE) in female SJL/J mice, an animal model for multiple sclerosis. The antisense ODN was administered intraventricularly to mice daily for 10 days beginning at the time of adoptive transfer of myelin basic protein-specific T lymphocytes. The antisense ODN treatment significantly reduced the clinical score of EAE and blocked iNOS mRNA and protein synthesis, as well as iNOS enzyme activity within the central nervous system. The levels of nitric oxide and cyclic guanosine monophosphate were also significantly reduced by the antisense ODN treatment. Neither sense nor random ODN affected clinical EAE or iNOS expression. Moreover, the protein and enzyme activity level of constitutive neuronal nitric oxide synthase was not affected by the antisense ODN. Thus, we have shown that the iNOS antisense ODN specifically blocked iNOS expression and ameliorated the induction of EAE.

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.

Testosterone therapy ameliorates experimental autoimmune encephalomyelitis and induces a T helper 2 bias in the autoantigen-specific T lymphocyte response.

Female SJL mice are more susceptible than male mice to experimental autoimmune encephalomyelitis (EAE) induced by myelin basic protein (MBP)-specific T lymphocytes. In the present study, we examined mechanisms involved in this gender-related difference in disease susceptibility. MBP-specific T lymphocytes derived from spleens of males during the effector phase of adoptive EAE produced significantly higher levels of IL-10, an anti-inflammatory cytokine in EAE. A protective effect of testosterone was then shown. Females implanted with dihydrotestosterone pellets demonstrated a significantly less severe course of EAE as compared with females implanted with placebo pellets. Finally, MBP-specific T lymphocytes derived from dihydrotestosterone-implanted females produced significantly higher levels of IL-10 than those from placebo. Together these data indicate that testosterone exerts a protective effect in EAE that is mediated at least in part by enhanced production of IL-10 by autoantigen-specific T lymphocytes.

Gender differences of inducible nitric oxide production in SJL/J mice with experimental autoimmune encephalomyelitis.

We identified gender related differences of inducible nitric oxide synthase (iNOS) expression and NO production in mice with experimental autoimmune encephalomyelitis (EAE). When myelin basic protein-specific T-lymphocytes derived from female mice were transferred, the female recipients developed more severe EAE and expressed higher levels of iNOS and NO than male recipients. When the T-lymphocytes derived from males were transferred, severe EAE was induced in neither female or male recipients and neither iNOS nor NO were detectable. These data show an association between No production and EAE severity, suggesting a possible role of NO in the pathogenesis of EAE.

Multiple sclerosis: re-expression of a developmental gene in chronic lesions correlates with remyelination.

Central nervous system tissue from multiple sclerosis and non-multiple sclerosis subjects was studied for the expression of exon 2 myelin basic protein gene products at the protein and message levels by immunocytochemistry and in situ hybridization, respectively. The exon 2-encoded protein sequence is normally expressed during development (myelination) within the 21.5- and 20.2-kd isoforms of myelin basic protein and is downregulated in the adult central nervous system where the 18.5- and 17.2-kd isoforms predominate, the latter devoid of exon 2 owing to alternative splicing. Exon 2 myelin basic protein gene products were readily demonstrable in multiple sclerosis samples, the highest levels correlating with remyelination in chronic lesions while normal adult central nervous system and non-multiple sclerosis material showed very low levels and fetal human central nervous system tissue (a positive control) showed high levels. We conclude that recapitulation of ontogenetic events during myelin repair accounts for the increased expression of the exon 2-encoded protein sequence in the adult central nervous system during multiple sclerosis, an event that might underly the previously observed T-cell activation to this protein sequence during relapses.

Exon 2 containing myelin basic protein (MBP) transcripts are expressed in lesions of experimental allergic encephalomyelitis (EAE).

Exon 2 containing myelin basic protein (MBP) transcripts are expressed during developmental myelination in mice and humans, and during remyelination subsequent to virally induced demyelination in adult mice. Since remyelination characterizes CNS lesions during experimental allergic encephalomyelitis (EAE) and multiple sclerosis (MS), we investigated whether exon 2 containing isoforms of MBP are expressed in EAE lesions during relapsing disease. Exon 2 containing MBP transcripts were detected by in situ hybridization in 17 of 52 EAE mice and in 16 of 30 mice at the peak of the first or second episode of paralysis. Thus exon 2 containing MBP transcripts are expressed in lesions of the CNS during active phases of chronic relapsing autoimmune disease. Implications of these findings with respect to future therapies aimed toward enhancing remyelination in EAE and, possibly MS, are discussed.

Antisense blockade of inducible nitric oxide synthase in glial cells derived from adult SJL mice.

Increasing evidence suggests a correlation between cytokine-induced nitric oxide synthase (iNOS) and demyelination in Multiple sclerosis (MS). Inhibition of iNOS may therefore be a novel therapeutic approach in MS. To test an antisense oligodeoxynucleotide (ODN) knockdown strategy for inhibiting iNOS, we used lipopolysaccharide (LPS) together with gamma-interferon (IFN-gamma) to induce iNOS in adult mouse mixed glial cell cultures. We administered an iNOS-derived antisense phosphorothiorate oligodeoxynucleotide (S-ODN) to block the induction. The antisense ODN treatment resulted in significant inhibition of LPS and IFN-gamma induced iNOS mRNA and protein expression. It also inhibited nitric oxide (NO) and cyclic GMP (cGMP) production in a dose dependent fashion. Sense and random S-oligo had no effect in any of these studies. These data indicate the efficacy and specificity of the antisense oligodeoxynucleotide approach in inhibiting iNOS in glial cells.

Epitope spreading occurs in active but not passive EAE induced by myelin basic protein.

Using experimental allergic encephalomyelitis, EAE, as a model for the study of autoimmune demyelinating disease in the CNS, previous studies have indicated that spread may occur with respect to the specificity of T cell responses during disease. This phenomenon, known as epitope spreading, is central to therapeutic strategies in multiple sclerosis (MS). However, in EAE, the clinical course, neuropathology and immunopathogenesis vary depending upon host factors and the method of disease induction. Since passive EAE in SJL/J mice resembles MS clinically and neuropathologically, this model was chosen to study the immune phenomenon of epitope spreading. T cells specific for whole 18.5 kDa MBP were used to initiate disease since MBP or one of its naturally occurring cleavage fragments may initiate a more physiological immune response than one generated to an artificially designed synthetic peptide. While a progressive increase in T cell responsiveness specific for the immunodominant MBP 87-106 region was observed during disease, there was no evidence of either intermolecular epitope spreading to the immunodominant region of proteolipid protein (PLP) 139-151 or of intramolecular epitope spreading to the exon 2 encoded region of MBP, which is spliced out of 18.5 kDa MBP. In addition there was no shift in immunodominance toward the subdominant MBP 16-35 region during disease. In contrast during active EAE induced by MBP, epitope spreading to the immunodominant epitope of PLP, 139-151, was observed. These data demonstrate that immune responses generated during passive versus active EAE may differ, and suggest that significant epitope spreading does not occur in chronic relapsing demyelinating disease initiated with T cells specific for whole MBP in the absence of exogenous antigen, complete Freund's adjuvant and pertussis. Implications of these findings with regard to epitope spreading in MS are discussed.

Human T lymphocytes specific for the immunodominant 83-99 epitope of myelin basic protein: recognition of golli MBP HOG 7.

Myelin basic protein (MBP) is a candidate autoantigen in the disease multiple sclerosis. Although MBP was thought to be sequestered behind the blood-brain barrier, isoforms of MBPs have recently been demonstrated in lymphoid tissues. These isoforms, termed golli MBPs, contain sequences that are shared with "classic" MBP within the CNS. In the present study, we have determined that epitopes within golli MBP isoforms may be recognized by human T lymphocyte clones specific for classic MBP. Ten of 12 T-cell clones recognized golli MBP. Although 11 clones were specific for the immunodominant 83-99 sequence, the clones differed with respect to human leukocyte antigen (HLA) restriction, T-helper phenotype, cytolytic activity, and T-cell receptor usage. Greater responses to classic MBP than to golli MBP suggested a difference in the ability of the two proteins to be processed and to present epitopes therein. These data advance the hypothesis that golli MBP sequences expressed within lymphoid tissues may be recognized by classic MBP-specific T lymphocytes during central or peripheral tolerance.