T-cell reactivity to multiple myelin antigens in multiple sclerosis patients and healthy controls

Myelin proteins, including myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) are candidate autoantigens in MS. It is not clear whether MS patients show a predominant reactivity to one or several myelin antigens. We evaluated the IFN-gamma production induced by MBP and MOG and selected MBP-, MOG- and PLP-peptides in MS patients and healthy controls using the IFN-gamma ELISPOT assay. Most MS patients and healthy controls showed a heterogeneous anti-myelin T-cell reactivity. Interestingly in MS patients a positive correlation was found between the anti-MOG and anti-MBP T-cell responses. No myelin peptide was preferentially recognized among the peptides tested (MBP 84-102, 143-168, MOG 1-22, 34-56, 64-86, 74-96, PLP 41-58, 184-199, 190-209). In addition the frequency of IL2R+ MBP reactive T-cells was significantly increased in blood of MS patients as compared with healthy subjects, indicating that MBP reactive T-cells exist in an in vivo activated state in MS patients. Most of the anti-MBP T-cells were of the Th1-type because reactivity was observed in IFN-gamma but not in IL-4 ELISPOT-assays. Using Th1 (IL-12) and Th2 (IL-4) promoting conditions we observed that the cytokine secretion pattern of anti-MBP T-cells still is susceptible to alteration. Our data further indicate that precursor frequency analysis of myelin reactive T-cells by proliferation-based assays may underestimate the true frequency of myelin specific T-cells significantly.     

Expression of only one myelin basic protein allele in mouse is compatible with normal myelination

Myelin deficiency (mld) is an autosomal recessive mutation in mice characterized by a severe myelin deficit in the central nervous system (CNS). The primary defect in mld is a reduction of the synthesis of the myelin basic protein (MBP) and probably lies in a regulatory element of the MBP gene. In young mld heterozygotes, the MBP mRNA and MBP levels are intermediate. In order to study whether reduced levels of MBP gene expression affect myelination, we determined the levels of MBP mRNA and MBP itself in mld heterozygous and control brains, at different ages during development. Total proteins and MBP were also measured in myelin isolated at 25 and 85 days of age. Myelin proteins were analyzed by SDS-PAGE. In addition, we carried out a morphometric analysis on 25- and 85-day-old optic nerves. Our results indicate that in spite of a roughly 50% reduction of MBP gene expression (compared to controls), the amounts of myelin isolated and the concentration of MBP in myelin were normal in heterozygous brains. Nevertheless, morphometric analyses of optic nerves, which myelinate later than the brainstem, showed thinner myelin sheaths in 25-day-old heterozygotes when compared to controls. This difference disappeared at 85 days of age. These results indicate that normal mice synthesize MBP in excess. The synthesis of this extramyelinic pool of MBP represents a safety factor allowing normal myelination to proceed even when MBP synthesis is severely reduced. In mld heterozygotes, a 30-50% reduction of this rate of synthesis can represent a limiting factor and locally delay myelin deposition without affecting the overall myelin content or myelin composition in heterozygous adult brains.     

Immunocytochemical localization of MAG, MBP and P0 protein in acute and relapsing demyelinating lesions of Theiler''s virus infection

Acute demyelinating and relapsing demyelinating lesions from spinal cords of mice infected with the WW strain of Theiler's encephalomyelitis virus (TMEV) were studied immunocytochemically with antisera to various myelin constituents. Acute lesions were studied for differences in the distribution of myelin basic protein (MBP) and myelin associated glycoprotein (MAG). Relapsing lesions, characterized by demyelination of areas previously remyelinated by Schwann cells, were studied for differences in the distribution of P0 and MAG. In both instances the earliest lesions were characterized by preferential disappearance of MBP and P0 respectively when compared to MAG. In well-developed lesions, MAG, MBP and P0 were absent in essentially equal proportion. These observations are in agreement with previous findings suggesting a primary loss of myelin rather than a direct attack on oligodendrocytes as the main pathogenetic mechanism of demyelination in this viral model.     

Myelin/oligodendrocyte glycoprotein expression during development in normal and myelin-deficient mice

The myelin/oligodendrocyte glycoprotein (MOG) is identified by monoclonal antibody 8-18C5. MOG is localized on the surface of myelin and oligodendrocyte processes. Recently, several studies have shown that MOG plays an important role as a target for antibody-induced demyelination. In the present study, we investigated MOG expression in the brains of normal and myelin-deficient (mld) mutant mice during development. By gel electrophoresis and immunoblotting, we observed the developmental pattern of two closely migrating bands, with apparent molecular masses of 26 and 28 kilodaltons. Their concentrations increased coordinately during the most active phase of myelin and myelin basic protein (MBP) synthesis. Between 20 and 25 days of age, the MOG developmental pattern superimposed that of MBP as well as myelin yields. In mld mutant mice, which are affected by a severe deficit of MBP synthesis, MOG was present at reduced levels (40% of controls at 60 days of age). At 85 days of age, mld mice exhibited increased concentrations of MBP, and myelin was better compacted. At this age, MOG concentrations increased and reached 70% of controls. These results suggest that MOG could play a role in the maintenance or completion of the myelin sheath. Its expression level may be modulated by the presence of compact myelin and/or MBP in the myelin sheath.     

Rag-1-dependent cells are necessary for 1,25-dihydroxyvitamin D(3) prevention of experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is a demyelinating disease involving genetic and environmental risk factors. Geographic, genetic, and biological evidence suggests that one environmental risk factor may be lack of vitamin D. Here, we investigated how 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) inhibits experimental autoimmune encephalomyelitis (EAE), an MS model. The experiments used adoptive transfer of TCR-transgenic (TCR1) cells specific for myelin basic protein (MBP) peptide into unprimed recipients. When unprimed TCR1 splenocytes were transferred, and the recipients were immunized with peptide, the mock-treated mice developed EAE, but the 1,25-(OH)(2)D(3)-treated recipients remained disease-free. Both groups had TCR1 T cells that proliferated in response to MBP Ac1-11 and produced IFN-gamma but not IL-4 in the lymph node. In the central nervous system (CNS), the mock-treated mice had activated TCR1 T cells that produced IFN-gamma but not IL-4, while the 1,25-(OH)(2)D(3)-treated mice had TCR1 T cells with a non-activated phenotype that did not produce IFN-gamma or IL-4. When activated TCR1 T cells producing IFN-gamma were transferred into unprimed mice, the mock-treated and the 1,25-(OH)(2)D(3)-treated recipients developed EAE. Likewise, the 1,25-(OH)(2)D(3) did not inhibit Th1 cell IFN-gamma production or promote Th2 cell genesis or IL-4 production in vitro. Finally, the 1,25-(OH)(2)D(3) inhibited EAE in MBP-specific TCR-transgenic mice that were Rag-1(+), but not in animals that were Rag-1-null. Together, these data refute the hypothesis that the hormone inhibits Th1 cell genesis or function directly or through an action on antigen-presenting cells, or promotes Th2 cell genesis or function. Instead, the evidence supports a model wherein the 1,25-(OH)(2)D(3) acts through a Rag-1-dependent cell to limit the occurrence of activated, autoreactive T cells in the CNS.     

Expression of citrullinated proteins in murine experimental autoimmune encephalomyelitis

In this study, we demonstrate for the first time the immunohistochemical expression of citrullinated proteins in the central nervous system (CNS) of mice with myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). By using an established monoclonal antibody (F95) against natural and synthetic citrullinated proteins (Nicholas and Whitaker [2002] Glia 37:328-336), numerous, small, previously unrecognized "patches" of citrullinated proteins were discovered throughout EAE brains, whereas EAE spinal cords showed similar but much larger lesions. On dual color immunofluorescence, these lesions were found to contain citrullinated myelin basic protein (MBP) and were surrounded by astrocytes immunoreactive for both glial fibrillary acidic protein (GFAP) and F95. These lesions became evident about the time when EAE mice became symptomatic and increased in size and number with increasing disease severity. In some sections of spinal cord but not brains of severely debilitated EAE mice, a widespread gliotic response was seen, with astrocytes containing citrullinated GFAP spread throughout the gray and white matter. Western blot analysis of acidic proteins from the brains and spinal cords of EAE mice had higher levels of multiple citrullinated GFAP isoforms compared with controls, with more F95-positive bands in the EAE brains vs. spinal cords. These results raise the possibility that citrullination of both GFAP and MBP may contribute to the pathophysiology of EAE and that the brains of EAE mice may contain more pathology than previously realized.     

Time course of T-cell responses to MOG and MBP in patients with clinically isolated syndromes

CD4(+) T-cell lines (TCLs) from patients with clinically isolated syndromes (CIS) were selected with purified human myelin basic protein (MBP) and recombinant human myelin oligodendrocyte glycoprotein (rhMOG), at onset of neurological symptoms and when patients developed clinically definite multiple sclerosis (CDMS). The epitope specificity of each TCL was mapped with overlapping synthetic peptides. TCLs were assessed for their ability to secrete IFN-gamma, IL-4, and IL-6. Diverse patterns of epitope recognition were observed: (a) recognition of a broad spectrum of MBP peptide epitopes with evidence of shifts over time; (b) an initial T-cell response focused to a restricted segment of the MBP molecule (83-102) that broadened over the course of disease; and (c) persistence of a focused anti-MOG T-cell response. CIS patients who failed to develop CDMS maintained a focused epitope response against two to six MBP epitopes. Most MBP peptide-specific TCLs secreted considerable amounts of IFN-gamma and low amounts of IL-4 and IL-6, whereas anti rhMOG(Igd) peptide-specific TCLs secreted preferentially IL-4 and IL-6. These data raise important issues for the pathogenesis and treatment of multiple sclerosis (MS).     

Quantitative analysis of myelin protein gene expression during development in the rat sciatic nerve.

We determined the temporal profile of expression of the genes encoding the P0 glycoprotein, the myelin-associated glycoprotein (MAG), the myelin basic protein (MBP), the proteolipid protein (PLP), and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in the sciatic nerve of rats. The level of expression of the MAG gene occurred maximally in animals 13 days of age, approximately one week earlier than the peak expression of the MBP and P0 genes. The genes encoding PLP and CNP were not expressed developmentally in a manner that correlated with the myelination of the sciatic nerve. Furthermore, using RNA synthesized in vitro, specific for each of the myelin protein genes, we have determined the absolute amounts of messenger RNA for the various myelin proteins in total RNA from sciatic nerves. P0 and MBP RNA were present at very high levels, whereas the amount of MAG, PLP and CNP RNA were much less.     

Dysmyelination and reduced myelin basic protein gene expression by oligodendrocytes of SHP-1-deficient mice

We have shown previously that myelin-forming oligodendrocytes express the protein tyrosine phosphatase SHP-1 and that myelin formation was decreased in SHP-1-deficient motheaten mice compared to that in normal littermates. These studies suggested a potential importance for SHP-1 in oligodendrocyte and myelin development. To address further this possibility, we analyzed myelin formation by microscopy and myelin basic protein (MBP) gene expression in motheaten mice at ages when myelination occurs in the developing central nervous system (CNS). Furthermore, we correlate these findings with MBP gene expression in oligodendrocytes grown in vitro. We have found that CNS myelination was significantly reduced in SHP-1-deficient mice relative to their normal littermates at multiple times during the active period of myelination. Under electron microscopy, greater numbers of axons in spinal cords of motheaten mice were either unmyelinated or had thinner myelin sheathes compared to those in matched areas of normal littermates. Accordingly, MBP protein and mRNA levels were reduced in SHP-1-deficient mice compared to that in the CNS of normal littermates. In vitro, O1(+) oligodendrocytes from motheaten mice expressed much less MBP than O1(+) oligodendrocytes of normal littermates indicating an alteration in oligodendrocyte differentiation. The latter correlated with reduced MBP mRNA relative to cerebroside galactosyl transferase (CGT) gene mRNA in SHP-1-deficient oligodendrocytes in purified cultures. We propose that SHP-1 is a critical regulator of developmental signals leading to terminal differentiation and myelin sheath formation by oligodendrocytes.     

T cell and antibody responses in remitting-relapsing experimental autoimmune encephalomyelitis in (C57BL/6 x SJL) F1 mice

To characterize T cell and antibody responses in remitting-relapsing experimental autoimmune encephalomyelitis (RR-EAE), we compared myelin oligodendrocyte glycoprotein (MOG)-induced RR-EAE in C57BL/6 (B6) x SJL (F1) mice and chronic-progressive EAE (CP-EAE) in B6 mice at week 8 p.i. when clinical scores were comparable. Although these two strains exhibited similar inflammation/demyelination pattern and MOG-induced T cell responses, RR-EAE mice produced significantly higher levels of anti-MOG IgG1/IgG2a antibodies. Further, lymphocytes of RR-EAE mice proliferated vigorously to the secondary epitope myelin basic protein (MBP) 1-11. These results support a potential involvement of anti-MOG antibodies and epitope spreading in T cell responses in the development of MOG-induced RR-EAE model.     

Inhibition of allergic encephalomyelitis in marmosets by vaccination with recombinant vaccinia virus encoding for myelin basic protein

A primary demyelinating form of experimental allergic encephalomyelitis (EAE) resembling human multiple sclerosis (MS) occurs in Callithrix jacchus marmosets following immunization with human white matter. Participation of a T-cell immune response against myelin basic protein (MBP) in this disease model is supported by observations of increased reactivity against MBP in PBMC and of adoptive transfer of an inflammatory form of EAE by MBP-reactive T-cells. To evaluate the effects of ectopic presentation of MBP on marmoset EAE, animals were vaccinated prior to induction of EAE by subcutaneous injection of attenuated strains of vaccinia virus genetically engineered to contain either the entire coding sequence for human MBP (vT15) or the equine herpes virus glycoprotein gH gene (vAbT249). Vaccination with vT15 was followed by transient cytoplasmic and surface membrane expression of MBP in circulating PBMC (15–45 days). The onset of clinical EAE after immunization (pi) was markedly delayed in vT15-vaccinated animals (37–97 days pi, n=4) compared to vAbT249-vaccinated controls (14–18 days pi, n=3). Proliferative responses against MBP but not against vaccinia antigens or phytohemagglutinin were suppressed in protected animals. Thus, development of attenuated live viruses carrying genes for myelin antigens could be useful for induction of immunologic tolerance and for modulation of autoimmune demyelination.     

Relative effectiveness of spinal cord and purified myelin basic protein in producing resistance to experimental allergic encephalomyelitis.

Isolated myelin basic protein (MBP) was less effective than an equivalent amount of spinal cord in inducing protection against experimental allergic encephalomyelitis produced by a challenge of either cord, purified myelin or MBP. Complete protection was only obtained when an MBP challenge was preceded by spinal cord treatment. There was a 100% incidence of disease in the guinea pigs pretreated with MBP before challenge with spinal cord or myelin, but the onset was delayed by 3--4 weeks and the disease was less severe than in the controls. Recurrent disease was seen in some control and pretreated animals challenged with spinal cord but not in animals challenged with MBP.     

Increase in anti-astrocyte antibodies in the serum of guinea pigs during active stages of experimental autoimmune encephalomyelitis.

From previous studies on the induction and treatment of experimental autoimmune encephalomyelitis (EAE) in guinea pigs and mice, antibodies have been implicated during both demyelination and remyelination. In the present study, sera from guinea pigs with acute, chronic and myelin basic protein/galactocerebroside (MBP/GC)-treated chronic EAE were evaluated for the presence of anti-glial cell antibodies by immunocytochemical techniques. Antigen specificity was confirmed by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The majority of sera from acute and chronic active EAE animals displayed intense labelling of astrocytes and only weak staining of oligodendrocytes when tested on sections of normal guinea pig brain tissue. In contrast, sera from animals with chronic EAE treated with MBP/GC gave strong labelling of oligodendrocytes and only minor staining of astrocytes. By immunoblotting, astrocyte staining was shown to be due to the presence of antiglial fibrillary acidic protein (GFAP) antibodies. The intense oligodendrocyte staining observed in sections reacted with sera from MBP/GC-treated guinea pigs corresponded well with high titers of serum anti-GC and anti-MBP antibodies measured by an ELISA. It was concluded that the presence of antibodies against astrocytes was possibly related to astrocytic antigens within the disease-inducing emulsion, at least during the initial phases of EAE, and not to their release from the central nervous system of affected animals.     

Myelin proteins in the CNS of 'shaking pups'

Myelin proteins were quantitated in whole tissue and isolated 'myelin fractions' from spinal cord, brainstem and hemispheres of 'shaking pups', a mutation in Springer-Spaniel dogs characterized by hypomyelination of the CNS. The amount of myelin basic protein (MBP) in the brainstem of affected 4-week-old pups was 2.6% of that in age-matched controls, while the levels of 2'3'-cyclic nucleotide phosphodiesterase (CNP) and myelin-associated glycoprotein (MAG) were 10% and 15% of the control levels, respectively. Similar results were obtained in the spinal cord and hemispheres, and the amounts of these proteins in the mutant pups did not change substantially between 4 and 16 weeks of age. The amount of the 21 kDa MBP compared to the 18 kDa MBP was relatively increased in the shaking pups, suggesting that the small amount of myelin formed was immature. The yields of myelin fractions from the mutant pups were very low; e.g., the yield from the brainstems of 4-week-old mutants was only 2.4% of that from age-matched controls and the yield did not increase by 16 weeks. The isolated myelin fractions contained very little MBP (less than 0.5% of total protein) or proteolipid protein, indicating that they were a very immature form of myelin or consisted largely of non-myelin contaminants. MAG in the 'myelin fractions' from the mutant brainstems were 9-15 fold higher and CNP levels were 2-3 fold higher than those in whole homogenates, suggesting that the isolated fractions were enriched in oligodendroglia-derived membranes. Overall, the biochemical results are consistent with a severe hypomyelination of the CNS in which a small amount of immature myelin is formed.     

Cell-mediated immunity to myelin-associated glycoprotein, proteolipid protein, and myelin basic protein in multiple sclerosis

Peripheral blood lymphocytes (PBL) from active and stable multiple sclerosis (MS) patients, patients with other neurologic diseases (OND), and control subjects were tested for sensitization to two myelin antigens not previously examined in multiple sclerosis, using a [3H]thymidine incorporation assay. The antigens investigated were myelin-associated glycoprotein (MAG) and proteolipid protein (PLP). In addition, sensitization to myelin basic protein (MBP) was also tested. Lymphocyte stimulation indices in active MS patients that were greater than 2 standard deviations above controls were as follows: 9/30 for MAG, 0/17 for PLP, and 8/81 for MBP. No control subjects responded to MAG or PLP, and only 1/29 control subjects responded to MBP. Three of the patients that responded to MAG also responded to MBP. Although the mean proliferative response to MAG and to MBP was greater in the population of active MS patients than in stable MS, ONDs, or controls, the difference was not statistically significant. The OND group was the only population which proliferated to PLP (6/16). The only statistically significant differences among the groups for all myelin antigens tested were the proportion of individuals with active MS vs. controls that responded to MAG (P less than 0.05), and OND vs. controls and active MS that responded to PLP (P less than 0.025). The greatest individual responses to the three antigens tested were to MBP in active MS patients. Elimination of the T8 (cytotoxic/suppressor) subset amplified the responses to myelin antigens in some patients and ONDs studied. These studies have demonstrated reactivity to MAG but not PLP in some patients with active MS, and reactivity to PLP in some patients with other neurologic diseases.     

Spontaneous development in vitro of a myelin basic protein-specific suppressor T cell line

T cell lines to myelin basic protein (MBP) developed following in vitro culture cause experimental allergic encephalomyelitis (EAE) upon transfer into naive recipient mice. We have, however, repeatedly observed that MBP-specific T cell lines lose their ability to transfer EAE after 40 days in culture. Analyses of such cell lines failed to show any differences in their proliferative responses to antigen, or in the secretion of interleukin-2 (IL-2) and/or IL-4 when compared to their encephalitogenic counterparts. In contrast, examinations of T cell receptor (TCR) beta-chain gene rearrangement patterns showed sequential changes in the clonal population of cells concomitant with the loss of encephalitogenic function. Furthermore, transfer of a non-encephalitogenic, genotypically altered cell line after long-term in vitro culture into mice challenged with MBP suppressed the development of EAE. These findings suggest that the development of such putative regulatory cells in vivo may be involved in the recovery in EAE.