Genetic susceptibility or resistance to autoimmune encephalomyelitis ...

International Immunology, Vol. 11, No. 9, pp. 1573–1580

© 1999 The Japanese Society for Immunology

Genetic susceptibility or resistance to autoimmune encephalomyelitis in MHC congenic mice is associated with differential production of pro- and anti-inflammatory cytokines Ruth Maron, Wayne W. Hancock1, Anthony Slavin, Maszakazu Hattori2, Vijay Kuchroo and Howard L. Weiner Center for Neurologic Diseases, Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, HIM 730, Boston, MA 02115, USA 1Leukosite, 215 First Street, Cambridge, MA 02142, USA 2Joslin Diabetes Center, 1 Joslin Place, Boston, MA 02115, USA

Keywords: autoimmunity, experimental allergic encephalomyelitis, genetics, NOD, transforming growth factor-β Abstract Experimental allergic encephalomyelitis (EAE) is a Th1-type cell-mediated autoimmune disease induced by immunization with myelin proteins and mediated by CD4F T cells. Although susceptibility to EAE is dependent largely on MHC background, the B10.S strain is resistant to induction of EAE despite sharing the I-As MHC locus with the susceptible SJL strain. Furthermore, NOD mice which spontaneously develop diabetes are susceptible to EAE induction with myelin oligodendrocyte glycoprotein (MOG) 35–55, whereas a MHC congenic strain, III, which also expresses I-Ag7 MHC haplotype does not develop diabetes and is also resistant to EAE induction. We induced EAE in these four strains of mice with MOG peptides 92–106 (for I-As strains) and 35–55 (for I-Ag7 strains) in complete Freund’s adjuvant. In the susceptible strains (SJL and NOD) in vitro, there are high levels of IFN-γ production, whereas the resistant strains (B10.S or III) secreted primarily IL-4/IL-10 and transforming growth factor (TGF)-β, and had decreased levels of IFN-γ. When brains from susceptible and resistant mice were examined by immunohistochemical methods for cytokine expression, the brains from resistant mice showed fewer infiltrates which predominantly expressed IL-4 and IL-10 and/or TGF-β. Brains from NOD and SJL with EAE showed mainly IL-2 and IFN-γ positive cells. Thus, resistance to MOG induced EAE in B10.S and III mouse strains is related to non-MHC genes and is associated with an altered balance of pro- and anti-inflammatory cytokines both in lymphoid tissue and in the brain following immunization with myelin antigens. Introduction Experimental allergic encephalomyelitis (EAE), a model for human multiple sclerosis, is a demyelinating inflammatory disease of the central nervous system (CNS) induced in a variety of animal species by injection of myelin proteins, peptides derived from myelin proteins or T cells specific for these antigens (1). It is mediated by CD41 T cells producing pro-inflammatory Th1-type cytokines such as IFN-γ, IL-2 and tumor necrosis factor (TNF)-β (2,3). In contrast, cells producing antiinflammatory cytokines such as IL-4, IL-10 and transforming growth factor (TGF)-β are non-encephalitogenic, and play an

important role in down-regulating inflammatory responses and inhibiting autoimmune injury (4). Although susceptibility to disease is largely dependent upon MHC background, susceptibility to EAE and diabetes varies among mouse strains that are MHC congenic, suggesting that genes outside the MHC contribute to disease susceptibility (5). Indeed, B10.S mice are resistant to both active and passive induction of EAE, despite bearing the same MHC haplotype as the SJL/J (H-2s) mouse, a prototypic strain used to study EAE and other autoimmune diseases. Similarly, NOD mice that spontaneously develop

Correspondence to: H. L. Weiner Transmitting editor: L. Steinman

Received 24 March 1999, accepted 8 June 1999

1574 Cytokines and genetic susceptibility in EAE autoimmune diabetes are also susceptible to EAE whereas H2 congeneic III mice that share MHC with NOD mice (I-Ag7) are resistant to diabetes (6) and, as shown in this study, are also resistant to EAE. Recently it has been suggested that the susceptibility of different mouse strains to infection and autoimmunity is influenced by the differential induction of Th1- versus Th2type responses. For example, mice producing a Th1-type response to L. major are resistant, while those producing a Th2 response, such as BALB/c, are susceptible (7). This paradigm can be reversed, however, by promoting a Th1type response by injecting IL-12 in the susceptible mice (8). This has also been demonstrated in EAE where myelin basic protein (MBP)-reactive T cells from the resistant B10.S mice when exposed to IL-12 to enhance Th1 responses were subsequently able to transfer disease to naive B10.S recipients (9). Thus, the development of the appropriate Th-type response is of crucial importance to confer upon the host the ability to discharge the pathogen or resist development of autoimmunity. The most dominant factor that influences development of Th1 or Th2 responses is the cytokine milieu during T cell differentiation. IL-12 promotes differentiation of Th1 cells (10) and IL-4 promotes differentiation of Th2 cells (11). The two cell types act antagonistically in that they inhibit expansion and function of each other. In addition to cytokines, other factors such as antigen dose (12,13), co-stimulatory molecules, and type and quality of antigen (14,15) may influence the Th1–Th2 differentiation process. We have been interested in studying the role of susceptible and resistant genetic backgrounds in H-2 congenic mice on the T cell differentiation, cytokine production and development of EAE. In order to investigate the effect of non-MHC genetic background in disease susceptibility or resistance we have characterized the cytokine production in MHC congenic mice that are either susceptible or resistant to the induction of EAE. In this study we have used two different sets of mice (SJL, B10.S and NOD, III) that are H-2 congenic but differ in their susceptibility to EAE. We found that EAE-resistant mice produce predominantly anti-inflammatory cytokines upon immunization with the myelin oligodendrocyte glycoprotein (MOG) myelin autoantigen both in the peripheral lymphoid tissue and in the nervous system, suggesting such responses may be involved in maintaining self-tolerance and in inhibiting EAE. Methods

Mice SJL/J and B10.S mice were purchased from Jackson Laboratory (Bar Harbor, ME). NOD mice were purchased from Taconic Farms (Germantown, NY). The mice were housed in a viral antibody-free (VAF) facility at the Harvard Medical School Animal Care Facilities. The breeding pairs for the III mice were originally obtained from the colony of Dr Makino (Aburahi Laboratories, Shionogi, Shiga, Japan), bred by brother–sister mating at the animal resources center and the radioisotope center of Kyoto University (Kyoto, Japan), and raised in the animal facility at the Joslin Diabetes Center. Antigens and chemicals MOG peptides 35–55 (MEVGWYRSPFSRVVHLYRNGK) and 92–106 (DEGGYTCFFRDHSYQ) were synthesized at the

Biopolymer Facilities, Howard Hughs Medical Institute, Harvard Medical School. Incomplete Freund’s adjuvant and Mycobacterium tuberculosis were purchased from Difco (Detroit, MI). Pertussis toxin was purchased from List Biological (Campbell, CA).

Immunization and EAE induction Mice were immunized in the footpad with 100 µg MOG peptide emulsified in complete Freund’s adjuvant (CFA) containing 200 µg M. tuberculosis H37 RA. Pertusis toxin 200 ng per mouse per injection was given i.v. at the time of immunization and 48 h later. Animals were observed daily and scored for EAE as follows: 0, no disease; 1, tail paralysis; 2, hind limb weakness; 3, hind limb paralysis; 4, hind and forelimb paralysis; 5, moribund.

Cytokine assays Draining popliteal lymph nodes, or splenocytes, were collected and pooled within each group 10 days post-immunization. Cells were isolated and red blood cells were removed by lysis with Tris-buffered ammonium chloride. Cells were cultured in X-VIVO 20 serum-free medium, purchased from Biowhittaker (Walkersville, MD). For proliferation assays, 53105 cells/well were cultured in 96 well plates with 50 µg/ ml antigen for 72 h. [3H]Thymidine (1 µCi /well ) was added for the last 8 h of culture. Cells were then harvested and incorporation of [3H]thymidine was measured using the LKB Betaplate liquid scintillation counter. For cytokine assays, culture supernatants were collected at 24 h (for IL-4) or at 40 h (for IL-10 and IFN-γ) and at 72 h for TGF-β. Cytokines ELISA were run as described previously (4). For cytokine quantification Maxisorp immunoplates (Nunc, Naperville, IL) were coated with capture mAb (1–5 µg/ml in carbonate buffer pH 8.2, 4°C overnight). Plates were washed, blocked with 10% BSA, washed again, and standards and samples added for another overnight incubation at 4°C. Wells were washed and cytokine levels determined by mAb and a peroxidase visualization system. Antibodies and reagents used in the assays were purchased from PharMingen (San Diego, CA). Avidin-peroxidase was purchased from Sigma (St Louis, MO). For the TGF-β assay, chicken anti-TGF-β was purchased from R & D systems (Minneapolis, MN) and murine anti-TGF-β mAb was purchased from Genzyme (Cambridge, MA). Peroxidase-labeled goat anti-mouse IgG was purchased from Kirkegaard & Perry (Gaithersburg, MD).

Immunopathology Immediately upon harvest of brains from each group of mice at day 30 post-immunization, samples were coded, subdivided, snap-frozen and stored at –70°C until sectioning or formalin-fixed and paraffin-embedded for light microscopy. Selected rat anti-mouse mAb were purchased from PharMingen. These consisted of rat mAb directed against all mouse leukocytes (CD45, 30F11.1), T cells (CD5, 53-7.3), monocytes (CD11b, M1/70), and the cytokines, IL-2 (S4B6), IL-4 (11B11), IFN-γ (R4-6A2) and IL-10 (JES5-2A5). TGF-β was detected using a goat polyclonal antibody which recognizes all TGF-β isoforms (R & D). Cryostat sections were fixed in paraformaldehyde–lysine–periodate for demonstration of cell surface antigens or in acetone for localization of cytokines and

Cytokines and genetic susceptibility in EAE 1575 incubated overnight at 4°C with primary antibodies; bound antibodies were detected using a peroxidase–antiperoxidase method and the substrate, diaminobenzidine, as described (16,17). The specificity of labeling was assessed using isotype-matched mAb (or purified goat Ig in the case of the anti-TGF-β antibody) and by overnight mAb absorption with recombinant cytokines (IL-2, IL-4, IL-10 and IFN-γ, obtained from PharMingen) prior to immunohistologic labeling (16,18). Sections were examined in a blinded manner by a pathologist (W. W. H.), focusing on the extent of submeningeal and perivascular inflammation characteristic of this model, as observed within 10 consecutive high-power fields (n 5 2–3 animals/group). Particular attention was given to assessment of the infiltrating cells and their cytokine products. The intensity of cytokine labeling was scored semiquantitatively as 0 (no staining), 6 (trace), 11 (weak), 21 (moderate) or 31 (strong or dense staining) and the area of cytokine staining within each section was determined by quantitative image analysis. For quantitative analysis, the area of brown substrate reaction product within five consecutive microscopic fields of superficial cortex/cytokine/sample was determined using scientific imaging software (IPLab Spectrum; Signal Analytics, Vienna, VA) and expressed as a percentage (mean 6 SD) of the total area of each section; data were evaluated by the Mann– Whitney U-test. Results

Resistance or susceptibility to EAE induction with MOG peptides in SJL, B10.S, NOD and III mice SJL and B10.S mice were immunized with MOG peptide 92– 106 in CFA. As shown in Fig. 1, SJL animals developed a mild initial episode of EAE followed by a very severe relapse, whereas no clinical disease developed in B10.S animals (Fig. 1A). A similar differential susceptibility or resistance to EAE was also observed in NOD and III animals immunized with MOG peptide 35–55 (Fig. 1B). NOD animals developed clinical signs of EAE, whereas III animals were completely resistant to clinical EAE. Cumulative data on incidence and severity of disease in all the four strains are shown in Fig. 1.

Cytokine patterns in animals immunized with MOG peptides Both SJL and NOD mice that develop EAE had a significantly higher IFN-γ response to immunization with MOG peptides than B10.S and III mice (NOD versus III P 5 0.0025 and SJL versus B10.S P 5 0.05) which were resistant to EAE (Fig. 2). Proliferative responses in the susceptible strains correlated with increased secretion of IFN-γ following antigen-specific activation of T cells from immunized mice (not shown). In marked contrast, lymph node cells from susceptible animals produced lesser amounts or no anti-inflammatory cytokines IL-4, IL-10 or TGF-β, whereas significant amounts of these anti-inflammatory cytokines were secreted by lymphocytes from resistant animals (P , 0.01). Similar effects were observed in spleen cell cultures (not shown). In B10.S, the major anti-inflammatory cytokine produced was TGF-β and in addition there was increased IL-10 secretion in these animals, whereas no TGF-β was induced in SJL mice. In the III strain, significant amounts of TGF-β and IL-4 were produced in

Fig. 1. Induction of EAE with MOG peptide in SJL, B10.S, NOD and III mice.

resistant animals, whereas minimal amounts of IL-4 and low levels of TGF-β were produced by the susceptible NOD strain. No IL-10 was observed in either the NOD or III strain.

Immunopathology of the central nervous system in resistant versus susceptible strains Intracerebral events associated with disease susceptibility versus resistance in animals that were immunized for the

1576 Cytokines and genetic susceptibility in EAE development of EAE were evaluated by immunohistology. Findings are summarized in Table 1, illustrated in Fig. 3 and the results of quantitative image analysis of cytokine protein expression are shown in Fig. 4. In susceptible animals, CNS infiltration consisted of inflammatory cells that predominantly secreted IL-2 and IFN-γ, whereas a markedly different pattern was observed in resistant animals. Resistant mice had fewer cellular infiltrates in the CNS and these infiltrates were mostly in the submeningeal areas. Furthermore, in contrast to susceptible mice the infiltrating cells produced little IL-2 or IFNγ, and showed significant expression of IL-4 and TGF-β. The most striking finding was a dense cellular infiltrate characterized by TGF-β secretion in B10.S animals. In addition, other anti-inflammatory cytokines were observed in resistant B10.S mice including IL-4, IL-10 and prostaglandin E2. Similar to the B10.S mice, III mice showed an increased production of IL-4, IL-10 and TGF-β in the CNS. The dissociation between expression of IL-4 and IL-10 in the brain and peripheral lymph nodes of some animals may relate to the different tissue microenvironments, types of antigen-presenting cells and that the lymph nodes were examined at 10 days, whereas the brains were examined at 30 days post-immunization.

Fig. 2. Proliferation and cytokine patterns following immunization with MOG peptides. Spleen and peripheral lymph nodes were harvested 10 days after immunization, pooled from three animals and cultured as described in Methods.

Discussion We have found that lymphocytes from B10.S mice which do not develop EAE upon immunization with MOG peptide 92– 106 secrete small amounts of IFN-γ when stimulated in vitro with MOG 92–106 and have low proliferative responses. However, lymphocytes from the B10.S mice immunized with the MOG peptide secrete high amounts of IL-10 and TGF-β. When brains of B10.S mice, immunized with MOG peptide, were evaluated for cytokine expression, markedly increased expression of TGF-β was seen. Analogous findings were observed in III animals which do not develop EAE upon immunization with MOG peptide 35–55. The cytokine profile in III mice is primarily a Th2-type pattern involving IL-4. Immunohistochemical staining of cytokines in the brains of III mice also reveals a Th2 cytokine pattern. In addition, III mice do not develop diabetes, in contrast to the NOD mice which spontaneously develop diabetes and can be induced to develop EAE when immunized with MOG peptide 35–55. SJL/J and B10.S mice have the same MHC complex; however, they respond differently to EAE induction with the MOG 92-106 peptide. Similarly, NOD and III carry the same H-2g7 haplotype and also respond differently to EAE induction. Thus, genes outside of the MHC complex contribute to disease susceptibility or resistance and our data indicate that background genes also affect the type of cytokine response to MOG peptide. Inbred strains of mice differ in their susceptibility to EAE induction and the MHC plays an important role in susceptibility. It is also clear that non-MHC genes also may play an important role. SJL and B10.S are both H-2s but B10.S are resistant to the development of EAE and as such provide an avenue to explore the effect of non-MHC genes and gene products or resistance versus susceptibility. There have been two reports suggesting that B10.S mice are susceptible to EAE induced with spinal cord homogenate (SCH). However, a closer examination of these data reveals that only two of two mice were sick in one report (19) and in the other report (20) some B10.S mice displayed histologic signs but no mice developed neurological signs of EAE when immunized with SCH. We have reported that B10.S mice immunized with proteolipid protein (PLP) 139–151 can occasionally develop EAE (5). However, only two of 53 (4%) of B10.S mice developed

Table 1. Immunopathology of CNS in mice immunized with MOG peptidesa Feature

SJL/J

B10.S

NOD

III

Cells

small perivascular and submeningeal groups of macrophages and 10–20% CD41 T cells 21 MNC 31 MNC 0 0 0

moderate to large collections of submeningeal macrophages and 10–20% CD41 T cells 6 6 21 MNC and focal EC 21 31 MNC and EC

small perivascular and submeningeal groups of ~50% macrophages and ~50% CD41 T cells 21 MNC 21 MNC negative 6 0

small perivascular and submeningeal groups of macrophages, with , 10% CD41 T cells 6 6 31 MNC and focal EC 21 31 MNC and focal EC

IL-2 IFN-γ IL-4 IL-10 TGF-β

aImmunohistologic findings in 10 adjacent sections/sample, with intensity of labeling scored semiquantitatively as 0–31 as described in Methods; the results of quantitative image analysis based on the proportion of the section stained are shown in Fig. 3. EC, endothelial cells; MNC, mononuclear cell.

Cytokines and genetic susceptibility in EAE 1577 EAE compared to 73 of 74 (99%) of SJL mice following immunization with PLP 139–151. In summary, the small number of reports suggesting B10.S mice are susceptible and the low incidence of EAE in B10.S mice in these reports indicates

that B10.S mice are primarily resistant to EAE induction with either SCH, MOG or PLP peptides. Segal and Shevach reported that the resistance of the B10.S strain to induction of EAE with guinea pig MBP was secondary to an antigen-

Fig. 3. Immunopathology of brains harvested at day 30 post-immunization from each experimental group; cryostat sections from each sample are arranged vertically. In each case, an asterisk indicates a small blood vessel and arrowheads indicate clumps of perivascular leukocytes present within serial sections, allowing direct comparison of the extent of cytokine expression. In SJL mice, small clumps of submeningeal leukocytes expressed IFN-γ and IL-2 but not IL-4 or TGF-β (a–d). By contrast, in B10.S mice, perivascular and submeningeal infiltrates lacked Th1 cytokines but showed dense expression of IL-4 and TGF-β (e–h). Similarly, NOD mice showed small clumps of perivascular leukocytes with a marked Th1 cytokine predominance (i–l), whereas III mice had only mild perivascular accumulation of Th2 and TGF-β cytokine-producing leukocytes (m–p). Hematoxylin counterstain, 350 original magnification.

1578 Cytokines and genetic susceptibility in EAE

Fig. 4. Quantitative image analysis of the percentage (mean 6 SD) of each brain section, involving inflammatory infiltrates within the superficial cortex and submeninges, which was stained brown for a given cytokine. IFN-γ (P , 0.0001) and IL-2 (P , 0.02) were statistically significantly increased in SJL and NOD mice versus B10.S and III animals; in contrast, IL-4 (P , 0.002) and TGF-β (P , 0.001) were significantly increased in B10.S and III mice versus the other two groups of mice.

specific defect in the generation of Th1 cells that produce IFN-γ and this could be overcome by exposure of MBPreactive cells to IL-12 in vitro (9). They also reported that the failure to produce IFN-γ in response to MBP was antigen specific and was not secondary to an ongoing Th2 response. However, whether this was due to secretion of TGF-β by T cells was not reported. Our results using MOG peptide suggest that the resistance to EAE may not be a passive process or lack of the induction of a Th1 response, but an active process in which TGF-β or Th2 cells are induced rather than IFN-γ upon immunization with the autoantigen. Although we used MOG as a disease inducing antigen in our study, we have also investigated murine MBP in the B10.S mouse and found that the B10.S mice also produce IL-4 and TGF-β in response to mouse MBP. This process does not appear unique to the B10.S as it was also seen in the III animals. Thus, the active induction of anti-inflammatory cytokines (IL-4/ IL-10 and TGF-β) may be an important general feature of resistance to autoimmunity. In other systems, expression of autoreactive TCR transgenic cells in susceptible and resistant backgrounds have led to differentiation of transgenic T cells into Th1 cells in the susceptible mice and Th2 cells on the resistant background, and thus demonstrating that even T cells expressing the same TCR can differentiate into Th1 or Th2 cells depending upon the genetic background (21). A number of studies have now shown that TGF-β is a major immunosuppressive cytokine which suppresses both Th1 and Th2 responses and regulates immune responses (22–25). A unique subset of regulatory T cells is induced following oral administration of antigen, which predominately produces TGF-β (4,26). Transfer of these TGF-β-producing T cells suppresses autoimmune responses upon adoptive transfer (4). Furthermore, TGF-β is induced in the CNS of mice undergoing recovery from EAE (27,28), thus showing a physio-

logical role of the immunosuppressive cytokine in regulation of autoimmune injury in the target tissue. A similar mechanism is most probably utilized in the genetic resistance to autoimmune diseases in that preferential production of TGFβ- and/or Th2-producing cells in the resistant mice upon immunization with an autoantigen may inhibit generation of pro-inflammatory cells and protect mice from development of autoimmunity. To address this issue, we tested the effect of anti-TGF-β antibody in vitro and in vivo on immune responses in B10.S mice. We found that in lymphocytes from B10.S mice immunized with MOG/CFA, the addition of neutralizing antiTGF-β antibody in vitro induced a shift towards IFN-γ and decreased IL-10 and TGF-β as compared to a control isotypematched antibody. The secretion of IFN-γ was increased from 540 to 4800 pg/ml, IL-10 decreased from 200 pg/ml to undetectable levels and as expected TGF-β was reduced (from 510 to 260 pg/ml). However, in vivo treatment with antiTGF-β antibody did not convert B10.S mice from being resistant to being susceptible to EAE. The inability to reverse disease may be secondary to a number of factors, including the need for neutralization of other anti-inflammatory cytokines such as IL-10, IL-4 and IL-13. In this regard, to reverse the inhibitory effect of IL-10-secreting TR1 cells required both anti-IL-10 and anti-TGF-β antibody (29). Furthermore, it may not be possible to reverse the phenotype of already committed cells in vivo. Of note, Strober, et al. have reported a reciprocal relation between IL-12 and TGF-β (30,31). An important observation in our study was that cells expressing TGF-β and IL-4 were observed in the brains of resistant mice with no clinical disease and were predominantly submeningeal. These cells presumably were induced in the peripheral lymphoid tissue upon immunization and migrated to the CNS. Our results in both the I-As and I-Ag7 strains are consistent with non-MHC effects in rat models of EAE and arthritis (32,33). We have undertaken a genetic analysis in a cross between SJL and B10.S mice to determine the genes and loci that may be responsible for susceptibility/resistance to EAE. In addition to multiple minor loci, significant linkage was found with two major loci, one on the telomeric end of chromosome 2 and another in the center of chromosome 3 (5). The relationship of these loci to the production of Th1 cytokines in SJL/NOD mice or induction of Th2/TGF-β in the B10.S is not clear at this stage. However, it is interesting to speculate that polymorphisms in CD40 and IL-2, IL-12 genes (between SJL and B10.S mice) which are located approximately at the same location on chromosome 2 and 3 respectively, may be responsible for enhanced Th1 responses in SJL mice. Our recent studies (34) further confirm that the autoimmune susceptible strains, SJL and NOD, share the same polymorphism in the IL-2 gene and the resistant strains express another allele. Whether this polymorphism in IL-2 which alters glycosylation and functional half-life of IL-2 affects T cell differentiation and genetic susceptibility to EAE and diabetes remains to be determined. Our findings have important implications for mechanism of tolerance in autoimmune disease both in animals and humans. It suggests that one mechanism by which the host does not develop an autoimmune disease when challenged with an autoantigen (either artificially in animals with Th1-inducing adjuvant such as CFA adjuvant) or in humans in association

Cytokines and genetic susceptibility in EAE 1579 with a viral or bacterial infection is the induction of an antiinflammatory Th2 or TGF-β response against the autoantigen, which serves as a protective response for autoantigens. Understanding how different genetic polymorphisms in certain individuals or animal strains contribute to the induction of a predominantly pro-inflammatory Th1 in response to the autoantigen may help decipher the mechanism of induction of autoimmune diseases.

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Acknowledgements Supported by NIH grant AI43458.

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Abbreviations CFA CNS EAE MBP MOG PLP SCH TGF TNF

complete Freund’s adjuvant central nervous system experimental allergic encephalomyelitis myelin basic protein myelin oligodendrocyte glycoprotein proteolipid protein spinal cord homogenate transforming growth factor tumor necrosis factor

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