Rat Myelin Basic Protein Research Articles

Identification and characterisation of a cDNA encoding a 17-kDa isoform of rat myelin basic protein

The myelin basic proteins (MBPs) are the major proteins of the myelin membrane. Multiple MBP mRNAs and protein isoforms are generated by alternative RNA splicing. Here we describe the isolation and characterisation of a cDNA clone encoding a 17-kDa MBP isoform from the rat ( Rattus norvegicus). The isoform is a 158-amino acid protein consisting of exons 1, 3, 4, 6 and 7 of the MBP gene. RT-PCR analysis of brain mRNA showed that transcripts encoding the 17-kDa isoform were expressed at higher levels early in post-natal development, up to 7 days post-partum.

Cloning and characterization of the rat myelin basic protein gene promoter

Expression of myelin basic protein in differentiating oligodendrocytes is mainly regulated at the transcriptional level. To better understand the regulation of myelin basic protein gene expression in mammalian cells, we cloned and characterized the rat myelin basic protein promoter by a genome walking technique. Extensive sequence homology has been found among mouse, rat and human MBP promoters. Alignment of the proximal core promoter of mouse and rat reveals highly conserved cis-elements that are important for regulating myelin basic protein gene transcription. One major transcription start site along with two minor sites have been identified in both mouse and rat myelin basic protein gene promoters using RNA ligase-mediated rapid amplification of 5′ cDNA ends. The amplified rat myelin basic protein promoter was cloned into a luciferase reporter construct. Transient transfection experiments show that both mouse and rat myelin basic protein promoters yield increased expression when oligodendrocytes differentiate. The sequence and characterization of the rat MBP promoter provide a useful tool to investigate MBP gene regulation in mammalian cells.

Early pregnancy factor suppresses the infiltration of lymphocytes and macrophages in the spinal cord of rats during experimental autoimmune encephalomyelitis but has no effect on apoptosis

Early pregnancy factor (EPF) is a secreted protein with immunosuppressive and growth factor properties that has been shown to suppress acute experimental autoimmune encephalomyelitis (EAE) induced with myelin basic protein (MBP) in Lewis rats. EAE is associated with infiltration of the central nervous system (CNS) with inflammatory cells. Spontaneous recovery involves the loss of T lymphocytes from the CNS and the selective apoptosis of Vβ8.2 + cells. In the present study, T cell, macrophage (CD11b/c +) and B cell (CD45RA +) populations in spinal cord and popliteal lymph nodes (LN) of Lewis rats with EAE were quantitated and apoptosis was studied. Rats were treated with EPF or vehicle. Following treatment on day 14 after inoculation with MBP, neither 1×100 μg nor 2×100 μg doses of EPF affected the total number of cells infiltrating the spinal cord on day 15, although the higher dose caused a decrease in the number of CD5 + and CD11b/c + cells. Treatment with 2×100 μg/day from days 10 to 14 decreased the total number of infiltrating cells, and the numbers of CD5 +, CD11b/c + and CD45RA + cells. Apoptosis was unaffected. No alteration on the number or type of inflammatory cells in the popliteal LN was observed after treatment on days 10–14. However, treatment with EPF from days 0 to 11 increased the total number of T and B cells and CD5 + T cells found on day 12 in the LN. Similarly, there was an increase in the frequency of MBP-reactive cells in the LN as determined by limiting dilution analysis. These results suggest that EPF treatment reduces the numbers of lymphocytes and macrophages in the CNS, possibly through an effect on cell trafficking.

Mutations in the rat myelin basic protein gene are associated with specific alterations in other myelin gene expression

The Long Evans shaker ( les) rat is a myelin basic protein (MBP) mutant that exhibits severe central nervous system (CNS) dysmyelination. We used a combination of immunohistochemistry, immunoblot and Northern blot analyses to determine the effect of MBP deficits on the expression of other CNS myelin genes in this mutant. Immunohistochemistry revealed a marked reduction in all major myelin proteins and differences in their intracellular distribution. Immunoblots confirmed the decreased expression of these proteins and indicated that relative levels of proteolipid protein (PLP) and DM20 were altered in this mutant. Quantitation of mRNA levels indicated that decreases in PLP and DM20 were a result of changes in mRNA levels but detected no change in other myelin gene transcripts.

Characterization of the optic nerve and retinal ganglion cell layer in the dysmyelinated adult Long Evans Shaker rat: evidence for axonal sprouting.

Myelin in the central nervous system (CNS) is hypothesized to help guide the growth of developing axons by inhibiting sprouting of aberrant neurites. Previous studies using animal models lacking CNS myelin have reported that increasing capacity for sprouting axons is negatively correlated with the degree of myelination. In the present study, we investigated the optic nerves of the recently identified Long Evans Shaker (LES) rat with prolonged dysmyelination of adult axons to determine whether the lack of myelin basic protein (MBP) in adult LES rats could manifest as increases in the population of CNS axons. We observed numerous small, unmyelinated axon profiles (<0.3 microm in diameter) clustered in bundles alongside normal caliber axons in dysmyelinated LES rats but not in normal myelinated Long Evans (LE) rats. These putative axon profiles resembled sprouting axons previously described in the CNS. Moreover, the high number of small putative axon profiles could not be accounted for by any significant increases in the number of ganglion cells and displaced amacrine cells in the ganglion cell layer when compared with normal rats as evaluated by using a variety of techniques. This finding suggests that the observed clusters of putative axon profiles were not due to developmental abnormalities in the retina but to the lack of myelin in the optic nerves of LES rats. The adult LES rat, therefore, may serve as a useful model to study the role of myelin in regulating axon development or axon regeneration after CNS injury in the adult mammalian system.

Acquisition of functional MHC class II/peptide complexes by T cells during thymic development and CNS-directed pathogenesis

This study provides evidence that both rat and mouse thymic and splenic T cells express significant levels of MHC class II glycoproteins (MHCII) in vivo. Derivation of rat and mouse chimeras revealed that a major source of MHCII on thymic T cells was acquired from radioresistant host APC. Expression of MHC on thymic T cells appeared physiologically relevant because presentation of rat myelin basic protein (RMBP) by nonadherent, radiosensitive thymic T cells was associated with the adoptive transfer of tolerance. Mature MBP-specific effector T cells isolated from the CNS in both rat and mouse models of EAE also expressed significant levels of MHCII. Adoptive transfer of activated B10.PL MBP/I-A u-restricted TCR transgenic T cells into F1(C57BL/6 × B10.PL) mice revealed acquisition of allogeneic I-A b on encephalitogenic CNS-derived T cells. Overall, this study indicates that immature and mature T cells in rats and mice acquire functional MHCII in vivo during thymic development and pathogenic inflammation.

Crucial Role of CCL3/MIP-1α in the Recurrence of Autoimmune Anterior Uveitis Induced with Myelin Basic Protein in Lewis Rats

In this study, we examined the role of CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, and CCL5/RANTES during recurrent anterior uveitis (RAU). LEW rats injected with myelin basic protein (MBP) developed experimental autoimmune encephalomyelitis (EAE) and associated anterior uveitis (AU), which was mediated by CD4+ T cells. After recovery, rats become resistant to EAE but developed RAU. Rats reinjected with MBP developed RAU without EAE. The chemokines tested were detected in the eye at RAU accelerated onset, increased as the disease progressed, and fell as clinical signs improved. At the same time, in the spinal cords of rats, these chemokines were still detected but at reduced levels. Administration of anti-MIP-1α neutralizing antibodies resulted in almost complete suppression of clinical RAU and significant reduction of inflammatory cell recruitment into the iris. Anti-MIP-1β and anti-MCP-1 antibodies were effective in suppression of RAU but to lesser degree. Treatment with anti-RANTES antibodies was not effective in protecting against the recurrent development of the disease. In the eyes, the message for CCR1 and CCR5 was considerably elevated prior to the onset of AU and decreased after treatment with anti-chemokine antibodies. Our results suggest a crucial role of CCL3/MIP-1α in the development of RAU in Lewis rats. In addition, CCL2/MCP-1 and CCL4/MIP-1β may also play a role in immunopathogenesis of RAU.

Dominant effects of mutations in the collagenous domain of mannose-binding protein.

Individuals heterozygous for mutant alleles encoding serum mannose-binding protein (MBP, also known as mannose-binding lectin) show increased susceptibility to infections caused by a wide range of pathogenic microorganisms. To investigate the molecular defects associated with heterozygosity, wild-type rat serum MBP polypeptides (MBP-A: 56% identical in sequence to human MBP) and rat MBP polypeptides containing mutations associated with human immunodeficiency have been coexpressed using a well-characterized mammalian expression system. The resulting proteins are secreted almost exclusively as heterooligomers that are defective in activating the complement cascade. Functional defects are caused by structural changes to the N-terminal collagenous and cysteine-rich domains of MBP, disrupting interactions with associated serine proteases. The dominant effects of the mutations demonstrate how the presence of a single mutant allele gives rise to the molecular defects that lead to the disease phenotype in heterozygous individuals.

Feedback activation of T-cell antigen-presenting cells during interactions with T-cell responders

Like many T cells in the myelin basic protein (MBP)-specific T-cell repertoire, CD4(-) GP2.3H3.16 (3H3) T cells recognize guinea pig MBP as an agonist but recognize autologous rat (R)MBP as a mixed agonist/antagonist. 3H3 T cells do not exhibit proliferative responses to RMBP but nonetheless respond to RMBP by accumulation of T-cell surface I-A/peptide complexes and generation of T-cell antigen-presenting cell (T-APC) activity. This study showed that presentation of RMBP by 3H3 T-APC is long-lived but is lost during interactions with cognate responders or on overt activation of T-APCs. Presentation of RMBP to encephalitogenic T cells resulted in the reciprocal activation of 3H3 T-APCs as evidenced by blastogenesis, proliferation, and induction of interleukin-2R and OX40 markers on 3H3 T-APC. These data indicate that T-APCs, like B-cell APCs, undergo clonal expansion after presentation of a cognate antigen to T-cell responders.

Genetic linkage analysis of the antibody responses to myelin basic protein and myelin oligodendrocyte glycoprotein in rats immunized with rat spinal cord homogenate

The genetic control of the antibody response to myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) was analysed in F1 and F2 crosses of DA and E3 rats, immunized with rat spinal chord homogenate. The DA rats were highly susceptible to encephalomyelitis and made antibody responses to both MBP and MOG, whereas the E3 rats were disease-resistant and responded only to MOG. The anti-MBP response was mainly controlled by the disease-promoting MHC region of the DA strain together with several disease loci outside MHC. In contrast, the anti-MOG response was associated with loci not related to or actually conferring resistance to disease.

T cell recognition of rat myelin basic protein as a TCR antagonist inhibits reciprocal activation of antigen-presenting cells and engenders resistance to experimental autoimmune encephalomyelitis.

The aim of this study was to assess whether T cell recognition of myelin basic protein (MBP) as a partially antagonistic self antigen regulates the reciprocal activation of professional antigen-presenting cells (APC). This study focused on the rat 3H3 T cell clone that recognized guinea pig (GP) MBP as a full agonist and self rat (R) MBP as a partial agonist. In cultures of 3H3 T cells and splenic APC, the agonist GPMBP elicited several responses by splenic APC, including production of nitric oxide, down-regulation of I-A, induction of B7.1 and B7.2, and prolongation of APC survival. RMBP stimulated a partial increase in production of nitric oxide, partially promoted survival of splenic APC, but did not alter expression of I-A, B7.1, or B7.2 on splenic APC. In the presence ofGPMBP, RMBP antagonized agonist-stimulated induction of B7 molecules, reversed the loss of I-A, and promoted the generation of I-A(+), costimulus-deficient APC. Furthermore, 3H3 T cells cultured with RMBP and irradiated splenocytes reduced the severity of EAE upon adoptive transfer into naive rat recipients subsequently challenged with an encephalitogenic dose of GPMBP/CFA. Overall, this study indicates that T cell receptor antagonism blocks T cell activation, inhibits feedback activation of splenic APC, and promotes T cell-dependent regulatory activities in EAE.

Interaction of Mannose-binding Protein with Associated Serine Proteases: EFFECTS OF NATURALLY OCCURRING MUTATIONS

Mannose-binding protein (MBP; mannose-binding lectin) forms part of the innate immune system. By binding directly to carbohydrates on the surfaces of potential microbial pathogens, MBP and MBP-associated serine proteases (MASPs) can replace antibodies and complement components C1q, C1r, and C1s of the classical complement pathway. In order to investigate the mechanisms of MASP activation by MBP, the cDNAs of rat MASP-1 and -2 have been isolated, and portions encompassing the N-terminal CUB and epidermal growth factor-like domains have been expressed and purified. Biophysical characterization of the purified proteins indicates that each truncated MASP is a Ca(2+)-independent homodimer in solution, in which the interacting modules include the N-terminal two domains. Binding studies reveal that both MASPs associate independently with rat MBP in a Ca(2+)-dependent manner through interactions involving the N-terminal three domains. The biophysical properties of the truncated MASPs indicate that the interactions with MBP leading to complement activation differ significantly from those between components C1q, C1r, and C1s of the classical pathway. Analysis of MASP binding by rat MBP containing naturally occurring mutations equivalent to those associated with human immunodeficiency indicates that binding to both truncated MASP-1 and MASP-2 proteins is defective in such mutants.

Regulation of encephalitogenic T cells with recombinant TCR ligands.

We have previously described recombinant MHC class II beta1 and alpha1 domains loaded with free antigenic peptides with potent inhibitory activity on encephalitogenic T cells. We have now produced single-chain constructs in which the peptide Ag is genetically encoded within the same exon as the linked beta1 and alpha1 domains, overcoming the problem of displacement of peptide Ag from the peptide binding cleft. We here describe clinical effects of recombinant TCR ligands (RTLs) comprised of the rat RT1.B beta1alpha1 domains covalently linked to the 72-89 peptide of guinea pig myelin basic protein (RTL-201), to the corresponding 72-89 peptide from rat myelin basic protein (RTL-200), or to cardiac myosin peptide CM-2 (RTL-203). Only RTL-201 possessed the ability to prevent and treat active or passive experimental autoimmune encephalomyelitis. Amelioration of experimental autoimmune encephalomyelitis was associated with a selective inhibition of proliferation response and cytokine production by Ag-stimulated lymph node T cells and a drastic reduction in the number of encephalitogenic and recruited inflammatory cells infiltrating the CNS. The exquisitely selective inhibition could be observed between molecules that differ by a single methyl group (the single amino acid residue difference between RTL-200 (threonine) and RTL-201 (serine) at position 80 of the myelin basic protein peptide). These novel RTLs provide a platform for developing potent and selective human diagnostic and therapeutic agents for treatment of autoimmune disease.

Selection of anti-myelin basic protein T-cell lines in the Lewis rat: V-β 8.2 dominance and conserved complementarity-determining-region-3 motifs are dependent on serine at position 78 of myelin basic protein

In the Lewis rat, the dominant T cell repertoire to myelin basic protein (MBP) is directed to the peptide 71–87 and the T cell receptors of pathogenic T cells are of the Vβ 8.2 genotype with short CDR3 sequences having a characteristic motif. However, this paradigm has been reached through analysis of long-term encephalitogenic lines and clones. We initiated the present study to examine the process of selection of the TCR Vβ 8.2 and characteristic CDR3 motifs upon immunization with guinea-pig MBP, and rat or guinea-pig 71–87 peptides. We found that the dominance of Vβ 8.2 developed progressively over 4–6 in vitro stimulations. Following immunization with rat 70–86, which differs from the guinea-pig peptide in one amino acid at position 78, the dominance of Vβ 8.2 and the characteristic CDR sequences are not seen. Thus, Vβ 8.2 dominance and specific CDR3 TCR motifs are seen with heterologous GpMBP but not with self rat MBP.

A rat model for investigation of bladder dysfunction associated with demyelinating disease resembling multiple sclerosis.

Myelin basic protein (MBP) can be used as an antigen for inducing experimental allergic encephalomyelitis (EAE). In various studies, EAE animals have been used as an experimental model of demyelinating diseases. The aim of this study was to determine whether EAE, induced by MBP in rats, can be useful for investigation of bladder dysfunction associated with demyelinating disease. Female Lewis rats were used. In Study 1, the time course of behavioral and cystometric changes were observed consecutively after MBP sensitization. In Study 2, the correlations between behavioral, cystometric, and histologic abnormalities were studied. The degree of paralysis and histologic findings were evaluated. In Study 1, transient hind limb paralysis was observed in all rats. Cystometric findings were characterized by three different patterns: 1) detrusor areflexia (DA), 2) detrusor hyperactivity (DH), and 3) normal. Ten (77%) of the 13 rats given MBP showed bladder dysfunction, including DA (seven), DA/DH (two) and DH (one). Study 2 showed DA in 10 rats, DH in one, and normal findings in nine animals. The difference in degree of paralysis between the DA and the cystometrically normal animals was statistically significant (P<0.01). The mean value of the degree of inflammation in the spinal cord (L6-S1) in the DA group was significantly (P<0.05) higher than that in the cystometrically normal group. The degrees of paralysis and spinal inflammation were weakly correlated (R = 0.47, P = 0.05). The present rat model seems useful for studies of bladder dysfunction associated with spinal myelitis/demyelinating diseases.

Fine T cell receptor repertoire analysis of spinal cord T cells responding to the major and minor epitopes of myelin basic protein during rat autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis is a disease induced by neuroantigen-reactive T cells bearing particular types of T cell receptor (TCR). Although the nature of TCRs of encephalitogenic T cells has been partially delineated using encephalitogenic T cell clones established in vitro, the entire TCR repertoire formed in situ after immunization with neuroantigen remains unclear. In the present study, we immunized Lewis rats with myelin basic protein (MBP) and its fragment peptides and determined the TCR repertoire of spinal cord T cells formed after the immunization by CDR3 spectratyping. It was revealed that the oligoclonal expansion of Vβ2, Vβ8.2, and Vβ17 spectratypes was detectable after immunization with guinea pig MBP and its immunodominant epitope, the 68–88 sequence, whereas immunization with a peptide containing a minor epitope induced Vβ10 expansion. Immunization with rat MBP induced much broader TCR Vβ expansion (all of the above Vβs plus Vβ3). These findings suggest that TCRs activated by immunization with guinea pig MBP used as heteroclitic immunogen recognize autoantigen, rat MBP. Furthermore, the strategy used in this study gives insight into the pathogenesis of autoimmune disease and provides useful information for designing TCR-based immunotherapy. J. Neurosci. Res. 59:145–152, 2000 © 2000 Wiley-Liss, Inc.

Fine T cell receptor repertoire analysis of spinal cord T cells responding to the major and minor epitopes of myelin basic protein during rat autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis is a disease induced by neuroantigen-reactive T cells bearing particular types of T cell receptor (TCR). Although the nature of TCRs of encephalitogenic T cells has been partially delineated using encephalitogenic T cell clones established in vitro, the entire TCR repertoire formed in situ after immunization with neuroantigen remains unclear. In the present study, we immunized Lewis rats with myelin basic protein (MBP) and its fragment peptides and determined the TCR repertoire of spinal cord T cells formed after the immunization by CDR3 spectra-typing. It was revealed that the oligoclonal expansion of Vbeta2, Vbeta8.2, and Vbeta17 spectratypes was detectable after immunization with guinea pig MBP and its immunodominant epitope, the 68-88 sequence, whereas immunization with a peptide containing a minor epitope induced Vbeta10 expansion. Immunization with rat MBP induced much broader TCR Vbeta expansion (all of the above Vbetas plus Vbeta3). These findings suggest that TCRs activated by immunization with guinea pig MBP used as heteroclitic immunogen recognize autoantigen, rat MBP. Furthermore, the strategy used in this study gives insight into the pathogenesis of autoimmune disease and provides useful information for designing TCR-based immunotherapy.

An autologous self-antigen differentially regulates expression of I-A glycoproteins and B7 costimulatory molecules on CD4- CD8- T helper cells.

During inflammation, T helper cells transiently express class II major histocompatibility complex (MHC) glycoproteins and present antigens to other T cells. To assess involvement of self-antigens in the generation of T cell antigen-presenting cell (T-APC) activity, rat (R) myelin basic protein (MBP) was used to stimulate a rat CD4-CD8- T cell clone. RMBP induced T cell surface expression of class II MHC glycoproteins and T-APC activity, although RMBP did not elicit interleukin (IL-2) production or proliferation. When added to culture with the strong agonist guinea pig (GP) MBP, RMBP acted as a partial antagonist and inhibited responses of IL-2 production, proliferation, and T cell expression of B7.1. RMBP did not, however, efficiently antagonize GPMBP-induced I-A expression on T cells. These findings indicate that the self-antigen RMBP specifically induces accumulation of I-A/peptide complexes at signaling thresholds that inhibit pathogenic autoimmune responses. Overall, this study suggests a role for self-antigens in the generation of B7-deficient T-APC activity as a mechanism of tolerance in experimental autoimmune encephalomyelitis.

Peritoneal antigen-presenting cells pulsed in vivo with myelin basic protein induce the suppression of experimental autoimmune encephalomyelitis (EAE) in Wistar rats

Suppression of Experimental Autoimmune Encephalomyelitis (EAE) can be achieved by i.p. administration of soluble myelin basic protein (MBP) in adult Wistar rats before the immunization. In the present work, we analyze the role of peritoneal antigen-presenting cells (APC) in the induction of tolerance to EAE. Peritoneal cells (PC) pulsed in vivo with MBP were obtained from rats that had been intraperitoneally injected 2 h previously with soluble MBP (MBP-PC) and then inoculated in recipient rats before the induction of EAE. Our findings show that the i.p. treatment of the animals with MBP-PC before the immunization was able to diminish the incidence and severity of the disease, reduce the histological alterations, abrogate the proliferative response against MBP and change the pattern of the humoral response to MBP. Moreover, when spleen mononuclear cells (MNC) from tolerant animals were cultured together with spleen MNC from sick animals, a dose-dependant inhibition of the proliferative response was observed, arguing for the presence of a regulatory cell population in the tolerant animals. It is also demonstrated that the MBP-PC are activated and their capability of inducing suppression of EAE is highly associated with the enhanced expression of MHC class II IA molecule. Our results show that peritoneal cells pulsed in vivo with MBP are able to induce tolerance and suggest that the up-regulation of MHC class II on MBP-PC is a necessary event for tolerance induction in our model.

Diversity of the B Cell Repertoire to Myelin Basic Protein in Rat Strains Susceptible and Resistant to EAE

Myelin basic protein (MBP) is a major protein of central nervous system myelin which can induce experimental autoimmune encephalomyelitis (EAE) in susceptible laboratory animals. The role of T cells in the induction of EAE has been extensively studied, but the antibody response to MBP has not been well characterized. In the present work, we immunized rats with encephalitogenic guinea-pig MBP and mapped autoreactive antibodies binding to peptides in the rat MBP sequence. We studied the responses of the Lewis rat strain, susceptible to EAE, and the responses of the Fischer and Brown–Norway (BN) rats, resistant to EAE. We found that Lewis rats immunized to guinea-pig MBP develop antibodies to a diversity of MBP epitopes with a dominance of MBP peptide p11–30 and peptides in the 71–140 region. Fischer rats showed a similar pattern of antibody specificities, but with higher titers than the Lewis rats. BN rats, in contrast, developed a very low titer of antibodies and lacked a response to p11–30. Thus, there is no clear correlation between the nature of the anti-MBP antibody response and the state of susceptibility or resistance to EAE induction in the different rat strains.