Myelin basic protein-stimulated rosette-forming T cells in multiple sclerosis

A subpopulation of T lymphocytes sensitized to human myelin basic protein in peripheral blood of patients with multiple sclerosis, central nervous system (CNS) tumors, and cerebrovascular accidents was demonstrated by the antigen-stimulated, rosette-forming T cell assay. A significant increase in the percent of active rosette-forming T cells was detected after in vitro exposure of peripheral blood lymphocytes to human myelin basic protein but not to histones. In contrast, peripheral blood lymphocytes from healthy controls and from patients with benign and malignant breast diseases were unresponsive to stimulation by either antigen. These results demonstrate a functionally active T-lymphocyte subpopulation sensitized to myelin basic protein in patients with multiple sclerosis and in patients with certain other CNS diseases.     

Cell lineage analysis of Schwann cells in the PNS determined by shiverer-normal mouse chimeras

The myelin of the peripheral nervous system from the shiverer mutant mice is characterized by the absence of myelin basic protein, while the other myelin protein components are present at normal levels. Myelin lamella formation is normal in the shiverer mutant. Therefore, by using antiserum against myelin basic protein, we can distinguish the shiverer from the wild-type control myelin immunohistochemically. To study the cell lineage of Schwann cells, chimeras produced by the aggregation of eight-cell embryos from wild-type mice and shiverer mice have been used. Using myelin basic protein as a marker, it was observed that Schwann cells in the sciatic nerve existed as patches of cells with like-genotype. The patches occurred in a linear array along the axons with some intermingling of Schwann cells. Complete randomization by intermingling of Schwann cells was not observed and clones of Schwann cells may persist as contiguous groups throughout peripheral nerve development.     

The role of serum 'adhesive factor' and leukocyte sensitization in pathogenesis of multiple sclerosis

The reactivity to the myelin basic protein, brain gangliosides purified derivative and also the influence of the serum on the cells adherence were determined by micromodification of the leukocyte adherence inhibition (LAI) test. 46 multiple sclerosis patients, 21 patients with hereditary disease and 41 donors were examined. The cellular sensitization to the myelin basic protein and brain gangliosides was revealed in 57% of the multiple sclerosis patients. The leukocyte adherence was increase in 53.5% patients when autologous serum was added. The adherence factor was most often found (90% case) in patients with severe neurological deficit. It is suggested that this factor is a connected t the increased levels of circulating adhesion molecules in multiple sclerosis patients.     

Production and Characterization of Monoclonal Antibodies to Peripheral and Central Nervous System Myelin

Monoclonal antibodies against P0, myelin basic protein, or myelin-associated glycoprotein were generated by fusing mouse myeloma cells with spleen cells from BALB/c mice immunized with central and peripheral nervous system myelin proteins. The antibodies secreted were either IgG, IgM, or IgA. Clone C6B5 (iso-type IgM) secreted antibody(ies) that bound to both myelin basic protein and myelin-associated glycoprotein, although binding of antibody to myelin basic protein as detected by the immunoblot technique appeared to be much less than to the myelin-associated glycoprotein. Antibodies were characterized in solid-phase radioimmunoassay for their species cross-reaction, and histologically for the specificity of binding to myelin in central and peripheral nervous system tissues. These monoclonal reagents should prove valuable in studying CSF and myelin-producing cells, since in both cases the concentration of myelin proteins is low.     

COMPARATIVE STUDIES ON THE MYELIN PROTEINS OF BOVINE PERIPHERAL NERVE AND SPINAL CORD

Abstract— (1) Two myelin fractions of bovine peripheral nerve and spinal cord have been studied comparatively. Cholesterol as well as cerebroside content per mg of protein in the peripheral nerve myelin was less than that in the spinal cord myelin, while no significant difference in the total phospholipid content was noted.
(2) The basic proteins in myelin fractions were quantitatively estimated by disc gel electrophoresis. Around one-fourth of the total myelin protein in the bovine peripheral nerve was a basic protein with a mobility of 1.07 relative to lysozyme by Reisfeld's disc gel electrophoresis.
(3) The myelin proteins in the peripheral nerve were less completely solubilized than those of the spinal cord by treatment with deoxycholate as well as by Triton-salt solution. The protein fractions obtained from the peripheral nerve myelin by techniques similar to that for obtaining the proteolipids from the spinal cord myelin, contained different types of protein.
(4) 2',3'-Cyclic nucleotide 3'-phosphohydrolase activity in the peripheral nerve myelin was only one tenth of that in the spinal cord myelin. The Triton-salt insoluble fraction showed remarkable high activity among subfractions of the spinal cord myelin.
(5) By immunological studies, it may be concluded that an antigenic substance for experimental allergic neuritis was localized in the peripheral nerve myelin, but not in its basic protein.     

Activation of regulatory cells suppresses experimental allergic encephalomyelitis via secretion of IL-10

Suppression of CD4+ Th1 cell-mediated autoimmune disease via immune deviation is an attractive potential therapeutic approach. CD4+ Th2 T cells specific for myelin basic protein, induced by immunization of young adult male SJL mice, suppress or modify the progression of CNS autoimmune disease. This report demonstrates that activation of non-neuroantigen-specific Th2 cells is sufficient to suppress both clinical and histological experimental allergic encephalomyelitis (EAE). Th2 cells were obtained following immunization of male SJL mice with keyhole limpet hemocyanin. Transfer of these cells did not modify EAE, a model of human multiple sclerosis, in the absence of cognate Ag. Disease suppression was obtained following adoptive transfer and subcutaneous immunization. Suppression was not due to the deletion of myelin basic protein-specific T cells, but resulted from the presence of IL-10 as demonstrated by the inhibition of Th2-mediated EAE suppression via passive transfer with either anti-IL-10 or anti-IL-10R mAb. These data demonstrate that peripheral activation of a CD4+ Th2 population specific for an Ag not expressed in the CNS modifies CNS autoimmune disease via IL-10. These data suggest that either peripheral activation or direct administration of IL-10 may be of benefit in treating Th1-mediated autoimmune diseases.     

Presentation of alpha B-crystallin to T cells in active multiple sclerosis lesions: an early event following inflammatory demyelination

In the development of multiple sclerosis (MS), (re)activation of infiltrating T cells by myelin-derived Ags is considered to be a crucial step. Previously, alpha B-crystallin has been shown to be an important myelin Ag to human T cells. Since alpha B-crystallin is an intracellular heat shock protein, the question arises at what stage, if any, during lesional development in MS this Ag becomes available for CD4+ T cells. In 3 of 10 active MS lesions, alpha B-crystallin could be detected inside phagocytic vesicles of perivascular macrophages, colocalizing with myelin basic protein and myelin oligodendrocyte glycoprotein (MOG). Although the detectability of MOG in phagosomes is considered as a marker for very recent demyelination, MOG was detected in more macrophages and in more lesions than alpha B-crystallin. The disappearance of alpha B-crystallin from macrophages even before MOG was confirmed by in vitro studies; within 6 h after myelin-uptake alpha B-crystallin disappears from the phagosomes. Alpha B-crystallin-containing macrophages colocalized with infiltrating T cells and they were characterized by expression of MHC class II, CD40, and CD80. To examine functional presentation of myelin Ags to T cells, purified macrophages were pulsed in vitro with whole myelin membranes. These macrophages activated both myelin-primed and alpha B-crystallin-primed T cells in terms of proliferation and IFN-gamma secretion. In addition, alpha B-crystallin-pulsed macrophages activated myelin-primed T cells to the same extent as myelin-pulsed macrophages, whereas myelin basic protein-pulsed macrophages triggered no response at all. These data indicate that, in active MS lesions, alpha B-crystallin is available for functional presentation to T cells early during inflammatory demyelination.     

The P2 protein of bovine root myelin: isolation and some clinical and immunological properties

Abstract— A small basic protein (mol.wt. 12,000), referred to as the P₂ protein, was extracted with dilute acid from delipidated bovine root myelin and purified by ion exchange chromatography on cellulose phosphate. It appeared homogeneous on polyacrylamide gel electrophoresis. The P₂ protein had a distinctly different amino acid composition than the larger basic protein (mol.wt. 18,000), referred to as the P₁ protein, that is also present in peripheral nerve myelin. It contained relatively more hydrophobic residues and much less histidine and proline. The P₂ protein conjugated with peroxidase was bound by lymph node cells and infiltrates in rabbits sensitized with whole bovine root myelin. No binding was evident with the bovine central nervous system myelin basic protein. Chemically and immunologically, the P₂ protein appears to be specific to peripheral nervous system myelin. The isolated P₂ protein produced mild clinical symptoms of experimental allergic neuritis, but no histological evidence of disease. It was suggested that the P₂ protein is an important antigen for experimental allergic neuritis, and that its antigenic determinants are likely to be conformation-dependent.     

Myelin basic protein and proteolipid protein reactivity of brain- and cerebrospinal fluid-derived T cell clones in multiple sclerosis and postinfectious encephalomyelitis

T cells were directly cloned from autopsied MS brain plaque tissue and reactivity was measured with the major encephalitogenic neuroantigens, myelin basic protein (MBP), and proteolipid protein (PLP). Control clones were simultaneously derived from the blood. The proportion of T4+ and T8+ T cell clones from the brain tissue differed from that of peripheral blood T cell clones derived at the same time, suggesting that the clones were not derived from the peripheral blood. None of 57 brain-derived T cell clones proliferated to either MBP or PLP, although they responded well to PHA and IL 2. An additional 235 clones derived from the cerebrospinal fluid and 126 clones from the peripheral blood of other subjects with multiple sclerosis also did not proliferate to MBP or PLP. In contrast, five of nine T4+ clones from the CSF of a subject with postinfectious encephalomyelitis exhibited low but clear reactivity to human MBP, supporting the possible role of MBP as the target antigen in this disease. These studies, the first to clone T cells directly from MS plaque tissue, suggest that the lack of consistent T cell reactivity to MBP or PLP in the peripheral blood of MS patients does not appear to be secondary to the sequestration of a large number of these cells in the brain.     

Interaction of membrane-spanning proteins with peripheral and lipid-anchored membrane proteins: perspectives from protein-lipid interactions (Review)

Studies of lipid-protein interactions in double-reconstituted systems involving both integral and peripheral or lipid-anchored proteins are reviewed. Membranes of dimyristoyl phosphatidylglycerol containing either myelin proteolipid protein or cytochrome c oxidase were studied. The partner peripheral proteins bound to these membranes were myelin basic protein or cytochrome c, respectively. In addition, the interactions between the myelin proteolipid protein and avidin that was membrane-anchored by binding to N-biotinyl phosphatidylethanolamine were studied in dimyristoyl phosphatidylcholine membranes. Steric exclusion plays a significant role when sizes of the peripheral protein and transmembrane domain of the integral protein are comparable. Even so, the effects on avidin-linked lipids are different from those induced by myelin basic protein on freely diffusible lipids, both interacting with the myelin proteolipid protein. Both the former and the cytochrome c/cytochrome oxidase couple evidence a propagation of lipid perturbation out from the intramembrane protein interface that could be a basis for formation of microdomains.     

HISTOCHEMISTRY OF MYELIN XIV PERIPHERAL NERVE MYELIN PROTEINS: ELECTROPHORETIC AND HISTOCHEMICAL CORRELATIONS

Abstract— Myelin from the peripheral nervous system has been shown to contain two basic protein components and an electrophoretically slower-moving major protein, the 'J' band. The 'J' band protein cannot be selectively removed by aqueous or organic solvents and does not correspond to proteolipid or acidic protein. Histochemical stains applied to peripheral nervous systems myelin proteins separated by polyacrylamide electrophoresis indicate that 'J' band protein is analogous with the neurokeratin of the nerve sheath. Trypanophilia observed histochemically in unfixed myelin is principally due to basic proteins. With prolonged tryptic digestion 'J' band protein is degraded. Thus, previous classifications of myelin proteins based on trypsin sensitivity have been modified. All peripheral nervous system myelin proteins should be regarded as trypsin-sensitive, the basic protein being relatively more and the 'J' band protein relatively less susceptible.     

Regulatory T cells control autoimmunity in vivo by inducing apoptotic depletion of activated pathogenic lymphocytes

Clinical autoimmunity requires both activation of self-reactive T cells as well as a failure of peripheral tolerance mechanisms. We previously identified one such mechanism that involves regulatory T cells recognizing TCR V beta 8.2 chain-derived peptides in the context of MHC. How this regulation affects the fate of target V beta 8.2(+) T lymphocytes in vivo that mediate experimental autoimmune encephalomyelitis has remained unknown. The present study using immunoscope and CFSE-labeling analysis demonstrates that the expansion of regulatory CD4 and CD8 T cells in vivo results in apoptotic depletion of the dominant, myelin basic protein-reactive V beta 8.2(+) T cells, but not subdominant V beta 13(+) T cells. The elimination of only activated T cells by this negative feedback mechanism preserves the remainder of the naive V beta 8.2(+) T cell repertoire and at the same time results in protection from disease. These studies are the first in clearly elucidating the fate of myelin basic protein-specific encephalitogenic T cells in vivo following regulation.     

Basic proteins of rodent peripheral nerve myelin: immunochemical identification of the 21.5K, 18.5K, 17K, 14K, and P2 proteins

Abstract: Proteins in peripheral nervous system and central nervous system myelin and homogenates of sciatic nerve and brain from young and adult mice and rats were characterized with affinity-purified anti-P₂ and anti-myelin basic protein sera after electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose sheets. Using this method we have identified a component of rodent peripheral nervous system myelin as P₂ protein. Peripheral nervous system myelin also showed the presence of four basic proteins in addition to P₂ protein. These were found to be analogous to the 14, 17, 18.5, and 21.5K species found in the central nervous system myelin. A number of high-molecular-weight proteins were also detected with anti-myelin basic protein serum in peripheral nervous system, as well as central nervous system myelin. In addition, we report the presence of a high-molecular-weight P₂ cross-reactive protein in rodent brain stem homogenates, but not in central nervous system myelin.     

Knockout of p75(NTR) impairs re-myelination of injured sciatic nerve in mice

Remyelination is an important aspect of nerve regeneration after nerve injury but the underlying mechanisms are not fully understood. The neurotrophin receptor, p75(NTR), in activated Schwann cells in the Wallerian degenerated nerve is up-regulated and may play a role in the remyelination of regenerating peripheral nerves. In the present study, the role of p75(NTR) in remyelination of the sciatic nerve was investigated in p75(NTR) mutant mice. Histological results showed that the number of myelinated axons and thickness of myelin sheath in the injured sciatic nerves were reduced in mutant mice compared with wild-type mice. The myelin sheath of axons in the intact sciatic nerve of adult mutant mice is also thinner than that of wild-type mice. Real-time RT-PCR showed that mRNA levels for myelin basic protein and P0 in the injured sciatic nerves were significantly reduced in p75(NTR) mutant animals. Western blots also showed a significant reduction of P0 protein in the injured sciatic nerves of mutant animals. These results suggest that p75(NTR) is important for the myelinogenesis during the regeneration of peripheral nerves after injury.     

Isolation of myelin basic protein-reactive T-cell lines from normal human blood

T-Cell lines which responded by proliferation to the autoantigen, myelin basic protein (MBP), were isolated from the blood of six of nine normal humans. These T-cell lines could be maintained in in vitro culture for up to 2 months through the use of Interleukin 2 and repeated MBP stimulation. Optimal antigen-induced proliferation required both antigen and antigen-presenting cells found in the adherent cell population of autologous peripheral blood mononuclear cells (PBM). The T-cell lines were predominantly of the helper phenotype (OKT3+, OKT4+, OKT8-) and responded to both human and guinea pig myelin basic protein.     

In vivo immunomodulation by monoclonal anti-CD4 antibody. II. Effect on T cell response to myelin basic protein and experimental allergic encephalomyelitis

In vivo administration of anti-CD4 mAb (GK1.5) has been shown to be effective in preventing acute and relapsing experimental allergic encephalomyelitis (EAE). In the present report we have studied the depletion of CD4+ cells by a single dose of GK1.5 on the immune response to myelin basic protein and in the development of EAE. Our studies show that depletion of CD4 cells in mice that had received encephalitogenic CD4+ T cells altered the kinetics of acute and relapsing EAE, but did not prevent disease altogether. The in vitro T cell proliferative response to myelin basic protein in lymph node cells was maintained in the presence of significant depletion of CD4+ cells. These studies indicate that the population of Ag-reactive cells to be large and relatively refractory to antibody therapy. The implication of these results to therapy of human autoimmune disease is discussed.     

Defective autoimmune regulator-dependent central tolerance to myelin protein zero is linked to autoimmune peripheral neuropathy

Chronic inflammatory demyelinating polyneuropathy is a debilitating autoimmune disease characterized by peripheral nerve demyelination and dysfunction. How the autoimmune response is initiated, identity of provoking Ags, and pathogenic effector mechanisms are not well defined. The autoimmune regulator (Aire) plays a critical role in central tolerance by promoting thymic expression of self-Ags and deletion of self-reactive T cells. In this study, we used mice with hypomorphic Aire function and two patients with Aire mutations to define how Aire deficiency results in spontaneous autoimmune peripheral neuropathy. Autoimmunity against peripheral nerves in both mice and humans targets myelin protein zero, an Ag for which expression is Aire-regulated in the thymus. Consistent with a defect in thymic tolerance, CD4(+) T cells are sufficient to transfer disease in mice and produce IFN-γ in infiltrated peripheral nerves. Our findings suggest that defective Aire-mediated central tolerance to myelin protein zero initiates an autoimmune Th1 effector response toward peripheral nerves.     

Cutting edge: in vitro-generated tolerogenic APC induce CD8+ T regulatory cells that can suppress ongoing experimental autoimmune encephalomyelitis

APC exposed to TGFbeta2 and Ag (tolerogenic APC) promote peripheral Ag-specific tolerance via the induction of CD8(+) T regulatory cells capable of suppressing Th1 and Th2 immunity. We postulated that tolerogenic APC might reinstate tolerance toward self-neuronal Ags and ameliorate ongoing experimental autoimmune encephalomyelitis (EAE). Seven days after immunization with myelin basic protein (MBP), mice received MBP-specific tolerogenic APC, and EAE was evaluated clinically. To test for the presence and the phenotype of T regulatory cells, CD4 and/or CD8 T cells from tolerogenic APC-treated mice were transferred to naive mice before their immunization with MBP. The MBP-specific tolerogenic APC decreased both the severity and incidence of ongoing EAE. Tolerance to self-neuronal Ags was induced in naive recipient mice via adoptive transfer of CD8(+), but not CD4(+) T cells. Rational use of in vitro-generated tolerogenic APC may lead to novel therapy for autoimmune disease.     

Regulated Galactolipid Synthesis and Cell Surface Expression in Schwann Cell Line D6P2T

Clonal cell line D6P2T, subcloned from an ethylnitrosourea-induced tumor line D6 of the rat peripheral nervous system, has been characterized with particular attention to galactolipid metabolism. Galactosylcerebroside and sulfatide synthesis and expression on the cell surface are highly regulated in D6P2T cells by mechanisms involving serum- and cyclic AMP-mediated pathways. These cells also express 2',3'-cyclic nucleotide 3'-phosphohydrolase (Wolfgram protein W1a) and laminin. In contrast, myelin basic protein and antigen HNK-1 were not detected. Line D6P2T appears to be a semi-differentiated Schwann cell model, which offers interesting possibilities for studies of galactolipid synthesis, transport, and sorting.