Phagocytosis of myelin by microglia in vitro

Previous experiments from this laboratory have shown that peritoneal macrophages in culture will phagocytize myelin. Myelin preopsonized with myelin antibodies is phagocytized to a much greater extent than untreated myelin, indicating that macrophages ingest myelin by an Fc receptor. The present work was undertaken to determine the characteristics of myelin phagocytosis by microglia, the resident macrophages of the central nervous system. Microglia isolated from 4–5 day primary cultures of newborn rat brains were shown to bind and phagocytize myelin labeled in the lipids by ¹⁴C-acetate. Both binding and phagocytosis as shown by the appearance of ¹⁴C-cholesterol ester were greatly increased if labeled myelin was preopsonized with antiserum to myelin basic protein or galactocerebroside. Both preopsonized and untreated myelin were phagocytized more actively by microglia than by peritoneal macrophages under the same culture conditions. Microglia cultured in the presence of GM-CSF showed slightly increased cholesterol ester production from opsonized myelin, but the effect of GM-CSF was significantly greater than myelin pretreated with control serum (34% increase) or untreated myelin (154% increase). There was no significant effect of GM-CSF on myelin phagocytosis by peritoneal macrophages. Cerebrospinal fluid containing immunoglobulin drawn from rabbits with acute EAE also opsonized myelin to increase phago cytosis by microglia, as has been previously shown with peritoneal macrophages. These results indicate that microglia may actively participate in myelin destruction in demyelinating diseases where myelin antibodies or a source of GM-CSF may be present. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Suppression of acute experimental allergic encephalomyelitis by intraperitoneal administration of synaptosomal antigens

The effect of a synaptosomal fraction isolated from bovine brain was examined on acute experimental allergic encephalomyelitis (EAE) in Wistar rats. Intraperitoneal administration of the animals with low doses of saline-soluble synaptosomal antigens 10 and 3 days previous to the active induction of the disease was an effective way of suppressing EAE. This treatment diminished the incidence and severity of EAE, reverted the appearance of central nervous system histological and biochemical alterations, and produced changes in the autoimmune humoral response against the encephalitogenic myelin basic protein. The phenomenon observed by treatment with synaptosomal fraction is similar to the previously described suppression mediated by myelin antigens. Taking into account that affinity-purified antibodies and T lymphocytes specific for myelin basic protein can also recognize several neuronal proteins, among them the specific synaptosomal protein synapsin I, can be suggested that antigen-driven bystander suppression could be a mechanism by which synaptosomal proteins suppress the response against myelin antigens. J. Neurosci. Res. 53:187–194, 1998. © 1998 Wiley-Liss, Inc.     

Antibodies to viral and non-viral antigens in subacute sclerosing panencephalitis and multiple sclerosis demonstrated by thin-layer polyacrylamide gel isoelectric focusing,antigen immunofixation and autoradiography

Antibody activity in IgG zones separated by thin-layer polyacrylamide gel isoelectric focusing (PAGIEF) was determined in 3 patients with subacute sclerosing panencephalitis (SSPE), 4 patients with multiple sclerosis (MS) and 4 subjects with psychosomatic disorders, using antigen immunofixation and autoradiography. Viral (measles, herpes simplex type 1, mumps) and non-viral (purified bovine myelin, bovine myelin basic protein, bovine oligodendrocytes, MS and normal human brain extract) were used as antigens. All oligoclonal and some of the polyclonal CSF IgG zones in the patients with SSPE contained measles virus antibodies, as did some of the oligoclonal and polyclonal CSF IgG zones in 3 of the patients with MS. No antibodies were detectable in CSF or serum IgG zones against any of the non-viral antigens tested.     

Chronic experimental autoimmune encephalomyelitis. Circulating autoantibodies bind predominantly determinants expressed by complexes of basic protein and lipids of myelin

Chronic relapsing experimental autoimmune encephalomyelitis (CREAE) was induced by immunising juvenile strain 13 guinea pigs with homologous spinal cord tissue in adjuvant. Thirteen animals were killed in the early chronic (5-12 weeks post immunisation) and 8 in the late chronic phase (after 15 weeks pi). Plasma titres of antibodies to an isolated myelin preparation were determined by enzyme linked immunoassay. Elevated titres of these antibodies were detected between 5-26 weeks pi, varied by 10-fold between different individuals, and had no direct relationship to clinical status or time pi. Of 20 CREAE plasma with anti-myelin immunoglobulins (Igs), only 3 contained substantial amounts of antibodies to myelin lipid and these were all from animals in the late chronic phase. By contrast 15/20 of the samples contained antibodies which appeared to require lipid-protein interactions for optimal binding to antigens in isolated myelin. There was a close correlation between plasma titres of antibodies to isolated myelin and to purified myelin basic protein (MBP). Even in samples with a moderately high lipid requirement for binding to isolated myelin, purified MBP could inhibit at least 50% of the binding. These observations suggest that MBP-lipid complexes are dominant immunogens in CREAE. Gross inflammation and myelin loss in spinal cords from these CREAE guinea pigs were determined by light microscopy. Substantial inflammation was apparent in some animals between 7 and 26 weeks pi. The most severe myelin loss was observed in late chronic phase animals having plasma anti-myelin Igs with a variety of specificities. The data suggest that circulating antibodies to myelin lipids or MBP-lipid complexes could contribute to demyelination in CREAE but their titres do not correlate with the extent of this process.     

In vitro cellular responsiveness in multiple sclerosis patients to a purified measles virus nuclear core and to other viral antigens

The Clausen modification of the peripheral blood buffy coat leukocyte migration test was used to test patients with multiple sclerosis, normal subjects and patients with other neurologic diseases for cell-mediated immunity to commercial measles virus, purified nuclear core material from a human neurotropic strain of measles virus, as well as to commercial preparations of rubella, mumps and parainfluenza HA₂. Leukocytes of multiple sclerosis patients showed significantly less mean inhibition of migration in the presence of both measles antigens, mumps and parainfluenza but no difference from controls when incubated with rubella. No correlation could be found between the degree of migration inhibition and concomitant serum anti-measles neutralization antibody titers to the same strain of virus. The use of purified viral antigens might result in more meaningful studies of the status of cell-mediated immunity to viral antigens in multiple sclerosis. The relationship of our findings to other studies in this area and to the pathogenesis of multiple sclerosis is discussed.     

Immune responses to myelin antigens in Guillain-Barré syndrome

Antibodies to nerve antigens were sought in the sera of 17 patients with acute Guillain-Barré syndrome (GBS), 11 with chronic relapsing demyelinating poly-radiculoneuropathy (CRP), 20 with other neuropathies (ON), 15 with other neurological diseases (OND) and 19 normal subjects. Complement-fixing antibodies to a suspension of human peripheral nerve tissue were identified in only 2 patients with GBS and 1 with chronic progressive neuropathy. Five GBS sera gave complement fixation reactions with rabbit sciatic nerve. The sera were also tested for galactocerebroside (Gal-C) binding activity using a solid phase assay. The range of values in all groups was the same, although the mean values for patients with GBS, ON and OND were higher than those of normal subjects. In a radioimmunoassay for antibodies to bovine P2 slightly more radiolabelled antigen was precipitated by the GBS group of sera than by sera from the other groups, but only one serum from the GBS and another from the CRP patients precipitated more than 10% of the label. Addition of bovine P2 to cultures of peripheral blood mononuclear cells from 11 patients with GBS did not cause significant stimulation. Immunoassay for antibody to myelin basic protein (MBP) showed an increased proportion of sera with low binding activity in the GBS and CRP groups. The results suggest that humoral immune responses to potentially neuritogenic antigens are found with marginally increased frequency in patients with GBS and CRP.     

Detection of antibodies to central nervous system antigens by solid phase radioimmunoassay

A solid phase radioimmunoassay is described which employs 125I-protein-A to detect the presence of antibodies against a panel of cellular and soluble central nervous system (CNS) specific antigens coated onto polyvinylchloride Microtiter plates. Serum antibodies from rabbits immunized against myelin, myelin basic protein (MBP), glial acidic fibrillary protein (GFAP), astroglioma cells, and cerebellar cells were easily detected, and high specificity for each antiserum and antigen was also demonstrable. The assay is applicable to cerebrospinal fluid (CSF) from patients with neurological diseases to detect antibodies against CNS-specific antigens. The assay should be useful for examining cell lines derived from CNS tissue for the presence of brain proteins.     

Characterization of membrane markers of isolated oligodendrocytes and clonal lines of the nervous system

We show here that the major glycolipid in myelin, galactocerebroside, is not only a useful marker for isolated bovine oligodendrocytes but that its quantitation can serve as a probe for cell differentiation. We have produced in the rabbit antisera to bovine oligodendroglia, bovine myelin and galactocerebroside. The binding of these antisera to membrane components of bovine oligodendroglia, bovine thymocytes, mouse glioma (oligodendroglioma) clonal cell strain G 26-24 and mouse neuroblastoma NB2 A was investigated with [¹²⁵I]protein A. We found major differences in immunological properties between the mature oligodendrocyte and clonal lines of oligodendroglioma cells. Differentiation appears to imply not only the expression of galactocerebroside but also of additional surface markers that are absent from the neurotumor cell lines.     

Expression of neuroectodermal antigens common to melanomas,gliomas, and neuroblastomas

Summary The reactivity spectrum of five different monoclonal anti-melanoma antibodies cross-reacting with gliomas and neuroblastomas and one monoclonal anti-glioma antibody cross-reacting with melanomas and neuroblastomas was investigated. Comparison of the binding activity of these monoclonal antibodies for 11 melanoma, seven glioma, and three neuroblastoma cell lines showed that each of these clones had a different pattern of cross-reactivity. The results indicated that the antigenic determinants detected by these antibodies were not associated with the same antigen and thus suggested the existence of at least six different antigens common to melanomas, gliomas, and neuroblastomas. Since all these tumors are known to derive from cells originating embryologically from the neural crest, it can be assumed that the antigens recognized by our monoclonal antibodies are neuroectodermal differentiation antigens.However, absorption with fetal brain homogenates abolished only the binding of monoclonal anti-glioma antibody, but did not modify the binding of monoclonal anti-melanoma antibodies.Abbreviations RIA radioimmunoassay - HAT medium containing 10^–4 M hypoxanthine, 5×10^–7 M aminopterin, 1.6×10^–5 M thymidin     

Mechanical stretch induces myelin protein loss in oligodendrocytes by activating Erk1/2 in a calcium-dependent manner

Myelin loss in the brain is a common occurrence in traumatic brain injury (TBI) that results from impact-induced acceleration forces to the head. Fast and abrupt head motions, either resulting from violent blows and/or jolts, cause rapid stretching of the brain tissue, and the long axons within the white matter tracts are especially vulnerable to such mechanical strain. Recent studies have shown that mechanotransduction plays an important role in regulating oligodendrocyte progenitors cell differentiation into oligodendrocytes. However, little is known about the impact of mechanical strain on mature oligodendrocytes and the stability of their associated myelin sheaths. We used an in vitro cellular stretch device to address these questions, as well as characterize a mechanotransduction mechanism that mediates oligodendrocyte responses. Mechanical stretch caused a transient and reversible myelin protein loss in oligodendrocytes. Cell death was not observed. Myelin protein loss was accompanied by an increase in intracellular Ca²⁺ and Erk1/2 activation. Chelating Ca²⁺ or inhibiting Erk1/2 activation was sufficient to block the stretch-induced loss of myelin protein. Further biochemical analyses revealed that the stretch-induced myelin protein loss was mediated by the release of Ca²⁺ from the endoplasmic reticulum (ER) and subsequent Ca²⁺-dependent activation of Erk1/2. Altogether, our findings characterize an Erk1/2-dependent mechanotransduction mechanism in mature oligodendrocytes that de-stabilizes the myelination program.     

Myelin basic protein and human coronavirus 229E cross-reactive T cells in multiple sclerosis

Multiple sclerosis (MS) is an inflammatory demyelinating neurological disease in which autoreactive T lymphocytes sensitized to myelin components of the central nervous system are postulated to contribute to pathogenesis. The possible relevance of molecular mimicry between a human coronavirus and the myelin basic protein component of myelin in the generation of this autoimmune reaction was evaluated. Myelin basic protein– and virus-reactive T-cell lines were established from 16 MS patients and 14 healthy donors and shown to be mostly CD4⁺. In contrast to healthy donors, several T-cell lines isolated from MS patients showed cross-reactivity between myelin and coronavirus antigens. Overall, 29% of T-cell lines from MS patients (10 donors) but only 1.3% of T-cell lines from normal control subjects (2 donors) showed an HLA-DR-restricted cross-reactive pattern of antigen activation after in vitro selection with either myelin basic protein or human coronavirus strain 229E antigens. Moreover, reciprocal reactivities were only observed in MS patients (4 donors). This establishes molecular mimicry between a common viral pathogen, such as this human coronavirus, and myelin as a possible immunopathological mechanism in MS and is consistent with the possible involvement of more than one infectious pathogen as an environmental trigger of disease.     

Detection of antibodies to myelin basic protein by solid-phase radioimmunoassay with [I]protein A

A solid phase radioimmunoassay (RIA) for the detection of antibodies to myelin basic protein (MBP) in sera has been developed employing MBP-coated flexible polyvinylchloride microtiter trays and [125I]protein-A as the radiolabel. [125I]Protein-A directly binds to the Fc region of serum IgG from several animal species and serves as an excellent reagent for detecting antibody. It can also be used to bind to a second antibody ligand, thereby making it useful even when it does not bind directly to the primary antibody Fc region. The use of one preparation of [125I]protein-A label allows sera from several species to be tested for antibodies to MBP simultaneously, thereby making this RIA technically simple, rapid and economical. This assay has been particularly useful in examining serum samples from animals with experimental autoimmune encephalomyelitis and hypomyelinogenisis congenita. In patients with multiple sclerosis low levels of antibody to MBP can be detected in cerebrospinal fluid (CSF) and acid-eluted extracts from brain plaque material; detection of low levels of antibody in human serum has not been possible due to non-specific binding of human serum Ig in this RIA.     

Myelin contains neutral sphingomyelinase activity that is stimulated by tumor necrosis factor-α

Purified myelin from mouse brain was found to contain two forms of neutral sphingomyelinase, one Mg²⁺ dependent and the other Mg²⁺ independent. The former had a pH optimum of 7.5 and K_m of 0.35 mM, whereas the corresponding values for the latter were pH 8.0 and K_m 3.03 mM. Specific activity of the Mg²⁺-dependent enzyme showed a rostral-caudal gradient, ranging from 75 nmol/mg protein/hr in myelin from cerebral hemispheres to 21 nmol/mg protein/hr in myelin from spinal cord. Relative specific activity was approximately 20% that of brain stem or cerebral hemisphere homogenate. Treatment of myelin with taurocholate or high salt concentration did not significantly reduce activity of the Mg²⁺-dependent enzyme. The activity of that enzyme did not change with time or in the presence or absence of protease inhibitors; by contrast, that of the Mg²⁺-independent enzyme decreased sharply in the absence of protease inhibitors but rose in their presence. To test for the effect of tumor necrosis factor-α (TNFα) on myelin sphingomyelinase, mouse brain myelin was labeled in vivo by intracerebral injection of [³H]acetate into 18–20-day-old mice. After 40 hr, brain stems were removed, minced, and treated with TNFα in Krebs-Ringer solution, after which myelin was immediately isolated. Separation and counting of individual lipids revealed TNFα treatment to cause increased labeling of myelin ceramide and cholesterol ester with concomitant decrease in myelin sphingomyelin. Western blotting of myelin proteins using antibodies to the two TNFα receptors as probes revealed the presence of the p75 receptor. Implications of these findings in relation to possible mechanisms of autoimmune demyelination are discussed. J. Neurosci. Res. 50:466–476, 1997. © 1997 Wiley-Liss, Inc.     

Humoral immune responses to myelin basic protein, cerebroside and ganglioside in chronic relapsing experimental allergic encephalomyelitis of the guinea pig

Titers of serum antibodies to myelin basic protein, cerebroside and ganglioside were determined in chronic relapsing experimental allergic encephalomyelitis in strain 13 guinea pigs at various intervals after inoculation with whole central nervous system (CNS) tissue. Levels of antibodies to cerebroside and ganglioside were higher in the animals with paralysis than those without paralysis during the early chronic stage. In the late chronic stage, these antibodies were still at high levels, but none of the levels correlated with clinical activity. Levels of antibody to cerebroside were significantly related to the amount of demyelination. The humoral response to the CNS antigens was monophasic, although the clinical course was polyphasic. Another factor seems to be required for clinical relapses in this animal model.     

Viral and bacterial antibody responses in multiple sclerosis

An imprint electroimmunofixation method (IEIF) was used to characterize antibodies to eight viral antigens (measles, mumps, rubella, herpes simplex type 1, varicella-zoster, vaccinia, cytomegalovirus, adenovirus) and four bacterial antigens (β-hemolytic streptococcus, Hemophilus influenzae type B, Escherichia coli, enterococcus) in serum and cerebrospinal fluid (CSF) of 12 patients with multiple sclerosis (MS). Twelve patients matched for age and sex served as controls. Evidence for intrathecal synthesis of oligoclonal antibodies to one or more antigens was found in all 12 MS patients and in 1 of the controls. In the MS group, antibodies to viruses with neurotropic properties were more frequently associated with local synthesis than antibodies to other viruses and bacteria. The types and number of locally synthesized antibodies showed no correlation with disease duration and severity. The antibodies were not associated with oligoclonal CSF IgG and appear to account for only a minor fraction of the locally synthesized CSF IgG in MS.     

A novel autoantibody from a rabbit preimmune serum that immunostains myelinated nerves of the brain

Preimmune serum from a rabbit was found to contain antibodies that selectively immunostained myelin in brain sections of the rat. It also reacted with oligodendroglial cells in culture as shown by double labeling with galactocerebroside. On Western blots of two-dimensional electrophoretic gels of rat cortex, this antiserum recognized a 46 kD protein with a basic pI. The presence of this protein within myelin may help identify possible cellular mechanisms of myelination and the autoimmune serum might be useful in structural studies of myelin.     

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.     

Monoclonal antibody production to human and bovine 2′:3′-cyclic nucleotide 3′-phosphodiesterase (CNPase): high-specificity recognition in whole brain acetone powders and conservation of sequence between CNP1 and CNP2

Monoclonal antibodies against human and bovine 2′:3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) were generated by fusing FOX-NY myeloma cells with spleen cells from RBF/Dn mice previously immunized with the purified brain antigens. The enzyme isolated from bovine brain was quite basic, with an isoelectric point of 9.71 and both the bovine and human enzymes consisted of a closely spaced doublet at approximately 44 and 46 kDa on SDS-PAGE. Six monoclonals were identified as strongly recognizing the enzyme on both ELISA plates and on immunoblots of whole brain protein. Four monoclonals very weakly cross-reacted with guinea pig myelin basic protein. In contrast with two previous reports, some of our monoclonal antibodies did immunostain 2 or 3 protein bands in peripheral nerve, two bands closely corresponding to those immunostained in central nervous system (CNS) myelin, the Wolfgram protein fraction and in acetone powders of whole brain. Each of the 6 monoclonals reacting strongly on immunoblots recognized the enzyme in from 2 to 5 of the species examined (human, bovine, rat, mouse and rabbit). In addition, all 6 monoclonals that immunostained the enzyme in whole brain, myelin and Wolfgram protein immunoblots recognized both CNP1 (44 kDa) and CNP2 (46 kDa). The two closely spaced protein bands observed on SDS-PAGE and previously stained on immunoblots of CNS CNPase using polyvalent rabbit anti-bovine CNPase antisera, and now different monoclonal antibodies, appear to be immunologically related and to contain highly conserved sequences.     

Cellular reaction to an acute demyelinating/remyelinating lesion of the rat brain stem: Localisation of GD3 ganglioside immunoreactivity

We describe a simple and reproducible acute demyelinating lesion of the rat brain stem induced by injection of ethidium bromide into the cisterna magna of young adult rats. Using immunofluorescence with a panel of antibodies to cell-specific antigens we have studied the changes in cell populations that occur at various stages during lesion progression and repair. In particular we localized the expression of ganglioside G_D3 immunoreactivity, a marker for oligodendroglial progenitors in developing brain. Both astroglia (GFAP⁺) and oligodendroglia (CNP⁺) were destroyed during the early response to the ethidium bromide although axons were spared. Splitting of myelin lamellae occurred as early as 4 days post-injection (DPI), with extensive demyelination of the inferior cerebellar peduncle following by 6 DPI. Large numbers of ED1⁺ and OX-42⁺ macrophages were present in the lesion site at this stage. Astrogliosis occurred around the perimeter of the lesions. Two populations of G_D3⁺ cells appeared within and around the lesion sites during the demyelination. One population was identified by the phenotype G_D3⁺ ED1⁺ and thus probably belonged to the macrophage/microglial lineage. In these cells both antigens appeared cytoplasmic. The second population of G_D3⁺ cells exhibited cell membrane G_D3 immunoreactivity but did not express the ED1 antigen. These cells are suggested to be oligodendroglial progenitors generated in response to the demyelination. No such cells were seen in control tissue. G_D3⁺ cells were present within the lesion sites from 6 DPI until 10–12. Following the clearance of myelin debris from the lesions, remyelination was a relatively rapid event with thin MBP⁺ myelin sheaths first seen at 11–12 DPI. Remyelination, which was extensive by 25 DPI, was predominantly oligodendroglial in origin (MBP⁺Po^? myelin) with only small pockets of peripheral myelin (MBP⁺Po⁺ myelin) observed. The present study, in addition to identifying putative glial progenitors within a demyelinated lesion, also demonstrates the difficulties in unambiguously identifying such cells in the normal and damaged adult CNS. © 1993 Wiley-Liss, Inc.