Human remyelination promoting antibody inhibits apoptotic signaling and differentiation through Lyn kinase in primary rat oligodendrocytes.

PURPOSE: Human remyelination promoting IgM mAbs target oligodendrocytes (OLs) and function in animal models of multiple sclerosis (MS). However, their mechanism of action is unknown. This study seeks to identify the cellular mechanism of action of a recombinant human IgM on OL survival. METHODS: Binding of rHIgM22 to the surface of rat OLs was studied by co-localization with various markers. RHIgM22-mediated effects on apoptotic signaling in OLs, differentiation markers, and signaling molecules were detected by Western blotting and immunoprecipitation. RESULTS: RHIgM22 co-localized with integrin ß3 but not other integrin ß-chains in OLs. Downstream of integrin ß3 we identified Src family kinase (SFK) Lyn as a key player of rHIgM22-mediated actions in OLs. Lyn immunoprecipitated in a complex together with integrin αvß3 and PDGFαR. Lyn expression was 9-fold up-regulated and Lyn activation was 3-fold higher inrHIgM22-treated OL cultures compared with controls. RHIgM22 inhibited apoptotic signaling by greater than 10-fold reduction of caspase-3 and capsase-9 cleavage and reduced by 4-fold expression of differentiation markers MBP and MOG in OLs. SFK inhibitors PP2 and SU6656 inhibited Lyn activity and restored caspase-cleavage in OLs. A human IgM that did not promote remyelination and medium wereused as controls. CONCLUSIONS: rHIgM22 prevented apoptotic signaling andinhibited OL differentiation by Lyn implying thatIgM-mediated remyelination is due toprotection of OPC and OLs rather than promotion of OPC differentiation.

Remyelination-promoting human IgMs: developing a therapeutic reagent for demyelinating disease.

Promoting remyelination following injury to the central nervous system (CNS) promises to be an effective neuroprotective strategy to limit the loss of surviving axons and prevent disability. Studies confirm that multiple sclerosis (MS) and spinal cord injury lesions contain myelinating cells and their progenitors. Recruiting these endogenous cells to remyelinate may be of therapeutic value. This review addresses the use of antibodies reactive to CNS antigens to promote remyelination. Antibody-induced remyelination in a virus-mediated model of chronic spinal cord injury was initially observed in response to treatment with CNS reactive antisera. Monoclonal mouse and human IgMs, which bind to the surface of oligodendrocytes and myelin, were later identified that were functionally equivalent to antisera. A recombinant form of a human remyelination-promoting IgM (rHIgM22) targets areas of CNS injury and promotes maximal remyelination within 5 weeks after a single low dose (25 microg/kg). The IgM isoform of this reparative antibody is required for in vivo function. We hypothesize that the IgM clusters membrane domains and associated signaling molecules on the surface of target cells. Current therapies for MS are designed to modulate inflammation. In contrast, remyelination promoting IgMs are the first potential therapeutic molecules designed to induce tissue repair by acting within the CNS at sites of damage on the cells responsible for myelin synthesis.

Immunoglobulin-mediated CNS repair.

Our view of the immune system continues to evolve from a system dedicated primarily to defense against pathogens to a system that monitors the integrity of the organism and aids in repair following damage. Repair following injury to the central nervous system (CNS) is facilitated by both cellular and humoral components of the immune system. Transfer of macrophages or T cells activated against CNS antigens promote axon regrowth and protect axons from further damage. Animals immunized with spinal cord antigens and subsequently challenged with demyelination or transection of the spinal cord demonstrate better repair than animals without prior immunization. In both experimental systems, antibodies are the biologically active immune component. Human mAbs reactive to oligodendrocytes that arise in the absence of neurologic injury promote remyelination. These data support the hypothesis that B-cell clones producing mAbs reactive to CNS epitopes are a normal part of the human antibody repertoire. They challenge the assertion that an immune response to CNS antigens is pathogenic. Treatment with CNS-reactive human mAbs following CNS disease may facilitate CNS regeneration.

Human monoclonal antibodies reactive to oligodendrocytes promote remyelination in a model of multiple sclerosis.

Promoting remyelination, a major goal of an effective treatment for demyelinating diseases, has the potential to protect vulnerable axons, increase conduction velocity, and improve neurologic deficits. Strategies to promote remyelination have focused on transplanting oligodendrocytes (OLs) or recruiting endogenous myelinating cells with trophic factors. Ig-based therapies, routinely used to treat a variety of neurological and autoimmune diseases, underlie our approach to enhance remyelination. We isolated two human mAbs directed against OL surface antigens that promoted significant remyelination in a virus-mediated model of multiple sclerosis. Four additional OL-binding human mAbs did not promote remyelination. Both human mAbs were as effective as human i.v. Ig, a treatment shown to have efficacy in multiple sclerosis, and bound to the surface of human OLs suggesting a direct effect of the mAbs on the cells responsible for myelination. Alternatively, targeting human mAbs to areas of central nervous system (CNS) pathology may facilitate the opsonization of myelin debris, allowing repair to proceed. Human mAbs were isolated from the sera of individuals with a form of monoclonal gammopathy. These individuals carry a high level of monoclonal protein in their blood without detriment, lending support to the belief that administration of these mAbs as a therapy would be safe. Our results are (i) consistent with the hypothesis that CNS-reactive mAbs, part of the normal Ig repertoire in humans, may help repair and protect the CNS from pathogenic immune injury, and (ii) further challenge the premise that Abs that bind OLs are necessarily pathogenic.

Oligodendrocyte precursor quantitation and localization in perinatal brain using a retrospective bioassay.

Several late stages of the oligodendrocyte (OL) developmental lineage can be identified immunologically in the newborn rat brain. However, OL lineage-specific markers are not available for the detection of the less mature, yet determined, OL precursors. We have developed a retrospective bioassay, combining limiting dilution analysis with a novel culture system, that quantitatively assesses the developmental potential in vivo of phenotypically undefined OL precursors in order to (1) demonstrate their existence, (2) estimate their total number in the premyelinated rat brain, and (3) demonstrate their presence in regions distal to germinal zones at times previously predicted to be devoid of such cells. Between embryonic day (E) 21 and postnatal day (P) 0, cells determined to become oligodendrocytes increase in frequency approximately 5-fold in the whole brain (from one precursor for every 365 cells to 1 in 74), and approximately 2.5-fold in the telencephalon (from 1 in 298 to 1 in 115). From these data it is calculated that a pool of approximately 10(6) phenotypically undefined cells are present in the newborn brain that are able to differentiate into OL in vitro. Further, by applying this assay to tissue samples of subdomains of the developing cerebellum, we have demonstrated that such cells are present in large numbers as early as E20 in regions sparsely populated with cells expressing the blastic neural cell marker ganglioside GD3, suggesting that they migrated to this position as a pre-GD3-expressing cell. These results significantly change the predicted ontogeny of the oligodendrocyte lineage and should fuel the ongoing search for these early OL precursors.

The oligodendrocyte and its many cellular processes.

The oligodendrocyte (OL) is increasingly providing a model system for probing central issues of cell biology. During development, OL progenitors undergo controlled migration, proliferation and differentiation, secrete and respond to a number of growth factors, and dramatically change their cellular architecture, culminating in the formation of the myelin sheath. This review examines some facets of the OL that make it an especially attractive tool for studying many basic questions in cell biology.

Differential myelinogenic capacity of specific developmental stages of the oligodendrocyte lineage upon transplantation into hypomyelinating hosts.

The capacity of oligodendrocytes (OLs) and their progenitors to migrate, proliferate, and differentiate in vivo was evaluated by transplanting highly enriched populations of sequential stages of the OL lineage (A2B5+O4-, O4+GalC-, and GalC+) into the telencephalon of the hypomyelinating mouse, shiverer. The shiverer mouse neither expresses the major myelin basic protein (MBP) nor makes normal myelin due to a large deletion in the gene for MBP. Thirty days after transplantation, serial 225 micron sections of the host brain were immunostained with antiserum to MBP and analyzed by confocal microscopy. The presence of MBP+ patches of myelin in the otherwise MBP- host brain allowed a retrospective analysis of the myelinogenic activity of the transplanted progenitors cells. Both the extent of MBP+ myelin and the location of MBP+ structures relative to the initial site of cell deposition were highly dependent on the developmental stage of the transplanted cells. Specifically, A2B5+O4- OL progenitors migrated distances of > or = 600 microns and produced MBP+ patches in nearly every slice of the host brain. An average of over 250 separate patches were found per host brain, some of which had cross-sectional areas of > 250,000 microns2 containing as many as 60 MBP+ OL cell bodies, and with densities of myelination rivaling that of normal brain. In marked contrast, transplantation of O4+GalC- cells produced only small (1,000-25,000 microns2), scattered (25-40 per brain) patches of MBP+ myelin containing one to five cell bodies, all of which were within 50 microns of the needle track or the nearest ventricular surface. GalC+ cells produced MBP+ myelin at a level similar to that of O4+CalC- cells. These data suggest that the developmental transition of OL progenitors from the O4- to the O4+ phenotype is accompanied by a dramatic reduction in the innate capacity of the cells to migrate and survive in vivo. The use of developmentally identified, enriched populations of OL progenitor cells offers the opportunity for more precise analyses of transplantation and remyelination behavior, and relates to clinically relevant studies indicating that contaminant cell types can seriously interfere with the stable integration of donor tissue into the host.

Proliferation and differentiation of O4+ oligodendrocytes in postnatal rat cerebellum: analysis in unfixed tissue slices using anti-glycolipid antibodies.

We report the study of the in vivo morphology, differentiation, and proliferation of oligodendrocytes (OLs) and their progenitors identified by the antiglycolipid antibodies O4, R-mAb, and O1 in postnatal rat cerebellum, using a novel immunocytochemical staining protocol which allows the analysis of the expression of OL-specific glycolipids in live, unfixed brain slices. An analysis of the individual cells identified in double label immunocytochemistry indicated that the order of antigen expression in OLs during in vivo development is, first, antigens recognized by O4, second, antigens recognized R-mAb, and third, antigens recognized by O1. This order of antigen expression is correlated with increasing morphological complexity and is a pattern mimicked in many culture systems. In vivo O4 identified 3 distinct stages of the OL lineage: (1) morphologically simple proligodendrocyte antigen+ (POA+) R-mAb- blast cells localized at the leading edge of myelinogenesis; (2) morphologically more complex R-mAb+O1- cells; and (3) actively myelinating O1+ [i.e., galactocerebroside+ (GalC)] OLs residing within the white matter. Only the POA+R-mAb- cells incorporated BrdU in animals that were prelabeled 3 hr before immunocytochemistry. We have demonstrated in vivo the subdivision of pre-GalC+ OL progenitors into shorter, biologically noteworthy, stages of maturation. A spatial comparison of the cell populations identified by O4, R-mAb, and O1 demonstrated a progressive wave of OL maturation from the base of the cerebellum toward the folia. The data are consistent with the hypothesis that multiprocessed O4+GalC- progenitors are the most mature stage of the OL lineage with significant proliferative capacity and the first postmigratory stage in normal development.

Stage specific, (O4+GalC-) isolated oligodendrocyte progenitors produce MBP+ myelin in vivo.

O4+/A007+GalC- proligodendroblasts represent a distinct stage of development in the oligodendrocyte lineage, occurring just prior to the appearance of postmitotic GalC+ oligodendrocytes. These cells, isolated directly from postnatal rat telencephalon by an immunopanning procedure, can terminally differentiate and myelinate axons when transplanted back into an in vivo environment. Specifically, after 30 days in the brain of newborn shiverer mouse hosts, O4+GalC- oligodendrocyte progenitors produced myelin basic protein positive (MBP+) patches. These MBP+ patches, examined by both light and confocal microscopy, contained oligodendrocyte cell bodies and ensheathed host shiverer axons morphologically similar to those found in normal rat brain at an analogous age. These results suggest that isolated O4+GalC- cells can become biochemically mature oligodendrocytes with the capacity to elaborate myelin sheaths, and further define the period of development during which oligodendrocytes retain their capacity to myelinate axons when given a receptive environment.

Multiple and novel specificities of monoclonal antibodies O1, O4, and R-mAb used in the analysis of oligodendrocyte development.

Three monoclonal antibodies that react with antigens on the surface of developing oligodendrocytes in a stage-specific manner, O1, O4 (Sommer and Schachner, 1981), and R-mAb (Ranscht et al., 1982), have been studied with respect to their specificities for a number of purified lipids. The observed specificities were consistent regardless of how the antigens were presented to the antibodies. O1 reacted with galactocerebroside, monogalactosyl-diglyceride, and psychosine and, in addition, labeled an unidentified species in rat brain extracts. R-mAb reacted with galactocerebroside, monogalactosyl-diglyceride, sulfatide, seminolipid, and psychosine; the reaction of R-mAb with sulfatide was nearly equal to that with galactocerebroside. O4 reacted with sulfatide, seminolipid, and to some extent with cholesterol. However, oligodendrocyte progenitor cells labeling with O4 that had not yet begun to express the O1 antigen failed to incorporate 35SO4 or [3H]galactose into sulfatide or seminolipid, the syntheses of which first appear in O1-positive cells. Therefore, O4 stains, in addition to sulfatide and seminolipid, and unidentified antigen that appears on the surface of oligodendrocyte progenitors prior to the expression of sulfatide and galactocerebroside. In primary cultures of rat brain, developing O4+ oligodendrocyte progenitors stained slightly earlier with R-mAb than with O1, and thus R-mAb transiently stained a larger population of oligodendrocytes than did O1. None of the three antibodies produced a detectable reaction on Western immunoblot after separation of brain proteins on reducing gels. In conclusion, the results show that O4, R-mAb, and O1 have multiple overlapping specificities, including previously unrecognized cross-reactions.

Direct microculture enzyme-linked immunosorbent assay for studying neural cells: oligodendrocytes.

Oligodendrocyte development has been studied in a standardized primary microculture system initiated from day 20-21 fetal rat brain using a solid-phase enzyme-linked immunosorbent assay (ELISA) carried out directly on fixed cells (direct microculture ELISA). A highly reproducible dissociation procedure is described that allows careful control of the number of cells seeded per culture. At a seeding density of 1 x 10(5) cells/culture, up to 250 oligodendrocyte-generating microcultures consisting of 10-12% oligodendrocytes can be prepared from a single fetal rat brain, thereby permitting the simultaneous assay of multiple developmental parameters in sibling cultures. The validity of this method for quantifying myelinogenesis was established by comparing the results obtained by direct microculture ELISA with immunocytochemical counting of cells in parallel cultures. As few as 200 oligodendrocytes could be detected using a biotinylated anti-Ig and an avidin-urease conjugate detection system; CNP immunoreactivity measured by ELISA was linearly proportional to the number of immunolabeled cells between 6 and 34 days in culture; the developmental time courses of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and myelin basic protein (MBP) expression determined by the two methods were very similar. Finally, cell suspensions were seeded at increasing dilution to determine the number of cells required to generate cultures that tested positive for oligodendrocytes by ELISA. As few as 9,000 cells were sufficient, predicting a minimum of 8,000 oligoprogenitors per 20-21 day fetal rat brain. The application of direct microculture ELISA for studying oligodendrocyte population size and myelinogenesis is discussed.