Immunological studies of the pathogenesis of multiple sclerosis Cell-mediated cytotoxicity by peripheral blood lymphocytes against basic protein of myelin, encephalitogenic peptide, cerebrosides and gangliosides

Cell-mediated cytotoxic reactions against myelin basic protein (MBP), encephalitogenic peptide, cerebrosides and gangliosides were studied in 165 MS cases. The cytotoxicity, which is detectable only in the autologous system, is mediated by T lymphocytes bearing antigens reactive with antibody OKT8. The cytotoxicity is highest during active MS regardless of whether it is relapsing or chronic progressive. During inactive MS cytotoxicity is significantly less and remains virtually unchanged at a low level over long periods. The degree of cytotoxicity is thus, predominantly, dependent on the activity stage of the disease. Further, there is a correlation with the severity of the clinical deficiencies. The pathogenetic significance of cytotoxic reactions can be recognized by the close correlation between the stage and course of MS, which can be demonstrated both statistically and individually.     

Influence of monensin on ganglioside anabolism and neurite stability in cultured chick neurons

We have examined the effects of monensin, a monovalent cationophore that disrupts exo- and endocytosis of membrane vesicles and diminishes Golgi anabolic function, on the incorporation of [3H]-galactose into glycosphingolipids in neurited primary cultures of chick embryo central nervous system neurons. A linear rate of incorporation into all ganglioside species from extracellular-labeled galactose was observed. Specific activity of anabolic labeling was markedly lower in GT1b and GQ1b than in the other major gangliosides of the embryonic neuron (GM3, GD3; GM2, GD2; GM1, GD1a, GD1b). With 100 nM monensin in the extracellular medium, the rate of labeling of GT1b diminished markedly to 20% of control; GD1a, GD1b, and GD2, to 35%; GQ1B to 48%; GD3 to 60%. Vigorous incorporation of label into GM3 was entirely undiminished by monensin. From these findings, it is suggested that ganglioside biosynthesis is compartmentalized in the cytodifferentiating embryonic neuron, with GM3 entirely, and GD3 and GQ1b partially, an extra-Golgi product. Extensive loss of neurites that occurred after several hours of exposure of the neurons to monensin could not be correlated directly with decreased ganglioside anabolism.     

Postnatal development of glycosidases and gangliosides in the rat central nervous system

The developmental profiles of sialidase, β-galactosidase, β-hexosaminidase and β-glucosidase were compared to those of the gangliosides in rat brain and spinal cord. The glucosidase activities (enzyme units/g wet tissue), except β-galactosidases, were found to be higher in brain than spinal cord, in adult rats. Among the hydrolases, β-hexosaminidase showed a higher level of activity in both brain and spinal cord. In brain, the hydrolases, except β-glucosidase, followed a similar developmental pattern, showing an increase from birth to 21 days, and then decreased to adult values by day 90. In the spinal cord, sialidase, β-galactosidase, pH 3.1, and β-hexosaminidase activities increased from birth to 21 days, reaching peak values. These activities then declined to adult values by 90 days of age. However, β-galactosidase, pH 4.5, and β-glucosidase activities showed a peak at day 14. Brain total ganglioside concentration (μg N-acetylneuraminic acid/g tissue) increased slowly between birth and 7 days of age, followed by a rapid phase of increase to attain a peak value by day 21. The concentration of total gangliosides in the spinal cord is less when compared to the brain. The proportions of individual gangliosides in the central nervous system also varied during development. The rapid phase of increase in enzyme activities between 0–7 and 14–21 days and a decrease thereafter is consistent with the turnover rate of gangliosides, which in rat brain is reported to be highest between 10 and 20 days.     

Gangliosides can activate human alternative complement pathway

The alternative complement pathway (ACP) in vertebrates is knownto be important in inflammatory reactions, and to be activatedby foreign substances such as bacterial lipopolysaccharide (LPS)and zymosan, although to date no intrinsic activators have beenidentified except complement receptor type 2. From the pointof the structural similarity of LPS to ganglioside, we haveinvestigated gangliosides which are abundantly present in animalcells for their activity on the human ACP. All of seven gangliosidestested were found to activate this pathway in a manner dependingon the number of sialic acids and neutral sugars contained inthe molecules. A dose - response study suggested a correlationof the threshold in ganglioside concentration with its criticalmicelle concentration. Gangliosides may thus serve as an intrinsicactivator for ACP in animals, thereby leading to inflammation.The possibility of the participation of sialidase in complementactivation is also discussed.     

Expression of GD3 ganglioside by developing rat cerebellar Purkinje cells in situ

GD3 is a major ganglioside of the immature vertebrate CNS, and its expression is suggested to be characteristic of immature neuroectodermal cells. Using immunocytochemistry on cryostat sections of developing rat cerebellum with a monoclonal antibody specific for GD3, we have found that GD3 begins to be expressed on the plasma membrane of Purkinje cell bodies and dendrites beginning at postnatal day 7. Staining became brighter as the dendritic tree of the cells enlarged. As the Purkinje cells began to mature in different folia, they became GD3+, until by 15 days postnatal all Purkinje cells were GD3+. Positive staining of the dendritic tree was still present in the adult cerebellum. Using a monoclonal antibody 7-8D2, which recognizes cerebellar granule cells and their axons (the parallel fibres), and polyclonal antibodies against a synaptic vesicle component synaptophysin, double-immunofluorescence staining together with anti-GD3 antibodies suggested that the appearance of GD3 immunoreactivity did not correlate either with the ingrowth of parallel fibres or the presence of their synapses on Purkinje cell dendrites. However, comparison with earlier morphological studies showed that the appearance of GD3 immunoreactivity correlated well with the formation of climbing fibre synapses on Purkinje cell dendrites and the onset of the rapid expansion of the dendritic tree. These results are in keeping with the idea that elevated GD3 concentrations are found in certain cell types during periods of rapid growth or high metabolic activity but also show that this is not only restricted to immature cells.     

Reversibility of ganglioside effects on astrocyte morphology

The B-subunit of cholera toxin (BCT) induces a morphological change in cultured rat cerebral astrocytes from flat (epithelioid) to stellate (process-bearing). This stellation is reversed by the gangliosides GM1 and GD1a at concentrations of 10 microM or higher. Upon changing to a ganglioside-free medium, the flat astrocytes reacquire the stellate morphology within 3 hr, indicating that the antistellation effect of gangliosides is reversible. The possibility that this reversibility was due to a loss of exogenously acquired gangliosides from the cell membrane can be ruled out since pretreatment with GM1, but not GD1a, which does not bind BCT, results in an increased responsiveness to BCT, which was identical whether measured immediately after withdrawal of the ganglioside or 3 hr later. Asialo-GM1, which neither binds BCT nor reverses BCT-induced stellation by itself, prevents the return to stellation after withdrawal of the gangliosides. These data suggest that while gangliosides remain associated with the cell, their effect on astrocytes can change from opposing to permitting the stellate morphology.     

Human monoclonal IgM with autoantibody activity against two gangliosides (GM1 and GD1b) in a patient with motor neuron syndrome.

Small amounts of oligoclonal immunoglobulins were detected by Western blotting in the serum from a patient with motor neuron syndrome. The prominent one, a monoclonal IgM lambda, reacted strongly with the gangliosides GM1 and GD1b and more weakly with asialo GM1, as shown by immunoenzymatic staining of thin-layer chromatograms of gangliosides, ELISA on purified glycolipid coats and immunoadsorption with purified GM1. Affinity-chromatography with purified GM1 resulted in the purification of monoclonal IgM lambda. This purified IgM and its Fab fragments showed the same pattern of reactivity with gangliosides as that observed with whole serum. Such monoclonal IgM could be responsible for motor neuron diseases in some patients with overt or barely detectable monoclonal gammopathies.     

Ganglioside–basic protein interaction: Protection of gangliosides against neuraminidase action

The ability of acidic phospho- and sphingolipids to interact with basic proteins was studied by double diffusion analysis. The phospholipids, tri- and diphosphoinositide, and the sphingolipid, sulfatide, interacted with myelin basic protein as evidenced by precipitin line formation. Of the sialoglycosphingolipids (gangliosides) tested, only the myelin-specific monosialoganglioside, G_M4

1 The ganglioside nomenclature used here is according to the system of Svennerholm [1963]. The gangliosides are designated as follows: G_M4 = I³NeuAc? GalCer; G_M3 = II³NeuAc? LacCer; G_M1 = II³NeuAc? GgOse₄Cer; G_D1a = IV³ NeuAc, II³NeuAc? GgOse₄Cer; G_D1b = II³(NeuAc)₂? GgOse₄Cer; and G_T1b = IV³NeuAc, II³(NeuAc)₂? GgOse₄Cer.

, formed a precipitin line with myelin basic protein. In addition, myelin basic protein retarded the activity of Clostridium perfringens neuraminidase against G_M4 and the disialoganglioside, G_D1b. Examination of purified rat brain myelin suggested the presence of a neuraminidase activity intrinsic to myelin. This finding, in concert with ganglioside-myelin basic protein complexes which selectively protect against neuraminidase, may provide a physiological explanation for the simplified ganglioside pattern found in myelin.