Characterization of anti -sulfatide autoantibodies in demyelinating diseases

Multiple sclerosis (MS) is the most common autoimmune disease of the central nerve system and Guillain Barré Syndrome (GBS) is an inflammatory neuropathy involving the peripheral nerves. Anti-myelin immunoglobins may play a role in the demyelination processes of the both diseases. Sulfatide is an abundant glycolipid on myelin and is a candidate target antigen for disease related autoantibodies. The objective of this study was to characterize anti-sulfatide antibodies and compare antibodies from GBS and MS patients with fetal antibodies. Our hypothesis is that some B cells producing disease-associated autoantibodies are derived from or related to B cells of the fetal repertoire. Here we report that reactivity of plasma IgM against sulfatide was elevated in twelve MS patients compared with twelve normal subjects. This result implies that anti-sulfatide antibodies are disease-related. A total of sixteen human B lymphocyte clones producing anti-sulfatide autoantibodies were isolated from MS patients, GBS patients and a human fetus. Seven of the clones were from three MS patients, four of the clones were from three GBS patients and five were from the spleen of a twenty-week human fetus. Sequences have been obtained for the heavy and light chain variable regions (VDJ and VJ regions) of all of the anti-sulfatide immunoglobulins. Seven of the sixteen antibodies used VH3 for the variable region gene of the heavy chain consistent with the rate of VH3 usage in randomly selected B cells. Somatic mutations were significantly more frequent in the patient antibodies than in the fetus and somatic mutations in CDR's (Complementarity Determining Region) were significantly more frequent than in framework regions. No significant difference was found between patients and fetus in length of VH CDRIII. However, it is reported that antibodies from randomly selected normal adult B cells have longer CDRIII lengths than those of the fetus (Sanz I, 1991 Journal of Immunology Sep 1;147(5):1720-9). Our results are consistent with derivation of the precursors of B cells producing these autoantibodies from B cells related to those of the fetal repertoire. These findings are consistent with a model in which quiescent B cells from clones produced early in development undergo proliferation in dysregulated disease states, accumulating somatic mutations and increasing in reactivity toward self-antigens. ^ Epitope mapping and molecular modeling were done to elucidate the relationships between antibody structure and binding characteristics. The autoantibodies were tested for binding activity to three different antigens: sulfatide, galactoceramide and ceramide. Molecular modeling suggests that antibodies with positive charge surrounded by or adjacent to hydrophobic groups in the binding pocket bind to the head of sulfatide via the sulfate group through electrostatic interactions. However, the antibodies with hydrophobic groups separated from positive charges appear to bind to the hydrophobic tail of sulfatide. This observation was supported by a study of the effect of NaCl concentration on antigen binding. The result suggested that electrostatic interactions played a major role in sulfate group binding and that hydrophobic interactions were of greater importance for binding to the ceramide group. Our three-dimensional structure data indicated that epitope specificity of these antibodies is more predictable at the level of tertiary than primary structure and suggested positive selection based on structure occurred in the. formation of those autoantibodies. ^