title: Stable expression of a recombinant human antinucleosome antibody to investigate relationships between antibody sequence, binding properties, and pathogenicity.
abstract: When purified under rigorous conditions, some murine anti-double-stranded-DNA (anti-dsDNA) antibodies actually bind chromatin rather than dsDNA. This suggests that they may actually be antinucleosome antibodies that only appear to bind dsDNA when they are incompletely dissociated from nucleosomes. Experiments in murine models suggest that antibody-nucleosome complexes may play a crucial role in the pathogenesis of glomerulonephritis in systemic lupus erythematosus. Some human monoclonal anti-DNA antibodies are pathogenic when administered to mice with severe combined immunodeficiency (SCID). Our objective was to achieve stable expression of sequence-altered variants of one such antibody, B3, in Chinese hamster ovary (CHO) cells. Purified antibodies secreted by these cells were tested to investigate whether B3 is actually an antinucleosome antibody. The pathogenic effects of the antibodies were tested by implanting CHO cells secreting them into SCID mice. Purified B3 does not bind to dsDNA unless supernatant from cultured cells is added, but does bind to nucleosomes. The strength of binding to dsDNA and nucleosomes is dependent on the sequence of the light chain. Mice that received CHO cells secreting wild-type B3 developed more proteinuria and died earlier than control mice that received nonsecreting CHO cells or mice that received B3 with a single light chain mutation. However, none of the mice had histological changes or deposition of human immunoglobulin G in the kidneys. Sequence changes may alter the pathogenicity of B3, but further studies using different techniques are needed to investigate this possibility.
authors.affiliation: Centre for Rheumatology, Division of Medicine, University College London, UK. l.mason@ucl.ac.uk
authors.forename: Lesley J
authors.initials: LJ
authors.lastname: Mason
authors.forename: Anastasia
authors.initials: A
authors.lastname: Lambrianides
authors.forename: Joanna D
authors.initials: JD
authors.lastname: Haley
authors.forename: Jessica J
authors.initials: JJ
authors.lastname: Manson
authors.forename: David S
authors.initials: DS
authors.lastname: Latchman
authors.forename: David A
authors.initials: DA
authors.lastname: Isenberg
authors.forename: Anisur
authors.initials: A
authors.lastname: Rahman
mesh_terms: D000818:Animals; D000974:Antibodies, Antinuclear / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D016466:CHO Cells / Q000276:immunology / Q000637:transplantation; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004247:DNA / Q000276:immunology; D004195:Disease Models, Animal; D005260:Female; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D007668:Kidney / Q000276:immunology / Q000473:pathology; D008181:Lupus Nephritis / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D016513:Mice, SCID; D009707:Nucleosomes / Q000276:immunology*; D011507:Proteinuria / Q000209:etiology* / Q000276:immunology / Q000473:pathology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D014162:Transfection
chemical_list: D000974:Antibodies, Antinuclear; D017077:Culture Media, Conditioned; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D009707:Nucleosomes; D011993:Recombinant Fusion Proteins; D004247:DNA