sepharose and Lupus-Nephritis

The relationship between chemical properties of anti-DNA antibodies (Abs) and lupus nephritis was investigated. The anti-DNA Abs in sera from systemic lupus erythematosus (SLE) patients were separated by two-dimensional electrophoresis (2-DE) and immunoblotting with goat anti-human IgG Abs. Highly cationic anti-DNA Abs were detected in deoxyribonuclease I (DNase I)-treated sera from patients with lupus nephritis (in 8 of 9 cases) but not in the sera from SLE patients without nephritis (in 0 of 9 cases), normal subjects, or patients with other renal diseases (in 0 of 7 cases). The mean titers of anti-dsDNA Abs in patients with lupus nephritis were not significantly different from those in SLE patients without nephritis. The highly cationic anti-DNA Abs in the sera disappeared after incubation with heparin-Sepharose. These results suggest that highly cationic anti-DNA Abs are specific for lupus nephritis and may be involved in development of lupus nephritis via the binding to glycosaminoglycans on the endothelial cell surface.

Topics: Adolescent; Adult; Antibodies, Antinuclear; Cations; Deoxyribonuclease I; DNA; Electrophoresis, Gel, Two-Dimensional; Female; Heparin; Humans; Immunoblotting; Immunoglobulin G; Lupus Nephritis; Male; Middle Aged; Sepharose

Previous studies have shown that the majority of C1q-binding IgG in patients with systemic lupus erythematosus (SLE) is composed of autoantibodies to the collagen-like region of C1q. Mice of the MRL/l strain are considered as a murine model of human SLE and possess autoantibodies to nuclear antigens as well as IgM and IgG rheumatoid factors (RF). This study was undertaken to characterize the C1q-binding IgG in MRL/l mice. In contrast to human SLE, C1q-binding IgG in MRL/l mice showed immunochemical characteristics of immune complexes rather than those of autoantibodies to C1q. Namely, C1q-binding IgG in MRL/l mice was large-sized upon HPLC gel filtration and abolished by digestion with pepsin or by high salt concentration, and bound to the globular region of C1q. The C1q-binding activity in MRL/l mice was absorbed by double-stranded DNA- or single-stranded DNA-cellulose. The medium-sized immune complexes containing RF have been well documented in MRL/l mice. In this study, however, mouse IgG-Sepharose failed to absorb fully C1q-binding IgG. We conclude that the majority of C1q-binding IgG in MRL/l mice consists of large-sized immune complexes containing antibodies to DNA.

Topics: Animals; Antibodies, Antinuclear; Antigen-Antibody Complex; Cellulose; Complement C1q; DNA; DNA, Single-Stranded; Female; Humans; Immunoglobulin G; Immunosorbent Techniques; Lupus Nephritis; Mice; Mice, Inbred BALB C; Mice, Mutant Strains; Protein Binding; Sepharose

An effort has been made to integrate insights on charge-based interactions in immune complex glomerulonephritis with nuclear antigen involvement in lupus nephritis. Attention was focussed on the histones, a group of highly cationic nuclear constituents, which could be expected to bind to fixed anionic sites present in the glomerular basement membrane (GBM). We demonstrated that all histone subfractions, prepared according to Johns (4), have a high affinity for GBM and the basement membrane of peritubular capillaries. Tissue uptake of 125I-labeled histones was measured by injecting 200 micrograms of each fraction into the left kidney via the aorta and measuring organ uptake after 15 min. In glomeruli isolated from the left kidneys, the following quantities of histones were found: f1, 13 micrograms; f2a (f2al + f2a2), 17 micrograms; f2b, 17 micrograms; and f3, 32 micrograms. Kinetic studies of glomerular binding showed that f1 disappeared much more rapidly than f2a. The high affinity of histones (pI between 10.5 and 11.0; mol wt 10,000-22,000) for the GBM correlates well with their ability to form aggregates (mol wt greater than 100,000) for comparison lysozyme (pI 11, mol wt 14,000), which does not aggregate spontaneously bound poorly (0.4 micrograms in isolated glomeruli). The quantity of histones and lysozyme found in the isolated glomeruli paralleled their in vitro affinity for a Heparin-Sepharose column (gradient elution studies). This gel matrix contains the sulfated, highly anionic polysaccharide heparin, which is similar to the negatively charged heparan sulfate present in the GBM. Lysozyme eluted with 0.15 M NaCl, f1 with 1 M NaCl, and f2a, f2b, and f3 could not be fully desorbed even with 2 M NaCl; 6 M guanidine-HCl was necessary. Two further findings of great relevance for the concept of induction of immune complex glomerulonephritis by histones were: (a) glomerular-bound histone was accessible for specific antibody given intravenously; and (b) prior binding of histones promoted glomerular deposition of anionic antigens, as could be shown with ssDNA fragments. These data justify the proposal that glomerular deposition of histones can induce immune complex formation, start an inflammatory process, and produce tissue damage.

Topics: Animals; Antigen-Antibody Complex; Basement Membrane; Binding Sites; Chromatography, Affinity; DNA; DNA, Single-Stranded; Heparin; Histones; Immune Sera; Injections, Intra-Arterial; Injections, Intravenous; Iodine Radioisotopes; Kidney Glomerulus; Lupus Nephritis; Macromolecular Substances; Male; Muramidase; Organ Specificity; Rats; Rats, Inbred Strains; Sepharose