muramidase and Lupus-Nephritis

Abrogation of peripheral tolerance in transgenic mice that express a uniform B-cell receptor may create a powerful tool to examine the molecular mechanisms that underlie the autoimmune response in B cells. Here we report that processes that induce a systemic lupus erythematosus-like syndrome in normal mice, namely chronic graft vs host reaction, trigger systemic autoimmunity in a well-established transgenic mice model of B cell receptor peripheral tolerance. The induction of graft vs host reaction in mice that carry both a rearranged B cell Ag receptors specific for hen egg lysozyme and expressing chronically circulating hen egg lysozyme Ag resulted in induction of high and sustained levels of circulating anti-hen egg lysozyme autoantibodies and glomerulonephritis with proteinuria. This was associated with marked changes in expression of cell-surface proteins, such as CD23 and complement receptor 2. B cells from the graft vs host-induced mice could proliferate in vitro in response to self-Ag, and upon stimulation with anti-IgD demonstrated rapid phosphotyrosine phosphorylation of specific proteins, which could not be induced in the anergic double transgenic B cells. Conversely, loss of tolerance was not associated with a higher induction in the level of Syk kinase phosphorylation following stimulation with anti-IgD. Taken collectively, these data establish that 1) processes that induce a systemic lupus erythematosus-like syndrome in normal mice can abrogate peripheral tolerance in transgenic mice expressing self-tolerized B cells, and that 2) loss of tolerance in this model is associated with marked changes in surface expression of B cell coreceptors as well as with selective changes in IgD-induced signaling by discrete tyrosine-phosphoproteins, but not Syk kinase.

Topics: Animals; Autoantibodies; Autoantigens; B-Lymphocytes; Disease Models, Animal; Enzyme Precursors; Graft vs Host Reaction; Immune Tolerance; Immunoglobulin D; Immunoglobulin M; Intracellular Signaling Peptides and Proteins; Lupus Erythematosus, Systemic; Lupus Nephritis; Lymphocyte Activation; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muramidase; Phosphoproteins; Phosphotyrosine; Protein-Tyrosine Kinases; Receptors, Antigen, B-Cell; Self Tolerance; Syk Kinase

We previously reported that lysozyme electrostatically inhibits the fibronectin-mediated DNA binding to the glomerular basement membrane (GBM) and reduces in situ DNA-anti-DNA complex formation in the GBM in NZB/W F1 mice [1]. In this study, we further noticed significant increases in urinary excretion of anti-DNA antibodies and immune complexes (IC) in lysozyme-treated NZB/W F1 mice. Their clearance ratios of IgG anti-DNA antibody to whole IgG were markedly high compared with those of saline-treated animals. A large number of IgG and C3 positive granules were observed in the tubular cells of NZB/W F1 mice treated with lysozyme. On the contrary, nil or only small amounts of anti-DNA antibodies were detected in the urine of NZB/W F1 mice without lysozyme administration despite a large amount of proteinuria, suggesting entrapment of the antibodies in lupus glomeruli. Lysozyme neither inhibited the binding of anti-DNA antibodies to DNA or heparan sulphate nor did it displace anti-DNA antibodies and IC from the kidney homogenates of lupus mice. It thus appears that the inhibition of DNA binding to the GBM due to lysozyme reduced the entrapment of anti-DNA antibodies in the GBM, resulting in urinary excretion of the antibodies.

Topics: Animals; Antibodies, Antinuclear; Antigen-Antibody Complex; Basement Membrane; Complement C3; DNA; Female; Immunoglobulin G; Kidney Glomerulus; Kidney Tubules; Lupus Nephritis; Mice; Mice, Inbred ICR; Mice, Inbred NZB; Muramidase

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

The effect of lysozyme on intraglomerular immune complex deposition was examined in NZB/W F1 mice undergoing unilateral nephrectomy. Unilateral nephrectomy enhanced the glomerular immune complex deposition and glomerular lesions, which were suppressed by repeated intraperitoneal injections of lysozyme, in spite of unaltered serum anti-DNA antibody titers. DNA binding to the glomerular basement membrane (GBM) examined in vitro and that to glomeruli examined in vitro were also suppressed by lysozyme. An increased survival rate and decreased proteinuria were also induced by this basic protein. The mechanisms of the ameliorative effect were studied in vitro. DNA was bound to the GBM only in the presence of serum, plasma, or fibronectin. A similar inhibitory effect on DNA binding was also obtained by another polycation, hexadimethrine, in place of lysozyme. The in vitro findings suggest that DNA binding to the GBM is mediated by fibronectin, and that lysozyme electrostatically inhibits this binding, thereby possibly reducing the in situ DNA-anti-DNA complex formation in the GBM.

Topics: Albuminuria; Animals; Antigen-Antibody Complex; Autoantibodies; Basement Membrane; DNA; Fibronectins; In Vitro Techniques; Kidney Glomerulus; Lupus Erythematosus, Systemic; Lupus Nephritis; Mice; Muramidase