Blood clearance kinetics and liver uptake of mononucleosomes in mice.

Nucleosomes generated by apoptosis have become of considerable interest in relation to pathogenesis of systemic lupus erythematosus in mice and humans. Therefore, the fate of circulating mononucleosomes was examined in normal C57Bl/6J mice. The mononucleosomes were prepared from chicken erythrocytes and radiolabeled on the histone component. The removal of nucleosomes from circulation at doses less than 11 micrograms of injected mononucleosomes was rapid, but with increasing doses of injected nucleosomes, the slopes of the removal curves decreased. Liver was the major organ for removal of circulating nucleosomes, accounting for 71.0 to 84.7% of nucleosomes removed from circulation at 10 min. After i.v. injection of nucleosomes, 0.52 +/- 0.15% localized in kidneys. With prior i.v. injection of histones, the glomerular localization of mononucleosomes increased threefold. The clearance of mononucleosomes was decreased sixfold by concurrent injection of ssDNA. These studies show that in mice, circulating mononucleosomes are handled similar to DNA, and they do not avidly localize in glomeruli unless histones have already bound to renal glomeruli.

Bone pain that responds to calcium channel blockers. A retrospective and prospective study of transplant recipients.

A severe episodic bone pain syndrome of unknown cause was first described in renal transplant recipients in France and associated with the use of cyclosporine. We have retrospectively and prospectively evaluated this pain syndrome in our transplant patients. This pain is bilateral, of acute onset and episodic, primarily involving the knees and/or ankles. It usually occurs at night or with recumbency, and is often relieved with elevation or walking. Physical examination of the affected areas is unrevealing. Kidney, liver, pancreas, heart, lung and combined organ transplant recipients on cyclosporine were available from the University of Washington Transplant Services and were retrospectively evaluated by chart review (n = 351) or prospective clinical evaluation (n = 38) for evidence of this clinical syndrome. In the retrospective chart review, 19.1% of patients had episodes of bone pain. The highest prevalence occurred in renal transplant recipients (27.7%). The bone pain syndrome was documented in the charts of 14 patients who subsequently developed significant osteonecrosis. Prospectively, 21 of 22 patients with the bone pain syndrome experienced complete relief of this pain upon treatment with calcium channel blockers (95.4% response rate). The pathophysiology of this bone pain syndrome is unknown, although its response to the vasodilatory effects of calcium channel blockers suggests a vascular etiology.

Antigens of varying size persist longer in subepithelial than in subendothelial immune deposits in murine glomeruli.

The kinetics of removal of immune deposits from the subendothelial and subepithelial areas of glomeruli were analyzed in mice. Radiolabeled, cationized Ag of different molecular size, including human serum albumin, bovine thyroglobulin, and human IgM, were used to form the immune deposits in mouse glomeruli with specific, purified rabbit antibodies to these proteins. The disappearance curves of the radiolabeled Ag from glomeruli consisted of two exponential components. The immune deposits and their location in glomeruli were identified by immunofluorescence and electron microscopy. The t1/2 of disappearance of immune deposits were assigned to subendothelial or subepithelial deposits on the basis of ultrastructural observations. The t1/2 of subendothelial immune deposits ranged from 0.6 to 1.9 days with the three different Ag-antibody systems. In contrast, the t1/2 of the subepithelial immune deposits ranged from 9.32 to 231 days. The cationized human serum albumin in subepithelial areas had the longest t1/2, and this was not altered by the endogenous immune response to the injected materials, as determined in studies with nude mice. The results constitute formal documentation of the prolonged t1/2 of an exogenous Ag in glomerular immune deposits. The described approach can serve to examine variables that alter this prolonged presence of subepithelial immune deposits in glomeruli.

Autoantibodies to the collagen-like region of C1Q deposit in glomeruli via C1Q in immune deposits.

The autoantibodies to the collagen-like region of C1q (CLR), purified from two patients with systemic lupus erythematosus, deposited in mouse glomeruli when human C1q was present in antigen-antibody complexes in glomeruli. The immune deposits with C1q in mouse glomeruli were achieved by the administration of cationized immune complexes containing human C1q. The presented data suggest that the autoantibodies to CLR could enhance the pathogenic role of immune complexes deposited in glomeruli by binding to C1q in immune deposits. These findings may explain the association of autoantibodies to CLR with proliferative lupus nephritis.

Glomerular subendothelial and subepithelial immune complexes, containing the same antigen, are removed at different rates.

To examine the persistence of immune deposits in the subendothelial and subepithelial areas of the glomerular basement membrane in rats, immune deposits were formed by injection of radiolabelled, cationized human serum albumin (HSA) as antigen, followed by rabbit antibodies to HSA. The disappearance of the radiolabelled antigen from immune deposits in glomeruli was described by a curve consisting of two exponential components. By electron microscopy, subendothelial and subepithelial immune deposits were initially present in glomeruli. At later time-points, only subepithelial immune deposits were present. The fast component of disappearance, attributed to subendothelial deposits, had a half-life of 3.89 +/- 0.32 h. The slow component of disappearance from glomeruli, attributed to subepithelial deposits, had a half-life of 85.5 +/- 3.1 h. Since some of the injected, radiolabelled antigen was sequestered in other compartments of the body, the possibility was raised that antigen from these sites might be released and contribute to the persistence of deposits in glomeruli. This possibility, however, was excluded when transplantation of kidneys with immune deposits to untreated recipients revealed no difference in the amount of antigen persisting in nontransplanted and transplanted kidneys.

A small proportion of cationic antibodies in immune complexes is sufficient to mediate their deposition in glomeruli.

Positively charged antibodies mediate enhanced deposition of circulating immune complexes at the glomerular basement membrane. The presented experiments demonstrate that when soluble immune complexes were prepared with a mixture of antibodies containing 10 to 25% cationic antibodies, then noncationic antibodies in the complexes were deposited in mouse glomeruli. One or two cationic antibodies in each immune complex sufficed for deposition of the complexes. Proof for this was obtained by two kinds of experiments. First, the injected immune complexes were prepared in Ag excess from mixtures of radiolabeled noncationic rabbit antibodies to human serum albumin (HSA) and unlabeled cationized rabbit antibodies to HSA, thus permitting the specific quantitation of the deposition of noncationic antibodies in glomeruli because of the presence of cationized antibodies within the same complexes. As a control experiment, immune complexes prepared only with noncationic antibodies resulted in very little deposition in kidneys over the same time period. Second, detection of the localization of the noncationic antibody in deposits in glomeruli by immunofluorescence microscopy was accomplished using immune complexes prepared with mixtures of noncationic goat antibodies to HSA and cationized rabbit antibodies to HSA. Thus, the synthesis of a small population of cationic antibodies during the immune response may lead to the formation of circulating immune complexes with enhanced propensity for deposition in glomeruli in patients with SLE or other immune complex diseases.

Deposition of immune complexes containing cationized antibodies in myocardial small blood vessels of mice.

Small myocardial blood vessels constitute a site for preferential deposition of preformed cationic immune complexes. This preferential deposition was demonstrated with a limited dose (100 micrograms) of cationized rabbit antibodies to human serum albumin, injected into C57B1/6J mice either alone or in the form of preformed immune complexes. Heart, kidney, liver, intestine, and skeletal muscle were examined for immune deposits by immunofluorescence microscopy. Highly cationized antibodies injected alone showed deposition in glomeruli and in the liver along the sinusoids but not in other tissues. Immune complexes containing native rabbit antibodies deposited only in liver in a Kupffer cell pattern. Moderate and highly cationized antibodies in immune complexes deposited in myocardial small blood vessels, liver, and glomeruli, but not in intestine or skeletal muscle. These complexes deposited via electrostatic interactions since unrelated polycationic molecules, protamine sulfate or cationized rabbit serum albumin, injected 1 min prior to cationic antibodies in immune complexes blocked deposition in myocardial small vessels, glomeruli, and liver. Administration of protamine or cationized rabbit serum albumin 1 min after deposition of cationized immune complexes resulted in displacement of the immune deposits in heart, kidney, and liver, but not when the injection was given 1 hr later. The presented data indicate that with passage of time the immune deposits rearrange and forces other than charge-charge interactions retain them in myocardial vessels.

Immune complexes with cationic antibodies deposit in glomeruli more effectively than cationic antibodies alone.

In previously published studies, highly cationized antibodies alone and in immune complexes bound to glomeruli by charge-charge interaction, but only immune complexes persisted in glomeruli. Because normal IgG does not deposit in glomeruli, studies were conducted to determine whether cationized antibodies can be prepared which deposit in glomeruli when bound to antigen but not when free in circulation. A series of cationized rabbit antiHSA was prepared with the number of added amino groups ranging from 13.3 to 60.2 per antibody molecule. Antibodies alone or in preformed soluble immune complexes, prepared at fivefold or 50-fold antigen excess, were administered to mice. With the injection of a fixed dose of 100 micrograms per mouse, antibodies alone did not deposit in glomeruli with less than 29.6 added amino groups by immunofluorescence microscopy. In contrast, 100 micrograms of antibodies with 23.5 added amino groups in immune complexes, made at fivefold antigen excess, formed immune deposits in glomeruli. With selected preparations of cationized, radiolabeled antibodies, deposition in glomeruli was quantified by isolation of mouse glomeruli. These quantitative data were in good agreement with the results of immunofluorescence microscopy. Immune complexes made at 50-fold antigen excess, containing only small-latticed immune complexes with no more than two antibody molecules per complex, deposited in glomeruli similar to antibodies alone. Selected cationized antibodies alone or in immune complexes were administered to mice in varying doses. In these experiments, glomerular deposition of immune complexes, made at fivefold antigen excess, was detected with five- to 10-fold smaller doses than the deposition of the same antibodies alone. These studies demonstrate that antibody molecules in immune complexes are more likely to deposit in glomeruli by charge-charge interactions than antibodies alone.

Only the initial binding of cationic immune complexes to glomerular anionic sites is mediated by charge-charge interactions.

The role of charge-charge interactions between cationic immune complexes and the anionic sites on the glomerular basement membrane was examined. For this purpose, soluble immune complexes at fivefold antigen excess were prepared with human serum albumin and cationized rabbit antibodies to this protein. When unrelated cationic proteins, protamine sulfate or cationized rabbit serum albumin, were given 1 min before the cationized immune complexes, glomerular immune deposits did not form. Cationic immune complexes allowed to deposit in glomeruli could readily be displaced by protamine sulfate or cationized rabbit serum albumin injected 1 min after the immune complexes. If the same cationic molecules were injected 1 hr after the immune complexes, the complexes could not be displaced from glomeruli. In contrast, cationic complexes that were deposited in glomeruli in the presence of a very high degree of antigen excess in circulation to prevent their condensation into larger complexes in glomeruli were readily displaced at 1 min and 1 hr with protamine sulfate or with cationized rabbit serum albumin. On the basis of these results, we concluded that the initial binding of cationic immune complexes to glomeruli occurs by charge-charge interactions. Once the immune complexes in glomeruli condense to larger deposits, forces other than charge-charge interactions are responsible for their retention in glomeruli.

Antibody localization in the glomerular basement membrane may precede in situ immune deposit formation in rat glomeruli.

The administration of cationized antibodies, specific to human serum albumin, into the renal artery of rats caused transient presence of IgG in glomeruli by immunofluorescence microscopy. Intravenous infusion of appropriate doses of antigen after the injection of cationized antibodies resulted in immune deposit formation in glomeruli that persisted through 96 hr. By electron microscopy, these deposits were located in the subepithelial area. The injection of large doses of antigen produced immune deposits which were present in glomeruli for only a few hours, presumably due to formation of only small-latticed immune complexes. The presented data indicate that cationic antibodies bound to the fixed negative charges of the glomerular basement membrane can interact with circulating antigen to form immune deposits in glomeruli. This mechanism may be important because anionic antigens have been shown to induce the synthesis of cationic antibodies.

Glomerular localization of preformed immune complexes prepared with anionic antibodies or with cationic antigens.

The glomerular basement membrane presents a highly anionic surface to circulation. The effects of anionic antibody and cationic antigen in preformed immune complexes prepared at 5-fold antigen excess were investigated in separate experiments in mice. Anionized antibodies (isoelectric point 4 to 6) to human serum albumin were prepared by acetylation and immune complexes produced in vitro. Blood clearance kinetics and glomerular immunofluorescence patterns of these immune complexes were not affected by anionization . Electron microscopy revealed mesangial deposits, indicating that the deposition of immune complexes in the mesangium occurs with highly anionic immune complexes. Cationic human serum albumin (AgED, isoelectric point 7.5 to 9.0) alone or as performed immune complexes ( AgEDAb ) showed rapid blood clearance (less than 1% remaining by 18 hours) and localized in renal glomeruli by immunofluorescence microscopy. AgED injected alone was present in glomeruli at 1 minute after injection but was absent at 12 hours. After injection of AgEDAb , both antigen and antibodies were present in glomeruli from 1 minute through 72 hours by immunofluorescence microscopy. Electron-dense deposits were seen at the anionic sites in the lamina rara interna and lamina rara externa at 1 minute and 1 hour after injection of AgED and AgEDAb containing free AgED. After AgEDAb injection electron-dense deposits were evident at 12 to 48 hours in the mesangium and in the subendothelial area, especially adjacent to the mesangium. By 72 hours after AgEDAb injections mesangial deposits predominated, although small subepithelial deposits were also present. An inflammatory reaction was noted in the glomeruli after administration of AgEDAb . Thus, preformed immune complexes containing cationized antigen show glomerular deposition, primarily in the subendothelial and mesangial regions and at later time points also in the subepithelial area.

Precipitating antigen-antibody systems are required for the formation of subepithelial electron-dense immune deposits in rat glomeruli.

This study was conducted to determine whether multivalent, precipitating antigens are required for formation of subepithelial electron-dense immune deposits in glomeruli. 2-nitro-4-azidophenyl (NAP) was conjugated with variable density to human serum albumin (HSA) to yield nonprecipitating (NAP3.1 X HSA and NAP11.4 X HSA) and precipitating (NAP19.7 X HSA) antigens with antibodies to the hapten. These antigen preparations were cationized with ethylene diamine to enhance deposition in renal glomeruli due to interaction with the fixed negative charges in the glomerular capillary wall. Following injection into the left renal artery of rats these antigens alone persisted in the glomeruli for a relatively short time by immunofluorescence microscopy. When antibodies to NAP were injected intravenously after the antigen injection, the nonprecipitating antigens and antibodies were detectable in the glomeruli by immunofluorescence microscopy up to 8 h, comparable to antigen alone. Electron-dense deposits were not formed in these glomeruli. In contrast, when the precipitating antigen was injected and followed by antibodies to the hapten, antigen and antibody were detected by immunofluorescence microscopy through 96 h. In these specimens electron-dense deposits were present from 40 min through 96 h and after 24 h the deposits were present only in the subepithelial area. The same results were obtained when the nonprecipitating hapten-carrier conjugates were followed with antibodies to the carrier molecule. These data indicate that the persistence of immune deposits by immunofluorescence microscopy and the formation of electron-dense deposits in the subepithelial area require a precipitating antigen-antibody system.

Effect of cationized antibodies in performed immune complexes on deposition and persistence in renal glomeruli.

To study the interaction of positively charged antibodies in immune complexes with the fixed negative charge on the glomerular capillary wall, chemical cationization of antibody was accomplished with the maintenance of antigen-binding activity. These cationized antibodies bound rapidly to glomeruli but did not persist. Large-latticed immune complexes formed with these cationic antibodies showed rapid deposition and persistence in renal glomeruli, even when administered in small doses. Electron-dense deposits were present at the anionic sites in the glomerular basement membrane at 1 min and 1 h, with extensive subendothelial deposits present from 12 to 72 h. By 14 d, the deposits were seen in the subepithelial region and the glomerular mesangium. The administration of small-latticed immune complexes prepared with cationized antibody revealed initial deposition without persistence in glomeruli in a manner similar to cationized antibodies alone. Thus, the positive charges on antibodies in immune complexes contribute to the deposition and persistence of the complexes in glomeruli, particularly in the subendothelial area.

Preparation and characterization of 4-azido-2-nitrophenyl human serum albumin as an antigen for covalent cross-linking of immune complexes.

Human serum albumin (HSA) was conjugated with 4-fluoro-3-nitrophenyl azide to yield varying density of the 4-azido-2-nitrophenyl haptenic group, useful for covalent cross-linking in the antibody-combining site. The epitope density of the antigen influenced several examined biologic properties. Precipitation in gel diffusion occurred when the average epitope density was 13 or above. Complement (C) activation was not found by incubation with guinea pig C, by binding to human Clq, or by conversion of the electrophoretic mobility of human C3 with epitope densities up to 13. Upon i.v. injection, rapid removal of the conjugated HSA occurred when more than seven 4-azido-2-nitrophenyl groups were present. This rapid removal was in part due to hepatic uptake. These studies point out the epitope density-dependent alterations of biologic properties of an antigen useful for preparation of immune complexes covalently cross-linked in the antibody-combining site.