Anti-DNA idiotypes deposited in renal glomeruli of patients with lupus nephritis

Idiotypes (Id) of human anti-DNA antibodies, designated as O-81 Id, were specifically detected on the immune deposits of renal glomeruli in 46% of patients with lupus nephritis. Id-binding to anti-Id antibodies was blocked by free O-81 Id and to some extent by free DNA. DNase or acid buffer treatment failed to reveal new Id determinants on the deposits. O-81 Id and NE-1 Id activity were also detected on the renal eluate-derived IgG, but not IgM from the autopsy cases with lupus nephritis. The incidences of O-81 Id were not associated with histological features in the glomeruli, but the distribution patterns were similar to those of IgG deposits. Our study also showed that 65% to 70% of patients with IgG deposits either in the subendothelium or in the subepithelial area of the glomerular basement membrane (GBM) showed positive tests for O-81 Id. It was also noted that most patients with massive proteinuria had O-81 Id in their glomeruli. It is concluded that O-81 Id deposits are relatively specific for active lupus nephritis and that immunofluorescence studies using anti-Id antibodies may be clinically useful for specifying the renal lesions of systemic lupus erythematosus (SLE).     

Anti-DNA autoantibodies initiate experimental lupus nephritis by binding directly to the glomerular basement membrane in mice

The strongest serological correlate for lupus nephritis is antibody to double-stranded DNA, although the mechanism by which anti-DNA antibodies initiate lupus nephritis is unresolved. Most recent reports indicate that anti-DNA must bind chromatin in the glomerular basement membrane or mesangial matrix to form glomerular deposits. Here we determined whether direct binding of anti-DNA antibody to glomerular basement membrane is critical to initiate glomerular binding of anti-DNA in experimental lupus nephritis. Mice were co-injected with IgG monoclonal antibodies or hybridomas with similar specificity for DNA and chromatin but different IgG subclass and different relative affinity for basement membrane. Only anti-DNA antibodies that bound basement membrane bound to glomeruli, activated complement, and induced proteinuria whether injected alone or co-injected with a non-basement-membrane-binding anti-DNA antibody. Basement membrane-binding anti-DNA antibodies co-localized with heparan sulfate proteoglycan in glomerular basement membrane and mesangial matrix but not with chromatin. Thus, direct binding of anti-DNA antibody to antigens in the glomerular basement membrane or mesangial matrix may be critical to initiate glomerular inflammation. This may accelerate and exacerbate glomerular immune complex formation in human and murine lupus nephritis.     

Expression of glomerular heparan sulphate domains in murine and human lupus nephritis.

BACKGROUND: Recently, we identified specific N- and 6-O-sulphated heparan sulphate (HS) domains on activated glomerular endothelial cells. In this study, we evaluated in lupus nephritis the expression of different HS domains on glomerular endothelium and in the glomerular basement membrane (GBM). METHODS: The expression of specific glomerular HS domains and the presence of immunoglobulins (Ig) were determined by immunofluorescence staining of kidney sections of patients with nephritis due to systemic lupus erythematosus (SLE) and MRL/lpr lupus mice. The expression/presence of glomerular HS domains and Ig was also evaluated after eluting Ig from renal sections of lupus mice using two elution methods, and in renal sections of lupus mice treated with heparinoids. RESULTS: Both MRL/lpr mice and patients with lupus nephritis showed a decreased expression of HS in the GBM. The expression of N- and 6-O-sulphated HS domains on glomerular endothelium was decreased in MRL/lpr mice, but increased in SLE patients. MRL/lpr mice had more extensive glomerular Ig deposits than SLE patients. After elution of Ig, the glomerular endothelial expression of N- and 6-O-sulphated HS domains in MRL/lpr mice was recovered and even increased above normal levels, while the expression of HS in the GBM was restored to normal levels. Treatment with heparinoids prevented Ig deposition and preserved the expression of glomerular HS domains at normal levels in lupus mice. CONCLUSION: The expression of specific HS domains on glomerular endothelium and in the GBM is changed during lupus nephritis due to masking by Ig deposits and induction of inflammatory N- and 6-O-sulphated HS domains.     

Glomerular binding of anti-dsDNA autoantibodies: The dispute resolved?

The binding of anti-double-stranded DNA (anti-dsDNA) autoantibodies to the glomerular basement membrane (GBM) in lupus nephritis can be explained by two mechanisms: (1) direct crossreactive binding to intrinsic glomerular antigens; (2) nucleosome-mediated binding to heparan sulfate in the GBM. Kalaaji et al. demonstrated using novel techniques that glomerular in vivo-bound antoantibodies bind to nucleosomes/dsDNA derived from apoptotic cells and not to intrinsic glomerular structures.     

Non-Goodpasture anti-GBM antibodies in patients with glomerulonephritis

The sera of 206 consecutive patients with biopsy-proven glomerulonephritis were tested by ELISA for the presence of Goodpasture and non-Goodpasture anti-GBM antibodies. Antigens were solubilised from human GBM with purified bacterial collagenase and with 6 mol/l guanidine-HCl respectively. Only 12 sera reacted when collagenase-resistant GBM proteins were used as antigens in ELISA. Sera from two of these patients also reacted with the Goodpasture antigen, that is the globular domain of collagen IV, purified from collagenase extracts of GBM. These two patients had classical Goodpasture syndrome with linear crescentic nephritis. The other ten sera did not react with the Goodpasture antigen and immunofluorescence microscopy showed granular glomerular immune deposits. Antibodies against antigens present in 6 mol/l guanidine-HCl extracts of human GBM were much more frequent, particularly in lupus nephritis and IgA nephropathy, but relatively common also in patients with glomerulonephritis associated with systemic connective tissue and systemic vasculitic disorders. In contrast, these non-Goodpasture antibodies were only sporadic in primary forms of glomerulonephritis such as minimal-change nephropathy, membranous glomerulopathy, or acute post-infectious glomerulonephritis. The presence of circulating IgG, IgA or IgM antibodies against 6 mol/l guanidine-HCl extractable GBM antigens correlated with granular deposits of corresponding immunoglobulins in both mesangial and capillary loop regions of glomeruli, indicating a possible pathogenic role for non-Goodpasture anti-GBM antibodies in several forms of glomerulonephritis.     

Anti-DNA antibodies can bind to the glomerulus via two distinct mechanisms.

It is generally assumed that antibodies to double stranded DNA (anti-DNA) play a pivotal role in the pathogenesis of SLE nephritis. Recently, we reported that anti-DNA antibodies can bind to heparan sulphate proteoglycan (HSPG), a constituent of the glomerular basement membrane (GBM), via histones and DNA. We postulated that these histone/DNA/anti-DNA complexes can bind via their histone part to the glomerulus in vivo. To test this hypothesis we performed in vitro binding studies with isolated GBM loops and renal perfusion studies in the rat using histones, DNA and an anti-DNA monoclonal antibody (mAb) with high avidity for dsDNA. A strong granular binding of anti-DNA mAb to isolated GBM loops occurred via histones and DNA and a moderate granular binding was found via DNA alone. Anti-DNA mAb alone did not bind to the GBM loops. After perfusion of histones, DNA and immediately thereafter anti-DNA, we found with immunoelectron microscopy (IEM) a strong binding to endothelial cells in the glomerulus and to a lesser extent in the GBM. When the anti-DNA mAb was injected i.v. one hour after perfusion of histones and DNA, we observed a strong fine granular binding to the capillary wall by immunofluorescence (IF) in a membranous pattern along with some minor mesangial deposits. After perfusion of DNA alone followed by anti-DNA mAb, binding in the glomerulus was less than with histones and DNA, and was more restricted to the mesangium. No direct binding to the glomerulus was observed after perfusion with anti-DNA mAb alone, histones and anti-DNA mAb, or histones, DNA and a control mAb.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunopathogenetic mechanisms in renal disease

This review deals with various immunopathogenetic mechanisms of renal diseases. Although immune-complex nephritis was long thought to be the result of deposition of circulating immune complexes, recent studies have revealed the importance of in situ formation. In experimental Heymann nephritis, an antigen (gp 330) has been localized on glomerular epithelial cells, and antibodies reactive with gp 330 have been eluted from glomeruli; deposits appear to result from shedding of complexes from the podocyte surface into the basement membrane. In human disease, it seems likely that mesangial and subendothelial deposits most often result from deposition of circulating immune complexes, whereas subepithelial deposits are formed mainly in situ. Cell-mediated immune mechanisms are capable of producing glomerular injury, as shown in experimental models. In humans, Wegener's granulomatosis and idiopathic crescentic glomerulonephritis may involve such mechanisms. In certain forms of experimental and human tubulointerstitial nephritis, cell-mediated immunity also seems to play an important role.     

Glomerular Autoimmune Multicomponents of Human Lupus Nephritis In Vivo (2): Planted Antigens

Glomerular planted antigens (histones, DNA, and C1q) are potential targets of autoimmunity in lupus nephritis (LN). However, the characterization of these antigens in human glomeruli in vivo remains inconsistent. We eluted glomerular autoantibodies recognizing planted antigens from laser-microdissected renal biopsy samples of 20 patients with LN. Prevalent antibody isotypes were defined, levels were determined, and glomerular colocalization was investigated. Renal and circulating antibodies were matched, and serum levels were compared in 104 patients with LN, 84 patients with SLE without LN, and 50 patients with rheumatoid arthritis (RA). Autoantibodies against podocyte antigens (anti–α-enolase/antiannexin AI) were also investigated. IgG2 autoantibodies against DNA, histones (H2A, H3, and H4), and C1q were detected in 50%, 55%, and 70% of biopsy samples, respectively. Anti-DNA IgG3 was the unique non-IgG2 anti-DNA deposit, and anti-C1q IgG4 was mainly detected in subepithelial membranous deposits. Anti-H3, anti-DNA, and anti-C1q IgG2 autoantibodies were also prevalent in LN serum, which also contained IgG3 against the antigen panel and anti-C1q IgG4. Serum and glomerular levels of autoantibodies were not strictly associated. High serum levels of all autoantibodies detected, including anti–α-enolase and antiannexin AI, identified LN versus SLE and RA. Anti-H3 and anti–α-enolase IgG2 levels had the most remarkable increase in LN serum and represented a discriminating feature of LN in principal component analysis. The highest levels of these two autoantibodies were also associated with proteinuria>3.5 g/24 hours and creatinine>1.2 mg/dl. Our findings suggest that timely autoantibody characterization might allow outcome prediction and targeted therapies for patients with nephritis.     

Internalization of antibodies by endothelial cells via fibronectin implicating a novel mechanism in lupus nephritis

BACKGROUND: One of the crucial events in lupus nephritis is the glomerular deposition of immunoglobulins (Igs), of which pathogenic properties have been proposed mostly to be either type IIor type III allergic reactions. Some of IgG3-producing hybridoma clones established from an MRL/MpTn-gld/gld (MRL/gld) lupus mouse generate wire loop-like lesions in glomeruli resembling lupus nephritis when injected into SCID mice. These clones are useful for analyzing the mechanisms of glomerular deposition of antibodies in lupus nephritis at the monoclonal level. METHODS: Glomerular lesions of SCID mice injected with the hybridoma clones, 17H8a or 1G3 as control were analyzed by light and electron microscopy. Interaction of the antibodies with human glomerular endothelial cells (HGECs) and human umbilical vein endothelial cells (HUVECs) in vitro was studied by fluorescence microscopy, electron microscopy, and flow cytometry. RESULTS: Both antibodies did not show any antigen specificity for mouse glomeruli. The glomerular lesions generated by 17H8a, but not by 1G3, contained electron-dense deposits not only in subendothelial regions but also in the cytoplasm of endothelial cells, suggesting internalization of the 17H8a antibodies by endothelial cells. In cell culture studies, internalization of only 17H8a antibodies by HGECs and HUVECs was observed, but the antibodies did not have antigen specificity for both types of endothelial cells. The internalization by HUVECs was mediated by actin polymerization, and it was inhibited by RGDS (Arg-Gly-Asp-Ser) tetrapeptide, antihuman fibronectin and antihuman integrin beta1 monoclonal antibodies. CONCLUSION: The interaction between particular antibodies and endothelial cell surface integrins via fibronectin may be involved in their subsequent internalization by endothelial cells leading to antibody deposition in glomeruli. This may be one of the mechanisms of glomerular injury in lupus nephritis.     

SLE nephritis: an ultrastructural immunogold study to evaluate the relationship between immune complexes and the basement membrane components type IV collagen, fibronectin and heparan sulphate proteoglycans

The nature of immune complexes and their relationship to the normal glomerular basement membrane (GBM) components type IV collagen, fibronectin, and heparan sulphate proteoglycans (HSPG) have been examined in the glomeruli of 7 cases of systemic lupus erythematosus (SLE) glomerulonephritis using an ultrastructural immunogold technique. In paraformaldehyde-fixed, Lowicryl resin-embedded tissue, the electron-dense deposits contained IgG, IgM, IgA, and C3 whether they were subepithelial, intramembranous, subendothelial, or mesangial and there was no particular relationship between the class of immunoglobulin and site of immune complex localization within the glomerulus. The normal GBM components type IV collagen, fibronectin, and HSPG were found within all the glomeruli, but did not have the same distribution. Type IV collagen and fibronectin were found predominantly on the inner aspect of the GBM and diffusely throughout the more central regions of the mesangial matrix. By contrast the HSPG was seen mainly on the outer aspect of the GBM and at the periphery of the mesangial matrix. In none of the cases were GBM antigens localized within the electron-dense deposits, results which suggest that autoantibodies to these GBM components may not play a role in the development of the glomerulonephritis.     

Mesangial cell-binding anti-DNA antibodies in patients with systemic lupus erythematosus

The mechanisms by which anti-DNA antibodies contribute to the pathogenesis of lupus nephritis (LN) remain to be elucidated. This study investigates the binding of polyclonal anti-DNA immunoglobulins from patients with systemic lupus erythematosus (SLE) to human mesangial cells (HMC) in vitro. Testing of cross-sectional serum samples from 280 LN patients (108 during active disease; 172 during remission), 35 SLE patients without renal involvement, 72 patients with non-lupus primary glomerular diseases, and 37 healthy subjects with a cellular enzyme-linked immunosorbent assay showed significant IgG mesangial cell-binding activity in patients with SLE, particularly those with active LN (P < 0.0001). Significant HMC-binding activity was demonstrated in 83.9%, 42.8%, and 47.1% of patients with active LN, inactive LN, and non-renal SLE, respectively. This was predominantly attributed to binding by anti-DNA antibodies, and immune complex binding accounted for 4.6%, 3.5%, and 2.8% of seropositive samples in the respective groups. Longitudinal studies in 27 LN patients demonstrated correlation between serial levels of anti-DNA antibodies, serum HMC-binding activity, and disease activity in 18 patients (66.7%). Affinity-purified polyclonal IgG anti-DNA antibodies from sera with HMC-binding activity showed significant binding to cultured HMC, and to a lesser extent glomerular and proximal tubular epithelial cells and human umbilical vein endothelial cells, but not tumor cell lines, peritoneal mesothelial cells, bronchial epithelial cells, or fibroblasts. The binding of anti-DNA antibodies to HMC was increased 1.47-fold (P = 0.0059) after the removal of Ig-associated DNA by DNase treatment, but it was unaffected by DNase treatment of HMC membrane. Controlled trypsinization of membrane proteins in HMC resulted in a 1.26-fold (P = 0.0025) increase in their binding by anti-DNA antibodies. In conclusion, subsets of anti-DNA antibodies from patients with SLE are capable of binding to HMC. The association of such binding with renal involvement and disease activity and its modulation by DNA concentration suggest that Ig binding to HMC can be a potential marker for disease activity in selected patients and that the binding of anti-DNA antibodies to HMC may be a pathogenetic mechanism in LN.     

Effect of antibody charge and concentration on deposition of antibody to glomerular basement membrane

Fixed anionic sites within the glomerular capillary wall influence the permeation of serum proteins, the localization of various antigens, and the deposition of antibody in the subepithelial space. In anti-GBM nephritis antibody deposition occurs very rapidly to antigenic sites located relatively proximal in the glomerular capillary wall. We examined the influence of the glomerular charge barrier on anti-GBM antibody deposition by comparing the rate of deposition of antibodies with cationic and anionic isoelectric points. Purified sheep anti-rat GBM IgG was isolated from acid eluates of kidneys obtained 24 hr after rats were injected with sheep antiserum to rat GBM. Anti-GBM IgG was separated into cationic (pI 6.4-8.5) and anionic (pI 4.2-6.8) fractions, which were radiolabelled with 131I and 125I, respectively, shown to have equal antibody contents measured by in vitro binding to normal glomeruli, mixed in equal amounts, and injected in incremental doses to ten rats. At 1 hr the glomerular antibody binding of each fraction was directly related to the blood level (r = 0.95, r = 0.97) and delivery of antibody (r = 0.98, r = 0.98). Glomerular binding of cationic antibody was four times greater than anionic antibody over the entire range of deliveries studied (P less than 0.001). We conclude that glomerular deposition of anti-GBM antibody is directly related to blood concentration and delivery of antibody. Furthermore, the deposition of cationic antibodies to GBM antigens was significantly greater than the deposition of anionic antibodies. The charge-selective glomerular filtration barrier may be an important determinant of the quantity and subclass composition of anti-GBM IgG deposits in glomeruli, and therefore of the severity of tissue injury produced.     

Experimental autoimmune glomerulonephritis induced by homologous and isologous glomerular basement membrane in Brown-Norway rats

In order to study disease mechanisms and potential forms of therapy in glomerulonephritis, a model of experimental autoimmune glomerulonephritis (EAG) has been developed in the rat. We have examined the response of Brown-Norway (BN) rats to a single i.m. injection of collagenase-solubilised homologous (Sprague-Dawley, SD) or isologous (BN) glomerular basement membrane (GBM), with and without complete Freund's adjuvant (CFA). There was a dose-dependent circulating anti-GBM antibody response to all preparations of rat GBM. Animals given either antigen alone at a dose of 2 mg/kg developed circulating anti-GBM antibodies, which reached peak values by 6 weeks (63 +/- 5% following SD GBM; 53 +/- 8% following BN GBM), but did not develop glomerular deposits of IgG or nephritis. Animals given 2 mg/kg SD GBM in CFA developed greater concentrations of anti-GBM antibody by 6 weeks (122 +/- 20%) together with linear deposits of IgG on glomerular and tubular basement membranes (TBM), albuminuria (mean 7 mg/24 h), and variable focal segmental necrotising glomerulonephritis with mild interstitial nephritis. The same dose of BN GBM in CFA produced similar concentrations of circulating antibody (144 +/- 26%), with linear deposits of IgG on GBM but rarely TBM, little albuminuria, and variable mild focal glomerulonephritis. Other strains injected with SD GBM in CFA showed a variable circulating anti-GBM antibody response, which was similar to that of BN rats in PVG and DA rats but lower in LEW and WAG rats. Linear deposits of IgG on the GBM were detected in a proportion of PVG and DA rats, but not in LEW or WAG rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleosomes and histones are present in glomerular deposits in human lupus nephritis

Background. Recently we showed that antinuclear autoantibodies Complexed to nucleosomes can bind to heparan sulphate (HS) in the glomerular basement membrane (GBM) via the histone part of the nucleosome. Histones have been identified in glomerular deposits in human and murine lupus nephritis. In addition, a decreased HS staining in the GBM was found, most probably due to masking by deposition of antibodies complexed to nucleosomes. Methods. In this study we first investigated whether histones or nucleosomes could be identified in glomerular deposits in human lupus nephritis, and secondly whether the presence of these nuclear components was correlated with absence of HS staining. Kidney biopsies of SLE patients (11 with diffuse proliferative glomerulonephritis (DPGN) and six with membranous glomerulonephritis (MGN)) and of non-SLE glomerular diseases were stained for histones, DNA, nucleosomes, IgG and HS. Results. Using a polyclonal anti-H3 1-21 antiserum, histones were detected in all patients with DPGN and in two of six patients with SLE-MGN (P <0.01(. Using a monoclonal antihistone antibody, histones were stained in three patients with DPGN, but in none of the biopsies with MGN. Using nucleosome specific monoclonal antibodies, nucleosomes were detected in five patients with DPGN, in two patients with MGN, but in none of the biopsies with non-SLE glomerulonephritis. HS staining was nearly absent in DPGN, whereas staining was only moderately reduced in patients with MGN and controls (P=0.001). Conclusion. Using polyclonal and monoclonal antihistone antisera, histones were identified in all patients with DPGN and their presence was associated with a decrease of HS staining. Nucleosomes were identified in five of 11 patients with DPGN and in two of six patients with MGN. This is the first demonstration of nucleosomes in glomerular deposits in SLE nephritis.     

Glomerular heparan sulfate alterations: mechanisms and relevance for proteinuria

Heparan sulfate (HS) is the anionic polysaccharide side chain of HS proteoglycans (HSPGs) present in basement membranes, in extracellular matrix, and on cell surfaces. Recently, agrin was identified as a major HSPG present in the glomerular basement membrane (GBM). An increased permeability of the GBM for proteins after digestion of HS by heparitinase or after antibody binding to HS demonstrated the importance of HS for the permselective properties of the GBM. With recently developed antibodies directed against the GBM HSPG (agrin) core protein and the HS side chain, we demonstrated a decrease in HS staining in the GBM in different human proteinuric glomerulopathies, such as systemic lupus erythematosus (SLE), minimal change disease, membranous glomerulonephritis, and diabetic nephropathy, whereas the staining of the agrin core protein remained unaltered. This suggested changes in the HS side chains of HSPG in proteinuric glomerular diseases. To gain more insight into the mechanisms responsible for this observation, we studied GBM HS(PG) expression in experimental models of proteinuria. Similar HS changes were found in murine lupus nephritis, adriamycin nephropathy, and active Heymann nephritis. In these models, an inverse correlation was found between HS staining in the GBM and proteinuria. From these investigations, four new and different mechanisms have emerged. First, in lupus nephritis, HS was found to be masked by nucleosomes complexed to antinuclear autoantibodies. This masking was due to the binding of cationic moieties on the N-terminal parts of the core histones to anionic determinants in HS. Second, in adriamycin nephropathy, glomerular HS was depolymerized by reactive oxygen species (ROS), mainly hydroxyl radicals, which could be prevented by scavengers both in vitro (exposure of HS to ROS) and in vivo. Third, in vivo renal perfusion of purified elastase led to a decrease of HS in the GBM caused by proteolytic cleavage of the agrin core protein near the attachment sites of HS by the HS-bound enzyme. Fourth, in streptozotocin-induced diabetic nephropathy and during culture of glomerular cells under high glucose conditions, evidence was obtained that hyperglycemia led to a down-regulation of HS synthesis, accompanied by a reduction in the degree of HS sulfation.     

Abnormal Podocyte CR-1 Expression in Glomerular Diseases: Association With Glomerular Cell Proliferation and Monocyte Infiltration

The expression of CR-1 complement receptors on glomerular epithelialcells, was studied in 77 renal biopsies from patients with (74)or without (3) glomerular diseases, employing an anti-CR-1 monoclonalantibody, and an indirect immunoperoxidase technique. Four patternsof CR-1 expression were recognised: normal (18); generally decreased(6); focal/segmental partial loss (44); and complete loss (9).Normal expression was detected in all three biopsies with non-glomerulardiseases, and in glomerular diseases with normal glomeruli onlight microscopy, but also in several glomerulonephritic biopsies(13) including diffuse proliferative lupus nephritis (1 of 7)and idiopathic membranous nephritis (5 of 14). However, themajority of biopsies from patients with glomerular diseasesshowed abnormal CR-1 expression (59 of 74), most evident inproliferative biopsies (43 of 49), with or without crescentformation (respectively, 18 of 20 and 25 of 29). Complete lossof CR-1 expression was almost restricted to crescentic biopsies(8 of 9). The abnormal CR-1 expression was unrelated to thepresence of capillary immune deposits of Ig or C. More intraglomerular monocytes, assessed by monoclonal antibodies,were encountered in glomerulonephritic biopsies with partialCR-1 loss (median 6.2, P<0.05) or complete loss (median 14,P<0.03), than in biopsies with normal receptor expression(median 1.4). Thus, changes in glomerular CR-1 expression are frequently seenin many glomerular diseases and are associated with glomerularproliferative changes and monocyte infiltration, but not withthe presence of capillary immunedeposits. This abnormal CR-1expression may be important considering the ability of CR-1receptors to down-regulate complement activation, and the importanceof podocyte function on the synthesis of GBM components.     

Oral administration of glomerular basement membrane prevents the development of experimental autoimmune glomerulonephritis in the WKY rat

Experimental autoimmune glomerulonephritis (EAG), an animal model of Goodpasture's disease, can be induced in Wistar Kyoto (WKY) rats by a single injection of collagenase-solubilized rat glomerular basement membrane (GBM) in adjuvant. EAG is characterized by circulating and deposited anti-GBM antibodies, accompanied by focal necrotizing glomerulonephritis with crescent formation. The inhibitory effect of orally administered antigens has been reported in various animal models of autoimmunity but not in EAG in the rat. The effects of feeding rat GBM by gavage, at total doses of 0.5, 2.5, or 5 mg, before immunization were examined. A dose-dependent effect was observed on the development of EAG. A dose of 0.5 mg of GBM had no effect on disease, 2.5 mg resulted in a moderate reduction in the severity of nephritis but no change in anti-GBM antibody production, and 5 mg resulted in a marked reduction in circulating and deposited anti-GBM antibodies, albuminuria, deposits of fibrin in the glomeruli, severity of glomerular abnormalities, and numbers of infiltrating T cells and macrophages. Animals that were fed 5 mg of GBM showed a significant reduction in IgG2a but not IgG1, anti-GBM antibody levels, suggesting downregulation of Th1 responses. There was also a dose-dependent reduction in the proliferative responses of splenic T cells from treated animals to GBM antigen in vitro. These results clearly demonstrate that mucosal tolerance can be induced by oral administration of GBM antigen and that this approach is effective in preventing EAG.     

Removal of subepithelial immune complexes with excess unaltered or cationic antigen

Since cationic molecules are known to pass through the lamina densa of the glomerular basement membrane at higher rates than anionic molecules, the ability of cationic antigen to disperse subepithelial glomerular immune deposits was investigated in rats. Subepithelial glomerular immune deposits were formed in rats by the sequential administration of cationized human serum albumin (HSAED) and purified rabbit antibodies to human serum albumin (HSA). In vitro the addition of fifty-fold excess HSA or HSAED to immune precipitates formed with HSAED and antibodies to human serum albumin (antiHSA) solubilized the precipitates to comparable degree. Excess HSA or HSAED was given intravenously to rats which already had HSAED-antiHSA immune deposits in glomeruli. Serial renal biopsies were obtained and examined. Control animals received saline or nonspecific cationic molecules, protamine sulfate or cationized rabbit serum albumin, without any effect on the persistence of immune deposits in glomeruli. The injection of 10 mg of HSAED caused complete disappearance of glomerular immune deposits by 48 hours. In contrast, 100 mg of HSA was required to achieve the same effect. Thus, cationic antigens are more efficient than anionic antigens in the removal of subepithelial glomerular immune deposits.     

Ultrastructural 'fingerprint' in cryoprecipitates and glomerular deposits: a clinicopathologic analysis of fingerprint deposits.

Organized glomerular electron-dense deposits with a fingerprint pattern are well known in some patients of lupus nephritis or cryoglobulinemia. In general, these two diseases are always discussed separately as the causes of such deposits. However, 3 of our 5 lupus patients with glomerular fingerprint deposits also had cryoglobulinemia. One of the remaining 2 patients died and the other was lost to follow-up. The purpose of our study was to seek an appropriate clinicopathologic assessment of fingerprint deposits. All these patients showed overt proteinuria, active urinary sediment, a high degree of activity of lupus nephritis, and diffuse proliferative glomerulonephritis (WHO class IV). Their cryoprecipitates and renal biopsy specimens were investigated by means of immunochemistry, immunofluorescence and electron microscopy. The ultrastructural 'fingerprint' structures were exactly the same in the cryoprecipitates and in the glomerular deposits in 2 of 3 lupus patients with cryoglobulinemia, as were IgG, IgM and IgA. Therefore, these observations furnish emerging morphologic evidence for the glomerular deposition of immune complexes of circulating cryoglobulins in lupus nephritis. In addition, electron microscopic fingerprint deposits on renal biopsy or cryoprecipitate can be regarded as a very sensible marker of concomitant or subsequent development of diffuse lupus nephritis. If the patient is accompanied by nephritic syndrome, an early trial of immunosuppressive therapy may be warranted.     

Monoclonal antibody analysis of glomerular hypercellularity in human glomerulonephritis

To determine the contribution of infiltrating circulating leucocytes to glomerular hypercellularity, and to further investigate the immune and inflammatory mechanisms involved in human glomerulonephritis, a series of renal biopsies were evaluated using cell-specific monoclonal antibodies. In ninety-three renal biopsies from patients with glomerulonephritis, intraglomerular leucocytes were identified by immunoperoxidase localization of monoclonal antibodies to the leucocyte-common antigen, and antigens characteristic of T-cell and T-cell subsets, B-cells, monocytes and granulocytes. Normal glomeruli contained a mean of 2 leucocytes, predominantly monocytes, per glomerular cross-section. No significant increase in leucocytes was found in 41 biopsies with non-proliferative types of glomerulonephritis. However, in renal biopsies from 22 of the 46 patients with proliferative forms of glomerulonephritis, there was a significant increase in glomerular leucocytes. These biopsies were from 5 patients with post-infectious glomerulonephritis (mean of 30 leucocytes per glomerulus), 11 patients with crescentic glomerulonephritis (mean of 16 leucocytes per glomerulus) and 6 patients with mesangial proliferative glomerulonephritis due to systemic lupus erythematosus (mean of 5 leucocytes per glomerulus). The increased intraglomerular leucocytes consisted of macrophages and granulocytes. T and B-cells were generally not found within glomeruli. Thus, glomerular hypercellularity in proliferative glomerulonephritis is in part due to infiltration by inflammatory cells. No evidence was found to directly incriminate cellular immune mechanisms in the pathogenesis of the glomerular lesions of glomerulonephritis since T-cells were not identified within glomeruli.