Passive Heymann nephritis induced by rabbit antiserum to membrane antigens isolated from rat visceral yolk-sac microvilli

Passive Heymann nephritis (PHN) is an animal model of immune-complex-induced renal disease resembling human membranous glomerulonephritis. It was induced in rats by injecting rabbit antiserum directed against glycoprotein antigens isolated from rat embryonic visceral yolk-sac microvilli (VYS-MV). The glycoprotein antigens were isolated by extracting the VYS-MV with detergent Nonidet P-40 followed by gel filtration in Sephacryl S-300 and finally by lectin affinity chromatography with Ricinus communis agglutinin I. In vitro immunofluorescent localization studies demonstrated that the nephritogenic antibodies were localized along the apical region of the visceral yolk-sac endodermal cells and the brush border of the proximal tubular cells of the kidney. Rats injected with a single dose of the antiserum manifested proteinuria. Indirect immunofluorescent studies showed that the injected rabbit IgG was localized in vivo along the capillary walls of the glomerulus in a granular fashion. Electron microscopic examination of the same kidney glomeruli revealed numerous electron-dense deposits along the lamina rara externa of the glomerular basement membrane. Fusion of the epithelial foot processes was also present. These findings represent the typical immunopathological characteristics of Heymann nephritis. Furthermore, with the aid of Ouchterlony analysis, the antiserum against the isolated VYS antigens exhibited an immunoprecipitin band which was in common with that formed by the antiserum against the homogeneous nephritogenic antigen (gp330) of renal brush border origin. Thus, the nephritogenic antigens which have been found to be associated with the brush border of the renal proximal tubules may also be present or cross-reacted in the microvilli of the rat embryonic visceral yolk-sac.     

Isolation and characterization of circulating immune complexes from rats with experimental membranous nephropathy

Circulating immune complexes (CIC) were isolated from serum from controls and rats with active Heymann nephritis (n = 31) by two methods. CIC detected by the fluid phase Clq binding assay were precipitated from serum using Clq and polyethylene glycol. CIC were also isolated by sequential chromatography with anion exchange and lectin affinity supports. The isolated material was analyzed by PAGE and immunoblotting. The immune complex material isolated by both methods from rats with Heymann nephritis contained the same 60/65-kDa tubular Ag. By immunoblotting, the 60/65-kDa tubular Ag-bound antibodies from rats with active Heymann nephritis, but not antibodies to gp330. Antibody bound to the 60/65-kDa tubular protein in the CIC was isolated. This antibody bound to a similar Ag in glomerular eluates from rats with active Heymann nephritis when tested by immunoblotting. These observations suggest that glomerular immune deposits and CIC in rats with Heymann nephritis contain the same tubular Ag. The 60/65-kDa Ag was isolated from CIC by HPLC using anion exchange and hydrophobic interaction columns. Rats immunized with this Ag developed Heymann nephritis. These studies suggest that CIC contribute to the development of glomerular subepithelial immune deposits in this model of membranous nephropathy. These studies do not exclude the participation of other Ag-antibody systems in Heymann nephritis, including gp330. This report describes methods for isolation and characterization of Ag-antibody components of CIC that might be useful to studies of other immune complex-mediated diseases.     

Titles of related papers in other sections

Previous results have shown that the autoantibody eluted from the glomeruli of rats with active Heymann nephritis contain a population of antibodies not only to the putative autoantigen of the disease, gp330, but alos to plasminogen. Since gp330 has been shown to serve as a receptor for plasminogen, we have analyzed the effects of autoantibody on plasminogen-binding to gp330 and activation of plasminogen to plasmin by urokinase. Autoantibody does not inhibit the binding of plasminogen to gp330. The change in the conformation of plasminogen when its lysine-binding sites are occupied or after conversion to plasmin results in a significant decrease in autoantibody-binding. The most significant effect of autoantibody on this system is the inhibition of plasminogen activation to plasmin by urokinase. The binding of autoantibody to plasminogen acts as a competitive inhibitor of the reaction by apparently blocking access of urokinase to plasminogen's activation site. These results indicate that autoantibody obtained from the immune deposits in the glomeruli of rats with active Heyman nephritis does not inhibit the binding of plasminogen to gp330 but does significantly alter the urokinase catalyzed activation of plasminogen to plasmin.     

Control of autologous immune complex nephritis. I. Suppression of the disease in the presence of T cell sensitization

Pretreatment of Lewis and Sprague-Dawley rats with the nephritogenic antigen, Fx1A, in incomplete Freund's adjuvant (IFA) reduced the incidence of autologous immune complex nephritis in rats subsequently challenged with Fx1A in complete Freund's adjuvant (CFA). The suppression was evidenced by a decrease in antibody production, in glomerular deposition of immunoglobulins, and in the incidence of proteinuria, and it was antigen specific. In vitro blastogenesis to Fx1A of lymphocytes from Fx1A-IFA-pretreated animals was normal. Rats pretreated with Fx1A-IFA initially developed a normal antibody response after challenge with Fx1A-CFA, but the response was not sustained. These results indicate that the Fx1A-IFA-induced suppressor mechanism does not inhibit sensitization, but rather modifies specific antibody production.     

Evaluation of sequential glomerular eluates from rats with Heymann nephritis

This study was undertaken to characterize the antigen-antibody content of sequential glomerular eluates from rats with Heymann nephritis. Serum and renal tissue were harvested every 2 wk after immunization with renal tubular antigen (Fx1A). Circulating antibody to the tubular antigen was detectable in the circulation from days 7 to 98. Direct immunofluorescence of renal tissue demonstrated an increase in IgG deposits through day 49 with stabilization thereafter. Tubular antigen deposits peaked at day 49 and then declined. One-hour and 3-hr acid eluates of isolated glomeruli were analyzed for IgG content, antibody specificity, and antigen content. Antibody from the 1-hr eluate bound to the tubular brush border but not the glomerulus, whereas the 3-hr eluate demonstrated binding to the glomerulus and not to the tubular brush border. In addition to rat IgG, the 1-hr eluate demonstrated a 70 kD band and the 3-hr eluate demonstrated a 45 kD band by polyacrylamide gel electrophoresis. By Western blot, antibody to the brush border bound to the 70 kD band. Anti-idiotypic antibody to anti-Fx1A, which binds to the glomerulus by indirect immunofluorescence, bound to the 45 kD band. The 3-hr eluate, but not the 1-hr eluate, precipitates radiolabeled F(ab')2 fragments from anti-Fx1A antibody but not from normal rat IgG. Quantitative analysis of the sequential eluates demonstrated that the 70 kD-anti-Fx1A system predominated early in the course of disease, whereas the 45 kD-anti-idiotype antigen-antibody system predominated late in the course of the disease. These observations confirm that two antigen-antibody systems contribute to the immune deposits in Heymann nephritis.     

Heymann antibodies induce complement-dependent injury of rat glomerular visceral epithelial cells

The aim of this study was to investigate the in vitro role of the complement membrane attack complex (MAC) in the injury induced by nephritogenic anti-brush border vesicle (Fx1A) antibodies on rat glomerular visceral epithelial cells (GEC). Both sheep and rabbit anti-rat brush border vesicle IgG-induced complement-dependent lysis of cultured GEC. Fab fragments of sheep anti-rat brush border vesicles and polyclonal or monoclonal gp330 IgG were devoid of lytic activity. Shedding of cell-surface antigens induced by sheep or rabbit anti-rat brush border vesicle IgG protected GEC from subsequent exposure to lytic antibodies and complement, an effect that was not obtained with Fab fragments. When GEC were incubated with sheep or rabbit anti-rat brush border vesicle IgG in capping conditions, the C3 component was co-redistributed with Heymann immune complexes; in contrast, the MAC remained diffusely bound to the cell surface, indicating that it was not associated with the antigen-antibody complexes. The MAC was demonstrated on the surface of GEC by immunofluorescence staining with anti-MAC neoantigen and by electron microscopy of negatively stained membranes showing focal clusters of 110 A MAC lesions. When GEC were treated with sheep IgG or rabbit IgG plus C6-deficient sera, the cells were not lysed and MAC was not demonstrable on the surface; however, lytic activity was restored when C6-deficient sera were reconstituted with purified C6. The results are consistent with the interpretation that injury induced by Heymann antibodies on GEC is MAC-dependent.     

Il-4 therapy prevents the development of proteinuria in active Heymann nephritis by inhibition of Tc1 cells

The role of IL-4, a key Th2 cytokine, in promoting or inhibiting active Heymann nephritis (HN) was examined. HN is induced by immunization with Fx1A in CFA, and proteinuria in HN is associated with subepithelial IgG and C3 deposition and infiltration of CD8(+) T-cytotoxic 1 (Tc1) cells and macrophages into glomeruli, as well as induction of Abs to Crry. Treatment with rIL-4 from the time of Fx1A/CFA immunization stimulated an earlier IgG1 response to Fx1A, induced anti-Crry Abs, and up-regulated IL-4 mRNA in lymphoid tissue, but did not alter proteinuria. Treatment with MRCOx-81, an IL-4-blocking mAb, resulted in greater proteinuria, which suggests endogenous IL-4 regulated the autoimmune response. Delay of rIL-4 treatment until 4 wk post-Fx1A/CFA immunization and just before the onset of proteinuria prevented the development of proteinuria and reduced Tc1 cell infiltrate in glomeruli. Delayed treatment with IL-4 had no effect on titer or isotype of Abs to Fx1A or on Ig, C3, and C9 accumulation in glomeruli. Treatment with rIL-13, a cytokine that alters macrophage function such as rIL-4, but has no direct effect on T or B cell function, reduced glomerular macrophage infiltrate, but did not prevent proteinuria or CD8+ T cell infiltrate. Anti-Crry Abs were paradoxically only induced with rIL-4 therapy, not in HN controls with proteinuria. It was concluded that the rIL-4 effect was probably by inhibition of Tc1 cells, which normally mediate the glomerular injury that results in proteinuria.     

An immunopathologic study of a 330-kD protein defined by monoclonal antibodies and reactive with anti-RTE alpha 5 antibodies and kidney eluates from active Heymann nephritis

There is evidence indicating that the glomerular Ig deposits of Heymann's nephritis (HN)--a model of epimembranous glomerulonephritis--may be formed at least in part in situ by binding of free circulating antibody with brush border (BB) antigen expressed by glomerular epithelial cells. In this work, we provide evidence that a 330-kD protein defined by seven monoclonal antibodies is responsible for HN. 1) Ig eluted from glomeruli of rats with HN induced classically with crude BB preparation bind specifically the 330-kD antigen; 2) passive immunization with monoclonal antibodies induces epimembranous glomerular Ig deposits; 3) active immunization with the 330-kD antigen induces proteinuric glomerulonephritis; 4) the 330-kD antigen was present in the nephritogenic preparation purified by Edgington, Glassock, and Dixon, because it was identified by the corresponding heterologous antisera. These results, obtained by a completely different approach, confirm and extend those of Kerjaschki and Farquhar and provide a link with the classical studies on HN.     

Microdomains of distinctive glycoprotein composition in the kidney proximal tubule brush border

Two membrane proteins, maltase and gp330 (the pathogenic antigen of Heymann nephritis), present in the proximal tubule brush border have recently been independently purified and found to be large glycoproteins of similar molecular weight (Mr = approximately 300,000) by SDS PAGE. To determine the relationship between the two, monoclonal antibodies raised against the purified proteins were used for comparative immunochemical analyses and immunocytochemical localization. When a detergent extract of [35S]methionine-labeled rat renal cortex was used for immunoprecipitation with monoclonal antimaltase IgG, a single band of approximately 300 kdaltons was precipitated, whereas a single 330-kdalton band was precipitated with monoclonal anti-gp330 IgG. Monoclonal antimaltase (gp300) IgG also immunoprecipitated maltase activity from solubilized renal maltase preparations, whereas monoclonal anti-gp330 IgG failed to do so. When cyanogen bromide-generated peptide maps of the two proteins were compared, there were many similar peptides, but some differences. When maltase and gp330 were localized by indirect immunofluorescence and by indirect immunoperoxidase and immunogold techniques at the electron microscope level, they were found to be differently distributed in the brush border of the initial (S1 and S2) segments of the proximal tubule: maltase was concentrated (approximately 90%) on the microvilli, and gp330 was concentrated (approximately 90%) in the clathrin-coated apical invaginations located at the base of the microvilli. We conclude that maltase (gp300) and the Heymann nephritis antigen (gp330) are structurally related membrane glycoproteins with a distinctive distribution in the proximal tubule brush border which may serve as markers for the microvillar and coated microdomains, respectively, of the apical plasmalemma.     

Analysis of the Heymann nephritogenic glycoprotein in rat, mouse, and human kidney

Passive Heymann nephritis is induced in rats by intravenous administration of antiserum raised against antigens of the renal proximal tubule. Evidence by Kerjaschki and Farquhar indicates that the critical nephritogenic is a high molecular weight glycoprotein (HMWgp) of rat renal brush border membrane. Their immunocytochemical studies also localize the nephritogenic antigen to the glomerular epithelial cell surface and may explain in situ formation of immune complexes at this locus in Heymann nephritis. We have confirmed the observations of Kerjaschki and Farquhar by demonstrating the HMWgp in extracts of rat brush border membrane and isolated glomeruli on sodium dodecyl sulfate-polyacrylamide (SDS-PA) (5%) gels. An antiserum raised to purified rat HMWgp identifies the antigen from rat or mouse kidney on Western blots. However, unlike rodent kidney, we were unable to detect a comparable HMWgp in extracts of human kidney on SDS-PA gels and found no cross-reactive material on Western blots of human brush border membrane proteins. Our observations suggest that human kidney lacks the nephritogenic antigen critical to initiation of Heymann nephritis in rodents.     

A possible ligand of serum origin for the kidney autoantigen of Heymann nephritis

Earlier studies have localized the Heymann nephritis (HN) autoantigen (gp330) in the coated pits of the plasma membrane and multivesicular bodies of the glomerular epithelial cell. Because of these locations in the glomerular epithelial cells, it has been suggested that the HN Ag may be a receptor. The aim of our study was to search for a ligand which can bind the HN autoantigen. Normal rat serum was subjected to SDS-PAGE under reducing and non-reducing conditions followed by Western analysis of the separated polypeptides. A reaction was revealed directly by autoradiography using 125I labeled HN autoantigen as a probe and indirectly by enzyme immunodetection using unlabeled nephritogenic autoantibody (anti-gp330) eluted from glomeruli of diseased rats followed by biotinylated rabbit anti-rat IgG avidin-peroxidase complex. A polypeptide of 76 kDa Mr was identified under non-reducing conditions as a serum protein reacting with the HN autoantigen. Reactivity of the 76-kDa polypeptide was lost when serum was electrophoresed under reducing conditions. Direct binding of the 76-kDa polypeptide obtained from serum to the HN autoantigen obtained from kidney suggests that the 76-kDa polypeptide may be a ligand for the autoantigen. This is the first documentation of a possible ligand for the HN autoantigen. Not only does this polypeptide bind to the HN autoantigen but it also shows direct binding with the nephritogenic autoantibody eluted from glomerular deposits. This characteristic of the 76-kDa polypeptide indicates that this serum protein may potentially play a role in the development of the glomerular lesion of active HN. Further analysis of this serum component should assist in understanding the normal function of the HN autoantigen.     

DNA vaccination against specific pathogenic TCRs reduces proteinuria in active Heymann nephritis by inducing specific autoantibodies

We have previously identified potential pathogenic T cells within glomeruli that use TCR encoding Vbeta5, Vbeta7, and Vbeta13 in combination with Jbeta2.6 in Heymann nephritis (HN), a rat autoimmune disease model of human membranous nephritis. Vaccination of Lewis rats with naked DNA encoding these pathogenic TCRs significantly protected against HN. Proteinuria was reduced at 6, 8, 10, and 12 wk after immunization with Fx1A (p < 0.001). Glomerular infiltrates of macrophages and CD8(+) T cells (p < 0.005) and glomerular IFN-gamma mRNA expression (p < 0.01) were also significantly decreased. DNA vaccination (DV) causes a loss of clonality of T cells in the HN glomeruli. T lymphocytes with surface binding of Abs were found in DNA vaccinated rats. These CD3(+)/IgG(+) T cells expressed Vbeta5 and Vbeta13 that the DV encoded. Furthermore, FACS shows that these CD3(+)/IgG(+) cells were CD8(+) T cells. Analysis of cytokine mRNA expression showed that IL-10 and IFN-gamma mRNA were not detected in these CD3(+)/IgG(+) T cells. These results suggest that TCR DNA vaccination produces specific autoantibodies bound to the TCRs encoded by the vaccine, resulting in blocking activation of the specific T cells. In this study, we have shown that treatment with TCR-based DV, targeting previously identified pathogenic Vbeta families, protects against HN, and that the mechanism may involve the production of specific anti-TCR Abs.     

Expression and modulation of surface antigens in cultured rat glomerular visceral epithelial cells

This study, using immunocytochemical light and electron microscopy techniques, characterizes the distribution of three antibodies bound to the surface of rat glomerular visceral epithelial cells (GEC) in culture, and tests their ability to redistribute corresponding antigens under conditions appropriate for antigenic modulation (antigen disappearance). At 4 degrees C or after fixation, anti-renal tubular brush border vesicle (BBV) IgG bound diffusely to the surface of GEC and to coated pits. Anti-gp330 IgG had a discrete distribution on the surface of GEC and reacted with coated pits. Anti-podocalyxin IgG was bound diffusely to the surface of GEC but not to coated pits. At 37 degrees C, anti-BBV IgG induced marked redistribution of immune complexes with both shedding and internalization. Anti-gp330 IgG induced weaker redistribution, with internalization of immune complexes predominating. Anti-podocalyxin IgG induced rapid redistribution of immune complexes and antigenic modulation but minimal internalization. Experiments of differential redistribution indicated that anti-BBV IgG modulated the expression of both gp330 and podocalyxin; anti-gp330 IgG had a weaker effect on BBV antigens and podocalyxin; and anti-podocalyxin failed to redistribute BBV antigens or gp330. The relevance of these immunocytochemical studies of antibody-cell surface antigen interaction in cultured GEC to understanding the pathogenesis of Heymann glomerulonephritis (HG) is discussed.     

Cytochrome P450 2B1 mediates complement-dependent sublytic injury in a model of membranous nephropathy

Membranous nephropathy is a disease that affects the filtering units of the kidney, the glomeruli, and results in proteinuria accompanied by loss of kidney function. Passive Heymann nephritis is an experimental model that mimics membranous nephropathy in humans, wherein the glomerular epithelial cell (GEC) injury induced by complement C5b-9 leads to proteinuria. We examined the role of cytochrome P450 2B1 (CYP2B1) in this complement-mediated sublytic injury. Overexpression of CYP2B1 in GECs significantly increased the formation of reactive oxygen species, cytotoxicity, and collapse of the actin cytoskeleton following treatment with anti-tubular brush-border antiserum (anti-Fx1A). In contrast, silencing of CYP2B1 markedly attenuated anti-Fx1A-induced reactive oxygen species generation and cytotoxicity with preservation of the actin cytoskeleton. Gelsolin, which maintains an organized actin cytoskeleton, was significantly decreased by complement C5b-9-mediated injury but was preserved in CYP2B1-silenced cells. In rats injected with anti-Fx1A, the cytochrome P450 inhibitor cimetidine blocked an increase in catalytic iron and ROS generation, reduced the formation of malondialdehyde adducts, maintained a normal distribution of nephrin in the glomeruli, and provided significant protection at the onset of proteinuria. Thus, GEC CYP2B1 contributes to complement C5b-9-mediated injury and plays an important role in the pathogenesis of passive Heymann nephritis.     

Serology and tissue lesions in rabbits immunized with Streptococcus mutans

Rabbits were immunized i.v. or i.d. with sterile suspensions of disrupted Streptococcus mutans strain MT703 or K1R. Indirect immunofluorescence assays indicated that sera from four of 10 rabbits immunized i.d. contained antibodies reactive with monkey and human heart and kidney components; 19 of 24 rabbits immunized i.v. had antibodies reactive with these tissues. Heart-reactive antibodies were also detected by immunoelectrophoresis and indirect radioimmunoassay. These antibodies were absorbed well by cytoplasmic membranes, a whole cell extract, and an alkali extract of S. mutans but only weakly by intact bacteria. Between 6 and 8 weeks after the first i.v. administration of S. mutans vaccines, rabbits developed proteinuria and hematuria with subsequent weight loss and lethargy. Approximately 25% of the animals died from illness between the fifth and sixth month of immunization. In 13 of 15 rabbits, immune deposits of C3 and IgG, IgM, or IgA and fibrinogen were seen in kidneys within the glomeruli, basement membranes of the peritubular capillaries, and in the interstitium. In the heart, deposits were seen along the capillaries of the myocardium. In 8 of 14 rabbits, focal deposits of S. mutans antigen were detected in glomeruli and in the kidney interstitium. The kidneys showed gross pathologic and histopathologic changes. Most kidneys were pale and enlarged. Microscopic examination revealed hypercellularity of the glomeruli, presence of neutrophils, thickening of glomerular and tubular basement membranes, tubular atrophy, edema, and fibrosis of the interstitium. The kidney disease presented features of poststreptococcal glomerulonephritis. Microscopic examination of heart sections revealed mild perivascular infiltration by polymorphonuclear leukocytes and plasma cells in some of the rabbits.     

Pathogenesis of passive Heymann glomerulonephritis: chlorpromazine inhibits antibody-mediated redistribution of cell surface antigens and prevents development of the disease

Two hypotheses were tested: first, that in LEW rats the interaction of sheep (or rabbit) anti-brush border antibodies with antigens (Heymann antigens) expressed on the plasma membrane of glomerular visceral epithelial cells is characterized by initial redistribution of immune complexes on the cell surface and by subsequent shedding of immune complexes in the subepithelial part of the capillary wall; and secondly, that this interaction is inhibited by chlorpromazine, a drug that displaces calcium ions from binding sites linking the plasma membrane to the cytoskeleton, and which blocks the redistribution of IgG on the surface of B lymphocytes exposed to anti-IgG antibodies. The studies were performed in vitro on cultured LEW glomerular epithelial cells and in vivo in LEW rats. In cultured glomerular epithelial cells exposed at 37 degrees C to anti-brush border IgG, chlorpromazine prevented, in a dose-dependent manner, the redistribution ("capping") of Heymann antigens and the fixation of complement. The renal glomeruli of chlorpromazine-treated LEW rats examined 6 and 48 hr after transfer of anti-brush border antibodies had punctate and, later, punctate and diffuse deposits of sheep (or rabbit) IgG on glomerular epithelial cells, but not similar deposits of rat C3. Moreover, granular subepithelial deposits of sheep (or rabbit) IgG and rat C3, characteristic of passive Heymann glomerulonephritis, did not develop, although deposits of sheep IgG were detected by immunoelectron microscopy on the microvilli of glomerular epithelial cells. Comparative studies on rats with similar reductions in glomerular filtration rates, produced by high doses of chlorpromazine or with renal artery stenosis, showed that the findings were not the consequence of insufficient delivery of antibody to glomerular epithelial cells. The results are consistent with the interpretation that Heymann glomerulonephritis is induced by mechanisms of redistribution of cell surface antigens comparable to those that govern the interaction of surface antigens (or receptors) with appropriate ligands in B lymphocytes and other classical in vitro systems.     

Course of transplanted Heymann nephritis kidney in normal host. Implications for mechanism of proteinuria in membranous glomerulonephropathy

Heymann nephritis [model of membranous glomerulonephropathy (MGN)] kidneys (n = 20) with proteinuria were transplanted into unilaterally nephrectomized normal syngeneic Lewis recipient rats to study the course of established MGN lesion in normal milieu. Concurrent with the loss of C3 staining from the MGN lesion at 2 to 4 wk after transplantation, the proteinuria decreased from the transplanted kidney (p less than 0.02). Thereafter, from 2 to 28 wk no further decrease was noted in proteinuria which stabilized at a lower but still abnormal level. No appreciable decrease in IgG deposits in MGN lesion was noted up to 12 wk but at 28 wk the deposits had decreased significantly (p less than 0.005). However, the deposits did not resolve completely even at 40 wk. The results indicate that in MGN one component of proteinuria is due to ongoing activation of C with the deposition of new antibody and the other is perhaps due to structural damage to glomerular filter. The former appears reversible and the later irreversible. Although the resolution of IgG deposits in the lesion is very slow significant improvement can occur with time (several months).     

Heymann antigen GP330 demonstrates affinity for fibronectin, laminin, and type I collagen and mediates rat proximal tubule epithelial cell adherence to such matrices in vitro.

We have utilized monoclonal antibodies directed against glycoproteins on the surface of proximal tubule epithelial cells (PTEC) to study their interaction with matrix components. PTEC exposed to monoclonal antibodies directed against a 330-kDa cell surface glycoprotein exhibited a significant epitope-specific inhibition of attachment and proliferation on type I collagen-, fibronectin-, laminin-, and gelatin-coated tissue culture surfaces. This effect was not due to antibody toxicity since such cells did not exhibit metabolic dysfunction in suspension cultures and the inhibition could be reversed upon removal of the antibody from the cell surface. Furthermore, detergent-solubilized gp330 demonstrated specific affinity for fibronectin, laminin, and type I collagen which was not inhibited by Arg-Gly-Asp-containing peptides. A monoclonal antibody directed against the receptor epitope was capable of promoting PTEC adherence and growth when such an antibody was immobilized on cell culture dishes. Although gp330 acted as a receptor for matrix proteins in primary cultures of freshly isolated PTEC, this effect was not demonstrable in established cultures. These results suggest that freshly isolated PTEC depend on gp330 for their attachment to matrix molecules while in vitro-adapted PTEC rely on other receptors activated by culture conditions. The affinity of gp330 for matrix molecules may be of pathogenic relevance in the persistence of gp330-containing immune complexes formed in the glomerular capillary wall in experimental membranous nephropathy (Heymann nephritis).     

The pathogenicity of T cell epitopes on human Goodpasture antigen and its critical amino acid motif

Goodpasture antigen, the non‐collagenous domain of α3 chain of type IV collagen [α3(IV)NC1], is the target antigen of anti‐glomerular basement membrane (GBM) antibodies. The pathogenicity of T cell epitopes is not elucidated clearly. In this study, we aim to define the nephritogenic T cell epitopes and its critical amino acid residues. Twenty‐four overlapping linear peptides were synthesized covering the whole sequence of human α3(IV)NC1. Wistar–Kyoto rats were immunized with linear peptides, and experimental autoimmune glomerulonephritis was evaluated. Critical amino acid was identified by the loss of nephritogenic function after each amino acid substitution by alanine. Of the 24 peptides, P14 (α3_127‐148) could induce 90.5% (19/21) of WKY rats developing anti‐GBM glomerulonephritis with proteinuria, elevated serum urea and creatinine, IgG linear deposit on GBM and substantial (in average 82.4 ± 5.6%) crescent formation in glomeruli. Lymphocytes of immunized rats proliferated in response to α3_127‐148 and α3(IV)NC1 in vitro. Sera of these rats recognized α3_127‐148 and later on together with intact human α3(IV)NC1. Antibodies towards α3_127‐148 and intact α3(IV)NC1 could also be detected from the kidney elutes. These antibodies showed no cross‐reaction with each other, which implies intramolecular epitope spreading during disease progress. After sequential amino acid substitution, the α3_127‐148 with substitution of tryptophan₁₃₆, isoleucine₁₃₇, leucine₁₃₉ or tryptophan₁₄₀ lost its nephritogenicity. Human α3_127‐148 is a nephritogenic T cell epitope in WKY rats, with the critical amino acids as W₁₃₆I₁₃₇xL₁₃₉W₁₄₀. These findings might facilitate future investigation on microbial aetiology and potential specific immunotherapy of anti‐GBM disease.     

Abrogation of mercuric chloride-induced nephritis in the Brown Norway rat by treatment with antibodies against TNFalpha

HgCl(2) induces an autoimmune disease in the Brown Norway rat characterized by synthesis of autoantibodies (mainly, anti-GBM Abs), severe proteinuria and interstitial nephritis. Also, HgCl(2)- injected rats develop glomerular cell infiltrates consisting of ED1(+) cells (monocyte/macrophage), starting on day 4 and reaching a maximum on day 8. Treatment with anti-TNF-alpha antiserum had preventative effects as it reduced the urinary protein levels to close to the normal range and also blocked the influx of inflammatory cells in the renal glomeruli and interstitium, but circulating anti-GBM and lineal glomerular IgG deposits were unmodified. In addition, whole isolated glomeruli from HgCl(2)-induced nephritis secreted TNF-alpha commencing on day 8, being maximally detected on day 11 and preceding, between 2 to 3 days, the development of proteinuria. The administration of anti-TNF-alpha antiserum or anti-alpha4 integrin mAb completely abrogated the synthesis of TNF-alpha in glomeruli isolated from the respective treated groups of animals, in addition to the proteinuria. Taken together our results confirm that TNF-alpha plays an important role in the induction and development of HgCl(2)-induced nephritis and highlights the pathogenic importance of the local release of TNF in those renal diseases in which prominent glomerular macrophage accumulation is a constant feature.