Lack of passive transfer of renal tubulointerstitial disease by serum or monoclonal antibody specific for renal tubular antigens in the mouse

Mice immunized with rabbit renal basement membranes form autoantibodies to their kidney glomerular and tubular basement membranes (GBM/TBM). Development of renal tubular disease (RTD) consists of deposition of autoantibodies along the GBM/TBM with the inter- and intratubular accumulation of lymphocytes and macrophages and destruction of the TBM. Transfer of this disease in mice with either serum or monoclonal antibodies, however, has been difficult to demonstrate and, therefore, attempts were made to confirm a report that RTD is passively transferred by anti-TBM autoantibodies. Using the revised protocol in this later report, we found that 12 weeks after transfer autoantibodies were deposited along the GBM and/or TBM of the recipients, yet RTD was not observed. Although qualitative and quantitative characteristics of the antibody may play a role in the pathogenesis in the murine model of RTD, we could not obtain evidence to support and confirm this study.     

Immunological studies of human placentae. Lectin binding to villous stroma and to trophoblastic basement membrane.

It has been demonstrated immunohistologically using cryostat sections that the lectins PHA and Con A bind to human placentae. Both lectins stain trophoblastic basement membrane (TBM), perivascular tissue and stroma. No staining of trophoblasts was observed. It has been shown by blocking experiments that the lectin binding sites on placental TBM are glycoproteins, whereas experiments involving pretreatment of placental sections with anti-collagen antisera or highly purified bacterial collagenase have indicated that lectin binding to stromal and perivascular structures is collagen-associated. The possible relation of TBM lectin-binding sites to immune response gene products is briefly discussed.     

A method for preparation of renal tubular basement membrane

A method is presented for the preparation of tubular basement membrane from renal cortex. The procedure utilizes a Polytron tissue disrupter, differential sieving, and for some species, sucrose density centrifugation. The procedure is especially useful for obtaining highly purified rabbit and bovine tubular basement membranes. Human tubular and glomerular basement membranes can be prepared with some cross contamination. The method offers the capability of rapidly generating large quantities of the basement membranes that retain the Goodpasture antigen and a TBM antigen associated with anti-TBM nephritis.     

Nephronophthisis. A primary tubular basement membrane defect

In order to characterize abnormalities in nephronophthisis, renal tissues from four patients were studied by light and electron microscopies and immunofluorescence using antibodies to laminin, type IV collagen, and tubular basement membranes (TBM). There were constant morphological alterations affecting TBM of all segments of the nephron, with or without cysts. These included extreme thinning and attenuation, layering, and thickening of these structures which ranged in size from 36 nm to 2000 nm. A combination of these features often affected the same TBM simultaneously, with abrupt transitions between different lesions. Although the ultrastructural TBM aberrations were observed in a wide variety of other chronic and acute renal disorders, they rarely occurred to the extent as in nephronophthisis or with abrupt transitions, both suggesting diagnostic significance. Laminin and type IV collagen were present in normal intensity and distribution, however, anti-TBM antibody staining was inconstantly reduced, perhaps signifying lack of a normal antigenic component in the TBM. These findings may well indicate the fundamental defect in nephronophthisis to be production of abnormal TBM, similar to the glomerular basement membrane lesions and consequences in Alport's syndrome.     

Role of sensitized cells in antitubular basement membrane interstitial nephritis.

The pathogenic effect of antitubular basement membrane (TBM) antibodies in anti-TBM interstitial nephritis has been demonstrated. To determine the relative role of sensitized cells in inducing this lesion, lymph node cells (LNC) alone or a combination of LNC and peritoneal exudate cells were transferred from Brown Norway (BN) rats, previously immunized with TBM into unimmunized BN-recipient rats. Cells were transferred directly under the recipients' renal capsules. Although significant tubular lesions were induced by the cells, the lesions were usually mild and always focal. Hence, sensitized cells do not appear to be central to the pathogenesis of anti-TBM nephritis.     

Isolation and characterization of the tubular basement membrane antigen associated with human tubulo-interstitial nephritis.

The target antigen, a 54-kD glycoprotein (gp54), reactive with sera from patients with anti-tubular basement membrane (anti-TBM) nephritis, was isolated from collagenase-digested (CD) bovine TBM. The purified gp54 was shown to be non-collagenous by amino acid analysis, and to be a unique basement membrane component by amino-terminal sequencing. The nephritogenicity of gp54 was demonstrated by immunizing strain XIII guineapigs with purified gp54, and producing anti-gp54 antibody and tubulo-interstitial nephritis. Anti-gp54 antibody, affinity-purified from sera of patients with anti-TBM nephritis, bound by immunoblotting to 54-kD and, to a lesser extent, 48-kD components of partially purified human CD-TBM. Indirect immunofluorescence showed that gp54 was present in the basement membrane of proximal tubules of the kidneys of normal human, cow, rabbit, guineapig and Brown-Norway rat but not in Lewis rat. Immunoelectron microscopy revealed localization of gp54 along the interstitial side of the TBM and its association with interstitial collagen fibres. These results indicate that gp54 is the nephritogenic antigen involved in tubulo-interstitial nephritis, and is unique in chemical characteristics and localization in the kidney.     

Inhibition of the neutrophilic infiltrate of experimental tubulointerstitial nephritis in the Brown-Norway rat by decomplementation

Anti-tubular basement membrane (TBM) antibody-associated tubulointerstitial nephritis (TIN) in Brown-Norway rats is induced by immunization with bovine TBM antigens and adjuvants. The lesion is characterized by linear deposition of IgG and C3 along the TBM with sequential neutrophil (Days 8-9)- and mononuclear (Day 10 and after)-dominated inflammatory infiltrates. To study the complement dependence of the infiltrative process, immunized rats were decomplemented with cobra venom factor (CVF). The CVF treatment did not affect the production or renal deposition of anti-TBM antibodies. CVF markedly reduced the neutrophilic inflammatory infiltrate. In rats immunized with suboptimal doses of soluble bovine TBM antigens to produce a mild lesion, decomplementation also decreased the mononuclear inflammatory infiltrates on Days 10-13. In rats immunized optimally with particulate TBM to induce maximally severe TIN, decomplementation did not affect the mild mononuclear cell infiltrate on Days 8 and 9 but did somewhat reduce the subsequent mononuclear infiltrate on Days 10 and 12. These results demonstrate that the anti-TBM antibody- and C3-associated neutrophilic inflammatory infiltrate is largely complement dependent. The early mononuclear cell infiltrate that was unmodified by CVF treatment may be dependent on complement-independent humoral events or related to cell-mediated immune events. A portion of the later mononuclear inflammatory infiltrate could be dependent on the preceding neutrophilic inflammatory phase.     

实验性肾小管间质性肾炎免疫发病机理的探讨

为探讨肾小管间质性肾炎的免疫发病机制，观察Ia抗原表达与肾小管间质性肾炎损害的关系，用兔肾小管基底膜（TBM）完全福氏佐剂及百白破疫苗，免疫雌性Wistar大鼠，成功地建立了肾小管间质性肾炎（TIN）模型。我们采用免疫组化和图像分析技术，研究TIN病变中单个核细胞的特征以及Ia抗原表达和TIN之间的关系。结果显示：在TIN发生时，TBM上有线型IgG沉积，这表明有抗TBM抗体产生。肾小管间质中渗出的单个核细胞，大多数为T细胞，部分有肉芽肿形成。B细胞逐渐减少，而单核细胞由少变多。肾间质损害表现为小管萎缩和间质纤维化。这些表明细胞免疫在TIN发病过程中起着重要作用。模型组中肾小管上皮细胞和Bowman囊Ia抗原表达强度比对照组高（P＜0．01），提示Ia抗原的表达可能影响TIN的发生和发展。     

Antibodies in guinea-pigs immunized with kidney and lung basement membranes

The cross-reaction of antibodies to tubular basement membrane (TBM) with alveolar basement membrane (ABM) has been studied in guinea-pigs with tubulointerstitial (TI) nephritis. Forty-three of fifty-two Hartley guinea-pigs immunized with rabbit TBM in complete Freund's adjuvant developed TI nephritis. In addition to linear deposits of guinea-pig IgG and C3 in the TBM, thirty-two of the nephritic animals showed linear immune deposits in ABM; twelve of these animals had thickened alveolar septa and increased numbers of polymorphonuclear (PMN) leucocytes in lung tissue. Sera and eluates of kidney and lung from nephritic guinea-pigs reacted strongly with TBM and more weakly with ABM of normal animals. Absorption experiments suggested that antibodies to TBM and ABM were closely related or identical. Only a minority of guinea pigs immunized with TBM showed in vivo binding of IgG to glomerular basement membrane (GBM). Immunization of guinea-pigs with lung-homogenate-induced antibodies binding to TBM and ABM (in approximately 45% of animals) and to GBM (in approximately 30% of animals). Immunization with crude GBM-induced antibodies which reacted preferentially with TBM and ABM. In contrast, collagenase-treated GBM-induced antibodies preferentially reactive with GBM. TI nephritis was induced in both Strain 13 and Strain 2 guinea-pigs, but the nephropathy developed much faster in Strain 13 animals. Immunization with rabbit TBM-induced antibodies reactive with ABM in 25% of Strain 13 guinea-pigs and in 50% of Strain 2 guinea-pigs, respectively.     

Tubulointerstitial nephritis induced by monoclonal anti-proximal tubular basement membrane antibodies in mice

Tubulointerstitial nephritis (TIN) was induced by monoclonal anti-tubular basement membrane (TBM) antibodies. Hybridomas producing anti-TBM antibodies were produced by the fusion of a mouse parental cell line with BALB/c mice which had been immunized with Wistar rat renal cortices. Three hybridoma cell lines were selected for production of antibodies against proximal TBM by indirect immunofluorescence. The isotypes of these monoclonal antibodies (MoAbs) were determined to be of the IgM class by double immunodiffusion. Subsequently, 6-week-old female BALB/c mice were injected intraperitoneally with cells (1 x 10(7)) of anti-TBM antibody-producing hybridomas, 39-1, 39-4, or 339-3. As a control, a monoclonal IgM-producing myeloma cell line was used. Proteinuria developed from Day 8, reaching 200 to 300 mg% in mice from the experimental groups, while in the control mice, urinary protein did not exceed 50 mg%. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the excreted urinary proteins proved to be of low molecular weight. An immunofluorescent study revealed a linear localization of IgM along the proximal TBM from Day 4, and light microscopy showed focal degenerative alteration in proximal tubules and focal round cell infiltration in the interstitium. Electron microscopy revealed dense deposits on some proximal TBM. These results indicate that monoclonal anti-TBM IgM antibodies can induce TIN as a result of persistent production of antibodies from intraperitoneally injected hybridomas.     

Experimental autoimmune tubulointerstitial nephritis in guinea-pigs: effects on renal lesions of cyclophosphamide administered before and after tubular basement membrane immunization

To evaluate the role of immune cells, including suppressor cells, in the tubular basement membrane model (TBM) of autoimmune tubulointerstitial nephritis (TIN) in guinea-pigs, a single high dose of cyclophosphamide (200 mg/kg body weight) was administered intraperitoneally 72 hr before or 72 hr after TBM/FCA (Freund's complete adjuvant, 500 μg/ml) immunization. Animals were divided into control, Group I (TBM immunized, no cyclophosphamide), Group II (cyclophosphamide 72 hr before TBM immunization) and Group III (TBM immunization, 72 hr later cyclophosphamide). Renal lesions in Groups II and III were either mild or absent and no linear deposits of IgG on tubular basement membrane were observed; in contrast Group I animals had renal interstitial infiltrate of mononuclear and giant cells and linear IgG deposits on tubular basement membrane.

Since cyclophosphamide pretreatment at high dosages can remove susceptible suppressor cells and their precursors and enhance some forms of delayed hypersensitivity, the lack of enhanced renal lesions with 72 hr cyclophosphamide pretreatment suggests that subsets of antigen-specific suppressor cells sensitive to cyclophosphamide are not significant in the pathogenesis of the renal lesions of TIN; whereas, in animals given cyclophosphamide after immunization, absence of lesions reflects effect of cyclophosphamide on rapidly dividing cells. The findings also support the central role of anti-TBM autoantibody in the pathogenesis of this model of tubulointerstitial nephritis.

     

A single autoantigen in Goodpasture's syndrome identified by a monoclonal antibody to human glomerular basement membrane

A mouse monoclonal antibody (P1) to the autoantigenic component of human glomerular basement membrane (GBM) was used to study the immunochemistry and tissue distribution of the Goodpasture antigen and the specificity of the human autoimmune response in Goodpasture's syndrome (anti-GBM disease). In solid phase assays, monoclonal antibody P1 bound to collagenase-solubilized human GBM (the ligand used in assays for human autoantibody), but not to other biochemically defined components of basement membrane. On Western blotting, P1 bound to the same 6 bands in solubilized GBM (between 26 and 58 kilodaltons with major bands at 26 and 54 kilodaltons) that were recognized by sera from all 42 patients studied with anti-GBM disease. Preincubation with sera from 8/8 patients blocked the subsequent binding of P1 from 83 to 89% on densitometer scanning of the Western blot; and preincubation with P1 blocked the binding of sera from 6/6 patients from 58 to 89%. Indirect immunofluorescence and immunoperoxidase studies revealed that the pattern of binding of P1 was identical to that of antibody eluted from the kidneys of a patient with Goodpasture's syndrome; there was linear binding to GBM, Bowman's capsule, and distal tubular basement membrane. In addition, P1 bound to basement membranes in lung and choroid plexus, and to membranes of the lens capsule, choroid, and retina of the eye and cochlea, but not to other organs studied. It is concluded that there is a single major autoantigenic component of human GBM (the Goodpasture antigen), which is present on fragments of different molecular weight in the collagenase digest. This antigen is distributed throughout well-defined basement membranes known to be involved in both Goodpasture's and Alport's syndromes. Human anti-GBM antibodies bind to the same (or closely related) determinants which are recognized by P1, demonstrating that the autoimmune response in Goodpasture's syndrome is of highly restricted specificity.     

Ultrastructural distribution of lectin-binding sites in the glomerular wall of streptozotocin-induced diabetic rats.

Since carbohydrates-containing molecules are known to be preferentially altered in diabetes mellitus and that major functional and morphological alterations do occur during diabetes in the renal tissue, we revealed in the present study various lectin-binding sites in the glomerular wall of control and long-term diabetic animals. Lectin-binding sites specific to N-acetyl-galactosamine, N-acetyl-glucosamine, sialic acid, galactose and fucose were revealed using the appropriate lectin and the lectin-gold complex at the electron microscope level. Differences in intensity of labeling as well as in distribution were detected for several lectin-binding sites particularly in the glomerular basement membrane, reflecting the presence of additional glycoconjugates and changes in the molecular organization of the basement membrane components during diabetes. Alterations in the glycocomponents and the glycoproteins of the glomerular basement membrane as well as non-enzymatic glycosylation of the basement membrane components have been described in diabetes, going along with our present results. The alteration in the distribution of some lectin-binding sites gives support to modifications in the three dimensional organization of some glycoproteins which could occur in diabetes. Since the glomerular wall is actively involved in blood filtration, these changes may either induce, or result from, the loss in selective permeability and the massive proteinuria occurring during diabetes.     

Development and heterogeneity of antigens in the immature nephron. Reactivity with human antiglomerular basement membrane autoantibodies

Indirect immunofluorescence microscopy was performed with 15 human anti-glomerular basement membrane (GBM) antibodies and mouse monoclonal antibodies to Type IV collagen (MBM4) and renal basement membranes (MBM15) on renal tissue from 6 fetuses (gestational age, 15-23 weeks), 8 infants (age, 1-21 days), and 8 children and adults (ages, 3-27 years). Of the 15 human anti-GBM antibodies that react with GBM in adult glomeruli, only 4 identified antigens in the GBM of fetal and infant glomeruli. In contrast, the monoclonal antibodies bound to basement membranes in the uninduced nephron and the GBM throughout all development stages of the fetal kidney. These studies demonstrate that the reactivity of human autoantibodies with GBM is developmentally and gestationally related--some identifying an antigen(s) in fetal glomeruli with early capillary loop formation and others reacting only with GBM in fully mature kidneys.     

Anti-glomerular basement membrane antibody disease: A rare autoimmune disorder affecting the kidney and the lung

Anti-glomerular basement membrane antibody disease is a rare, but well characterized cause of glomerulonephritis. By definition serum anti-GBM antibody and/or a linear binding of IgG detected by direct immunofluorescence (IF) in a histological specimen of the kidney or the lung have to be detected. These antibodies can lead to acute rapid progressive glomerulonephritis(RPGN) and/or pulmonary hemorrhage (PH) because of collagen similarities in the basement membrane.Principally anti-GBM antibody disease can be divided into two groups: anti-GBM antibody disease without PH was regarded as renal-limited anti-GBM antibody disease and that with PH was defined as Goodpasture's syndrome (GPS).The important determinant for the response of therapy and long term diagnosis on anti-GBM disease is early diagnosis to prevent endstage renal disease. Therefore, standard treatment is a combined therapy of plasmapherisis, prednisolone and cyclophosphamide.The aim of this review is an overview of the pathogenesis, clinical presentation, diagnosis and treatment of anti-GBM disease.     

Experimental autoimmune glomerulonephritis induced by anti-glomerular basement membrane antibody. II. Effects of injecting heterologous, homologous, or autologous glomerular basement membranes and complete Freund's adjuvant into sheep.

The effects of injecting human, rabbit, rat, or single-kidney homologous glomerular basement membrane (GBM) or autologous GBM, each in complete Freund's adjuvant (CFA), into 15- to 18-month-old sheep are compared. All sheep receiving heterologous GBM and 3 of 6 sheep receiving homologous GBM had anti-GBM nephritis, but such sheep did not bind autoantibodies or have Goodpasturelike lesions in their lungs. Sheep given injections of human GBM had autoantibodies to antigenic determinants shared by fetal or adult sheep and human GBM, by lung basement membranes, and by certain nonvascular basement membranes. Sheep given homologous GBM had two populations of autoantibodies: one was neither species- nor organ-specific; the other was sheep-specific. No sheep given autologous GBM had any evidence of anti-GBM autoantibodies or nephritis. Their kidneys were indistinguishable by histologic, immunohistologic, and functional studies from CFA-treated controls. Thus, sheep seem very tolerant to autologous GBM. These findings suggest that human anti-GBM nephritis may occur if the GBM is altered so that it becomes cross-reacting and induces autoantibodies, as does homologous GBM.     

Studies on the pathogenesis of experimental anti-tubular basement membrane nephritis in the guinea pig.

Using the model of renal disease induced in guinea pigs by immunization with bovine TBM preparations in adjuvant, the following observations were made. Animals with activity induced disease show bright staining for IgG along the TBM and only faint, inconstant staining along the GBM. Following transfer of serum animals with anti-TBM disease to normal recipients, accumulation of IgG was found predominantly in glomeruli at 4 hours, but at Days 3 and 5, IgG was seen predominantly along the TBM. There was no appreciable accumulation of neutrophils in the kidneys of recipients of anti-TBM serum, even at early intervals (4 and 24 hours) after transfer. However, within 2 days, small numbers of mononuclear cells were found. By Day 3, mononuclear cells were numerous, and multinucleate giant cells and tubular cell damage were present. After that, the lesions increased in severity and by 10 days were indistinguishable from those found in actively immunized animals at 14 to 21 days. Study of frozen section of kidneys obtained from animals with active disease at 14 days, employing sheep cells coated with rabbit antibody (IgG EA) revealed rosettes around many of the mononuclear cells in the infiltrate, indicating that they are mononuclear phagocytes (monocytes or macrophages). IgM complexed with sheep cells and complement (EAC) did not react and thus failed to provide evidence for the presence of B lymphocytes. Transfer of 7 X 10(8) lymph node cells from the TBM-immunized Strain 13 donors to normal Strain 13 recipients failed to result in renal lesions. The findings are interpreted as indicating that anti-TBM antibodies mediate the renal disease without the participation of cell-mediated immunity and further that these antibodies bring about an influx of ciculating mononuclear cells, predominantly monocytes, without attracting appreciable numbers of neutrophils.     

Identification of the hepatic asialo-glycoprotein receptor (hepatic lectin) as a component of liver specific membrane lipoprotein (LSP).

Liver specific membrane lipoprotein (LSP), the target for anti-LSP antibodies in various liver diseases, is thought to be comprised of fragments of the hepatocellular plasma membrane. In the present study, therefore, evidence has been sought for the presence in LSP of the hepatocyte surface receptor (hepatic lectin) that binds desialylated glycoproteins. Eight guinea-pig anti-LSP antisera (four anti-human and four anti-rabbit LSP) were found to react by ELISA and/or RIA against affinity purified human and rabbit hepatic lectin. Binding of the antisera to 125I-hepatic lectins was inhibited by the unlabelled lectins, by human and rabbit LSP and by purified rabbit liver plasma membranes but not by a 50,000-fold excess of kidney homogenate. The results indicate that hepatic lectin is a liver specific, species cross-reactive antigen comprising about 0.25% of the protein in LSP.     

Isologous monoclonal antibodies can induce anti-GBM glomerulonephritis in rats.

Injection of isologous monoclonal antibodies (SR2, SR3) caused anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM nephritis) in WKY/NCrj rats. The antibodies were obtained from hybridoma cells derived from fusion of the spleen of a nephritic WKY/NCrj rat injected with rat solubilized renal basement membranes with adjuvant, and mouse SP2-myeloma cells. They belonged to the rat IgG2a subclass and bound to rat kidney in a linear pattern along the glomerular and tubular basement membranes. Histological changes in glomeruli were detected at day 1 after the injection; proteinuria with haematuria appeared on day 2; and proteinuria became severe and reached a plateau by day 5. These results demonstrate that anti-GBM nephritis can even be induced by an isologous monoclonal antibody and that the rat IgG2a subclass is at least nephritogenic. The experimental model of anti-GBM nephritis with isologous monoclonal antibodies makes it possible and easier to analyse further the mechanism of anti-GBM nephritis.     

Lactosamine type asialooligosaccharide recognition in NK cytotoxicity

Inhibition of pig NK cell activity by asialooligosaccharides (aOS) isolated from human serum glycoproteins was investigated. Train-tennary aOS (aOSIII) of ceruloplasmin was found to be the most potent inhibitor up to the concentration 0.1 micrograms/ml, which is in agreement with its highly specific binding to NK-activity-enriched pig lymphocytes (with a morphology similar to human large granular lymphocytes (LGL]. Only lectins with the specificity to Gal(beta 1----4)GlcNAc or Gal(beta 1----3)GalNAc structures exhibited inhibition of NK cytotoxicity. F(ab)2 fragments of rabbit antibodies against pig spleen membrane lectin cross-reacting with the pig liver membrane lectin completely inhibited NK activity when preincubated with the effectors or present in the incubation mixture during the assay. These data suggest that lectin receptors on cells of pig NK-activity-enriched fraction specific for aOSIII and antigenically related to membrane lectins isolated from pig spleen and liver, are involved in the NK recognition of several xenogeneic targets.