Renal Section, Division of Medicine, Imperial College School of Medicine, Hammersmith Hospital, London, UK
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Abstract |
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Methods. To this end, we have cloned and sequenced rat 3(IV)NC1 and expressed it in COS-7 cells. Recombinant rat
3(IV)NC1, secreted into the COS-7 cell supernatant, was purified on an anti-M2 FLAG affinity column and characterized by western blotting. Recombinant antigen was then used to immunize WKY rats, in order to induce EAG.
Results. The recombinant material was antigenic as judged by binding to sera from patients with Goodpasture's disease and a mAb to 3(IV)NC1. Immunization of WKY rats (n=5), with recombinant rat
3(IV)NC1 in FCA at a dose of 1 mg/kg resulted in circulating anti-GBM antibodies directed towards
3(IV)NC1, linear deposits of IgG on the GBM, albuminuria, deposits of fibrin in the glomeruli, severe focal necrotizing glomerulonephritis with crescent formation, and glomerular influx of CD8+ T cells and macrophages. Western blot analysis demonstrated that sera from these rats bound strongly to recombinant rat
3(IV)NC1, as well as to collagenase-solubilized human and rat GBM. The pattern of binding was indistinguishable from that of sera from patients with Goodpasture's disease.
Conclusions. This purified recombinant rat 3(IV)NC1, which is both antigenic and nephritogenic, will be of value in analysing autoimmune responses in experimental anti-GBM disease.
Keywords: COS-7 cells; experimental autoimmune glomerulonephritis; Goodpasture's disease; purified recombinant antigen; rat 3(IV)NC1
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Introduction |
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In order to explore the mechanisms of autoimmunity in Goodpasture's disease, several animal models have been developed in which autoimmunity is induced by the injection of heterologous, homologous or isologous GBM. The original studies by Steblay [13] demonstrated that sheep immunized with human GBM developed crescentic glomerulonephritis. The induction of experimental autoimmune glomerulonephritis (EAG) in the genetically susceptible Wistar Kyoto (WKY) rat, using various preparations of GBM, has proved to be a good model of the human disease. Sado et al. first showed that WKY rats immunized with bovine GBM in FCA developed circulating and deposited anti-GBM antibodies on the GBM, accompanied by crescentic glomerulonephritis. Subsequently they showed that immunization with an affinity purified preparation of homologous GBM resulted in a very similar disease, implying the presence of a similar antigenic epitope in both preparations [14]. The same group has been able to transfer disease using antibodies purified from the urine of nephritic rats [15]. However, Bolton et al. [16] found that EAG in the WKY rat could only be induced by reducing the pH of bovine GBM preparations, and that Bordetella pertussis was required as an adjuvant in addition to FCA. These rats showed delayed type hypersensitivity to GBM, and both T cells and macrophages were present on renal histology.
The model currently used in our laboratory involves the induction of EAG in the WKY rat by a single injection of homologous GBM in FCA [17]. This results in sustained anti-GBM antibody synthesis, linear deposits of IgG on the GBM, deposits of fibrin in the glomeruli, haematuria, albuminuria, and focal necrotizing crescentic glomerulonephritis, with variable lung haemorrhage. Even though 3(IV)NC1 is a minor component of GBM, evidence from our laboratory and others suggests that
3(IV)NC1 is a major autoantigen in EAG. One- and two-dimensional western blotting has shown that anti-GBM antibodies from these rats bind to cationic NC1 domains of human and rat type IV collagen, similar to those recognized by patients' sera. Other studies have reported the unique capacity of
3(IV)NC1 dimers, extracted from bovine kidney, to induce EAG in rabbits and, latterly, in WKY rats [18]. Recently, Sado et al. [19] reported that a recombinant form of human
3(IV)NC1 was capable of inducing severe anti-GBM nephritis in WKY rats.
As in the human disease, it is clear that EAG in the rat is a T cell dependent phenomenon. We have shown that T cells from WKY rats with EAG proliferate in response to cationic monomer and dimer NC1 domains of rat GBM. Immunohistology of kidneys in EAG demonstrates that an influx of T cells precedes macrophage infiltration and glomerular injury. Transfer of CD4+ T helper cells from spleens of rats with EAG can prime naive recipients for the disease [20], while in vitro stimulation of these T cells by GBM, before transfer, induces anti-GBM antibody production in recipients. Previously, we have demonstrated prevention of EAG in the Brown Norway rat by the administration of cyclosporin A and anti-CD4 mAb [21]. More recently, we have found that anti-CD8 mAb is effective at preventing the development of crescentic nephritis in the WKY rat [J. Reynolds, unpublished observation]. The role of T cells in the pathogenesis of EAG was further elucidated in studies showing abrogation of disease in WKY rats receiving CTLA4-Ig [22], a fusion protein which prevents the ligation of CD28 on T cells by B7.1 and B7.2 on antigen presenting cells.
The induction of EAG in WKY rats using preparations of homologous GBM, of which 3(IV)NC1 is only a minor component, is not the ideal approach for assessing the specificity of the autoimmune response. Immunization with a purified recombinant form of the antigen would confer advantages for the investigation of autoimmune mechanisms in EAG, particularly with regard to exploration of the T cell response. In this study, we have cloned, sequenced and expressed the NC1 domain of the rat
3(IV) chain. Subsequently, we have shown that WKY rats immunized with a purified recombinant form of rat
3(IV)NC1 develop severe anti-GBM nephritis, similar to that observed in Goodpasture patients.
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Subjects and methods |
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Expression and purification of rat 3(IV)NC1
The following primers were designed to amplify, by PCR, a cDNA encoding the full-length rat 3(IV)NC1, together with some 5' collagenous sequence, and incorporated, respectively, the restriction enzyme sites BamHI and XhoI: 5' CG GGA TTC ATG GGA CCG CCA GGT TTA 3' and 5' T CCC CTC GAG TCA TGA CTT TGA ATA 3'. The BamHI/XhoI fragment of the amplified product was cloned into the pUC19 plasmid. Individual clones were assessed for the correct rat
3(IV)NC1 as above. The HindIII/KpnI fragment from a genuine pUC19 rat
3(IV)NC1 clone was subsequently cloned into the pFLAG CMV-1 expression vector (Kodak). COS-7 cells were transfected with plasmid DNA as previously described [5]. Recombinant rat
3(IV)NC1 was secreted into the COS supernatant as a fusion protein with the FLAG epitope. Recombinant material was purified in a single affinity step on an anti-FLAG agarose column. Eluates were collected from the column and protein was detected on a UV spectrophotometer. Determination of the protein concentration was based on the calibration that an OD 280 measurement of one was equivalent to 0.76 mg/ml of native GBM.
SDS-PAGE and western blotting
Recombinant material was analysed by electrophoresis on 12.5% polyacrylamide gels containing SDS. Gels were electroblotted onto nitrocellulose and membranes blocked with PBS/0.5% Tween (Tw) for 12 h. Recombinant protein was detected using human or rat sera containing anti-GBM antibodies, followed by alkaline phosphatase conjugated anti-human or anti-rat IgG, respectively. The mAb W17 [24], a kind gift from Professor J. Wieslander (University of Lund, Sweden), was used as a positive control for the detection of 3(IV)NC1. Colour was developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate (Sigma).
ELISA for detection of recombinant 3(IV)NC1
An ELISA for the detection of recombinant rat 3(IV)NC1, using anti-GBM antibodies, was developed for use in 96-well microtitre plates. Purified collagenase-solubilized rat GBM, coated at a concentration of 40 µg/ml in guanidine thiocyanate, was used as a positive control. Recombinant protein was diluted in 50 mM sodium carbonate buffer, pH 9.8, and 100 µl were used to coat each well in duplicate, at a concentration of 4 µg/ml. After overnight incubation at 4°C, the plate was washed three times in PBS/Tw and then blocked for 1 h in 100 µl of blocking buffer containing PBS/Tw/1% bovine serum albumin. Primary antibody, in the form of sera from Goodpasture patients or from rats immunized with recombinant rat
3(IV)NC1, was diluted in blocking buffer and 100 µl were incubated in each well at 37°C for 1 h. The plate was again washed three times, and 100 µl of 1:1000 dilution of secondary antibody, alkaline phosphatase conjugated goat anti-IgG, in blocking buffer, were added to each well and incubated for 1 h at 37°C. The plate was washed three times, and developed with phosphatase substrate tablets; one tablet was dissolved in 5 ml carbonate buffer containing 5 mM MgCl2 and 100 µl of this were added to each well. Colour was allowed to develop and the optical density (OD) was read at 405 nm using an Anthos htII ELISA plate reader.
Experimental animals
Male WKY rats, aged 812 weeks and weighing 120150 g, were purchased from Charles River, Margate, UK. All animals were housed in standard conditions and had free access to normal laboratory diet and water.
Experimental protocol
Groups of WKY rats (n=5) were given a single i.m. injection of: (i) recombinant rat 3(IV)NC1 in FCA, at a dose of 1 mg/kg body weight, or (ii) a similar volume of buffer with FCA. Serial blood samples were taken by tail artery puncture under light anaesthesia (Isofluorane), and 24 h urine specimens were obtained by placing animals in metabolic cages. Kidneys were removed at the time of sacrifice (4 weeks).
Assessment of EAG
ELISA for anti-3(IV)NC1 antibodies
Circulating anti-3(IV)NC1 antibodies were measured in sera from animals with EAG by a solid-phase enzyme linked immunosorbent assay (ELISA), based on that previously described [17]. Briefly, purified recombinant rat
3(IV)NC1 was coated, at a concentration of 4 µg/ml, on to microtitre plates (Gibco, Paisley, UK) by overnight incubation at 4°C, and test or control sera were applied for 1 h at 37°C. Bound anti-
3(IV)NC1 antibody was detected by horseradish peroxidase conjugated sheep anti-rat IgG (Sigma), and developed using the substrate ortho-phenylenediamine dihydrochloride (OPD, Sigma). The absorbance for each well was read at 492 nm using a Titertek Multiskan ELISA plate reader (Lab Tech International, Uckfield, UK), and the results expressed as the mean OD of triplicate wells.
Direct immunofluorescence
Direct immunofluorescence (IF) studies to assess deposits of IgG and fibrin within the glomeruli, were carried out on kidneys obtained at sacrifice by a method similar to that previously described [17]. Tissue was embedded in OCT II embedding medium (Miles) on cork discs, snap frozen in isopentone (BDH) pre-cooled in liquid nitrogen, and stored at 70°C. Cryostat sections were cut at 4 µm and incubated with fluorescein isothiocyanate (FITC) labelled rabbit anti-rat IgG (Serotec), or goat anti-rat fibrin (Nordic). The degree of immunostaining was assessed blind and graded from 0 to 3+.
Rocket immunoelectrophoresis.
Urinary albumin concentrations were measured in 24 h collections from animals with EAG by rocket immunoelectrophoresis (Pharmacia, St Albans, UK) as described previously [17]. Briefly, urine samples from experimental animals were subjected to immunoelectrophoresis at 60 V in an electrophoresis tank containing Barbitone buffer (BDH), pH 9.5, for 6 h, using a 1% agarose gel containing rabbit antisera to rat albumin raised in our laboratory. Results were calculated using rat serum albumin standards (which were run at the same time), and expressed in mg per 24 h.
Creatinine clearance.
Creatinine levels in serum and urine from animals at week 4 (the time of sacrifice) were measured by spectrometry using standard techniques. Creatinine clearance was then calculated for each animal and expressed as ml/min.
Light microscopy.
Kidney tissue, taken at the time of sacrifice, was fixed in 10% neutral buffered formalin, processed and embedded in paraffin wax for light microscopy by standard techniques. Briefly, 3 µm sections were stained with haemotoxylin and eosin, and periodic acid-Schiff. Fifty consecutive glomeruli per section were assessed blind and graded as: severe (>50% of the glomerulus affected by necrosis/crescent formation), abnormal (<50% of the glomerulus affected), or normal.
Immunohistology.
Formalin fixed kidney sections, obtained at sacrifice, were stained with monoclonal antibodies ED1 (for macrophages), W3/13 (for T lymphocytes), W3/25 (for CD4+ lymphocytes) and OX8 (for CD8+ lymphocytes), by standard indirect immunoperoxidase staining techniques. The cellular infiltrate was assessed blind by counting the number of positively stained cells per 50 consecutive glomeruli.
Statistical analysis
Differences between data were determined by the MannWhitney U test.
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Results |
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Production and characterisation of recombinant rat 3(IV)NC1
Tissue culture flasks (T 162 cm2) of sub-confluent COS-7 cells were transfected with 20 µg of plasmid DNA using standard DEAE-dextran techniques as described previously [5]. Recombinant material was secreted into the COS-7 supernatants as a fusion protein in which the FLAG peptide was linked to the rat 3(IV)NC1 domain by a short collagenous segment of rat
3(IV). Supernatants were collected 72 h, 144 h and 216 h post-transfection. Recombinant material was purified on an anti-FLAG M2 affinity column and examined by SDS-PAGE and western blotting. Sera from rats immunized with recombinant rat
3(IV)NC1 bound strongly to the characteristic dimer and monomer components of human and rat GBM (Figure 3
). Recombinant rat
3(IV)NC1 was detected as two monomeric isoforms at the predicted molecular weight of approximately 28 kD by antibodies from Goodpasture patients as well as from rats immunized with rat
3(IV)NC1. The observation that higher molecular weight recombinant material was also recognized by autoantibodies suggests the spontaneous formation of dimers, possibly via interactions between the short collagenous segments of recombinant monomers (Figure 3
). This supposition finds support in the fact that such putative dimeric components can be digested by collagenase. Western blot analysis also showed that recombinant rat
3(IV)NC1 was strongly recognized by mAb 17 (Figure 3
), an antibody raised to bovine GBM and shown to bind to native human
3(IV)NC1 [24]. By ELISA, recombinant rat
3(IV)NC1 was recognised both by sera from rats immunized with rat
3(IV)NC1 and by sera from Goodpasture patients (Figure 4
).
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Assessment of EAG
Circulating anti-3(IV)NC1 antibodies.
All animals immunized with recombinant rat 3(IV)NC1 in FCA produced detectable circulating anti-
3(IV)NC1 antibodies by week 2, which increased to high levels by week 3 and peaked at week 4. Control animals given FCA alone showed no increase in circulating anti-
3(IV)NC1 antibodies (Figure 5a
).
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Direct immunofluorescence for IgG.
Direct IF for IgG on kidney tissue at 4 weeks revealed that animals immunized with recombinant rat 3(IV)NC1 in FCA showed strong linear deposits of IgG along the GBM, and to a lesser extent the TBM. Control animals given FCA alone showed no antibody binding. Results are summarized in Table 1
and illustrated in Figure 6a
.
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Albuminuria.
All animals immunized with recombinant rat 3(IV)NC1 in FCA produced detectable levels of albuminuria by week 2, which increased by week 3 and peaked at week 4. Control animals given FCA alone did not develop albuminuria (Figure 5b
).
Creatinine clearance.
All control animals given FCA alone showed a normal creatinine clearance, in the range of 1.01.2 ml/min. All animals immunized with recombinant rat 3(IV)NC1 in FCA showed a marked reduction in creatinine clearance at 4 weeks (Table 2
).
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Direct immunofluorescence for fibrin.
Direct IF for fibrin on kidney tissue at 4 weeks revealed that all animals immunized with recombinant rat 3(IV)NC1 in FCA showed large fibrin deposits within the glomeruli. Control animals given FCA alone showed no binding. Results are summarized in Table 1
.
Light microscopy.
Light microscopy of kidney tissue at week 4 revealed that all animals immunized with recombinant rat 3(IV)NC1 in FCA showed severe focal necrotizing glomerulonephritis with crescent formation. Control animals given FCA alone showed normal renal histology. Results are shown in Table 2
and illustrated in Figure 6b
.
Immunohistology.
Immunohistology of kidney tissue at week 4 showed that all animals immunized with recombinant rat 3(IV)NC1 in FCA had a glomerular infiltrate of T lymphocytes (which were predominantly CD8+) and macrophages. Control animals given FCA alone showed no inflammatory infiltrate (Table 2
).
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Discussion |
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The present study demonstrates that immunization with purified recombinant rat 3(IV)NC1 can induce severe anti-GBM nephritis in WKY rats. These animals developed circulating anti-GBM antibodies directed towards
3(IV)NC1, linear deposits of IgG on the GBM, albuminuria, deposits of fibrin in the glomeruli, severe focal necrotizing glomerulonephritis with crescent formation, and glomerular influx of CD8+ T cells and macrophages. Such disease features are similar to those observed in models of autoimmune nephritis in which rats have been immunized with native GBM, and also approximate to the clinical features of Goodpasture's disease. It is noteworthy that antibodies from these rats shared similar binding characteristics with human autoantibodies. The induction of anti-GBM disease by immunization with recombinant antigen is not unprecedented. Our findings are in general agreement with a report showing that rats immunized with human recombinant
3(IV)NC1 developed severe anti-GBM nephritis [19]. That study also showed that human
4(IV)NC1 was nephritogenic, but that other recombinant type IV collagen NC1 domains associated with the GBM were not. Our contention is that immunization with a homologous form of the antigen is more appropriate in studies of autoimmunity, since it might avoid the broader immune response that could be elicited by heterologous or foreign antigen. This approach would confer certain advantages for the investigation of autoimmune mechanisms in experimental anti-GBM disease, particularly in delineating the T cell response, which may depend on specific amino acid residues.
The induction of nephritis using a purified form of rat 3(IV)NC1 provides a framework for understanding the molecular basis of autoimmunity in Goodpasture's disease. Even though the
3(IV) chain is a minor component of the GBM, it appears that this molecule has a particular capacity to induce autoimmune nephritis. The humoral aspects of Goodpasture's disease are well recognized and a consensus is now forming as to the localization of the major B cell epitope. Studies using synthetic peptides to localize the major epitope within the
3(IV)NC1 domain have given varying results [25,26]. More recently, our group has shown, using chimeric NC1 domains of human
3(IV)NC1 and
1(IV)NC1, that the major antibody binding site is contained largely within the amino terminal portion of
3(IV)NC1 [5]. This conclusion has been supported by subsequent studies of the immunoreactivity of chimeric NC1 domains, which have also defined the major conformational epitope(s) in greater detail [6,7]. Given the established nephritogenicity of recombinant rat
3(IV)NC1, the capacity of such chimeric NC1 domains to induce nephritis will be of considerable interest.
The analysis of T cell responses in Goodpasture's disease has been slow because of the difficulty in isolating relevant T cell clones. It is also conceivable that by the time patients present with the disease, the original autoreactive T cell response may have been diluted by epitope spreading, thus compounding this difficulty. Our model of EAG induced by native rat GBM [17,22] has proved to be an accurate model of Goodpasture's disease in many respects. However, in order to explore the T cell response and define autoreactive epitopes, there are obvious advantages to a model developed using a single specific autoantigen, as described here. The development of purified recombinant rat 3(IV)NC1 will advance the study of EAG in the rat, and allow the exploration of cell mediated immunity in an accurate model of Goodpasture's disease.
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Acknowledgments |
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Notes |
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References |
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