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. 2012 Feb;93(1):11–17. doi: 10.1111/j.1365-2613.2011.00792.x

Production of heterologous IgG antibody against Heymann nephritis antigen by injections of immune complexes

Arpad Z Barabas *, Chad D Cole †, Maria Sensen ‡, Rene Lafreniere *
PMCID: PMC3311017  PMID: 22103575

Abstract

Heterologous IgG antibody (ab) can be produced against Heymann nephritis (HN) antigen (ag) in rabbits by administering it in Freund's complete adjuvant. The developing abs reacted at high titre with rat kidney brush border (BB) regions of the renal proximal tubules in an indirect fluorescence ab test. A single IV injection of the heterologous ab into a susceptible strain of rat resulted in the localization of IgG ab to glomerular fixed ags, producing immune complex glomerular nephritis. The injected ab also reacted with the BB region of the renal proximal tubules. The aim of this experiment was to find out whether heterologous IgG ab against the HN ag can also be produced in recipient rabbits by injecting immune complexes (ICs) composed of a rat kidney tubular preparation [rat kidney fraction 3 (rKF3)] and donor rabbit–derived rabbit anti-rKF3 IgG ab. We found that anti-rKF3 IgG ab – against the BB region of the renal proximal tubules – could be induced in rabbits injected with ICs, and the resulting ab was able to initiate passive HN in rats. This was the first time a pathogenic IgG ab was produced against HN ag in rabbits without the use of adjuvant. Ab responses in recipient rabbits were achieved by ab information transfer. Recipient rabbits injected with the IC produced the same class of immunoglobulin with the same specificity against the target ag rKF3, as was present in the innoculum, namely rabbit anti-rKF3 IgG ab.

Keywords: immune complex, membranous glomerulonephritis, modified vaccination technique, passive Heymann nephritis, pathogenic

Heymann nephritis (HN), an experimental autoimmune kidney disease of rats, was first produced by Heymann et al. (1959) by repeated IP injections of renal tubular antigens (ags) in Freund's complete adjuvant (FCA). After four weekly injections of the antigenic preparation, rats developed progressive proteinuria and immune complex glomerulonephritis (ICGN). The disease was morphologically and functionally similar to membranous glomerulonephritis (MGN) of humans, representing a typical pathogenic autoantibody-(aab) initiated and aab-maintained autoimmune disease (Edgington et al. 1968; Barabas & Lannigan 1969; Fleuren et al. 1980; Mendrick et al. 1980; Noble et al. 1982; Andres et al. 1986; Salant & Cybulsky 1988).

Another variant of HN, that is considered not to be a true autoimmune disease, is passive HN (PHN). This experimental disease model was produced by Barabas et al. (1970) by a single IV injection of a heterologous (rabbit) anti-rat kidney fraction 3 (arKF3) IgG antibody (ab) in rats. Rats developed ICGN, characterized by beaded depositions around the glomerular capillary loops, staining for rabbit IgG first (heterologous phase) and a week or so after the injection of the heterologous ab glomerular capillaries also stained for rat IgG (autologous phase) (Couser et al. 1978; Van Damme et al. 1978; Salant et al. 1989). The initial reactivity of the injected heterologous IgG ab was with the glomerular-associated nephritogenic ag (Salant et al. 1989). It was also demonstrated that the glomerular-associated nephritogenic ag was found on the podocytes (Van Damme et al. 1978). The nephritogenic ag was produced in the clathrin-coated pits of the glomerular epithelial cell's foot processes and distributed as small granules along the podocytes’ surfaces and at the base of the foot processes touching the glomerular basement membrane (GBM) (Kerjaschki & Farquhar 1983).

The PHN experimental kidney disease model is useful to study the initial and continuous development of immune complexes (ICs) in the glomeruli, especially during the early phase of the disease (Challice et al. 1986; Salant et al. 1989). It was observed that IC formation was because of in situ formation of ICs around the glomerular capillary loops (Couser et al. 1978; Fleuren et al. 1978; Van Damme et al. 1978). The heterologous arKF3 or anti-Heymann nephritis ag (derived from renal tubules) IgG abs (Edgington et al. 1967) reacted with the GBM-associated nephritogenic ags and not as previously believed that ICs were formed in the circulation and deposited on the epithelial side of the GBM (Dixon et al. 1961; Edgington et al. 1968). This was the first time that a podocyte-produced nephritogenic autoantigen was identified and shown to be present along the foot processes (Kerjaschki & Farquhar 1983). Such glomerular-associated nephritogenic ag has not been identified in humans with MGN by some (Whitworth et al. 1976; Collins et al. 1981), but not all, investigators (Naruse et al. 1973, 1974; Pardo et al. 1975; Strauss et al. 1975; Shwayder et al. 1976; Gilboa et al. 1977; Douglas et al. 1981; Zanetti et al. 1981; Gonzalez-Cabrero et al. 1992). Recently, Ronco and colleagues have shown that a podocyte-associated ag, neutral endopeptidase (NEP), in the new born can be a target by transplacentally transferred maternal anti-NEP IgG ab (Debiec et al. 2004; Ronco & Debiec 2006). Pathogenic ab reacting with NEP at the sole of foot processes initiated and maintained IC depositions in the glomeruli resulting in MGN. Treatments by ag-specific immunotherapies of HN ag- and NEP ag-induced MGN in rats and humans, respectively, are thought to provide eventual curative solutions.

Recently, Barabas and colleagues have shown that slowly progressive HN (SPHN) can be prevented and, when present specifically, terminated by an ag-specific treatment modality with minimal side effects in 100% of a genetically susceptible strain of rat. This was achieved using a new vaccination method called “modified vaccination technique” (MVT) (Barabas et al. 2004b, 2006a,b). They also suggested that the MVT – with appropriate modifications – could provide specific preventive and curative solutions for other autoimmune disorders as well (Barabas et al. 2007a,b).

In a previous experiment using the same immunization method, we have shown that rats could produce a high-titred ab response against a soluble exogenous ag as well (Barabas et al. 2007c).

The present experiment investigated whether the application of the MVT using ICs could evoke a predetermined immune response outcome against a particulate ag as well.

Material and methods

Preparation of renal tubular ag

Kidneys were obtained from normal adult male Sprague-Dawley rats. Immediately after euthanasia, rats were bled out and their blood vessels were thoroughly washed out with chilled saline until the kidneys were pale. Renal capsules and the peri-renal fat were removed, and kidneys were placed into 0.25 M buffered sucrose solution pH 7.3 at 4 °C and cut-up into small pieces with a pair of scissors. Kidney preparations were homogenized first with a Cyclone Vertishear (Virtis) and then by a Potter-Elverhjem homogenizer. Rat kidney fraction 3 was obtained by differential centrifugation (Hübscher et al., 1965), using a Beckman Model J2.21 centrifuge (Barabas et al. 2004b).

Preparation of rabbit arKF3 IgG antiserum using rKF3 in FCA

Four adult female New Zealand white rabbits were injected with 1 ml of 5 mg rKF3 ag in FCA at multiple IM sites on 0, 4, 8 and 10 weeks. Five milligram of aqueous rKF3 ag was also injected in 2 ml volumes at multiple SC routes at 12, 13 and 17 weeks. Serum was obtained from each immunized rabbit at 18 weeks. Donor rabbit sera – having ab activities against the brush border (BB) region of the renal proximal convoluted tubules in an indirect fluorescence ab test – were used in the experiment.

Preparation and injection schedule of ICs made up of rabbit arKF3 IgG ab X rKF3 ag at ag excess

Four adult female New Zealand white rabbits were injected with ICs [(rabbit arKF3 IgG ab X rKF3) rKF3]. Immune complexes were prepared as follows: 3 ml of donor rabbit arKF3 IgG (6 mg/ml) and 0.5 ml of rKF3 ag (40.6 mg/ml) were made up to 12 ml with saline and rotated gently at RT° for one and half hours. After the completion of the ag/ab reaction, ICs were centrifuged at 90,000 g for half an hour at 4 °C and subsequently washed twice with saline to eliminate irrelevant un-rKF3 ag–reacting rabbit IgG. Sediment containing the IC was re-suspended with 8 ml of saline. Two millilitres prepared IC was injected at SC sites into each rabbit at 0, 2, 8, 12 and 16 weeks. Rabbits also received 4 mg of aqueous rKF3 ag by multiple SC injections at 18 weeks. Rabbits were bled for sera 4 days after the last injection.

Two adult female New Zealand white rabbits (controls) were also injected with the same doses of rKF3 ag (5 mg) in an aqueous solution at the same time intervals as rabbits earlier, and sera were obtained from them 4 days after the last injection.

Preparation of rabbit IgG

Centrifuged and filtered samples of sera from rabbits injected with rKF3 ag in FCA and IC and from sera of rabbits injected with aqueous rKF3 ag were applied to HiTrap® affinity columns (HiTrap® protein A; Amersham Pharmacia Biotech AB, SE-751 84, Uppsala, Sweden) to obtain IgG preparations. One-millilitre columns were washed out with five column volumes of start buffer (20 mM sodium phosphate pH 7.0); and the serum samples were adjusted to pH 7.0 prior to applications using syringes. After applying 1 ml of samples, columns were washed with five column volumes of the start buffer to eliminate irrelevant serum protein components. Rabbit IgG preparations were eluted into test tubes containing 0.5 ml of 1 M Tris–HCl, pH 9.0 (to obtain samples at neutral pH), using two column volumes of elution buffer (0.1 M citric acid, pH 3.2). Serum samples were applied to HiTrap® protein A 1-ml columns several times to obtain the required ags of rabbit IgG preparations. Collected samples were dialysed during a 24-h period against three changes of saline at 4 °C and thereafter reduced in volumes by Polyethylene glycol 8000. Rabbit IgG preparations were analysed for protein contents by the Biuret protein estimation (Weichelbaum 1946), and their final concentrations were adjusted to 6 mg/ml with saline.

Indirect fluorescence ab tests – ab activity of rabbit IgG preparations against the BB-associated nephritogenic ag

Dilutions of IgG abs [obtained from individual rabbits immunized with rKF3 ag in FCA, IC and aqueous rKF3 ag; and from IgG preparations absorbed with rKF3 (used for injecting groups 2 and 4 rats)] were tested for ab activity against rat kidney tubular ags on 3-μm-thick frozen sections of normal Sprague-Dawley rat kidneys. Dilutions of IgG preparations were incubated on marked kidney sections for 30 min in a wet box at RT°. Following incubation, washed kidney sections were counter-stained with Alexa Fluor® 488 goat anti-rabbit IgG (H + L; Molecular Probes Inc., Eugene, OR, USA) for half an hour prior to mounting and viewing sections with a Zeiss Axioscop microscope. Final dilutions of abs reacting with renal tubular BB regions were recorded.

Experimental design

Individually identified adult male Sprague-Dawley rats were assigned into five experimental groups.

Group 1: six rats received a single IV injection of 1 ml of rabbit arKF3 IgG ab (produced by conventional means by injecting rKF3 ag in FCA into rabbits as described in Material and Methods).

Group 2: six rats received a single IV injection of 1 ml of centrifuged supernatant of rabbit arKF3 IgG ab, used in group 1 rats, but mixed for one and half hours with rKF3 ag in ag excess prior to injection (to absorb out abs against the nephritogenic ag).

Group 3: six rats received a single IV injection of 1 ml of rabbit arKF3 IgG ab obtained from rabbits injected with ICs [(rabbit arKF3 IgG ab X rKF3) rKF3] to produce the ab.

Group 4: six rats received a single IV injection of 1 ml of centrifuged supernatant rabbit arKF3 IgG ab, used in group 3 rats, but mixed for one and half hours with rKF3 ag in ag excess prior to injection (to absorb out abs against the nephritogenic ag).

Group 5: three rats received a single IV injection of 1 ml of rabbit arKF3 IgG ab obtained from rabbits immunized with aqeous rKF3 ag.

Direct fluorescence ab tests

Kidney samples were obtained from each rat on day 3 under general anaesthesia (renal biopsies) and at the end of the experiment on day 7 from euthanized rats (whole kidneys). Frozen sections were cut at 3 μm thickness on a Microm HM 500M cryostat at −22 °C and processed prior to staining the kidney sections with Alexa Fluor® 488 goat anti-rat and goat anti-rabbit IgG (H + L) abs, as previously described (Barabas et al. 2004b). Kidney sections were viewed and pictures taken with a Zeiss Axioscope microscope.

Results

Indirect fluorescence ab test results

Rabbits injected with rKF3 ag in FCA followed by injections of aqueous rKF3 ag produced high-titred pathogenic rabbit arKF3 IgG abs against the BB regions of the proximal convoluted tubules. On average, the ab response against the BB-related nephritogenic ag was detectable at 34×103 dilution of IgG preparations. Rabbits injected with ICs also produced pathogenic IgG abs against the injected nephritogenic ag (Table 1). On average, an ab response against the BB region–associated nephritogenic ag was detectable at 1:9×103 up to 9000 dilution. Rabbit arKF3 IgG preparations – absorbed with rKF3 ag – used for injecting groups 2 and 4 rats did not react with the BB-associated zone of the renal proximal convoluted tubules nor did the IgG preparation from the aqueous rKF3 ag–immunized rabbits.

Table 1.

Indirect fluorescence ab test results. Antibody titres against the BB ag of the renal proximal convoluted tubules in rabbit IgG preparations

Rabbits injected with Titre of rabbit IgG ab against the BB ag (in thousands) Average ab titre (in thousands)
rKF3 ag in FCA (4 R) R1 = 35 R2 = 30 34
R3 = 30 R4 = 40
IC composed of (R arKF3 IgG ab X rKF3) rKF3 (4 R) R5 = 7 R6 = 7 9
R7 = 13 R8 = 7
Pooled rabbit IgG preparations from R1–4 and R5–8 absorbed with rKF3 ag and used in groups 2 and 4 rats (8 R) R1–4 = 0 R5–8 = 0 0
Aqueous rKF3 (2 R) R9 = 0 R10 = 0 0
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ab, antibody; ag, antigen; arKF3, anti-rat kidney fraction 3; BB, brush border; FCA, Freund's complete adjuvant; IC, immune complex; rKF3, rat kidney fraction 3; (R), number of rabbits.

In addition, the rabbit arKF3 IgG ab (obtained from groups 1 and 3 rabbits) reacted faintly with the glomerular capillary loop–localized nephritogenic ag and with the mesangial-trapped nephritogenic ag more intensely in the indirect fluorescence ab test (Figure 1).

Figure 1.

Figure 1

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Indirect fluorescence antibody (ab) test. Pathogenic ab activity against kidney components. 1:5000 dilution of serum from a group 1 rabbit immunized with rat kidney fraction 3 antigen (ag) in Freund's complete adjuvant. Pathogenic IgG ab reacts with normal rat kidney section's brush border zone of cells located in the renal proximal convoluted tubules (white arrow) and faintly with the glomerular-localized nephritogenic ags (red arrow). Mesangial cells, where the nephritogenic ag is also present, stain intensely. Sera of group 3 rabbits (immunized with immune complex) stained normal rat kidney sections similarly (not illustrated). White scale bar = 25 μm.

Direct fluorescence ab test results

Intravenously injected rabbit arKF3 IgG ab (having ab activity against the BB-associated ags of the renal proximal convoluted tubules, mesangial-trapped and glomerular-localized nephritogenic ags) (Figure 1) produced typical PHN kidney lesions in group 1 rats by days 3 and 7. The lesions were characterized by beaded deposits staining for the heterologous IgG ab around the glomerular capillary loops (Figure 2a). The rabbit arKF3 IgG ab was also present in the tubular basement membrane (TBM) and the BB-associated regions of the renal proximal tubules (directed against the nephritogenic ag) (Figure 2b). Glomerular deposits did not stain for rat IgG on day 3; however, they stained 7 days after the ab injection. Group 2 rats injected with the same volume of rabbit arKF3 IgG ab as group 1 rats, but absorbed with rKF3 ag prior to injection, showed no localization of rabbit and rat IgG in any of the aforementioned renal sites on days 3 and 7. Group 3 rats immunized with the IC-generated rabbit arKF3 IgG ab had the same typical PHN kidney lesions as group 1 rats staining for rabbit IgG (Figure 3a,b), but showed no staining for rat IgG on day 3 post-ab injections (Figure 4a); however, by day 7, kidney sections showed staining of the glomeruli with a beaded pattern for rat IgG (Figure 4b). Group 4 rats injected with the same IC-generated rabbit arKF3 IgG ab (rKF3 ag absorbed) showed no typical renal lesions on days 3 and 7. Group 5 rats injected with IgG preparations from aqueous rKF3 ag–immunized rabbits did not produce typical PHN either.

Figure 2.

Figure 2

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Direct fluorescence antibody (ab) test. Kidney section of a group 1 rat injected with rabbit anti-rat kidney fraction 3 (arKF3) IgG ab (obtained from a rabbit immunized with rKF3 antigen in Freund's complete adjuvant). Kidney biopsy was obtained 3 days after the injection of the ab. Glomerular capillary loops show intense staining for rabbit IgG with fine beaded deposits (a) [red arrow]. Staining of the brush border zone of cells (b) [white arrow] and beaded staining of the tubular basement membrane (a) [pink arrow] can also be observed; staining of the glomeruli for rat IgG was not present (not illustrated). Staining of rat kidney sections of group 1 rats for rabbit IgG on day 7 was similar to day 3 results (not illustrated). White scale bar = 25 μm.

Figure 3.

Figure 3

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Direct fluorescence antibody (ab) test. Kidney section of a group 3 rat injected with rabbit anti-rat kidney fraction 3 IgG ab (obtained from a rabbit immunized with immune complex at antigen excess). Kidney biopsy was obtained 3 days after the injection of the ab. The glomerular capillary loops show intense staining for rabbit IgG characterized by fine beaded deposits (a) [red arrow]. The brush border zone of cells show intense fluorescence staining for rabbit IgG (b) [white arrow] and the tubular basement membrane stains with a beaded pattern (b) [pink arrow]. Staining of the glomeruli for rat IgG was not present (not illustrated). Staining of rat kidney sections of group 3 rats for rabbit IgG on day 7 was similar to day 3 results (not illustrated). White scale bar = 25 μm.

Figure 4.

Figure 4

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Direct fluorescence antibody (ab) test. Kidney section of a group 3 rat injected with rabbit anti-rat kidney fraction 3 IgG ab (obtained from a rabbit immunized with immune complex at antigen excess). Kidney biopsy was obtained 3 days after the injection of the ab. The glomerular capillary loops, the brush border zone of cells and the tubular basement membrane did not stain for rat IgG (a) (i.e. rat IgG ab – directed against the rabbit IgG – is not present at an early stage). Kidney sections of both group 1 (illustrated) and 3 (not illustrated) rats’ showed beaded depositions staining for rat IgG around the glomerular capillary loops by day 7 (b). White scale bar = 25 μm.

Discussion

The fact that we produced PHN by a single IV injection of a heterologous IgG ab, directed against the nephritogenic ag, is not novel. Barabas and colleagues have shown as early as 1970 and others subsequently (Barabas et al. 1970; Barabas & Lannigan 1974; Feenstra et al. 1975; Couser et al. 1978; Van Damme et al. 1978) that PHN can be induced in rats by such injections. What is novel in our experiment is how we produced a heterologous ab against the nephritogenic ag. The nephritogenic ag (obtained from rat kidneys and injected into rabbits) was an exogenous ag, as far as the rabbits were concerned. Routinely, heterologous pathogenic IgG ab against the rat nephritogenic ag (derived from the renal proximal convoluted tubules) is produced by injecting it in adjuvants (most often in FCA) into rabbits or sheep (Feenstra et al. 1975; Kerjaschki & Farquhar 1982; Madaio et al. 1983; Quigg et al. 1988). Repeated injections of the preparation after 2 months yielded high-titred specific pathogenic IgG abs that were directed against the nephritogenic ag residing in the BB region of the renal proximal convoluted tubules of normal rat kidneys abundantly and sparsely around the glomerular capillary loops in association with the epithelial cell's foot processes (Barabas et al. 2004a).

We have utilized a new vaccination technique that we have developed – and call MVT (Barabas et al. 2007a,b) – for the production of rabbit arKF3 IgG ab without using adjuvants. ICs were prepared: of the rKF3 ag and rabbit arKF3 IgG ab (obtained from rKF3 ag in FCA-injected donor rabbits), at ag excess. Repeated injections of this IC into recipient rabbits initiated and maintained the production of the same class of ab with the same specificity against the target ag as resided in the inoculum, namely, rabbit arKF3 IgG ab. The MVT provides a predetermined tailor-made immune response in 100% of animals receiving the appropriately assembled IC (Barabas et al. 2004b, 2007c). The vaccination is safe and initiates specific immune response outcomes without interfering with normal functions of the immune system.

The MVT promises to be a versatile immunization method for prophylactic and therapeutic application for the prevention or termination of exogenous and endogenous ag-initiated mishaps by redirecting immune response outcomes.

The other observation that we have made in this experiment relates to the inability of rKF3 ag–absorbed rabbit arKF3 IgG abs to induce PHN. This provides further support for the notion that the podocyte and renal proximal tubule BB-located nephritogenic ag is identical in every respect (Barabas et al. 2008). The nephritogenic ag in the proximal convoluted tubule is, therefore, the source of the nephritogenic ag that contributes to the podocyte-localized ag in normal rat kidneys and also to the continuous build-up of glomerular ICs together with rat arKF3 IgG abs and complement components in HN and SPHN (Barabas et al. 2003, 2004a).

Recently, a podocyte-produced and podocyte-localized target ag – in acute neonatal MGN – was identified to be NEP (Ronco & Debiec 2007). This is the first target ag identified as causing and contributing to IC depositions in the glomeruli in human MGN. ICGN is because of a NEP-negative mother's pathogenic IgG ab response against the podocyte-localized NEP of the foetus (Debiec et al. 2004; Ronco & Debiec 2005, 2006, 2007, 2010). A second podocyte-related target ag identified was the phospholipase A2 receptor (PLA2R): it was shown to play a role in glomerular IC depositions in the kidneys of patients with idiopathic membranous nephropathy (Beck, Jr. et al. 2009; Ronco & Debiec 2010). ICs form in situ on the epithelial aspect of the GBM and are composed of the podocyte-associated PLA2R ag, anti-PLA2R IgG4 ab and complement components. Recently, a new immunofluoresence test and a Western blot immunoassay were developed for measuring circulating PLA2R ab levels (Hofstra et al. 2011; Hoxha et al. 2011) that could assist in diagnosing primary MGN and, during treatment (Beck, Jr. et al. 2011), monitor improvements in kidney function.

Several publications reveal that the HN ag is not present in the glomeruli of normal and diseased human kidneys (Whitworth et al. 1976; Collins et al. 1981). However, during certain pathological events, glomerular lesions and circulating aabs against the BB region of the renal proximal tubules have been observed just as in rats with HN (Naruse et al. 1973, 1974; Gilboa et al. 1977; Douglas et al. 1981; Zanetti et al. 1981; Gonzalez-Cabrero et al. 1992). There is no doubt that other endogenous and exogenous ags exist in the circulation as well as podocyte-localized ones, and that, under certain circumstances could evoke pathogenic IgG ab responses causing MGN. It is imperative to identify these podocyte-localized nephritogenic MGN-causing ags to institute an ag-specific treatment programme (Beck, Jr. & Salant 2010; Ronco & Debiec 2010). When such nephritogenic ags are identified and prepared ex vivo, then by the application of the MVT, we shall have the potential of terminating immunopathological events that initiate and maintain MGN in humans.

Acknowledgments

We acknowledge the assistance of our research associate, Zoltan B. Kovacs, in computer and laboratory-related work.

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