Immunology 1979 36 759

Induction of autoimmunity to antigens of the glomerular basement membrane in inbred Brown-Norway rats

MARIANNE STUFFERS-HEIMAN, E. GUNTHER* & L. A. VAN ES Department of Nephrology, University Hospital, Leiden, The Netherlands and *Max-Planck-Institut far Immunbiologie, Freiburg, West Germany

Received 27 June 1978; acceptedfor publication 2 August 1978

Summary. Induction of autoimmune antibodies against antigens of glomerular basement membrane (GBM) was studied in nine inbred strains of rats each with a different major histocompatibility complex H-1. Brown-Norway (BN) (H-ln), Lewis (H-I'), PVG/c (H-1c), AS2 (H- I), AVN (H-la), BD V (H-ld), DA (H- la) and F344 (H- 1) rats were immunized with bovine GBM and Freund's complete adjuvent (CFA). A pronounced linear deposition of host IgG (IgG 1 and IgG2a) along the GBM was found in BN rats. No deposition of C3 could be detected in the glomeruli nor did the animals develop proteinuria. The quantity of autoimmune antibodies fixed to the GBM was low (48 pg ± 14) which could explain the absence of C3 deposition and proteinuria. The antigenic specificity of the antibodies deposited along the GBM in BN rats was shown by the fixation in vitro of the eluted antibodies to the GBM and tubular basement membrane (TBM) of normal kidneys. A much weaker and irregular deposition of host IgG along the GBM was observed in PVG/c, AS2. AVN, BD V, DA and F344 rats. Of these strains, eluates from the glomeruli of PVG/c, AVN, BD V and DA rats fixed very weakly to the GBM of normal kidneys whereas eluates from AS2 and F344 rats did not fix to

GBM or TBM. No deposition of host IgG was found in Lewis rats, and the eluates did not fix to normal kidneys. Congenic L.BN rats with the BN H-1 n haplotype and a Lewis background did not respond. This study shows a genetic predisposition in rats to an autoimmune anti-GBM response which is not, or not exclusively, controlled by genes linked to the H-1 histo-compatibility complex.

INTRODUCTION Experimental allergic glomerulonephritis (EAG) has been successfully induced in several outbred species, such as sheep (Steblay, 1962), rabbits (Unanue & Dixon, 1967), goats (Williams & Steblay, 1965) and monkeys (Steblay, 1963) by immunization with heterologous glomerular basement membrane (GBM) and Freund's complete adjuvant (CFA). Attempts to induce EAG in inbred rats, however have been complicated by the development of autologous immune complex nephritis (AICN) owing to the extreme susceptibility of rats to renal tubular epithelial (RTE) antigens present in the immunogen (R. J. Glassock, personal communication). Other studies have shown that the H- I major histocompatibility complex plays a role in the induction of several autoimmune phenomena in the rat, such as experimental allergic encephalomyelitis (Gasser, Newlin, Palm & Gonatas,

Correspondence: Marianne Stuffers-Heiman, Dept of Nephrology, University Hospital, Leiden, The Netherlands. 00 1 9-2805/79/0400-0759$02.00

©) 1978 Blackwell Scientific Publications 759

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Marianne Stuffers-Heiman, E.

1975; Williams & Moore, 1973), experimental thyroiditis (Penhale, Farmer, Urbaniak & Irvine, 1975), AICN (Stenglein, Thoenes & Gunther, 1978), HgCl2induced anti-GBM nephritis (Druet, Sapin, Giunther, Feingold & Druet, 1977) and anti-TBM nephritis (Lehman, Wilson & Dixon, 1974). Therefore a study was initiated to investigate the genetic predisposition of various strains of rats to an autoimmune anti-GBM response after immunization with bovine GBM and CFA. Contamination of the immunogen by RTE was prevented by using sodium desoxycholate for the isolation of GBM (Meezan, Hjelle, Brendel & Carlson, 1976). This detergent solubilizes RTE without affecting the immunogenicity of the GBM. Furthermore, the development of AICN was obviated by investigating strains that were resistant to the development of AICN (Stenglein, Thoenes & Gunther, 1975; 1978). We observed that the tolerance for GBM antigens was lost in the BN strain, the loss being characterized by a linear deposition of host IgG along the GBM in the kidneys of immunized rats and by linear fixation of acid eluates from these kidneys to the GBM and TBM of normal kidneys in vitro.

MATERIALS AND METHODS Animals Brown-Norway (BN) rats were purchased from the Institute for Hygiene in Amsterdam and bred in our laboratory at the University Hospital, Leiden, The Netherlands. L.BN rats, which carry the H-l haplotype of BN rats on the Lewis strain genetic background, AS2, AVN, DA and BD V rats were bred at the Max-Planck-Institut fur Immunbiologie, Freiburg, West Germany. Lewis rats were purchased from the Department of Pathology, University Hospital, Leiden, The Netherlands, PVG/c rats were a gift from Dr F. W. J. Gribnau of the Radboud Hospital, Nijmegen, The Netherlands. Eight-week-old female rats (125-150 g) were used. No spontaneous glomerulonephritis was observed in our rats during the first year of life as assessed by the absence of deposits of host IgG in the glomeruli and the absence of proteinuria. Preparation of GBM Glomeruli were isolated according to a modification of the method of Krakower & Greenspon (1951).

Gunther & L. A. van Es

Minced renal cortex of bovine kidneys was pushed through a 100-mesh stainless steel sieve placed on top of a 150-mesh sieve. The latter retained glomeruli, tubular segments and connective tissue fragments, whereas cellular debris passed through both sieves. The glomeruli were suspended in cold isotonic saline and repeatedly washed by centrifugation (90 s, 120 g until the supernatant remained clear. The white layer of tubules on top of the pellet of glomeruli and the supernatant were removed by aspiration. To remove remaining tubular segments and connective tissue fragments, the final sediment was suspended in isotonic saline and subsequently forced through a 120-mesh nylon sieve placed above a 150-mesh nylon sieve. The first sieve retained the larger fragments of tubules and connective tissue, whereas the glomeruli were collected on the second. Only preparations containing less than 2% renal tubules were used for isolation of GBM. GBM was isolated from the glomeruli in 4% sodium desoxycholate according to the method of Meezan, Hjelle, Brendel & Carlson (1976). The isolated glomeruli were suspended in a large volume (100: 1) of distilled water containing 0 1% sodium azide, and were magnetically stirred overnight to lyse the cells and release their intracellular contents. The tissue suspension was then centrifuged (5 min, 120 g) and the supernatant discarded. The pellet was suspended in 40 ml of 1 M NaCl and 2000 kunitz units of DNA-ase were added. The suspension was stirred for 4 h. The mixture was again centrifuged (5 min, 120 g). The pellet was then suspended and stirred overnight in 40 ml of 4% sodium desoxycholate containing 0 1% sodium azide. The mixture was centrifuged (5 min, 120 g) and the pellet washed several times by means of centrifugation and resuspension. Immunization of rats with heterologous GBM Bovine GBM was sonicated for 5 min at a concentration of 24 mg (dry weight) per ml of isotonic saline. The homogenized suspension was added to CFA containing 8 mg Mycobacterium butyricum per ml to form a 1: I water-in-oil emulsion. Rats were immunized with 0-4 ml (4-8 mg GBM) of this emulsion; 0 05 ml was injected into each front foot pad, and 0 3 ml intracutaneously at three sites in the neck. Two weeks later, a total of 0-3 ml (3-6 mg GBM) was injected intracutaneously at three different sites in the neck, and 12 mg GBM in 0-1 ml 0-9°% NaCI was injected intraperitoneally. Each experimental group consisted of ten animals. Control groups received saline and CFA containing 8 mg M. butyricum per ml.

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Demonstration of anti-GBM antibodies Four weeks after primary immunization, wedge biopsies were taken from the kidneys of immunized and control rats; the animals were killed 8 weeks later. At 4 and 12 weeks, renal tissue was examined by direct immunofluorescence for glomerular deposits of rat IgG and rat C3 with a Leitz-Orthoplan microscope equipped with an epi-illuminator (Kijlstra, Van der Lelij, Knutson, Fleuren & van Es, 1978). Immunoglobulin (sub)classes were determined by indirect immunofluorescence before and after elution procedures. The anti-rat immunoglobulin (sub)class specific antisera (anti-a, anti-e, anti-y, anti-yI, anti-y2a, anti-y2J) were provided by Dr H. Bazin of the Catholic University of Louvain, Brussels, Belgium. Rabbit anti-rat C3 (Ra a-Rt C3) was prepared by immunizing rabbits with highly purified rat C3 (Daha, Stuffers-Heiman, Kijlstra & van Es, 1978). The IgG fraction was isolated from the sera by ammonium sulphate precipitation and ion exchange chromatography (Reif, 1969) and conjugated with FITC according to The & Feltkamp (1970). The Ra IgG a-Rt C3 was monospecific for rat C3 as determined by immunoelectrophoresis and Ouchterlony analysis with whole rat serum and highly purified rat C3. Furthermore, with FITC-Ra IgG a-Rt C3 a pronounced linear deposition of rat C3 could be detected along the GBM of rats in the heterologous phase of nephrotoxic neph-

rntis. Immunoglobulins, deposited in the kidneys of immunized animals, were eluted from the isolated glomeruli by the method of Lemer, Glassock & Dixon (1967). All eluates were tested on sections of normal kidney by indirect immunofluorescence. All sections used for the immunofluorescence studies were coded, and scored individually by two persons who were not familiar with the code.

Quantification of glomerular-bound antibodies in immunized BN rats The amount of host IgG fixed to the GBM of immunized BN rats was calculated by comparing the maximum amount ofheterologous (rabbit) anti-GBM antibodies that could be fixed to the GBM of nonimmunized and immunized animals. These antibodies were raised by intraperitoneal immunization of rabbits with rat GBM (Daha, Blok, de Graeff & van Es, 1977). The rabbit sera were inactivated at 560 for 30 min. The IgG fraction was isolated by ammonium sulphate precipitation in a final concentration of 40% and DEAE-cellulose chromatography (Reif, 1969)

and labelled with 125I according to McConahey & Dixon (1966). Rabbit IgG from non-immunized rabbits was labelled with 1311 and given simultaneously as a control for non-specific binding of rabbit IgG to the rat tissues. The amount of kidney-fixing antibody (KFAb) in the rabbit IgG preparation was calculated according to McPhaul & Dixon (1970). The maximum amount of KFAb that could fix to the GBM of rats was determined by administration of increasing amounts of KFAb. Quantitative excretion ofprotein Urine was collected for 24 h every 2 weeks from 4 to 12 weeks after primary immunization. The protein content was determined by the biuret method with human serum as a standard. An excretion of more than 10 mg/24 h was considered abnormal.

RESULTS Table 1 presents the results of immunofluorescence studies of kidney sections taken from various strains 4 weeks after primary immunization with GBM and CFA. A pronounced linear staining for rat IgG along the GBM was observed in BN rats (Fig. 1). A weak linear deposition of IgG was noted in PVG/c rats, 80% of the F344 and 30% of the DA rats. No deposition of IgG was seen in the glomeruli of the other strains (Lewis, L.BN, AS2, AVN and BD V). After 12 weeks (Table 2) a marked linear deposition of rat IgG was still present along the GBM of BN rats. A weak and irregular deposition of host IgG along the GBM was seen in all PVG/c, AS2, DA, AVN, BD V and F334 rats (Fig. 2). The glomeruli of Lewis and L.BN rats showed no staining. No fixation of host IGg was found along the tubular basement membrane of any of the strains. The antibodies deposited in a linear fashion along the GBM of BN kidneys belonged to the IgGl and IgG2a immunoglobulin subclasses, as determined by indirect immunofluorescence. There was no positive immunofluorescence for IgA, IgG2c, IgE or IgM. Rat C3 could not be detected in the glomeruli of any of the strains nor did any of the animals develop proteinuria in the first 3 months after immunization. Control animals did not show deposition of either rat IgG or rat C3 in the kidneys. Studies with kidney eluates The specificity of the IgG deposited along the GBM of

Marianne Stuffers-Heiman, E. Gunther & L. A. van Es

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Table 1. Deposition of host IgG along the GBM 4 weeks after primary immunization of 9 different inbred strains of rats with bovine GBM and CFA, as detected by immunofluorescence

Deposition of rat IgG along GBM at 4 weeks Strain

Number of positive rats*

BN L.BN Lewis PVG/c AS2 AVN BD V DA F344

10/10 0/10 0/10 10/10 0/10 0/10 0/10 3/10 8/10 *

Intensity

Pattern

++

linear

+

linear

+ +

linear linear

Number positive/number tested

immunized rats was further investigated by acid elution of immunoglobulin from glomeruli isolated from kidneys of immunized animals. The BN kidney eluate showed pronounced fixation not only to the GBM and TBM of sections of normal BN kidneys in vitro (Table 3, Fig. 3) but also to the GBM and TBM of normal Lewis, L.BN, PVG/c, AS2, AVN, DA, BD V and F344 kidneys, thus demonstrating that the antigen against which the autologous antibodies were directed was present in these strains. After absorption of the BN eluate with purified BN GBM, fixation to GBM and TBM was no longer seen. A weak linear fixation to the GBM of normal kidney sections was observed for PVG/c, AVN, DA and BD V eluates. Lewis, L.BN, F344 and AS2 eluates did not fix to either GBM or TBM of normal kidneys. The auto-antibodies eluted from the kidneys of BN rats belonged to the same subclasses as the immunoglobulins deposited in vivo along the GBM of immunized animals, as determined by indirect immunofluorescence.

Figure 1. Cryostat section of kidney from a BN rat obtained 12 weeks after primary immunization. Staining with fluoresceinconjugated rabbit antibody to rat IgG. Note the continuous linear deposition of IgG along the basement membrane of the glomerular capillary walls ( x 400).

Anti-GBM antibodies in inbred rats Table 2. Deposition of host IgG along the GBM 12 weeks after primary immunization of 9 different inbred strains of rats with bovine GBM and CFA, as detected by immunofluorescence

Deposition of rat IgG along GBM at 12 weeks Strain

Number of positive rats*

BN L.BN Lewis PVG/c AS2 AVN BD V DA F344

10/10 10/10 10/10 10/10 10/10 10/10 10/10 9/10 10/10

Intensity ++ -

+ + + + + +

Pattern linear

irregular irregular irregular irregular irregular irregular

* Number positive/number tested

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Quantification of glomerular-bound antibodies in immunized BN rats The amount of host antibody fixed to GBM was calculated by measuring the maximum quantity of heterologous rabbit anti-rat GBM antibodies that fixed to the GBM of immunized BN rats compared with normal BN rats. For this purpose increasing amounts of 1251-labelled rabbit IgG anti-rat GBM mixed with unlabelled rabbit IgG anti-rat GBM, which was shown by immunofluorescence to fix only to GBM, were given intravenously to normal rats. Equivalent amounts of '3ll-labelled IgG from non-immunized rabbits were given simultaneously. The rats were killed I h after the injection and perfused with 150 ml isotonic saline. Kidneys were removed and weighed, and the radioactivity was measured. The radioactive counts were corrected for background activity and non-specific binding of rabbit IgG to rat tissues. Fig. 4 shows the relationship between the amount of KFAb

Figure 2. Cryostat section of kidney from a BD V rat obtained 12 weeks after primary immunization. Staining with fluorescein-conjugated rabbit antibody to rat IgG. Note the irregular pattern of staining on the basement membrane of glomerular capillary walls ( x 400).

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Marianne Stuffers-Heiman, E. Gunther & L. A. van Es

Table 3. Fixation of acid eluates of kidneys from immunized BN rats to sections of normal kidneys

Fixation to normal section of: Acid eluate BN L.BN Lewis PVG/c AS2 AVN BD V DA F344

BN kidney

Isologous kidney

++

++

-

-

-

-

+

+

-

-

-

+ + +

-

301 + 27 pg. Five BN rats, immunized twice with BoGBM were injected i.v. with 800 pg rabbit KFAb 4 weeks after primary immunization. Instead of 300 pg, only 253 + 8 pg rabbit KFAb fixed to the kidneys of the immunized BN rats. On the basis of this experiment, we conclude that the amount of rabbit KFAb fixed to the GBM of immunized BN rats was 48 + 14 pg less than that for normal BN rats. Presumably this was due to the presence of an equivalent quantity of autoantibodies on the GBM.

DISCUSSION This study shows that the tolerance to GBM antigens can be terminated in BN rats after immunization with bovine GBM and CFA. Linear deposition of host

~~~~I----- I ._. . * A Figure 3. Cryostat section of kidney from a normal BN rat, incubated with acid eluate from kidneys of immunized BN rats. Staining with fluorescein-conjugated rabbit antibody to rat IgG. Glomerular and tubular basement membranes are stained in a linear pattern ( x 200).

injected and the amount of KFAb fixed to the kidney. When more than 500 pig rabbit KFAb was injected, the antigenic sites in the kidneys of normal BN rats were saturated with immunoglobulin. The maximum amount of KFAb fixed to the GBM of normal rats was

immunoglobulin was found exclusively along the GBM. The anti-GBM activity of the immunoglobulin could be demonstrated after acid elution by the fixation in vitro to the GBM and TBM of normal BN kidney sections.

Anti-GBM antibodies in inbred rats 400 r 300

u

'

x____ __________ 0

..-1

200

1001

200

400 500 Injected F

Induction of autoimmunity to antigens of the glomerular basement membrane in inbred Brown-Norway rats.

Immunology 1979 36 759 Induction of autoimmunity to antigens of the glomerular basement membrane in inbred Brown-Norway rats MARIANNE STUFFERS-HEIMA...
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