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J Am Soc Nephrol 12:61-70, 2001
© 2001 American Society of Nephrology

Oral Administration of Glomerular Basement Membrane Prevents the Development of Experimental Autoimmune Glomerulonephritis in the WKY Rat

JOHN REYNOLDS and CHARLES D. PUSEY

Renal Section, Division of Medicine, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom.

Correspondence to Dr. John Reynolds, Renal Section, Division of Medicine, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London, W12 ONN, United Kingdom. Phone: 44 20 8383 3152; Fax: 44 20 8383 2062; E-mail: john.reynolds{at}ic.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Abstract. Experimental autoimmune glomerulonephritis (EAG), an animal model of Goodpasture's disease, can be induced in Wistar Kyoto (WKY) rats by a single injection of collagenase-solubilized rat glomerular basement membrane (GBM) in adjuvant. EAG is characterized by circulating and deposited anti-GBM antibodies, accompanied by focal necrotizing glomerulonephritis with crescent formation. The inhibitory effect of orally administered antigens has been reported in various animal models of autoimmunity but not in EAG in the rat. The effects of feeding rat GBM by gavage, at total doses of 0.5, 2.5, or 5 mg, before immunization were examined. A dose-dependent effect was observed on the development of EAG. A dose of 0.5 mg of GBM had no effect on disease, 2.5 mg resulted in a moderate reduction in the severity of nephritis but no change in anti-GBM antibody production, and 5 mg resulted in a marked reduction in circulating and deposited anti-GBM antibodies, albuminuria, deposits of fibrin in the glomeruli, severity of glomerular abnormalities, and numbers of infiltrating T cells and macrophages. Animals that were fed 5 mg of GBM showed a significant reduction in IgG2a but not IgG1, anti-GBM antibody levels, suggesting downregulation of Th1 responses. There was also a dose-dependent reduction in the proliferative responses of splenic T cells from treated animals to GBM antigen in vitro. These results clearly demonstrate that mucosal tolerance can be induced by oral administration of GBM antigen and that this approach is effective in preventing EAG.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The inhibitory effect of orally administered autoantigens has been widely reported in studies using known autoantigens in experimental models of autoimmune disease in rodents, including encephalomyelitis, uveitis, collagen-induced arthritis, thyroiditis, diabetes, and myasthenia gravis (1,2,3,4,5,6). Recently, similar findings have been reported in glomerulonephritis in SJL/J mice (7) and in tubulointerstitial nephritis in Brown Norway (BN) rats (8). Nasal administration of lower doses of antigen or of immunodominant synthetic peptides has also been shown to induce mucosal tolerance in various models of autoimmunity, such as encephalomyelitis (9,10), collagen-induced arthritis (11,12), and myasthenia gravis (13). The last is of particular relevance to our work, because autoantibodies are clearly pathogenic in myasthenia gravis, as in Goodpasture's disease. In addition, coupling the antigen to the cholera toxin B subunit (CTB) has been shown to enhance development of mucosal tolerance (14), possibly because of more efficient induction of regulatory cells as a result of the binding properties of CTB. There is still controversy as to the mechanisms involved in the induction of mucosal tolerance. There is evidence that high doses of antigen lead to clonal deletion or anergy, whereas lower doses favor the development of suppressor mechanisms (1,2,3,4,5,6). In some models, this suppression may be due to the stimulation of Th2 cells, which downregulate the inflammatory response involving Th1 cells (15). The balance between Th1 and Th2 cells may be controlled by a regulatory subset of T cells, which has been shown to suppress immune responses in vitro and in vivo by release of the immunosuppressive cytokine transforming growth factor-ß (TGF-ß) (16). This subset of T cells has been termed Th3 by some authors (17). There is also evidence that T cells that bear the {gamma}{delta} T-cell receptor are involved (18).

Experimental autoimmune glomerulonephritis (EAG) is an experimental model of Goodpasture's disease that can be induced in genetically susceptible strains of rats by immunization with heterologous or homologous preparations of glomerular basement membrane (GBM) in adjuvant (19,20,21,22,23,24). The development of nephritis is associated with both cell-mediated and humoral immunity to the noncollagenous (NC1) domain of the {alpha}3 chain of type IV collagen [{alpha}3(IV)NC1] (25,26,27,28,29), which is also the autoantigen in Goodpasture's disease (30,31). In the model used in this study, Wistar Kyoto (WKY) rats that were given a single injection of collagenase-solubilized rat GBM in Freund's complete adjuvant (FCA) develop sustained anti-GBM antibody synthesis, linear deposition of IgG on the GBM, deposits of fibrin in the glomeruli, albuminuria, focal necrotizing glomerulonephritis with crescent formation, and variable lung hemorrhage (24). Anti-GBM antibodies from rats and mice with EAG have been shown to be pathogenic in passive transfer experiments (7,32). However, there is also increasing evidence for the role of T lymphocytes in the pathogenesis of EAG (33,34,35,36,37,38,39,40,41). In the present model, EAG is characterized by an influx of T cells that precedes macrophage infiltration and glomerular injury (33). T cells from animals with EAG proliferate in response to cationic monomer and dimer NC1 domains of rat GBM (34). Transfer of Th lymphocytes purified from the spleens of rats with EAG can prime naive recipients for the disease (35), and in vitro stimulation of these cells by GBM, before transfer, confers the ability to induce anti-GBM antibody production in recipients (Reynolds et al., unpublished observation). We have previously shown that cyclosporin A (36) and anti-CD4 monoclonal antibody (mAb) (37) can prevent EAG in the BN rat, and have recently found that anti-CD8 mAb (38) and blockade of the CD28-B7 co-stimulatory pathway (39,40) are effective in EAG in the WKY rat. It has also been demonstrated that antibodies to intercellular adhesion molecule-1 and lymphocyte function-associated antigen-1 are effective in both the prevention and the treatment of EAG in the WKY rat (41).

The present model of EAG is highly appropriate for studying mechanisms of oral tolerance because of the close similarities to human anti-GBM disease in both the immune response and the glomerular pathology. We therefore examined the effects of feeding different doses of collagenase-solubilized rat GBM on the development of EAG. The results obtained demonstrate for the first time that mucosal tolerance can be induced in EAG in the WKY rat in a dose-dependent manner by oral administration of GBM antigen.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental Animals
Male WKY rats, ages 8 to 10 wk and weighing 120 to 150 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. All experimental procedures were conducted in accordance with the UK Animals (Scientific Procedures) Act.

Preparation of GBM Antigen
Collagenase-solubilized GBM was prepared from Sprague Dawley (SD) rat kidneys, as described previously (21,24). Briefly, the kidneys were decapsulated, the medulla was partly removed, and the cortex was forced through sieves to isolate the glomeruli. After examination by light microscopy, the glomeruli were disrupted ultrasonically and the resulting material was lyophilized and digested with purified type I collagenase (Sigma-Aldrich Company Ltd., Poole, UK) for 1 h at 37°C.

Induction of EAG
EAG was induced in WKY rats by a single intramuscular injection of collagenase-solubilized rat GBM in an equal volume of FCA, at a dose of 5 mg/kg body wt (24). Serial blood samples were taken by tail artery puncture under light anesthesia (Isoflurane), and 24-h urine specimens were obtained by placing animals in metabolic cages.

Experimental Protocol
Collagenase-solubilized rat GBM was administered orally to groups of WKY rats (n = 5 to 6) by gavage, at total doses of 0.5, 2.5, or 5 mg, before immunization. In addition, positive control groups, immunized with GBM in FCA, were fed bovine serum albumin (5 mg), and negative control groups, injected with FCA alone, were fed rat GBM (5 mg). All groups of animals were fed five times at intervals of 2 to 3 d with the last feeding 2 d before immunization.

Assessment of EAG
Enzyme-Linked Immunosorbent Assay. Circulating anti-GBM antibody concentrations were measured in sera from animals with EAG by a solid-phase enzyme-linked immunosorbent assay (ELISA), as described previously (24). Collagenase-digested rat GBM was coated onto microtiter plates (Life Technologies, Paisley, UK) by overnight incubation at 4°C, and test or control sera were applied for 1 h at 37°C. Bound anti-GBM antibody was detected by horseradish peroxidase conjugated sheep anti-rat IgG (Sigma-Aldrich Company Ltd.) and developed using the substrate orthophenylenediamine dihydrochloride (Sigma-Aldrich Company Ltd.). The absorbances for each well were read at 492 nm using an Anthos Multiskan ELISA plate reader (Lab Tech International, Uckfield, UK), and the results initially were calculated as mean optical density for each triplicate sample. To compare different experimental groups, results were finally expressed as a percentage of the binding obtained with a positive reference serum (24).

Subclass ELISA. Circulating levels of IgG1 and IgG2a anti-GBM antibodies were measured in sera from animals with EAG at week 4 after immunization, by an indirect ELISA similar to that previously described (39,42). Briefly, collagenase-digested rat GBM was coated onto microtiter plates and test or control sera were applied, as described above. The isotypes of circulating anti-GBM antibodies were detected by mouse mAb specific for rat IgG1 and IgG2a (Serotec Ltd., Oxford, UK), followed by goat anti-mouse IgG (Serotec Ltd.). Levels of bound IgG1 and IgG2a anti-GBM antibodies were detected by alkaline phosphatase conjugated rabbit anti-goat IgG (Sigma-Aldrich Company Ltd.) and developed using the substrate p-nitrophenyl phosphate (Sigma-Aldrich Company Ltd.). The absorbances for each well were read at 405 nm, and the results were expressed as mean optical density for each triplicate sample.

Rocket Immunoelectrophoresis. Urinary albumin concentrations were measured in 24-h collections from animals with EAG by rocket immunoelectrophoresis (Amersham Pharmacia Biotech, St Albans, UK) as described previously (43). Briefly, urine samples from experimental animals were subjected to immunoelectrophoresis at 60 v in an electrophoresis tank containing Barbitone buffer (BDH Laboratory Supplies, Poole, UK), pH 9.5, for 6 h, using a 1% agarose gel (BDH Laboratory Supplies) 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 milligrams per 24 h.

Creatinine Clearance. Creatinine levels in serum and urine from animals at week 4 were measured by spectrometry using standard techniques. Creatinine clearance was then calculated for each animal and expressed as milliliters per minute.

Direct Immunofluorescence. Deposits of IgG and fibrin within the glomeruli were detected by direct immunofluorescence, as described previously (44). Tissue was embedded in OCT II embedding medium (Miles Inc., Elkhart, IN) on cork discs, snap-frozen in isopentane (BDH Laboratory Supplies), precooled in liquid nitrogen, and stored at -70°C. Cryostat sections were cut 4 µm thick and were incubated with FITC-labeled rabbit anti-rat IgG (Serotec Ltd.) or goat anti-rat fibrin (Nordic Immunology, Tilburg, The Netherlands). The extent and the intensity of immunostaining were assessed and graded from 0 to 3+ by a blinded observer.

Light Microscopy. Kidney tissue 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 hematoxylin and eosin and periodic acid-Schiff. Fifty glomeruli per section were assessed and graded by a blinded observer as normal, abnormal, or severe (>50% of the glomerulus affected by necrosis/crescent formation) and expressed as a percentage of glomeruli examined (24).

Immunohistology. Formalin-fixed kidney sections were stained with mAb ED1 (for macrophages) and OX8 (for CD8+ lymphocytes) (Serotec Ltd.) by standard indirect immunoperoxidase staining techniques. The cellular infiltrate was assessed by a blinded observer by counting the number of positively stained cells per 50 consecutive glomeruli in cross section (45).

T-Cell Proliferation Assay
Spleens were dissociated into a single cell suspension (34) and cultured with GBM antigen as described previously (22,35,46). Spleen cells from positive and negative controls or animals fed with GBM were suspended in RPMI 1640 medium (supplemented with 10% fetal calf serum, 100 µg/ml of penicillin, 100 µg/ml of streptomycin, and 2 mM glutamine) and cultured in 96-well plates (Life Technologies) at a concentration of 2 x 105 cells/well. Collagenase-solubilized rat GBM (10 µg/ml) was added, and spleen cells were incubated at 37°C in a humidified environment with 5% CO2 for 7 d. As a control, spleen cells were also cultured with the mitogen Concanavalin A (2 µg/ml; Sigma-Aldrich Company Ltd.) for 3 d. Tritiated thymidine (Amersham International, Amersham, UK) was added at 1 µCi/well 16 h before harvesting, and thymidine incorporation was measured using an automated ß counter (Amersham Pharmacia Biotech, St. Albans, UK). Results were expressed as a stimulation index, which was calculated by dividing the cpm in wells cultured with antigen by cpm in wells with no antigen.

Statistical Analyses
Differences between data were determined by the Mann-Whitney U test. ANOVA was used to confirm differences between multiple data.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Circulating Anti-GBM Antibody Concentrations
All positive control rats immunized with rat GBM in FCA and fed bovine serum albumin orally at a total dose of 5 mg produced detectable circulating anti-GBM antibody levels by week 2, which increased to high levels by week 3 and peaked at week 4. Oral administration of GBM had a dose-dependent effect on circulating antibody levels. A total dose of 0.5 mg had no effect, whereas 2.5 mg led to a slight, though not significant, reduction and 5 mg resulted in a marked reduction in antibody concentration. Negative control animals injected with FCA alone and fed GBM at a total dose of 5 mg did not develop circulating antibody. Results are shown in Figure 1.



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  		Figure 1. Effect of oral administration of glomerular basement membrane (GBM) on circulating anti-GBM antibody concentrations in groups of WKY rats (n = 5 to 6) with experimental autoimmune glomerulonephritis (EAG). Results shown represent the mean of each group: positive control ([UNK]), GBM 0.5 mg ({blacksquare}), GBM 2.5 mg ({square}), or GBM 5 mg ({blacktriangleup}), and negative control ({circ}). *, P < 0.01 (positive control versus GBM 5 mg).

 

Circulating Anti-GBM Antibody Isotypes
All positive control rats immunized with rat GBM in FCA produced detectable levels of both IgG1 and IgG2a anti-GBM antibodies by week 4. Animals that were given GBM orally at a dose of 0.5 mg showed no reduction in either IgG1 or IgG2a antibodies, whereas 2.5 mg showed a slight reduction in IgG2a antibodies and 5 mg led to a marked reduction in the levels of IgG2a but not IgG1 antibodies. Negative control animals that were given FCA alone did not develop detectable levels of IgG1 or IgG2a antibodies. The ratio of IgG1/IgG2a anti-GBM antibodies was significantly increased in the 5 mg group compared with positive controls. Results are shown in Figure 2.



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  		Figure 2. Effect of oral administration of GBM on circulating levels of IgG1 (A), IgG2a (B), and ratio of IgG1/IgG2a anti-GBM antibodies (C) in groups of WKY rats (n = 5 to 6) with EAG. Results shown represent the mean ± SD of each group at week 4 after immunization. *, P < 0.01 (positive control versus GBM 5 mg).

 

Direct Immunofluorescence for IgG
Direct immunofluorescence for IgG on kidney tissue at 4 wk revealed that positive control animals that were given rat GBM in FCA showed strong linear deposits of IgG along the GBM and, to lesser extent, the TBM. Animals that were given GBM orally at 0.5 mg showed no reduction in IgG deposits, whereas those that were given 2.5 mg showed a moderate reduction and those that were given 5 mg showed a marked reduction in intensity of staining. Negative control animals that were given FCA alone showed no IgG deposition. Results are summarized in Table 1 and illustrated in Figure 3.


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  		Table 1. Effects of oral administration of GBM to WKY rats with EAG on deposition of IgG on the GBM and deposition of fibrin in the glomeruli. Results are expressed as intensity of fluorescence at week 4 in individual animals
 


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  		Figure 3. Direct immunofluorescence of kidney tissue at week 4 in WKY rats with EAG showing strong linear deposition of IgG along the GBM in a positive control animal (A), marked reduction in the deposition of IgG in an animal given GBM orally at 5 mg (B), large deposits of fibrin within the glomeruli of a positive control animal (C), and negative findings for fibrin in an animal given GBM orally at 5 mg (D). Magnification, x300.

 

Albuminuria
All positive control rats that were immunized with rat GBM in FCA produced detectable levels of albuminuria by week 2, which increased further by week 3 and peaked at week 4. Animals that were given GBM orally at 0.5 mg showed no reduction in albuminuria, whereas those that were given 2.5 mg showed a moderate reduction and those that were given 5 mg showed a marked reduction in levels at all time points. Negative control animals that were given FCA alone did not develop albuminuria. Results are shown in Figure 4.



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  		Figure 4. Effect of oral administration of GBM on albuminuria in groups of WKY rats (n = 5 to 6) with EAG. Results shown represent the mean of each group: positive control ([UNK]), GBM 0.5 mg ({blacksquare}), GBM 2.5 mg ({square}), or GBM 5 mg ({blacktriangleup}), and negative control ({circ}). *, P < 0.01 (positive control versus GBM 2.5 mg); **, P < 0.001 (positive control versus GBM 5 mg).

 

Creatinine Clearance
Negative control animals that were given FCA alone showed a normal creatinine clearance in the range of 1.2 to 1.5 ml/min. Positive control rats that were immunized with rat GBM in FCA showed a marked reduction in creatine clearance. Animals that were given GBM orally at 0.5 mg or 2.5 mg showed a moderate reduction in creatinine clearance, whereas those that were given 5 mg had clearances similar to negative control animals. Results are shown in Figure 5.



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  		Figure 5. Effect of oral administration of GBM on creatinine clearance in groups of WKY rats (n = 5 to 6) with EAG. Results shown represent the mean ± SD of each group at week 4 after immunization. *, P < 0.01 (positive control versus GBM 5 mg).

 

Direct Immunofluorescence for Fibrin
Direct immunofluorescence for fibrin on kidney tissue at 4 wk revealed that positive control animals that were given rat GBM in FCA showed large areas of fibrin deposits within the glomeruli. Animals that were given GBM orally at 0.5 mg showed no reduction in fibrin deposits, whereas those that were given 2.5 mg showed a moderate reduction and those that were given 5 mg showed a marked reduction in extent and intensity of staining. Negative control animals that were given FCA alone showed no antibody binding. Results are summarized in Table 1 and illustrated in Figure 3.

Light Microscopy
Light microscopy of kidney tissue at 4 wk revealed that positive control animals that were given rat GBM in FCA showed diffuse necrotizing glomerulonephritis affecting 100% of glomeruli, with severe segmental necrosis/crescent formation (>50% of the glomerulus) affecting 65% of glomeruli. Animals that were given GBM orally at a total dose of 0.5 mg also showed glomerulonephritis in 100% of glomeruli, with severe changes in 50% of glomeruli, whereas those that were given 2.5 mg showed glomerulonephritis in 75% of glomeruli, with severe changes in 30% of glomeruli, and those that were given 5 mg showed glomerulonephritis only in 30% of glomeruli, with no histologic evidence of segmental necrosis/crescent formation. Negative control animals that were given FCA alone showed normal renal histology. Results are shown in Figure 6 and illustrated in Figure 8.



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  		Figure 6. Effect of oral administration of GBM on renal histology at week 4 in groups of WKY rats (n = 5 to 6) with EAG. Results shown represent the severity of glomerular abnormalities, which were graded as normal, abnormal, or severe (>50% of the glomerulus affected by necrosis/crescent formation). *, P < 0.01 (positive control versus GBM 2.5 mg; **, P < 0.001 (positive control versus GBM 5 mg).

 


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  		Figure 8. Light microscopy of kidney tissue at week 4 in WKY rats with EAG showing marked segmental necrosis of the glomerular tuft with crescent formation in a positive control animal (A; hematoxylin and eosin), mild segmental proliferation in an animal given GBM orally at 5 mg (B; hematoxylin and eosin), large numbers of macrophages infiltrating the glomeruli and interstitium in a positive control animal (C; immunoperoxidase), and reduced numbers of macrophages in an animal given GBM orally at 5 mg (D; immunoperoxidase). Magnification, x300.

 

Immunohistology
Immunohistology of kidney tissue at 4 wk showed that positive control animals that were given rat GBM in FCA had glomerular infiltration with CD8+ T lymphocytes and macrophages. Animals that were given GBM orally at 0.5 mg showed no reduction in the numbers of T cells and macrophages infiltrating the glomeruli, whereas those that were given 2.5 mg showed a moderate reduction and those that were given 5 mg showed a marked reduction. Negative control animals that were given FCA alone showed no cellular infiltrate. Results are shown in Figure 7 and illustrated in Figure 8.



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  		Figure 7. Effect of oral administration of GBM on the numbers of CD8+ T cells (A) and macrophages (B) detected by immunoperoxidase staining of kidney tissue in groups of WKY rats (n = 5 to 6) with EAG. Results shown represent the mean ± SD of each group at week 4 after immunization. *, P < 0.01 (positive control versus GBM 5 mg).

 

T-Cell Proliferation
Spleen cells from positive control animals that were given rat GBM in FCA showed a significant T-cell proliferative response (stimulation index = 9.5) when cultured with GBM in vitro for 7 d at a concentration of 10 µg/ml. Spleen cells from animals that were given GBM orally at 0.5 or 2.5 mg showed a proliferative response similar to that of positive control animals, whereas splenocytes from animals that were fed 5 mg of GBM showed a marked reduction in T-cell proliferation, which was similar to that of negative control animals. Results are shown in Figure 9.



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  		Figure 9. Effect of oral administration of GBM on in vitro proliferation of splenic T cells pooled from different groups of WKY rats with EAG. Results shown represent the mean ± SD of each group at week 4 after immunization. *, P < 0.01 (positive control versus GBM 5 mg).

 


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The inhibitory effect of orally (1,2,3,4,5,6,7,8) and nasally (9,10,11,12,13) administered antigens in various animal models of autoimmunity has been widely reported, but there is still much controversy as to the mechanisms involved. Furthermore, there are no reported studies in EAG in the rat. To investigate this, we examined the effects of feeding different doses of collagenase-solubilized rat GBM on the development of EAG in the WKY rat. This resulted in a dose-dependent effect on the development of EAG. A total dose of 0.5 mg of GBM had no effect on disease, 2.5 mg resulted in a moderate reduction in the severity of nephritis but no significant change in anti-GBM antibody production, and 5 mg resulted in a marked reduction in circulating and deposited anti-GBM antibodies and in the severity of glomerular abnormalities. In addition, animals that were fed 5 mg of GBM showed a significant reduction in IgG2a (but not IgG1) anti-GBM antibody levels and a reduced T-cell proliferative response to GBM antigen in vitro. Our results clearly demonstrate that oral administration of GBM antigen at a total dose of 5 mg before immunization reduces the autoimmune response and completely prevents the development of crescents in this model of EAG.

Previous studies in other animal models of nephritis, although limited, have demonstrated that oral administration of renal antigens can be successful in prevention or amelioration of disease (7,8). In a mouse model of anti-GBM disease, induced in SJL mice by immunization with bovine {alpha}3(IV)NC1, oral administration of {alpha}3(IV)NC1 dimers, before immunization, resulted in a substantial decrease in numbers of crescents and prevention of interstitial inflammation (7). In addition, a decrease in serum titers of IgG2a anti-GBM antibodies and a reduction of IL-12 expression in the kidney was observed in mice that were fed {alpha}3(IV)NC1. In autoimmune interstitial nephritis, induced in BN rats by immunization with renal tubular antigen, oral feeding of renal tubular antigen reduced the severity of interstitial nephritis and renal function, but this was not found to be associated with the suppression of delayed-type hypersensitivity responses or antigen-specific IgG titers (8).

The primary mechanism by which mucosal tolerance to autoantigens is mediated is still unclear, but the determining factor seems to be the dose of the antigen fed. High-dose antigen seems to induce systemic anergy or deletion, whereas a regimen of multiple feedings with low-dose antigen favors the induction of regulatory T cells, whose suppressive activities are mediated through the production of anti-inflammatory cytokines such as interleukin-4 (IL-4), IL-10, and TGF-ß (1,2,3,4,5,6). TGF-ß—secreting cells seem to form a unique subset that has been termed Th3 cells (17). It is now widely believed that cytokines associated with the Th1 phenotype (interferon-{gamma}, tumor necrosis factor-{alpha}, and IL-12) promote inflammation in the target organ, whereas cytokines associated with Th2 (IL-4, IL-10) and Th3 (TGF-ß) responses have a role in suppressing disease (15,16).

The finding that Th1 and Th2 subsets of CD4+ T cells can cross regulate each other, via production of distinct cytokine profiles, supports the idea that oral tolerance induced by low doses of antigen reflects the downregulation of Th1 cells by Th2 cells. In the present study, we examined only the effect of low-dose tolerance, i.e., oral administration of up to 5 mg of GBM, as compared with an immunizing dose of 5 mg/kg. On the basis of the literature (7,47), we would need to give at least 10 times more GBM antigen to examine the effect of high-dose tolerance. The results from our study are consistent with the hypothesis of low-dose tolerance, because we found a decrease in IgG2a anti-GBM antibodies, suggesting downregulation of Th1 cells. In addition, we showed a marked decrease in the deposits of fibrin within the glomeruli and a decrease in the number of infiltrating glomerular T cells and macrophages, which are regarded as markers of a Th1-like response. Furthermore, we demonstrated a reduced proliferative response of splenic lymphocytes to the GBM antigen in vitro. This confirms that tolerance has been induced at the level of autoreactive T cells against the GBM antigen. Further work is required to determine whether this is due to production of anti-inflammatory cytokines such as IL-4, IL-10, and TGF-ß.

Another route to inducing mucosal tolerance in autoimmune disease is by nasal administration of autoantigens or immunodominant synthetic peptides. This approach has been shown to be very effective in both the prevention (9,10,11,12,13) and the treatment (47,48) of various experimental models of disease. Low doses of nasally administered peptides are effective in prevention of disease, whereas high doses seem to be necessary for treatment. In addition, conjugation of the antigen with CTB greatly increases the development of mucosal tolerance (14), allowing considerably lower doses of antigen to be used. If mucosal tolerance is to be used clinically for autoimmune renal disease, then we must first demonstrate it to be effective in treating established disease in an appropriate animal model. The use of nasally administered recombinant rat {alpha}3(IV)NC1 (29) or synthetic peptides based on its sequence is likely to be required to achieve high-dose tolerance. These studies are in progress in our laboratory.

In conclusion, we demonstrated for the first time that mucosal tolerance can be induced in EAG in the rat. In particular, oral administration of GBM antigen before immunization can completely prevent the development of crescentic nephritis. In addition, we have demonstrated a significant reduction in IgG2a anti-GBM antibody levels and a reduced T-cell proliferative response to GBM antigen in vitro. The most likely explanation for these findings is that oral administration of GBM antigen has resulted in the induction of regulatory T cells whose suppressive activities are mediated through the production of anti-inflammatory cytokines that suppress the Th1-like response in EAG. However, further work, including analysis of cytokine profiles from autoreactive T cells and analysis of glomerular expression of selected cytokines in EAG, is clearly required. The results from this study may have implications for the development of specific immunotherapy for human glomerulonephritis.


   Acknowledgments
 
This work was presented in part at the 29th Annual meeting of the American Society of Nephrology 1996 (Reynolds J, Newman VO, Pusey CD: Oral administration of GBM to WKY rats prevents the development of EAG. J Am Soc Nephrol 7: 1717, 1996). We are grateful to Vanessa Newman, Vicki Norgan, and Mark Duda for technical assistance and to Action Research, United Kingdom, for supporting this study.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication March 16, 2000. Accepted for publication June 10, 2000.




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