Suppression of Systemic Lupus Erythematosus Disease in Mice by Oral Administration of Kidney Extract

Article No. jaut.1999.0334, available online at http://www.idealibrary.com on

Journal of Autoimmunity (1999) 13, 405–414

Suppression of Systemic Lupus Erythematosus Disease in Mice by Oral Administration of Kidney Extract William Ofosu-Appiah, George Sfeir, Dana Viti and Elena Burashnikova Department of Immunology, Masonic Medical Research Laboratory, Utica, NY 13501, USA

Received 26 April 1999 Accepted 3 September 1999 Key words: SLE, oral tolerance, kidney extract, autoimmunity

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the increased production of antibodies reactive with a variety of self and non-self antigens. A number of immunomodulatory therapies have been investigated for the disease process. Intragastric administration of low dose kidney extract (KE) three times weekly for 5 weeks and then weekly until 6 months of age in SLE mice, showed decreased anti-dsDNA antibody levels, less kidney damage and significantly prolonged survival compared with control phosphate buffered saline (PBS)-fed mice. The KE-fed mice also exhibited reduced T cell proliferative response to KE in comparison with PBS-fed controls. Serum isotype distribution of the anti-dsDNA antibodies revealed a marked reduction of IgG1 and IgG3 responses in the KE-fed mice. While the renal inflammatory cell infiltration and expression of interleukin-4 (IL-4) and IL-10 were markedly suppressed, no local enhancement of transforming growth factor-â (TGF-â) was detected. Oral administration of low dose KE, however, upregulated expression of IL-2, IFN-ã and TNF-á in the kidneys and suppressed glomerulonephritis. These findings suggest that oral KE affects the disease process in SLE and raise the possibility that oral administration of KE or other potential autoantigens may provide a new approach for the treatment of SLE. © 1999 Academic Press

Introduction

side effects including generalized suppression of the immune system. The ideal treatment, if possible, should specifically eliminate the autoimmune T and B cell responses against autoantigens without disabling the rest of the immune system. One way to approach this goal would be to induce immunological tolerance to the putative autoantigen in SLE. Oral tolerance is a recognized procedure for inducing antigen-specific peripheral immune tolerance [8]. This procedure has been successfully utilized for some experimental autoimmune diseases, including EAE [9], collagen-induced arthritis [10] and diabetes [11]. We have been investigating antigendriven peripheral immune tolerance as a means to suppress autoimmune processes in SLE, using the oral route of antigen exposure. Oral tolerance as a means to treat SLE is attractive because of its virtual lack of toxicity and its inherent clinical applicability. The rationale for using kidney extract as the source of the autoantigen is that anti-DNA antibodies have been shown to react directly with renal antigen. We therefore reasoned that the kidneys must be the source of the autoantigens that drive the pathogenic autoimmune response in SLE. One of the primary goals for the immunotherapy of SLE is to develop nontoxic, antigen-specific therapies that can be administered early in the course of the disease. In the present study, we report successful suppression of anti-DNA

Systemic lupus erythematosus (SLE) is an autoimmune disease, characterized by the increased production of autoantibodies [1] and defective T cellmediated responses [2]. The deposition of anti-DNA antibodies in the kidney and other organs suggests the involvement of autoantibody-producing B cells in the pathogenesis of SLE [1]. Moreover, it is has been shown that SLE B cell autoantibody production is regulated by CD4 + T cells [3]. A variety of immunomodulatory treatments have been studied in NZB/W F1 mice. In general, treatments that affect T cell function or are immunosuppressive have been effective, such as in vivo treatment with anti-CD4 antibody [4] and anti-CD40 Ab [5]. Unfortunately, the use of anti-CD4 Ab in human autoimmune disorders might lead to prolonged depletion of this important T cell subset in some cases [6]. In autoimmune diseases such as rheumatoid arthritis, the efficacy of anti-CD4 Ab has been disappointing [7]. As to the treatment of SLE, it often involves the use of immunosuppressive drugs. Although effective, these drugs may produce adverse Correspondence to: William Ofosu-Appiah, Dept. of Immunology, Masonic Medical Research Laboratory, 2150 Bleecker Street, Utica, NY 13501–1787, USA. Fax: (315) 735 5648. E-mail: [email protected] 405 0896–8411/99/080405+10 $30.00/0

© 1999 Academic Press

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antibody production, reduced renal disease, and prolonged survival in SLE mice by oral administration of kidney extract

Materials and Methods Animals Female 8-week-old NZB×NZW F1 (NZB/W F1) mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA). Mice were housed three to a cage and maintained on Purina mice chow and water ad libitum. The animal room was maintained at 22°C with automatic 12-h light/dark cycles. The animals were acclimatized at least 1 week after receipt before use in any experiment. The studies reported here conformed to the principles for laboratory animal research outlined by the Animal Welfare Act and the Department of Health, Education and Welfare (National Institutes of Health) guidelines for the experimental use of animals.

Antigens and reagents Preparation of double stranded DNA (dsDNA) To prepare dsDNA, calf thymus DNA (CTDNA) was treated with purified S1 nuclease (Sigma Chemical Co. Ltd., St. Louis, MC, USA) as previously described [12]. Briefly, CTDNA dissolved in 33 mM sodium acetate, 50 mM NaCl, and 0.03 mM zinc chloride, pH 4.5, was incubated with S1 nuclease at 0.1 U/mg of DNA for 45 min at 37°C. The reaction was stopped by addition of NaEDTA to a final concentration of 10 mM. An equal volume of isoamyl alcohol/chloroform (24:1) was then added to remove protein. After centrifugation, the top layer was removed and the DNA was precipitated with cold 95% ethanol. The DNA was dissolved in SSC (0.1 M NaCl, 0.015 M Na citrate, pH 8). The concentration of DNA was determined by absorbance measurement at 260 and 280 nm. The 260/280 ratio for the DNA preparation was routinely >1.9. The preparation was filtered through 0.45 nitrocellulose filters to remove residual single stranded DNA (ssDNA) contamination. The dsDNA was resuspended to 500 ìg/ml in phosphate buffered saline (PBS) and frozen in small aliquots at −20°C. For assays, each tube was thawed once and unused dsDNA was discarded.

Preparation of kidney extract (KE) The KE was prepared according to the method of Freytag et al. [13] with some modifications. Briefly, cortices from 20 rat kidneys were separated from the medulla using razor blades. The tissue was homogenized in ice-cold 1 M NaCl, 0.05 M Tris-HCl, pH 7.4, containing 1 mM EDTA and protease inhibitors using Brinkman Polytron PT 3000. The homogenate was sonicated for 10 min at 140 watts at 4°C. The homogenate was passed through sieves of 80-ìm and 200-ìm

pore size to remove DNA and any tissue fragment contamination, which are resistant to sonication. The isolated glomeruli were collected by gently washing the 200-ìm sieve with ice-cold 1 M NaCl containing protease inhibitors, and separate harvests were pooled. Glomeruli proteins were prepared by homogenization of glomeruli diluted 1:1 in ice-cold 1 M NaCl containing protease inhibitors. The homogenate was then centrifuged for 60 min at 10,000×g in at 4°C. The pellet was washed five times with ice-cold 1 M NaCl+inhibitors by resuspending the pellet at 500×g for 10 min, discarding the supernatant. Finally, the pellet was washed three times with ice-cold distilled water and used as the kidney extract. After determining the protein concentration, the supernatant was aliquoted at 50 mg/ml and lyophilized. For use in oral tolerance studies, an aliquot of the lyophilized material was reconstituted with 1 ml sterile PBS.

Induction of oral tolerance to kidney extract NZB/W F1 mice (8-week-old) were fed low-dose (1 mg) of kidney extract (KE) in 0.5 ml phosphate buffered saline (PBS; GIBCO BRL, Grand Island, NY, USA) by gastric intubation with a 18-gauge stainless steel ball-tipped feeding needle (Thomas Scientific, Swedenboro, NJ, USA). Animals were fed three times weekly for 5 weeks and then weekly until 6 months of age. Control mice received PBS alone. In some experiments, mice tolerized with KE were immunized with egg ovalbumin (OVA), grade VI (Sigma). In addition, some mice were fed 1 mg of OVA in PBS following the same schedule as outlined above for KE to determine whether the tolerance to KE is specific. Blood was withdrawn at 1-month intervals for the determination of serum anti-dsDNA antibody levels.

Assessment of renal disease The development of renal disease was assessed by proteinuria, which was measured biweekly using dipsticks (Albustix, Miles, IN, USA) according to manufacturer’s instructions. A protein concentration of >100 mg/dl urine was defined as positive.

Serum IgG anti-dsDNA isotypes Serum IgG anti-dsDNA isotypes were measured using isotype-specific ELISA. Microtiter plates were coated with 10 ìg/ml dsDNA in 50 mM carbonate buffer, pH 9.6 overnight at 4°C. After 1 h blocking with 2% BSA, serial dilutions of sera starting from 1:100 to 1:25,600 were applied to the wells. Goat anti-mouse IgG1, IgG2a, IgG2b, and IgG3 Abs linked to alkaline phosphatase (Southern Biotechnology Associates, Birmingham, AL, USA) diluted at 1:10,000 were added to the wells. After 1 h incubation at 37°C, the plates were washed and developed using p-nitrophenyl phosphate (GIBCO), at a concentration of 1 mg/ml as substrate. The optical density (O.D.) at 405 nm was

Oral tolerance to kidney extract modulates SLE

read using an Bio-Rad automatic plate reader. The titer was calculated as the dilution giving an O.D. of 0.2 over background as estimated by a regression analysis of plots of the O.D. against the reciprocal value of the dilution.

KE-induced T cell proliferation Single cell suspensions were prepared from spleens using on Ficoll-Paque gradient (Pharmacia LKB Biotechnology, Piscataway, NJ, USA) as previously described [14]. The cells were suspended in complete RPMI 1640 medium (GIBCO) containing 10% fetal bovine serum (FBS), 20 mM HEPES, 2 mM L-glutamine, 100 (ìg/ml penicillin, 100 ìg/ml streptomycin and 5×10 −5 M 2-mercaptoethanol (2-ME). Splenic T cells (2×105/well) were cultured using specific (KE), SLE related antigen, dsDNA or non-specific (OVA) antigen at (10 ìg/ml). Some wells were coated with purified anti-CD3 Ab (Pharmingen, San Diego, CA, USA; 10 ìg/ml) as a positive control. After 72-h incubation, the cultures were labeled with 1 ìCi of 3H-TdR (Amersham Corp., Arlington Heights, IL, USA) for 18 h, harvested onto glass-fiber filters and the radioactivity measured in a Beckman liquid scintillation spectroscopy.

Histopathologic studies of kidney specimens Mice were killed only when it was obvious that they would not survive more than 24 h. Some of the mice died naturally and our animal care administrator found them before rigor mortis set in, and thus we were able to obtain the kidneys. For the long-term survivors of KE-fed mice, they were killed at the end of the experiment at 18 months despite being healthy. Kidneys were immediately removed from the mice and fixed using Histochoice, a non-cross-linking fixative (Amresco, Solon, OH, USA) for 24 h and embedded in paraffin. Several sections of 5 ìm thickness were cut and stained with hematoxylin and eosin according to standard procedure [15]. Renal histopathologic changes were graded by using a scale ranging from 0 to 4 as described previously by Berden et al. [16] 0: normal 1: lesions corresponding to minimal thickening of the mesangium: 2: lesions contained noticeable increases in both mesangial and glomerular cellularity; 3: lesions characterized by the inflammatory exudates and capsular adhesions and 4: lesions in which the glomerular architecture is destroyed in >70% of the glomeruli, and tubular cast formation is extensive.

RNA extraction and assessment of cytokine mRNA in renal tissue Total RNA was isolated from kidney and spleen from individual animals using TriPure (Boehringer Mannheim, Indianapolis, IN, USA). Five micrograms of total RNA were reverse transcribed to cDNA using Superscript II reverse-transcriptase kit (Life

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Technologies, Grand Island, NY, USA) by using digoxigenin-labeled dNTP mix, oligo (dT)12–18 and cloned Moloney murine leukemia virus reverse transcriptase (20 U) in a 20 ìl reaction at 42°C for 60 min. The PCR was conducted under hot-start conditions. In a total volume of 50 ìl PCR buffer, 2 ìl of cDNA (corresponding to 0.2 ìg of total RNA) were incubated with 2 U Taq DNA polymerase (Life Technologies), 0.44 ìg/ml of TaqStart antibodies (Clontech Labs., Palo Alto, CA, USA), 0.5 mM deoxynucleotide triphosphates, and 0.2 ìM sense and anti-sense primers. Each sample was incubated in a thermocycler (Oncor, Gaithersburg, MD, USA) using one cycle at 94°C for 5 min; this was followed by 25 cycles for cyclophilin or 35 cycles for cytokine genes. Each cycle consisted of 94°C for 45 s, 60°C for 45 s and 72°C for 60 s. The specific oligonucleotide primer pairs were purchased from Clontech Labs. Cyclophilin was used as a housekeeping gene to verify that similar amounts of RNA were amplified. For each gene product, the optimum number of cycles was determined experimentally, and was defined as that number of cycles that would achieve a detectable concentration that was well below saturating conditions. PCR products were analyzed by electrophoresis through 1.5% agarose gels containing 0.25 ìg/ml of ethidium bromide and visualized under UV light. In addition, the integrity of the cytokine primers was verified using cytokine gene containing plasmid. RNA isolated from Concanavalin A (Con-A)-stimulated spleen cells served as a positive control in all PCR runs. The PCR products were confirmed to be the expected mouse cytokine or cyclophilin sequences by Southern blot analysis using probes with sequences internal to those of the primers used for the PCR. An additional negative control containing water instead of RNA was used throughout. PCR products were not obtained when reverse transcriptase was omitted, indicating that there was no genomic DNA contamination. All PCR results shown are representative of at least five separate experiments, each performed using RNA obtained from organs and tissues from individual mice. All PCR products compared were produced in the same PCR to avoid interassay variations

Statistical analyses Differences between groups were evaluated by the Student’s t-test for unpaired or paired variables. All data are expressed as mean±standard error of mean (SEM), and P<0.05 was accepted as a statistically significant difference.

Results Effects of oral tolerance on T cell function in vitro The influence of a low dose KE on T cell responses was assessed after oral administration of KE in

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1.6 Anti-dsDNA antibodies (O.D. 405 nm)

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40 30 20 10

1.4 1.2 1.0 0.8 0.6

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Figure 1. Suppression of antigen-specific proliferative responses in spleen cells from orally tolerized mice. Oral tolerance was induced by repeated feedings of low dose (1 mg) KE or OVA. Non-tolerized control mice were fed PBS. Lymphocytes from spleens of KE-, OVA- or PBS-fed mice at 10 months of age were cultured in vitro in the presence or absence of KE, dsDNA or OVA at 10 ìg/ml. The results are displayed as the mean±SEM of triplicate wells. Proliferation in response to medium alone ranged from 560 to 1080 cpm in all groups. Three independent experiments produced comparable results. PBS-fed T cells tested with KE (– –); KE-fed T cells tested with KE (– –); KE-fed, OVA primed T cells tested with OVA (– –); KE-fed cells tested with OVA (– –). Table 1. Suppression of T cell function by oral tolerance to KEa Treatment PBS-fed KE-fed OVA-fed

In vitro stimulation

Proliferation (×10 −3 cpm)b

Medium Anti-CD3 Ab Medium Anti-CD3 Ab Medium Anti-CD3 Ab

1.1±0.4 71.3±1.7 0.7±0.2 69.7±0.2 0.6±0.1 70.4±3.5

a

Splenic cells were prepared from mice that died at 9 months of age. Results are expressed as mean counts per minute (cpm) [standard error of mean (SEM)].

0

Total Ig IgG1 IgG2a IgG2b IgG3 Serum IgG isotypes

Figure 2. Effect of oral tolerance to KE on total IgG anti-dsDNA antibody and isotype production in vivo. Sera were collected from PBS-( ) and KE-fed (") mice individually every month. Total IgG anti-dsDNA antibody and isotypes levels were analyzed in the sera of 9-month-old SLE mice by total IgG and isotype-specific ELISA. Data represent the mean OD±SEM from 10 mice. Note that the data shown are for sera diluted 1:400.* Indicates P<0.005; KE-fed mice compared with PBS-fed mice.

from KE- and PBS-fed mice. No significant suppression of proliferative response of T cells to dsDNA was detected (5,884±1,018 cpm in comparison with non-tolerant PBS-fed controls, 6,002±1,216 cpm at 10 ìg/ml; P>0.2). T cells from PBS-fed, KE-fed and OVA-fed groups responded similarly to anti-CD3 Ab after 72 h of culture (Table 1), suggesting that all of the cell populations were viable and contained similar numbers of potentially responsive T cells, emphasizing the specificity of the non-responsiveness to KE seen in Figure 1. The anti-CD3 response from KE-fed mice declined more rapidly than PBS-fed mice by 5 days in culture, perhaps reflecting enhanced susceptibility of orally tolerized lymphocytes to cell death in vitro [18] (data not shown).

b

NZB/W F1 mice. A marked suppression of T cell proliferation to KE was detected in the KE-fed mice (Figure 1) (6,459±1,123 cpm) in comparison with nontolerant PBS-fed controls, 35, 341±1,123 cpm at 10 ìg/ ml; P<0.005. The tolerance was KE-specific, since KE-fed mice responded normally when immunized with OVA (38,112±2312 cpm compared with 3,552±981 cpm in PBS control; P<0.005). The induction of suppression was KE-specific since spleen T cells from OVA-fed mice proliferated in response to KE (32,588±1355 cpm compared with 2,922±cpm in PBS control; P<0.005). However, when OVA was added to the culture, suppression was observed (4,867±978 cpm compared with 34,277±2138 in KE-fed OVA-primed T cells). Since DNA is believed to be the autoantigen that drives the autoimmune response in SLE [17], we tested dsDNA against T cells

Feeding with KE significantly reduces the levels of anti-dsDNA Abs in NZB/W F1 mice A number of autoantigens could be a potential target antigen of an autoimmune attack that leads to the development of SLE. The rationale for using KE as potential source of autoantigen in SLE is that the glomeruli appear to serve as the target for the pathogenic anti-DNA antibodies. Whole renal antigens were used because it is likely contain both disease-inducing and non disease-inducing epitopes. Levels of total IgG anti-dsDNA Ab in the sera were examined by ELISA. As shown in Figure 2, mice fed with low dose KE had significant reduction in the levels of IgG anti-dsDNA Abs compared with those fed with PBS. The mean IgG anti-dsDNA Ab levels differed significantly in the two groups from age 6 months to 10 months (P<0.001, Student’s t-test). After 10 months of age there were no mice in the PBS-fed group for statistical comparison (Figure 2).

Oral tolerance to kidney extract modulates SLE

PBS-fed

KE-fed

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Figure 3. Cytokine mRNA expression in the kidneys by RT-PCR. Total RNA was prepared from kidneys of PBS-fed or KE-fed SLE mice at 10 months of age and was assayed for the indicated cytokines by RT-PCR as described in Materials and Methods. PCR products were analyzed by electrophoresis on 2% agarose gels and visualized by ethidium bromide staining under UV illumination.

Levels of anti-dsDNA-specific IgG isotypes As indicators of the activity of Th1 and Th2 cells, we compared the levels of IgG anti-dsDNA-specific isotypes in the sera of KE- and PBS-fed mice at 10 months of age. It can be seen in Figure 2 that control animals fed with PBS produced significant levels of IgG1 and IgG3, and both isotypes were significantly reduced in mice fed with 1 mg KE. We detected a slight enhancing effect on IgG2a levels in the KE-fed mice, probably because of the increased production of IFN-ã, which is known to induce IgG2a differentiation. The levels of serum IgG2b response did not show any statistically significant differences between the KE-fed and the control PBS-fed groups. Thus, the profile of the IgG isotype responses suggest that Th2-dependent Ab responses appear to be susceptible to tolerance induced by low dose KE, whereas non-tolerant PBS-fed mice exhibited augmented Th2-dependent Ab responses.

Oral administration of KE selectively inhibits Th2 cytokine production in the kidneys Both Th1 and Th2 cytokines have been implicated in the pathogenesis of SLE. To determine whether oral tolerance to KE impacted on renal cytokine levels, cytokine mRNAs in renal tissues were examined in KE- and PBS-fed mice. As shown in Figure 3, renal tissues from PBS-fed mice displayed increased expression of Th2 (IL-4 and IL-10) cytokine mRNA with minimal expression of Th1 (IL-2, IFN-ã and TNF-á). Oral administration of low dose KE, however, virtually eliminated mRNA expression of IL-4 and IL-10 with upregulation of Th1 cytokines. Surprisingly, oral

Age of mice (months)

Figure 4. Development of proteinuria in PBS-(– –), OVA(–.–) and KE-fed (– –) SLE mice. Groups of 10 SLE mice fed with PBS or KE from age 2 months until age 6 months. Development of proteinuria was monitored at different ages using Albustix dipsticks. Data represent the percentage of mice with >100 mg/dl protein in their urine.* Indicates P<0.005; KE-fed mice compared with PBS-fed mice.

administration of low dose KE did not result in detectable increases in renal TGF-â as determined by RT-PCR and protein staining with anti-TGF-â monoclonal antibody (mAb) (data not shown). Cytokine mRNA expression was also examined in the spleen, which served as a representative peripheral lymphatic organ from which T cells, could migrate to the kidneys. It can be seen in Figure 3 that spleens from KE-fed mice also exhibited marked inhibition in Th2 cytokine mRNA expression with increased message for IL-2 and IFN-ã cytokines. These findings are consistent with the inhibition of IL-4-induced serum IgG1 and IgG3 antibody levels in KE-fed mice.

Oral tolerance to KE reduces renal damage in NZB/W F1 mice To follow the progression of renal disease in the PBS-fed and KE-fed mice, the mice were tested biweekly for the presence of protein in their urine. A 3+reading by dipstick indicates the presence of >100 mg/dl of proteinuria, consistent with severe renal disease or nephritis. Figure 4 shows that feeding mice with low dose KE significantly inhibited renal disease. By 10 months, proteinuria (>100 mg/dl) had developed in only 20% of the KE-fed mice, whereas all the PBS-fed mice developed severe renal disease (>100 mg/dl). To accurately reflect the development of renal disease in all mice, each point reflects the current level of proteinuria in surviving mice, as well as the last measurement of proteinuria in deceased mice. These results suggest that tolerance to KE reduced the development of proteinuria. This improvement coincided with a significant improvement in survival at 18 months of age (Figure 5). In contrast, oral administration of an irrelevant antigen, ovalbumin, could not prevent the development of nephritis suggesting that a specific antigen present in

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120

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6 8 10 12 14 16 18 20 Age of mice (months)

Figure 5. Survival in SLE mice fed with PBS ( ) or KE ( ) from age 2 months until age 6 months. It can be seen that survival rate in KE-fed mice was significantly greater than PBS- or OVA-fed mice (P<0.001).

the KE was responsible for the inhibition of renal disease.

Oral tolerance to KE prolongs survival in NZB/W F1 mice Since oral tolerance to KE was accompanied by suppression of anti-dsDNA Ab production and significant reduction in renal damage, we were interested to see whether oral tolerance to KE could enhance the survival of NZB/W F1 mice. At 12 months of age, no control PBS-fed mice were alive, but six of 10 (60%) KE-fed mice were alive (Figure 5). Survival rate in KE-fed mice were significantly greater than in PBS-fed mice (P<0.001) respectively by Student’s t-test. Oral administration of an irrelevant antigen, ovalbumin, did not prolong survival suggesting that the beneficial effect of KE is unique to the kidneys. To verify the reduction of renal disease in the KE tolerized mice, histological analysis was performed on the kidneys of mice in all the groups. As shown in Figure 6A, PBS-fed mice developed severe glomerulonephritis with intense tubulointerstitial infiltration of inflammatory cells. In contrast, the kidneys from long-term surviving KE-fed mouse that died at 16 months of age showed no overt glomerular disease, with a minimal tubulointerstitial cellular infiltration (Figure 6B).

Suppression of established glomerulonephritis by oral administration of low dose KE Because of the profound tolerance induced by low dose of KE on the development of glomerulonephritis (GN) when feeding was initiated before the onset of GN, we next assessed whether oral administration of KE could influence the course of GN after its onset. Oral administration of KE was started on 9-month-old NZB/W F1 mice with established GN. The mice were fed 1 mg KE daily for 1 month. The inhibition of GN was assessed by proteinuria, which was measured biweekly on urine samples as previously described in

Figure 6. Oral tolerance to KE prevents kidney damage in SLE mice. A Renal pathology from 10-month-old PBS-fed mice showing intense tubulointerstitial cellular infiltration and B 16-month-old low dose KE-fed mice showing no overt nephritis with a minimal tubulointerstitial cellular infiltration. Results are representative of several mice studied. Original magnification,×40.

this manuscript. Animals receiving oral KE after the onset of GN exhibited a modest decrease in the severity of GN (Figure 7). This therapeutic suppression persisted for at least 120 days until disease monitoring was stopped. All the mice fed with PBS died between 10–12 months of age. These data suggest that timing of oral feeding is critical, but that suppression can readily be achieved even after GN has been established with long-lasting effects.

Splenomegaly is reduced in KE-fed NZB/W F1 mice Splenomegaly, a hallmark of disease in NZB/W F1 mice, was diminished in KE-fed mice (Figure 8). Spleen weight was reduced five-fold in KE-fed mice compared with PBS-fed mice (0.31±0.09) versus 1.52±0.2 g, respectively). The spleens were representative samples taken from PBS-fed mice that died at 10 and 16 months of age, respectively. In some experiments, KE-fed mice were killed at 10 months to directly compare the spleen size with the PBS-fed mice that died at 10 months, and similar size spleens were observed.

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Proteinuria (%)

100 80 60 40 20 0

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10 12 Age of mice (months)

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Figure 7. Survival in SLE mice with established glomerulonephritis fed with PBS (– –) or KE (– –) from 9 to 14 months of age. Inhibition of renal damage in the mice was monitored by measurement of urinary protein levels weekly using Albustix dipsticks.

PBS-fed spleen

KE-fed spleen

Figure 8. Oral tolerance to KE prevents splenomegaly in SLE mice. It can be seen that spleen from PBS-fed mouse at 10 months of age was large presumably as a result of intense infiltration of inflammatory cells, whereas the spleen from a KE-fed mouse that died at 16 months of age had a normal size spleen. KE-fed SLE mice killed at 10 months of age also had normal size spleen.

Discussion For the past several years, many studies have demonstrated that oral administration of autoantigen induces tolerance in experimental autoimmune diseases by downregulating both humoral and cellmediated responses [19]. In the present study, we directly determined whether feeding NZB/W F1 mice with low dose KE could induce oral tolerance in those mice and prevent renal damage. Data presented here demonstrate that oral administration of low dose KE results in a marked suppression of renal damage and inhibition of dsDNA-specific serum IgG (especially IgG1 and IgG3 isotype responses) in KE-fed but not in PBS-fed mice. T cell proliferation to KE was diminished in mice fed KE. This tolerance is clearly consistent with other disease models [20]. Our findings are in agreement with previous studies showing that mice fed low dose (1 mg) of hen egg lysozyme (HEL) secreted less IL-4 than mice fed high dose HEL [21]. One of the important findings in the current study is the time dependence for optimal tolerance induction.

Consistent with prior protocols for oral tolerance induction through feeding the animals before the disease is initiated, we have observed a reproducible inhibition of renal damage in lupus mice. However, one question that we sought to address was whether oral tolerance could be induced and sustained after the onset of renal damage in the lupus mice. In this study, we address this question by feeding 9-month-old lupus mice daily for 1 month. The results clearly suggest that oral administration of KE after the onset of renal damage was unable to significantly suppress renal damage as judged by histology, but it did inhibit further renal damage as monitored by proteinuria thereby prolonging the survival of the mice. Our findings are reminiscent of a previous study that showed that oral feeding renal tubular antigen abrogated interstitial nephritis and renal failure in rats [22]. In systemic lupus erythematosus (SLE) patients, potential tolerance induction and therapeutic manipulation with oral antigen could generate regulatory T cells that may shift the balance of the autoimmune response toward suppression. To determine whether oral KE-induced tolerance does, in fact, shift cytokine balance, we monitored local (i.e. renal) and systemic (spleen) cytokine levels in the KE-fed mice. Oral administration of low dose KE virtually eliminates expression of Th2 cytokines, IL-4 and IL-10, which may contribute to the pathogenesis of renal damage. In contrast to oral tolerance in other animal models such as EAE [23], however, Th2-like cytokine such as IL-4, IL-10 and TGF-â are upregulated in the tolerized mice. TGF-â is associated with negative immunoregulatory functions, suppressing the growth of T cells [24] and B cells [25]. It was rather surprising that we did not see enhanced TGF-â production in the kidneys since previous studies have suggested that oral tolerance induction by low doses of antigen is mediated by inhibitory cytokines, particularly, TGF-â. However, there are reports suggesting that low dose oral tolerance can be generated in mice in the absence of TGF-â [26]. This suggests that factors other the production and secretion of TGF-â are involved in the induction of low dose tolerance. It is also possible that the amount of KE-fed (1 mg, several times) was too high to be considered a low dose in NZB/W F1 mice. Our data support the findings of others, which indicate that clonal anergy is the primary mechanism involved in inducing oral tolerance regardless of whether low or high doses of antigen are fed [27]. The down-regulation of Th2 responses in the low dose KE-fed mice was surprising since low dose antigen feeding is usually associated with preferential suppression of Th1 responses [28]. The finding of preferential suppression of Th2 responses and up-regulation of Th1 responses in low dose KE-fed mice suggest that Th2 responses are more relevant to lupus pathogenesis. Our results are consistent with studies in murine SLE that indicated that both Th1 and Th2 cells contribute to IgG production, and IL-4 and IL-12 play key roles in the complexity of cytokine regulation in the pathogenesis of SLE [29]. However, when the same authors looked at the roles of IL-4 and IL-12 in the

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development of nephritis, they concluded that IL-4 plays a more critical role in the disease process [29]. Since IgG3 autoantibody is regulated mainly by IL-4 in NZB/W F1 mice [29], it is possible that the increased production of Th1 cytokines in the KE-fed mice down-regulated IL-4 and IL-10 production leading to suppression of anti-dsDNA IgG1 and IgG3 Ab isotypes. Indeed, it has been demonstrated that IFNã-producing Th1 cells can inhibit Th2 cell function for IgG1 and IgG3 responses [30]. Several studies have shown that NZB/W F1 mice produce very low levels of TNF-á [31], and that therapy with TNF-á significantly delays the development of nephritis [32]. It is possible that the enhanced induction of Th1 cells by oral tolerance to KE in the SLE mice could exacerbate glomerulonephritis since previous studies by Adams et al. [33] have demonstrated that NZB/W F1 mice treated with IFN-ã showed accelerated disease manifestation. However, the generated Th1 cells will also produce IL-2 and TNF-á, which have been shown to prevent the development of SLE and glomerulonephritis [32, 34]. Therefore, high levels of IL-2 expression and to a lesser extent, TNF-á expression may block the diseaseenhancing effect of IFN-ã. Since IL-2 is a Th1 autocrine factor, the suppression of anti-dsDNA Ab production in the KE-fed mice could be due to restoration of the balance in favor of Th1 cells, resulting in suppression of Th2 cell activity. On the other hand, IFN-ã is also a potent inhibitor of macrophage and T cell proliferation [35] and, therefore, could be responsible for thwarting the development of nephritis in the KE-fed mice. Indeed, IFN-ã has been shown to prevent MRLFas1pr autoimmune interstitial nephritis by inhibiting macrophage growth and increasing macrophage apoptosis [36]. Clearly, the role of IFN-ã in SLE is complex. It would be important to rigorously establish the point in the pathogenesis and the amount of IFN-ã that provide protection or facilitate glomerulonephritis. We postulate that the induction of regulatory Th1 cells by oral tolerance to low dose KE may control the development of glomerulonephritis by the release of IFN-ã within the renal microenvironment that provides a self-regulating mechanism to limit autoreactive T cell expansion in the kidney. The majority of autoimmune diseases are mediated by Th1 CD4 + T cells; for example native type II collagen-induced arthritis, experimental autoimmune encephalomyelitis, and nonobese diabetic mouse [37]. In contrast, most cases of SLE appear to be mediated by Th2-type cytokines [38]. In SLE patients and in NZB/W F1 mice, there is an increase in Th2-type cytokine production and a decrease in Th1-type cytokine production or both [39, 40]. MRL/1 pr and (SWR×NZB) F1 mice are the only exception where Th1-type cytokines have been implicated in the disease [41, 42]. Experiments are in progress to examine the effect of high dose KE in the prevention of renal damage in NZB/W F1 mice as wells to characterize the nature of the antigen (s) in the KE responsible for the prevention of renal damage in NZB/W F1 mice. A recent study in (SWR×NZB) F1 mice model of SLE showed that intravenous injection

of nucleosomal peptide H416–39 could delay the development of lupus nephritis and prolonged the survival of older mice with established nephritis [43]. In summary, our study is the first to demonstrate that oral administration of low dose KE induces Th1 T cells/cytokines which provide a negative regulatory pathway capable of preventing renal damage in lupus mice. We suggest that interfering with activation of an important part of the T cell repertoire that is naturally activated in NZB/W F1 mice (i.e. T cells reactive with kidney antigens) via oral tolerance to KE would prevent or inhibit activation of the inflammatory cascade culminating in renal damage. One of the primary goals for the immunotherapy of SLE is to find nontoxic antigen-specific therapies that could be administered early in the course of the disease. Our results in the NZB/W F1 model raise the possibility that orally administered renal antigen and/or other autoantigens could provide a new approach for the prevention of renal failure due to glomerulonephritis, which is the major cause of death in SLE.

Acknowledgements We thank Ms. Sharon Hailston for excellent secretarial assistance and Dr Charles Antzelevitch for critical review of the manuscript. This work was supported by grant from the Lupus Foundation of America, Marguerite Curri Chapter, Utica, NY, USA.

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