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The Journal of Immunology, 2005, 175: 3577-3583.
Copyright © 2005 by The American Association of Immunologists

Enhanced Oral Tolerance in Transgenic Mice with Hepatocyte Secretion of IL-101

Rifaat Safadi*, Carlos E. Alvarez*, Masayuki Ohta*, Jens Brimnes{dagger}, Thomas Kraus{dagger}, Wajahat Mehal§, Jonathan Bromberg{ddagger}, Lloyd Mayer{dagger} and Scott L. Friedman2,*

* Division of Liver Diseases and {dagger} Immunobiology Center, {ddagger} Recanati Miller Transplantation Institute and Carl C. Icahn Center for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, New York, NY 10029; and § Section of Digestive Diseases, Yale University School of Medicine, New Haven, CT 06510


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Several cytokines derived from Th3 and Tr1 cells, including IL-10, are believed to regulate oral tolerance, but direct evidence is lacking. We have explored the potential role of IL-10 by generating transgenic (TG) mice with sustained hepatocyte-specific expression of rat IL-10. TG mice expressed rat IL-10 downstream of a transthyretin promoter, which led to serum levels that were increased 10- to 100-fold compared with normal animals. Animals were orally administered 1 mg of whole OVA for 5 consecutive days, with control animals receiving PBS. There were six animal groups: Either OVA or PBS were fed orally to rat IL-10 TG mice, non-TG wild-type mice without IL-10 administration, and non-TG wild-type mice administered rat IL-10 systemically. On day 8, all mice were immunized with two injections of OVA, and then analyzed on day 18. T cell proliferation responses were reduced by 65.8 ± 14.3% after feeding of OVA in rIL-10 TG animals, compared with 39.4 ± 15.6% in the non-TG mice (p = 0.02). Anti-OVA titers were expressed as fold increase over naive non-TG mice. After feeding, titers decreased by ~33% (from 3- to 2-fold) in TG animals and, to a lesser extent, in non-TG animals. IFN-{gamma} secretion by cultured popliteal lymphocytes decreased in TG animals by 83% after feeding and by 69% in non-TG animals. IL-4 secretion increased 4-fold in TG-fed mice, but did not significantly change in non-TG OVA-fed animals. In contrast to hepatic TG expression of rIL-10, systemic administration of rIL-10 had only a modest effect on tolerance. IL-10, when transgenically expressed in the liver enhances mucosal tolerance to an oral Ag.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
It is essential for the intestinal immune system to discriminate between potentially harmful and harmless foreign proteins. Exposure to harmless Ags through the oral route eventually results in immunologic hyporesponsiveness or oral tolerance (1, 2, 3, 4, 5). The primary mechanisms underlying oral tolerance are thought to include T cell deletion, anergy, and suppression (6, 7). The dose of Ag may dictate the type of tolerance inducted. For example, oral administration of low doses of Ag results in the suppression of both Th1 and Th2 (Th1 > Th2) responses through the induction of Ag-specific regulatory T cells (8, 9, 10), whereas high doses of Ag can induce tolerance by either anergizing Ag-specific T cells or via clonal deletion (11). Efforts to stimulate oral tolerance have been exploited for the treatment of autoimmune disease in animal studies and clinical trials to dampen the systemic immune response, but pathways regulating this response are not fully understood.

Studies using either neutralizing Abs to cytokines and specific mediators or selective genetic knockout (KO)3 mouse models have provided conflicting data regarding the mechanisms of tolerance induction. Earlier studies implicated CD8+ T cells as suppressor cells in oral tolerance; however, it appears that CD4+ T cells can fulfill this role as well, and can transfer oral tolerance in vivo (12, 13). This response was previously attributed to down-regulation of Th1 CD4+ cells by Th2 CD4+ cells. However, oral administration of OVA results in suppression of both Th1 and Th2 responses (14), and normal oral tolerance can be induced in the absence of Th2 cells in KO mice lacking either IL-4 or STAT6 (14, 15). These results indicate that both Th1 and Th2 subsets are targets of the induction of tolerance to specific oral Ags, which can be induced in both a Th1- or Th2-deficient environment.

The emerging role of CD4+ T cells as mediators of oral tolerance has been explained by the presence of regulatory T cell subpopulations. Specific T cell populations such as Th3, Tr1, or CD25+CD4+ T cells produce suppressive cytokines or cell surface molecules that can inhibit Th1-mediated immunopathologies such as experimental autoimmune encephalomyelitis and colitis (10, 16, 17).

Among the suppressive cytokines produced by regulatory T cells, IL-4, IL-10, and TGF-{beta}1 have received the most attention. IL-4 appears to be dispensable, because normal oral tolerance can be induced in its absence (14, 15). In contrast, IL-10 is an attractive candidate for mediating oral tolerance, because it suppresses Th1 activity via down-regulation of both costimulatory molecules and IL-12 production by APCs (18).

IL-10 is a regulatory cytokine produced by numerous cell types including activated Th0, Th1 (in humans), and Th2 CD4+ Th cells, cytotoxic CD8+ T cells, monocytes, B cells, Kupffer cells (19, 20), hepatocytes (19), and hepatic stellate cells (21). IL-10 inhibits Ag-specific activation, proliferation, and production of cytokines by Th0, Th1, and Th2 clones by reducing the Ag presenting capacity of monocytes and dendritic cells, associated with the down-regulation of MHC class II molecules and CD80/86 expression on their surface (22, 23). In contrast, in B lymphocytes, IL-10 stimulates Ig secretion (24, 25). IL-10 also exerts potent anti-inflammatory effects. It down-regulates the synthesis of proinflammatory cytokines and chemokines by monocytes and Kupffer cells stimulated by endotoxin, including IL-1, TNF-{alpha}, IL-6, IL-8, and IL-12, and up-regulates the synthesis of the IL-1R antagonist (20, 26, 27). Neutrophil chemotaxis and chemokine expression are also down-regulated (28).

The liver has long been implicated in immunoregulatory functions. It is the largest reticulo-endothelial organ in the body, and several subpopulations of its cells are involved in Ag presentation and/or processing (29). For example, portocaval shunts or blockade of Kupffer cell function may attenuate induction of oral tolerance in animal models (30). Also, Ab titers to intestinal flora are elevated in humans with chronic liver diseases who undergo portocaval shunts (30, 31). Finally, portal vein administration of donor cells can promote allospecific hyporesponsiveness (31). Thus, the liver may be necessary for peripheral immune tolerance induction through first pass clearance of Ags.

Despite the growing familiarity with the activities of IL-10, its role in mediating oral tolerance remains unclear. To address this issue, and to explore the potential role of hepatic IL-10 in the oral tolerance response, we generated transgenic (TG) mice with hepatocyte-specific expression of IL-10 by expressing an active form of rat IL-10 driven by the transthyretin (TTR) promoter (32). Our results suggest that IL-10 expressed in liver in this model can significantly augment oral tolerance to a foreign Ag. This enhancement of tolerance is greater than that seen by systemic administration of IL-10 at comparable levels, suggesting that the liver and IL-10 may be important in tolerance induction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation of rat IL-10 TG mice

TG mice expressing rat IL-10 mainly in hepatocytes were generated under the control of the TTR promoter as recently reported (32). A rat IL-10 cDNA fragment was inserted into an StuI site of the expression vector that contains a TTR minigene construct consisting of the 3-kb TTR promoter region upstream of the rat IL-10 transgene. The construct was linearized with BglI and microinjected into oocytes by standard methods in the Mount Sinai Mouse Genetics Shared Research Facility as described previously (32). Rat IL-10 cDNA shares significant homology with mouse IL-10 and is equally as potent, but can be discriminated by a rat-specific ELISA. Two of the 14 founder (F0) mice carried the TTR-rIL-10 transgene as identified by PCR analysis of genomic DNA extracted from mouse tails using rat-specific IL-10 primers (32). The two individual male F0 TTR-rIL-10 TG mice (nos. 1 and 3) were generated on a mixed C57BL/6 and C3H/HeNHsd background and then were mated with female wild-type C57BL/6 mice to generate F1 TG mice. TG founders were backcrossed onto the C57BL/6 background three times, generating F1, F2, and F3 mice. As a result, two TG lines were characterized and were selected for this study, lines 1 (F0 no. 1 progeny) and 2 (F0 no. 3 progeny). Non-TG offspring were used as control non-TG mice in our experiments.

DNA extraction and rIL-10 genotyping

Genomic DNA was extracted from tail clippings after digestion with proteinase K using Wizard SV 96 Genomic DNA Purification System (Promega) according to the manufacturer’s instructions. Oligonucleotide primers for rIL-10 were used in a PCR (sense, 5'-AGT GAA GAC CAG CAA AGG CCA TTC-3' and antisense, 5'-CAT TTT GAG TGT CAC GTA GGC TTC-3'). These primers were able to distinguish between the TG rat and murine IL-10 sequences.

Tissue RNA extraction and rIL-10 mRNA detection

Total cellular RNA was extracted from target tissues using TRIzol reagent (Invitrogen Life Technologies) and DNA digestion. RNA was then used as a template for reverse transcription into single-strand cDNA using the Reverse Transcriptase System (Promega). Synthesized rat IL-10 cDNA was detected by PCR with the primers mentioned above.

Tolerance protocol

Eight-week-old mice were used in this study, both IL-10 TG animals (fourth generation on a mixed C57BL/6 and C3H background) and their non-TG littermates as a control. Tolerance induction was performed by repeated oral low-dose administration of OVA (Sigma-Aldrich). The Ag administration schedule was 1 mg of whole OVA by gastric intubation for 5 consecutive days (days 1–5). To compare the effect of systemic vs hepatic IL-10 on oral tolerance, we separately treated non-TG animals with i.p. rIL-10 injections (65 ng/day for 14 days) that achieved serum concentrations similar to those see in IL-10-TG mice. There were six animal groups: Either OVA or PBS were fed orally to rat IL-10 TG mice, non-TG wild-type mice without IL-10 administration, and non-TG wild-type mice administered rat IL-10 systemically. Each animal subgroup contained 8 male mice in the first experiment (total of 32 mice) and 5 in the second independent experimental set. Only male mice were used, similar to prior studies (33).

On day 8, all mice were immunized with two footpad injections of a total of 200 µg of OVA emulsified in IFA (Sigma-Aldrich) at a 1:1 ratio. On day 18, mice were sacrificed, blood was collected, and serum was separated. Spleens and the popliteal lymph nodes (PLN) were aseptically removed and teased into single-cell suspensions. Each mouse was analyzed as a separate data point.

Serum anti-OVA Ab measurements

Anti-OVA serum Ab titers were measured by ELISA. Diluted serum samples were incubated for 1 h on ELISA plates (Nalge Nunc International) previously coated with 5 µg/ml OVA in 0.1 M carbonate buffer (pH 9.5). Plates were washed and incubated for 1 h with 100 µl/well HRP-conjugated goat anti-mouse IgG (Roche Molecular Biochemicals) diluted to 0.2 µg/ml. A/B substrate solution (BD Pharmingen) was added and colorimetric analysis was performed on an ELISA reader (Bio-Tek Instruments) at a wavelength of 650 nm. As controls, nonimmunized (normal mouse sera) and OVA-immunized (i.p.) mouse sera were used at concentrations identical to the samples.

In vitro analysis of T cell anti-OVA responses

The spleen and PLN from each mouse were analyzed separately. Splenocytes and cells from the PLN were teased into single-cell suspensions and washed twice with PBS. Cells were then cultured at 2 x 106 cells/ml in RPMI 1640 supplemented with 10% FCS (Atlantic Biologicals), 2-ME (5 x 10–5 M) and 1% penicillin-streptomycin-glutamine (Invitrogen Life Technologies). Cultured cells were analyzed for Ag-specific cytokine secretion and T cell proliferation.

Cytokine measurements

Cells (2 x 106/ml) were cultured in the presence or absence of OVA (25 µg/ml), and supernatants were collected at intervals of 24 h, 3 days, and 5 days. Culture supernatants were analyzed for the presence of murine IL-10, IL-4, and IFN-{gamma} by OptEIA ELISA kits (BD Pharmingen) according to the manufacturer’s protocol. A standard curve was generated using recombinant cytokines and concentrations of samples were determined by a polynomial curve fit analysis. A similar kit and measurements were used for determination of rIL-10 serum levels. Cells were also exposed separately to Con A (Sigma-Aldrich) stimulation in vitro (1 µg/ml) as a positive control.

T cell proliferation

Splenocytes and PLN cells were cultured in the above medium in the presence or absence of OVA (10 and 100 µg/ml) for 72 h, followed by an 18-h pulse with 1 µCi of [3H]thymidine (ICN). Incorporated radioactivity was measured on a flatbed MicroBeta-counter (Wallac). As a positive control for T cell proliferative capacity, cells were stimulated with Con A at 1 µg/ml. T cell proliferation was quantified as cpm. T cell proliferation after OVA stimulation was divided by that obtained from nonstimulated lymphocytes, and were represented as the fold increase in proliferation. These fold increase values were used to calculate the percentage of proliferation reduction after feeding. Final data were calculated as the relative fold increase of fed vs nonfed mice.

Cell isolation, staining, and flow cytometric analysis

The spleen and Peyer’s patches were teased, and lymphocytes were washed and counted before staining for FACS analysis. Intrahepatic lymphocytes (IHL) were isolated by perfusion of the liver with digestion buffer (3 ml of medium (in 1 min) containing collagenase (2 mg/10 ml) and DNase I (0.2 mg/10 ml) at 37°C). After perfusion, the liver was homogenized with an additional 10 ml of digestion buffer, completed to 40 ml by RPMI 1640 plus 5% FBS, and then incubated under constant shaking (Hot Shaker; 1 cycle per second) at 37°C for 30 min. The digested liver cell suspension was centrifuged to remove hepatocytes and cell clumps at 30 x g for 3 min at 4°C. The supernatant was then centrifuged to obtain a pellet of cells depleted of hepatocytes to a final volume of 1 ml. Lymphocytes were then isolated from this cell suspension using 24% metrizamide gradient separation (34).

Cells were adjusted to 2 x 107/ml in staining buffer (saline with 1% bovine albumin). Fifty micoliters of the cell suspension were incubated with fluorescein-labeled Abs on ice for 30 min, washed with staining buffer, and fixed with 2% paraformaldehyde. FACS data were acquired using a FACSCalibur flow cytometer (BD Biosciences), set to acquire all events. Abs used for staining were rat anti-mouse CD4, CD8, TCR {alpha}/{beta}, and TCR {gamma}/{delta} Abs, conjugated to FITC, PE, CyChrome, and APC, respectively. Abs were purchased from BD Biosciences, Transduction Laboratories. Intracellular staining of splenocytes with anti-IFN-{gamma} and IL-4 (FITC and PE conjugated, respectively) was performed according to the manufacturer’s protocol (BD Biosciences). Specific cytokine production by lymphocytes was visualized after stimulation with PMA and ionomycin then the addition of a Golgi transport inhibitor, Brefeldin A. FACS data were analyzed using CellQuest software (BD Biosciences).

Statistical analysis

Lymphocyte subsets, thymidine incorporation, cytokine concentrations, and anti-OVA titers were compared by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Characterization of rIL-10 TG mice

Untreated rat IL-10 TG mice were completely healthy and displayed similar survival rates to non-TG wild-type littermate animals for up to 12 mo (32). They all demonstrated a normal blood profile, including peripheral blood counts, electrolytes, and liver biochemistries (including alanine aminotransferase, aspartate aminotransferase, and bilirubin). Moreover, the volume and histologic features of liver, spleen, kidneys, colon, intestine, heart, lungs, thymus, and brain were indistinguishable from controls (data not shown). Using a rat-IL-10-specific ELISA, serum rIL-10 levels were below detectable levels in all non-TG animals. Average rat IL-10 serum levels in the TG nonfed group were 147 ± 222 and 202 ± 301 pg/ml (p = NS) in the TG-fed animals (Fig. 1). Levels of rIL-10 in liver homogenates from TG mice were always elevated and varied between 465 and 625 pg/ml (0.017–0.024% of total liver protein extract), even in those TG mice with normal serum levels. However, hepatic rIL-10 levels from mice of the systemic rIL-10 experiment were undetectable. Levels of rIL-10 were also elevated in gallbladder bile in the TG animals (90–231 pg/ml). Detection of rIL-10 mRNA in the liver by semiquantitative RT-PCR was apparent in both TG lines (Fig. 2). The same size product was also seen in the spleen and intestine, whereas less expression was observed in lungs of line 1 TG animals.



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  		FIGURE 1. Serum rIL-10 levels. Serum rat IL-10 levels were determined by ELISA in all 52 animals (expressed as {circ}). Mean levels illustrated as (–) were 147 ± 222 pg/ml in the rIL-10 TG nonfed (left column) and 202 ± 301 pg/ml in the OVA-fed (second column) TG animals. Twenty percent of IL-10 TG animals had undetectable serum rIL-10 levels. Concentrations in bile and liver extracts of TG animals were also elevated.

 


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  		FIGURE 2. Rat IL-10 mRNA transgene expression in different tissues from two lines of rIL-10 TG animals. RNA expression of rIL-10 was assessed by RT-PCR as described in Materials and Methods, and demonstrated significant transgene expression in the liver, spleen, and intestinal mucosa, but not the lungs of line 1 TG animals. In line 2 animals, transgene expression was restricted to the liver. Because the primers only amplify TG rat IL-10 there was none detected in wild-type non-TG animals (data not shown).

 
The composition of lymphocyte subsets from spleens, IHL, lymph nodes, and Peyer’s patches among naive rIL-10 TG vs non-TG animals is illustrated in Table I. Importantly, both intrahepatic CD4 and TCR {alpha}/{beta} cells were significantly reduced in rIL-10 TG mice compared with non-TG mice. Although CD4 subsets from peripheral lymph nodes demonstrated the same pattern, total CD4 cells were significantly increased in the TG Peyer’s patch lymphocytes. Significant decreases were seen in total CD8 lymphocytes obtained from TG spleens and livers. No significant alterations were seen in NK cells obtained from all organs in both TG and non-TG mice. Unlike IL-4-secreting CD4 lymphocytes, significant changes were recorded in IFN-{gamma} CD4 splenocytes. They significantly decreased in the TG splenocytes, reflecting attenuation of the Th1 profile. Results were similar in both TG lines 1 and 2 (data not shown). Although line 1 appeared to have more promiscuous rIL-10 mRNA expression than line 2 (Fig. 2), the distribution of lymphocyte subsets from the Peyer’s patches was similar in both TG lines.


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  		Table I. The composition of lymphocyte subsets from spleens, IHL, Payer’s patches, and lymph nodes

 
Because the rIL-10 TG mice contain the LPS-responsive C3H/HeN genetic background, they are an appropriate model to investigate the response to oral tolerance. In contrast to these LPS-responsive C3H/HeN mice, lack of oral tolerance was recently reported in the LPS nonresponsive C3H/HeJ strain (35).

Oral tolerance to OVA in rIL-10 TG and wild-type mice

Gastric feeding of OVA significantly reduced PLN T cell proliferative responses to OVA in vitro (Fig. 3). After feeding and systemic immunization, in vitro proliferation of T cells stimulated with 10 µg of OVA per milliliter was reduced 39.4 ± 15.6% in non-TG groups (p = 0.044 between fed and nonfed states), compared with 65.8 ± 14.3% in line 1 TG animals and 72 ± 8% in line 2 TG mice (p < 0.0001 between fed and nonfed states; and p = 0.02 between reduction in non-TG and TG rodents. No significant differences were found between both TG lines). To compare the effect of systemic vs hepatic TG rIL-10 on oral tolerance, non-TG animals were treated with daily i.p. rIL-10 injections. Mean serum rIL-10 by ELISA in rIL-10-treated mice was 128 ± 53 and 176 ± 58 pg/ml within OVA-fed and nonfed groups, respectively. After feeding and systemic immunization, in vitro proliferation of PLN T cells stimulated by 10 µg of OVA per milliliter was reduced 47.5 ± 17.8% in non-TG groups with IL-10 administration (p = 0.02). Subsequently, reduction of T cell proliferation after OVA feeding was significantly enhanced in IL-10 TG mice (65.8 ± 14.3%) compared with 47.5 ± 17.8% (p = 0.05) and 39.4 ± 15.6% (p = 0.02) in non-TG groups treated with or without systemic IL-10 administration, respectively (Fig. 3). Thus, the effect of hepatic IL-10 on enhanced tolerance was not necessarily due to increased cytokine (IL-10) levels in the blood.



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  		FIGURE 3. Ag-specific T cell proliferation. PLN cells isolated 10 days after footpad immunization with OVA/adjuvant were cultured in the presence or absence of OVA (10 µg/ml) for 72 h, followed by a 16-h pulse with 1 µCi of [3H]thymidine. T cell proliferation was reduced after feeding from 45,018 ± 18,452 to 25,836 ± 16,304 cpm in wild-type mice (p = 0.027), from 51,542 ± 24,302 to 17,563 ± 6,017 cpm in all TG animals (p = 0.001) and from 51,342 ± 10,722 to 38,553 ± 39,854 cpm in systemic IL-10-treated groups (p = 0.05). T cell proliferative responses were significantly reduced after feeding by 65.8 ± 14.3% in line 1 rIL-10 TG animals and 72 ± 8% in line 2 (p < 0.0001 between fed and nonfed states, no significant differences were seen between both TG lines) compared with a less significant (p = 0.02) reduction (39.4 ± 15.6%, p = 0.044 between fed and nonfed states) in the non-TG mice. To compare the effect of systemic vs the hepatic IL-10 on oral tolerance, non-TG animals were treated with daily i.p. rIL-10 injections instead of using the rIL-10 TG mice. After feeding, in vitro proliferation of T cells stimulated by 10 µg of OVA per milliliter was reduced 47.5 ± 17.8% in non-TG groups (p = 0.02 between fed and nonfed states), which was less than IL-10 TG (p = 0.05) and similar to the reduction seen in the non-TG that were not treated with IL-10. Results were generated in two independent experiments (eight mice in each subgroup).

 
The same pattern was seen in cells stimulated with 100 µg of OVA per milliliter in vitro (data not shown). No significant differences were observed in proliferation of splenocytes. After nonspecific in vitro stimulation by Con A, T cell proliferative responses from PLN and splenocytes were identical in all non-TG and TG-fed or nonfed mice (data not shown). Results were similar in two independent experiments (eight animals in each subgroup). Animals were sacrificed on day 18, because earlier or later time points failed to show significant changes (data not shown).

Cytokine production

Cells from the PLN were cultured in vitro for 1, 3, and 5 days in the presence or absence of 25 µg of OVA per milliliter. After OVA feeding, both non-TG and rIL-10 TG mice exhibited significantly reduced IFN-{gamma} secretion at day 5 in the presence (Fig. 4A) but not in the absence (data not shown) of OVA stimulation in vitro. Serum IFN-{gamma} secretion was decreased from 7536 ± 3325 to 2142 ± 1402 pg/ml in TG mice (p = 0.02), and from 9731 ± 3315 to 3819 ± 3843 pg/ml (p = 0.04) in non-TG animals. However, the reduction in IFN-{gamma} at day 3 (Fig. 4B) was significant only in the TG animals (from 3569 ± 1642 to 1521 ± 276 pg/ml, p = 0.045) and not non-TG mice.



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  		FIGURE 4. Cytokine production in low-dose OVA oral tolerance in rIL-10 TG mice. Cells from the PLNs of unfed or OVA-fed and from rIL-10 TG and wild-type OVA-immunized mice were cultured with 10 µg of OVA for 24–120 h ({blacksquare}) or without (data not shown), and cytokine secretion was measured by ELISA. Although results indicate that fed mice had a decrease of IFN-{gamma} at 120 and 72 h (A and B) and increased secretion of IL-4 at 24 h (C), rIL-10 TG mice showed enhanced changes compared with non-TG animals. Mouse IL-10 secretion at 72 h (D) was increased in fed mice compared with unfed mice, but was less prominent in the rIL-10 TG, compared with non-TG immunized mice, possibly due to an inhibitory effect of TG rIL-10 on mouse IL-10 secretion. Results were similar in two independent experiments of 32 mice (eight each subgroup).

 
After feeding, in vitro stimulation of PLN with OVA in the TG mice induced a 4-fold increase in IL-4 secretion on day 1 (p = 0.05), but showed no change in the non-TG-fed animals (Fig. 4C). Murine IL-10 secretion after OVA feeding was higher (8.1-fold, p = 0.026) in non-TG but not in TG mice (1.3-fold). This may reflect feedback inhibition of IL-10 by high levels of TG rIL-10 (Fig. 4D).

All animal groups displayed similar cytokine responses after nonspecific stimulation by Con A (data not shown). Results of cytokines were similar in two independent experiments of 32 mice (eight in each subgroup). Cytokine measurements from all other time points fail to show significant differences between animal groups (data not shown).

Serum IgG anti-OVA titers were reduced after OVA feeding in both non-TG and TG groups (Fig. 5). At a 1/1000 dilution, the anti-OVA titer in non-TG animals decreased from 1 ± 0.27 to 0.54 ± 0.24 OD after OVA feeding (p = 0.02). Anti-OVA Ab titers followed the same pattern but were lower in both TG groups, as titers decreased from 0.85 ± 0.32 to 0.48 ± 0.16 OD after OVA feeding (p = 0.05). Results were similar in two independent experiments in a total of 52 mice (13 in each subgroup). Results were not significant at other dilutions including 1/5,000, 1/25,000 (Fig. 5), 1/2,000, or 1/125,000 (data not shown). Animals were sacrificed on day 18, because earlier or later time points failed to show significant changes (data not shown).



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  		FIGURE 5. Anti-OVA serum levels are reduced in fed mice. Serum from wild-type and IL-10 TG mice that were or were not fed OVA before OVA immunization were diluted and analyzed for the presence of anti-OVA IgG by ELISA. Serum anti-OVA IgG titers were reduced after feeding and toleration induction among non-TG ({blacksquare} and {blacktriangleup} in nonfed and fed mice, respectively) and TG groups ({square} and {triangleup} in nonfed and fed mice, respectively) at 1/1000 and 1/5000 dilutions, compared with a nonimmunized animal as a negative control and an immunized animal as a positive control. Results were similar in two independent experiments of a total of 52 mice (13 each subgroup).

 
All tolerization findings in TG line 1 were similar to those found in line 2, including T cell proliferation (Fig. 3), cytokines, and anti-OVA Abs (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Accumulating evidence has identified a role for IL-10 in mediating oral tolerance. Initial reports suggested that the production of IL-10 was enhanced in low-dose oral tolerance (12, 13, 36), and IL-10-producing clones have been isolated from animals tolerized by feeding myelin basic protein (12, 13, 36). Moreover, mucosal administration of IL-10 orally enhances tolerance after mucosal autoantigen exposure in an experimental model of autoimmune encephalomyelitis (37). IL-10-dependent, OVA-specific TCR TG T lymphocytes can also mediate nonspecific bystander suppression of experimental colitis when adoptively transferred in vivo followed by oral administration of OVA (10). Similarly, there is now solid evidence for the existence of CD4+ cells (Th3, Tr1) that produce IL-4, IL-10, and TGF-{beta}. Analogous populations of IL-10-dependent regulatory cells (Tr1) that produce TGF-{beta} are capable of suppressing a murine model of inflammatory bowel disease (38), further highlighting a role of IL-10 in mucosal immune regulation. Finally, in low-dose oral tolerance to Ag in a uveitis model, both IL-4 and IL-10 are required for induction of protective oral tolerance that is dependent upon regulatory cytokines (34).

In contrast, some studies have refuted a role for IL-10 in oral tolerance. For example, marked suppression of IL-10 occurs in mice fed OVA (14), and anti-IL-10 treatment did not block either the induction or the maintenance of orally induced tolerance to OVA (39). Studies of oral tolerance in IL-10 KO animals have shown that IL-10 is required for oral tolerance induction (3). Although IL-10 is not required for experimental autoimmune uveitis induction in IL-10 KO animals, both IL-10 and IL-4 are required for the induction of protective oral tolerance (34).

One interpretation of these conflicting results is that the effects of IL-10 might either depend on the site of production and/or whether elevated levels are intermittent (for example, after exogenous administration) or sustained. In the present study we have assessed the impact of IL-10 on oral tolerance to OVA when secreted from TG hepatocytes in a sustained manner. We find a significant reduction of T cell proliferation in TG animals tolerized to OVA, compared with non-TG mice. This reduction was less clear when IL-10 was administered systemically instead (Fig. 3) (p = 0.20, NS). Moreover, these TG mice displayed more marked suppression of proinflammatory responses by switching to an anti-inflammatory cytokine profile, manifested by a more significant decrease in IFN-{gamma} secretion and a greater enhancement of IL-4 secretion.

IL-10 may enhance oral tolerance in our model by a number of mechanisms, but the hepatic secretion of the cytokine appears the most important because systemic administration of IL-10 did not fully replicate the findings in TG IL-10 mice. There may also be indirect actions from the systemic effects of high circulating levels of rIL-10 and/or after Ag processing and exposure to especially high concentrations of IL-10 in the liver.

Mucosal tolerance is thought to reflect the impact of the local mucosa-associated lymphoid tissue environment that influences Ag presentation where regulatory cells are being generated. Alternatively, mucosal tolerance might be mediated by modulating MHC class-II expression on APCs or effects upon costimulatory molecules (CD80/86). Mucosal tolerance is also regulated by the cytokine microenvironment as well as the immunogenic properties of the specialized cells that line the intestinal surface, the M cell and the absorptive epithelial cell (40, 41). In humans, these absorptive epithelial cells express MHC class I, class II, and nonclassical class I molecules (42), as well as a unique costimulatory molecule that induces the proliferation of CD8+ T cells in culture (43). Treatment of dendritic cells with IL-10 decreases their capacity to stimulate CD4+ T cells in a MLR. IL-10 also stimulates immature DC to become tolerogenic APCs (44) and preferentially primes naive T cells to generate Th2-type cells in vitro and in vivo (45). More recent studies suggest that production of IL-10 by lamina propria APCs might participate in local immunoregulation in the gut (46, 47).

Our model allows us to address the liver’s role in oral tolerance and to examine the impact of high local concentrations of IL-10. The liver is increasingly recognized as an immunoregulatory organ that participates in oral tolerance (29). Induction of peripheral immune tolerance appears to require first-pass clearance of specific subpopulations of cells or peptides via the portal circulation from the intestine through the liver (30, 48). As noted above, diversion of portal blood from the liver by portacaval shunt, or blockade of Kupffer cell function abrogates oral tolerance in animal models (30). In support of this conclusion, elevated titers of Abs to intestinal flora have been reported in humans with chronic liver diseases who have undergone portocaval shunts (30, 31). Additionally, by administering properly modified alloantigen into the hepatic environment by portal vein inoculation, prolonged T cell anergy can be induced, which allows for the indefinite acceptance of donor-specific heterotopic cardiac allografts (31).

Although our data cannot conclusively distinguish between effects of circulating vs local hepatic rat IL-10, serum rIL-10 levels were not elevated in all TG mice. Despite this, effects on oral tolerance were consistently observed in all animals. In fact, 20% of rIL-10 TG mice had normal IL-10 serum levels (Fig. 1), yet all had tolerance responses indistinguishable from animals with high serum IL-10 levels, including equivalent T cell proliferation, anti-OVA Abs and cytokine alterations (data not shown).

Based on these data, locally enhanced hepatic expression of rIL-10 is likely to play an important role in the enhanced oral tolerance in our TG mice. This conclusion is supported by evidence of altered IHL composition in TG animals. IHL normally include {alpha}{beta} TCR (TCR {alpha}{beta}+) cells, T cells with the TCR {gamma}{delta}+, classic NK cells (NK), NK cells with the TCR (NK-T), and dendritic cells (49). The hepatic CD4 to CD8 ratio is the opposite of the lymph node, with CD8+ cells predominating, but ~20% of TCR {alpha}{beta}+ cells are TCR {alpha}{beta} low and negative for CD4 and CD8 (TCR low-DN). The liver also contains small numbers of a lin–c-kit+ population, which may have pluripotent potential (48, 49, 50). In contrast, IL-10 hepatocyte TG mice displayed a significant decrease of intrahepatic CD4 and TCR {alpha}{beta} compared with non-TG animals (Table I). These changes would favor the persistence of oral tolerance by down-regulation of Th1 CD4+ cells or CD4+ T-regs as a result of prolonged exposure to high IL-10 levels. Similar localized anti-proliferative changes were also reported in an IL-10 gene transfer model in transplanted hearts, which showed an increase of Fas/FasL mediated apoptosis of CD4 and CD8 cells, accompanied by decreased apoptosis of myocytes and a decrease in rejection episodes (51).

The data presented in this paper are in line with a recent study (52), which reported that the liver plays an important role in inducing peripheral tolerance in a mucosal tolerance model, especially after feeding high-dose OVA. In this report, the livers from BALB/c mice fed with OVA at either a low or high dose were transplanted into syngeneic recipients. Nonfed recipients were controls. Orthotopic liver transplantation was followed by OVA immunization and delayed-type hypersensitivity challenge. Livers from all OVA-fed mice after 10 days transferred tolerance to OVA-naive mice. The in vitro proliferative response of the liver nonparenchymal cells to OVA revealed a decreased response in both dosage groups over the control group.

The pharmacokinetics of IL-10 secretion in rIL-10 TG mice might also favor development of oral tolerance. Specifically, elevated levels of IL-10 are sustained through constant production by TG hepatocytes, in contrast to intermittent administration of exogenous IL-10, as previously evaluated (37, 46). The specific impact of sustained elevations of IL-10 merits further evaluation.

In summary, our data establish IL-10 as an important immunomodulatory protein that enhances oral tolerance toward OVA when expressed locally via hepatocyte secretion. In this model, IL-10 modifies cytokine secretion (with less proinflammatory responses in TG animals), reduces T cell proliferation, and decreases anti-OVA titers. Based on these data and related studies (39, 50), as well as the acceptable safety profile of IL-10 in human studies, in vivo augmentation of IL-10 might enhance the effectiveness of oral tolerance regimens currently being tested in a variety of chronic diseases. Additionally, our model of hepatic overexpression of TG rat IL-10 may prove useful for exploring the role of IL-10 in other hepatic responses, including immunologic injury, cholestasis, metabolic derangements, or hepatic fibrosis.


   Acknowledgments
 
We acknowledge the Mount Sinai Mouse Genetics Shared Research Facility for the production of the founder TG animals.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by funds from the Artzt Primary Biliary Fibrosis Program of the Mount Sinai School of Medicine, the Juvenile Diabetes Research Foundation International (to J.B.) and National Institutes of Health Grants DK56621 (to S.L.F.); AI23504, AI 24671, AI44236, and AI 07605 AI61093 (to L.M.); and R01 AI41428 and R01 AI44929 (to J.B.). The Mount Sinai Mouse Genetics Shared Research Facility is partially supported by National Institutes of Health Grant 1R24 CA88302-01. Back

2 Address correspondence and reprint requests to Dr. Scott L. Friedman, Box 1123, Mount Sinai School of Medicine, 1425 Madison Avenue, Room 11-70C, New York, NY 10029. E-mail address: Scott.Friedman{at}mssm.edu Back

3 Abbreviations used in this paper: KO, knockout; TG, transgenic; TTR, transthyretin; PLN, popliteal lymph node; IHL, intrahepatic lymphocyte. Back

Received for publication October 29, 2004. Accepted for publication June 22, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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