- Email: [email protected]
Treatment of experimental colitis by oral tolerance induction: a central role for suppressor lymphocytes
THE AMERICAN JOURNAL OF GASTROENTEROLOGY © 2000 by Am. Coll. of Gastroenterology Published by Elsevier Science Inc.
Vol. 95, No. 4, 2000 ISSN 0002-9270/00/$20.00 PII S0002-9270(00)00727-9
Treatment of Experimental Colitis by Oral Tolerance Induction: A Central Role for Suppressor Lymphocytes Yaron Ilan, M.D., Sarah Weksler-Zangen, Ph.D., Shomron Ben-Horin, M.D., Judith Diment, M.D., Bernhard Sauter, M.D., Elazar Rabbani, Ph.D., Dean Engelhardt, Ph.D., Namita Roy Chowdhury, Ph.D., Jayanta Roy Chowdhury, M.D., and Eran Goldin, M.D. Gastroenterology and Liver Units, Department of Medicine, and Department of Pathology, Hadassah University Hospital, Jerusalem, Israel; Marion Bessin Liver Research Center, Albert Einstein College of Medicine, New York; and ENZO Bichem Inc., New York
OBJECTIVE: Inflammatory bowel diseases (IBD) are immune-mediated disorders wherein an imbalance between proinflammatory (Th1) and antiinflammatory (Th2) cytokines is thought to play a role in the pathogenesis. The aim of this study was to test whether induction of oral tolerance to proteins extracted from inflammatory colon alleviates experimental colitis, and whether oral tolerization mediated by suppressor cells can induce immune tolerance. METHODS: Colitis was induced in rats by intracolonic instillation of trinitrobenzenesulfonic acid (TNBS). Rats received five oral doses of colonic proteins extracted from TNBS-colitis colonic wall. Splenocytes harvested from tolerized and control rats were transplanted into irradiated naive rats. RESULTS: Feeding of colitis-extracted proteins ameliorated colonic inflammation, as shown by reduction of colonic ulcerations, as well as decreased diarrhea, intestine and peritoneal adhesions, wall thickness, and edema. A marked reduction of the fraction of injured colonic area and colon weight, and decrease in colon weight, were observed in tolerized rats versus controls. Histological parameters for colitis were markedly improved in tolerized animals that showed significant reduction in inflammatory response and mucosal ulcerations. Tolerized rats developed an increase in TGF1 and a decrease in IFN␥ serum levels. TNBS-induced colitis was significantly attenuated in naive recipients of splenocytes from tolerized rats, compared with rats that received splenocytes from control donors. CONCLUSIONS: Induction of oral tolerance to colitis-extracted proteins downregulates the anticolon immune response, thereby ameliorating experimental colitis. Suppressor lymphocytes mediate the tolerance by induction of a shift from a proinflammatory to an antiinflammatory immune response. (Am J Gastroenterol 2000;95:966 –973. © 2000 by Am. Coll. of Gastroenterology)
INTRODUCTION Inflammatory bowel diseases (IBD) are common GI disorders. Although the cause of these disorders remains unknown, various features suggest immune system involvement in their pathogenesis. Several extraintestinal manifestations that accompany IBD are autoimmune phenomena and immunosuppressive agents are used to alleviate the disease (1, 2). Patients with IBD have antibodies against components of colon cells and several different bacterial antigens (3, 4). Abnormalities of T-cell–mediated immunity, including cutaneous anergy and diminished responsiveness to T-cell stimuli, have also been described in these patients (5, 6). In addition, changes in mucosal-cell–mediated immunity were identified, including increased concentrations of mucosal IgG cells and changes in T-cell subsets, suggesting antigen stimulation (7, 8). Exposure of target antigens after infectious, immune, or toxic damage leads to activation of mucosal immune cells, resulting in cytokines that lead to mucosal inflammatory response (9). Secretion of proinflammatory cytokines such as IFN␥ contributes to an increase in mucosal permeability, and has been described in animal models of IBD (10). Several animal models of chronic inflammatory immune response have been established. These include rats carrying the transgenes for HLA B27 and 2-microglobulin, and mice in which genes for IL2, IL10, and ␣ and  chains of T-cell receptors are absent (11–14). A Th1-mediated granulomatous colitis model has been established by the adoptive transfer of normal CD45RB T-cells from Balb/C mice into CB-17 scid mice (15). Thus, IBD can be envisioned as an imbalance between proinflammatory and antiinflammatory cytokines. Oral tolerance is the induction of immunological hyporesponsiveness toward specific antigens or, through a bystander effect, toward other antigens present at the site, via secretion of immunosuppressive cytokines (16). The mechanism of tolerance may depend on antigen dose. Lower doses induce tolerance through generation of negative immunoregulatory cells, whereas higher doses lead to clonal deletion (17). Both in animals and in humans, tolerance
AJG – April, 2000
Oral Tolerance for Experimental Colitis
Table 1. Experimental and Control Groups Group
Antigen Fed
Donor Splenocytes
A B C D E F
CEP CEP BSA BSA None None
None None None None Group B Group D
CEP ⫽ colitis-extracted proteins; BSA ⫽ bovine serum albumin.
induction is associated with a Th2-type immune response, leading to secretion of immunosuppressive cytokines such as TGF1, IL4, and IL10 (18). In the present study we used a model system that employs normal rats treated with 2,4,6trinitrobenzene sulfonic acid (TNBS) (19). This type of experimental colitis, similar to the human disease, is a Th1-mediated immune disorder resulting in life-long inflammatory response against the colon. We have recently shown that oral tolerance can abrogate antigen-specific preexisting immunity (20). Therefore, we sought to evaluate whether induction of oral tolerance toward colitis-extracted proteins could be used to abrogate host anticolon immune response for treating experimental colitis.
MATERIALS AND METHODS Animals Normal inbred, male Sabra rats (200 –300 g) were obtained from the Animal Core of the Hadassah-Hebrew University Medical School. Rats were maintained on standard laboratory chow and kept in 12-h light/dark cycles. Induction of Colitis We induced TNBS-colitis by rectal instillation of TNBS, 25 mg/rat, dissolved in 1 ml of 50% ethanol (19). Preparation and Administration of the Oral Antigen Colon was removed from TNBS-induced– colitis rats, and was cut into small strips and mechanically homogenized. After filtration through a 40-m nylon cell strainer, the intact cells were spun down and removed. Proteins were quantified using protein assay kit (Biorad, Munchen, Germany). Colitis-extracted proteins (CEP) were introduced to the experimental groups described, using a feeding atraumatic needle every other day for 11 days (a total of five doses). Experimental Groups Six groups of rats, consisting of 10 animals each, were studied (Table 1). Groups A and B included rats fed with colitis-extracted proteins (500 g/rat) every other day starting 24 h after colitis induction. Two groups of rats, Groups C and D, were used as controls and were fed a similar dose of bovine serum albumin (BSA) alone. Adoptive transfer experiments were performed to evaluate the mechanism of tolerance, and distinguish between induction of suppressor cells and clonal inactivation or deletion. Rats in groups B
967
and D served as tolerized and control (nontolerized) splenocyte donors for recipient animals in groups E and F, respectively, as described later. Evaluation of the Effects of Tolerance Induction on Experimental Colitis CLINICAL ASSESSMENT OF COLITIS. Diarrhea was followed daily throughout the study. MACROSCOPIC SCORE OF COLITIS. Colitis assessment was performed 10 days after colitis induction using the following standard parameters (19). Rats were killed and colon was removed. The percentage of the total colonic wall that was injured and colon weight were measured. Four additional parameters were determined, namely, degree of colonic ulcerations; intestine and peritoneal adhesions; wall thickness; and degree of mucosal edema. Each parameter was graded on a scale from 0 (completely normal) to 4 (most severe) by two experienced blinded examiners. GRADING OF HISTOLOGICAL LESIONS. For histological evaluation of inflammation, distal colonic tissue (last 10 cm) was removed and fixed in 10% formaldehyde. Five paraffin sections from each rat were then stained with hematoxylin-eosin according to standard techniques. The degree of inflammation on microscopic cross sections of the colon was graded semiquantitatively from 0 to 4 as follows (9, 19): Grade 0: normal with no signs of inflammation; Grade 1: very low level of leukocyte infiltration; Grade 2: low level of leukocyte infiltration; and Grade 3: high level of infiltration with high vascular density, and thickening of the bowel wall; Grade 4: transmural infiltrates with loss of goblet cells, high vascular density, wall thickening, and disruption of normal bowel architecture. Grading was performed by two experienced blinded pathologists. Evaluation of the Mechanism of Immune Tolerance Induction ADOPTIVE TRANSFER OF TOLERANCE. To determine whether splenic lymphocytes from tolerized rats are capable of producing tolerance upon transplantation into naive rats, donor rats from groups B and D (five rats from each group) were killed 10 days after induction of colitis, and single suspensions of lymphocytes derived from the spleen were prepared as described (21, 22). Cells were resuspended in phosphate-buffered saline (PBS) immediately before transplantation. Recipient rats were sublethally irradiated with 300 rad total body irradiation, 24 h before intravenous injection of 1 ⫻ 108 donor cells in 0.5 ml PBS. Twenty rats were studied. Ten rats (Group E) received cells from group B donor rats, and 10 rats (Group F) received splenocytes from group D rats. All rats were treated with TNBS enemas, 24 h after cell transplantation. Clinical, macroscopic, and histological parameters for colitis were determined 10 days after colitis induction, as described earlier.
968
Ilan et al.
Figure 1. Effect of tolerance induction toward colitis-extracted proteins on experimental colitis (10 days after colitis induction). Significant decrease in percentage of colonic area exhibiting signs of injury, as well as colon weight, was observed in tolerized groups A and B (white bars), compared with nontolerized controls C and D (black bars, p ⫽ 0.14, and p ⫽ 0.02, respectively).
SERUM TGF1 AND IFN␥ LEVELS. TGF1 and IFN␥ levels were measured by a so-called “sandwich” enzymelinked immunosorbent assay (ELISA) using Genzyme Diagnostics kits (Genzyme Diagnostics, MA) according to the manufacturer’s instructions. Serum levels were measured in all rats from tolerized and nontolerized control rats 10 days after colitis induction.
RESULTS Oral Tolerization Toward Colitis-Extracted Proteins Alleviates Experimental Colitis CLINICAL ASSESSMENT. A marked decrease in diarrhea was observed daily in tolerized rats from groups A and B as compared with nontolerized controls in groups C and D throughout the study period. MACROSCOPIC GRADING. The percentage of colonic area exhibiting macroscopic injury decreased to 57% in tolerized rats versus 72% in controls (p ⫽ 0.14), and the mean colon weight was 1.6 g compared with 2.2 g in controls (p ⫽ 0.02, Fig. 1). In tolerized rats, degree of colonic ulceration, intestine and peritoneal adhesions, and wall thickness improved significantly (p ⬍ 0.05, using Mann-Whitney Rank Sum test, Fig. 2). The total scores of all macroscopic parameters tested were 1.01 in tolerized rats, compared with 2.11 in nontolerized controls (p ⫽ 0.001, Fig. 2). HISTOLOGICAL LESIONS. Histological evaluation of bowel tissues by standard pathological scoring, 10 days after induction of colitis, showed marked reduction in inflammatory response and mucosal ulceration (1.34 vs 3.66 in tolerized vs nontolerized controls, receptively). In four of 20 recipients tolerized by enteral administration of colitis-extracted proteins the colons were nearly normal by histological examination. In the rest of these rats, only minimal lymphocytic infiltration was detected (Groups A and B, Fig.
AJG – Vol. 95, No. 4, 2000
Figure 2. Effect of tolerization on macroscopic grading of colitis. Parameters tested were graded on a scale from 0 (completely normal) to 4 (most severe), including degree of colonic ulceration, intestine and peritoneal adhesions, wall thickness, and degree of mucosal edema. All tested parameters had less inflammation in tolerized rats than in controls (p ⬍ 0.05). The average score of all macroscopic parameters improved from 2.11 in nontolerized controls (black bars) to 1.01 in tolerized animals (white bars, p ⫽ 0.001).
3 A, B). In contrast, severe inflammatory reaction (grade 3– 4) was observed in bowel specimens taken from nontolerized control rats (Groups C and D, Fig. 3 C, D). Suppressor Cells Mediate Immune Tolerance Via Secretion of Immunosuppressive Cytokines ADOPTIVE TRANSFER OF TOLERANCE. Splenic lymphocytes from tolerized rats were capable of adoptively transferring the immune tolerance upon transplantation into naive rats. A total of 20 naive sublethally irradiated rats were infused with splenic lymphocytes taken from rats from each of donor groups B and D. Adoptive transfer of tolerance was seen only in rats (group E) that received splenocytes from donor rats fed with colitis-extracted proteins (group B). After rectal TNBS administration, these rats (group E) developed a much lesser degree of colitis, similar to that observed in tolerized rats from group A. By contrast, when lymphocytes from vehicle-fed control rats (group D) were used as donors for group F recipients, adoptive transfer of tolerance did not occur. A marked decrease in diarrhea was observed in tolerized rats from group E as compared with nontolerized controls from group F, throughout the study period. The percentage of colonic area exhibiting signs of injury was 33% versus 80%, in group E versus group F rats, respectively (p ⫽ 0.07, Fig. 4). All tested individual parameters, including intestinal and peritoneal adhesions, degree of mucosal edema, degree of colonic ulcerations, and colonic wall thickness, improved (Fig. 5). The average score of all macroscopic parameters tested improved from 2.65 in nontolerized rats (group F) to 0.62 in tolerized rats (group E, p ⫽ 0.003, Fig. 5). Furthermore, in tolerized rats from group E inflammatory response and
AJG – April, 2000
Oral Tolerance for Experimental Colitis
969
Figure 3. Effect of tolerization histological evaluation bowel mucosa 10 days after induction of colitis, showing marked reduction in inflammatory response and mucosal damage in tolerized (groups A, B) versus nontolerized controls (groups C, D). In 4/20 recipients tolerized by enteral administration of colitis-extracted proteins from group A, normal sections were observed. In the rest, only minimal lymphocytic infiltration was detected (3A, B). In contrast, severe inflammatory reaction (grade 3– 4) was observed in bowel specimens taken from nontolerized control rats from group B (3C, D). The sum of the pathological score was 1.34 versus 3.66 using standard pathological scores in tolerized versus nontolerized controls.
mucosal ulceration were minimal 10 days after colitis induction. In contrast, severe inflammatory reactions (grade 3– 4) were observed in bowel specimens from group F rats. The standard pathological score was 1.12 versus 3.86 in tolerized (group E, Fig. 6 A, B) versus nontolerized controls (group F, Fig. 6 C, D). SERUM TGF1 AND IFN␥ LEVELS. Serum TGF1 levels increased to 81 ⫹ 16 and 96 ⫹ 21 ng/ml 10 days after induction of colitis in tolerized rats from groups A and E, respectively, compared with 24 ⫹ 6 and 19 ⫹ 6 ng/ml in nontolerized control rats (Groups C and F, Fig. 7, p ⬍ 0.0005). Levels in normal untreated rats were 17–22 ng/ml. In contrast, serum IFN␥ levels markedly decreased in tolerized rats from groups A and E (37.8 ⫹ 4 and 28.7 ⫹ 8 pg/ml, respectively), compared with 154 ⫹ 28 and 182 ⫹ 23 pg/ml, in nontolerized rats from groups C and F, respectively (Fig. 7, p ⫽ 0.006). Levels in normal untreated rats were 11–20 pg/ml.
DISCUSSION In the present study, we used a model system for experimental colitis that employs rats treated with TNBS. We demonstrate that oral administration of colitis-extracted pro-
Figure 4. Adoptive transfer of tolerance. Naive sublethally irradiated rats were infused with splenic cell lymphocyte suspensions from five rats taken from donor rats in groups B and D. Adoptive transfer of the tolerance was seen only in rats from group E (white bars), which received splenocytes from rats fed with colitis-extracted proteins (group B). After rectal TNBS administration, these rats showed marked macroscopic improvement similar to that observed in the tolerized rats from group A. In contrast, when group D nontolerized lymphocytes were used as donors for group F (black bars), adoptive transfer of tolerance did not occur. Percentage of colonic area exhibiting signs of injury, as well as colon weight, decreased in tolerized versus nontolerized control rats (p ⫽ 0.07).
970
Ilan et al.
Figure 5. Effect of adoptive transfer of tolerance on macroscopic grading of colitis. Tested individual parameters included degree of colonic ulceration, intestine and peritoneal adhesions, wall thickness, degree of mucosal edema, and the sum of all macroscopic parameters, improved in rats from group E (white bars), which received splenocytes from tolerized donor rats fed with colitisextracted proteins (group B), compared with rats from group F (black bars), for which nontolerized rats from group D were used as lymphocyte donors (p ⫽ 0.003 for the sum of scores).
teins induces immune tolerance, downregulates the inflammatory immune response, and alleviates the colitis. A bystander effect, or the effect of surrogate antigens, cannot be
AJG – Vol. 95, No. 4, 2000
Figure 7. Effect of oral tolerization on serum TGF1 and IFN␥ levels. TGF1 levels increased to 81 ⫹ 16 ng/ml 10 days after induction of colitis in rats fed with colitis-extracted-proteins from group A (white bars), compared with 24 ⫹ 6 ng/ml in nontolerized control rats from Group C (black bars, p ⬍ 0.0005). Serum IFN␥ levels markedly decreased in orally tolerized rats from group C, 37.8 ⫹ 4 pg/ml (white bars), compared with 154 ⫹ 28 pg/ml in nontolerized controls from group E (black bars, p ⫽ 0.006).
ruled out, however, as normal colon proteins were not evaluated. Inflammatory bowel diseases are major illnesses of the gastrointestinal tract, the exact pathogenesis of which is unknown. The initial mucosal insult leads to production and release of proinflammatory cytokines with attraction of more immune cells and disruption of mucosal integrity. Exposure of specific epitopes on diseased bowel mucosa
Figure 6. Tolerized rats from group E (6A, B) exhibited none or only minimal histological evidence for disease 10 days after induction of colitis, with marked reduction in inflammatory response and mucosal ulceration, compared with nontolerized rats from group F (6C, D). The pathological score was 1.12 versus 3.86 in tolerized (group E) versus nontolerized controls (group F), respectively.
AJG – April, 2000
through toxic, infectious, or immune-mediated effects is a possible mechanism in the pathogenesis of IBD (1, 2). Patients with IBD have antibodies that react with colon tissue extracts, and specific colonic proteins are recognized by such antibodies (3, 8). Autoantibodies specific to a 95-kD microvillar actin-bundling protein, villin, are highly prevalent in IBD patients (23). Preferential expression of the chemokine ENA-78 in the intestinal epithelium of patients with IBD may result in attracting inflammatory cells to the diseased bowel (24). The immune response is the result of a balance between Th1 and Th2 subtypes of responses (25). The Th1-type response is involved in the pathogenesis of several autoimmune and chronic inflammatory disorders such as Crohn’s colitis (10, 26). It was recently shown, both in animals and humans, that antiinflammatory cytokines such as IL10 downregulate the proinflammatory effects of Th1-mediated cytokines, thereby alleviating the disease (27, 28). Thus IBD can be perceived as an imbalance between proinflammatory and antiinflammatory cytokines. Immunosuppressive regimens have shown clinical and histological improvement in IBD patients (1). However, these drugs are associated with short- and long-term complications. In contrast, induction of specific tolerance toward colitis antigens could potentially allow long-term alleviation of the disease, leaving the general immunological defense of the recipient intact. Oral tolerance is a recognized procedure for induction of antigen-specific peripheral immune hyporesponsiveness (17). Oral administration of antigens has been shown, both in animals and humans, to prevent or alleviate several autoimmune disorders such as collageninduced arthritis and experimental allergic encephalomyelitis (29 –32). A bystander effect involving reactivities to multiple autoantigens and surrogate antigens was shown to play a role in oral tolerance induction in several models (17). It involves regulatory cells secreting nonantigen-specific cytokines that suppress inflammation in the microenvironment, where the fed antigen is localized. A similar approach has been used in clinical trials in patients with multiple sclerosis, diabetes, and rheumatoid arthritis (25, 26, 33–34). Surrogate antigens, related to the disease-target epitopes, may have a similar immunomodulating effect. They imply that closely related proteins are being presented and processed by gut-associated lymphoid tissue in a similar way. Therefore, administration of an antigenically similar epitope— or of an epitope distinct from the disease-target antigen, but found in the target organ— can downregulate peripheral immune activation. When suppressor T-cells activated by APC presentation of these proteins encounter similar epitopes in the colon, they secrete antiinflammatory cytokines. These cytokines are not antigen-specific. Geographical proximity and/or antigenic similarity to the disease-target antigen may be held responsible for these effects. As normal colonic proteins were not evaluated in the present study, a bystander or surrogate antigen effect cannot be ruled out. We have previously shown that oral tolerance toward
Oral Tolerance for Experimental Colitis
971
adenoviral antigens effectively prevents the antivirus immune response (35). Furthermore, we have demonstrated that oral tolerance prevents secondary immune response in the presence of preexisting antiadenovirus immunity in animals. This approach was more potent than other modes of immune tolerance induction to adenoviral antigens (20, 35– 39). Mucosal Th2/Th3-like cells, secreting TGF, can be generated by intermittent feeding with low doses of antigen, and have been considered to play a role in antigen-specific suppressor activity and bystander tolerance (16, 17). On the other hand, we have shown that the liver, and particularly Kupffer cells, are required for tolerance induction (41). The suppressor cells responsible for maintenance of tolerance reside in the peripheral lymphatic system rather than in the bowel wall (35, 41). Administration of anti-TGF1 has been shown to abrogate oral tolerance in several model systems (40). The results of the present study, using low-dose antigen feeding, favor tolerance induction by an active suppression mechanism. Immune tolerance was achieved by generation of immunomodulatory cells secreting cytokines such as TGF, along with downregulation of IFN␥ and other Th1type cytokine secretion. Adoptive transfer of tolerance by transplantation of splenocytes from tolerized donors to sublethally irradiated recipients confirms the presence of suppressor cells in this setting. The immune response to rectal administration of TNBS was shown to be driven by a Th1 cell response. Previous studies showed that in vitro stimulated cells from inflamed mucosa produce increased amounts of IFN␥ and IL2, and reduced amounts of IL4 (9, 19). Moreover, it was shown that if anti-IL12, anti-␣ or anti-IFN␥ antibodies were administered at the time of colitis induction, the disease could be prevented (19, 42). Our results show that induction of oral tolerance using colitisextracted protein leads to an immunological shift from a Th1- to a Th3-type immune response. This shift causes a change in cytokine secretion from proinflammatory to antiinflammatory ones, with marked alleviation of clinical, macroscopic, and microscopic manifestations of IBD. In several models such as in uveitis, induction of a bystander effect was difficult to achieve (43– 45). In the experimental colitis model, tolerance induced toward haptenized proteins, or toward TNBS, which are bystander antigens, had some beneficial effect (19, 46). The present study shows for the first time that feeding of colon antigens extracted from a diseased bowel induced anticolon immunodownregulation, with marked alleviation of clinical and histological parameters of colitis. These results suggest that TNBS instillation exposed or generated target epitopes that are important in the pathogenesis of experimental colitis induction. Feeding these antigens induced immune tolerance, while shifting the immune response from a Th1 to a Th3 type, thus changing the proinflammatory/antiinflammatory cytokine balance and alleviating the colitis. As the CEP being fed includes many unidentified proteins, the results of
972
Ilan et al.
the present study do not enable us to draw final conclusions as to the exact mechanism of tolerance induction. In conclusion, induction of immune tolerance against colitis-target antigens can be achieved by feeding rats with colitis-extracted proteins. Immune tolerance induction downregulates the inflammatory immune response, permitting long-term alleviation of experimental colitis. This mode of tolerance induction is mediated by negative immunoregulatory cells secreting inhibitory cytokines. This method has the potential to be used in clinical practice to tolerize IBD patients against colon antigens associated with the disease. It presents the possibility of providing effective long-term therapy for these disorders, and for understanding the immune target antigens involved in the pathogenesis of IBD.
ACKNOWLEDGMENTS This work was supported in part by grants from The United States-Israel Binational Science Foundation 0060 (to Y.I. and J.R.C.). Additional support was provided by a grant from Hadasit-Yissum (to Y.I.), a grant from ENZO Biochem Inc., NY (to Y.I.); and grants ROI-DK 46057 (to J.R.C.), RO1-DK 39137 (to N.R.C.), and P30-DK 41296 (Liver Research Core Center, to J.R.C.). Reprint requests and correspondence: Yaron Ilan, M.D., Liver Unit, Department of Medicine, Hadassah University Hospital, P.O.B 12000 Jerusalem, Israel IL-91120. Received Mar. 16, 1999; accepted Aug. 18, 1999.
REFERENCES 1. Podolsky DK. Inflammatory bowel disease. N Engl J Med 1991;325:928 –35. 2. Strober W, Neurath MF. Immunological diseases of the gastrointestinal tract. In: Rich RR, ed. Clinical immunology. St. Louis: CV Mosby, 1995:1401–28. 3. Hibi S, Also N, Ishikawa M, et al. Circulating antibodies to the surface antigens on colon epithelial cells in ulcerative colitis. Clin Exp Immunol 1983;54:163– 8. 4. Das KM, Vecchi M, Sakemaki S. A shared and unique epitope(s) on human colon, skin and biliary epithelium detected by a monocoloncal antibody. Gastroenterology 1990; 98:464 –9. 5. Chiba M, Bartnik W, Remine SG, et al. Human colonic intraepithelial and lamina peripheral lymphocytes: Cytotoxicitiy in vitro and the potential effects of the isolation method on their functional properties. Gut 1981;22:177– 82. 6. Raedler A, Fraenkel S, Klose G, et al. Increased numbers of peripheral T cells in inflammatory bowel disease displaying the T9 antigen and Fc ␥ receptors. Clin Exp Immunol 1985; 60:518 –26. 7. Dasgupta A, Mandel A, Das KM. Circulating immunoglobulin G1 in patients with ulcerative colitis against the colonic epithelial protein detected by a novel monoclonal antibody. Gut 1994;35:1712–7. 8. Takahashi F, Das KM. Isolation, and characterization of a colonic autoantigen specifically recognized by colon tissuebound immunoglobulin G from idiopathic ulcerative colitis. J Clin Invest 1985;76:311– 8.
AJG – Vol. 95, No. 4, 2000
9. Neurath M, Kelasall BL, Presky DH, et al. Experimental granulomatous colitis in mice is abrogated by induction of TGF-mediated oral tolerance. J Exp Med 1996;183:2605–16. 10. Strober W, Kelsall B, Fuss L, et al. Reciprocal IFN-␥, and TGF- responses regulate the occurrence of mucosal inflammation. Immunol Today 1997;18:61– 4. 11. Hollander GA, Si SJ, Mizoguchi E, et al. Severe colitis in mice with aberrant thymic selection. Immunity 1995;3:27–38. 12. Sadlack B, Merz H, Schorle H, et al. Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell 1993;75:253– 61. 13. Hammer RE, Maika SD, Richardson JA, et al. Spontaneous inflammatory disease in transgenic rats expressing HLA B27 and human 2m: An animal model of HLAB-27-associated human disorders. Cell 1990;63:1099 –1112. 14. Kuhn RI, Lohler D, Rennick. K, et al. Interleukin 10 deficient mice develop chronic enterocolitis. Cell 1993;75:263–74. 15. Powrie F, Leach MW, Mauze S, et al. Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4⫹T cells. Immunity 1994; 1:553– 62. 16. Weiner H. Oral tolerance. Proc Natl Acad Sci USA 1994;91: 10762–5. 17. Weiner HL. Oral tolerance: Immune mechanisms and treatment of autoimmune diseases. Immunol Today 1997;18:335– 43. 18. Yoshida T, Hachimura S, Kaminogawa S. The oral administration of low-dose antigen induces activation followed by tolerization, while high-dose antigen induces tolerance without activation. Clin Immunol Immunopathol 1997;82:207–15. 19. Neurath MF, Fuss I, Kelsall BL, et al. Antibodies to IL-12 abrogate established experimental colitis in mice. J Exp Med 1995;182:1281–90. 20. Ilan Y, Sauter B, Roy Chowdhury N, et al. Oral tolerization to adenoviral proteins permits repeated adenovirus-mediated gene therapy in rats with pre-existing immunity to adenovirus. Hepatology 1998;27:1368 –76. 21. Shouval D, Adler R, Ilan Y. Adoptive transfer of immunity to hepatitis B virus in nude mice following T-cell depleted bone marrow transplantation. Hepatology 1993;17:955–9. 22. Ilan Y, Prakash R, Jona VK, et al. Adoptive transfer of tolerance from rats tolerized to adenoviral antigens allows long term adenovirus-mediated gene therapy in Gunn rats. Hepatology 1996;24:304A. 23. Rimm DL, Holland E, Tracey E, et al. Autoantibodies specific for villin found in patients with colon cancer and other colitides. Digest Dis Sci 1995;40:389 –95. 24. Z’graggen K, Walz A, Mazzucchelli L, et al. The C-X-O chemokine ENA-78 is preferentially expressed in intestinal epithelium in inflammatory bowel disease. Gastroenterology 1997;113:808 –16. 25. Adorini L, Francesco S. Pathogenesis and immunotherapy of autoimmune diseases. Immunol Today 1997;18:209 –11. 26. Mizoguchi A, Mizoguchi E, Chiba C, et al. Cytokine imbalance and autoantibody production in T cell receptor-␣ mutant mice with inflammatory bowel disease. J Exp Med 1996;183: 847–56. 27. Madsen KL, Lewis Simon A, Tavernini MM, et al. Interleukin 10 prevents cytokine-induced disruption of T84 barrier integrity and limits chloride secretion. Gastroenterology 1997;113: 151–9. 28. Van Deventer Sander J, Charles O, Fedorak RN. Multiple doses of intravenous interleukin 10 in steroid refractory Crohn’s disease. Gastroenterology 1997;113:383–9. 29. Esbjorn T, Karlsson M, Dahlman-Hoglund A, et al. Oral tolerance in experimental animals. Int Arch Allergy Immunol 1997;113:219 –23.
AJG – April, 2000
30. Von Herrath MG, Dyrberg T, Oldstone MBA. Oral insulin treatment suppresses virus-induced antigen-specific destruction of  cells and prevents autoimmune diabetes in transgenic mice. J Clin Invest 1996;98:1324 –31. 31. Hancock W, Polansli M, Zhang J, et al. Suppression of insulitis in NOD mice by oral insulin administration is associated with selective expression of IL-4 –10, TGF- and prostaglandine-E. Am J Path 1993;147:1193–7. 32. Javed NH, Gienapp IE, Cox KL, et al. Exquisite peptide specificity of oral tolerance in experimental autoimmune encephalomyelitis. J Immunol 1995;155:1599 –1605. 33. Weiner HL, Mackin GA, Matsui M, et al. Double-blind pilot trial of oral tolerization with myelin antigen in multiple sclerosis. Science 1993;261:1321– 4. 34. Trentham DE, Dynesius-Trentham RA, Orav EJ, et al. Effects of oral administration of type II collagen on rheumatoid arthritis. Science 1993;261:1727–30. 35. Ilan Y, Prakash R, Davidson A, et al. Oral tolerization to adenoviral antigens permits long term gene expression using recombinant adenoviral vectors. J Clin Invest 1997;99:1098 – 1106. 36. Takahashi M, Ilan Y, Sengupta K, et al. Induction of tolerance to recombinant adenoviruses by injection into newborn rats: Long-term amelioration of hyperbilirubinemia in Gunn rats. J Biol Chem 1996;271:26536 – 42. 37. Ilan Y, Jona VK, Sengupta K, et al. Transient immunosuppression with FK506 permits long term expression of therapeutic genes introduced into the liver using recombinant adenoviruses. Hepatology 1997;26:949 –56.
Oral Tolerance for Experimental Colitis
973
38. Ilan Y, Droguett G, Roy Chowdhury N, et al. Insertion of the adenoviral E3 region into a recombinant viral vector prevents antiviral humoral and cellular immune responses and permits long term gene expression. Proc Nat Acad Sci USA 1997;94: 2587–92. 39. Ilan Y, Attavar P, Takahashi M, et al. Induction of central tolerance by intrathymic inoculation of adenoviral antigens into the host thymus permits long term gene therapy in Gunn rats. J Clin Invest 1996;98:2640 –7. 40. Fontana A, Sonstan K, Frei U, et al. Modulation of the immune response by TGF. Int Arch Allergy Immunol 1992;99:1–7. 41. Ilan Y, Roy Chowdhury N, Sauter BS, et al. A critical role of the liver in inducing oral tolerance to adenoviral vectors for long term gene therapy. Gastroenterology 1998;114:260A. 42. Qiu CJ. Pfeiffer H, Keith JC. Protection by recombinant human interleukin-11 against experimental TNB-induced colitis in rats. Dig Dis Sci 1996;41:1625–30. 43. Carvalho BA, Verdolin SA, Vaz NM. Indirect effects of oral tolerance cannot be ascribed to bystander suppression. Scand J Immunol 1997;45:276 – 81. 44. Blanas E, Carbone FR, Allison J, et al. Induction of autoimmune diabetes by oral administration of autoantigen. Science 1996;274:1707–9. 45. Christian K, Hiroshi K, Carbone FR, et al. Class I-restricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive CD8⫹T cells. J Exp Med 1997;186:239 – 45. 46. Sharmanov AT, Elson CO, Tennyson GS, et al. TNBS-specific oral tolerance protects from hapten induced colitis. Gastroenterology 1995;106:335A.
Vol. 95, No. 4, 2000 ISSN 0002-9270/00/$20.00 PII S0002-9270(00)00727-9
Treatment of Experimental Colitis by Oral Tolerance Induction: A Central Role for Suppressor Lymphocytes Yaron Ilan, M.D., Sarah Weksler-Zangen, Ph.D., Shomron Ben-Horin, M.D., Judith Diment, M.D., Bernhard Sauter, M.D., Elazar Rabbani, Ph.D., Dean Engelhardt, Ph.D., Namita Roy Chowdhury, Ph.D., Jayanta Roy Chowdhury, M.D., and Eran Goldin, M.D. Gastroenterology and Liver Units, Department of Medicine, and Department of Pathology, Hadassah University Hospital, Jerusalem, Israel; Marion Bessin Liver Research Center, Albert Einstein College of Medicine, New York; and ENZO Bichem Inc., New York
OBJECTIVE: Inflammatory bowel diseases (IBD) are immune-mediated disorders wherein an imbalance between proinflammatory (Th1) and antiinflammatory (Th2) cytokines is thought to play a role in the pathogenesis. The aim of this study was to test whether induction of oral tolerance to proteins extracted from inflammatory colon alleviates experimental colitis, and whether oral tolerization mediated by suppressor cells can induce immune tolerance. METHODS: Colitis was induced in rats by intracolonic instillation of trinitrobenzenesulfonic acid (TNBS). Rats received five oral doses of colonic proteins extracted from TNBS-colitis colonic wall. Splenocytes harvested from tolerized and control rats were transplanted into irradiated naive rats. RESULTS: Feeding of colitis-extracted proteins ameliorated colonic inflammation, as shown by reduction of colonic ulcerations, as well as decreased diarrhea, intestine and peritoneal adhesions, wall thickness, and edema. A marked reduction of the fraction of injured colonic area and colon weight, and decrease in colon weight, were observed in tolerized rats versus controls. Histological parameters for colitis were markedly improved in tolerized animals that showed significant reduction in inflammatory response and mucosal ulcerations. Tolerized rats developed an increase in TGF1 and a decrease in IFN␥ serum levels. TNBS-induced colitis was significantly attenuated in naive recipients of splenocytes from tolerized rats, compared with rats that received splenocytes from control donors. CONCLUSIONS: Induction of oral tolerance to colitis-extracted proteins downregulates the anticolon immune response, thereby ameliorating experimental colitis. Suppressor lymphocytes mediate the tolerance by induction of a shift from a proinflammatory to an antiinflammatory immune response. (Am J Gastroenterol 2000;95:966 –973. © 2000 by Am. Coll. of Gastroenterology)
INTRODUCTION Inflammatory bowel diseases (IBD) are common GI disorders. Although the cause of these disorders remains unknown, various features suggest immune system involvement in their pathogenesis. Several extraintestinal manifestations that accompany IBD are autoimmune phenomena and immunosuppressive agents are used to alleviate the disease (1, 2). Patients with IBD have antibodies against components of colon cells and several different bacterial antigens (3, 4). Abnormalities of T-cell–mediated immunity, including cutaneous anergy and diminished responsiveness to T-cell stimuli, have also been described in these patients (5, 6). In addition, changes in mucosal-cell–mediated immunity were identified, including increased concentrations of mucosal IgG cells and changes in T-cell subsets, suggesting antigen stimulation (7, 8). Exposure of target antigens after infectious, immune, or toxic damage leads to activation of mucosal immune cells, resulting in cytokines that lead to mucosal inflammatory response (9). Secretion of proinflammatory cytokines such as IFN␥ contributes to an increase in mucosal permeability, and has been described in animal models of IBD (10). Several animal models of chronic inflammatory immune response have been established. These include rats carrying the transgenes for HLA B27 and 2-microglobulin, and mice in which genes for IL2, IL10, and ␣ and  chains of T-cell receptors are absent (11–14). A Th1-mediated granulomatous colitis model has been established by the adoptive transfer of normal CD45RB T-cells from Balb/C mice into CB-17 scid mice (15). Thus, IBD can be envisioned as an imbalance between proinflammatory and antiinflammatory cytokines. Oral tolerance is the induction of immunological hyporesponsiveness toward specific antigens or, through a bystander effect, toward other antigens present at the site, via secretion of immunosuppressive cytokines (16). The mechanism of tolerance may depend on antigen dose. Lower doses induce tolerance through generation of negative immunoregulatory cells, whereas higher doses lead to clonal deletion (17). Both in animals and in humans, tolerance
AJG – April, 2000
Oral Tolerance for Experimental Colitis
Table 1. Experimental and Control Groups Group
Antigen Fed
Donor Splenocytes
A B C D E F
CEP CEP BSA BSA None None
None None None None Group B Group D
CEP ⫽ colitis-extracted proteins; BSA ⫽ bovine serum albumin.
induction is associated with a Th2-type immune response, leading to secretion of immunosuppressive cytokines such as TGF1, IL4, and IL10 (18). In the present study we used a model system that employs normal rats treated with 2,4,6trinitrobenzene sulfonic acid (TNBS) (19). This type of experimental colitis, similar to the human disease, is a Th1-mediated immune disorder resulting in life-long inflammatory response against the colon. We have recently shown that oral tolerance can abrogate antigen-specific preexisting immunity (20). Therefore, we sought to evaluate whether induction of oral tolerance toward colitis-extracted proteins could be used to abrogate host anticolon immune response for treating experimental colitis.
MATERIALS AND METHODS Animals Normal inbred, male Sabra rats (200 –300 g) were obtained from the Animal Core of the Hadassah-Hebrew University Medical School. Rats were maintained on standard laboratory chow and kept in 12-h light/dark cycles. Induction of Colitis We induced TNBS-colitis by rectal instillation of TNBS, 25 mg/rat, dissolved in 1 ml of 50% ethanol (19). Preparation and Administration of the Oral Antigen Colon was removed from TNBS-induced– colitis rats, and was cut into small strips and mechanically homogenized. After filtration through a 40-m nylon cell strainer, the intact cells were spun down and removed. Proteins were quantified using protein assay kit (Biorad, Munchen, Germany). Colitis-extracted proteins (CEP) were introduced to the experimental groups described, using a feeding atraumatic needle every other day for 11 days (a total of five doses). Experimental Groups Six groups of rats, consisting of 10 animals each, were studied (Table 1). Groups A and B included rats fed with colitis-extracted proteins (500 g/rat) every other day starting 24 h after colitis induction. Two groups of rats, Groups C and D, were used as controls and were fed a similar dose of bovine serum albumin (BSA) alone. Adoptive transfer experiments were performed to evaluate the mechanism of tolerance, and distinguish between induction of suppressor cells and clonal inactivation or deletion. Rats in groups B
967
and D served as tolerized and control (nontolerized) splenocyte donors for recipient animals in groups E and F, respectively, as described later. Evaluation of the Effects of Tolerance Induction on Experimental Colitis CLINICAL ASSESSMENT OF COLITIS. Diarrhea was followed daily throughout the study. MACROSCOPIC SCORE OF COLITIS. Colitis assessment was performed 10 days after colitis induction using the following standard parameters (19). Rats were killed and colon was removed. The percentage of the total colonic wall that was injured and colon weight were measured. Four additional parameters were determined, namely, degree of colonic ulcerations; intestine and peritoneal adhesions; wall thickness; and degree of mucosal edema. Each parameter was graded on a scale from 0 (completely normal) to 4 (most severe) by two experienced blinded examiners. GRADING OF HISTOLOGICAL LESIONS. For histological evaluation of inflammation, distal colonic tissue (last 10 cm) was removed and fixed in 10% formaldehyde. Five paraffin sections from each rat were then stained with hematoxylin-eosin according to standard techniques. The degree of inflammation on microscopic cross sections of the colon was graded semiquantitatively from 0 to 4 as follows (9, 19): Grade 0: normal with no signs of inflammation; Grade 1: very low level of leukocyte infiltration; Grade 2: low level of leukocyte infiltration; and Grade 3: high level of infiltration with high vascular density, and thickening of the bowel wall; Grade 4: transmural infiltrates with loss of goblet cells, high vascular density, wall thickening, and disruption of normal bowel architecture. Grading was performed by two experienced blinded pathologists. Evaluation of the Mechanism of Immune Tolerance Induction ADOPTIVE TRANSFER OF TOLERANCE. To determine whether splenic lymphocytes from tolerized rats are capable of producing tolerance upon transplantation into naive rats, donor rats from groups B and D (five rats from each group) were killed 10 days after induction of colitis, and single suspensions of lymphocytes derived from the spleen were prepared as described (21, 22). Cells were resuspended in phosphate-buffered saline (PBS) immediately before transplantation. Recipient rats were sublethally irradiated with 300 rad total body irradiation, 24 h before intravenous injection of 1 ⫻ 108 donor cells in 0.5 ml PBS. Twenty rats were studied. Ten rats (Group E) received cells from group B donor rats, and 10 rats (Group F) received splenocytes from group D rats. All rats were treated with TNBS enemas, 24 h after cell transplantation. Clinical, macroscopic, and histological parameters for colitis were determined 10 days after colitis induction, as described earlier.
968
Ilan et al.
Figure 1. Effect of tolerance induction toward colitis-extracted proteins on experimental colitis (10 days after colitis induction). Significant decrease in percentage of colonic area exhibiting signs of injury, as well as colon weight, was observed in tolerized groups A and B (white bars), compared with nontolerized controls C and D (black bars, p ⫽ 0.14, and p ⫽ 0.02, respectively).
SERUM TGF1 AND IFN␥ LEVELS. TGF1 and IFN␥ levels were measured by a so-called “sandwich” enzymelinked immunosorbent assay (ELISA) using Genzyme Diagnostics kits (Genzyme Diagnostics, MA) according to the manufacturer’s instructions. Serum levels were measured in all rats from tolerized and nontolerized control rats 10 days after colitis induction.
RESULTS Oral Tolerization Toward Colitis-Extracted Proteins Alleviates Experimental Colitis CLINICAL ASSESSMENT. A marked decrease in diarrhea was observed daily in tolerized rats from groups A and B as compared with nontolerized controls in groups C and D throughout the study period. MACROSCOPIC GRADING. The percentage of colonic area exhibiting macroscopic injury decreased to 57% in tolerized rats versus 72% in controls (p ⫽ 0.14), and the mean colon weight was 1.6 g compared with 2.2 g in controls (p ⫽ 0.02, Fig. 1). In tolerized rats, degree of colonic ulceration, intestine and peritoneal adhesions, and wall thickness improved significantly (p ⬍ 0.05, using Mann-Whitney Rank Sum test, Fig. 2). The total scores of all macroscopic parameters tested were 1.01 in tolerized rats, compared with 2.11 in nontolerized controls (p ⫽ 0.001, Fig. 2). HISTOLOGICAL LESIONS. Histological evaluation of bowel tissues by standard pathological scoring, 10 days after induction of colitis, showed marked reduction in inflammatory response and mucosal ulceration (1.34 vs 3.66 in tolerized vs nontolerized controls, receptively). In four of 20 recipients tolerized by enteral administration of colitis-extracted proteins the colons were nearly normal by histological examination. In the rest of these rats, only minimal lymphocytic infiltration was detected (Groups A and B, Fig.
AJG – Vol. 95, No. 4, 2000
Figure 2. Effect of tolerization on macroscopic grading of colitis. Parameters tested were graded on a scale from 0 (completely normal) to 4 (most severe), including degree of colonic ulceration, intestine and peritoneal adhesions, wall thickness, and degree of mucosal edema. All tested parameters had less inflammation in tolerized rats than in controls (p ⬍ 0.05). The average score of all macroscopic parameters improved from 2.11 in nontolerized controls (black bars) to 1.01 in tolerized animals (white bars, p ⫽ 0.001).
3 A, B). In contrast, severe inflammatory reaction (grade 3– 4) was observed in bowel specimens taken from nontolerized control rats (Groups C and D, Fig. 3 C, D). Suppressor Cells Mediate Immune Tolerance Via Secretion of Immunosuppressive Cytokines ADOPTIVE TRANSFER OF TOLERANCE. Splenic lymphocytes from tolerized rats were capable of adoptively transferring the immune tolerance upon transplantation into naive rats. A total of 20 naive sublethally irradiated rats were infused with splenic lymphocytes taken from rats from each of donor groups B and D. Adoptive transfer of tolerance was seen only in rats (group E) that received splenocytes from donor rats fed with colitis-extracted proteins (group B). After rectal TNBS administration, these rats (group E) developed a much lesser degree of colitis, similar to that observed in tolerized rats from group A. By contrast, when lymphocytes from vehicle-fed control rats (group D) were used as donors for group F recipients, adoptive transfer of tolerance did not occur. A marked decrease in diarrhea was observed in tolerized rats from group E as compared with nontolerized controls from group F, throughout the study period. The percentage of colonic area exhibiting signs of injury was 33% versus 80%, in group E versus group F rats, respectively (p ⫽ 0.07, Fig. 4). All tested individual parameters, including intestinal and peritoneal adhesions, degree of mucosal edema, degree of colonic ulcerations, and colonic wall thickness, improved (Fig. 5). The average score of all macroscopic parameters tested improved from 2.65 in nontolerized rats (group F) to 0.62 in tolerized rats (group E, p ⫽ 0.003, Fig. 5). Furthermore, in tolerized rats from group E inflammatory response and
AJG – April, 2000
Oral Tolerance for Experimental Colitis
969
Figure 3. Effect of tolerization histological evaluation bowel mucosa 10 days after induction of colitis, showing marked reduction in inflammatory response and mucosal damage in tolerized (groups A, B) versus nontolerized controls (groups C, D). In 4/20 recipients tolerized by enteral administration of colitis-extracted proteins from group A, normal sections were observed. In the rest, only minimal lymphocytic infiltration was detected (3A, B). In contrast, severe inflammatory reaction (grade 3– 4) was observed in bowel specimens taken from nontolerized control rats from group B (3C, D). The sum of the pathological score was 1.34 versus 3.66 using standard pathological scores in tolerized versus nontolerized controls.
mucosal ulceration were minimal 10 days after colitis induction. In contrast, severe inflammatory reactions (grade 3– 4) were observed in bowel specimens from group F rats. The standard pathological score was 1.12 versus 3.86 in tolerized (group E, Fig. 6 A, B) versus nontolerized controls (group F, Fig. 6 C, D). SERUM TGF1 AND IFN␥ LEVELS. Serum TGF1 levels increased to 81 ⫹ 16 and 96 ⫹ 21 ng/ml 10 days after induction of colitis in tolerized rats from groups A and E, respectively, compared with 24 ⫹ 6 and 19 ⫹ 6 ng/ml in nontolerized control rats (Groups C and F, Fig. 7, p ⬍ 0.0005). Levels in normal untreated rats were 17–22 ng/ml. In contrast, serum IFN␥ levels markedly decreased in tolerized rats from groups A and E (37.8 ⫹ 4 and 28.7 ⫹ 8 pg/ml, respectively), compared with 154 ⫹ 28 and 182 ⫹ 23 pg/ml, in nontolerized rats from groups C and F, respectively (Fig. 7, p ⫽ 0.006). Levels in normal untreated rats were 11–20 pg/ml.
DISCUSSION In the present study, we used a model system for experimental colitis that employs rats treated with TNBS. We demonstrate that oral administration of colitis-extracted pro-
Figure 4. Adoptive transfer of tolerance. Naive sublethally irradiated rats were infused with splenic cell lymphocyte suspensions from five rats taken from donor rats in groups B and D. Adoptive transfer of the tolerance was seen only in rats from group E (white bars), which received splenocytes from rats fed with colitis-extracted proteins (group B). After rectal TNBS administration, these rats showed marked macroscopic improvement similar to that observed in the tolerized rats from group A. In contrast, when group D nontolerized lymphocytes were used as donors for group F (black bars), adoptive transfer of tolerance did not occur. Percentage of colonic area exhibiting signs of injury, as well as colon weight, decreased in tolerized versus nontolerized control rats (p ⫽ 0.07).
970
Ilan et al.
Figure 5. Effect of adoptive transfer of tolerance on macroscopic grading of colitis. Tested individual parameters included degree of colonic ulceration, intestine and peritoneal adhesions, wall thickness, degree of mucosal edema, and the sum of all macroscopic parameters, improved in rats from group E (white bars), which received splenocytes from tolerized donor rats fed with colitisextracted proteins (group B), compared with rats from group F (black bars), for which nontolerized rats from group D were used as lymphocyte donors (p ⫽ 0.003 for the sum of scores).
teins induces immune tolerance, downregulates the inflammatory immune response, and alleviates the colitis. A bystander effect, or the effect of surrogate antigens, cannot be
AJG – Vol. 95, No. 4, 2000
Figure 7. Effect of oral tolerization on serum TGF1 and IFN␥ levels. TGF1 levels increased to 81 ⫹ 16 ng/ml 10 days after induction of colitis in rats fed with colitis-extracted-proteins from group A (white bars), compared with 24 ⫹ 6 ng/ml in nontolerized control rats from Group C (black bars, p ⬍ 0.0005). Serum IFN␥ levels markedly decreased in orally tolerized rats from group C, 37.8 ⫹ 4 pg/ml (white bars), compared with 154 ⫹ 28 pg/ml in nontolerized controls from group E (black bars, p ⫽ 0.006).
ruled out, however, as normal colon proteins were not evaluated. Inflammatory bowel diseases are major illnesses of the gastrointestinal tract, the exact pathogenesis of which is unknown. The initial mucosal insult leads to production and release of proinflammatory cytokines with attraction of more immune cells and disruption of mucosal integrity. Exposure of specific epitopes on diseased bowel mucosa
Figure 6. Tolerized rats from group E (6A, B) exhibited none or only minimal histological evidence for disease 10 days after induction of colitis, with marked reduction in inflammatory response and mucosal ulceration, compared with nontolerized rats from group F (6C, D). The pathological score was 1.12 versus 3.86 in tolerized (group E) versus nontolerized controls (group F), respectively.
AJG – April, 2000
through toxic, infectious, or immune-mediated effects is a possible mechanism in the pathogenesis of IBD (1, 2). Patients with IBD have antibodies that react with colon tissue extracts, and specific colonic proteins are recognized by such antibodies (3, 8). Autoantibodies specific to a 95-kD microvillar actin-bundling protein, villin, are highly prevalent in IBD patients (23). Preferential expression of the chemokine ENA-78 in the intestinal epithelium of patients with IBD may result in attracting inflammatory cells to the diseased bowel (24). The immune response is the result of a balance between Th1 and Th2 subtypes of responses (25). The Th1-type response is involved in the pathogenesis of several autoimmune and chronic inflammatory disorders such as Crohn’s colitis (10, 26). It was recently shown, both in animals and humans, that antiinflammatory cytokines such as IL10 downregulate the proinflammatory effects of Th1-mediated cytokines, thereby alleviating the disease (27, 28). Thus IBD can be perceived as an imbalance between proinflammatory and antiinflammatory cytokines. Immunosuppressive regimens have shown clinical and histological improvement in IBD patients (1). However, these drugs are associated with short- and long-term complications. In contrast, induction of specific tolerance toward colitis antigens could potentially allow long-term alleviation of the disease, leaving the general immunological defense of the recipient intact. Oral tolerance is a recognized procedure for induction of antigen-specific peripheral immune hyporesponsiveness (17). Oral administration of antigens has been shown, both in animals and humans, to prevent or alleviate several autoimmune disorders such as collageninduced arthritis and experimental allergic encephalomyelitis (29 –32). A bystander effect involving reactivities to multiple autoantigens and surrogate antigens was shown to play a role in oral tolerance induction in several models (17). It involves regulatory cells secreting nonantigen-specific cytokines that suppress inflammation in the microenvironment, where the fed antigen is localized. A similar approach has been used in clinical trials in patients with multiple sclerosis, diabetes, and rheumatoid arthritis (25, 26, 33–34). Surrogate antigens, related to the disease-target epitopes, may have a similar immunomodulating effect. They imply that closely related proteins are being presented and processed by gut-associated lymphoid tissue in a similar way. Therefore, administration of an antigenically similar epitope— or of an epitope distinct from the disease-target antigen, but found in the target organ— can downregulate peripheral immune activation. When suppressor T-cells activated by APC presentation of these proteins encounter similar epitopes in the colon, they secrete antiinflammatory cytokines. These cytokines are not antigen-specific. Geographical proximity and/or antigenic similarity to the disease-target antigen may be held responsible for these effects. As normal colonic proteins were not evaluated in the present study, a bystander or surrogate antigen effect cannot be ruled out. We have previously shown that oral tolerance toward
Oral Tolerance for Experimental Colitis
971
adenoviral antigens effectively prevents the antivirus immune response (35). Furthermore, we have demonstrated that oral tolerance prevents secondary immune response in the presence of preexisting antiadenovirus immunity in animals. This approach was more potent than other modes of immune tolerance induction to adenoviral antigens (20, 35– 39). Mucosal Th2/Th3-like cells, secreting TGF, can be generated by intermittent feeding with low doses of antigen, and have been considered to play a role in antigen-specific suppressor activity and bystander tolerance (16, 17). On the other hand, we have shown that the liver, and particularly Kupffer cells, are required for tolerance induction (41). The suppressor cells responsible for maintenance of tolerance reside in the peripheral lymphatic system rather than in the bowel wall (35, 41). Administration of anti-TGF1 has been shown to abrogate oral tolerance in several model systems (40). The results of the present study, using low-dose antigen feeding, favor tolerance induction by an active suppression mechanism. Immune tolerance was achieved by generation of immunomodulatory cells secreting cytokines such as TGF, along with downregulation of IFN␥ and other Th1type cytokine secretion. Adoptive transfer of tolerance by transplantation of splenocytes from tolerized donors to sublethally irradiated recipients confirms the presence of suppressor cells in this setting. The immune response to rectal administration of TNBS was shown to be driven by a Th1 cell response. Previous studies showed that in vitro stimulated cells from inflamed mucosa produce increased amounts of IFN␥ and IL2, and reduced amounts of IL4 (9, 19). Moreover, it was shown that if anti-IL12, anti-␣ or anti-IFN␥ antibodies were administered at the time of colitis induction, the disease could be prevented (19, 42). Our results show that induction of oral tolerance using colitisextracted protein leads to an immunological shift from a Th1- to a Th3-type immune response. This shift causes a change in cytokine secretion from proinflammatory to antiinflammatory ones, with marked alleviation of clinical, macroscopic, and microscopic manifestations of IBD. In several models such as in uveitis, induction of a bystander effect was difficult to achieve (43– 45). In the experimental colitis model, tolerance induced toward haptenized proteins, or toward TNBS, which are bystander antigens, had some beneficial effect (19, 46). The present study shows for the first time that feeding of colon antigens extracted from a diseased bowel induced anticolon immunodownregulation, with marked alleviation of clinical and histological parameters of colitis. These results suggest that TNBS instillation exposed or generated target epitopes that are important in the pathogenesis of experimental colitis induction. Feeding these antigens induced immune tolerance, while shifting the immune response from a Th1 to a Th3 type, thus changing the proinflammatory/antiinflammatory cytokine balance and alleviating the colitis. As the CEP being fed includes many unidentified proteins, the results of
972
Ilan et al.
the present study do not enable us to draw final conclusions as to the exact mechanism of tolerance induction. In conclusion, induction of immune tolerance against colitis-target antigens can be achieved by feeding rats with colitis-extracted proteins. Immune tolerance induction downregulates the inflammatory immune response, permitting long-term alleviation of experimental colitis. This mode of tolerance induction is mediated by negative immunoregulatory cells secreting inhibitory cytokines. This method has the potential to be used in clinical practice to tolerize IBD patients against colon antigens associated with the disease. It presents the possibility of providing effective long-term therapy for these disorders, and for understanding the immune target antigens involved in the pathogenesis of IBD.
ACKNOWLEDGMENTS This work was supported in part by grants from The United States-Israel Binational Science Foundation 0060 (to Y.I. and J.R.C.). Additional support was provided by a grant from Hadasit-Yissum (to Y.I.), a grant from ENZO Biochem Inc., NY (to Y.I.); and grants ROI-DK 46057 (to J.R.C.), RO1-DK 39137 (to N.R.C.), and P30-DK 41296 (Liver Research Core Center, to J.R.C.). Reprint requests and correspondence: Yaron Ilan, M.D., Liver Unit, Department of Medicine, Hadassah University Hospital, P.O.B 12000 Jerusalem, Israel IL-91120. Received Mar. 16, 1999; accepted Aug. 18, 1999.
REFERENCES 1. Podolsky DK. Inflammatory bowel disease. N Engl J Med 1991;325:928 –35. 2. Strober W, Neurath MF. Immunological diseases of the gastrointestinal tract. In: Rich RR, ed. Clinical immunology. St. Louis: CV Mosby, 1995:1401–28. 3. Hibi S, Also N, Ishikawa M, et al. Circulating antibodies to the surface antigens on colon epithelial cells in ulcerative colitis. Clin Exp Immunol 1983;54:163– 8. 4. Das KM, Vecchi M, Sakemaki S. A shared and unique epitope(s) on human colon, skin and biliary epithelium detected by a monocoloncal antibody. Gastroenterology 1990; 98:464 –9. 5. Chiba M, Bartnik W, Remine SG, et al. Human colonic intraepithelial and lamina peripheral lymphocytes: Cytotoxicitiy in vitro and the potential effects of the isolation method on their functional properties. Gut 1981;22:177– 82. 6. Raedler A, Fraenkel S, Klose G, et al. Increased numbers of peripheral T cells in inflammatory bowel disease displaying the T9 antigen and Fc ␥ receptors. Clin Exp Immunol 1985; 60:518 –26. 7. Dasgupta A, Mandel A, Das KM. Circulating immunoglobulin G1 in patients with ulcerative colitis against the colonic epithelial protein detected by a novel monoclonal antibody. Gut 1994;35:1712–7. 8. Takahashi F, Das KM. Isolation, and characterization of a colonic autoantigen specifically recognized by colon tissuebound immunoglobulin G from idiopathic ulcerative colitis. J Clin Invest 1985;76:311– 8.
AJG – Vol. 95, No. 4, 2000
9. Neurath M, Kelasall BL, Presky DH, et al. Experimental granulomatous colitis in mice is abrogated by induction of TGF-mediated oral tolerance. J Exp Med 1996;183:2605–16. 10. Strober W, Kelsall B, Fuss L, et al. Reciprocal IFN-␥, and TGF- responses regulate the occurrence of mucosal inflammation. Immunol Today 1997;18:61– 4. 11. Hollander GA, Si SJ, Mizoguchi E, et al. Severe colitis in mice with aberrant thymic selection. Immunity 1995;3:27–38. 12. Sadlack B, Merz H, Schorle H, et al. Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell 1993;75:253– 61. 13. Hammer RE, Maika SD, Richardson JA, et al. Spontaneous inflammatory disease in transgenic rats expressing HLA B27 and human 2m: An animal model of HLAB-27-associated human disorders. Cell 1990;63:1099 –1112. 14. Kuhn RI, Lohler D, Rennick. K, et al. Interleukin 10 deficient mice develop chronic enterocolitis. Cell 1993;75:263–74. 15. Powrie F, Leach MW, Mauze S, et al. Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4⫹T cells. Immunity 1994; 1:553– 62. 16. Weiner H. Oral tolerance. Proc Natl Acad Sci USA 1994;91: 10762–5. 17. Weiner HL. Oral tolerance: Immune mechanisms and treatment of autoimmune diseases. Immunol Today 1997;18:335– 43. 18. Yoshida T, Hachimura S, Kaminogawa S. The oral administration of low-dose antigen induces activation followed by tolerization, while high-dose antigen induces tolerance without activation. Clin Immunol Immunopathol 1997;82:207–15. 19. Neurath MF, Fuss I, Kelsall BL, et al. Antibodies to IL-12 abrogate established experimental colitis in mice. J Exp Med 1995;182:1281–90. 20. Ilan Y, Sauter B, Roy Chowdhury N, et al. Oral tolerization to adenoviral proteins permits repeated adenovirus-mediated gene therapy in rats with pre-existing immunity to adenovirus. Hepatology 1998;27:1368 –76. 21. Shouval D, Adler R, Ilan Y. Adoptive transfer of immunity to hepatitis B virus in nude mice following T-cell depleted bone marrow transplantation. Hepatology 1993;17:955–9. 22. Ilan Y, Prakash R, Jona VK, et al. Adoptive transfer of tolerance from rats tolerized to adenoviral antigens allows long term adenovirus-mediated gene therapy in Gunn rats. Hepatology 1996;24:304A. 23. Rimm DL, Holland E, Tracey E, et al. Autoantibodies specific for villin found in patients with colon cancer and other colitides. Digest Dis Sci 1995;40:389 –95. 24. Z’graggen K, Walz A, Mazzucchelli L, et al. The C-X-O chemokine ENA-78 is preferentially expressed in intestinal epithelium in inflammatory bowel disease. Gastroenterology 1997;113:808 –16. 25. Adorini L, Francesco S. Pathogenesis and immunotherapy of autoimmune diseases. Immunol Today 1997;18:209 –11. 26. Mizoguchi A, Mizoguchi E, Chiba C, et al. Cytokine imbalance and autoantibody production in T cell receptor-␣ mutant mice with inflammatory bowel disease. J Exp Med 1996;183: 847–56. 27. Madsen KL, Lewis Simon A, Tavernini MM, et al. Interleukin 10 prevents cytokine-induced disruption of T84 barrier integrity and limits chloride secretion. Gastroenterology 1997;113: 151–9. 28. Van Deventer Sander J, Charles O, Fedorak RN. Multiple doses of intravenous interleukin 10 in steroid refractory Crohn’s disease. Gastroenterology 1997;113:383–9. 29. Esbjorn T, Karlsson M, Dahlman-Hoglund A, et al. Oral tolerance in experimental animals. Int Arch Allergy Immunol 1997;113:219 –23.
AJG – April, 2000
30. Von Herrath MG, Dyrberg T, Oldstone MBA. Oral insulin treatment suppresses virus-induced antigen-specific destruction of  cells and prevents autoimmune diabetes in transgenic mice. J Clin Invest 1996;98:1324 –31. 31. Hancock W, Polansli M, Zhang J, et al. Suppression of insulitis in NOD mice by oral insulin administration is associated with selective expression of IL-4 –10, TGF- and prostaglandine-E. Am J Path 1993;147:1193–7. 32. Javed NH, Gienapp IE, Cox KL, et al. Exquisite peptide specificity of oral tolerance in experimental autoimmune encephalomyelitis. J Immunol 1995;155:1599 –1605. 33. Weiner HL, Mackin GA, Matsui M, et al. Double-blind pilot trial of oral tolerization with myelin antigen in multiple sclerosis. Science 1993;261:1321– 4. 34. Trentham DE, Dynesius-Trentham RA, Orav EJ, et al. Effects of oral administration of type II collagen on rheumatoid arthritis. Science 1993;261:1727–30. 35. Ilan Y, Prakash R, Davidson A, et al. Oral tolerization to adenoviral antigens permits long term gene expression using recombinant adenoviral vectors. J Clin Invest 1997;99:1098 – 1106. 36. Takahashi M, Ilan Y, Sengupta K, et al. Induction of tolerance to recombinant adenoviruses by injection into newborn rats: Long-term amelioration of hyperbilirubinemia in Gunn rats. J Biol Chem 1996;271:26536 – 42. 37. Ilan Y, Jona VK, Sengupta K, et al. Transient immunosuppression with FK506 permits long term expression of therapeutic genes introduced into the liver using recombinant adenoviruses. Hepatology 1997;26:949 –56.
Oral Tolerance for Experimental Colitis
973
38. Ilan Y, Droguett G, Roy Chowdhury N, et al. Insertion of the adenoviral E3 region into a recombinant viral vector prevents antiviral humoral and cellular immune responses and permits long term gene expression. Proc Nat Acad Sci USA 1997;94: 2587–92. 39. Ilan Y, Attavar P, Takahashi M, et al. Induction of central tolerance by intrathymic inoculation of adenoviral antigens into the host thymus permits long term gene therapy in Gunn rats. J Clin Invest 1996;98:2640 –7. 40. Fontana A, Sonstan K, Frei U, et al. Modulation of the immune response by TGF. Int Arch Allergy Immunol 1992;99:1–7. 41. Ilan Y, Roy Chowdhury N, Sauter BS, et al. A critical role of the liver in inducing oral tolerance to adenoviral vectors for long term gene therapy. Gastroenterology 1998;114:260A. 42. Qiu CJ. Pfeiffer H, Keith JC. Protection by recombinant human interleukin-11 against experimental TNB-induced colitis in rats. Dig Dis Sci 1996;41:1625–30. 43. Carvalho BA, Verdolin SA, Vaz NM. Indirect effects of oral tolerance cannot be ascribed to bystander suppression. Scand J Immunol 1997;45:276 – 81. 44. Blanas E, Carbone FR, Allison J, et al. Induction of autoimmune diabetes by oral administration of autoantigen. Science 1996;274:1707–9. 45. Christian K, Hiroshi K, Carbone FR, et al. Class I-restricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive CD8⫹T cells. J Exp Med 1997;186:239 – 45. 46. Sharmanov AT, Elson CO, Tennyson GS, et al. TNBS-specific oral tolerance protects from hapten induced colitis. Gastroenterology 1995;106:335A.