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Responses to self and non-self intestinal microflora in health and inflammatory bowel disease
TOLERANCE
AND IMMUNITY
IN THE MUCOSAL
IMMUNE
SYSTEM
589
Responses to self and non-self intestinal microflora in health and inflammatory bowel disease R. Duchmann I. Medical
Clinic,
(*I, M.F. Neurath,
University
The number of bacteria within the normal intestinal flora has been estimated to be 1013, i.e., 10 times more than the estimated number of total body cells. Antigens from the normal intestinal flora therefore constitute a quantitatively important form of “beneficial” antigen present along the entire intestinal canal. Although these antigens do not constitute self antigens in the proper sense, it is obvious that immunizing immune responses to these permanent antigens would result in chronic inflammation. As a consequence, antigens from normal intestinal flora have generally been assumed to induce some kind of immune tolerance. This, however, is intriguing, since orally administered live bacteria have been shown to prime active immune responses. Studies to confirm immune tolerance to antigens from normal intestinal flora have rarely been performed (Berg and Savage, 1975 ; Foo and Lee, 1974), and it is thus not surprising that there is also very incomplete information on the mechanisms that might contribute to the generation of immune tolerance to live commensal bacteria and their products. However, there is indirect evidence for tight regulation of the immune response to normal intestinal flora from recent studies in different animal models, which clearly demonstrated that an immune system troubled by defined disruptions of immunoregulatory molecules unleashes the potential of the normal flora to induce chronic intestinal inflammation (Aranda et al., 1997 ; Rath et al., 1996 ; Strober and Ehrhardt, 1993). To provide for a better understanding of immune responses to normal intestinal flora, it is of interest that the context in which the immune system encounters antigens from the intestinal flora bears a number of distinctive features. First, bacteria are known to produce a variety of non-specific products which exhibit profound irnrnunomodulatory effects.
(*) For correspondence.
3 1, 1997.
Meyer
of Mainz, LangenbeckstraJe,
Introduction
Received December
K.H.
zum Biischenfelde D-55101
Mainz (Germany)
As a consequence, involution of the intestinal immune system with a loss of cellularity and a loss of immune function involving DTH responses and oral tolerance induction was observed in germ-free mice, but reversed by reconstitution with bacteria or administration of LPS (MacDonald and Carter, 1979 ; Wannemuehler et al., 1982). This emphasizes that the intestinal flora not only constitutes an important reservoir for antigen but also functions as a source for endogenous stimuli that are crucial for the development of a normal intestinal immune system and function. These immunomodulators might also have a role in chronic intestinal inflammation, e.g. by altering the immune response to antigens from the intestinal flora. Second, colonization of the intestinal tract by the normal flora during infancy is not random but a plastic process which involves both host and bacterial factors and ultimately leads to the stable expression of an autochthonous intestinal flora, common to all members of an animal species (Berg, 1996). Furthermore, indicating that the intestinal flora may be genetically tailored for each individual, it has been reported that the bacterial flora of monozygotic twins is more similar than that of dizygotic twins (Van de Merwe et al., 1983). This apparent influence of genetic factors on selection of the normal intestinal flora and the evident permanence of its antigens renders them quite similar to self antigens. Increasing evidence suggests that an abnormal immune response to constituents of the normal intestinal flora may be crucial for the development and/or maintenance of chronic intestinal inflammation. We therefore decided to examine immune responses of control and IBD individuals to the potentially relevant antigens within their own intestinal flora. In the current article we first summarize our studies which demonstrate that the normal immune system is tolerant to antigens from its own (self) intestinal flora but not (non-self) intestinal
72nd FORUM
590
IN IMMUNOLOGY
flora of a foreign individual. We then discuss the evidence that patients with IBD have an abnormal immune response to their own (self) intestinal flora and demonstrate that this abnormality can be therapeutically manipulated in a mouse model of chronic intestinal inflammation resembling Crohn’s disease. Finally, we discuss studies concerned with T-cell specificity and cross-reactivity to intestinal flora antigens. Analysis of the immune response to self and nonself intestinal flora in humans When we started to stimulate mononuclear cells from peripheral blood (PBMC) and intestinal lamina propria (LPMC) from control individuals with bacterial sonicates derived from their own (self) intestinal flora or the (non-self) intestinal flora of a foreign individual, it was a first observation (fig. 1) that PBMC and LPMC from control individuals showed very low proliferation in response to self intestinal flora, but substantial proliferation to foreign intestinal flora (Duchmann et al., 1995). Hyporesponsiveness or tolerance to sonicates from self intestinal flora was not due to a defective stimulatory capacity
of the sonicates or a defective proliferative capacity of the cellular preparations. This was demonstrated in cross-mixing studies in which we used the same sonicates and cells in heterologous combination and observed substantial proliferation. Similar to these results, Berg et al. had demonstrated in mice that i.p. injection with autochthonous Escherichia coli or Bacteroides ssp. induced a decreased humoral immune response compared to i.p. injection with non-autochthonous E. coli or Bucteroides ssp. (Berg and Savage, 1975). Our in vitro studies extend this observation to cell-mediated immune responses and indicate that responses to antigens from the intestinal flora are already different at the interindividual level. Because abnormal T-cell responses and a loss of tolerance to normal intestinal flora are increasingly incriminated in the pathogenesis of chronic intestinal inflammation (Aranda et al., 1997; Ma et al., 1993, we next (fig. 1) stimulated mononuclear cells from peripheral blood, inflamed and non-inflamed colonic mucosa of IBD patients with self or non-self intestinal flora (Duchmann et al., 1995). As we had observed with control cells, incubation with non-self intestinal flora induced significant proliferation in cells from all three sources, whereas mononuclear
BsH
y,Control m Medium w PHA q BsA
n q q
0
10
20
30
40
cpm/103 Fig. 1. Tolerance Mononuclear control individuals autologous (BsA) involved intestine Proliferation was
Medium PHA BsA
to self intestinal
cpm/103 flora is broken in inflammatory
bowel disease (IBD).
cells from peripheral blood (PBMC) and intestinal lamina propria (LPMC) of and patients with IBD were stimulated with bacterial sonicates from flora of or heterologous (BsH) intestine. LPMC from IBD patients were isolated from non(LPMC-) and involved intestine (LPMC+). Cultures were incubated for 6 days. measured by 3H-thymidine incorporation.
TOLERANCE
AND IMMUNITY
IN THE MUCOSAL
cells from peripheral blood and non-inflamed colonic IBD mucosa of the same patients were hyporesponsive to stimulation with self intestinal flora. In interesting contrast, incubation of mononuclear cells from inflamed colonic mucosa with self intestinal flora was clearly abnormal and resulted in significant proliferation. Further studies showed that proliferation to intestinal flora was associated with production of IL1 2, IFNy and IL10 protein. Increased expression of activation markers on CD4+ and CD8+ T cells, CD8+ T-cell expansion and inhibition of the proliferative response by anti-MHC class II indicated a specific involvement of T cells in the proliferative response to intestinal flora. Altogether, the data indicated that infiltrating IBD T lymphocytes are not only hyperresponsive to standard microbial antigens (Pirzer et al., 1991), but are also hyperresponsive to the more relevant antigens within their own intestinal flora. Treatment with IL10 and antibodies to IL12 therapeutically modulates the hyperreactive immune response to self intestinal flora in a murine model of Crohn’s disease Confirming the studies performed with human mononuclear cells, we have shown (fig. 2) that mononuclear cells from mouse spleen, small intestine and large intestine of different mouse strains also exhibit a specific hyporesponsiveness or tolerance to self intestinal flora (Duchmann et al., 1996a). Since rectal application of the hapten reagent TNBS results in a chronic granulomatous and transmural colitis which resembles human Crohn’s disease in many clinical and immunologic aspects (Neurath et al., 1995) and also induces a loss of tolerance to self intestinal flora (Duchmann et al., 1996a), we used this animal model to explore the regulation of the immune response to antigens from self versus non-self intestinal flora. In that, our interest was focused on two cytokines, IL10 and IL12. First, IL10 is a well studied inhibitor of many immune responses and a potent downregulator of IL12 and IFNy which are important mediators of antibacterial immune responses. In addition, the role of IL10 in preventing chronic intestinal inflammation in response to normal flora has been nicely demonstrated in IL10 KO mice (Kuhn et d., 1993). These mice develop spontaneous enterocolitis in a dirty environment and significantly improve under specific pathogen-free and germ-free conditions. On the other hand, bacteria or their products very potently induce IL12 production in macrophages. This is known to constitute an important signal for the development of IFNy-producing Thl cells. The relevance of IL10 and IL1 2 as regulators of intestinal inflammation was shown in further experiments which demonstrated that treatment with IL10 and
IMMUNE
SYSTEM
591
antibodies to IL12 was effective in TNBS-induced murine colitis (Duchmann et ul., 1996a) as well as other animal models of chronic intestinal inflammation (Ehrhardt et al., 1997). With regard to its effect on the immune response to self or non-self intestinal flora, we found that treating mice with TNBSinduced colitis with IL10 and antibodies to IL12 restored tolerance to self intestinal flora, but did not downregulate proliferation to foreign intestinal flora (fig. 2). Responsiveness to IL10 and anti-IL12 thus emerges as a key feature of the immune response to self intestinal flora. T-cell specificity bacteria
and cross-reactivity
to intestinal
To investigate the T-cell response to intestinal bacteria in detail, we used an antigen non-specific protocol for the isolation of T-cell clones from human peripheral blood and intestine, and then restimulated the T-cell clones with a variety of different bacteria. Using this approach (Duchmann et al., 1996b and submitted), we could demonstrate that T cells responding to non-self Enterobacteria were more frequently isolated from inflamed IBD intestine than from control or non-inflamed IBD intestine. In addition, the experiments showed that proliferation of CD4+ TCRc#+ T cells induced by different species of non-self Enterobacteriu like Sulmonellu, Yersiniu or E. coli, common intestinal anaerobes or heterogenous mixtures of self or nonself intestinal flora is selective (fig. 3), inhibitable by anti-MHC class II and thus involves discrete antigens and not polyclonal T-cell activators. Although most of the T cells selectively responded to only one of the bacteria studied, we also isolated cross-reactive T-cell clones, indicating the presence of shared antigens. Of potential relevance to IBD pathogenesis, T-cell clones responding to discrete antigens of self intestinal flora were thus far only isolated from IBD patients. Purification of bacterial antigens recognized by human flora-specific T cells indicated that the relevant antigens are soluble antigens expressed in glycoproteins (Duchmann et al., submitted). Conclusion
and future directions
Antigens that encounter the intestinal immune system can initiate two different types of immune responses, leading to the induction of immunity or tolerance. Our studies emphasize that the normal immune system provides for mechanisms to differentially regulate the response to antigens from self and non-self intestinal flora along these pathways. In detail, we have shown that human T-cell clones are stimulated by discrete antigens present within nor-
Control
TNBS
Spleen
I
Spleen
q Medium n PHA n BsA Small Bowel q BsHl
Small Bowel
n PHA n BsA q BsHl
t!ii BsH2 Large Bowel
65 BsH2 LargeBowel 0
lo&o
2oooo
cpm TNBS+PBS
4oooo
TNBS+ILlO
Spleen
SmallBowel
3oooo
cm
0
Medium
w
PHA
m
BsA
0
BsHl
0
BsH2
LargeBowel
Spleen 0
Medium
n PHA n BsA
Small Bowel
0
BsHl
q BsH2 Large Bowel 0
loo00
cpm
h
4t
TNBS+ anti-IL12
Spleen
Spleen
SmallBowel
q Medium n PHA SmallBowel n BsA id
BsHl
Q
BsH2
0 Medium m PHA
n BsA q BsHl q BsH2
LargeBowel
wm Fig. 2. Treatment
3oooo
cpm
TNBS+ control IgG
LargeBowel
2oom
with IL10 and antibodies to IL12 restores tolerance murine model of Crohn’s disease.
cw to self intestinal
flora in a
Mononuclear cells were isolated from indicated organs of control mice or mice that had received a rectal application of TNBS alone or TNBS and a simultanous i.p. injection with IL10 (or PBS control) or antibodies to IL12 (or rat IgG control). Cells from these mice (n=5 per group) were then stimulated with flora of autologous (BsA) or heterologous (BsH) intestine. Cultures were incubated for 6 days. Proliferation was measured by ‘H-thymidine incorporation.
TOLERANCE
AND IMMUNITY
IN THE MUCOSAL
cm
IMMUNE
SYSTEM
593
wm
Fig. 3. T-cell specificity for intestinal bacteria. T-cell clones were isolated from peripheral blood, non-involved intestine and involved intestine of control individuals and IBD patients by cloning with PHA. They were then restimulated for 42 h with sonicates from indicated bacteria, mixtures of bacteria isolated from autologous (BsA) or heterologous (BsH) intestine, PHA and tetanus toxoid. Proliferation was measured by 3H-thymidine incorporation. Results shown are from a BsH-specific and an E. c&specific T-cell clone.
ma1 intestinal flora and that the normal immune response to self but not non-self intestinal flora in vitro is characterized by tolerance, as indicated by a lack of proliferation or cytokine production. In contrast, and this was shown in IBD patients and a murine model of Crohn’s disease, tolerance to self intestinal flora was abrogated in involved intestine and restored in mice by treatment with IL10 or antibodies to IL12. These data describing the cell-mediated immune response to normal flora are complemented by recent studies from van der Waaij et al., who determined the antibody coating of the normal intestinal flora and provided evidence that the humoral tolerance to self flora in IBD is also disturbed (van der Waaij et al., 1997). Mechanisms of tolerance induction to antigens from the normal intestinal flora are still unclear, but may at least in part be similar to the tolerance elicited by luminal antigens from ingested dietary products or other sources whose capacity to induce regulatory pathways of peripheral (oral) tolerance is well documented (Mowat, 1987) and was shown to be mediated by T-cell anergy/deletion or the induction of Th2/Th3 regulatory cells (Weiner, 1997). Our finding that antibodies to IL12 restore tolerance to self intestinal flora in experimental murine colitis indicates that IL12 is not only an important counterregulator of TGFP-mediated oral tolerance (Neuratb er ul., 1996; Marth et (II., 1996), but also counterregulates tolerance to normal intestinal flora. Abnormal production of IL12 as a consequence of a disturbed discriminatory capacity between antigens from self and non-self intestinal flora by primi-
tive receptors on antigen-presenting cells has thus been suggested by us to be a mechanism explaining the loss of tolerance to own intestinal flora in involved IBD intestine. In addition, there is evidence for genetically controlled selection of “self’ flora during colonization (Van de Merwe et al., 1983), as well as cross-tolerization between self-antigens and antigens from normal intestinal flora (Foo and Lee, 1974), indicating that tolerance of an individual to his own intestinal flora may share aspects of self tolerance. Development of such self tolerance and/or development of a fully competent immune system seem to be necessary to generate tolerance to one’s own intestinal flora, since it was not observed in healthy young mice (unpublished data). The concept that IBD is driven by a dysregulated T-cell response towards components from normal intestinal flora is in concert with experiments in different animal models (Aranda et al., 1997; Cong et al., 1997; Rath et al., 1996; Strober and Ehrhardt, 1993) of IBD and human studies which demonstrated that IBD T cells exhibit increased expression of activation markers and abnormal cytokine production. However, very little is known about the antigen specificity of intestinal T cells. Our analysis of clonal CD4+ TCRa@+ T cells suggests that human flora-specific T cells in the intestine are activated by discrete glycoprotein antigens and that at least some of these T cells cross-react with different bacterial species, By precisely defining the relevant antigens. determining their distribution among bacteria from the normal intestinal flora and analysing the molecular structure of the corresponding T-cell receptors, we will hopefully improve our approach to IBD pathogenesis and treatment.
72nd
FORUM
References Aranda, R., Sydora, B.C., McAllister, P.L., Binder, S.W., Ying Yang, H., Targan, S.R. & Kronenberg, M. (1997), Analysis of intestinal lymphocytes in mouse colitis mediated by transfer of CD4+, CD45RB”@’ T cells to SCID recipients. J. Immurwl., 158, 3464-3473. Berg, R.D. & Savage, D.C. (1975), Immune response of specific pathogen-free and gnotobiotic mice to antigens of indigenous and non-indigenous microorganisms. Zfect. Immun., 11, 320-329. Berg, R.D. (1996), The indigenous gastrointestinal microflora. Trends Microbiology, 4, 430-435. Cong, Y., McCabe, R.P., Seibold, F., Harmon, S., Lazenby, A., Sundberg, J.P. & Elson, C.O. (1997), Clonal restriction of colitis-inducing, enteric bacterial antigenspecific CD4+ T cells. Gastroenterology, 112, A:951. Duchmann, R., Kaiser, I., Hermann, E., Mayet, W., Ewe, K. & Meyer zum Biischenfelde, K.H. (1995), Tolerance exists towards resident intestinal flora but is broken in active inflammatory bowel disease. Clin. Eq Immunol., 102, 448. Duchmann, R., Schmitt, E., Knolle, P., Meyer zum Bi.ischenfelde, K.H. & Neurath, M. (1996a), Tolerance towards resident intestinal flora in mice is abrogated in experimental colitis and restored by treatment with interleukin-10 or antibodies to interleukin-12. Eur. J. Immunol., 26, 934-938. Duchmann, R., Mtirker-Hermann, E. & Meyer zum Bi.ischenfelde, K.H. (1996b), Bacteria-specific T-cell clones are selective in their reactivity towards different Enterobacteria or H. pylori and increased in inflammatory bowel disease. Stand. J. Immunol., 44, 71-79. Ehrhardt, R.O., Ludviksson, B.R., Gray, B., Neurath, M. & Strober, W. (1997). Induction and prevention of colonic inflammation in IL-2-deficient mice. J. Immunol., 158, 566-573. Foo, M.C. & Lee, A. (1974), Antigenic cross-reaction between mouse intestine and a member of the autochthonous microflora. Infect. Immun., 9, 1066-1069. Kiihn, R., Lbhler, J., Rennick, D., Rajewski, K. h Miiller, W. (1993), Interleukin-lo-deficient mice develop chronic enterocolitis. Cell, 75, 263-274. Ma, A., Datta, M., Margosian, E., Chen, J. & Horak, I. (1995), T cells, but not B cells, are required for bowel inflammation in interleukin 2-deficient mice. J. Exp. Med., 182, 1567-1572. MacDonald, T.T. & Carter, P.B. (1979), Requirement for a bacterial flora before mice generate cells capable of mediating the delayed hypersensitivity reaction
IN IMMUNOLOGY to sheep red blood cells. J. Immunol., 122, 26242629. Marth, T., Strober, W. & Kelsall, B.L. (1996), High dose oral tolerance in ovalbumin TCR-transgenic mice: systemic neutralization of IL-12 augments TGF-beta secretion and T cell apoptosis. J. Immunol., 157, 2348-2357. Mowat, A.M. (1987), The regulation of immune responses to dietary protein antigens. Immunol. Today, 8, 9398. Neurath, M., Fuss, I., Kelsall, B.L., Stiiber, E. & Strober, W. (1995), Antibodies to IL-12 abrogate established granulomatous colitis in mice. J. Exp. Med., 182, 1281-1290. Neurath, M., Fuss, I., Kelsall, B.L., Presky, D.H., Waegell, W. & Strober, W. (1996), Experimental granulomatous colitis in mice is abrogated by induction of TGFP-mediated oral tolerance. J. Exp. Med., 183, 26052616. Pirzer, U., Schiinhaar, A., Fleischer, B., Hermann, E. & Meyer zum Biischenfelde, K.H. (1991), Reactivity of infiltrating T lymphocytes with microbial antigens in Crohn’s disease. Lancet, 338, 1238-1239. Rath, H.C., Herfarth, H.H., Ikeda, J.S., Grenther, W.B., Hamm, T.E.J., Balish, E., Taurog, J.D., Hammer, R.E., Wilson, K.H. & Sartor, R.B. (1996). Normal luminal bacteria, especially bacteroides species, mediate chronic colitis, gastritis, and arthritis in HLA-B271human p2 microglobulin transgenic rats. J. Clin. Invest., 98, 945-953. Strober, W. & Ehrhardt, R.O. (1993), Chronic intestinal inflammation : an unexpected outcome in cytokine or T cell receptor mutant mice. Cell, 75, 203-205. Van de Merwe, J.P., Stegeman, J.H. & Hazenberg, M.P. (1983), The resident fecal flora is determined by genetic characteristics of the host. Implications for Crohn’s disease? Antonie van Leeuwenhook, 49, 119124. Van der Waaij, L.A., Kroese, F.G.M., Jansen, P.L.M., Visser, A. & Hunter, J.O. (1997), Anaerobic bacteria in feces of patients with IBD are coated with immunoglobulins: loss of immunological tolerance? Gur, 41, A:P161A. Wannemuehler, M.J., Kiyono, H., Babb, J.L., Michalek, S.M. & McGhee, J.R. (1982), Lipopolysaccharide (LPS) regulation of the immune response: LPS converts germfree mice to sensitivity to oral tolerance induction. J. Immunol., 129, 959-965. Weiner, H.L. (1997), Oral tolerance: immune mechanisms and treatment of autoimmune diseases. Immunol. Today, 18, 335-343.
AND IMMUNITY
IN THE MUCOSAL
IMMUNE
SYSTEM
589
Responses to self and non-self intestinal microflora in health and inflammatory bowel disease R. Duchmann I. Medical
Clinic,
(*I, M.F. Neurath,
University
The number of bacteria within the normal intestinal flora has been estimated to be 1013, i.e., 10 times more than the estimated number of total body cells. Antigens from the normal intestinal flora therefore constitute a quantitatively important form of “beneficial” antigen present along the entire intestinal canal. Although these antigens do not constitute self antigens in the proper sense, it is obvious that immunizing immune responses to these permanent antigens would result in chronic inflammation. As a consequence, antigens from normal intestinal flora have generally been assumed to induce some kind of immune tolerance. This, however, is intriguing, since orally administered live bacteria have been shown to prime active immune responses. Studies to confirm immune tolerance to antigens from normal intestinal flora have rarely been performed (Berg and Savage, 1975 ; Foo and Lee, 1974), and it is thus not surprising that there is also very incomplete information on the mechanisms that might contribute to the generation of immune tolerance to live commensal bacteria and their products. However, there is indirect evidence for tight regulation of the immune response to normal intestinal flora from recent studies in different animal models, which clearly demonstrated that an immune system troubled by defined disruptions of immunoregulatory molecules unleashes the potential of the normal flora to induce chronic intestinal inflammation (Aranda et al., 1997 ; Rath et al., 1996 ; Strober and Ehrhardt, 1993). To provide for a better understanding of immune responses to normal intestinal flora, it is of interest that the context in which the immune system encounters antigens from the intestinal flora bears a number of distinctive features. First, bacteria are known to produce a variety of non-specific products which exhibit profound irnrnunomodulatory effects.
(*) For correspondence.
3 1, 1997.
Meyer
of Mainz, LangenbeckstraJe,
Introduction
Received December
K.H.
zum Biischenfelde D-55101
Mainz (Germany)
As a consequence, involution of the intestinal immune system with a loss of cellularity and a loss of immune function involving DTH responses and oral tolerance induction was observed in germ-free mice, but reversed by reconstitution with bacteria or administration of LPS (MacDonald and Carter, 1979 ; Wannemuehler et al., 1982). This emphasizes that the intestinal flora not only constitutes an important reservoir for antigen but also functions as a source for endogenous stimuli that are crucial for the development of a normal intestinal immune system and function. These immunomodulators might also have a role in chronic intestinal inflammation, e.g. by altering the immune response to antigens from the intestinal flora. Second, colonization of the intestinal tract by the normal flora during infancy is not random but a plastic process which involves both host and bacterial factors and ultimately leads to the stable expression of an autochthonous intestinal flora, common to all members of an animal species (Berg, 1996). Furthermore, indicating that the intestinal flora may be genetically tailored for each individual, it has been reported that the bacterial flora of monozygotic twins is more similar than that of dizygotic twins (Van de Merwe et al., 1983). This apparent influence of genetic factors on selection of the normal intestinal flora and the evident permanence of its antigens renders them quite similar to self antigens. Increasing evidence suggests that an abnormal immune response to constituents of the normal intestinal flora may be crucial for the development and/or maintenance of chronic intestinal inflammation. We therefore decided to examine immune responses of control and IBD individuals to the potentially relevant antigens within their own intestinal flora. In the current article we first summarize our studies which demonstrate that the normal immune system is tolerant to antigens from its own (self) intestinal flora but not (non-self) intestinal
72nd FORUM
590
IN IMMUNOLOGY
flora of a foreign individual. We then discuss the evidence that patients with IBD have an abnormal immune response to their own (self) intestinal flora and demonstrate that this abnormality can be therapeutically manipulated in a mouse model of chronic intestinal inflammation resembling Crohn’s disease. Finally, we discuss studies concerned with T-cell specificity and cross-reactivity to intestinal flora antigens. Analysis of the immune response to self and nonself intestinal flora in humans When we started to stimulate mononuclear cells from peripheral blood (PBMC) and intestinal lamina propria (LPMC) from control individuals with bacterial sonicates derived from their own (self) intestinal flora or the (non-self) intestinal flora of a foreign individual, it was a first observation (fig. 1) that PBMC and LPMC from control individuals showed very low proliferation in response to self intestinal flora, but substantial proliferation to foreign intestinal flora (Duchmann et al., 1995). Hyporesponsiveness or tolerance to sonicates from self intestinal flora was not due to a defective stimulatory capacity
of the sonicates or a defective proliferative capacity of the cellular preparations. This was demonstrated in cross-mixing studies in which we used the same sonicates and cells in heterologous combination and observed substantial proliferation. Similar to these results, Berg et al. had demonstrated in mice that i.p. injection with autochthonous Escherichia coli or Bacteroides ssp. induced a decreased humoral immune response compared to i.p. injection with non-autochthonous E. coli or Bucteroides ssp. (Berg and Savage, 1975). Our in vitro studies extend this observation to cell-mediated immune responses and indicate that responses to antigens from the intestinal flora are already different at the interindividual level. Because abnormal T-cell responses and a loss of tolerance to normal intestinal flora are increasingly incriminated in the pathogenesis of chronic intestinal inflammation (Aranda et al., 1997; Ma et al., 1993, we next (fig. 1) stimulated mononuclear cells from peripheral blood, inflamed and non-inflamed colonic mucosa of IBD patients with self or non-self intestinal flora (Duchmann et al., 1995). As we had observed with control cells, incubation with non-self intestinal flora induced significant proliferation in cells from all three sources, whereas mononuclear
BsH
y,Control m Medium w PHA q BsA
n q q
0
10
20
30
40
cpm/103 Fig. 1. Tolerance Mononuclear control individuals autologous (BsA) involved intestine Proliferation was
Medium PHA BsA
to self intestinal
cpm/103 flora is broken in inflammatory
bowel disease (IBD).
cells from peripheral blood (PBMC) and intestinal lamina propria (LPMC) of and patients with IBD were stimulated with bacterial sonicates from flora of or heterologous (BsH) intestine. LPMC from IBD patients were isolated from non(LPMC-) and involved intestine (LPMC+). Cultures were incubated for 6 days. measured by 3H-thymidine incorporation.
TOLERANCE
AND IMMUNITY
IN THE MUCOSAL
cells from peripheral blood and non-inflamed colonic IBD mucosa of the same patients were hyporesponsive to stimulation with self intestinal flora. In interesting contrast, incubation of mononuclear cells from inflamed colonic mucosa with self intestinal flora was clearly abnormal and resulted in significant proliferation. Further studies showed that proliferation to intestinal flora was associated with production of IL1 2, IFNy and IL10 protein. Increased expression of activation markers on CD4+ and CD8+ T cells, CD8+ T-cell expansion and inhibition of the proliferative response by anti-MHC class II indicated a specific involvement of T cells in the proliferative response to intestinal flora. Altogether, the data indicated that infiltrating IBD T lymphocytes are not only hyperresponsive to standard microbial antigens (Pirzer et al., 1991), but are also hyperresponsive to the more relevant antigens within their own intestinal flora. Treatment with IL10 and antibodies to IL12 therapeutically modulates the hyperreactive immune response to self intestinal flora in a murine model of Crohn’s disease Confirming the studies performed with human mononuclear cells, we have shown (fig. 2) that mononuclear cells from mouse spleen, small intestine and large intestine of different mouse strains also exhibit a specific hyporesponsiveness or tolerance to self intestinal flora (Duchmann et al., 1996a). Since rectal application of the hapten reagent TNBS results in a chronic granulomatous and transmural colitis which resembles human Crohn’s disease in many clinical and immunologic aspects (Neurath et al., 1995) and also induces a loss of tolerance to self intestinal flora (Duchmann et al., 1996a), we used this animal model to explore the regulation of the immune response to antigens from self versus non-self intestinal flora. In that, our interest was focused on two cytokines, IL10 and IL12. First, IL10 is a well studied inhibitor of many immune responses and a potent downregulator of IL12 and IFNy which are important mediators of antibacterial immune responses. In addition, the role of IL10 in preventing chronic intestinal inflammation in response to normal flora has been nicely demonstrated in IL10 KO mice (Kuhn et d., 1993). These mice develop spontaneous enterocolitis in a dirty environment and significantly improve under specific pathogen-free and germ-free conditions. On the other hand, bacteria or their products very potently induce IL12 production in macrophages. This is known to constitute an important signal for the development of IFNy-producing Thl cells. The relevance of IL10 and IL1 2 as regulators of intestinal inflammation was shown in further experiments which demonstrated that treatment with IL10 and
IMMUNE
SYSTEM
591
antibodies to IL12 was effective in TNBS-induced murine colitis (Duchmann et ul., 1996a) as well as other animal models of chronic intestinal inflammation (Ehrhardt et al., 1997). With regard to its effect on the immune response to self or non-self intestinal flora, we found that treating mice with TNBSinduced colitis with IL10 and antibodies to IL12 restored tolerance to self intestinal flora, but did not downregulate proliferation to foreign intestinal flora (fig. 2). Responsiveness to IL10 and anti-IL12 thus emerges as a key feature of the immune response to self intestinal flora. T-cell specificity bacteria
and cross-reactivity
to intestinal
To investigate the T-cell response to intestinal bacteria in detail, we used an antigen non-specific protocol for the isolation of T-cell clones from human peripheral blood and intestine, and then restimulated the T-cell clones with a variety of different bacteria. Using this approach (Duchmann et al., 1996b and submitted), we could demonstrate that T cells responding to non-self Enterobacteria were more frequently isolated from inflamed IBD intestine than from control or non-inflamed IBD intestine. In addition, the experiments showed that proliferation of CD4+ TCRc#+ T cells induced by different species of non-self Enterobacteriu like Sulmonellu, Yersiniu or E. coli, common intestinal anaerobes or heterogenous mixtures of self or nonself intestinal flora is selective (fig. 3), inhibitable by anti-MHC class II and thus involves discrete antigens and not polyclonal T-cell activators. Although most of the T cells selectively responded to only one of the bacteria studied, we also isolated cross-reactive T-cell clones, indicating the presence of shared antigens. Of potential relevance to IBD pathogenesis, T-cell clones responding to discrete antigens of self intestinal flora were thus far only isolated from IBD patients. Purification of bacterial antigens recognized by human flora-specific T cells indicated that the relevant antigens are soluble antigens expressed in glycoproteins (Duchmann et al., submitted). Conclusion
and future directions
Antigens that encounter the intestinal immune system can initiate two different types of immune responses, leading to the induction of immunity or tolerance. Our studies emphasize that the normal immune system provides for mechanisms to differentially regulate the response to antigens from self and non-self intestinal flora along these pathways. In detail, we have shown that human T-cell clones are stimulated by discrete antigens present within nor-
Control
TNBS
Spleen
I
Spleen
q Medium n PHA n BsA Small Bowel q BsHl
Small Bowel
n PHA n BsA q BsHl
t!ii BsH2 Large Bowel
65 BsH2 LargeBowel 0
lo&o
2oooo
cpm TNBS+PBS
4oooo
TNBS+ILlO
Spleen
SmallBowel
3oooo
cm
0
Medium
w
PHA
m
BsA
0
BsHl
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with IL10 and antibodies to IL12 restores tolerance murine model of Crohn’s disease.
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Mononuclear cells were isolated from indicated organs of control mice or mice that had received a rectal application of TNBS alone or TNBS and a simultanous i.p. injection with IL10 (or PBS control) or antibodies to IL12 (or rat IgG control). Cells from these mice (n=5 per group) were then stimulated with flora of autologous (BsA) or heterologous (BsH) intestine. Cultures were incubated for 6 days. Proliferation was measured by ‘H-thymidine incorporation.
TOLERANCE
AND IMMUNITY
IN THE MUCOSAL
cm
IMMUNE
SYSTEM
593
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Fig. 3. T-cell specificity for intestinal bacteria. T-cell clones were isolated from peripheral blood, non-involved intestine and involved intestine of control individuals and IBD patients by cloning with PHA. They were then restimulated for 42 h with sonicates from indicated bacteria, mixtures of bacteria isolated from autologous (BsA) or heterologous (BsH) intestine, PHA and tetanus toxoid. Proliferation was measured by 3H-thymidine incorporation. Results shown are from a BsH-specific and an E. c&specific T-cell clone.
ma1 intestinal flora and that the normal immune response to self but not non-self intestinal flora in vitro is characterized by tolerance, as indicated by a lack of proliferation or cytokine production. In contrast, and this was shown in IBD patients and a murine model of Crohn’s disease, tolerance to self intestinal flora was abrogated in involved intestine and restored in mice by treatment with IL10 or antibodies to IL12. These data describing the cell-mediated immune response to normal flora are complemented by recent studies from van der Waaij et al., who determined the antibody coating of the normal intestinal flora and provided evidence that the humoral tolerance to self flora in IBD is also disturbed (van der Waaij et al., 1997). Mechanisms of tolerance induction to antigens from the normal intestinal flora are still unclear, but may at least in part be similar to the tolerance elicited by luminal antigens from ingested dietary products or other sources whose capacity to induce regulatory pathways of peripheral (oral) tolerance is well documented (Mowat, 1987) and was shown to be mediated by T-cell anergy/deletion or the induction of Th2/Th3 regulatory cells (Weiner, 1997). Our finding that antibodies to IL12 restore tolerance to self intestinal flora in experimental murine colitis indicates that IL12 is not only an important counterregulator of TGFP-mediated oral tolerance (Neuratb er ul., 1996; Marth et (II., 1996), but also counterregulates tolerance to normal intestinal flora. Abnormal production of IL12 as a consequence of a disturbed discriminatory capacity between antigens from self and non-self intestinal flora by primi-
tive receptors on antigen-presenting cells has thus been suggested by us to be a mechanism explaining the loss of tolerance to own intestinal flora in involved IBD intestine. In addition, there is evidence for genetically controlled selection of “self’ flora during colonization (Van de Merwe et al., 1983), as well as cross-tolerization between self-antigens and antigens from normal intestinal flora (Foo and Lee, 1974), indicating that tolerance of an individual to his own intestinal flora may share aspects of self tolerance. Development of such self tolerance and/or development of a fully competent immune system seem to be necessary to generate tolerance to one’s own intestinal flora, since it was not observed in healthy young mice (unpublished data). The concept that IBD is driven by a dysregulated T-cell response towards components from normal intestinal flora is in concert with experiments in different animal models (Aranda et al., 1997; Cong et al., 1997; Rath et al., 1996; Strober and Ehrhardt, 1993) of IBD and human studies which demonstrated that IBD T cells exhibit increased expression of activation markers and abnormal cytokine production. However, very little is known about the antigen specificity of intestinal T cells. Our analysis of clonal CD4+ TCRa@+ T cells suggests that human flora-specific T cells in the intestine are activated by discrete glycoprotein antigens and that at least some of these T cells cross-react with different bacterial species, By precisely defining the relevant antigens. determining their distribution among bacteria from the normal intestinal flora and analysing the molecular structure of the corresponding T-cell receptors, we will hopefully improve our approach to IBD pathogenesis and treatment.
72nd
FORUM
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