Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121
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ABSTRACT |
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Inflammatory bowel disease (IBD) is a multifactorial immune disorder of uncertain etiology. The advent of several mouse models of mucosal inflammation that resemble IBD has provided insight into the mechanisms governing both normal and pathological mucosal immune function. In a widely used adoptive transfer model, the injection into immunodeficient mice of a subset of CD4+ T lymphocytes, the CD4+CD45RBhigh cells, leads to inflammation of the intestine. Pathogenesis is due in part to the secretion of proinflammatory cytokines. The induction of colitis can be prevented by cotransfer of another CD4+ subpopulation, the CD4+CD45RBlow T cells. This population behaves analogously to the CD4+CD45RBhigh population in terms of the acquisition of activation markers and homing to the host intestine. However, their lymphokine profile when activated is different, and anti-inflammatory cytokines secreted and/or induced by CD4+CD45RBlow T cells prevent colitis. In this themes article, a description of the adoptive transfer model is given, the factors that promote and prevent colitis pathogenesis are discussed, and some controversial aspects of the model are addressed.
adoptive transfer; immunodeficient mice; inflammatory bowel disease; CD4+CD45RBhigh cells
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INTRODUCTION |
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INFLAMMATORY BOWEL DISEASE (IBD) in humans, mainly
encompassing Crohn's disease and ulcerative colitis, is a complex
multifactorial immunological disorder. Studies of immunoreactive cells
and proinflammatory mediators in the intestinal tissue of patients have
suggested that IBD is the result of dysregulated immune responses to
enteric antigens. Although there is now evidence that Crohn's disease is driven by the secretion of the so-called Th1 or proinflammatory cytokines, including interleukin (IL)-12, interferon- (IFN-
), and
tumor necrosis factor (TNF), it has been proposed that ulcerative colitis is better classified as an IL-4- and IL-5- or a Th2-driven inflammation (26).
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COLITIS CAN BE INDUCED FOLLOWING TRANSFER OF SYNGENEIC T CELLS |
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Several mouse models of experimentally induced or spontaneously occurring colitis are now available for examination of the pathogenesis of IBD. One of these, which involves the adoptive transfer of the CD4+CD45RBhigh T cell subpopulation to immunodeficient recipients, is the topic of this themes article. This rodent model is particularly attractive for study of the role of T cells in the induction of intestinal inflammation because it is reproducible, it is relatively easy to induce, it has a predictable time course, and it is highly subject to manipulation from a genetic and immunological viewpoint. Furthermore, it bears some resemblance to the clinical course, the therapeutic response, and the inflammatory mediator profile of some forms of IBD.
The transfer of spleen- or lymph node-derived syngeneic or coisogeneic
CD4+CD45RBhigh
T cells from wild-type donor mice into immunodeficient severe combined
immunodeficient (SCID) or recombination activating gene (RAG)/
recipients leads to wasting and
intestinal inflammation (17), starting 3-5 wk after transfer. In
contrast, in most cases, the transfer of total, unfractionated
CD4+ T cells, or transfer of the
CD4+CD45RBlow
subset, does not induce wasting. Moreover, the
CD4+CD45RBlow
subset inhibits the potential of the
CD4+CD45RBhigh
population to induce disease (15) (see Fig.
1). Although most groups working with this
transfer model obtained comparable results when transferring
CD4+CD45RBhigh
and
CD4+CD45RBlow
T cells, some controversy exists, as one group consistently reports the
induction of colitis by transferring total, unfractionated CD4+ T cells, albeit later than
when
CD4+CD45RBhigh
cells are transferred (19). Most likely the
CD4+CD45RBhigh
population is enriched for the pathogenic precursors, and environmental conditions can influence the number of these precursors present in the
total donor population. It should be emphasized that the transfers in
this model do not cross a major histocompatibility barrier leading to
graft vs. host disease. Taken together, the data imply that normal mice
have subsets of T lymphocytes that are capable of causing colitis and
that these cells are kept in check by other T lymphocytes. After
transfer into an immunodeficient recipient lacking the inhibitory T
cell subsets, the pathogenic T cells cause disease due to their
expansion and cytokine secretion.
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HISTOPATHOLOGIC CHANGES FOLLOWING T CELL TRANSFER |
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Histopathological inflammation in diseased recipients extends diffusely
from the cecum to the rectum. It is limited to the mucosa of mildly
affected mice but is transmural in severely affected mice. Lesions
consist of mucin depletion, epithelial hyperplasia, and increases in
intraepithelial lymphocyte numbers. Less frequent findings include
ulceration with fibrosis, crypt abscesses, crypt loss, and
granulomatous inflammation. Furthermore, there is increased epithelial
and lamina propria expression of major histocompatibility class II
antigens (13). This increased expression is apparently due to IFN-
and TNF production by Th1 cells. In general, these lesions have some
features in common with ulcerative colitis, including their diffuse
distribution, restriction primarily to the large intestine, and mucin
depletion. On the other hand, they also share features with Crohn's
disease, including the transmural nature of the infiltration, the
predominance of lymphocytes and macrophages, and the presence of rare
crypt abscesses.
The injected T cells do not migrate exclusively to the intestine.
Histological analysis of
CD4+CD45RBhigh-transferred
SCID mice revealed inflammatory lesions in the stomach (84%), liver
(44%), and lungs (26%) (13). In one set of experiments, the
reconstitution of SCID mice resulted in skin alterations that strikingly resemble human psoriasis both clinically and
histopathologically and with regard to cytokine expression (20).
Furthermore, in our own recent experiments, we obtained a substantial
fraction of RAG /
recipients whose primary site of inflammation
is in the lungs (De Winter and Cheroutre, unpublished observations). The occurrence of inflammation in different sites in the
immunodeficient recipients is likely to reflect environmental
differences in animal colonies. For example, as in other animal models
of colitis, bacterial flora are necessary for pathogenesis in this
transfer model (2) and alterations in the flora in different vivaria
could be critical for determining both the predominant site and
severity of the resulting inflammation.
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PHENOTYPIC SIMILARITIES BETWEEN PATHOGENIC AND DISEASE-PREVENTING T CELL POPULATIONS |
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What distinguishes the
CD4+CD45RBhigh
T cells from their
CD4+CD45RBlow
counterparts? At the phenotypic level, after transfer, surprisingly little distinction is present. Both donor populations can colonize the
host intestine, although the
CD4+CD45RBhigh
T cells are slightly more efficient at doing this. Regardless of the
donor population, the donor T cells in the intestine have an activated
phenotype, including low levels of expression of CD45RB, even when the
cells originally injected were
CD45RBhigh, low levels of
expression of the lymph node homing receptor CD62L, and high levels of
expression of the activation marker CD69 (2). Many of the donor T cells
found in the intestine also express a mucosal-specific integrin
composed of E- and
7-subunits. After adoptive transfer, both the large and small
intestines are extensively colonized, although inflammation is seen
predominantly in the large intestine.
The level of expression of CD45RB on
CD4+ T cells is widely believed to
distinguish naive (CD45RBhigh)
from activated or memory
(CD45RBlow) cells in normal
mice. The suitability of CD45RBlow
as a stable marker of activation or memory is questionable because T
cells can both increase and decrease their level of CD45RB expression. The results from some studies have indicated that there may be two
distinct types of CD4+ memory T
cells: the CD45RBlow and
CD45RBhigh "revertant" (3).
The
CD4+CD45RBhigh
memory cells produce high levels of proinflammatory Th1 cytokines, such
as IL-2, IFN-, and TNF, but little IL-4 in response to a variety of
mitogenic stimuli, whereas the
CD45RBlow population tends to
produce Th2-type cytokines, such as IL-4 (14). This is consistent with
the difference between the two T cell subpopulations in the
pathogenesis of colitis.
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THE ROLE OF PROINFLAMMATORY CYTOKINES IN COLITIS PATHOGENESIS |
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To dissect the mechanisms of colitis induction, it is important to
establish which cytokines are involved in the pathogenic pathway. There
is broad consensus that the colitis induced by the
CD4+CD45RBhigh
population is due to a proinflammatory, Th1-type immune response by the
transferred CD4+ T cells. In this
regard, the
CD4+CD45RBhigh
adoptive transfer model resembles several other models for intestinal inflammation, including the colitis induced by administration of
trinitrobenzene sulfonic acid, the disease induced by bone marrow
reconstitution of T cell- and natural killer (NK)
cell-deficient Tg26 transgenic mice, the colitis in mice deficient
for the trimeric G protein
G
i-2, and the spontaneous
colitis that occurs in IL-10-deficient mice (22). Whereas the results
from clinical trials suggest that TNF is critically important for the
pathogenesis of Crohn's disease in some patients (24), in the adoptive
transfer model the respective contributions to intestinal inflammation
of the key players in a typical Th1 response (such as IFN-
, TNF, and others) remain to be determined. IL-12, produced by activated macrophages and dendritic cells, is the principal Th1-inducing cytokine. Many microbial products stimulate IL-12 production. IL-12 in
turn activates three transcription factors: signal transducer and
activator of transcription (STAT)-1, STAT3, and STAT4. Of these, STAT4
seems to be exclusively activated by IL-12 and no other cytokine.
Consistent with the importance of an IL-12- and STAT4-mediated pathway
for Th1 cytokine induction, it has been found that mice deficient for
either the IL-12 or STAT4 genes have markedly reduced Th1 responses
(1). It is surprising, therefore, that reconstitution of
immunodeficient mice with
CD4+CD45RBhigh
T cells in the presence of anti-IL-12 monoclonal antibody (MAb), or
with T cells from STAT4-deficient mice, does not abrogate the disease
completely. The disease that results is much milder, however, with
lower frequencies of IFN-
producing T cells in the mesenteric lymph
nodes and colonic lamina propria (21). These studies suggest that
factors outside the IL-12/STAT4 pathway contribute to the development
of the more chronic forms of intestinal inflammation. IL-18, the
IFN-
-inducing factor, is a potential candidate because this cytokine
has been shown to have a significant effect on the long-term ability of
T cells to produce IFN-
and because it has its highest expression in
the intestinal epithelial cells (11, 23). Furthermore, it has been
shown to synergize with IL-2 in Th1 cytokine induction.
Although the results from a number of experiments indicate that IFN-
is important for the induction of colitis in animal model systems, it
is not clear if it acts directly, by causing damage to colonic
epithelial cells, or if it acts primarily to activate macrophages with
the subsequent production of inflammatory mediators such as reactive
oxygen and nitrogen intermediates (IL-1, IL-6, IL-12, and TNF), some of
which have been shown to cause intestinal damage. Moreover, IFN-
may
influence cell trafficking by regulating adhesion molecule expression.
The potential importance of this cytokine in contributing to
pathogenesis is suggested by the finding that neutralization of IFN-
using MAbs significantly attenuates colitis development in mice
reconstituted with
CD4+CD45RBhigh
T cells. Unfortunately, the results obtained from transfers of T cells
from IFN-
-deficient donors do not bring more clarity. One group
reported that the adoptive transfer of
CD4+CD45RBhigh
cells from IFN-
knockout mice does not induce colitis (10). Surprisingly, this is in flagrant contradiction to the findings of
another group, who reported that when
CD4+CD45RBhigh
T cells from IFN-
knockout donors are transferred large numbers of
reconstituted recipients develop wasting and colitis, often of
comparable severity to that seen in animals reconstituted with wild-type cells (21). It is therefore likely that Th1 cells can mediate
intestinal inflammation via several mechanisms, not all of which result
from the production of IFN-
and which are mostly but not entirely
governed by the ability to produce and respond to IL-12 (see Fig. 1).
Ultimately, this is most likely achieved by the concerted expression of
several cytokines and cytotoxic molecules. In colitic mice, T cells
express TNF at a similar frequency, regardless of whether or not
IFN-
is expressed by the same cells, and treatment with anti-TNF MAb
also can ameliorate disease, although it is not as effective as IFN-
MAb treatment (15).
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HOW DO CD4+CD45RBLOW T CELLS PREVENT DISEASE? |
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Downregulation of pathogenesis by
CD4+CD45RBlow
T cells may be due to the production or induction of a cytokine
repertoire that antagonizes either the expansion, localization,
differentiation, or effector function of the T cells involved in the
inflammatory response. Because of the well-documented anti-inflammatory
properties of IL-10 and the spontaneous colitis that develops in
IL-10-deficient mice (12), it would seem likely that IL-10 would play
an important role in the inhibition of colitis mediated by
CD4+CD45RBlow
T cells. Consistent with this hypothesis, the systemic administration of recombinant IL-10 significantly inhibits the development of colitis
due to T cell transfer (18). Likewise, cotransfer of T cells from
transgenic mice that produce IL-10 under the control of the IL-2
promoter also prevents the induction of colitis by the
CD4+CD45RBhigh
population (9). Furthermore, regulatory T cells called Tr1 cells, which
primarily secrete IL-10 as a result of prolonged culture in high levels
of IL-10, also can prevent disease on cotransfer with
CD4+CD45RBhigh
T cells (8). It is therefore surprising that the protection from
colitis mediated by the
CD4+CD45RBlow
population was not diminished by neutralization of IL-10 with specific
MAbs, although it remains possible in this case that the neutralization
of IL-10 was not complete (16). In the same experiment, however, the
neutralization of transforming growth factor- (TGF-
)
with specific MAbs did abrogate the disease-preventing effect of the
CD4+CD45RBlow
population. Several questions remain unresolved concerning the mechanism whereby the
CD4+CD45RBlow
T cells exert their anti-inflammatory effects. First, it remains uncertain if TGF-
and IL-10 act in a single pathway, perhaps by the
TGF-
-mediated induction of IL-10 synthesis, or if they prevent
inflammation through independent pathways. Furthermore, it is not known
whether the anti-inflammatory cytokines are produced primarily by the
CD4+CD45RBlow
T cells themselves or if they are induced in other cell types. The
results from the IL-10 transgenic mice and the transfer of Tr1 cells,
however, suggest that T cell-derived IL-10 is of critical importance. Third, it is not known if the
CD4+CD45RBlow
T cells prevent disease by acting locally in the intestine or elsewhere. Finally, additional mechanisms for the prevention
of disease pathogenesis must be considered. For example, host NK cells
have recently been demonstrated to inhibit colitis induction by
transfer of
CD4+CD45RBhigh
T cells, apparently in a perforin-dependent way (7).
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THE ROLE OF BACTERIAL PRODUCTS IN THE INDUCTION OF COLITIS |
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The antigen specificity and the original site of encounter with antigen by the transferred CD4+CD45RBhigh population remain unknown. Published evidence suggests that T cell expansion is occurring in the lamina propria and the epithelial layer of the small and large intestine (19). T cell homing to the intestine is however believed to require previous antigen contact; therefore, it is likely that the initial priming with antigen occurred in the spleen or some other site. The published data also suggest that the donor-derived T lymphocytes are polyclonally activated, as reflected by a diverse T cell repertoire. This suggests the presence of a polyclonal mitogen or a polyclonal response against different gut-derived antigens (19).
Any infectious agent or luminal toxin that is capable of breaking the mucosal barrier may be able to create an inflammatory response that can become chronic in the genetically susceptible host. The normal tolerance (or ignorance) that exists toward the indigenous intestinal flora is broken in experimental colitis (6). The finding that significant bowel pathology is limited to the large intestine supports the hypothesis that bacteria play a crucial role in pathogenesis, as the colon contains the bulk of the flora. Consistent with this, germ-free housing of the SCID recipients significantly attenuates the extent of disease and colitis can be ameliorated by antibiotic treatment of the SCID recipients (2).
The dramatic skewing toward a Th1 response in the SCID recipients could reflect IL-12 secretion and polyclonal activation of T cells, possibly induced by superantigens, mitogens, or other cell wall products derived from luminal bacteria of the colon. In particular, Helicobacter hepaticus has been shown to cause colitis in immunodeficient mice but causes no intestinal pathology in immunocompetent animals (25). Furthermore, when CD4+CD45RBhigh T cells are injected into Helicobacter-infected animals, the resulting intestinal inflammation is more severe than the one resulting from T cell transfer or Helicobacter infection alone (5).
Finally, the question remains as to why do the SCID mice become ill
following
CD4+CD45RBhigh
T cell transfer. The clear implication from the
CD4+CD45RBlow
cotransfer studies is that the SCID recipient lacks an important regulatory or anti-inflammatory T cell population. It remains possible,
however, that the SCID mouse has additional defects as a result of its
total immunodeficiency. For example, -intraepithelial lymphocytes
might function to maintain the epithelial barrier through the synthesis
of keratinocyte growth factor and other cytokines (4), and the absence
of this lymphocyte population in immunodeficient recipients could
contribute to disease susceptibility by causing impaired barrier
function or an impaired healing response to local inflammation.
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CONCLUSIONS |
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In summary, the adoptive transfer of CD4+CD45RBhigh T cells to immunodeficient recipients provides an attractive and highly versatile model for the study of the etiopathogenesis of mouse intestinal inflammation and of the factors that prevent inflammation. With the use of genetically altered donors or recipients, such as animals deficient in cytokine genes, and with the use of treatments with neutralizing antibodies or with recombinant cytokines, the exact role of particular mediators in pathogenesis can in principle be unraveled. Such an analysis should aid in understanding both the etiopathogenesis and the treatment of human IBD, in particular Crohn's disease, which is mediated in part by an analogous proinflammatory cytokine cascade.
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ACKNOWLEDGEMENTS |
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The title of this article was inspired by a review from J. P. Allison and M. F. Krummel (The yin and yang of T cell costimulation, Science 270: 932-933, 1995).
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FOOTNOTES |
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* Second in a series of invited articles on Mucosal Immunity and Inflammation.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-46763 (M. Kronenberg) and DK-54451 (H. Cheroutre). This is publication number 292 from the La Jolla Institute for Allergy and Immunology.
Address for reprint requests and other correspondence: M. Kronenberg, Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121 (E-mail: mitch{at}liai.org).
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