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Eß7 Prevents and Ameliorates Immunization-Induced Colitis in IL-2-/- Mice
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Mucosal Immunity Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, and
Protein Design Laboratories, Inc., Mountain View, CA 94043
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Eß7 is highly expressed on colonic
intraepithelial lymphocytes and has been suggested to function as a
homing or retention molecule for intraepithelial lymphocytes. To
evaluate the role of Eß7 in colitis, we
administered a mAb against Eß7 to
IL-2-/- mice that were immunized at the same time with
TNP-OVA in CFA. To our surprise, this treatment resulted in a
significantly reduced colitis severity score, 0–2 vs 3–4, that was
associated with a significant reduction in CD4+ lamina
propria lymphocyte subpopulation (p < 0.01). In
contrast, the total number of splenic CD4+ T cells of
treated animals was significantly elevated compared with that of
untreated animals (3.2 ± 0.6 x 107 vs 1.2
± 0.2 x 107; p < 0.05).
Similarly, functional studies revealed that IFN-
production by
lamina propria lymphocytes isolated from IL-2-/-
TNP-OVA-immunized mice treated with
anti-Eß7 was significantly lower than
in untreated IL-2-/- TNP-OVA-immunized mice. In contrast,
IFN- production by splenic cells isolated from treated
IL-2-/- TNP-OVA-immunized mice was significantly higher
than in untreated mice. Finally, TNP-OVA-immunized
IL-2-/- mice that were treated after the colitis had been
established also showed a significant decrease in mucosal inflammation
after Eß7 mAb administration. Thus, the
above findings demonstrate that the onset and maintenance of
inflammatory bowel disease depends on the colonic localization of
lamina propria CD4+ lymphocytes expressing
Eß7. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß7, on
circulating lymphocytes and the addressin mucosal addressin cell
adhesion molecule-1 (MAdCAM-1) on endothelial cells (5, 6, 7, 8, 9, 10). The
"homing" interactions governing the entry of cells into tissues are
accompanied by additional adhesion molecule-ligand interactions that
ensure the retention of cells in the target tissue (11). In the case of
a particular subclass of mucosal lymphocytes, the intraepithelial
lymphocytes (IELs) located among the epithelial cells above the mucosal
basement membrane, it has been shown that the integrin,
Eß7, interacts with the ligand E-cadherin
on epithelial cells, and it has been postulated that this
interaction serves to retain IELs in the intraepithelial site (12, 13, 14, 15, 16).
Since Eß7 is also expressed on lamina
propria lymphocytes (LPL), the question arises as to its function in
the lamina propria (LP). Eß7 is
up-regulated on mucosal cells in general, i.e., those in the LP as well
as those in the intraepithelial compartment, perhaps as a result of
exposure to increased concentrations of TGF-ß, a cytokine known to
stimulate Eß7 expression (9, 17). This
leads to the speculation that it may have regulatory functions, such as
homing or retention, not limited to the epithelial layer.
With respect to chronic inflammation of the mucosa, previous studies on
both cotton-top tamarin monkeys and on the SCID CD45Rbhigh
transfer murine colitis model have demonstrated that treatments
directed against the 4ß7/MAdCAM-1
interaction diminish the extent of colitis in both of these IBD models
(18, 19, 20). However, the role of Eß7 in this
organ-specific inflammation is currently unclear. In the present work
we set out to determine whether Eß7
participates in the development of colitis by assessing the effect of
the administration of anti-Eß7, and
for comparison, anti-4ß7, on
immunization-induced colitis in IL-2-/- mice.
Recent studies have shown that IL-2-/- mice do not develop significant colitis when maintained in a pathogen-free environment, but can be induced to develop severe colitis rapidly and predictably following i.p. injection of 2,4,6-trinitrophenol (TNP)-substituted protein in CFA (21). Such induced colitis is a Th1 CD4+ lymphocyte-driven inflammation that is similar to the spontaneous colitis developing in IL-2-/- mice maintained in a conventional environment (21, 22, 23). We have postulated that in the immunization-induced colitis, the TNP-substituted protein administration induces T cells in the thymus and spleen that cross-react with Ags in the endogenous colonic microflora; thus, when such T cells are stimulated by the microflora in the colon, they are induced to produce inflammatory cytokines. That this occurs in IL-2-/- mice and not in normal mice may be due to their lack of counterregulatory responses, such as the production of regulatory cytokines like TGF-ß (24).
Since the immunization-induced colitis found in the
IL-2-/- mice depends on the extravasation of activated
CD4+ lymphocytes or their precursors into the colonic
compartment, it provides a unique opportunity to analyze the role of
Eß7 in this experimental model of chronic
intestinal inflammation. We report here that the administration of
either anti-4ß7 or
anti-Eß7 prevents colonic inflammation
and that anti-Eß7 reverses
pre-existing inflammation, indicating that both entry and retention of
cells are necessary for the induction and maintenance of colitis in
IL-2-/- mice. These results provide strong support for
the notion that Eß7 plays an important
functional role in LP cells, perhaps in their retention within the LP.
Furthermore, these observations may have an important implication for
the treatment of human IBD.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice rendered IL-2 deficient via gene targeting (25) were graciously donated by W. Paul (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD). Colonies of mice were generated by breeding heterozygous parents, and 4 wk after birth, homozygous mice were identified by PCR analysis of DNA isolated from digested tail snips as described (21). Mice were housed in a specific pathogen-free animal facility on the Bethesda campus of the National Institutes of Health. Before study, all mice were handled with gloves under a class II hood, fed sterile food and water ad libitum, and maintained in sterile microisolators that were changed weekly. Selected tissues of various mice were collected for histopathology and endotape testing; cecum and colons were collected for parasitology. Such tissues tested negative for mouse adenoviruses, carbocillus, ectromelia, epizootic diarrhea of infant mice, George disease VII, lymphocytomegalovirus, mouse hepatitis virus, Mycoplasma, mouse pneumonia virus, reovirus, sendavirus, mouse cytomegalovirus, and parvovirus. Mice of both sexes age 5–8 wk were used.
Immunization and treatment of mice
Mice (4–7 wk old) were immunized once with the TNP conjugate of OVA in CFA. The conjugate was prepared as follows. Twenty milligrams of OVA (Pierce Chemical, Rockford, IL) reconstituted in 2.0 ml of distilled water was added to 2.0 ml of potassium borate buffer (pH 9.0–9.3) and then mixed with 3.0 ml of 1 mg/ml solution of TNBS (a gift from Dr. J. Inman, National Institute of Allergy and Infectious Diseases, National Institutes of Health) in distilled water, and 8 µl of 1 M Na2CO3 was added. The mixture was allowed to sit in the dark for 8 h at room temperature and dialyzed 12 h in 1x PBS, after which the concentration of the conjugate was determined via bicinchoninic acid (BCA) protein assay reagent (Pierce Chemical). Mice were injected i.p. with 100 µg of TNP-OVA emulsified in CFA (Sigma, St. Louis, MO). Control mice were injected with PBS emulsified in CFA.
To determine the role of 4ß7 and
Eß7 in the development of colitis in
IL-2-/- mice, the mice were treated with 0.5 mg of
anti-4ß7 mAb, reacting to the
ß7 chain of the molecule (rat IgG2a mAb, clone DATK32,
PharMingen, San Diego, CA), or 0.5 mg of
anti-Eß7 mAb, reacting to the
E chain of the molecule (rat IgG2a mAb, clone M290,
PharMingen), or rat-IgG2a 
control Ab (PharMingen) i.p. 1 h
before TNP-OVA immunization (day 0), and the same amount of each mAb
was readministered on days 2, 4, and 6 until mice were sacrificed. In
some studies, groups of mice that had previously been immunized with
TNP-OVA and had developed colitis were injected with 0.5 mg of
anti-Eß7 every other day for a total
of three times and evaluated on the 7th day from the first
anti-Eß7 mAb injection. FACS analysis
of double staining of LP cells with
anti-4ß7 and
anti-Eß7 mAb revealed no
cross-reactivity between the anti-4ß7
and anti-Eß7.
Grading of histology changes
Colons were dissected from immunized mice and processed for the preparation of frozen sections. Briefly, small sections, approximately 3–5 mm, were cut from each tissue and submerged in dry ice-cooled OCT Embedding Compound (Tissue Tek; Miles, Elkhart, IN). After complete solidification of the embedding media, samples were stored at -80°C.
Tissues were removed at indicated time points, cryopreserved, fixed, and embedded in paraffin. The embedded tissues were sectioned and stained with hematoxylin and eosin (American Histolabs, Rockville, MD). The degree of inflammation of the colon was graded semiquantitatively from 0 to 4 in a blinded fashion using the following criteria: 0, no signs of inflammation; 1, very low-level inflammation indicated by increased mononuclear cell infiltration in <10% of the higher (x50) examination field; 2, low-level inflammation indicated by increased mononuclear cell infiltration in 10–20% of the examination field; 3, moderate inflammation indicated by diarrhea, increased level of mononuclear cell infiltration in 30–50% of the examination field, high vascular density, and thickening of the colon wall; and 4, severe inflammation indicated by >50% transmural mononuclear cell infiltration of the examination field, loss of goblet cells, high vascular density, greatly increased thickening of the colon wall, severe diarrhea, weight loss, and death if left untreated.
IEL and LP cell isolation
Preparation of IEL and LP cells has been previously described (21). Briefly, detritus was squeezed out of the colon lumen with forceps, the tissue was minced with sterile scissors, and the pieces were washed with HBSS over a 100 µm nylon mesh bowl to remove remaining detritus and metabolites. IEL were liberated by incubating the cut pieces in 25 ml of warm (37°C) HBSS media without Ca/Mg (Biofluids, Rockville, MD), supplemented with 25 mM HEPES buffer (National Institutes of Health Media Unit) and 10% FCS (BioWhittaker, Walkersville, MD) containing 0.1 M EDTA, for 20 min at 37°C. Filtered cells were enriched for lymphocytes by Percoll-gradient centrifugation. For colonic LPL isolation, the remaining pieces of colon were washed over nylon mesh; resuspended in RPMI 1640 (BioWhittaker) augmented with 25 mM HEPES, 10% FCS, 400 U/ml DNase (Boehringer Mannheim, Indianapolis, IN), and 400 U/ml collagenase (Boehringer Mannheim); and incubated for 1.5 h at 37°C on a rocker. The resultant cell suspension was filtered sequentially through 100 µm and 40 µm nylon mesh filters and then washed two times in RPMI 1640 supplemented with 25 mM HEPES and 10% FCS.
In vitro cell stimulation and cytokine assay
Isolated cells were stimulated in RPMI 1640 supplemented with
10% FCS, 5% NCTC 109 (Biofluids, Rockville, MD), 1%
penicillin/streptomycin/0.25% fungisozone (Life Technologies,
Gaithersburg, MD), 2 mM glutamine (Life Technologies), 5 x
105 M 2-ME (Sigma), and 15 mM HEPES at a density of
1 x 106 cells/1.0-ml final volume. Cells were
cultured in 24-well tissue culture plates (Costar, Cambridge, MA) in
the presence of plate-bound anti-CD3 (10 µg/ml; PharMingen) with
soluble anti-CD28 (1 µg/ml; PharMingen) and IL-2 (50 U/ml;
Chiron, San Diego, CA) at 37°C in humidified 6% CO2.
Culture supernatants were harvested after 48 h and frozen at
-20°C until assayed. IFN- production in culture supernatants was
assayed by ELISA using the PharMingen protocol in association with
coating, standard, and biotinylated secondary Abs obtained from
PharMingen and horseradish peroxidase-streptavidin obtained from Zymed
(South San Francisco, CA).
Flow cytometry
To characterize T cell phenotypes in recovered LPL, IEL, and
splenocytes, flow cytometric analysis with Abs to murine cell surface
determinants (PharMingen) was employed. For this purpose, cells were
washed two times in FACS buffer (National Institutes of Health Media
Unit), resuspended at 107 cells/ml in FACS buffer, and
transferred to FACS tubes (Becton Dickinson, Mountain View, CA). To
prevent nonspecific FcR-mediated binding of Abs, 1–10 µg/ml of
anti-FcR (2.4G2; PharMingen) was added to each tube 3 min before
staining. Cell suspensions were stained with 1 µg/ml for 30 min on
ice with FITC-conjugated anti-CD3, FITC-conjugated anti-CD4,
phycoerythrin-conjugated anti-CD8, biotin-conjugated
anti-ICAM-1 (CD54), biotin-conjugated anti-CD69, or
biotin-conjugated anti-Eß7
(PharMingen). Cells stained with biotin-conjugated Abs were
subsequently washed once in FACS buffer and incubated on ice for 30 min
with streptavidin-phycoerythrin at 1 µg/ml. All stained cells were
washed two times in FACS buffer, resuspended at 1 x
106 cells/ml, and examined on a FACScan analyzer with Lysis
II software (Becton Dickinson). Nonviable cells were excluded by
forward angle scatter or by propidium iodide uptake.
Statistical analysis
Descriptive statistics and testing for significance of differences were assessed by Student’s t test using the StatWorks and Microsoft Excel statistical analysis computer programs.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eß7 or
anti-4ß7 mAb is associated with
reduction of LPL and IEL but increase of splenic cell numbers
To evaluate the role of Eß7 vs
4ß7 in the TNP-OVA/IL-2-/-
model of colitis, IL-2-/- mice maintained under specific
pathogen-free conditions were immunized i.p. with TNP-OVA in CFA and
treated with either anti-Eß7 or
anti-4ß7 mAbs at the same time of
immunization. As shown previously and again in Fig. 1
, severe colitis was seen in mice
injected with TNP-OVA (21). However, when TNP-OVA was coinjected with
anti-Eß7 mAb, the colitis was
prevented and microscopic examination of the colonic mucosa revealed
mucosa resembling the one seen in PBS-injected mice. This was
reflected by significant reduction in colitis severity score (CSS). CSS
for IL-2-/- mice injected with TNP-OVA was 3–4; whereas
that for TNP-OVA + anti-Eß7-injected
mice was 0–2 (Table I). Furthermore,
this inhibition of colitis was associated with a fourfold reduction in
LPL numbers and twofold reduction in IEL numbers in mice that were
coinjected with anti-Eß7 mAb (for
IL-2-/- LPL, 27.1 ± 11.8 x 106
(TNP-OVA) vs 6.5 ± 0.6 x 106 (TNP-OVA
coinjected with anti-Eß7);
p < 0.01; for IL-2-/- IEL, 5.2 ±
1.8 x 106 (TNP-OVA) vs 2.4 ± 0.9 x
106 (TNP-OVA coinjected with
anti-Eß7); p <
0.05). Interestingly, when we looked at the total number of cells
within the spleen, there was a slight increase of total cell
numbers following anti-Eß7 mAb treatment
(Table I). Furthermore, TNP-OVA-induced colitis was significantly
reduced when mice with established colitis were treated with three
anti-Eß7 mAb injections (0.5 mg i.p.
each); CSS for TNP-OVA CSS was 3–4 and that for TNP-OVA +
anti-Eß7 mAb injections was 1–2. As
shown in Table II, such treatment of
established colitis also led to a significant reduction of LPL and IELs
(p < 0.01). To evaluate the role of
4ß7 in this model of colitis, we also
coinjected IL-2-/- mice with TNP-OVA and
anti-4ß7. As shown in Table I,
injection of anti-4ß7 also prevented
the development of TNP-OVA-induced colitis in IL-2-/-
mice. Therefore, the above data demonstrate that not only is the
injection of anti-Eß7 mAb capable of
preventing TNP-OVA-induced colitis in IL-2-/- mice, but
it also can also directly ameliorate established colitis.
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Eß7 mAb in
IL-2-/- mice reduces the number of infiltrating
CD4+ T cells within the LPL but increases CD4+
T cells within the spleen
We have previously demonstrated that CD4+ T
cells are the principal effector cells involved in the pathogenesis of
immunization-induced colitis in IL-2-/- mice (21, 26). We
were therefore interested to see whether the administration of
anti-Eß7 vs
anti-4ß7 had different effects on
lymphoid organ compartmentalization of CD4+ and
CD8+ T cells. As shown in Table I
, injection of
anti-Eß7 mAb led to the most
noticeable reduction of infiltrating lymphocytes within the LP
compartment. This reflects our previous observation (demonstrated here
in Table I and Fig. 1) that the most severe inflammation is found
within the LP of the colon in this murine colitis model. As shown in
Fig. 2,
anti-Eß7 mAb injection led to a
fivefold reduction of LP CD4+ lymphocytes; total number of
CD4+ LPL in TNP-OVA was 21.7 x 106, and
that in TNP-OVA + anti-Eß7 was
4.7 x 106 (p < 0.01). Within
the IEL compartment, anti-Eß7 treatment
was associated with a modest reduction of CD8+ T cells:
TNP-OVA, 4 x 106; TNP-OVA +
anti-Eß7, 1.6 x 106
(Fig. 3). Interestingly, the total number
of CD4+ T cells within the intraepithelial compartment was
not significantly altered by the injection of
anti-Eß7 mAb into
IL-2-/- mice. Furthermore, when we analyzed similarly
treated IL-2+/+ mice, no changes in the total number of
CD4+ T cells were observed within any of the compartments
evaluated (Table I and data not shown).
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Eß7 on
CD3+ LPL and IEL. As shown in Table II, immunization of
TNP-OVA led to a significant increase of
Eß7-expressing lymphocytes both by total
number of cells and, more importantly, by the percentage of
CD3+ lymphocytes within the LP compartment: for
TNP-OVA-immunized IL-2-/- mice,
CD3+/Eß7+ LPL,
63.3 ± 6% (total, 17.2 x 106); for the
PBS-injected control group, 35.5 ± 2.1 (total, 1.6 x
106); p < 0.05. Furthermore, treatment
with anti-Eß7 mAb of established
colitis led to a significant reduction of infiltrating
CD3+/Eß7+
lymphocytes within the LP compartment: for
CD3+/Eß7+ LPL,
25.6 ± 3.4% (total, 2.6 x 106);
p < 0.002. As expected, the majority of
CD3+ IEL were expressing the
Eß7 integrin. In contrast to the LPL,
treatment with anti-Eß7 mAb did not
significantly alter the ratio of
Eß7+ lymphocytes within this
compartment. However, a noticeable reduction of
Eß7-expressing CD3+ IEL was
found within the large granular lymphocyte gate (blasts, Table II)
following anti-Eß7 mAb treatment: for
TNP-OVA-immunized IL-2-/- mice, 53 ± 3%; for the
anti-Eß7 mAb-treated group, 31 ±
2%; p < 0.002).
To further evaluate whether the reduction of CD4+
lymphocytes in the LP of TNP-OVA-immunized IL-2-/- mice
following anti-Eß7 mAb treatment was
due to the deletion of lymphocytes or to an effect on lymphocyte
distribution, we also evaluated the number of T cells within the
peripheral lymphoid organs. As shown in Fig. 4, injection of
anti-Eß7 was associated with
significantly increased numbers of CD4+ T cells within the
spleen: TNP-OVA, 13.5 x 106; TNP-OVA +
anti-Eß7, 32.4 x
106; p < 0.01. Within the mesenteric lymph
node compartment, total CD4+ T cell numbers were similar in
TNP-OVA-immunized IL-2-/- mice whether or not they had
received anti-Eß7 (data not shown).
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Eß7 mAb results in
decreased IFN- production within LPL but increased IFN-
production within the spleen
Since it is now well established that the severity of chronic
mucosal inflammation correlates with the level of IFN- production by
inflammatory CD4+ T cells in many murine models of colitis,
we further analyzed whether the treatment with
anti-Eß7 mAb affects IFN- levels.
Thus, in initial studies, we compared the IFN- production of LPL
isolated from the various treatment groups. After isolation, LPL were
directly stimulated in vitro with anti-CD3 and anti-CD28 mAbs,
and IFN- production in the culture supernatants was analyzed. As
demonstrated in Fig. 5, T cells from
anti-Eß7 mAb-treated
IL-2-/- mice manifested a fivefold reduction of IFN-
when compared with untreated TNP-OVA-immunized mice: for TNP-OVA +
anti-Eß7 mAb, 46 ± 15 U/ml
IFN-; for TNP-OVA alone, 225 ± 72 U/ml IFN-
(p = 0.003). In a similar series of studies, we
evaluated splenic T cell IFN- production from treated vs untreated
IL-2-/- mice. As shown in Fig. 5, TNP-OVA immunization
alone induced a threefold increase in IFN- production in the splenic
T cells when compared with PBS-injected control IL-2-/-
mice. Furthermore, splenic IFN- secretion was even higher, following
coinjection of TNP-OVA and anti-Eß7:
for TNP-OVA alone, 97 ± 20 U/ml IFN-; for TNP-OVA +
anti-Eß7 mAb, 202 ± 14 U/ml
IFN- (p = 0.005). Of note, in none of the
above conditions could we find significant IL-4 production (data not
shown).
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Eß7 mAb is preventing the influx
and/or retention of T cells that are committed to the Th1 cytokine
pathway into the colonic LP.
Anti-Eß7 mAb treatment prevents
accumulation of CD3+/CD54+ LPL in
TNP-OVA-immunized IL-2-/- mice
The expression of adhesion molecules is undoubtedly an important
regulatory element during the induction of chronic mucosal
inflammation. ICAM-1 (CD54) is one member of the Ig superfamily that is
significantly up-regulated on T cells during Th1-mediated inflammation,
and the binding to its ligand very late Ag-1 (VLA-1) has been
demonstrated not only to enhance T cell adhesion but also to induce
intracellular T cell activation (27, 28, 29, 30, 31, 32). Furthermore, we have
previously shown that one of the characteristics of the TNP-OVA-induced
inflammatory response in IL-2-/- mice is the induction of
CD54 and CD69 (21, 26). Since anti-Eß7
mAb treatment led only to decreased IFN- production within the LP
compartment but not in the spleen, we were interested to see whether
this was reflected by the expression of the above-described T cell
activation markers. As demonstrated in Table III, there was a significant decrease of
CD3+/CD54+ LPL following coinjection of TNP-OVA
and anti-Eß7 compared with TNP-OVA
alone, 2.6 x 106 and 17.9 x 106,
respectively, and this was also evident by fewer percent of LPL
expressing CD54 following anti-Eß7
treatment. However, when we evaluated the T cell expression of CD54
within the spleen, there was a notable increase of
CD3+/CD54+ lymphocytes following
anti-Eß7 treatment: total number of
CD3+/CD54+ lymphocytes following coinjection of
TNP-OVA + anti-Eß7 mAb was 165.6
x 106; that for TNP-OVA alone was 135 x
106. Similar findings were seen for the expression of
another, but less characterized, activation marker, CD69 (Table II).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eß7, an integrin formerly associated with
the retention of IEL within the mucosal epithelium, in maintaining
inflammatory lesions in the LP of the colon. Blocking this integrin
with a specific mAb that reacts with E was surprisingly
as effective as treatment with
anti-Eß7 mAb, an Ab known to block
mucosal T cell homing, in preventing colitis. Moreover,
anti-Eß7 treatment not only was able
to prevent colitis but was also effective in ameliorating existing
inflammation in the colon. The effects observed manifested themselves
in a significant reduction of IFN- production of colonic LP cells
with a corresponding increase of IFN- secretion in the spleen. In previous studies we have demonstrated that the colitis induced in IL-2-/- mice by administration of TNP-protein depends, at least during its initiation, on the influx of T cells from inductive sites of initial Ag presentation to sites in the colonic LP, where T cells are presumably cross-reacting with mucosal Ag. The inductive sites from which the necessary cellular migration originates fall into two potential categories. One is the spleen, or lymph nodes, i.e., the "peripheral" lymphoid tissues. Another site is the thymus, the "central" lymphoid tissue; the latter may also be exposed to TNP-protein and can potentially utilize TNP-protein as an Ag of cellular selection (2). The possible involvement of the thymus in the induced colitis of IL-2-/- mice is supported by recent studies that show quite clearly that thymic development is significantly disrupted in the IL-2-/- mouse and that thymocytes developing in IL-2-/- mice after TNP-protein administration are themselves able to induce colitis when transferred to normal recipient mice (26, 33, 34).
Recent publications have demonstrated that in the mouse, homing of
lymphocytes into the gut is mediated by the interaction of the
4ß7 integrin with MAdCAM-1 in both
Peyer’s patch high endothelial venules (HEVs) and postcapillary
venules in LP (6, 35). Given these findings, it was not surprising that
the combination of two Abs reacting with the ß7 integrin
and MAdCAM-1 prevented the development of colitis in the SCID T cell
transfer colitis model (20). In another model of IBD, mAbs blocking
both 4ß1 and
4ß7 attenuated established colitis in
cotton-top tamarin monkey (18, 19).
Another molecule, besides 4ß7, that
is highly specific for intestinal lymphocytes (lymphocyte Peyer’s
patch adhesion molecule-1; LPAM-1) is
Eß7 (CD103), which is expressed on almost
all intestinal epithelial lymphocytes and seems to be responsible for
the retention of these cells (11). Thus, based on the assumption that
retention of lymphocytes within the mucosal compartment might be also a
crucial mechanism for organ-specific inflammation, we sought to
determine whether Eß7 has a role in
maintaining colonic inflammation as well. As mentioned above, a mAb
against the E portion of the
Eß7 molecule was not only able to prevent
TNP-OVA-induced colitis but also was able to inhibit established
colitis. However, in contrast to anti-IL-12 treatment, which led to
a generalized deactivation of peripheral lymphocytes in several organs
as well as colonic LPL, the anti-inflammatory effect of
anti-Eß7 treatment was only found
within the intraepithelial and more profoundly in the LP compartments
of the colon. This is to our knowledge the first time that
Eß7 has been shown to play a functional
role in activated LP lymphocytes.
The results from this study suggest several possible mechanisms for
Eß7 and its role in down-regulating
mucosal inflammation. First, it is possible that
Eß7+ T cells play an important
role in inducing and maintaining mucosal inflammation, and
cross-linking Eß7 on the surface of
intraepithelial T cells could significantly alter their function as an
effector cell and thus could lead to the down-regulation of LP T cell
activation. This, however, is not very likely, since it could not
explain our finding that anti-Eß7
treatment not only led to an increase of
Eß7 and 4ß7
CD4+ cells in the spleen (data not shown), but also to an
increase in IFN- production as well. Such accumulation of activated
LP T cells in the spleen could be explained by the fact that we and
others have shown that the colonic inflammation in IL-2 knockout mice
requires a cellular migration from the thymus and spleen to the colon
mucosa (26, 33). Thus, it is possible that the disruption of cellular
traffic from other organs leads to a decreased influx of inflammatory T
cells into the mucosal tissue while resulting in the accumulation of
such cells in other nonmucosal tissues. In this case,
Eß7 would function as a homing molecule
rather than a retention molecule in the mucosal immune system. This
possibility is unlikely on several grounds. The percentage of
Eß7+ cells in the circulation
is only 2%, far lower than that of 4ß7,
35%; thus, if Eß7 were a homing integrin
similar to 4ß7, one would expect the
percentage of cells in the circulation to be higher (36). Moreover,
Eß7 expression increases dramatically
after cells enter the LP (9), suggesting that the function of
Eß7 is relevant to cells already in the LP
rather than to those entering the site. That
Eß7 serves rather as a retention than a
homing molecule is also supported by the findings of Austrup et al.
(37) that in vitro induction of Eß7
expression on naive splenic T cells did not lead to their
preferentially homing into the gut. However, these experiments were not
performed under in vivo inflammatory condition, so it is possible that
under those conditions a different homing pattern would have been
observed (37). Thus, the final and most likely explanation in our
opinion is that the treatment of colitic animals with an
Eß7 mAb disrupts the interaction of
Eß7+ LP cells as well as
Eß7+ IELs with their ligand
and such disruption leads to the failure of retaining inflammatory
cells at this site. This hypothesis, however, is clearly problematic
under the current knowledge, that E-cadherin serves as a ligand for
Eß7 on IEL in the intraepithelial space of
the mucosa and its expression is absent from the LP (12, 14, 15, 38, 39). However, the possibility that this integrin interacts with a
second ligand is reasonable, particularly since other integrins and
selectins bind with several different ligands and, in fact, it has been
shown that only 40% of Eß7+
cells bind to cells bearing E-cadherin, suggesting that another ligand
may exist (12). Furthermore, it has been demonstrated that
4ß7 can serve as a ligand for itself, and
since Eß7 may induce homotypic aggregation
of Eß7+ lymphocytes, it is not
inconceivable that it could, similarly to
4ß7, serve as a ligand to itself (40, 41).
A final mechanism by which treatment with intact mAb against
Eß7 could be working is the formation of
immune complexes on cell surfaces followed by complement-mediated lysis
and/or removal of Eß7+
lymphocytes by the reticuloendothelial immune system. We think that
this mechanism is unlikely in view of the fact that an increased number
of Eß7+ lymphocytes
accumulates in the spleen of anti-Eß7
mAb-treated mice. However, it could be further examined more directly
with the use of mAb lacking Fc (Fab Ab).
While anti-Eß7 administration greatly
reduced the accumulation of CD4+ T cells in the LP, it had
little effect on CD8+ T cells at this site. In contrast, it
did reduce the number of CD8+ T cells otherwise
accumulating in the intraepithelial compartment (as well as the
CD4+ T cells in this compartment), suggesting that
CD8+ T cells do not require
Eß7 for retention in the LP. However, the
number of CD8+ T cells under study in the LP was quite
small, and additional studies will be necessary to validate this point.
Previous studies of induced colitis in IL-2 knockout mice employing
systemically administered anti-CD4 and anti-CD8 Abs indicated
that CD4+ T cells and not CD8+ T cells are the
key effector cells in IL-2-/- colitis (21). The studies
here showing that prevention of colitis by
anti-Eß7 administration affects the
infiltration and retention of LP CD4+, but not
CD8+ T cells, offers additional corroboration of this
point.
In summary, in contrast to anti-IL-12 treatment, the administration
of anti-Eß7 mAb leads to a redirection
of activated mucosal CD4+ T cells from the LP into the
spleen, and thus only affects mucosa-specific T cells. Thus, one
important advantage of treating colitis with
anti-Eß7 mAb is that it is not likely
to have a general immunosuppressive effect, as seen with
anti-IL-12, during the period of treatment. However, on a
cautionary note, anti-Eß7 treatment
led to the induction of inflammation at different sites, since such
treatment clearly seems to affect only trafficking and not
down-regulation of T cell activation. Translating this to the treatment
of human IBD, the use of Abs to prevent the retention of inflammatory
cells may be very beneficial in conjunction with T cell down-regulatory
treatment to achieve complete remission of chronic colonic inflammatory
diseases.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: IBD, inflammatory bowel disease; MAdCAM-1, mucosal addressin cell adhesion molecule-1; IEL, intraepithelial lymphocyte; LPL, lamina propria lymphocyte; LP, lamina propria; TNP, 2,4,6-trinitrophenol; CSS, colitis severity score.
Received for publication August 21, 1998. Accepted for publication January 22, 1999.
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References |
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