1 Department of Internal Medicine, School of Medicine, Keio University, Tokyo 160-8582; 2 Second Department of Internal Medicine, National Defense Medical College, Saitama 359-8513, Japan; and 3 Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71110
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ABSTRACT |
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Relatively little is known
about how recirculation of lymphocytes through the inflamed intestinal
mucosa is regulated. The aim of this study was to investigate the
dynamic process of T lymphocyte-endothelial cell adhesion in
TNF--challenged murine colonic mucosa by intravital microscopy. T
lymphocytes from spleen (SPL) and intestinal lamina propria (LPL) were
fluorescence labeled, and their adhesion to microvessels in the colonic
mucosa was observed. In TNF-
(25 µg/kg)-stimulated colonic
venules, an enhanced adhesion of SPL and LPL was demonstrated, with
dominant recruitment of LPLs. The magnitude of the increased LPL
adhesion was more significant in the colon than in the small intestine.
These T lymphocyte interactions in the colonic mucosa were
significantly reduced by blocking MAbs against either mucosal addressin
cell adhesion molecule-1 (MAdCAM-1), VCAM-1,
4-integrin,
or
7-integrin but not by anti-ICAM-1.
Immunohistochemistry revealed significant MAdCAM-1 expression in the
lamina propria and VCAM-1 expression in the submucosa of
TNF-
-treated colon. Spatial heterogeneity of MAdCAM-1 and VCAM-1
activation following TNF-
challenge may promote specific T
lymphocyte recruitment in the inflamed colonic mucosa.
microcirculation; inflammatory bowel disease; 4-integrin;
7-integrin; lamina proprial
lymphocytes
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INTRODUCTION |
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RECIRCULATION
OF LYMPHOCYTES through the body is critical for immune
regulation. Although the regulation of lymphocyte migration from blood
into the lymphoid tissues has been studied extensively in recent years
(6, 9, 41), relatively little is known about how the
process of lymphocyte migration through the intestinal mucosa is
regulated at the local level, especially during an inflammatory response. Lymphocyte homing from blood to lymphoid tissue and at
inflammatory sites depends on the interaction between lymphocytes and
venular endothelial cells. It is becoming increasingly apparent that
inflammation is associated with enhanced expression of endothelial cell
adhesion molecules (ECAMs) in the intestine of both experimental animals and humans (8, 10, 21, 30). Lymphocyte homing to
Peyer's patches and mucosal sites is thought to be regulated by
4
7-integrin and its ligand, mucosal
addressin cell adhesion molecule-1 (MAdCAM-1) (5).
Increased MAdCAM-1 expression has been demonstrated in the colonic
microvasculature of different animal models of colitis (19,
21). We recently demonstrated that enhanced expression of
MAdCAM-1 in inflamed intestinal mucosa plays a significant role in the
development of dextran sulfate sodium-induced colitis
(18). ICAM-1 and VCAM-1 are other important ECAMs that are
induced within the inflamed colon. In clinical studies, these three
molecules, ICAM-1, VCAM-1, and MAdCAM-1, were reported to be
upregulated in the intestinal mucosa during the active stages of
ulcerative colitis and Crohn's disease (17, 27, 36).
Anti-ICAM-1 MAb and anti-ICAM-1 antisense oligonucleotide were shown to
prevent acute dextran sulfate sodium-induced colitis in both rats and
mice (3, 15, 38). Anti-VCAM-1 antibody was also reported
to abrogate the increased leukocyte adhesion in colonic venules during
trinitrobenzene sulfonic acid-induced colitis in rats
(32). However, relatively little is known about the exact
location of adhesion molecule upregulation within the inflamed
intestine. Furthermore, there is a paucity of information regarding how
the different ECAMs contribute to T lymphocyte recruitment in the
colonic mucosa under inflammatory conditions.
Recent studies indicate that immune regulation in the colonic mucosa is different from that in the small intestine. For example, intraepithelial lymphocytes (IELs) of the murine colon are mostly CD4+ cells, and 30% of IELs in the colon express L-selectin, whereas IELs in the small intestine are predominantly CD8+ cells, which are devoid of L-selectin (2, 33). Colonic IELs and lamina proprial lymphocytes (LPL) appear to be less activated than their small intestinal counterparts. These differences may be related to the luminal environment of the bowel at these anatomic sites. Although there also appear to be differences in the trafficking of lymphocytes between small intestine and colonic mucosa, there is relatively little quantitative information related to lymphocyte homing, especially for the colonic mucosa (34). Furthermore, the relative contributions of various ECAMs to lymphocyte migration have not been compared between small intestinal and colonic mucosa under both control and inflammatory conditions.
The diverse biological actions of TNF- contribute to inflammatory
responses, to antibacterial activity, and to T cell-mediated immunity
in the intestinal mucosa. TNF-
is produced mainly by T cells and
macrophages. It is a growth factor for T cells, B cells, and natural
killer cells, and the cytokine promotes inflammation by triggering
chemotaxis of neutrophils and monocytes and by increasing the
expression of ECAMs (13, 22, 42). TNF-
production in the mucosa has been studied most extensively in inflammatory bowel disease, with some reports describing increased cytokine levels in
diseased lamina propria compared with adjacent uninflamed bowel (7, 26, 31). The increased expression of adhesion
molecules on endothelial cells is known to be mediated by
proinflammatory cytokines such as IL-1 and TNF-
(28, 29,
40), suggesting an enhanced emigration of lymphocytes to the
inflamed gut of patients with inflammatory bowel disease. Antibody to
TNF-
improves symptoms and induces remission in Crohn's disease,
which strongly implicates TNF-
in the pathophysiology of Crohn's
disease (1, 39). Although TNF-
has wide-ranging effects
on T lymphocytes in normal and inflamed intestine, there has been no
direct in vivo observation of the effects of TNF-
treatment on
lymphocyte interactions with microvessels of the colonic mucosa.
In this study, an intravital microscopic procedure for monitoring the
dynamic process of lymphocyte migration was employed to 1)
characterize and quantify T lymphocyte-endothelial cell adhesion in
control and TNF--challenged mucosae of the colon and small intestine
and 2) define the localization and contribution of three
different ECAMs (MAdCAM-1, VCAM-1, and ICAM-1) to TNF-
-induced T
cell recruitment in the colon and small bowel.
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METHODS |
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Collection and separation of lymphocytes. Female BALB/c mice at 8 wk of age were used. They were maintained on standard laboratory chow. The care and use of laboratory animals were in accordance with the guidelines of the Keio University Animal Research Committee. Splenic lymphocytes (SPL) were isolated from spleen. Briefly, spleens were mechanically dissociated and red blood cells were lysed in ammonium phosphate/chloride lysis buffer. Cell suspensions were washed and stored in RPMI 1640 medium (pH 7.4; GIBCO, Grand Island, NY) with 5% fetal calf serum on ice until used. LPLs were isolated from mice by the modified procedures as described by Davies and Parrot (11). Briefly, inverted small intestine was cut into four segments, and all Peyer's patches were removed. These segments were transferred to a 50-ml conical tube containing 45 ml of 5% fetal calf serum in Ca2+- and Mg2+-free Hanks' balanced salt solution (HBSS; GIBCO) and shaken at 150 rpm in the horizontal position in an orbital shaker for 45 min at 37°C. Cell suspensions were removed, and the remaining fragments were then transferred to flasks containing HBSS with 90 U/ml of collagenase type I (Sigma, St Louis, MO) and stirred gently for 25 min at 37°C. Cell suspensions containing LPL were filtered through nylon mesh and then centrifuged. LPL were purified by using a 44-70% discontinuous Percoll (Pharmacia) gradient. After centrifugation at 600 g for 20 min at 20°C, cells at the interface were collected, washed, and resuspended in RPMI medium.
A whole cell population of 2 × 107 lymphocytes in 3 ml RPMI medium with 1% fetal calf serum was incubated in 1 g of nylon wool (Wako Pure Chemical, Osaka, Japan) in a column for 1 h at 37°C, and the eluted fraction was designated as the T cell.Lymphocyte labeling with carboxyfluorescein diacetate
succinimidyl ester.
Carboxyfluorescein diacetate succinimidyl ester (CFDSE; Molecular
Probes, Eugene, OR) was dissolved in dimethylsulfoxide to 15.6 mM, and
a small aliquot (300 µl) was stored in a cuvette sealed with argon
gas at 80°C until the experiments. Lymphocytes (1 × 107) were incubated in a CFDSE solution (20 µl of
stock solution was diluted with 20 ml RPMI) for 30 min at 37°C. The
labeled lymphocytes were immediately centrifuged through a cushion of
heat-inactivated fetal bovine serum and washed twice with a cold
suspension medium. The cells were resuspended in 0.2 ml of the medium
and used within 30 min.
Intravital observation of lymphocyte migration in murine colonic mucosa and small intestinal mucosa. After an intraperitoneal injection of pentobarbital sodium (50 mg/kg), the abdomen was opened via a midline incision. For observation of colonic mucosa, a 3-cm colonic segment (1 cm distal to the cecal valve) was chosen and was gently placed onto the plate. A longitudinal incision was made by microcautery along its antimesenteric border. The intestine was kept warm and moist by continuous superfusion with physiological saline warmed to 37°C. Krebs-Ringer solution (pH 7.4) was infused into the lumen to flush away food residue. The adjacent intestinal segment and mesentery were covered with absorbent cotton soaked with Krebs-Ringer solution. Suitable areas of the microcirculation in colonic mucosa were observed from the mucosal surface by an inverted fluorescence microscope (Diaphot TMD-2S; Nikon, Tokyo, Japan) and recorded by a television videotape recording system. The behavior of fluorescently-labeled lymphocytes was visualized on the television monitor by a fluorescence microscope equipped with a silicon-intensified target camera with a contrast-enhancing unit (C-2400-08; Hamamatsu Photonics, Shizuoka, Japan) according to previously described methods (14, 23). Epi-illumination was achieved with filters of excitation at 470-490 nm and emission at 520 nm. Lymphocytes (1 × 107 dissolved in 0.2 ml RPMI) were injected into the jugular vein of the recipient mice for 3 min. The cell kinetics of infused lymphocytes and the interaction with microvascular beds of the colonic mucosa were monitored and continuously recorded on S-VHS videotapes for the first 20 min and then at 10-min intervals for 60 min. The lymphocytes adherent to the venular wall without movement for >30 s were defined as sticking lymphocytes. In this setting, changing the focal plane allowed the investigator to observe at the depth of the submucosa. The number of sticking lymphocytes was normalized to 1 mm2 of the area observed on the video image.
In another set of experiments, a 5-cm ileal segment was chosen for observation of the small intestinal mucosa and placed gently on a plastic plate. The microcirculation of small intestinal villi was also observed from the mucosal surface, and lymphocyte migration was observed through a fluorescence microscope as previously described (24).Administration of agents and antibodies.
TNF- was administered (25 µg/kg) at 5 h before intravital
observation of lymphocyte migration according to previous reports (16). In some experiments, lymphocytes were preincubated
with MAbs that functionally block adhesion molecules. Antibodies
against
7-integrin (Fib27), L-selectin (MEL-14), and
CD11a (M17/4) were purchased from PharMingen (San Diego, CA). Antibody
against
4-integrin (PS/2) was obtained from the American
Type Culture Collection (Manassas, VA). As a control, rat IgGa
(R35-95, PharMingen) was used under the same conditions. Cells
(1 × 107) were incubated with 100 µg/ml of MAbs for
30 min before infusion of T lymphocytes. In some experiments,
anti-MAdCAM-1 MAb (2 mg/kg MECA-367; PharMingen) dissolved in 0.2 ml
saline was infused from jugular vein at 30 min before the injection of
T lymphocytes. The effect of F(ab')2 fragments of the anti-MAdCAM-1 MAb
was also examined. As a control, nonblocking antibody of MAdCAM-1
(MECA-89; PharMingen) was used. In another set of experiments,
anti-VCAM-1 MAb (429; PharMingen) and anti-ICAM-1 MAb (3E2; PharMingen)
were administered under identical conditions.
Histological study of ECAM expression. Localization of ECAM expression in the intestinal mucosa was assessed by immunohistochemistry by using the labeled streptavidin biotin method. Colon and small intestine of mice were removed and fixed in periodate lysine paraformaldehyde (PLP) solution. Thereafter, they were embedded in optimum cutting temperature compound (Miles, Elkhart, IN) before being frozen in dry ice and acetone. Cryostat sections of 6 µm were transferred to poly-L-lysine-coated slides and air dried for 1 h at 20°C. After they were washed in PBS (pH 7.4) containing 1% Triton X-100 for 5 min, sections were incubated in 5% normal goat serum in PBS. Monoclonal antibodies against MAdCAM-1 (MECA-367; 0.5 mg/ml), VCAM-1 (429; 0.5 mg/ml), and ICAM-1 (3E2; 0.5 mg/ml) were diluted 50 times with PBS, layered onto the section, and were incubated overnight at 4°C. Sections were incubated with secondary antibody, biotinylated anti-rat IgG antibody (Amersham, Little Chalfont, UK) for 1 h at room temperature. Then, sections were incubated with FITC-conjugated streptavidin (Amersham) for 30 min at room temperature. Rinsing with PBS containing 1% bovine serum albumin was performed between each step. A coverslip was applied by using glycerol jelly. These sections were observed under a fluorescent microscope (BX60; Olympus, Tokyo, Japan). The ECAM-positive vessels in the colonic and ileal tissues were quantified by using an image analyzer and expressed as area of positively stained vessels per millimeter muscularis mucosa.
Analysis of surface adhesion molecules by fluorescence-activated
cell sorter.
The T cell suspensions were washed in HBSS containing 0.2% bovine
serum albumin and 0.1% NaN3. This medium was used
throughout the staining procedure, and all cells were kept at 4°C
during the experimental procedure. For immunofluorescence staining,
2 × 105 lymphocytes in 25 µl medium were first
incubated with 1 µg of anti-mouse MAbs to characterize and quantify
adhesion molecules. Antibodies against mouse 4-integrin
(PS/2),
1-integrin (9EG7),
7-integrin
(FIB27), CD11a (M17/4), L-selectin (MEL-14), and CD3 (145-2C11)
(PharMingen) were used for this characterization. After incubation for
30 min, the cells were washed in 400 µl HBSS and centrifuged at 1,500 g for 5 min three times. Cells were washed twice and
resuspended for analysis. For controls, lymphocytes were preincubated
with isotype-matched, irrelevant antibodies. Positively stained cells
were detected with FITC-conjugated anti-rat/hamster IgG. Flow
cytometric analysis was performed by using FACSort (Becton Dickinson,
Mountain View, CA), and dead cells were excluded from analysis on the
basis of performed iodide dye exclusion.
Statistics. All results were expressed as means ± SE. Differences among groups were evaluated by one-way ANOVA and Fisher's post hoc test. Statistical significance was set at P < 0.05.
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RESULTS |
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T lymphocyte migration in colonic mucosa and the effect of TNF-
treatment.
The movement and interaction of CFDSE-labeled SPL and LPL within
microvessels were visualized by fluorescence microscopy in the colonic
mucosa. In control mice, the total number of lymphocytes that entered
colonic microvessels was not significantly different between SPL and
LPL at 10 min after infusion (SPL, 9.9 ± 2.2/min vs. LPL,
9.0 ± 2.1/min). Furthermore, the total number of lymphocytes that
entered colonic microvessels was not altered by TNF-
treatment relative to control mice (SPL, 9.9 ± 2.0/min vs. LPL, 11.0 ± 2.3/min at 10 min). Figure 1 shows
microscopic images of the distribution of adherent T lymphocytes (LPL)
in colonic mucosa 60 min after cell infusion, and Fig.
2 shows the changes in adherent T cells over time in this area. In control mice, a few SPL were shown to adhere
in colonic microvessels. In TNF-
-treated mice, SPL adherence was
significantly increased at 20 min (compared with control mucosa) and
reached 11.4 ± 3.9/mm2 at 60 min (Fig.
2A). For LPL, there was a small number of adherent LPL in
colonic microvessels under control conditions, and their number reached
15.0 ± 3.0/mm2 at 60 min (Fig. 1A and Fig.
2B). Similarly, in TNF-
-treated mice, a significant
increase in LPL adherence was observed in the colonic mucosa (compared
with control mucosa) that reached 29.4 ± 7.2/mm2 at
60 min (Fig. 1B and Fig. 2B).
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Lymphocyte migration in colon vs. small intestine.
Figure 3 shows the number of T
lymphocytes migrating into venules of small intestinal mucosa in
control and TNF--treated animals at 60 min. In the small intestine
of control mice, sticking of both SPL and LPL was observed, with the
dominant accumulation of LPL in the mucosa, as seen in the colonic
mucosa. The migration of SPL and LPL was significantly enhanced in
TNF-
-treated small intestinal mucosa, like in the colonic mucosa.
However, in the small intestine, the magnitude of the increase in
lymphocyte adhesion was more significant for SPL than for LPL, such
that the maximal number of adherent cells elicited by TNF-
for both
cell populations reached a comparable level (SPL, 32.4 vs. LPL, 35.4),
with no significant difference between the two populations. When the
two cell populations (LPL and SPL) were compared in TNF-
-treated colonic mucosa, the numbers of adherent SPL and LPL were ~2.43 and
~2.23 times (respectively) larger than those obtained in control mucosa at 60 min, maintaining LPL as the dominant cell population in
these vessels (Fig. 2). The increase in LPL adhesion was more significant in the colonic mucosa (2.23 times) than in the small intestinal mucosa (1.73 times).
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Effect of antibodies against adhesion molecules.
We examined the effects of antibodies against adhesion molecules on T
lymphocyte accumulation in the colonic mucosa of TNF--treated animals. In these experiments, the number of adherent lymphocytes entering colonic microvessels was not significantly altered among the
different treatment groups at 10 min after infusion (control, 11.2 ± 2.1/min; anti-
7 treatment, 9.8 ± 3.5/min;
anti-
4 treatment, 10.4 ± 2.2/min; anti-VCAM-1
treatment 9.3 ± 2.8; anti-MAdCAM-1 treatment, 9.8 ± 2.7/min). Figure 4 demonstrates the
inhibitory effect of function-blocking MAbs against adhesion molecules
on the adhesion of T lymphocytes in TNF-
-treated colonic
microvessels at 60 min. The numbers of sticking SPL and LPL in
TNF-
-treated colonic vessels were 10.2 ± 1.6 and 29.4 ± 7.2 cells/mm2, respectively. However, the number of
adherent SPL was significantly reduced by pretreatment with MAbs that
block either
4-integrin or
7-integrin
(Fig. 4A). Although not shown in this figure, MAbs that
block either L-selectin or CD11a did not blunt SPL adhesion. Preinfusion of anti-MAdCAM-1 [either MECA-367 whole or F(ab')2 portion] or anti-VCAM-1 antibody to mice also significantly attenuated TNF-
-stimulated SPL interactions. Combination of anti-MAdCAM-1 and
anti-VCAM-1 further drastically inhibited the SPL sticking. However,
preinfusion of an anti-ICAM-1 MAb has no effect (Fig. 4A).
For LPL, the TNF-
-induced adhesion was remarkably reduced by
pretreatment with MAbs that block
4-integrin or
7-integrin (Fig. 4B). Antibodies that blocked
either L-selectin or CD11a did not affect LPL adhesion (data not
shown). Similar to SPL, preinfusion of either an anti-MAdCAM-1 or
anti-VCAM-1 significantly attenuated LPL sticking, and combination of
these antibodies could almost completely inhibit LPL adhesion, whereas
an anti-ICAM-1 MAb had no effect. Preinfusion of control anti-MECA-89
MAb did not affect TNF-
-stimulated SPL and LPL adhesion (data not
shown).
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Histological study on colonic and ileal mucosa.
To examine the changes in expression of ECAM in the inflamed colonic
mucosa, the ECAM expression was quantified before and after TNF-
treatment. All three ECAMs (ICAM-1, MAdCAM-1, and VCAM-1) were not
strongly expressed in venules of the colonic mucosa in control mice
(Fig. 5, A-C),
but after TNF-
treatment, an increased expression of these ECAMs was
observed. For MAdCAM-1, an increased expression was mainly noted in the
lamina propria (Fig. 5D), but it was not uniformly found in
the microvessels of colonic mucosa; only some venules were MAdCAM-1
positive. VCAM-1 was dominantly induced in the submucosa of the colon,
with some induction in venules of the lamina propria and subserosa
(Fig. 5E). ICAM-1 was also observed mainly in the large
venules of the colonic submucosa after TNF-
treatment (Fig.
5F). Adhesion site of infused FITC-labeled T lymphocytes and
the expression of these ECAMs were also histologically determined.
After infusion of CFDSE-labeled LPL, these cells were primarily
observed in the lamina propria of colonic mucosa. The site of LPL
adhesion coincided with MAdCAM-1 expression in venules of the lamina
propria as determined by a laser-scanning microscope (Fig.
6A). Both CFDSE-labeled SPL
and LPL were localized in submucosal venules of the colonic mucosa, where VCAM-1 was significantly expressed after TNF-
treatment (Fig.
6B). A quantitative comparison of the expression of ECAMs between small intestinal mucosa and colonic mucosa is shown in Fig.
7. There was low-level constitutive
expression of MAdCAM-1 in the colonic mucosa as well as in the ileal
mucosa of control mice. After TNF-
treatment, there was a remarkable
increase in MAdCAM-1 expression in both small intestinal and colonic
mucosa, although the magnitude of the increase in MAdCAM-1 expression in colonic mucosa (7.95 times) was larger than that of small intestinal mucosa (2.50 times). Almost no significant expression of VCAM-1 was
observed in the colonic as well as in the ileal mucosa; however, TNF-
treatment remarkably increased VCAM-1 expression at both sites.
At that time, the increased expression of VCAM-1 in the small
intestinal mucosa (23.9 times) was comparable to that observed in the
colonic mucosa (22.3 times). There was weak expression of ICAM-1 in the
unstimulated mucosa of both small intestine and colon, but TNF-
treatment enhanced the expression of ICAM-1, although the extent of
increase did not reach a statistically significant level in the colonic
mucosa.
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Analysis by fluorescence-activated cell sorter.
The expression of various adhesion molecules
(4-integrin,
1-integrin,
7-integrin, L-selectin, and CD11a) on the surface of T
cell population of SPL and LPL was determined by flow cytometric analysis using specific MAbs (Fig. 8).
Cell surface expression of
4-integrin,
1-integrin,
7-integrin, L-selectin, and
CD11a was noted in SPL. Control LPL also exhibited the expression of these adhesion molecules, with stronger expression of
7-integrin and weaker expression of L-selectin compared
with SPLs.
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DISCUSSION |
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In the present study, we demonstrated that when intestinal
tissues (colon and small intestine) are exposed to an intense
inflammatory stimulus (TNF-), intense lymphocyte-endothelial
interactions are elicited. There have been few reports describing the
direct observation of lymphocyte trafficking in the inflamed mucosa. We
have previously reported an increased migration of gut-derived T
lymphocytes in venules of lymphoid (Peyer's patches) as well as
nonlymphoid (villus mucosa) regions of small intestine after endotoxin
stimulation in rats (24). In the present study, we have
also demonstrated a TNF-
-induced increase in T cell adhesion in
venules of colonic as well as small intestinal mucosa accompanied by an
increased expression of several ECAMs including MAdCAM-1 and VCAM-1.
ICAM-1 was also increased in small intestine but did not exhibit a
significant increase in colon, different from our previous report
(15). The exact reason for the discrepancy is not known,
but this might result from a difference in evaluation method
(radiolabeled MAb technique vs. immunohistochemistry) or a difference
in mouse species. The functional significance of the elevated
expression of these ECAMs was evidenced by the reduced lymphocyte-endothelial cell adhesion after immunoneutralization of
these adhesion molecules with specific monoclonal antibodies. In this
study, we clearly demonstrated that MAdCAM-1 and VCAM-1, but not
ICAM-1, mediate T lymphocyte recruitment into the inflamed gut in this
model. Sans et al. (32) reported that immunoneutralization of ICAM-1 significantly attenuated the increased leukocyte adhesion in
rat colonic venules induced by trinitrobenzene sulfonic acid. It is
possible that leukocyte subpopulations other than T cells (possibly
neutrophils) may play important roles in this ICAM-1-dependent recruitment process. We speculate that upregulation of MAdCAM-1 or
VCAM-1 is particularly important for the lymphocyte-endothelial interactions observed in acute and chronic inflammation, especially when TNF-
is involved, which is in accordance with the observation of Connor et al. (10). The results of this study also
revealed a significant heterogeneity in the distribution of MAdCAM-1
and VCAM-1 in the inflamed intestinal mucosa, which may possibly induce T lymphocyte recruitment to the lamina propria via MAdCAM-1 and to the
submucosa via VCAM-1. Our findings therefore suggest that the three
ECAMs may play different roles in mediating the leukocyte-endothelial interactions observed in the inflamed intestinal mucosa.
In this study, we found that LPL adhered to venules of the colonic
mucosa, whereas only a few SPL migrated to this site under control
conditions. The basal level of sticking T lymphocytes in untreated mice
may be due to a constitutively expressed MAdCAM-1 in colon. In
TNF--stimulated venules, however, we demonstrated an enhanced
adhesion of both SPL and LPL, maintaining the dominance of LPL as the
major adherent population at this site. The exact reason for the
dominant migration of LPL to the colonic mucosa compared with SPL
remains unclear, but it may relate to the different degrees of adhesion
molecule expression between the two leukocyte populations. The analysis
of various adhesion molecules on the surface of the T cell population
showed similar results, as previously reported (14). LPL
strongly express
4
7- and
4
1-integrins as well, whereas SPL express
both
4
1- and
4
7-integrins, with a lesser degree of
4
7-integrin expression than LPL. Because MAdCAM-1 is known to be a specific ligand for
4
7-integrin, but not for
4
1-integrin (5), the
preferential migration of LPL into colonic mucosa may be mainly due to
the dominant role of
4
7-integrin-MAdCAM-1
interactions in vivo. On the other hand, in TNF-
-stimulated venules
SPL adherence was also significantly inhibited by antibody against
7-integrin, suggesting that the level of
7-integrin expression may not be an accurate indicator of function at the lymphocyte level. We also observed in this study
that both MAdCAM-1 and VCAM-1 are upregulated after TNF-
stimulation. Berlin et al. (5) demonstrated that
4
7-integrin can also recognize VCAM-1
under flow conditions and that the activation of
4
7-integrin cells enhances MAdCAM-1 and
VCAM-1 binding. Hence, there is a possibility that, in addition to
MAdCAM-1, VCAM-1 may contribute to the increased adhesion of LPL in
TNF-
-stimulated colonic mucosa. The
4
1- or
4
7-integrin-VCAM-1 and
4
7-integrin-MAdCAM-1 pathways may be
equally important for T lymphocyte adhesion to stimulated colonic
venules, because antibodies against VCAM-1 and MAdCAM-1 were equally
effective in either case of SPL or LPL. Furthermore, the importance of
the combined function of MAdCAM-1 and VCAM-1 in T lymphocyte adhesion
to inflamed colonic mucosa was revealed by the almost complete
inhibition of residual lymphocytes after treatment with both antibodies.
We also demonstrated that, under control conditions, there was a
relatively substantial migration of LPL to the small intestinal mucosa,
whereas only a few of these cells adhered to unstimulated venules in
the colonic mucosa. We also demonstrated that, after TNF-
stimulation, the interaction of lymphocytes with endothelial cells in
mucosal microvessels was significantly enhanced in both the colon and
the small intestine, but the extent of the leukocyte recruitment
differed between these two sites. The exact reason for the difference
in lymphocyte interaction with microvessels between small and large
intestine is not known, but it is presumably due to differing
expressions of adhesion molecules at both sites and/or different
environmental factors, such as residing enteric microflora that may
also affect cytokine or chemokine production in the mucosa. Although
MAdCAM-1 is constitutively expressed on endothelial cells in the lamina
propria of small as well as large intestine (37), MAdCAM-1
expression in normal small intestine appeared to be greater than that
in colon. VCAM-1 expression was almost negligible at both sites. These
results are in accordance with the significant migration of LPLs to the
small intestine in the control situation (14). After
TNF-
stimulation, however, a more significant enhancement of
MAdCAM-1 expression was observed in the colonic mucosa than in the
ileal mucosa, which likely explains the dominant increase of LPL
adherence to the colonic mucosa. After TNF-
stimulation in the small
intestine, the increase in VCAM-1 expression was more drastic than that
of MAdCAM-1, which is in accordance with the preferential increase in
SPL sticking to this site. On the other hand, because VCAM-1 and
MAdCAM-1 expression were significantly enhanced to a comparable degree
in the colonic mucosa, we speculate that the enhanced expression of
both molecules is an important determinant of the dominant adhesion of
LPLs, rather than SPLs, to this site.
TNF- plays a key role in inflammation by triggering chemotaxis and
upregulating adhesion molecules. Secretion of TNF-
by IEL increases
epithelial cell permeability and causes epithelial cells to produce
chemokines (20, 25). Production of TNF-
by LPL should
recruit cells to the lamina propria and activate resident cells, such
as mesenchymal cells and endothelial cells (13, 22),
thereby facilitating local inflammation in the intestinal mucosa.
Increased expression of MAdCAM-1 and VCAM-1 on murine endothelial cells
can be induced after stimulation with IL-1 and TNF-
(35). TNF-
and IL-1 are each capable of increasing the expression of all of the ECAMs through direct activation of endothelial cells (16). There is a possibility that the
TNF-
-stimulated T cells liberate additional TNF-
as well as other
cytokines that can exert either additive (IL-1) or synergistic
(IFN-
) effects on TNF-
, thereby enhancing T cell actions on
different vascular beds to express more ECAMs (12) and
producing a vicious inflammatory cycle. From the present result, we can
speculate that cytokine-induced systemic inflammation may
preferentially promote the migration of gut-derived T cells to the
colonic mucosa. The nature of gut tropism of T lymphocytes during
TNF-
-induced systemic inflammation and their regulatory mechanisms
should be further investigated in vivo from the standpoint of
anticytokine therapy.
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ACKNOWLEDGEMENTS |
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This study was supported in part by Grants-in-Aid for Scientific Research from the Japanese Ministry of Health, Labour and Welfare and by grants from Keio University School of Medicine and the National Defense Medical College. D. Neil Granger was supported by a grant from the National Heart, Lung, and Blood Institute (HL-26441).
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FOOTNOTES |
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Address for correspondence: S. Miura, Second Dept. of Internal Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa City, Saitama 359-8513, Japan (E-mail: miura{at}me.ndmc.ac.jp).
Address for reprint requests: H. Ishii, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan (E-mail: hishii{at}sc.itc.keio.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
May 29, 2002;10.1152/ajpgi.00026.2002
Received 18 January 2002; accepted in final form 13 May 2002.
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