(Journal of Leukocyte Biology. 2001;70:896-902.)
© 2001
by Society for Leukocyte Biology
Intercellular cell adhesion molecule-1 regulates lymphocyte movement into intestinal microlymphatics of rat Peyer’s patches
Ryota Hokari*,
Soichiro Miura*,
Hiroshi Nagata
,
Hitoshi Fujimori,
Seiichiro Koseki,
Shingo Kato*,
Iwao Kurose,
Eiichi Sekizuka
,
D. Neil Granger
and
Hiromasa Ishii
* Second Department of Internal Medicine, National Defense Medical College, Saitama, Japan;
Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan;
Department of Internal Medicine, National Saitama Hospital, Saitama, Japan; and
Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport
Correspondence: Soichiro Miura, M.D., Professor, Second Department of Internal Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan. E-mail: miura{at}me.ndmc.ac.jp
 |
ABSTRACT
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The objective of this study was to determine whether specific adhesion
molecules modulate lymphocyte movement from Peyer’s patches into
intestinal microlymphatics. The fluorochrome acridine orange was
injected via a micropipette into Peyer’s patches to fill lymphatics.
The flux of labeled lymphocytes into intestinal microlymphatics was
monitored with intravital fluorescence microscopy. The lymphatic
microvessels in the perifollicular area of Peyer’s patches were filled
with lymphocytes, most of which remained within the lymphatics. Some
lymphocytes became detached and were drained into intestinal lymph.
Administration of antibodies directed against ICAM-1 significantly
increased lymphocyte flux into interfollicular lymphatics. The
immunohistochemical study showed intense ICAM-1 expression on the
lymphocytes densely packed in the lymphatics surrounding follicles in
Peyer’s patches. A large number of lymphocytes are normally
sequestered in the lymphatic network of Peyer’s patches. This
sequestration of lymphocytes is largely mediated by ICAM-1-dependent
cell-cell interactions.
Key Words: CD18 • acridine orange • collecting lymphatics • mesenteric lymphadenectomy
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INTRODUCTION
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Lymphocytes continuously move throughout the body in search of
foreign antigens to facilitate immune responses to these antigens. If
immunologically unengaged, lymphocytes continue their migration through
the parenchyma of lymphoid tissues and exit via efferent lymphatic
vessels. The lymphocytes in Peyer’s patches are thought to follow a
migratory pathway through mesentric lymphatic vessels, the mesentric
lymph nodes, and the thoracic duct before reentry into the blood
circulation [1
]. The regulation of lymphocyte
trafficking within blood vessels of lymphoid tissues and at sites of
inflammation has been studied extensively. These studies have revealed
that lymphocytes establish adhesive interactions with endothelial cells
that enable them to cross the endothelial lining [2
,
3
]. In Peyer’s patches, lymphocytes have been shown to
preferentially extravasate across morphologically distinct
postcapillary venules called high endothelial venules (HEV)
[4
]. In several species, a variety of lymphocyte
adhesion molecules, including L-selectin [5
], CD44
[6
], and 
4ß7 [7
8
9
], have been
implicated in the organ-specific homing of lymphocytes.
Although there are a number of published studies that address the
molecular mechanisms that regulate the entrance of lymphocytes into
lymphoid organs, relatively little is known about the signals that
regulate the exit of these lymphocytes from lymphoid tissue into
lymphatics. Antigen stimulation or local infusion of vasoactive
neurotransmitter substances is known to produce prompt and marked
changes in the emigration of lymphocytes into efferent lymph in lymph
nodes [10
11
12
]. Furthermore, we have previously
demonstrated that absorption of long-chain fatty acids in the small
intestine increases the traffic of lymphocytes through
mesenteric-collecting lymphatics [13
]. Therefore,
although factors such as lipid absorption, antigen stimulation, and
neuropeptide release appear to influence the output of lymphocytes into
mesenteric lymphatics, the mechanism that regulates the exit of
lymphocytes from gut lymphoid tissue is mostly unexplored.
Peyer’s patches appear to be the major source of lymphocytes in
mesentric lymph [14
]. We have demonstrated an immense
population of lymphocytes in the lymphatic microvessels of rat Peyer’s
patches [15
] and that most of the lymphocytes in the
thymus-dependent region of Peyer’s patches remain for extended periods
in lymphatic microvessels before they drain into larger intestinal
lymphatics. The long residence time of lymphocytes within this
well-developed system of microlymphatics in Peyer’s patches appears to
represent a large storage pool that may play a rate-determining role in
the exit of lymphocytes from this lymphoid tissue. The distribution of
human Peyer’s patch efferent microlymphatics has been characterized
recently [16
]. It was shown that the microlymphatics
contain numerous lymphoid cells, with occasional clustering of lymphoid
cells. Immunostaining revealed that most of these lymphocytes were
naive T and B lymphocytes, with a few memory T lymphocytes that
strongly expressed 4ß7. These observations suggest that in the
perifollicular and interfollicular areas of the Peyer’s patch, there
is a large resident pool of lymphocytes that is readily available for
mobilization. However, the factors that promote the sequestration of
these lymphocytes in the microlymphatics and mechanisms that regulate
their exit from this lymphatic reservoir remain poorly understood.
We recently developed an intravital microscopic method for monitoring
the dynamic process of lymphocyte movement within lymphatic
microvessels of rat Peyer’s patches [15
]. In the
present study, we used this method to assess the contribution of
specific adhesion molecules to the regulation of lymphocyte efflux from
the lymphatic microvessels of Peyer’s patches. Immunohistochemistical
techniques were also used to localize the expression of intercellular
cell adhesion molecule-1 (ICAM-1) within the Peyer’s patch.
|
MATERIALS AND METHODS
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Visualization of lymphatic microvessels and local administration of
adhesion molecules-directed antibodies
Male Wistar rats, weighing about 200 g, were maintained on
a diet of standard laboratory chow. The care and use of laboratory
animals were in accordance with National Institutes of Health
guidelines. The rats were anesthetized with sodium pentobarbital (45
mg/kg) intraperitoneally. Rectal temperature was monitored and
maintained at 37°C by a heating pad. The surgical procedures used
were as described previously [15
]. A segment of ileum
was exposed, and two small incisions were made with a microcautery.
Krebs’ solution was infused into the bowel lumen to flush away food
residue. A cold glass light rod with a small, mirrored prism cemented
to the end was passed into the intestinal lumen through one of the
incisions. A Peyer’s patch was then transilluminated from the luminal
side.
Under microscopic observation, a glass micropipette was inserted
through the serosa into the Peyer’s patch, avoiding blood vessels. The
micropipette was filled with the fluorochrome acridine orange, which
was dissolved in physiological saline at a concentration of 10 µg/ml.
Using a microinjector, the solution was injected (2 µl for 5 s)
into the Peyer’s patch to fill the lymphatic microvessels and to stain
lymphocytes within these microvessels. Lymphatics within the
interfollicular region were observed using a charged-coupled devise
(CCD) or silicon-intensified target image tube camera connected
to the intravital fluorescence microscope. The behavior of labeled
lymphocytes was visualized on a TV monitor and quantified as previously
described [17
]. We achieved epi-illumination with
filters of excitation at 420–490 nm and emission at 520 nm.
Measurements of lymphocyte efflux from microlymphatics were performed
by counting the total flux of lymphocytes passing through the draining
lymphatics connected to the interfollicular lymphatics. The entire
procedure was videotaped for later playback and analysis. Diameters and
lengths of the vessels were measured using a video-measuring gauge (FOR
A TV-560, Tokyo, Japan).
In some experiments, anti-adhesion molecule antibodies (at a
concentration of 0.25 mg/ml) were injected along with acridine orange
into Peyer’s patches using the micropipette described above.
Anti-L-selectin monoclonal antibody [mAb; HRL-3; hamster
immunoglobulin G (IgG), Seikagaku-Kogyo, Japan], anti-4-integrin mAb
(MR4-1; mouse IgG, Pharmingen, San Diego, CA), anti CD11a mAb
(LFA-1; mouse IgG, Seikagaku-Kogyo), anti-ICAM-1 mAb (1A29; mouse IgG,
Pharmingen), and anti-CD-18 mAb (CL26; mouse IgG, provided by D. C. Anderson, Pharmacia-Upjohn Laboratories, Kalamazoo, MI) were used
for these experiments. Time course of change on lymphocyte outflux to
interfollicular lymphatics was determined after administration of
antibodies directed to various adhesion molecules.
In vivo visualization of collecting lymphatics of rat mesentry
Collecting lymphatics of the rat mesentry were observed, and
lymphocyte flux was determined using the method described previously
[13
]. Briefly, after laparotomy, an intestinal loop
corresponding to two-thirds of the small intestine was gently
exteriorized, and the mesentery was spread over a glass plate equipped
with a small water bath that was maintained at 38°C. Collecting
lymphatics, 80–150 µm in diameter, were observed under a high-speed
microscope system consisting of an inverted microscope (Nikon
Diaphot-TMD, Tokyo, Japan), a high-speed video camera, and a recorder
(Kodak Ekta Pro 1000, San Diego, CA). Measurements of lymphocyte flux
and diameter of lymphatics were performed by video image analysis. An
antibody directed against ICAM-1 (1A29) was administered intravenously
(i.v.) from the jugular vein at the dose of 2 mg/kg, and the change in
lymphocyte flux in collecting lymphatics was examined. To assess the
effects of administration of anti-ICAM-1 mAb on lymph flow, thoracic
duct was cannulated with a polyethylene tube 6 weeks after mesenteric
lymphadenectomy (MLNX). In these rats, lymph flow from intestine can be
directly determined without influence of mesenteric lymph nodes. Lymph
flow was measured before and after the administration of anti-ICAM-1
mAb (2 mg/kg). Saline was infused through a duodenal tube at a rate of
2.4 ml/h to replenish the fluid and electrolyte loss associated with
lymphatic drainage.
Flow cytometric analysis of lymphocytes in intestinal lymph
To analyze the expression of adhesion molecules on lymphocytes,
which drained out from Peyer’s patches, rats were
mesenteric-lymphadenectomized. After 6 weeks, the thoracic duct was
cannulated to permit healing, and intestinal lymph was collected to
obtain lymphocytes, which were drained from Peyer’s patches. For
immunofluorescence staining, 1 x 106 of lymphocytes
in 25 µl medium were incubated with fluorescein isothiocyanate
(FITC)-conjugated anti-rat ICAM-1 antibody (1A29) and phycoerythrin
(PE)-labeled anti-CD4 (W3/25, Serotec, Oxford, UK), anti-CD8 (OX-8,
Serotec), anti-B cell (RLN-9D3, Serotec), anti-dendritic cell (OX-62,
Serotec), or anti-CD45 RC (OX-22, Serotec) for 30 min on ice. Cells
were analyzed on a FACSCalibur cytofluorimeter using CellQuest software
(Becton Dickinson, San Jose, CA).
Immunohistochemistry
The expression of adhesion molecules ICAM-1 and CD18 in rat
Peyer’s patches was assessed by immunohistochemistry using labeled
streptavidin biotin. Ileal Peyer’s patches were embedded in OCT
(optimum cutting temperature) compound (Miles Inc., Elkhart, IN) before
being frozen in dry ice and acetone. Cryostat sections of 6 µm were
transferred to APS-coated slides and fixed in cold acetone (-20°C)
for 2 min, followed by air drying. After they were washed in
phosphate-buffered saline (PBS; pH 7.4) containing 1% Triton X for 5
min, sections were incubated in 5% normal goat serum in PBS. The mAbs
anti-ICAM-1 (1A29), anti-CD18 (CL26), and anti-human factor VIII (DAKO,
High Wycome, Buchs, UK) were diluted 50–100 times with PBS and layered
on the section overnight at 4°C. The sections were incubated with a
second antibody, biotinylated anti-mouse IgG class antibody (Amersham
International plc, Backinghamshire, U.K.) for 1 h at 37°C. Then,
sections were incubated with FITC-conjugated streptavidin
(Streptavidin-fluorescein; Amersham) for 30 min at room temperature.
With each step, the sections were rinsed with PBS containing 1% bovine
serum albumin. A coverslip was applied using glycerol jelly, and the
sections were observed using a fluorescent microscope (Olympus, Tokyo,
Japan).
Statistics
Results are expressed as the mean ± SE.
Differences among groups were evaluated using a one-way analysis of
variance (ANOVA) and Fisher’s protected least-significant difference
test. P values of .05 or less were considered to be
statistically significant.
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RESULTS
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Collecting lymphatics of rat mesentery
Lymphocyte fluxes were determined in collecting lymphatics of the
mesentery under light microscopy. The flux of lymphocytes in these
vessels remained constant throughout the observation period, even after
injection of the control (nonbinding) antibody. Lymphatic pump
activity, e.g., lymphatic contractility, was not significantly altered
by the administration of control or anti-ICAM-1 antibody. However,
treatment with an anti-ICAM-1 antibody increased the number of
lymphocytes transported in the lymphatics. Figure 1
presents the time course of changes in lymphocyte flux within
three, collecting lymphatics after administration of an anti ICAM-1
antibody. The number of lymphocytes began to increase about 30–50 min
after ICAM-1 immunoneutralization, and the flux remained elevated for
up to 80 min. However, the amplitude and onset of the lymphocyte
response to the ICAM-1 antibody exhibited substantial variability among
the vessels examined.
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Figure 1. Time-course changes of lymphocyte flux in collecting lymphatics of rat
mesentery after i.v. administration of antibody against ICAM-1 (1A29; 2
mg/kg). Collecting lymphatics of rat mesentry were observed under an
inverted microscope equipped with a high-speed video camera system.
Results of three representative experiments were shown. As controls,
indifferent mouse antibody against rat IgG was used.
|
|
To exclude the possibility that anti-ICAM-1 mAb affects the lymphocyte
outflux secondary by increasing lymph flow, we measured the intestinal
lymph flow of rats, which were mesenteric-lymphadenectomized. The lymph
flow appeared not to be significantly different between before
(22.8±8.5 µl/min) and after (25.0±9.4 µl/min) the administration
of anti-ICAM-1 mAb.
Lymphatic microvessels in Peyer’s patches
Acridine orange injection into lymphatics of the Peyer’s patches
allowed for ready recognition of the dense lymphatic microvessels in
the lymphatic plexus located between follicles (interfollicular
lymphatics) and perifollicular areas (perifollicular lymphatics)
(Fig. 2 A
). We observed that perifollicular lymphatics were filled with
numerous lymphocytes, which were often aggregated (Fig. 2B)
. The
lymphocytes remained in perifollicular lymphatics and were not washed
away by an additional injection of saline. Occasionally, a few
lymphocytes would detach from a cluster in the perifollicular
lymphatics and would drain into interfollicular lymphatics under
control conditions.
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Figure 2. Representative image of lymphatic plexus in rat Peyer’s patches.
Acridine orange was injected into Peyer’s patches tissue by
micropipette to visualize lymphatics. (The injection site was seen in
the right border.) The lymphatic network, filled with acridine orange,
encircles the germinal center (GC) and is located in the perifollicular
(PF) and interfollicular areas (IF). Original bar = 200 µm (A).
A lymphatic microvessel under higher magnification. The lymphatic
microvessel contains numerous lymphocytes that are attached to each
other and stayed in the lymphatic microvessels. Original bar = 100
µm (B).
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Time-course changes of lymphocyte efflux from interfollicular
lymphatics were determined after administration of various adhesion
molecule-directed mAbs. As illustrated in Figure 3
, the lymphocyte flux after injection of control (mouse IgG)
antibody was 12 ± 4.2/min. Anti-CD18 antibody slightly increased
the flux of lymphocytes, but this response was not statistically
significant. Conversely, after administration of anti-ICAM-1 antibody,
packed lymphocytes in lymphatics began to flow away; thus, lymphatics
were vacant in a few minutes. As a result, the lymphocyte flux
remarkably increased after anti ICAM-1 Ab injection. The increased
outflux because of anti-ICAM-1 mAb reached its peak at 20 s after
antibody injection and decreased gradually thereafter because a store
of lymphocytes in lymphatics had run out. However, the other antibodies
against adhesion molecules (anti-4-integrin, CD11a, and L-selectin)
did not significantly alter lymphocyte flux in the interfollicular area
of the Peyer’s patch. Simultaneous administration of anti-ICAM-1 and
anti-CD18 antibodies tended to further increase lymphocyte efflux above
ICAM-1 mAb treatment alone, however this tendency was not significant.
After anti-ICAM-1 treatment, the formation of lymphocyte clusters was
frequently observed in interfollicular lymphatics (Table 1
). The ICAM-1 mAb caused aggregates of lymphocytes in
perifollicular lymphatics to loosen and form a number of smaller
clusters (more than 20/min), which were transported in microlymphatics.
The combination of anti-ICAM-1 and anti-CD18 also promoted the
appearance of lymphocyte clusters, however anti-CD18 alone did not
elicit such a response. Antibodies against other adhesion molecules
such as CD11a, 4-integrin, or L-selectin did not induce the
appearance of clustered lymphocytes. Administration of the various
anti-adhesion molecule antibodies did not alter the contractile
activity or diameter of the lymphatic microvessels in the
interfollicular areas.
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Figure 3. Changes over time in lymphocyte efflux after in situ administration of
mAbs against adhesion molecules into interfollicular lymphatics of
Peyer’s patches. Lymphocyte flux was expressed as number of
lymphocytes passing through the certain portion of interfollicular
lymphatics per second. As controls, indifferent mouse antibody against
rat IgG was used. mAbs against alpha4-integrin (MR4-1), L-selectin mAb
(HRL-3), CD-18 (CL26), CD11a (LFA-1), and ICAM 1 (1A29) were used for
this study. Values are means ± SD of six animal
experiments. *, P < 0.05 compared with the values of
controls.
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Histological expression of ICAM-1 and CD18
The expression of ICAM-1 and CD18 in rat Peyer’s patches was
determined by immunohistochemistry. The expression of ICAM-1 was
observed in the germinal center (possibly corresponding follicular
dendritic cells), in some microvessels in the margin of follicles, and
the surface of lymphocytes within the lamina propria of the villi. As
shown in Figure 4 A
, ICAM-1 expression surrounding the follicular area appeared to
correspond to the lymphatic networks as indicated by acridine-orange
staining in vivo (see Fig. 2
). At a higher magnification, the intense
staining of ICAM-1 that surrounded the follicules was seen on the
surface of aggregated lymphocytes (Fig. 4B)
. The leukocytes in Peyer’s
patches did not express significant levels of ICAM-1 except this area,
whereas nearly all of the leukocytes in Peyer’s patches expressed CD18
on their surface.
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Figure 4. Immunohistochemical assessment of ICAM-1 expression in rat Peyer’s
patch. Localization of ICAM-1 was achieved using the labeled
streptavidin-biotin method with an antibody against ICAM-1 (1A29).
ICAM-1 expression was located in the lymphatic-like structure, which
surrounds the follicular area (F: follicle; IF: interfollicular area).
Original bar = 200 µm (A). At higher magnification, ICAM-1
expression was observed on the surface of aggregated cells (arrows).
Original bar = 50 µm (B).
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As shown in Figure 5 A
, there was significant expression of factor VIII on the surface
of some vessels within the interfollicular area, suggesting that these
vessels were venules. Numerous ICAM-1-presenting cells were seen in the
immediate proximity to the factor VIII-positive venules (Fig. 5B)
,
suggesting that the ICAM-1-positive cells were primarily located in the
lymphatics surrounding the follicles. No ICAM-1-positive blood cells
were observed in the venules.
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Figure 5. Immunohistochemical localization of factor VIII (A) and ICAM-1 (B) in
rat Peyer’s patch using serial cryosections. Factor VIII-positive
venules are located in the interfollicular area (arrows). Original
bar = 50 µm (A). ICAM-1-positive cells were observed adjacent to
the factor VIII-positive venules (arrows) but not inside the venules
(V). Original bar = 50 µm (B).
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Analysis by fluorescence-activated cell sorter (FACS)
To examine whether the lymphocytes, which are present in efferent
lymphatics of Peyer’s patches, actually express ICAM-1, FACS analysis
was performed on leukocytes from intestinal lymph after mesenteric
lymphadenectomy. Lymph cells (36%) were shown to express ICAM-1. Next,
we characterized the subset of these ICAM-1-expressing cells by
two-color flow cytometry. ICAM-1-expressing cells consisted of B cells
(75%), CD8 cells (12%), dendritic cells (1%), and CD4 cells (1%).
Among lymphocyte subsets, only a small fraction of CD4 cells expressed
ICAM-1 (6%), suggesting that most CD4 cells are naive cells. However,
other subsets of leukocytes expressed ICAM-1 more frequently. For
example, the rate of ICAM-1 expression was as follows: 82% of B cells,
100% of dendritic cells, and 40% of CD8 cells. Most of the
ICAM-1-positive cells (92%) co-expressed CD45 RC, known as an
activation marker (Fig. 6
).
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Figure 6. Flow cytometric analysis on ICAM-1 expression in different
subpopulations of leukocytes collected from intestinal lymph of rats
that received mesenteric lymphadenectomy. Cells (1x106) in
25 µl medium were incubated with FITC-conjugated anti-rat ICAM-1
antibody (1A29) and PE-conjugated anti-CD4 (W3/25), anti-CD8 (OX-8),
anti-B cell (RLN-9D3), anti-dendritic cell (OX-62), or anti-CD45 RC
(OX-22, Serotec) for 30 min on ice. Cells were analyzed on a
FACSCalibur cytofluorimeter using CellQuest software (Becton
Dickinson).
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DISCUSSION
|
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Peyer’s patches are generally considered an important source of
migrating lymphocytes, particularly the B and T lymphocytes that appear
in mesenteric lymphatics [14
]. The critical importance
of Peyer’s patches in lymphocyte transport is exemplified by studies
demonstrating a significant reduction in lymphocyte transport through
intestinal lymph (without changing lymph flow) upon removal of Peyer’s
patches in rat intestine [18
]. Although the pivotal role
of Peyer’s patches in lymphocyte transport is now well-appreciated,
relatively little is known about the factors that govern the movement
of lymphocytes into the lymphatic system that drains Peyer’s patches.
Efferent lymphatics begin blindly as vessels lined by fenestrated
endothelium that allow for the passive and nonselective entry of
lymphocytes. Recently, we demonstrated, using intravital microscopy,
that transport of T lymphocytes into the microlymphatics of Peyer’s
patches is significantly increased during absorption of long-chain
fatty acids [19
] or inhibition of nitric oxide synthesis
in the intestinal mucosa [20
]. These increases in
lymphocyte transport were associated with the adhesion of lymphocytes
to the high endothelial venules of Peyer’s patches. Administration of
vasoactive intestinal peptide into the mesenteric artery significantly
blunted the appearance of T lymphocytes into interfollicular lymphatics
without altering the adhesion of lymphocytes in postcapillary venules
of Peyer’s patches [12
]. Furthermore, it has been shown
that for a given number of cells migrating into the Peyer’s patch, T
cells are preferentially transported over B cells into intestinal
lymphatics [17
]. Overall, these findings suggest that
entry and transport of lymphocytes in intestinal lymphatics are not
simple, passive processes but a selective phenomenon that is governed
by factors that are intrinsic to Peyer’s patches.
In the present study, we examined whether specific adhesion molecules
are involved in gating the output of lymphocytes in efferent
microlymphatics of the Peyer’s patch. Although cells homing to mucosal
effector sites such as the gut lamina propria primarily use 4ß7 to
extravasate [8
], we found that blocking 4-integrin
does not affect lymphocyte traffic in efferent lymphatics. Instead, we
found that administration of an anti-ICAM-1 antibody significantly
increased the lymphocyte efflux from Peyer’s patches. This effect
appeared to be a direct action on the adhesion molecule because
anti-ICAM-1 treatment did not significantly affect other variables such
as intestinal peristalsis or lymphatic contractility, which are known
to influence lymph propulsion [21
]. An explanation for
the increased lymphocyte efflux from Peyer’s patches is not readily
available, however there are possibilities worthy of discussion.
Visualization of lymphatic microvessels in Peyer’s patches revealed
that microvessels in the perifollicular area were normally filled with
lymphocytes. Most of the lymphocytes in these perifollicular lymphatics
remain within the microvessels for extended periods, and some
lymphocytes become detached and drain into interfollicular lymphatics.
These findings suggest that perifollicular lymphatics in Peyer’s
patches may function as a reservoir for lymphocytes before they enter
the larger intestinal lymphatics.
In this study, we found that ICAM-1 is strongly expressed on the
surface of lymphocytes within the microlymphatcis that surround
follicles in Peyer’s patches. Our histochemical analysis also revealed
an intense expression of ICAM-1 on the surface of aggregated
lymphocytes, which contrasts with the low-level expression of ICAM-1 in
lymphocytes located outside the microlymphatics of the Peyer’s
patches. Lymphocytes expressed ICAM-1 when they were activated
[22
, 23
]. Homotypic aggregation of
lymphocytes has been described following stimulation with specific
antigens or with phorbol esters in vivo. The lymphocytes form large
cell clusters, and this homotypic aggregation can be completely and
immediately inhibited (leading to disaggregation) by antibodies
directed against ICAM-1 or LFA-1 [24
, 25
].
Hence, it appears likely that lymphocytes located in perifollicular
lymphatics aggregate with one another through ICAM-1-dependent
interactions, forming a storage pool for emigrating lymphocytes.
Anti-ICAM-1 antibody, in the presence of some mechanical fluid shear
forces, may act to promote disaggregation of these lymphocytes, which
results in an increased lymphocyte efflux. The occasional appearance of
lymphocyte clusters after anti-ICAM-1 treatment supports this
hypothesis. Because neither anti-CD18 nor anti-LFA-1 was effective,
possible counter-ligands of ICAM-1 are thought to be CD43 or
fibrinogen. CD43 is expressed on leukocytes, tissue macrophage,
dendritic cells, epithelium, and endothelium, and CD43 was shown to
block forming clusters of dendritic cells and lymphocytes
[26
]. Recently, interaction between fibrinogen on
endothelium and ICAM-1 on leukocytes was also shown to be important for
cell migration [27
]. From these observations, these
interactions might also be involved in sequestration of lymphocytes in
lymphatics.
Although the vast majority of lymphocytes in efferent lymph are
considered to be naive cells, in FACS analysis of Peyer’s
patches-derived leukocytes, about 36% of them expressed
ICAM-1. Actually ICAM-1-positive cells almost co-expressed CD45 RC
(92%), known as an activation marker. By two-color flow cytometry, we
demonstrated that these ICAM-1-positive leukocytes in efferent lymph
consisted of B cells, dendritic cells, and CD8 cells, and few CD4 cells
expressed ICAM-1. The presence of activated lymphocytes in efferent
lymphatics is in accordance with studies from other laboratories.
Farstad et al. [16
, 28
] demonstrated in
humans that CD45RO+ T cells and sIgD B cells, which express high levels
of alpha-4 beta-7-integrins, are often located near putative efferent
lymphatics. Only a small fraction (<20%) of such memory cells express
L-selectin.
In this study, we demonstrated that the exit of lymphocytes from gut
lymphoid tissue is largely associated with ICAM-1-dependent cell-cell
interactions under physiological condition. The exploration of
lymphocyte mobilization in efferent lymphatics under pathologic
conditions should be investigated further.
|
ACKNOWLEDGEMENTS
|
|---|
This study was supported in part by grants from the National
Defense Medical College and Keio University, School of Medicine.
D. N. G. is supported by a grant from the National Institutes
of Health (HL26441).
Received October 2, 2000;
revised June 18, 2001;
accepted June 27, 2001.
|
REFERENCES
|
|---|
-
Brandtzaeg, P., Farstad, I. N., Haraldsen, G. (1999) Regional specialization in the mucosal immune system: primed cells do not always home along the same track Immunol. Today 20,267-277[Medline]
-
Warnock, R. A., Askari, S., Butcher, E. C., von Andrian, U. H. (1998) Molecular mechanisms of lymphocyte homing to peripheral lymph nodes J. Exp. Med. 187,205-216[Abstract/Free Full Text]
-
Butcher, E. C., Picker, L. J. (1996) Lymphocyte homing and homeostasis Science 272,60-66[Abstract]
-
Girard, J-P., Springer, T. A. (1995) High endothelial venules (HEVs): specialized endothelium for lymphocyte migration Immunol. Today 16,449-457[Medline]
-
Gallatin, W. M., Weissman, I. L., Butcher, E. C. (1983) A cell-surface molecule involved in organ specific homing of lymphocytes Nature 304,30-34[Medline]
-
Jalkanen, S. T., Bargatze, R. F., Herron, L. R., Butcher, E. C. (1986) A lymphoid cell surface glycoprotein involved in endothelial cell recognition and lymphocyte homing in man Eur. J. Immunol. 16,1195-1202[Medline]
-
Holzmann, B., Weissman, I. L. (1989) Peyer’s patch-specific lymphocyte homing receptors consist of a VLA-4-like a chain associated with either of two integrin beta chains, one of which is novel EMBO J 8,1735-1741[Medline]
-
Hamann, A., Andrew, D. P., Jablonski-Westrich, D., Holzmann, B., Butcher, E. C. (1994) Role of 4 integrins in lymphocyte homing to mucosal tissues in vivo J. Immunol. 152,3282-3293[Abstract]
-
Bargatze, R. F., Jutila, M. A., Butcher, E. C. (1995) Distinct roles of L-selectin and integrins 4ß7 and LFA-1 in lymphocyte homing to Peyer’s patch-HEV in situ: the multistep model confirmed and refined Immunity 3,99-108[Medline]
-
Moore, T. C. (1984) Modification of lymphocyte traffic by vasoactive neurotransmitter sustances Immunology 52,511-518[Medline]
-
Mackay, C. R., Marston, W., Dudler, L. (1992) Altered patterns of T cell migration through lymph nodes and skin following antigen challenge Eur. J. Immunol. 22,2205-2210[Medline]
-
Miura, S., Serizawa, H., Tsuzuki, Y., Kurose, I., Suematsu, M., Higichi, H., Shigematsu, T., Hokari, R., Hirokawa, M., Kimura, H., Ishii, H. (1997) Vasoactive intestinal peptide modulates T lymphocyte migration in Peyer’s patches of rat small intestine Am. J. Physiol. 272,G92-G99[Abstract/Free Full Text]
-
Miura, S., Sekizuka, E., Nagata, H., Oshio, C., Minamitani, H., Suematsu, M., Suzuki, M., Hamada, Y., Kobayashi, K., Asakura, H., Tsuchiya, M. (1987) Increased lymphocyte transport by lipid absorption in rat mesentric lymphatics Am. J. Physiol. 253,G596-G600[Abstract/Free Full Text]
-
Steer, H. W. (1980) An analysis of the lymphocyte content of rat lacteals J. Immunol. 125,1845-1848[Abstract]
-
Nagata, H., Miyairi, M., Sekizuka, E., Morishita, T., Tatemichi, M., Miura, S., Tsuchiya, M. (1994) In vivo visualization of lymphatic microvessels and lymphocyte migration through rat Peyer’s patches Gastroenterology 106,1548-1553[Medline]
-
Farstad, I. N., Norstein, J., Brandtzaeg, P. (1997) Phenotypes of B and T cells in human intestinal and mesenteric lymph Gastroenterology 112,163-173[Medline]
-
Miura, S., Tsuzuki, Y., Fukumura, D., Serizawa, H., Suematsu, M., Kurose, I., Imaeda, H., Kimura, H., Nagata, H., Tsuchiya, M., Ishii, H. (1995) Intravital demonstration of sequential migration process of lymphocyte subpopulations in rat Peyer’s patches Gastroenterology 109,1113-1123[Medline]
-
Serizawa, H., Miura, S., Tashiro, H., Imaeda, H., Shiozaki, H., Tanaka, S., Yoshioka, M., Ohara, M., Hibi, T., Yamashita, A., Tsuchiya, M. (1993) Significant changes in intestinal lymphatic system and immune response elicited by Peyer’s patch excision in adult rats J. Gastroenterol. Hepatol. 8,63-69[Medline]
-
Tsuzuki, Y., Miura, S., Kurose, I., Suematsu, M., Higuchi, H., Shigematsu, T., Kimura, H., Serizawa, H., Hokari, R., Akiba, Y., Yagita, H., Okumura, K., Tso, P., Granger, D. N., Ishii, H. (1997) Enhanced lymphocyte interaction in postcapillary venules of Peyer’s patches during fat absorption in rats Gastroenterology 112,813-825[Medline]
-
Hokari, R., Miura, S., Fujimori, H., Tsuzuki, Y., Shigematsu, T., Higuchi, H., Kimura, H., Kurose, I., Serizawa, H., Suematsu, M., Yagita, H., Granger, D. N., Ishii, H. (1998) Nitric oxide modulates T lymphocytes migration in Peyer’s patches and villous submucosa of rat small intestine Gastroenterology 115,618-627[Medline]
-
Unthank, J. L., Bohlen, H. G. (1988) Lymphatic pathways and role of valves in lymph propulsion from small intestine Am. J. Physiol. 254,G389-G398[Abstract/Free Full Text]
-
Pocsik, E., Mihalik, R., Gyodi, E., Reti, M., Paloczi, K., Petranyi, G. G., Benczur, M. (1990) Activation of lymphocytes after platelet allotransfusion possessing only class I MHC product Clin. Exp. Immunol. 82,102-107[Medline]
-
Wang, P., Vanky, F., Li, S. L., Patarroyo, M., Klein, E. (1990) Functional characteristics of the intercellular adhesion molecule-1 (CD54) expressed on cytotoxic human blood lymphocytes Cell. Immunol. 131,366-380[Medline]
-
Rothlein, R., Springer, T. A. (1986) The requirement for lymphocyte function-associated antigen-1 in homotypic leukocyte adhesion stimulated by phorbol ester J. Exp. Med. 163,1132-1149[Abstract/Free Full Text]
-
Dustin, M., Rothlein, R., Bhan, A. K., Dinarello, C. A., Springer, T. A. (1986) Induction by IL-1 and interferon-gamma: tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1) J. Immunol. 137,245-254[Abstract]
-
Fanales-Belasio, E., Zambruno, G., Cavani, A., Girolomoni, G. (1997) Antibodies against sialophorin (CD43) enhance the capacity of dendritic cells to cluster and activate T lymphocytes J. Immunol. 159,2203-2211[Abstract/Free Full Text]
-
Sans, E., Delachanal, E., Duperray, A. (2001) Analysis of the roles of ICAM-1 in neutrophil transmigration using a reconstituted mammalian cell expression model: implication of ICAM-1 cytoplasmic domain and Rho-dependent signaling pathway J. Immunol. 166,544-551[Abstract/Free Full Text]
-
Farstad, I. N., Halstensen, T. S., Kvale, D., Fausa, O., Brandtzaeg, P. (1997) Topographic distribution of homing receptors on B and T cells in human gut-associated lymphoid tissue: relation of L selectin and integrin alpha 4 beta 7 to naive and memory phenotypes Am. J. Pathol. 150,187-199[Abstract]
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ABSTRACT
INTRODUCTION
