* 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
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: CD18 • acridine orange • collecting lymphatics • mesenteric lymphadenectomy
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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.
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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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 View larger version (17K): [in a new window] |
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.
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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.
 View larger version (49K): [in a new window] |
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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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.
 View larger version (24K): [in a new window] |
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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View this table: [in a new window] |
Table 1. Appearance of Lymphocyte Clusters in Interfollicular Lymphatics: Effect
of Adhesion Molecule Antibodies
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 View larger version (30K): [in a new window] |
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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 View larger version (36K): [in a new window] |
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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 View larger version (39K): [in a new window] |
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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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.
Received October 2, 2000; revised June 18, 2001; accepted June 27, 2001.
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4 integrins in lymphocyte homing to mucosal tissues in vivo J. Immunol. 152,3282-3293[Abstract]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]This article has been cited by other articles:
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