Altered migration of gut-derived T lymphocytes after
activation with concanavalin A
Ryota
Hokari1,
Soichiro
Miura2,
Hitoshi
Fujimori1,
Seiichiro
Koseki1,
Yoshikazu
Tsuzuki1,
Hiroyuki
Kimura1,
Hajime
Higuchi1,
Hiroshi
Serizawa1,
D. Neil
Granger3, and
Hiromasa
Ishii1
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
Medical Center, Shreveport, Louisiana 71130-3932
 |
ABSTRACT |
Although activation of lymphocytes is known to
be associated with profound changes in homing behavior, it remains
unclear how activation alters migration of gut-derived lymphocytes in lymphoid and nonlymphoid organs. The objectives of this study were
1) to compare migration of naive and
concanavalin A (ConA)-activated T lymphocytes into the gut mucosa,
spleen, and liver and 2) to define
the role of specific adhesion molecules in this homing process.
Fluorescently labeled T lymphocytes collected from rat intestinal lymph
were injected into the jugular vein, and the kinetics of appearance of
the infused lymphocytes were monitored in ileal Peyer's patches,
spleen, and liver. The migration of naive and ConA-activated T
lymphocytes into microvessels were compared using an intravital
microscope. ConA stimulation significantly increased the rolling
velocity of T lymphocytes in postcapillary venules of Peyer's patches,
and ConA-stimulated lymphocytes exhibited a loss of the selective
adherence properties in Peyer's patches that is normally observed with
naive T cells. ConA activation also suppressed the accumulation of T
cells in the spleen. On the other hand, the adherence of T cells to
hepatic sinusoidal endothelium was significantly increased after ConA
activation, especially in the periportal area, and this increase was
attenuated by an anti-intercellular adhesion molecule (ICAM)-1
antibody. Flow cytometry analysis revealed a decline in
L-selectin expression and an increase in CD11a expression and ICAM-1 on
the surface of ConA-treated T cells. In conclusion, activation of
gut-derived T lymphocytes with ConA significantly alters their
migration path, with a diminished localization to Peyer's patches and
spleen and a preferential accumulation in hepatic sinusoids. This
altered migration pattern likely results from changes in the expression of leukocyte adhesion molecules such as L-selectin and CD11a.
Peyer's patches; spleen; adhesion molecules; intestinal lymph; CD11a; intercellular adhesion molecule-1
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INTRODUCTION |
EFFECTIVE IMMUNE surveillance is achieved by the
continuous migration of lymphocytes between lymphoid and nonlymphoid
organs. This process enables naive cells to increase the frequency of encounters with cognate antigens (9, 26). These lymphocytes have the
capacity to migrate very efficiently from the blood into secondary
lymphoid tissues, such as lymph nodes and Peyer's patches, by
extravasating through the endothelium of specialized postcapillary or
high endothelial venules (HEVs) (11). In several species, a variety of
lymphocyte adhesion molecules, namely L-selectin (10), CD44 (19), and
4
7
(2, 12, 17), are considered to play a role as organ-specific homing
receptors. Recently, we have reported that
4-integrins make a critical
contribution to the rolling and sticking of T cells and their
subsequent transendothelial migration in postcapillary venules (PCVs)
of Peyer's patches exposed to physiological shear rates (22).
There is evidence that the homing behavior of lymphocytes can be
profoundly altered on activation and differentiation. The migration
properties of activated lymphocytes appear to be both more selective
and more diverse than those of naive lymphocytes; indeed, the concept
of "organ-specific" homing originated from studies on lymphoblast
traffic. Some migration properties of "memory" lymphocytes more
closely resemble those of activated lymphocytes (7, 15, 21, 31). Most
memory and effector lymphocytes probably traffic through lymphoid
organs, but, unlike naive cells, they can also access and recirculate
through extralymphoid immune effector sites such as the
intestinal lamina propria or inflamed skin and joints (9, 21, 30). In
humans, for example, CD4 cells that express both cutaneous
lymphocyte-associated antigen and L-selectin preferentially accumulate
in inflamed skin (28). Although large numbers of gut-derived
lymphocytes are known to traffic through the liver and spleen, where
they contribute to immunologic defense, it remains unclear whether the
nature and intensity of lymphocyte-endothelial cell adhesion in these
vascular beds are altered following lymphocyte activation (15).
Moreover, it is not known how activation of lymphocytes affects their
trafficking within microvessels supplying blood to either lymphoid
(Peyer's patch) or nonlymphoid (lamina propria) regions of the gut mucosa.
Concanavalin A (ConA), a plant lectin from jack beans, is known to
mitogenically activate T lymphocytes via the antigen receptor. This
lectin, along with intravital microscopic procedures for monitoring the
dynamic process of lymphocyte migration, was employed in the present
study to address three major objectives:
1) to compare the migration of naive
and ConA-activated gut T lymphocytes into lymphoid (Peyer's patches)
and nonlymphoid regions of the gut mucosa,
2) to assess the influence of ConA
activation on the recruitment of lymphocytes in the vascular beds of
the spleen and liver, and 3) to
determine the contribution of
4-integrin, LFA-1
,
intercellular adhesion molecule (ICAM)-1, and L-selectin to the
adhesive interactions between activated lymphocytes and endothelial
cells in the gut, spleen, and liver. We found that activation of
gut-derived T lymphocytes with ConA significantly alters their
migration path, with a diminished localization to Peyer's patches and
spleen and a preferential accumulation in hepatic sinusoids, and found
that this altered migration pattern likely results from changes in the
expression of leukocyte adhesion molecules including L-selectin and CD11a.
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METHODS |
Experimental setup for microvascular studies.
Male Wistar rats weighing 250-300 g were maintained on standard
laboratory chow (Oriental Yeast, Tokyo, Japan). The care and use of
laboratory animals were in accordance with the National Institutes of
Health guidelines. Under anesthesia with 50 mg/kg of pentobarbital
sodium, the abdomen was opened via a midline incision. Twelve
centimeters of the ileal segment ending at the cecal valve were chosen
for observation and placed on a plastic plate. The intestine was kept
warm and moist by continuous superfusion with physiological saline
warmed to 37°C. Two small incisions in the bowel wall were made
with a microcautery. Krebs-Ringer solution (pH 7.4) was
infused into the lumen to flush away food residue. The luminal pressure
of the gut loop was maintained at 15 cmH2O with warm Krebs-Ringer
solution, which was instilled into loops through vinyl tubes from the
proximal end to obtain appropriate resolution and to minimize
intestinal motility. Suitable areas of the microcirculation in Peyer's
patch and villus mucosa were observed through the serosa by an inverted
type fluorescence microscope (Diaphot TMD-2S, Nikon, Tokyo) equipped
with a silicon-intensified target image tube camera with a
contrast-enhancing unit (C-2400-08, Hamamatsu Photonics, Shizuoka,
Japan) via a ×10 or ×20 objective lens. In separate groups,
the microcirculation of intestinal villi was also observed from the
mucosal surface after the intestine was cut along its antimesenteric
border. The adjacent intestinal segment and mesentery were covered with
absorbent cotton soaked with Krebs-Ringer solution. The behavior of
fluorescently labeled lymphocytes was visualized on a television
monitor through a fluorescence microscope according to a previously
described method (22). Epi-illumination was achieved with filters of
excitation at 470-490 nm and emission at 520 nm.
In another set of experiments, the liver and spleen were placed on a
plastic stage with a nonfluorescent coverglass and carefully adjusted
to minimize respiratory movements. The exposed portions of the liver or
spleen were immediately covered with a transparent plastic film. In
vivo microcirculatory images of the surface of the liver and spleen
were observed after lymphocyte injection through a fluorescence
television microscope as described above. After observation of
lymphocyte adherence, FITC-labeled BSA (5 mg/ml; Sigma Chemical, St.
Louis, MO) was injected (as a bolus) from the jugular vein to evaluate
the distribution of microvascular blood flow in the liver and spleen.
The femoral artery was cannulated for measurement of systemic arterial
pressure, using a Statham P23A pressure transducer (Oxnard, CA).
Systemic arterial pressure was then continuously recorded with a Grass
polygraph recorder (Grass Instruments, Quincy, MA). The femoral vein
was also cannulated for administration of monoclonal antibodies (MAbs).
In some experiments, an optical light rod (3.2 mm outer diameter,
Sugiura, Tokyo, Japan) connected with a xenon cold light source
(Olympus, Tokyo, Japan) was inserted into the intestinal loop through
the output vinyl tube. A high-speed video camera system equipped with a
recorder (Kodak Ekta Pro 1000) was used for measurement of the velocity
of red blood cells (RBCs) as reported previously (23). The observed
area was recorded using a high-speed video recording system on an S-VHS
videotape at a speed of 1,000 frames/s. The videotape was replayed at
30 frames/s. Changes in the RBC velocity of each microvascular branch
were determined as time course changes with a video-measuring gauge
(For A, Houei, Tokyo, Japan).
Collection and separation of lymphocytes.
After an intraperitoneal injection of pentobarbital sodium (50 mg/kg),
the main mesenteric lymphatic duct was cannulated as described by
Bollman et al. (5). When the surgical procedure was completed, animals
were maintained in Bollman's cages and saline was infused
intravenously from the jugular vein at a flow rate of 2.4 ml/h to
replenish the fluid and electrolyte loss associated with lymphatic
drainage. Lymph samples were collected in ice-cold vials containing 6 U/ml heparin, fetal bovine serum, and RPMI 1640 medium (pH 7.4; GIBCO,
Grand Island, NY). Lymphocytes from mesenteric lymph were washed three
times with working medium [RPMI 1640 (pH 7.4), penicillin and
streptomycin (GIBCO), and 0.1% BSA (Sigma)] before separation
and labeling.
A T cell-rich fraction of mesenteric lymphocytes was first obtained
using a nylon-wool column. The whole cell population of 1 × 108 lymphocytes in 20 ml of RPMI
1640 medium with 1% fetal bovine serum was incubated in 1 g of
nylon-wool (Kanto Kagaku, Tokyo, Japan) in a column, and the
pass-through fraction was designated as the T cell-rich fraction after
incubation for 1 h at 37°C. The T cell-rich suspension was then
passed through an IgG-coated Immulan (Biotecx, Houston, TX) column to
obtain negatively selected T cells. These manipulations had no
significant effect on lymphocyte viability as assessed by trypan blue
exclusion. The purity of T cells was evaluated by a
fluorescence-activated cell sorter (FACS; Becton-Dickinson, Mountain
View, CA). An MAb against rat CD3 (W3/13) (Serotec, Kidlington, UK) was
used for FACS analysis. The purity of the isolated T cells was shown to
be at least 95%.
Lymphocyte stimulation and labeling with carboxyfluorescein
diacetate succinimidyl ester.
T cells were cultured at a concentration of 2 × 106 cells/ml in RPMI 1640 medium
supplemented with 10% fetal bovine serum, 5 × 10
5 M 2-mercaptoethanol,
penicillin, and streptomycin. The T cells were stimulated with several
concentrations of ConA (Sigma) and incubated in 96-well flat bottom
microtiter plates (0.2 ml/well) for 48 h at 37°C in 5%
CO2-95% air. To see the T cell
activation, 1 µCi of
[3H]thymidine (ICN
Biochemicals, Costa Mesa, CA; 6.7 Ci/mM) was added to each well 6 h
before the end of incubation. Cultures were harvested onto glass filter
papers (LKB, Gaithersburg, MD) using a Skatron cell plate counter
(LKB). T cells were washed extensively to remove the activating
stimulus before use in the presence of 25 mM methyl
-D-mannoside (Sigma) to block
any residual ConA that may have been transferred with the lymphocytes.
Cultured lymphocytes not exposed to ConA were used as controls.
Carboxyfluorescein diacetate succinimidyl ester (CFDSE; Molecular
Probes, Eugene, OR) was dissolved in DMSO 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 × 108) in 20 ml of RPMI 1640 were
incubated with 20 µl of CFDSE solution for 30 min at 37°C. After
hydrolysis by cytoplasmic esterases, CFDSE forms a stable fluorochrome
carboxyfluorescein succinimidyl ester (CFSE). Intracellular
fluorophores react with lysine residues of intracellular proteins and
remain within cells as long as the membrane is intact (32).
Data analysis of lymphocyte behavior.
Lymphocytes (3 ×107 cells in
1 ml RPMI 1640) were injected into the jugular vein of recipient rats
over a 3-min period. The traffic of CFSE-labeled T lymphocytes in the
microvasculature of Peyer's patches and villus mucosa was continuously
monitored and recorded on S-VHS video tape for 60 min after infusion of the cells. The locomotive behavior of individual lymphocytes was evaluated by replaying the video recording on a four-head video cassette recorder with a shuttle wheel single frame forward and reverse
control (GT-4W, NV-FS70, Panasonic). To determine the distribution of
lymphocytes at different depths, the focusing plane was placed at
5-µm intervals from the surface of the intestine. Lymphocytes
adhering to the venules with occasional movement along the wall were
defined as "rolling" lymphocytes. Those adhering to the wall
without movement after exhibiting transient rolling were defined as
"adherent" lymphocytes. They remained in the same position
throughout each observation period (30 s). The average lymphocyte
rolling velocity was plotted for at least 50 lymphocytes in more than
six independent distinct venules (total of >300 lymphocytes).
Because T lymphocytes are known to selectively adhere to 25- to
500-µm-diameter PCVs of Peyer's patches, which correspond to second-
or third-order branches of large interfollicular veins (22), we
assessed T lymphocyte behavior using microvessels in this portion of
Peyer's patches. In the villus mucosa, the submucosal arteriolar and
venular branches were identified and lymphocyte adherence was studied
along third-order branches of venules. Villus tip capillaries with
arcade vessels were identified by observation from the mucosal side.
The number of adherent cells in PCV of Peyer's patches and in
unbranched straight venules and capillaries of villus mucosa was
normalized to a 1-mm2 observation field.
The migration of CFSE-labeled T lymphocytes through the
microvasculature of the liver or spleen was continuously monitored and
recorded on video tape for up to 50 min after injection. In the liver,
suitable images including several units of hepatic lobules were
selected and the lobular landmarks, the terminal portal venules (TPVs)
and terminal hepatic venules (THVs) were identified when the replayed
images of the FITC-labeled hepatic microangiograph were evaluated. The
location of migrated lymphocytes was arbitrarily divided into three
zones of the liver acinus, namely, periportal, midzonal, and
perivenular. The number of T lymphocytes draining into THVs of liver
was also compared between control and ConA-treated cells and expressed
as lymphocyte flux per 10-min observation period.
Agents studied.
A mouse IgG2a MAb functionally blocking the rat integrin
4-chain
(MR
4-1) was purified
from ascites on a protein G column as described previously (36). WT-3,
an MAb that functionally blocks CD11a (the leukocyte
function-associated molecule-1
; LFA-1
) was obtained from
Seikagaku Kogyo (Tokyo, Japan).
F(ab')2 fragments were
obtained by incubation of 2.0 µg of pepsin/1.0 mg of IgG for 2 h at
37°C, pH 3.5 (24). The digestate was then dialyzed to neutral pH,
and samples were examined on gel electrophoresis for characterization
of the fragments. CFSE-labeled T lymphocytes were preincubated with
MR
4-1 or WT-3 in vitro for
20 min [3 × 107 lymphocytes in 10 ml RPMI 1640 containing 0.5 mg F(ab')2
fragments of antibody] and then infused into the recipient rats
via the femoral vein. The antibody functionally blocking L-selectin
(HRL3, IgG1) was purchased from Seikagaku Kogyo. In the case of HRL3, lymphocytes (3 × 107) were
preincubated with 0.6 mg of HRL3 in 10 ml of calcium-free Hanks' for
20 min. In control animals, lymphocytes were preincubated with
nonbinding antibodies (P6H6 or HRL4) for 20 min before infusion. P6H6
was provided by Cytel (San Diego, CA), and HRL4 was obtained from
Seikagaku Kogyo. In some experiments, animals were pretreated (30 min
before lymphocyte infusion) with an MAb directed against ICAM-1 (1A-29,
2 mg/kg). 1A-29 was purchased from Seikagaku Kogyo. As controls,
nonbinding antibody P6H6 was used, and the same protocol was used.
In a separate series of experiments, gadolinium III chloride
hexahydrate (GdCl3; Aldrich
Chemical, Brussels, Belgium), which depletes Kupffer cells and inhibits
their function (16), was administered intravenously [4 mM
solution in 0.9% NaCl (1.0 ml)/250 g body wt] 24 h before
lymphocyte infusion, and the same protocol was employed in liver microcirculation.
Analysis of cell surface adhesion molecules of T cells.
Selected single cell suspensions of T cells from intestinal lymph were
washed in Hanks' balanced salt solution containing 0.2% BSA and 0.1%
NaN3. This medium was used
throughout the staining procedure. All incubations with antibodies were
performed at 4°C for 30 min. For immunofluorescence staining, 1 × 106 lymphocytes were first
incubated with mouse anti-rat MAbs to characterize and quantify
4-integrin and CD11a
expression. After incubation, the cells were washed in 400 µl of
Hanks' balanced salt solution and centrifuged three times at 1,500 g for 30 s. The cells were then
incubated with 1 ml of FITC-labeled anti-rat IgG. Cells were washed
twice and resuspended for analysis. The antibody against L-selectin
(HRL3, IgG1) was detected by an FITC-conjugated anti-hamster antibody
(Cappel, West Chester, PA). For controls, lymphocytes were preincubated
with isotype-matched, irrelevant antibodies. The lymphocytes were
analyzed with an EPICS Elite flow cytometer (Coulter, Hialeah, FL).
Data were obtained using CONSORT software on viable cells, as
determined by forward light scatter intensity. Representative data from
at least four individual measurements are shown.
Statistics.
All results are expressed as means ± SE of 6 rats. Differences
among groups were evaluated by ANOVA and Fisher's post hoc test. For
the comparison of histogram profiles of lymphocyte rolling velocity
between different groups, the mean value and distribution were
statistically evaluated by a nonparametric Mann-Whitney U-test and
Kolmogorov-Smirnov test, respectively. Statistical significance was set
at P < 0.05.
 |
RESULTS |
Lymphocyte stimulation and surface expression of adhesion molecules
on T lymphocytes.
Activation of T lymphocytes during blastogenesis was achieved by ConA
treatment for 48 h. The maximum proliferation of T cells was induced at
a concentration of 2.5 µg/ml as determined by
[3H]thymidine
incorporation (Table 1). Therefore, in all
subsequent experiments, ConA was employed at a concentration of 2.5 µg/ml.
The expression of various adhesion molecules (CD11a,
4-integrin, L-selectin, and
ICAM-1) on the surface of T lymphocytes was determined using specific
MAbs. FACS analysis revealed that the expression of L-selectin was
downregulated on activation with ConA (Fig.
1). ConA treatment slightly increased the
intensity of CD11a and ICAM-1 on T lymphocytes, but it did not alter
the expression of
4-integrin.

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Fig. 1.
Expression of adhesion molecules L-selectin
(A),
4-integrin
(B), CD11a
(C), and intercellular adhesion
molecule (ICAM)-1 (D) on T
lymphocytes determined by flow cytometric analysis. Effect of
concanavalin A (ConA) treatment is shown. Control, naive T lymphocytes
from intestinal lymph. ConA, T cells stimulated with ConA (2.5 µg/ml)
for 48 h. Lymphocytes (1 × 106) were first incubated with
anti-rat monoclonal antibodies against CD11a (WT-3), 4-integrin
(MR 4-1), L-selectin
(HRL3), and ICAM-1 (1A-29). Cells were incubated with 1 ml FITC-labeled
anti-rat IgG or anti-hamster IgG. Flow cytometry analysis was performed
using FACScan (Epics Elite, Coulter). Data were obtained using CONSORT
software on viable cells, as determined by forward light scatter
intensity. Representative data from at least 4 individual measurements
are shown.
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In vivo study of T lymphocyte migration into intestine.
Characteristic lymphocyte-endothelium interactions were observed in
PCVs of Peyer's patches after infusion of T lymphocytes. At an early
stage (10 min after cell administration), some lymphocytes transiently
interacted with the vessel wall by rolling and then soon detached and
returned to the blood stream. During the period of interaction, these
lymphocytes rolled on the endothelium of PCVs at various speeds and for
varying distances. The velocity of rolling lymphocytes, which slowly
travel along the venules, was calculated and compared between control
and ConA-stimulated T cells. The mean flow velocity of RBCs in
30-µm-diameter venules was 3.0 ± 0.8 mm/s in the control group,
and there was no significant change in RBC velocity when ConA-treated T
lymphocytes were administered. A histogram summarizing lymphocyte
rolling shows that 60-80 µm/s was the most frequent rolling
velocity in the control group. A significant increase in rolling
velocity was observed after ConA treatment, with the most frequent
rolling velocity being 140-160 µm/s in the ConA-treated group.
Adherent lymphocytes gradually increased in number in the middle-sized
(25-50 µm) PCVs of Peyer's patches during the observation period, especially during the initial 30- to 40-min period (Fig. 2). Figure
3A
illustrates the time course of changes in the number of adherent
lymphocytes in PCVs of Peyer's patches and the effect of ConA
activation. Lymphocytes located both inside and along (extravasated)
microvessels are also illustrated. The number of adherent lymphocytes
in PCVs of the control group was 165.0 ± 18.0 and 195.2 ± 14.8 cells/mm2 at 20 and 30 min,
respectively. ConA treatment significantly inhibited the number of
adherent lymphocytes, particularly within the first 30 min (Fig. 2),
with the number of these lymphocytes being 18.3 ± 4.0 and 25.1 ± 5.1 cells/mm2 at 20 and 30 min, respectively (Fig. 3A). In
vitro pretreatment of lymphocytes with antibody functionally blocking
4-integrin (MR
4-1) almost
completely abolished the adherence of ConA-activated lymphocytes to
PCVs of Peyer's patches, but anti-L-selectin antibody (HRL3) did not
affect these interactions (Fig. 3A).

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Fig. 2.
Representative images of the distribution of carboxyfluorescein
succinimidyl ester (CFSE)-labeled naive T lymphocytes (A)
and ConA-stimulated T lymphocytes (B) in postcapillary
venules of Peyer's patches at 20 min after infusion. Treatment with
ConA remarkably inhibited the number of sticking lymphocytes compared
with control. Bar = 100 µm.
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Fig. 3.
Effect of ConA activation on the time course of T lymphocyte sticking
in postcapillary venules of rat Peyer's patches
(A), third-order submucosal venules
of villi (B), and villus tip
capillaries (C) and the effect of
monoclonal antibodies against
4-integrin
(MR 4-1) or L-selectin
(HRL3) on ConA-induced changes of T lymphocyte sticking in these area.
Lymphocytes were incubated with ConA (2.5 µg/ml). Lymphocytes located
both inside and along venules were counted in the
1-mm2 observation field.
Peyer's patches and submucosal venules were observed from the serosal
side, and villus tip capillaries were observed from the mucosal side.
* P < 0.05 compared with
controls. # P < 0.05 compared with ConA alone. Values are means ± SE from 6 animals.
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Although the density of sticking lymphocytes was quite low in the
villus (nonlymphoid) mucosa compared with PCVs in Peyer's patches, we
constantly observed a number of adherent lymphocytes in these tissues.
In submucosal venules (third-order branches) of villi, the number of
lymphocytes adherent to venular endothelium gradually increased during
the observation period (Fig. 3B). In contrast to the PCVs of Peyer's patches, ConA treatment did not significantly decrease the number of adherent lymphocytes in this region (12.8 ± 1.8 in control vs. 11.5 ± 1.0 cells/mm2 in ConA treated at 40 min). In vitro pretreatment with
MR
4-1 but not HRL3
significantly decreased the adherence of ConA-activated lymphocytes to
submucosal venules (Fig. 3B). Figure
3C illustrates the number of adherent
lymphocytes in villus tip capillaries as observed from the mucosal
surface. In the control group, the adherent lymphocytes in villus tip
capillaries amounted to 11.0 ± 2.0 cells/mm2 at 10 min, but their
number did not significantly change during the 40-min observation
period (12.0 ± 1.3 cells/mm2
at 40 min). ConA treatment did not induce a different level or time
course of lymphocyte accumulation in villus tip capillaries compared
with naive lymphocytes. Treatment with
MR
4-1 did not affect the
lymphocyte accumulation in this area.
In vivo study of T lymphocyte migration into spleen and liver and
the effect of antiadhesion molecules.
The interaction of CFSE-labeled T lymphocytes with microvessels was
visualized in the spleen. There was almost no significant lymphocyte
rolling along the endothelium of these microvessels, and T lymphocytes
appeared to adhere immediately on entering the splenic
microcirculation. The time course of accumulation of adherent lymphocytes in splenic microvessels is shown in Fig.
4. In control animals, the number of
adherent T lymphocytes gradually increased over the 60-min period. In
contrast, treatment of T cells with ConA significantly inhibited the
accumulation of adherent lymphocytes in the spleen (133.5 ± 7.3 in
control vs. 37.9 ± 7.2 cells/mm2 in ConA treated at 30 min), although the total flux of appearing lymphocytes in spleen was
not significantly different between the two groups. To determine which
adhesion molecules were involved in T lymphocyte adherence within the
splenic microcirculation, several MAbs against adhesion molecules were
studied. As shown in Fig. 4, all of the adhesion molecule-specific
antibodies tested (anti-L-selectin, anti-CD11a, and
anti-
4-integrin) did not alter the accumulation of unstimulated (Fig.
4A) as well as ConA-activated (Fig.
4B) T lymphocytes in the splenic
microcirculation, suggesting that these adhesion molecules are not
involved in the splenic T cell migration observed under normal
physiological conditions.


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Fig. 4.
Time course changes of T lymphocyte sticking in microvessels of rat
spleen. Effects of adhesion molecule antibodies on unstimulated
lymphocytes (A) and ConA-activated
lymphocytes (B) are shown. ConA (2.5 µg/ml) was used for stimulation of T lymphocytes. In some
experiments, lymphocytes were treated with a monoclonal antibody
against CD11a (WT-3), L-selectin (HRL3), and
4-integrin
(MR 4-1) before infusion.
Lymphocytes located in the 1-mm2
observation field were determined.
* P < 0.05 compared with
controls (unstimulated lymphocytes). Values are means ± SE from 6 animals.
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In the liver, most of the infused, labeled T lymphocytes emerged
(appeared) vertically from the periportal area (PVs), crossed the
hepatic sinusoid, and drained into the THVs (Fig.
5). Flow velocity of these lymphocytes was
256 ± 28 µm/s in venules of the midzonal area and 320 ± 30 µm/s in venules of the perivenular area. ConA treatment did not
significantly affect the flow velocity of T lymphocytes in either the
midzonal or perivenular area. Some lymphocytes attached to the hepatic
sinusoids especially in the portal area and perivenular area after
transient rolling. The percentage of rolling flux was significantly
greater in activated lymphocytes (53.3 ± 5.0%) than in
unstimulated lymphocytes (37.5 ± 3.5%) in the midzonal area at 10 min after infusion. The time course of accumulation of adherent
lymphocytes in hepatic sinusoids was determined in both periportal and
perivenular areas, and the effects of treatment of T cells with ConA
are shown in Fig. 6. The number of adherent
T lymphocytes in the periportal area reached a maximum within
10-20 min (295 ± 52 and 310 ± 44 cells/mm2 at 10 and 20 min,
respectively). ConA stimulation significantly enhanced the number of
adherent lymphocytes in the periportal zone, with remarkable increases
observed at 10 min (779 ± 129 cells/mm2) and 20 min (960 ± 138 cells/mm2) after cell
activation (Fig. 6A). However, the
number of T lymphocytes adherent to hepatic sinusoids of the
perivenular area was greater for naive vs. ConA-treated lymphocytes
during the first 10 min (Fig. 6B).
Thereafter, activated T lymphocytes accumulated in sinusoids of the
perivenular area, whereas the number of adherent naive lymphocytes
diminished. Consequently, the number of adherent ConA-treated T
lymphocytes exceeded that of controls at 20 min (Fig.
6B). T lymphocytes drained into
the THV, with 280 ± 28 cells · mm
2 · 10 min
1 observed at 10 min
after the infusion of naive T lymphocytes. On the other hand, ConA
stimulation remarkably inhibited the lymphocyte flux into THVs (35 ± 8 cells · mm
2 · 10 min
1) at 10 min because
many activated lymphocytes were trapped in the periportal zone.

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Fig. 5.
Representative image of the distribution of CFSE-labeled naive T
lymphocytes (A) and ConA-stimulated T lymphocytes
(B) in rat hepatic sinusoid at 30 min after infusion. Most
of the infused, labeled T lymphocytes appeared vertically from the
periportal area (PV), crossed the hepatic sinusoid, and drained into
the terminal hepatic venules (THV). Some lymphocytes attached to the
hepatic sinusoids, especially in the portal area and perivenular area.
ConA stimulation particularly enhanced the number of adherent
lymphocytes in the periportal zone.
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Fig. 6.
Effect of ConA activation on time course of T lymphocyte sticking in
periportal area (A) and perivenular
area (B) of hepatic sinusoids.
Lymphocytes located in the 1-mm2
observation field were counted.
* P < 0.05 compared with
controls. Values are means ± SE from 6 animals.
|
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To determine which adhesion molecule is involved in the enhanced
adherence of activated T lymphocytes within hepatic sinusoids, we
assessed the actions of the adhesion molecule-specific MAbs on the
number of adherent lymphocytes (Fig.
7A) and
the lymphocyte flux into THVs (Fig.
7B) at 30 min after the T lymphocyte
infusion. Values are expressed as a percentage of the adherent
unstimulated or ConA-treated T lymphocytes not exposed to an antibody.
Anti-ICAM-1 (1A-29) significantly decreased lymphocyte adherence to
67.7 ± 7.0% of that of unstimulated lymphocytes and 59.3 ± 11.3% of that of ConA treated (Fig.
7A). However, although not shown in
Fig. 7, no further decrease in lymphocyte adherence was observed when in vitro pretreatment of lymphocytes with 1A-29 in conjunction with
1A-29 systemic injection was compared with the 1A-29 infusion alone. In
vitro pretreatment of lymphocytes with
anti-
4-integrin (MR
4-1) did not
significantly attenuate lymphocyte sticking (98.0 ± 9.0% and 84.8 ± 9.2% of the antibody untreated values in unstimulated and
ConA-treated lymphocytes, respectively). Lymphocyte sticking was
reduced to 76.0 ± 11.0% and 74.3 ± 13.0% by treatment with
anti-CD11a (WT-3) in unstimulated and ConA-treated lymphocytes, respectively, although these decreases were not statistically significant. Pretreatment of lymphocytes with HRL3 did not
significantly affect the adherence of these lymphocytes (Fig.
7A). In accordance with the results
of the effect of MAbs on lymphocyte adherence in the periportal area,
treatment with 1A-29 significantly increased the flux of T
lymphocytes into THVs and this increase was especially remarkable for
ConA-activated lymphocytes (Fig.
7B). WT-3 treatment also
significantly accelerated the flux of both unstimulated and ConA-treated lymphocytes into THVs, whereas
MR
4-1 and HRL3 did not
significantly alter lymphocyte flux of these lymphocytes (Fig.
7B).


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Fig. 7.
Effect of monoclonal antibodies against CD11a (WT-3),
4-integrin
(MR 4-1), L-selectin
(HRL3), or ICAM-1 (1A-29) and
GdCl3 treatment of T lymphocyte
sticking in the periportal area of hepatic sinusoids
(A) and lymphocyte flux into THVs
(B). Effects of various treatments
on sticking or flux of unstimulated cells (solid bars) as well as of
ConA-induced T lymphocytes (hatched bars) are demonstrated. Lymphocytes
were treated with monoclonal antibody against CD11a (WT-3),
L-selectin (HRL3), or
4-integrin
(MR 4-1) before infusion.
In some experiments, animals were pretreated (30 min before lymphocyte
infusion) with a monoclonal antibody directed against ICAM-1 (1A-29, 2 mg/kg). In other series of experiments,
GdCl3 was administered
intravenously [4 mM solution in 0.9% NaCl (1.0 ml)/250 g body wt
at 24 h before lymphocyte infusion] and the same protocol was
employed in liver microcirculation. Values are expressed as percentage
of unstimulated control groups or ConA-treated groups and are means ± SE of 6 animal experiments.
# P < 0.05 compared with the
values of control or ConA-treated alone.
|
|
The depletion and inhibition of Kupffer cells in rat liver by treatment
with GdCl3 significantly
attenuated lymphocyte adherence to 55.5 ± 5.4% of that observed
with ConA treatment, but this was not the case for unstimulated
lymphocytes (86.0 ± 10.0%) (Fig. 7A). Furthermore,
GdCl3 drastically increased the
flux of activated T lymphocytes in THVs as shown in Fig.
7B.
 |
DISCUSSION |
The migration of lymphocytes through different lymphoid organs and
other tissues of the body is a carefully controlled process that
enables immune cells to encounter the cognate antigen and to
disseminate memory and effector cells for immunologic surveillance (7).
In this study, we have demonstrated, using intravital video microscopy,
that activation of T lymphocytes by ConA leads to profound alterations
in the sequential migration of these cells in the microcirculation of
different lymphoid and nonlymphoid organs. The activated T cells either
lose or exhibit a diminished capacity to enter Peyer's patches and the
spleen, whereas they exhibit a preferential distribution to the liver.
A similar behavior of activated lymphocytes in lymph nodes or Peyer's
patches has been reported in previous studies, although these were in
vivo homing studies that used
51Cr-labeled lymphocytes or an in
vitro binding assay of frozen sections (8, 13, 14). Our study also
clearly demonstrates that stimulation of T lymphocytes leads to a
selective suppression of lymphocyte binding to PCV of Peyer's patches
in the intestinal mucosa. The altered homing of activated lymphocytes
may reflect changes in the expression of cell surface adhesion
molecules. Recently, we have demonstrated that
4-integrins mediate the rolling and sticking of T cells in PCVs of rat Peyer's patches (34). In this
study, we were unable to detect any significant changes in the surface
expression of
4-integrins on T
lymphocytes after ConA treatment. Instead, we showed (using flow
cytometric analysis) that the expression of L-selectin was
downregulated when activated with ConA. Data suggest that the decreased
L-selectin expression of ConA-treated lymphocytes is responsible for
increased rolling velocity; other work shows that even a 50% reduction
in L-selectin expression can dramatically reduce homing
(33). Lymphocytes from L-selectin knockout mice show
impaired homing to Peyer's patches, and in vivo homing studies have
shown that an antibody to L-selectin partially blocks lymphocyte
migration to Peyer's patches (1, 12). Our present results show that
blocking of
4 integrin of
ConA-treated lymphocytes almost completely inhibited the
lymphocyte-endothelial interaction in Peyer's patches, suggesting the
concerted action of L-selectin and
4-integrin molecules in this
region. It appears likely that L-selectin plays a primary role in the
initiation of lymphocyte contact (rolling and adherence) with Peyer's
patch HEVs in association with
4-integrin (6), whereas
4
7
and LFA-1
sequentially participate in processes of firm
adhesion and transendothelial migration (2).
In the present study, we observed profound differences in the magnitude
and kinetics of T lymphocyte recruitment into lymphoid (Peyer's patch)
and nonlymphoid (villus mucosa) regions of the intestinal
microcirculation. In contrast to Peyer's patch HEVs, we were unable to
demonstrate significant differences in the accumulation of stationary
naive vs. ConA-activated lymphocytes in submucosal venules and villus
tip capillaries of the rat intestinal mucosa. These observations are
somewhat different from previously published reports of an enhanced
migration of activated lymphocytes into the gut mucosa (3, 4, 7, 27,
30). Berlin et al. (4) reported an increased in situ interaction of
activated murine lymph node lymphocytes with venules in the small
intestinal lamina propria compared with resting lymph node cells. They
also found that
4
7
can mediate the direct L-selectin-independent interactions of activated
lymph node cells with mucosal address in cell adhesion molecule-1. The
mucosal immunoblast is considered to traffic efficiently to the
intestine (27), and recent studies indicate that mucosal T cell blasts
preferentially bind to human mucosal HEVs, primarily via
4
7,
but with contributions from CD44 and LFA-1
(30). This
characteristic gut tropism of gut-derived activated T cells was not
observed in the present study. The exact reason for the difference
between previous reports and our observations is not known but may
relate to the use of different cell sources (cells from intestinal
lymph vs. lymph node cells or lamina propria cells), type of
stimulation (ConA vs. other activators), and the species difference
(rats vs. mice or humans). On the other hand, Salmi et al. (30) also
showed that lamina propria lymphocytes (LPLs) stimulated in vitro with
phytohemagglutinin and interleukin-2 did not bind any more avidity than
the "not-as-activated" small LPLs. Hamann et al. (13) suggested
that in vitro activation by mitogen induces only one peculiar type of
migration behavior. It may be that in vitro stimulation and
differentiation may not mimic the in vivo situation very closely. In
our present results, the accumulation of activated T lymphocytes to
microvessels of villus tips was not inhibited by
anti-
4-integrin antibodies. We
also recently demonstrated that T lymphocyte adhesion in periglandular and villus tip capillaries is significantly increased by endotoxin, but
these interactions were independent of
4-integrins or
2-integrins (23). Additional
work is needed to more fully characterize the homing of activated
lymphocytes to different sites within the intestinal mucosa, such as
lymphoid vs. nonlymphoid regions, as well as submucosal venules vs.
mucosal capillaries.
The predominant role of the spleen in lymphocyte migration has been
postulated because higher numbers of lymphocytes enter this regional
circulation than enter the thoracic duct (25) of rats. Lymphocyte
homing to the spleen does not involve HEVs, and the adhesion of these
leukocytes in the splenic microcirculation may depend on the expression
of different lymphocyte surface molecules. Our results confirm the view
that T lymphocytes readily adhere to splenic microvascular endothelium
and further demonstrate that none of the known adhesion molecules
(L-selectin,
4-integrin, and
CD11a/ICAM-1) is involved in this process. Activated lymphocytes, in
contrast, have a strongly reduced capacity to enter the spleen compared
with resting lymphocytes, which is consistent with previous findings
(15). The reduced capacity of activated lymphocytes to enter the spleen
is not likely a consequence of L-selectin shedding because treatment of
T lymphocytes with an anti-L-selectin MAb did not induce a significant
reduction in adhesion, which is comparable to the behavior of activated
lymphocytes. These findings suggest that unknown alterations in the T
cell phenotype occur on activation, which contribute to the reduced
ability of these lymphocytes to reach the spleen.
In this study, we demonstrated a significant accumulation of activated
T lymphocytes in the liver mirocirculation, particularly in the
periportal region, although nonactivated lymphocytes interact less
effectively with hepatic sinusoids. Our results from flow cytometric
analyses and blocking studies performed using MAbs against CD11a and
ICAM-1 suggest the possibility that an interaction between
LFA-1
/ICAM-1 accounts for the lymphocyte accumulation in hepatic sinusoids. There is histochemical evidence that ICAM-1 is
expressed on liver endothelium but mainly in the portal area (35).
Together, these results indicate that activated T lymphocytes may
preferentially adhere in hepatic sinusoids of the periportal region
using an ICAM-1-dependent process. A common picture emerges that
activation seems to induce a general change in the migration properties
of lymphocytes, diminishing their entry into lymphoid tissue while
enhancing their transit through nonlymphoid sites such as the liver
(13, 15). There is some evidence that a major portion of blasts remain
in the liver and die there. Hence, the liver may constitute a temporary
depository for activated lymphocytes within the body. Recently, Huang
et al. (18) demonstrated that, as injected T cells expressing the
transgene T cell receptor disappear from lymph nodes and spleen after
administration of an antigenic peptide, a massive accumulation of these
cells occurs in the liver, where they undergo apoptosis.
Our study also demonstrates that inhibition and depletion of Kupffer
cells in the liver by GdCl3
treatment significantly attenuate the accumulation of activated T
lymphocytes in the periportal area, suggesting that Kupffer cells
contribute to lymphocyte recruitment in the liver microcirculation.
Recently, we have demonstrated that Kupffer cell-mediated cytotoxicity
against hepatoma cells occurs through cell-cell adhesion via
ICAM-1/CD18, causing calcium mobilization and oxidative activation of
nuclear factor-
B, which may lead to the increased production of
nitric oxide in Kupffer cells (20, 29). Thus there is also a
possibility that activated T cells could be retained by Kupffer cells
when they enter the hepatic microcirculation through an
ICAM-1-dependent interaction. The dominant distribution of Kupffer
cells in the portal area supports this possibility. Once activated
lymphocytes interact with Kupffer cells or endothelial cells, an
increased synthesis of tumor necrosis factor-
may occur, which in
turn could enhance the expression of ICAM-1 expression in hepatic
sinusoids. The molecular basis for the increased migration of activated
lymphocytes to the liver and the significance of this process to the
immunologic response warrant further attention.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Hideo Yagita (Department of Immunology, Juntendo
University, School of Medicine, Tokyo, Japan) for the generous donation
of MR
4-1.
 |
FOOTNOTES |
This study was supported in part by Grants-in-Aid for Scientific
Research from the Japanese Ministry of Education, Science, and Culture
of Japan and by a grant from Keio University, School of Medicine. This
study was also supported by Funds for Food Allergy from the Ministry of
Welfare of Japan. D. N. Granger was supported by National Heart, Lung,
and Blood Institute Grant HL-26441.
Present address of R. Hokari: Second Dept. of Internal Medicine,
National Defense Medical College, Saitama 359-8513, Japan.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. Ishii, School
of Medicine, Keio Univ., 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
Received 14 January 1999; accepted in final form 13 July 1999.
 |
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