| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Biomedical Engineering, University of Virginia School of Medicine, Charlottesville, VA 22908;
Department of Immunology, Duke University Medical Center, Durham, NC 27710; and
Institute for Genetics, University of Cologne, Cologne, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Several molecules have been implicated in mediating lymphocyte recruitment into Peyer’s patches. L-selectin is a Ca2+-dependent mammalian lectin expressed constitutively on granulocytes and monocytes as well as on most circulating lymphocytes, including naive and certain effector T cells (2). Although L-selectin-deficient (L-/-) mice have morphologically normal Peyer’s patches, they exhibit a significant reduction in short term homing of exogenous lymphocytes to Peyer’s patches (3). Subsequent data have shown that this reduction in homing is most likely the result of a deficit in leukocyte rolling when L-selectin function is blocked, because lymphocytes treated with an L-selectin mAb and injected i.v. fail to roll in Peyer’s patch HEV (4), and functional blockade of L-selectin with a mAb in vivo reduces the number of rolling leukocytes in Peyer’s patch HEV by up to 90% (5). However, the normal size and cellularity of Peyer’s patches in L-/- mice (3) suggest that other important adhesion molecules can bypass the requirement for L-selectin in lymphocyte recruitment to Peyer’s patches. In fact, the absence of L-selectin delays, but does not prevent, lymphocyte homing to Peyer’s patches (3).
The most important adhesion molecule in lymphocyte recruitment to
Peyer’s patches is 
4ß7, an integrin
expressed at low levels on naive T and B cells and at high levels on
effector and memory T cells within the gut (6). This integrin can
mediate rolling interactions in vitro independent of L-selectin (7).
Treatment of lymphocytes with a mAb specific for the 4
chain of this integrin results in a twofold increase in the rolling
velocity of exogenous lymphocytes passing through HEV of wild-type
Peyer’s patches in vivo (4). Recently, ß7
integrin-deficient (ß7-/-) mice
have been generated (5) to address the role of
4ß7 under physiologic conditions. These
mice have severely reduced Peyer’s patch cellularity and decreased
lymphocyte adhesion to Peyer’s patch HEV. However, the number of
leukocytes rolling in Peyer’s patch HEV of
ß7-/- mice was normal, although
the leukocyte rolling velocity was increased. Lymphocyte rolling in
Peyer’s patch HEV of both wild-type and
ß7-/- mice was inhibited by
>90% with an L-selectin mAb (5). Here, we examine ß7
integrin/L-selectin double mutant (ß7/L-/-)
mice that lack both adhesion molecules known to be important in
lymphocyte homing to Peyer’s patches. We hypothesize that in the
absence of these two adhesion molecules, other, previously unknown,
adhesion mechanisms in Peyer’s patch HEV may be unmasked.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Mice deficient for L-selectin (3) or ß7 integrin (5) expression were generated as described previously. Mice deficient for both L-selectin and ß7 integrin were generated by breeding homozygous L-/- mice and homozygous ß7-/- mice and testing the progeny for both mutated genes by Southern blotting. The absence of cell surface expression of L-selectin and ß7 integrins was confirmed using two-color immunofluorescence flow cytometry4,5. ß7-/- mice were generated on a C57BL/6 background, and L-/- mice were at least a fifth generation backcross onto a C57BL/6 background. Control experiments were performed in age-matched C57BL/6 mice bred at the University of Virginia (breeders purchased from Hilltop Farms, Scottdale, PA). All experiments were performed on healthy mice that were at least 8 wk of age. All mice were housed in a conventional facility. All animal experiments were approved by the institutional animal care and use committee.
Monoclonal Abs
The P-selectin mAb RB40.34 (rat IgG1, 30 µg/mouse) was a gift
from Dr. Dietmar Vestweber at the University of Munster (Munster,
Germany). This Ab blocks P-selectin-dependent adhesion in vitro (8),
P-selectin-dependent leukocyte rolling in vivo (9), and
P-selectin-dependent leukocyte recruitment in vivo (8). The L-selectin
mAb MEL-14 (rat IgG2a; 100 µg/mouse) was purified from hybridoma
supernatant (American Type Culture Collection, Manassas, VA). This Ab
blocks L-selectin-dependent lymphocyte homing to peripheral lymph nodes
(10) and L-selectin-dependent leukocyte rolling in vivo (9, 11). The
mAb PS/2 specific for the 4 integrin chain (rat IgG2b;
100 µg/mouse) was also purified from hybridoma supernatant (American
Type Culture Collection). This Ab blocks
4ß7-dependent binding of lymphocytes to
recombinant MadCAM-1 in vitro (7) and lymphocyte homing to Peyer’s
patches in vivo (12).
Animal preparation and epifluorescence intravital microscopy
Mice were premedicated with an i.p. injection of a mixture of 30 mg/kg sodium pentobarbital (Nembutal, Abbott Laboratories, North Chicago, IL) and 0.1 mg/kg atropine (Elkins-Sinn, Cherry Hill, NJ) in isotonic saline. Mice were then anesthetized with an i.p. injection of 100 mg/kg ketamine hydrochloride (Ketalar, Parke-Davis, Detroit, MI) in isotonic saline. The trachea, carotid artery, and jugular vein were cannulated, and mice were thermocontrolled at 37°C using a small animal heating pad (model 50-7503, Harvard, South Natik, MA). Blood pressure was monitored continuously (model BPMT-2, Stemtech, Menomonee Falls, WI).
A Peyer’s patch was prepared for intravital microscopy as described previously (4, 5). Briefly, a 1.5-cm incision was made along the linea alba to open the peritoneal cavity and expose the cecum and small intestine. The mouse was then turned on its side, and the intestines were pushed out of the peritoneal cavity by gently pressing on the back. The intestines were superfused with thermocontrolled (37°C) bicarbonate-buffered saline as previously described (13). To immobilize the small intestine and allow for viewing of the Peyer’s patch microvasculature, a plastic coverslip (Baxter, Deerfield, IL) cut to approximately 1 cm2 was overlaid with two parallel strips of clear silicone, high vacuum grease (Dow Corning, Midland, MI). This coverslip was then laid over the preparation, with the small intestine lying parallel between the two rows of grease and the Peyer’s patch flattened against the coverslip to allow better microscopic viewing. To visualize intravascular leukocytes by epifluorescent microscopy, each mouse was given a 0.15-ml venous injection of 1 mg/ml acridine red (Chroma, Stuttgart, Germany) 30 min before intravital observation.
Microscopic observations were made using an intravital microscope (Axioskop, Carl Zeiss, Thornwood, NY) modified for stroboscopic (60/s; Strobex 236, Chadwick Helmuth, Mountain View, CA) epifluorescence illumination (filter block Zeiss 9) with a saline immersion objective (SW 40, 0.75 numerical aperture). HEV were observed and recorded (S-VHS recorder, Panasonic, Osaka, Japan) through a CCD camera system (model VE-1000CD, Dage-MTI, Michigan City, IN). Throughout the experiment, 10-µl blood samples were withdrawn at 45-min intervals from the carotid catheter, diluted 1/9 with Kimura (11 ml of 5% (w/w) toludine blue, 0.8 ml of 0.03% light green SF yellowish, 0.5 ml of saturated saponin, and 5 ml of 0.07 M phosphate buffer, pH 6.4; all reagents were obtained from Sigma, St. Louis, MO), and analyzed for leukocyte concentration (expressed as number of leukocytes per microliter of whole blood). Additional blood samples were taken, using the above procedure, before and after administration of mAb and at the termination of the experiment.
Paraffin section histology
Two Peyer’s patches per mouse (one being the observed Peyer’s patch) were excised from the small intestine and fixed in 4% (w/w) paraformaldehyde (Sigma) for at least 4 h but not >24 h. The tissues were then dehydrated in increasing concentrations of ethanol (75% (v/v), 95% (v/v), and absolute), cleared in xylene, and soaked in warm (56°C) liquid paraffin for several hours to allow saturation. The tissues were embedded by cooling until the paraffin hardened. Ten-micron serial transverse sections were cut (Leitz microtome, Wetzlar, Germany), floated out in a water bath onto poly-L-lysine (Sigma)-coated slides, and dried at room temperature. Sections were cleared in xylene, rehydrated with decreasing concentrations of ethanol (absolute, 95% (v/v), 70% (v/v), and distilled water), stained with hematoxylin and eosin (Sigma), cleared in absolute ethanol and xylene, and mounted using Permount (Fisher Scientific, Pittsburgh, PA).
In vivo labeling of P-selectin
Initial attempts at staining Peyer’s patch HEV with indirect immunofluorescence of peroxidase-conjugated secondary Abs yielded unsatisfactory results, most likely because of low expression of P-selectin (data not shown). Therefore, we developed a new staining technique employing fluorescent beads. Recombinant protein G (Sigma) was covalently coupled to fluorescent-carboxylated (2-µm diameter) YG microspheres (Polysciences, Warrington, PA) using a carbodiimide coupling kit according to the manufacturer’s directions (Polysciences). Beads were stored in the storage buffer provided and used within 1 wk. Thirty minutes before observation of the Peyer’s patch, 200 µl of protein G-coupled beads were mixed with 200 µg of either a nonbinding rat IgG (Pierce, Rockford, IL) or the P-selectin mAb RB40.34. The mixture was allowed to incubate for 30 min before the beads were washed in isotonic saline with 1% BSA three times. The beads were resuspended in isotonic saline with 1% BSA and vortexed to break up any aggregates. Animals were injected with three sequential 50-µl boluses of the rat IgG-coupled beads over 5 min followed by a 10-min resting period to allow for removal of any circulating beads. Subsequently, the RB40.34-coupled beads were injected in the same manner, and any bound beads were observed by intravital fluorescence microscopy.
Determination of P-selectin ligand activity on Peyer’s patch lymphocytes
Indirect immunofluorescence flow cytometry was used to examine binding of a recombinant human P-selectin-IgG chimera (gift from S. R. Watson, Genentech, South San Francisco, CA) to suspensions of Peyer’s patch lymphocytes. Human P-selectin can bind to mouse PSGL-1 (14). Peyer’s patches were isolated from wild-type, ß7-/-, L-/-, and ß7/L-/- mice and suspended in PBS, pH 7.4, with 2 mM CaCl2, 0.02% sodium azide, and 5% goat serum. Lymphocytes from Peyer’s patches were then released by gentle syringe aspiration. A multivalent P-selectin complex (15) was preformed by mixing recombinant P-selectin-IgG chimera and FITC-conjugated goat F(ab')2 anti-human IgG (Fc-specific) Ab (Caltag, Burlingame, CA) in a 1/2 (w/w) ratio followed by incubation at 4°C for 30 min. Aliquots of the P-selectin-IgG FITC complex (2 µg P-selectin-IgG/4 µg goat anti-human IgG FITC) were added to 1 x 106 cells and incubated for 20 min on ice. Lymphocytes in the Peyer’s patch preparation were identified by coincubation with 1 µg each of phycoerythrin-conjugated mAb against the CD3 complex on T cells (clone 17A2, rat anti-mouse IgG2b) and CD45R/B220 on B cells (clone RA3-6B2; rat anti-mouse IgG2a; PharMingen, San Diego, CA). Selectin-specific adhesion was confirmed by inhibition of P-selectin complex binding to Peyer’s patch lymphocytes by treating cell suspensions with 5 mM EDTA. Cell suspensions were analyzed on a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ) using CellQuest software (version 1.2).
Data analysis
Microvessel diameters were measured using a digital image
processing system (16). Centerline blood flow velocity was determined
after i.v. injection of 2-µm diameter fluorescent YG microspheres
(Polysciences, Warrington, PA) by measuring frame-to-frame displacement
(three microspheres per venule). Average blood flow velocity was
determined by dividing the measured centerline velocity by a factor of
2. The rolling leukocyte flux fraction was determined as the number of
rolling leukocytes expressed as a percentage of all leukocytes passing
through the venule per unit time. In Peyer’s patch HEV, the number of
rolling leukocytes was consistently higher (by 
60%) than the
product of the flow rate and the systemic leukocyte concentration, a
parameter used in previous studies (9, 11) as the denominator for the
rolling flux fraction. An increased concentration of leukocytes in
microvessels can occur as a consequence of multiple flow partition at
bifurcations in the microvascular network (17, 18), leading to
leukocyte accumulation in the most distal branches of the arteriolar
tree. Consequently, venules fed by these branches also carry blood
containing a higher leukocyte concentration. Therefore, the flux
fraction in wild-type mice was set at 100%, and the flux fractions in
the other genotypes were adjusted accordingly. The leukocyte rolling
flux fraction was also corrected for variations in centerline velocity
and venular diameter as described previously (9). Individual leukocyte
rolling velocities were measured from video recordings by analyzing 5
to 15 leukocytes/venule and measuring the time necessary to travel a
fixed distance (50–80 µm) using a digital image-processing system
(16).
Statistical analysis
All statistical comparisons were conducted using an analysis of variance with a post-hoc Student-Newman-Keuls multiple comparison procedure. SPSS software (SPSS, Chicago, IL) was used for all statistical analyses. Statistical significance was set at p < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
All mice used in this work appeared healthy and of normal size and
weight for their ages. During the intubation surgery, no enlarged
cervical lymph nodes were observed in any mice. Similar to what has
been previously reported, the systemic leukocyte counts in
L-/- mice (3) were slightly higher than those
in wild-type mice (Table I
). Although
previous work has not identified an increase in circulating counts in
ß7-/- mice (5), we found that
circulating counts in these mice were also higher than those in
wild-type mice (Table I). Similarly, the circulating leukocyte counts
in ß7/L-/- mice were elevated
(Table I). The percentages of granulocytes and mononuclear leukocytes
were similar in each genotype (Table I).
|
We first examined the morphology of Peyer’s patches from
wild-type, L-/-, and
ß7/L-/- mice (Fig. 1). The number of Peyer’s patches
visible along the small intestine was reduced from 8 to 10 in
wild-type mice to 1 to 2 in both the
ß7-/- and
ß7/L-/- mice (data not shown).
The Peyer’s patches that could be located using a dissecting
microscope on the wall of the small intestine in
ß7/L-/- mice were small and
hypocellular (Fig. 1C) compared with wild-type Peyer’s
patches (Fig. 1A). The number of Peyer’s patches in
L-/- mice was not different from that in
wild-type mice, and they were similar in size and cellularity to those
in their wild-type counterparts (Fig. 1B). Concomitant with
reduced Peyer’s patch size in
ß7-/- mice, the number of HEV
observed through intravital observation was reduced compared with that
in wild-type mice (data not shown) and the average diameter of those
seen was slightly smaller (Table II).
|
|
We used blocking mAb to determine which adhesion molecules
were mediating the leukocyte rolling in each genotype (Fig. 2). As shown previously (4, 5), leukocyte
rolling in Peyer’s patches from wild-type mice was predominantly
mediated by L-selectin, since the L-selectin function-blocking mAb
MEL-14 blocked most of the observed rolling (Fig. 2A).
However, injection of the mAb RB40.34 significantly reduced the rolling
flux fraction by about 29% in wild-type mice (Fig. 2A).
Interestingly, the percentage of granulocytes in the wild-type mice
investigated here was about 29 to 38% of the total circulating
leukocyte count (Table I
), suggesting that many of the cells rolling
using P-selectin may be granulocytes. Neutrophils are known to express
a constitutively functional ligand for P-selectin called PSGL-1 (19).
However, effector T cells also express functional PSGL-1 (20, 21) and
may be rolling via P-selectin in Peyer’s patches.
|
B). Residual rolling
in these mice was largely mediated by P-selectin (the mAb RB40.34 to
P-selectin blocked almost all leukocyte rolling), but the rolling flux
was unaffected by the mAb PS/2 to the 4 integrin chain
(Fig. 2B). Similar to wild-type mice and consistent with
previous results (5), leukocyte rolling in
ß7-/- mice was mediated by
L-selectin (Fig. 2C). P-selectin blockade with the mAb
RB40.34 in ß7-/- mice led to a
slight (10%), but not significant, reduction in the rolling flux
fraction (Fig. 2C).
ß7/L-/- mice showed a low rolling
flux fraction similar to that found in L-/-
mice. This residual leukocyte rolling was totally P-selectin dependent
(Fig. 2D).
Leukocyte rolling in vivo is known to be affected by geometric
and hemodynamic conditions such as venular surface-to-volume ratio and
average blood flow velocity (9, 22). Therefore, we measured several
parameters that could affect leukocyte rolling in these experiments
(Table II). Fluorescent beads were used to measure the centerline blood
flow velocity in each observed venule. The data from all genotypes were
stratified for venules with similar average wall shear rates.
Leukocyte rolling velocities
Adhesion molecules involved in leukocyte rolling may change the
total number of rolling leukocytes, their rolling velocity, or both
parameters (11). Therefore, we also analyzed the velocities of rolling
leukocytes in each genotype (Fig. 3). In
HEV of wild-type mice, leukocyte rolling velocities ranged from <10 to
>100 µm/s (Fig. 3A) with a median velocity of 43 µm/s
(Fig. 3E and Table III). In
L-/- mice, the population of faster rolling
leukocytes was missing (Fig. 3B), which significantly
reduced (p < 0.05 vs wild-type mice) the
median rolling velocity to 30 µm/s (Fig. 3E and Table III). Conversely, slow rolling leukocytes were missing in
ß7-/- mice (Fig. 3C),
which significantly increased (p < 0.05 vs
wild-type and L-/- mice) the median velocity to
85 µm/s (Fig. 3E and Table III). Taken together, these
findings indicate that the leukocyte rolling velocity histogram in
wild-type mice is composed of two distinct populations: slower
ß7 integrin-dependent rolling and more rapid
L-selectin-dependent rolling. It should be noted, however, that the
rolling flux fraction in L-/- mice is much
lower (Fig. 2). This suggests that L-selectin may be important in
capturing leukocytes from the flowing blood in Peyer’s patches in
addition to mediating rolling. It therefore appears that efficient
leukocyte attachment requires L-selectin even in the presence of
ß7 integrin. In
ß7/L-/- mice (Fig. 3D), only a residual population of rolling leukocytes was
detected (Fig. 2), which rolled at an intermediate velocity with a
median of 56 µm/s (Fig. 3E and Table III), not
significantly different from the velocity distribution in wild-type
mice. Rolling in ß7/L-/- mice was
mediated by P-selectin (Fig. 2D). The observed velocity
distribution is similar to that seen in venules of the mouse cremaster
muscle where rolling is dominated by P-selectin (23).
|
|
). Since
blocking P-selectin in wild-type mice also causes an 30% reduction
in the rolling flux fraction (Fig. 2A), the velocity
increase may be due to the lack of a population of leukocytes that
rolls more slowly using P-selectin. In
ß7-/- mice, addition of RB40.34
did not significantly alter the rolling flux fraction (Fig. 2B) and also did not affect the median rolling velocity
(Table III). As shown above, P-selectin mediated almost all the rolling
in L-/- mice (Fig. 2C).
Interestingly, the median velocity of the few remaining rolling
leukocytes in these mice was very low (Table III). P-selectin expression in Peyer’s patch HEV
mAb blocking data (Fig. 2) and rolling velocity data (Fig. 3) showed that P-selectin contributes to leukocyte rolling in Peyer’s
patches. To directly visualize P-selectin expression, we attempted to
use standard immunoperoxidase staining using biotinylated secondary Ab
and streptavidin-peroxidase conjugate as described previously (24).
This method was not sensitive enough to detect P-selectin in Peyer’s
patch HEV. To increase the sensitivity, we developed a bead-based
assay. Fluorescent beads were coated with the P-selectin mAb RB40.34
and injected i.v. (Fig. 4). Injection of
control beads coated with an irrelevant rat IgG Ab showed low, but
detectable, background binding to the Peyer’s patch venular
endothelium of wild-type mice (Fig. 4A), whereas the
RB40.34-coated beads bound in much greater quantities (Fig. 4B). Binding was specific for P-selectin on the endothelium
because i.v. pretreatment of the mouse with RB40.34 reduced the binding
of the RB40.34-coated beads to levels comparable to that of the control
beads (Fig. 4C). As a positive control, the binding of
RB40.34-coated beads to the venular endothelium of the acutely
exteriorized mouse cremaster muscle was investigated. Under these
conditions, cremaster muscle venules, but not arterioles, express
P-selectin (24). RB40.34-coated beads bound avidly to the venular (Fig. 4D), but not arteriolar (data not shown), endothelium in the
mouse cremaster muscle. HEV in Peyer’s patches of
L-/- and
ß7/L-/- mice also bound
RB40.34-coated beads (data not shown), consistent with the observed
P-selectin-dependent rolling in these vessels (Fig. 2).
|
Peyer’s patch lymphocytes express P-selectin binding activity
Because we observed that P-selectin was involved in
leukocyte rolling in all genotypes (Figs. 2 and 3) and was expressed on
the endothelium of Peyer’s patch HEV (Fig. 4), we determined whether
purified Peyer’s patch lymphocytes from each genotype exhibited
P-selectin ligand activity. A multivalent P-selectin complex was formed
by cross-linking a P-selectin-IgG chimera with a FITC-conjugated
secondary Ab (15) and was used to investigate P-selectin binding
activity of purified Peyer’s patch lymphocytes from wild-type,
L-/-,
ß7-/-, and
ß7/L-/- mice by flow cytometry
(Fig. 5). Approximately 26% of wild-type
Peyer’s patch lymphocytes bound multivalent P-selectin (Fig. 5A). A similar number (32%) of Peyer’s patch
lymphocytes from L-/- mice also stained
positively (Fig. 5C). In contrast, 45% of lymphocytes from
ß7-/- mice (Fig. 5B)
and 57% of lymphocytes from
ß7/L-/-mice (Fig. 5D)
stained positively for P-selectin ligand activity. Binding was selectin
specific, as shown by complete inhibition in the presence of 5 mM EDTA
(dotted lines in Fig. 5). As a positive control, we used HL-60 cells, a
myeloid precursor cell line known to bind to P-selectin (27, 28). All
HL-60 cells stained positively with the P-selectin complex (data not
shown).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Although we observed P-selectin-dependent leukocyte rolling in
all mouse genotypes studied in this work (i.e., wild-type,
L-/-,
ß7-/-, and
ß7/L-/-), P-selectin has never
before been implicated in participating in the recruitment of
lymphocytes to Peyer’s patches. In fact, no evidence exists that
suggests that P-selectin is even expressed on Peyer’s patch HEV.
P-selectin has, however, been shown to be expressed on HEV in inflamed
as well as noninflamed palantine and adenoid tonsils (29, 30).
Additionally, P-selectin expression has been detected in the small
intestine using 125I-labeled RB40.34 (31), but was not
localized to any specific region (e.g., Peyer’s patches or lamina
propria). By using fluorescent beads coupled to the P-selectin mAb
RB40.34, we show that P-selectin is indeed expressed on the vascular
endothelium of Peyer’s patch HEV under the conditions studied. Our
data represent the first evidence for P-selectin expression in Peyer’s
patch HEV. More importantly, we also show that P-selectin is functional
in these vessels and can mediate rolling after minimal trauma
(exteriorization). P-selectin expression is known to occur in response
to mediators such as histamine (26, 32) or during inflammation in
response to cytokines such as TNF- and IL-1 (33, 34). Since
RB40.34-coated beads did not bind to the vascular endothelium in
Peyer’s patches before surgery was initiated, we conclude that this
expression is due to up-regulation of surface expression of this
molecule after surgical manipulation of the small intestine. Therefore,
P-selectin may not be involved in the recruitment of lymphocytes to
Peyer’s patches under in situ conditions, but may play a role in
trafficking of effector T lymphocytes during trauma or inflammation.
Many types of leukocytes express P-selectin ligand activity.
Neutrophils (19), monocytes (35), and eosinophils (36) express a
constitutively functional P-selectin ligand (PSGL-1) that mediates at
least 90% of the P-selectin binding activity during inflammation in
vivo (37). Although all lymphocytes express PSGL-1 (38), only certain
subsets, including both effector ß and 
T-cells (20) and
memory T cells (39), can bind to P-selectin. PSGL-1 functionality is
regulated by an inducible fucosyl transferase called FucT-VII (40).
Thus, for a T cell to bind to P-selectin, PSGL-1 must have the proper
post-translational modifications (41). P-selectin binding to PSGL-1
appears to mediate Th1 cell recruitment into inflamed skin in vivo
(42). Of the leukocyte subsets present in the circulation under normal
conditions, neutrophils, eosinophils, and effector T cells all can bind
to and roll on P-selectin. We therefore suspect that neutrophils,
eosinophils, monocytes, and effector T cells are rolling through
Peyer’s patch HEV using P-selectin expressed on the HEV endothelium
after surgical manipulation.
In vivo differentiation of leukocyte subsets in Peyer’s patch HEV is not achievable with current intravital microscopic tools, so we are unable to determine which types of leukocytes are rolling via P-selectin in this model. Ex vivo labeling of isolated lymphocytes would allow differentiation of lymphocytes from granulocytes, as shown previously (43). However, excessive handling and manipulation of isolated cells may lead to partial activation and phenotypic changes. We were interested in observing leukocyte-endothelial interactions under relatively unperturbed conditions, so isolated, labeled lymphocytes were not used in this study. Nevertheless, we found that a significant number of isolated Peyer’s patch lymphocytes from all genotypes express P-selectin ligand activity. Even though P-selectin expression in Peyer’s patch HEV is below the detection limit of our current methods under in situ conditions, significant numbers of T and B lymphocytes in the Peyer’s patch can bind to this molecule (26–57%). Naive lymphocytes may enter a Peyer’s patch through the use of L-selectin and ß7 integrin and may acquire P-selectin binding activity after differentiation into Ag-specific effector lymphocytes. These T lymphocytes would then be able to traffic to mucosal inflammatory sites using P-selectin, similar to what has been observed for Th1 cell recruitment to inflamed skin (42). The increased fraction of P-selectin-binding lymphocytes found in Peyer’s patch preparations from ß7-/- mice and ß7/L-/- mice may be due to an enrichment in the number of effector T lymphocytes (which can bind to P-selectin) in Peyer’s patches of mice lacking ß7 integrin, since fewer naive lymphocytes can enter this tissue when this molecule is absent. A similar enrichment in effector T lymphocytes has been observed in peripheral lymph nodes of L-/- mice, since few naive lymphocytes can traffic through this tissue in the absence of L-selectin (44). This interpretation is consistent with the recent observation of a shift in lymphocyte subsets in ß7/L-/- mice4.
Our data support a role for ß7 integrin in
mediating leukocyte rolling. This evidence comes from the shift in the
rolling velocity distribution observed in the absence of
ß7 integrin. An increase in rolling velocity when
4ß7 integrin has been blocked (4) or when
ß7 integrin is knocked out (5) has been reported. Our
present analysis confirms and expands these data to show that at least
three molecules are involved in modulating leukocyte rolling velocity
in Peyer’s patches: L-selectin, ß7 integrin, and
P-selectin. The rolling velocity distribution in wild-type mice appears
to be a superposition of three distributions: a population of
leukocytes that rolls rapidly (80 µm/s) mainly via L-selectin
(seen in the ß7-/- mice), a
population of leukocytes that rolls more slowly (30 µm/s) mainly
using ß7 integrin (seen in the
L-/- mice), and a smaller population of
leukocytes that rolls with an intermediate range of velocities via
P-selectin (seen in the ß7/L-/-
mice). These three distributions superimposed can replicate the
wild-type distribution measured here. Pure ß7
integrin-dependent leukocyte rolling can be seen in
L-/- mice treated with a P-selectin mAb. In
these mice, the residual rolling flux is only 5% of that in wild-type
mice, suggesting that L-selectin is important for efficient leukocyte
capture in Peyer’s patch HEV. The median rolling velocity in
L-/- mice treated with a blocking P-selectin
mAb is on the order of 20 µm/s. A small rolling flux fraction in
conjugation with a low rolling velocity suggests that ß7
integrin mainly functions to strengthen rolling interactions (i.e.,
reduce the rolling velocity), but is unable to efficiently mediate
leukocyte capture and initiate rolling.
In conclusion, we present evidence that P-selectin can be induced on Peyer’s patch venular endothelium but is probably not involved in the recruitment of naive lymphocytes to Peyer’s patches. Our data showing P-selectin ligand activity in lymphocytes isolated from Peyer’s patches suggests that effector lymphocytes can bind to P-selectin and may use this molecule for recruitment to mucosal sites of inflammation. Under physiologic conditions, L-selectin and ß7 integrins appear to account for all lymphocyte rolling and adhesion in Peyer’s patch HEV.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Klaus Ley, Department of Biomedical Engineering, University of Virginia School of Medicine, Box 377, Health Sciences Center, Charlottesville, VA 22908. E-mail address:
3 Abbreviations used in this work: HEV, high endothelial venule; L-/-, L-selectin deficient; ß7-/-, ß7 integrin deficient; ß7/L-/-, ß7 integrin/L-selectin double knockout.
4 D. A. Steeber, M. L. K. Tang, X.-Q. Zhang, N. Wagner, T. F. Tedder. Lymphocyte migration across high endothelial venules of mouse Peyer’s patches requires L-selectin or ß7 integrin expression. Submitted for publication.
5 N. Wagner, J. Lohler, T. F. Tedder, K. Rajewsky, W. Müller, D. A. Steeber. L-selectin and ß7 integrin synergistically mediate lymphocyte migration to mesenteric lymph nodes. Submitted for publication.
Received for publication February 2, 1998. Accepted for publication April 23, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
4 ß7 and LFA-1 in lymphocyte homing to Peyer’s patch-HEV in situ: the multistep model confirmed and refined. Immunity 3:99.[Medline]
4 Integrins mediate lymphocyte attachment and rolling under physiologic flow. Cell 80:413.[Medline]
4-integrins in lymphocyte homing to mucosal tissues in vivo. J. Immunol. 152:3282.[Abstract]
/ß and / T lymphocyte subsets in shear flow with E-selectin and P-selectin. J. Exp. Med. 183:1193. in vivo. Cell Adhes. Commun. 2:7.[Medline]
-activated vascular endothelium under flow in vitro. J. Immunol. 157:326.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  A. Martin-Fontecha, D. Baumjohann, G. Guarda, A. Reboldi, M. Hons, A. Lanzavecchia, and F. Sallusto CD40L+ CD4+ memory T cells migrate in a CD62P-dependent fashion into reactive lymph nodes and license dendritic cells for T cell priming J. Exp. Med., October 27, 2008; 205(11): 2561 - 2574. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. E. Eriksson No detectable endothelial- or leukocyte-derived L-selectin ligand activity on the endothelium in inflamed cremaster muscle venules J. Leukoc. Biol., July 1, 2008; 84(1): 93 - 103. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. V. Ostanin, K. L. Furr, K. P. Pavlick, L. Gray, C. G. Kevil, D. Shukla, D. D'Souza, J. M. Hoffman, and M. B. Grisham T cell-associated CD18 but not CD62L, ICAM-1, or PSGL-1 is required for the induction of chronic colitis Am J Physiol Gastrointest Liver Physiol, June 1, 2007; 292(6): G1706 - G1714. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Rydstrom and M. J. Wick Monocyte Recruitment, Activation, and Function in the Gut-Associated Lymphoid Tissue during Oral Salmonella Infection J. Immunol., May 1, 2007; 178(9): 5789 - 5801. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Harakawa, A. Shigeta, M. Wato, G. Merrill-Skoloff, B. C. Furie, B. Furie, T. Okazaki, N. Domae, M. Miyasaka, and T. Hirata P-selectin glycoprotein ligand-1 mediates L-selectin-independent leukocyte rolling in high endothelial venules of peripheral lymph nodes Int. Immunol., March 1, 2007; 19(3): 321 - 329. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Rivera-Nieves, T. L. Burcin, T. S. Olson, M. A. Morris, M. McDuffie, F. Cominelli, and K. Ley Critical role of endothelial P-selectin glycoprotein ligand 1 in chronic murine ileitis J. Exp. Med., April 17, 2006; 203(4): 907 - 917. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Zhou, Y. Luo, C. D. Kaplan, J. A. Kruger, S.-H. Lee, R. Xiang, and R. A. Reisfeld A DNA-based cancer vaccine enhances lymphocyte cross talk by engaging the NKG2D receptor Blood, April 15, 2006; 107(8): 3251 - 3257. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. R. Mangan, D. O'Quinn, L. Harrington, C. S. Bonder, P. Kubes, D. F. Kucik, D. C. Bullard, and C. T. Weaver Both Th1 and Th2 Cells Require P-Selectin Glycoprotein Ligand-1 for Optimal Rolling on Inflamed Endothelium Am. J. Pathol., December 1, 2005; 167(6): 1661 - 1675. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Rivera-Nieves, T. Olson, G. Bamias, A. Bruce, M. Solga, R. F. Knight, S. Hoang, F. Cominelli, and K. Ley L-Selectin, {alpha}4{beta}1, and {alpha}4{beta}7 Integrins Participate in CD4+ T Cell Recruitment to Chronically Inflamed Small Intestine J. Immunol., February 15, 2005; 174(4): 2343 - 2352. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Van Assche and P. Rutgeerts Physiological Basis for Novel Drug Therapies Used to Treat the Inflammatory Bowel Diseases I. Immunology and therapeutic potential of antiadhesion molecule therapy in inflammatory bowel disease Am J Physiol Gastrointest Liver Physiol, February 1, 2005; 288(2): G169 - G174. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Bistrup, D. Tsay, P. Shenoy, M. S. Singer, N. Bangia, S. A. Luther, J. G. Cyster, N. H. Ruddle, and S. D. Rosen Detection of a Sulfotransferase (HEC-GlcNAc6ST) in High Endothelial Venules of Lymph Nodes and in High Endothelial Venule-Like Vessels within Ectopic Lymphoid Aggregates: Relationship to the MECA-79 Epitope Am. J. Pathol., May 1, 2004; 164(5): 1635 - 1644. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Hiraoka, H. Kawashima, B. Petryniak, J. Nakayama, J. Mitoma, J. D. Marth, J. B. Lowe, and M. Fukuda Core 2 Branching {beta}1,6-N-Acetylglucosaminyltransferase and High Endothelial Venule-restricted Sulfotransferase Collaboratively Control Lymphocyte Homing J. Biol. Chem., January 23, 2004; 279(4): 3058 - 3067. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Ley Sulfated Sugars for Rolling Lymphocytes J. Exp. Med., November 3, 2003; 198(9): 1285 - 1288. [Full Text] [PDF]
|
|
|
|
|
|
A. van Zante, J.-M. Gauguet, A. Bistrup, D. Tsay, U. H. von Andrian, and S. D. Rosen Lymphocyte-HEV Interactions in Lymph Nodes of a Sulfotransferase-deficient Mouse J. Exp. Med., November 3, 2003; 198(9): 1289 - 1300. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Haddad, C. J. Cooper, Z. Zhang, J. B. Brown, Y. Zhu, A. Issekutz, I. Fuss, H.-o. Lee, G. S. Kansas, and T. A. Barrett P-Selectin and P-Selectin Glycoprotein Ligand 1 Are Major Determinants for Th1 Cell Recruitment to Nonlymphoid Effector Sites in the Intestinal Lamina Propria J. Exp. Med., August 4, 2003; 198(3): 369 - 377. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. H. Grayson, R. S. Hotchkiss, I. E. Karl, M. J. Holtzman, and D. D. Chaplin Intravital microscopy comparing T lymphocyte trafficking to the spleen and the mesenteric lymph node Am J Physiol Heart Circ Physiol, June 1, 2003; 284(6): H2213 - H2226. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. M. Moore, W. B. Shirah, P. L. Khimenko, P. Paisley, R. N. Lausch, and A. E. Taylor Involvement of CD40-CD40L signaling in postischemic lung injury Am J Physiol Lung Cell Mol Physiol, December 1, 2002; 283(6): L1255 - L1262. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Redlin, J. Werner, H. Habazettl, W. Griethe, H. Kuppe, and A. R. Pries Cariporide (HOE 642) Attenuates Leukocyte Activation in Ischemia and Reperfusion Anesth. Analg., December 1, 2001; 93(6): 1472 - 1479. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Shigematsu, R. D. Specian, R. E. Wolf, M. B. Grisham, and D. N. Granger MAdCAM mediates lymphocyte-endothelial cell adhesion in a murine model of chronic colitis Am J Physiol Gastrointest Liver Physiol, November 1, 2001; 281(5): G1309 - G1315. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Sperandio, S. B. Forlow, J. Thatte, L. G. Ellies, J. D. Marth, and K. Ley Differential Requirements for Core2 Glucosaminyltransferase for Endothelial L-Selectin Ligand Function In Vivo J. Immunol., August 15, 2001; 167(4): 2268 - 2274. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Singbartl, J. Thatte, M. L. Smith, K. Wethmar, K. Day, and K. Ley A CD2-Green Fluorescence Protein-Transgenic Mouse Reveals Very Late Antigen-4-Dependent CD8+ Lymphocyte Rolling in Inflamed Venules J. Immunol., June 15, 2001; 166(12): 7520 - 7526. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. H. von Andrian and C. R. Mackay T-Cell Function and Migration -- Two Sides of the Same Coin N. Engl. J. Med., October 5, 2000; 343(14): 1020 - 1034. [Full Text] [PDF]
|
|
|
|
|
|
R.A. Warnock, J.J. Campbell, M.E. Dorf, A. Matsuzawa, L.M. McEvoy, and E.C. Butcher The Role of Chemokines in the Microenvironmental Control of T versus B Cell Arrest in Peyer's Patch High Endothelial Venules J. Exp. Med., January 3, 2000; 191(1): 77 - 88. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Jung and K. Ley Mice Lacking Two or All Three Selectins Demonstrate Overlapping and Distinct Functions for Each Selectin J. Immunol., June 1, 1999; 162(11): 6755 - 6762. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. Steeber, M. L. K. Tang, X.-Q. Zhang, W. Muller, N. Wagner, and T. F. Tedder Efficient Lymphocyte Migration Across High Endothelial Venules of Mouse Peyer's Patches Requires Overlapping Expression of L-Selectin and {beta}7 Integrin J. Immunol., December 15, 1998; 161(12): 6638 - 6647. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
