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*
Department of Immunology, Duke University Medical Center, Durham, NC 27710; and
Institute for Genetics, University of Cologne, Cologne, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß7 are predominantly
hypothesized to direct the selective migration of lymphocytes to
peripheral lymph nodes and the gut-associated lymphoid tissues,
respectively. To further characterize interactions between L-selectin
and ß7 integrins during lymphocyte recirculation, mice
deficient in both receptors (L-selectin/ß7
integrin-/-) were generated. The simultaneous loss of
L-selectin and ß7 integrin expression prevented the
majority of lymphocytes (>95% inhibition) from attaching to high
endothelial venules (HEV) of Peyer’s patches and other lymphoid
tissues during in vitro binding assays. Moreover, the inability to bind
HEV eliminated the vast majority of L-selectin/ß7
integrin-/- lymphocyte migration into Peyer’s patches
during short-term and long-term in vivo migration assays (>99%
inhibition, p < 0.01). The lack of lymphocyte
migration into Peyer’s patches correlated directly with the
dramatically reduced size and cellularity (99% reduced) of this tissue
in L-selectin/ß7 integrin-/- mice. High
numbers of injected L-selectin/ß7
integrin-/- lymphocytes remaining in the blood of
wild-type mice correlated with markedly increased numbers of
circulating lymphocytes in L-selectin/ß7
integrin-/- mice. Loss of either L-selectin or the
ß7 integrins alone resulted in significant but incomplete
inhibition of Peyer’s patch migration. Collectively, the phenotype of
L-selectin/ß7 integrin-/- mice demonstrates
that these two receptors primarily interact along the same adhesion
pathway that is required for the vast majority of lymphocyte migration
into Peyer’s patches. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß7 (5, 7, 8, 9, 10, 11). Lymphocyte attachment to
Peyer’s patch HEV during in vitro assays is specifically and
completely blocked by Abs against 4 or ß7
integrin chains (12). However, lymphocyte migration into mouse
Peyer’s patches is partially inhibited by Fab of the MEL-14 mAb
(13), and mouse Peyer’s patch HEV express low but functional
levels of L-selectin carbohydrate ligands (14). The generation of
L-selectin-deficient (L-selectin-/-) mice confirmed
that L-selectin significantly influences lymphocyte migration into
Peyer’s patches and MLNs (6). This has been further supported by
intravital microscopic analysis of isolated Peyer’s patches
demonstrating that both L-selectin, and to a lesser degree,
4ß7 integrins participate in the initial
interaction between lymphocytes and HEV as well as in subsequent
rolling (15, 16). Consistent with this,
4ß7 and 4ß1
integrins can each mediate rolling and firm adhesion of lymphocytes
during in vitro assays (17, 18, 19).
L-selectin-/- mice have a significant reduction
(70–90%) in the number of resident lymphocytes within PLNs, but are
viable, have no developmental defects, and do not succumb to multifocal
infections (6, 20, 21). Lymphocytes from L-selectin-/-
mice are completely deficient in their ability to attach to PLN HEV
during in vitro binding assays and are unable to migrate across HEV of
resting or responsive PLNs in normal mice during short-term (1 h) or
long-term (48 h) in vivo migration experiments (6, 20, 21, 22). Migration
of L-selectin-/- lymphocytes into Peyer’s patches and
MLNs is also dramatically reduced in short-term migration assays, but
this deficit largely recovers during long-term assays (6, 22). The
ability of L-selectin-/- lymphocytes to reconstitute
Peyer’s patches to near wild-type levels, and MLNs to a lesser extent,
in long-term migration assays suggests that either
4ß7 mediates a low level of
L-selectin-independent cell capture/entry from the blood or
alternatively that adhesion molecules other than
4ß7 substitute for L-selectin in these
tissues. L-selectin-/- mice also demonstrate decreased
leukocyte rolling at sites of inflammation, decreased leukocyte
recruitment into an inflamed peritoneum, decreased delayed-type
hypersensitivity responses, delayed rejection of allogeneic skin
transplants, and resistance to LPS-induced septic shock, but have
augmented humoral and cellular immune responses (6, 23, 24, 25, 26, 27).
Lymphocyte migration into lymphoid tissues is also influenced by the
ß7 integrins: the ß7 chain associates with
two -chains to generate 4ß7 and
Eß7 (7, 12, 28, 29).
Eß7 is primarily expressed on
intraepithelial lymphocytes and lymphocytes migrating to the mucosa
(30, 31, 32, 33). By contrast, the majority of mouse lymphocytes express
4ß7, which interacts with the mucosal
addressin cell adhesion molecule-1 (MAdCAM-1) to mediate Peyer’s patch
and MLN migration (27, 34, 35, 36). MAdCAM-1 is expressed on HEV located
within Peyer’s patches and MLN, on vessels within the lamina propria,
and on follicular dendritic cells within Peyer’s patches and
chronically inflamed PLN and spleen (37, 38, 39). ß7
integrin-deficient (ß7 integrin-/-)
mice are healthy, viable, and develop normally (16). These mice have
dramatically hypocellular Peyer’s patches that contain only
rudimentary follicles, and have lamina propria and intraepithelial
lymphocyte numbers that are significantly reduced. Migration of
ß7 integrin-/- lymphocytes into Peyer’s
patches is severely reduced during short-term in vivo assays, while
lymphocyte migration into MLNs is diminished and migration into PLNs is
normal (16). Studies using function-blocking mAbs have demonstrated
that L-selectin and 4ß7 initiate
lymphocyte interactions with Peyer’s patch HEV, and L-selectin and
4ß7 both participate in rolling during
acute migration assays in situ (15). Furthermore,
4ß7 dramatically reduces lymphocyte
rolling velocities, an apparent requirement for LFA-1 engagement. In
additional studies, L-selectin was necessary for 80–90% of lymphocyte
rolling in Peyer’s patch HEVs of wild-type and ß7
integrin-/- mice, while ß7 integrins were
required for adhesion and emigration during acute migration assays in
situ (16, 40). Furthermore, leukocyte rolling on Peyer’s patch HEV
occurs at different characteristic velocities with slow rolling
predominating in L-selectin-/- mice and fast rolling
characterizing ß7 integrin-/- mice
(40). Therefore, both L-selectin and the ß7 integrins
serve major roles in lymphocyte migration to gut-associated lymphoid
tissues.
A dual migratory specificity for Peyer’s patches and MLNs involving
L-selectin and 4ß7 integrins establishes a
framework for understanding lymphocyte migration into these tissues.
Because most circulating lymphocytes express both L-selectin and
ß7 integrins (27, 41), 4ß7
could mediate selectin-independent cell capture from the blood, thus
explaining the ability of L-selectin-/- lymphocytes to
enter Peyer’s patches and MLNs in long-term migration assays. However,
it is relevant that significantly more L-selectin-/-
lymphocytes enter the spleen than wild-type lymphocytes during
migration assays and that L-selectin-/- mice have
30–50% larger spleens than their wild-type littermates. By contrast,
Peyer’s patches and MLNs are not increased in size in
L-selectin-/- mice (6, 22). Thus, it remains
unresolved as to whether these two molecules account for all lymphocyte
migration into Peyer’s patches and MLNs or whether these two
receptors have additive functions. Therefore, mice deficient in
both L-selectin and ß7 integrins
(L-selectin/ß7 integrin-/-) were generated
to further characterize interactions between these adhesion molecules
during lymphocyte recirculation. Lymphocyte migration into Peyer’s
patches was examined in these studies because the lack of afferent
lymphatics precludes lymphocyte entry though other routes (42), which
thereby provides an excellent tissue for examining lymphocyte/HEV
interactions. The phenotype of L-selectin/ß7
integrin-/- mice demonstrated a requirement for both
L-selectin and ß7 integrin expression for optimal
lymphocyte migration into Peyer’s patches with lymphocyte migration
into this tissue being eliminated in L-selectin/ß7
integrin-/- mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L-selectin-/- mice were generated as described (6) and backcrossed onto the C57BL/6 background for 7–10 generations. ß7 integrin-/- mice were generated as described (16). Mice lacking both L-selectin and ß7 integrins were generated by crossing F1 offspring from crosses of homozygous L-selectin-/- mice with homozygous ß7 integrin-/- mice as described (43). Cell surface L-selectin or ß7 integrin expression by lymphocytes was assessed by two-color fluorescence cytometry. All mice used were 2–3 mo of age and were housed in a specific pathogen-free barrier facility. Control mice were age-matched wild-type mice generated from heterozygous breedings of L-selectin+/-/ß7 integrin+/- mice or were C57BL/6 mice purchased from The Jackson Laboratory (Bar Harbor, ME). Equivalent results were obtained for both groups of control mice, and both groups are referred to subsequently as wild-type mice. All studies and procedures were approved by the Animal Care and Use Committee of Duke University.
Lymphocyte isolation, mAbs, and flow cytometry
Blood was aspirated from the retroorbital venous plexus of anesthetized mice. Single-cell suspensions from spleen, PLN (bilateral axillary and inguinal nodes were pooled), MLN (superior mesenteric cords were pooled), and Peyer’s patches were prepared as described (22) and filtered through nylon gauze to remove debris before washing twice in PBS. Erythrocytes in splenocyte suspensions were lysed with Tris-buffered 0.1 M ammonium chloride solution before labeling. Total cell numbers were enumerated using a hemocytometer. For counting blood leukocytes, erythrocytes were lysed with 2% acetic acid.
Abs used in these studies included unconjugated, FITC-, or
biotin-conjugated mAbs reactive with L-selectin (LAM 1–110, 44 ,
ß7 integrin (M293; PharMingen, San Diego, CA), and
4ß7 integrin (DATK-32; American Type
Culture Collection, Manassas, VA). Phycoerythrin (PE)-conjugated
streptavidin was used to reveal biotinylated Ab staining and
PE-conjugated goat anti-rat IgG Abs (both from Southern
Biotechnology, Birmingham, AL) were used to detect
4ß7 integrin labeling. Isotype-matched rat
IgG Abs (PharMingen) were used as controls. Following staining, blood
erythrocytes were lysed with Coulter whole-blood Immuno-Lyse kit
according to the manufacturer’s instructions (Coulter, Miami, FL). Ten
to 30,000 cells with the forward and side light scatter properties of
lymphocytes were analyzed on a FACScan flow cytometer (Becton
Dickinson, San Jose, CA) with fluorescence intensity shown on a
4-decade log scale. Fluorescence contours are shown as 50% log density
plots.
Stamper-Woodruff frozen section assay
Lymphocyte binding to PLN, MLN, and Peyer’s patch HEV was performed as previously described (22, 45). Briefly, splenocytes were isolated from wild-type, L-selectin-/-, ß7 integrin-/-, and L-selectin/ß7 integrin-/- mice. Following lysis of erythrocytes, cells were washed three times in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 5% FCS and 10 mM HEPES, adjusted to 25 x 106 cells/ml, and kept on ice until used. Then, 200 µl of the above cell suspension was overlayered onto 12-µm thick frozen sections of PLN, MLN, and Peyer’s patches from wild-type mice. The slides were rotated at 64 rpm for 30 min at 4°C. Cells were then gently dumped off the slides and the slides were placed vertically into 2.4% buffered glutaraldehyde overnight at 4°C. The slides were washed in PBS, counterstained in hematoxylin, and mounted under glass coverslips. For each experiment, splenocytes from each of the four groups of mice were assayed for binding to at least two sections from each tissue.
In vivo lymphocyte migration assays
Two-color lymphocyte migration experiments were as described (6, 22). Briefly, single-cell suspensions were prepared from the spleens of donor mice and labeled with calcein-AM (Molecular Probes, Eugene, OR). Splenocytes (5–10 x 107) were incubated in 2 ml of RPMI 1640 medium containing 1 µM calcein on ice for 30 min with gentle mixing every 5 min. Cells were then washed twice in PBS and counted. Internal control wild-type splenocytes were labeled with PKH26 (Sigma, St. Louis, MO). Splenocytes (5 x 107) were resuspended in 1 ml of PKH26 diluent, immediately added to 1 ml of a 3 µM PKH26 dye solution, and incubated for 2 min at room temperature. For some experiments, ß7 integrin-/- splenocytes were used as an internal control. Labeling was stopped by the addition of 2 ml of FCS. Cell suspensions were washed twice with PBS and counted. Equal numbers (2 x 107) of calcein-labeled and PKH26-labeled cells were mixed, pelleted, and resuspended in a total volume of 400 µl of PBS for injection into the lateral tail veins of individual wild-type mice. An aliquot of the injected cell mixture was analyzed by flow cytometry to calculate the injected ratio of calcein- to PKH26-labeled cells (Ri). After either 1 or 48 h of migration, single-cell suspensions of tissues and blood were prepared, and the percentage of calcein- and PKH26-labeled cells was determined by flow cytometric analysis. Two to 5000 PKH26-labeled cells were collected for each sample. The ratio of calcein-/PKH26-labeled cells within tissues or blood (Ro) was calculated, and results were expressed as the ratio of Ro/Ri in each tissue, as described (6, 22). The total number of calcein- or PKH26-labeled cells recovered from individual lymphoid tissues or blood was determined by multiplying the total cell counts for individual tissues by the frequency of labeled cells.
Statistical Analysis
All data are shown as mean values ± SEM. Comparisons between groups were conducted using the Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L-selectin-/- and ß7
integrin-/- mice were crossed to generate mice homozygous
deficient at both loci (L-selectin/ß7
integrin-/-) as described (43). Each group of mice was
free from obvious signs of pathology or disease for up to 1 yr of age.
Leukocytes from L-selectin/ß7 integrin-/-
mice completely lacked cell surface expression of both L-selectin and
ß7 integrins (Fig. 1
,
A and B). Both circulating and splenic leukocytes
from L-selectin-/- mice expressed wild-type levels of
ß7 integrins, while leukocytes from ß7
integrin-/- mice expressed wild-type levels of L-selectin
(Table I). Significant differences in the
percentage of circulating or splenic leukocytes expressing either
L-selectin or ß7 integrins were not observed in
ß7 integrin-/- or
L-selectin-/- mice, respectively, compared with wild-type
mice (Table I). Thus, the loss of one adhesion molecule did not lead to
significant changes in expression intensity or frequency of the other
adhesion molecule. However, circulating leukocytes expressing both
L-selectin and ß7 integrins were present at a higher
frequency compared with spleen cells in wild-type mice as previously
reported (27).
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95% reduction in lymphocyte binding to HEV of each lymphoid tissue
(Fig. 1
C). L-selectin loss resulted in an 
30% decrease
in splenocyte binding to HEV of Peyer’s patch, a 75% decrease in
binding to MLN HEVs, and a 98% decrease in binding to PLN HEVs. Loss
of ß7 integrin expression resulted in a 97% and 46%
reduction in splenocyte binding to Peyer’s patch and MLN HEVs,
respectively, without a significant loss in binding to PLN HEVs (Fig. 1C). Therefore, L-selectin and ß7 integrins
collectively mediate the vast majority of lymphocyte binding to HEV
under these in vitro conditions. Peyer’s patch development in L-selectin/ß7 integrin-/- mice
The loss of both L-selectin and ß7 integrins
severely affected the distribution of lymphocytes within Peyer’s
patches from 2-mo-old L-selectin/ß7
integrin-/- mice. Most Peyer’s patches from
L-selectin/ß7 integrin-/- mice were
virtually undetectable macroscopically with an average of 3.5 ±
0.4 (range 2–5; p < 0.001 vs wild-type;
n = 12) Peyer’s patches being identified in each
mouse. The most easily identified Peyer’s patches showed no protrusion
from the gut wall and were nearly translucent in color. This was
consistent with a significant reduction in numbers of lymphocytes
present in histologic sections of these Peyer’s patches (Fig. 2). However, despite the small size and
significant reduction in cellularity of Peyer’s patches from
L-selectin/ß7 integrin-/- mice, rudimentary
follicles were still observed (Fig. 2). Microscopic examination of
serial sections across the entire gut revealed an equal number of
Peyer’s patches in both wild-type and L-selectin/ß7
integrin-/- mice, as described for ß7
integrin-/- mice (16). Macroscopic examination revealed
equal numbers (range of 6–12) of readily detectable Peyer’s patches
that were microscopically similar protruding from the gut wall in both
wild-type and L-selectin-/- mice (Fig. 2). Normal
numbers of Peyer’s patches were visually identified and harvested from
ß7 integrin-/- mice (7.7 ± 0.6 per
mouse, range 6–10, n = 6). However, Peyer’s patches
of ß7 integrin-/- mice were markedly
reduced in size and protruded only minimally from the gut wall, making
them macroscopically less apparent (Fig. 2, and 16 . Thus, the
combined loss of L-selectin and ß7 integrin expression
had a major effect on Peyer’s patch cellularity, but not its overall
gross architecture.
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The numbers of lymphocytes within Peyer’s patches of
L-selectin/ß7 integrin-/- mice were reduced
significantly beyond the decreases resulting from the loss of either
L-selectin or ß7 integrin expression alone. Specifically,
Peyer’s patches of L-selectin/ß7
integrin-/- mice were reduced in cellularity by >99% in
comparison with wild-type mice (Table II). This may be a slight underestimate
of the effect of L-selectin/ß7 integrin loss because the
small size of Peyer’s patches from these mice resulted in a higher
recovery of intraepithelial lymphocytes that surrounded the Peyer’s
patches and contaminated the Peyer’s patch cell populations to varying
degrees (data not shown). However, as stated above, the smallest of the
Peyer’s patches from the L-selectin/ß7
integrin-/- mice were not identified macroscopically and
thus were not harvested or included in the cell counts. Peyer’s
patches in ß7 integrin-/- mice were reduced
by 90% as previously described (16), which was significantly
different from wild-type or L-selectin-/- mice. These
results indicate that L-selectin and ß7 integrins account
for the vast majority of lymphocyte migration into Peyer’s patches.
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, and 43 . L-selectin-/-
mice had an 45% reduction, while ß7
integrin-/- and wild-type mice had similar numbers of MLN
lymphocytes. By contrast, the loss of L-selectin expression resulted in
a 95% decrease in PLN lymphocytes, but the number of resident
lymphocytes within PLNs of L-selectin-/- mice tended to
be increased with the additional loss of ß7 integrin
expression (Table II). ß7 integrin loss also resulted in
a 10% increase in PLN cellularity. These results are consistent with
overlapping functions for L-selectin and ß7 integrins
during lymphocyte entry into MLNs, but point out that ß7
integrin expression does not contribute substantially to lymphocyte
migration into PLNs.
Circulating leukocyte numbers were markedly increased in
L-selectin/ß7 integrin-/- mice relative to
wild-type mice (by 260%, Table II). By contrast, circulating leukocyte
numbers were not significantly different between wild-type and
L-selectin-/- mice and were only slightly elevated in
ß7 integrin-/- mice. Increased lymphocyte
numbers accounted for the vast majority of the increase in circulating
leukocytes in L-selectin/ß7 integrin-/-
mice. Smaller, yet significant, relative increases in numbers of
circulating neutrophils, monocytes, and eosinophils were also observed
in these mice compared with wild-type controls.
L-selectin-/- mice had significantly increased numbers of
circulating monocytes (130% increase) compared with wild-type
controls as previously described (22). Mice deficient in
ß7 integrins alone had slightly increased numbers of
circulating neutrophils, lymphocytes, and eosinophils relative to
wild-type mice (Table II).
The loss of L-selectin resulted in an 60% increase in spleen
lymphocytes, which was further increased by 49% with the additional
loss of ß7 integrin expression (Table II). There was a
trend toward increased spleen cellularity with the loss of
ß7 integrin expression, although this was a variable
finding. Therefore, the combined loss of L-selectin and the
ß7 integrins had an additive effect on the number of
circulating leukocytes and the number of lymphocytes that accumulated
within the spleen.
Migration of L-selectin/ß7 integrin-/- lymphocytes into lymphoid tissues
The extent that L-selectin and the ß7 integrins regulate lymphocyte entry into lymphoid tissues was assessed in short-term (1 h) in vivo migration assays. Splenocytes from wild-type, L-selectin-/-, ß7 integrin-/-, and L-selectin/ß7 integrin-/- mice were labeled with calcein, mixed with an equal number of PKH26-labeled wild-type splenocytes (internal control), and injected i.v. into wild-type mice. The frequency of labeled lymphocytes within tissues was determined after 1 h. Differences in migration patterns were assessed by comparing the ratio of calcein-labeled test cells to PKH26-labeled internal control cells present within each tissue (Ro) with the ratio of calcein-labeled test cells to PKH26-labeled control cells that were injected (Ri). Splenocytes were used for these studies because they represent mixed populations of T and B cells, contain both naive and memory cell populations, and have the most heterogeneous distribution of L-selectin and ß7 integrin expression (27). Nonetheless, the distribution of lymphocyte populations present within the spleens of 2-mo-old L-selectin/ß7 integrin-/- mice was not dramatically different from that of wild-type mice (D.A.S., X.-Q.Z., N.W., and T.F.T., manuscript in preparation), ß7 integrin-/- mice (16) or L-selectin-/- mice (22).
The migration of L-selectin/ß7 integrin-/-
lymphocytes into Peyer’s patches was dramatically decreased compared
with wild-type (99% decrease, p < 0.001),
L-selectin-/- (96% decrease, p <
0.001), or ß7 integrin-/- lymphocytes (85%
decrease, p < 0.01) (Fig. 3). Similarly, the migration of
double-deficient lymphocytes into MLNs was significantly decreased
compared with wild-type (by 99%, p < 0.001),
L-selectin-/- (by 77%, p < 0.01), or
ß7 integrin-/- (by 98%, p
< 0.001) lymphocytes (43). The combined loss of L-selectin and
ß7 integrin expression resulted in a reduction of
lymphocyte migration to PLNs similar to the decrease observed for
L-selectin-/- lymphocytes (Fig. 3). The increase in
L-selectin/ß7 integrin-/- lymphocyte
migration to the spleen was also similar to that seen for
L-selectin-/- lymphocytes (Fig. 3). The combined loss of
both L-selectin and ß7 integrin expression increased the
number of lymphocytes remaining in the blood after 1 h by 25%
(p < 0.01) compared with wild-type
lymphocytes. Therefore, the L-selectin/ß7
integrin-/- deficiency significantly reduced lymphocyte
migration into Peyer’s patches and MLNs beyond the level seen in the
single-deficient lymphocytes, while enhancing blood and spleen
localization.
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). There was also a 97% decrease in
L-selectin/ß7 integrin-/- lymphocyte
migration into MLNs and a 98% reduction of migration into PLNs as
compared with ß7 integrin-/- lymphocytes
(p < 0.01). Therefore, L-selectin is necessary
for the efficient migration of wild-type and ß7
integrin-/- lymphocytes into Peyer’s patches and MLN and
the combined effect of these deficiencies is more than additive.
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To assess how L-selectin and ß7 integrin loss
affected the accumulation of recirculating lymphocytes within tissues
after the cells had the opportunity to reach equilibrium, in vivo
migration assays were conducted as described above except lymphocyte
localization within tissues was assessed at 48 h. Lymphocytes
deficient in both L-selectin and ß7 integrin expression
were excluded from entering Peyer’s patches (>99% decrease,
p < 0.001) relative to wild-type lymphocytes (Fig. 5). Furthermore, lymphocyte localization
within MLNs (>99% decrease, p < 0.001) and PLNs
(98% decrease, p < 0.001) was also significantly
affected when compared with wild-type lymphocytes (Fig. 5). By
contrast, ß7 integrin-/- lymphocytes showed
a 94% decrease in Peyer’s patch migration and a modest 34% decrease
in MLN migration when compared with wild-type splenocytes (Fig. 5). The
number of circulating L-selectin/ß7
integrin-/- lymphocytes remained increased at 48 h
(by 60%, p < 0.01) relative to splenocytes from
all other groups of mice (Fig. 5). The migration of
L-selectin/ß7 integrin-/- lymphocytes into
the spleen was increased by twofold relative to wild-type lymphocytes
(p < 0.001) and tended to be increased
slightly relative to L-selectin-/- lymphocytes (Fig. 5).
These results show that the L-selectin/ß7 integrin
deficiency eliminated the vast majority of lymphocyte recirculation
through Peyer’s patches, MLNs, and PLNs.
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Conclusions similar to those discussed above were obtained when
the numbers of splenocytes that migrated into tissues at 1 and 48
h were determined (Table III). Within
Peyer’s patches, L-selectin/ß7 integrin-/-
lymphocyte migration was <1% of wild-type levels at 1 h and
0.2% at 48 h. Within MLNs, L-selectin/ß7
integrin-/- lymphocyte migration was <2% of wild-type
levels at 1 h and <1% at 48 h. Similarly,
L-selectin/ß7 integrin-/- lymphocyte
migration into PLNs was 6% of wild-type levels at 1 h and
1.5% at 48 h. These results indicate that the
L-selectin/ß7 integrin deficiency affects the majority of
splenocyte migration including both naive and memory subpopulations.
Two additional observations were of interest. First, despite the
L-selectin/ß7 integrin deficiency a very small number of
lymphocytes were nonetheless able to enter Peyer’s patches and MLNs at
1 h (Table III). These emigrant cells were lymphocytes, although
their identity or whether they represented lymphocytes present within
the vasculature of the harvested tissues was not assessed. However, the
number of labeled L-selectin/ß7
integrin-/- lymphocytes within Peyer’s patches and MLNs
did not increase over the subsequent 48 h. This is in stark
contrast to results obtained using splenocytes of wild-type or
single-deficient mice. Specifically, migration of wild-type lymphocytes
to Peyer’s patches and MLNs at 48 h increased by four- and
threefold, respectively, relative to 1 h numbers
(p < 0.001, Table III). Similar increases in
numbers of L-selectin-/- lymphocytes migrating to
Peyer’s patches and MLNs (6.3- and 3.7-fold, respectively,
p < 0.05) were observed over this time period.
ß7 integrin-/- lymphocytes showed
significant but smaller increases in numbers of migrating lymphocytes
over the 48-h time period (2.3-fold, p < 0.02)
relative to wild-type or L-selectin-/- cells. Therefore,
in contrast to L-selectin/ß7 integrin-/-
lymphocytes, these results indicate that wild-type and single-deficient
lymphocytes accumulate within these tissues.
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). This observation appears valid because the number of resident
lymphocytes within PLNs of L-selectin/ß7
integrin-/- mice tended to be greater than in
L-selectin-/- littermates (Table II). Although the
numbers of L-selectin-/- or L-selectin/ß7
integrin-/- lymphocytes migrating into PLNs did not
increase from 1 to 48 h, numbers of wild-type or ß7
integrin-/- cells increased 3.5-fold (Table III,
p < 0.01). Within the circulation, the number of
wild-type or single-deficient lymphocytes decreased by 40–50% over
the 48-h time period (Table III). By contrast, the number of
circulating L-selectin/ß7 integrin-/-
lymphocytes only decreased a modest 20% from 1 to 48 h. These
results further support the observations that lymphocytes lacking
L-selectin and ß7 integrin expression are unable to leave
the circulation and enter lymphoid tissues such as Peyer’s patches,
PLNs, and MLNs and are consistent with the significantly elevated
numbers of circulating lymphocytes observed in
L-selectin/ß7 integrin-/- mice (Table II). L-selectin and ß7 integrin expression by resident Peyer’s patch lymphocytes
Whether the loss of L-selectin or ß7 integrin
expression skewed Peyer’s patch lymphocyte expression of
ß7 integrins or L-selectin, respectively, was assessed in
adhesion molecule-deficient mice. As observed for circulating
lymphocytes (Fig. 1A), L-selectin loss did not affect the
level of ß7 integrin expression by lymphocytes within
Peyer’s patches (Fig. 6). Most
lymphocytes within Peyer’s patches of wild-type (94 ± 1%
positive, n = 4) and L-selectin-/-
(92 ± 1%, n = 4) mice expressed
4ß7 at comparable intensities. By
contrast, the frequency of L-selectin expressing lymphocytes in
ß7 integrin-/- mice (45 ± 5%,
n = 4) was significantly lower than in wild-type mice
(75 ± 4%, n = 3, p < 0.002),
although L-selectin was expressed at similar intensities by most
L-selectin-bearing lymphocytes (Fig. 6). The lower frequency of
L-selectin expressing lymphocytes in Peyer’s patches of
ß7 integrin-/- mice compared with blood
lymphocytes (Fig. 1) is likely to reflect the slower entry of
circulating lymphocytes into this tissue in the absence of
ß7 integrin expression. As a result, the majority of
Peyer’s patch lymphocytes in ß7
integrin-/- mice are likely to have been long-term
residents within this tissue or may have arisen from in situ
proliferation. This alters the lymphocyte subset distribution within
Peyer’s patches of ß7 integrin-/- mice
compared with wild-type mice, but these changes are relatively minor
(D.A.S., X.-Q.Z., N.W., and T.F.T., manuscript in preparation). An
additional previously discussed influence on L-selectin expression is
that Peyer’s patch preparations from L-selectin/ß7
integrin-/- mice contain some degree of contaminating gut
lymphocytes that typically do not express L-selectin.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Moreover, the inability to bind HEV eliminated
the vast majority of L-selectin/ß7
integrin-/- lymphocyte migration into Peyer’s patches
during short-term in vivo migration assays (>99% inhibition,
p < 0.01, Fig. 3, Table III) as well as the long-term
localization of lymphocytes in these tissues (>99% inhibition,
p < 0.01, Fig. 5, Table III). The lack of lymphocyte
migration into Peyer’s patches correlated directly with the
dramatically reduced size (Fig. 2) and cellularity (99% reduced) of
this tissue in L-selectin/ß7 integrin-/-
mice (Table II). In addition, the increased numbers of injected
L-selectin/ß7 integrin-/- lymphocytes
remaining in the blood of wild-type mice after 48 h (Fig. 5)
correlated with the markedly increased numbers of circulating
lymphocytes found in L-selectin/ß7
integrin-/- mice (Table II). While loss of either
L-selectin or, to a greater extent, the ß7 integrins
alone resulted in significant inhibition of Peyer’s patch migration,
neither produced complete inhibition. Collectively, these results
indicate that L-selectin and ß7 integrins primarily
interact along the same adhesion pathway to mediate the vast majority
of lymphocyte migration into Peyer’s patches, although each molecule
can function independent of the other, albeit less efficiently.
The current study of lymphocyte migration into Peyer’s patches extends
prior in situ studies (15, 16, 40) by demonstrating that the combined
absence of L-selectin and ß7 integrins eliminates the
vast majority of lymphocyte migration into mouse Peyer’s patches
(Figs. 3 and 5). Because the current studies circumvent the
inflammatory influences introduced by the invasive surgical procedures
necessary to view leukocyte/endothelial interactions in situ (15, 16, 40), it is likely that L-selectin and the ß7 integrins
are the essential receptors for normal migration during noninflammatory
situations. The absence of lymphocyte migration into Peyer’s patches
even after 48 h suggests that other adhesive mechanisms are
unlikely to compensate significantly for the loss of both L-selectin
and ß7 integrins during migration into reactive Peyer’s
patches because other adhesion molecules are unable to compensate for
L-selectin loss during lymphocyte entry into reactive PLNs across HEVs
(22). Therefore, a remaining issue is the identity of the adhesion
molecules that facilitate lymphocyte migration into Peyer’s patches in
the absence of L-selectin or ß7 integrin expression.
P-selectin has recently been shown to support a low level of leukocyte
rolling along Peyer’s patch venules from L-selectin/ß7
integrin-/- mice following exteriorization (40). It has
also been postulated that P-selectin facilitates the entry of
lymphocytes into lymphoid tissues across HEVs by the formation of
lymphocyte-platelet aggregates (46, 47). However, because lymphocytes
do not migrate across HEVs of resting or activated PLNs in the absence
of L-selectin expression (22) and circulating lymphocyte-platelet
aggregates are not encountered under normal physiologic conditions,
this mechanism may only contribute modestly to lymphocyte entry into
lymphoid tissues. Therefore, it is likely that
4ß7 integrins mediate lymphocyte capture
and rolling on Peyer’s patch HEV in the absence of L-selectin
expression, albeit at much lower levels of efficiency than the
combination of L-selectin and ß7 integrins. Previous
studies have documented a role for LFA-1 during lymphocyte migration
into lymphoid tissues (48), and L-selectin and ICAM-1 function
cooperatively to mediate leukocyte rolling at sites of inflammation
(26). Consistent with this, migration of ß7
integrin-/- lymphocytes into Peyer’s patches is further
significantly reduced with the additional loss of ICAM-1 expression
(D.A.S., X.-Q.Z., and T.F.T., unpublished observations).
Therefore, it is likely that these molecules retard lymphocyte rolling
velocities sufficiently to permit the formation of firm adhesive
interactions that proceed lymphocyte migration into Peyer’s patches in
the absence of ß7 integrin expression. However, in the
absence of L-selectin and ß7 integrin expression, these
alternative adhesive mechanisms are inadequate to mediate lymphocyte
migration into Peyer’s patches.
T cells migrate into Peyer’s patches and all other lymphoid tissues at
a faster tempo than B cells, in part, due to their expression of
50–100% higher levels of L-selectin compared with B cells (27).
Although B cells express 4ß7 integrins at
twofold higher levels than T cells, more T cells still migrate into
Peyer’s patches. In the absence of L-selectin expression, the vast
majority of PLN lymphocytes (which are predominantly T cells) are
unable to enter Peyer’s patches (22) despite the fact that the
majority of these cells express 4ß7
integrins (27). In fact, even in the absence of L-selectin expression,
spleen T cells migrate into Peyer’s patches at a fivefold higher rate
than spleen B cells (27). However, ß7 integrin loss
appeared to have a more significant effect on splenocyte/Peyer’s patch
HEV interactions than the loss of L-selectin in the current studies
(Figs. 3 and 5). This apparently contradictory finding points out a
need for further examination of the differential roles for L-selectin
and ß7 integrins in short-term T and B cell subset
migration. For example, a loss of ß7 integrin expression
by B cells in combination with their lower levels of L-selectin
expression may significantly diminish or preclude their entry into
Peyer’s patches. Although many of the effects of adhesion receptor
loss described in the current study were quite large and thereby were
likely to involve most or all lymphocyte subsets, heterogeneity of
L-selectin and ß7 integrin expression on some minor
lymphocyte subsets may be of substantial importance in directing their
trafficking to selective tissues (49). Nonetheless, the results
discussed above demonstrate that adhesion molecule requirements during
migration may not be generally equivalent between lymphocytes isolated
from different lymphoid tissues.
In addition to facilitating lymphocyte/HEV interactions, we have
postulated previously that the ß7 integrins retain
lymphocytes within Peyer’s patches subsequent to their entry across
HEV, particularly B cells (27). The current studies validate this
hypothesis. The entry of L-selectin-/- and
ß7 integrin-/- lymphocytes into Peyer’s
patches at 1 h is inhibited by 48–70% and 89% relative to
lymphocytes from wild-type mice, respectively (Table III and 27 .
However, by 48 h the number of emigrant
L-selectin-/- lymphocytes within Peyer’s patches is
similar to that of wild-type lymphocytes (Table III). Peyer’s patches
in L-selectin-/- and wild-type mice are also similar in
cellularity (6, 22). Therefore, although the loss of L-selectin greatly
reduces the tempo of lymphocyte migration into Peyer’s patches,
emigrant L-selectin-/- lymphocytes can populate this
tissue to near wild-type levels by 48 h. By contrast, the number
of newly emigrated ß7 integrin-/-
lymphocytes within Peyer’s patches by 48 h is 93% below that of
wild-type lymphocytes (Table III). Similarly, the cellularity of
Peyer’s patches in ß7 integrin-/- mice is
reduced by 90% (Table II and 16 . Therefore, the bulk of
lymphocytes that enter Peyer’s patches are not retained in the absence
of ß7 integrin expression. This is perhaps consistent
with the recent observation that MAdCAM-1 is expressed at sites within
lymphoid tissues in addition to its display by HEV (37).
L-selectin and 4ß7 have been predominantly
hypothesized to direct the selective migration of lymphocytes to
peripheral lymph nodes and the gut, respectively (reviewed in 50).
However, this conclusion is based in part on the prediction that
lymphocytes selectively recirculating through an organ will
preferentially express adhesion receptors for organ-specific
endothelial ligands that allowed their entry into that organ. Thus,
when lymphocytes exiting a given tissue expressed high or low levels of
a particular adhesion molecule, this has been felt to provide strong
support for that molecule being either involved or not involved,
respectively, in lymphocyte entry into the tissue. However, the current
studies demonstrate a variable and heterogeneous pattern of L-selectin
expression on Peyer’s patch lymphocytes in ß7
integrin-/- mice (Fig. 6) despite the fact that
L-selectin is critically involved in lymphocyte migration into this
tissue in the absence of ß7 integrin expression (Figs. 3 and 5). Conversely, the majority of PLN lymphocytes express
4ß7 (27), although the ß7
integrins play no apparent role in lymphocyte migration into PLNs
(Figs. 3 and 5). Collectively, these findings force the conclusion that
adhesion molecule expression by lymphocytes within or exiting a given
tissue is likely to reflect processes or developmental programs ongoing
within that tissue rather than emulate the display of adhesion
molecules necessary for lymphocyte entry into that tissue.
Although it has been well recognized that L-selectin and the
ß7 integrins play major roles in the migration of
lymphocytes into secondary lymphoid tissues it was surprising that the
loss of these two adhesion molecules eliminated the vast majority of
lymphocyte migration into Peyer’s patches. Although concomitant
L-selectin/ß7 integrin loss also dramatically
inhibited lymphocyte migration into other HEV-bearing lymphoid tissues,
PLNs and MLNs retained 7–8% of their wild-type levels of cellularity
(Table II), presumably due to lymphocyte entry through their afferent
lymphatics. Therefore, whether the deficiency in Peyer’s patches has
deleterious or selective effects on mucosal immunity will be an
important issue to resolve. Moreover, because lymphocyte entry into
PLNs, MLNs, and Peyer’s patches represents only a small portion of
lymphocyte migration (51), it will be important to assess whether the
loss of L-selectin/ß7 integrins has additional
detrimental effects on lymphocyte migration to other extralymphoid
sites of lymphocyte accumulation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Douglas A. Steeber, Department of Immunology, Box 3010, Duke University Medical Center, Durham, NC 27710. E-mail address:
3 Abbreviations used in this paper: HEV, high endothelial venule; PLN, peripheral lymph node; MLN, mesenteric lymph node; MAdCAM-1, mucosal addressin cell adhesion molecule-1; L-selectin-/-, L-selectin-deficient; ß7 integrin-/-, ß7 integrin-deficient; L-selectin/ß7 integrin-/-, L-selectin/ß7 integrin-deficient; PE, phycoerythrin; MFI, mean fluorescence intensity; Ri, the ratio of calcein-labeled test cells to PKH26-labeled internal control cells injected into mice for migration assays; Ro, the ratio of calcein-labeled test cells to PKH26-labeled internal control cells within each tissue after migration assays.
Received for publication June 12, 1998. Accepted for publication August 28, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß7 and LFA-1 in lymphocyte homing to Peyer’s patch-HEV in situ: the multistep hypothesis confirmed and refined. Immunity 3:99.[Medline]
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