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Department of Immunology, Duke University Medical Center, Durham, NC 27710
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß7 integrin. Whether L-selectin
also regulates lymphocyte subset-specific migration into specific
lymphoid tissues was examined in this study by comparing the migration
of CD4+ T cells, CD8+ T cells, and B cells from
L-selectin-deficient and wild-type mice. T cells were the predominant
lymphocyte subset entering PLN, MLN, Peyer’s patches, and spleen
during short term (1-h) migration assays. However, both B cell and
CD4+ and CD8+ T cell entries into PLN, MLN, and
Peyer’s patches were dramatically impaired (73–98%) by loss of
L-selectin. Lymphocyte expression of 4ß7
integrin did not compensate for the loss of L-selectin, since both B
and T cells predominantly migrated into the spleen in the absence of
L-selectin. The more efficient migration of T cells into peripheral
lymphoid tissues relative to that of B cells was partly explained by
the finding that T cells expressed L-selectin at 50 to 100% higher
levels than B cells. In addition, a 50% reduction in L-selectin
expression by lymphocytes from hemizygous L-selectin+/-
mice resulted in a 50 to 70% decrease in short term lymphocyte
migration into peripheral lymphoid tissues relative to that of
wild-type lymphocytes. Thus, the differential migration of T and B
lymphocyte subsets to lymphoid tissues is regulated in part by
subset-specific differences in L-selectin expression levels. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß7 integrin
also expressed by lymphocytes interacts with mucosal HEV determinants
to mediate lymphocyte migration into Peyer’s patches and MLN (5, 6, 7).
Migration of lymphocytes into PLN, MLN, and Peyer’s patches is reduced
markedly in L-selectin-deficient mice (4, 8, 9, 10), while lymphocyte
migration into MLN and Peyer’s patches is diminished markedly in
ß7 integrin-deficient mice (11). These tissue-specific
lymphocyte recirculation pathways establish a framework for
understanding lymphocyte migration into individual lymphoid tissues,
since most blood-derived T and B cells express both L-selectin and
4ß7 (12). However, the preferential
migration of lymphocyte subsets into specific peripheral lymphoid
tissues suggests that subset-specific mechanisms regulate the
extraction of lymphocyte subsets into given tissues rather than the
mere presence or absence of tissue-specific lymphocyte-endothelial
adhesion receptors (12, 13). Studies of lymphocyte migration in sheep indicate that different lymphocyte subsets have selective affinities for individual lymphoid tissue vascular beds. Specifically, CD4+ cells appear to enter PLN from the blood via HEV more efficiently than other lymphocyte subsets (13, 14, 15, 16). Similarly, CD4+ cells reportedly recirculate much more efficiently than CD8+ cells under physiologic conditions in vivo (13, 16, 17, 18). Based on the different migration properties of CD4+ cells, CD8+ cells, and B cells, the existence of subset-specific lymphocyte-endothelial cell recognition systems has been postulated (13, 14, 15, 16, 17, 19, 20, 21). Studies in humans and rats have also indicated subset-specific differences in lymphocyte migration (22, 23, 24, 25, 26).
Studies in mice further support the existence of lymphocyte subset-specific adhesion mechanisms that regulate the differential entry of lymphocyte subsets into lymphoid tissues. In mice, T cells distribute preferentially to PLN, whereas B cells are reported to distribute preferentially to Peyer’s patches and spleen (27, 28, 29). Similar proportions of CD4+ and CD8+ cells migrate into PLN, although CD4+ T cells migrate into Peyer’s patches more efficiently than CD8+ T cells (29). The different migration characteristics of T and B cells are evident regardless of whether the cells are from spleen, PLN, MLN, or Peyer’s patches (27). Although previous studies of L-selectin-deficient mice have documented L-selectin’s role in tissue-specific lymphocyte migration into secondary lymphoid tissues (4, 8, 9, 10), such studies have not evaluated whether the absence of L-selectin reduces the migration of all lymphocyte populations or only the migration of a specific lymphocyte subset(s). The finding that the tissue distribution of CD4+ cells and memory T cells is affected more severely than that of other lymphocyte subsets in L-selectin-deficient mice (8) suggests that L-selectin could be involved in lymphocyte subset-specific migration. Therefore, the role of L-selectin in the regulation of lymphocyte subset migration was examined in the current study. Our findings demonstrate that CD4+ and CD8+ T cells have the highest propensity to migrate into PLN, MLN, Peyer’s patch, and spleen and that L-selectin regulates this migration. Furthermore, intrinsic differences in expression levels of cell surface L-selectin were found to regulate stoichiometrically the differential migration of T and B cell subsets into lymphoid tissues.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L-selectin-/- mice were generated as previously described (4) and backcrossed onto the C57BL/6 background for seven to nine generations. Lack of cell surface L-selectin expression was verified by immunofluorescence staining of blood leukocytes with a FITC-conjugated anti-mouse L-selectin mAb (30). All mice used were 2 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+/- 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 Medical Center (Durham, NC).
Lymphocyte isolation, mAbs, and flow cytometry
Blood was aspirated from the retro-orbital venous plexus of anesthetized mice. Single-cell suspensions from spleen, PLN (bilateral axillary, inguinal, and brachial nodes were pooled), MLN (superior mesenteric cords were pooled), and Peyer’s patches were prepared as previously described (8) and filtered through nylon gauze to remove debris before washing twice in PBS. Erythrocytes in splenocyte suspensions and peripheral blood were lysed with 0.83% ammonium chloride solution or 2% acetic acid, respectively. Total cell numbers were enumerated using a hemocytometer.
Abs used in these studies included unconjugated and FITC-,
phycoerythrin (PE)-, or biotin-conjugated mAbs reactive with L-selectin
(LAM 1-116 mouse mAb) (30), 4ß7
(DATK-32, American Type Culture Collection, Rockville, MD) (31), Thy1.2
(5a-8; Caltag, San Francisco, CA), B220 (RA3-6B2; Caltag), CD4 (L3T4;
PharMingen, San Diego, CA), and CD8 (53-6.72; PharMingen). Secondary
Abs used were FITC-conjugated goat anti-rat IgG (Southern
Biotechnology Associates, Birmingham, AL) and streptavidin PerCP
(Becton Dickinson, San Jose, CA). Isotype-matched rat IgG Abs
(PharMingen) were used as controls.
Expression of L-selectin or 4ß7 by
lymphocyte subsets was assessed by two-color fluorescence cytometry.
Single cell suspensions of lymphocytes were incubated either with
unconjugated DATK-32 mAb followed by FITC-conjugated goat anti-rat
IgG secondary Ab or with FITC-conjugated LAM1–116 mAb. Cells labeled
with DATK-32 mAb and goat anti-rat IgG secondary Ab were incubated
with PBS containing 5% normal rat serum to block secondary Ab binding
sites. Subsequently, all cell preparations were stained with
PE-conjugated Abs against Thy1.2, CD4, CD8, or B220. Mean log
fluorescence intensities of L-selectin and
4ß7 expressed by gated T cells and B cells
were then determined by flow cytometry.
Concurrent expression of 4ß7 and
L-selectin on T and B cell subsets was examined by three-color
fluorescence cytometry. Single cell suspensions were incubated
sequentially with DATK-32 mAb, PE-conjugated goat anti-rat IgG Abs,
5% normal rat serum as a blocking agent, FITC-conjugated LAM1-116 mAb,
and biotinylated mAbs against CD4, CD8, Thy1.2, or B220, followed by
streptavidin PerCP. Concurrent L-selectin and
4ß7 expression was determined for gated
subsets of PerCP-labeled lymphocytes by flow cytometric analysis.
In all cases, after blood leukocytes were labeled, the erythrocytes
were lysed with a Coulter wholeblood Immuno-Lyse kit according to the
manufacturer’s instructions (Coulter, Miami, FL). For phenotype
experiments, 10,000 cells with the forward and side light scatter
properties of mononuclear cells were analyzed on a FACScan flow
cytometer (Becton Dickinson) with fluorescence intensity shown on
either a 3-decade (Figs. 3
and 4) or a 4-decade (Fig. 2) log scale.
Fluorescence contours are shown as 50% log density plots.
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For single-color migration assays, single-cell suspensions of splenocytes from wild-type or L-selectin-/- mice were labeled with calcein-AM (Molecular Probes, Eugene, OR) as previously described (32). Briefly, cells (5–10 x 107) were incubated in 2 ml of RPMI 1640 medium (Life Technology, Gaithersburg, MD) containing 1 µM calcein-AM on ice for 30 min, with gentle mixing every 5 min. Cells were then washed twice in PBS, counted, and resuspended at 1 x 108 cells/ml in PBS. In most experiments, calcein-labeled splenocytes (1–4 x 107 cells in 250–400 µl) were injected into the lateral tail vein of individual wild-type mice. At the appropriate times, single-cell suspensions of tissues were prepared, and aliquots of cells were labeled with PE-conjugated Abs against Thy1.2, B220, CD4, or CD8. Five thousand calcein-labeled cells with the forward and side light scatter properties of mononuclear cells were analyzed by flow cytometry, although for some tissues smaller numbers of L-selectin-deficient cells were collected for analysis because of decreased numbers of migrating cells within these tissues. The total number of calcein-labeled cells recovered from individual lymphoid tissues was determined by multiplying the total cell counts for individual tissues by the frequency of labeled cells. The percentage of injected calcein-labeled cells that migrated to individual tissues was then determined.
Two-color migration experiments were performed as previously described (4, 8). Test cells (either L-selectin+/- or wild-type splenocytes) were labeled with 1 µM calcein-AM as described above. Internal control wild-type splenocytes were labeled with PKH26 (Sigma, St. Louis, MO). Briefly, splenocytes (5 x 107) were resuspended in 1 ml of PKH26 diluent, immediately added to an equal volume of a 3-µM PKH26 dye solution, and allowed to incubate at room temperature for 2 min. Labeling was stopped by the addition of 2 ml of FCS. Cell suspensions were washed twice with PBS, counted, and then stored on ice. PKH26-labeled cells (2 x 107) and calcein-labeled cells (2 x 107) were mixed in a total volume of 400 µl for injection into individual wild-type mice. An aliquot of the injected cell mixture was also analyzed by flow cytometry to calculate the injected ratio of calcein- to PKH26-labeled cells (Ri). After 1 h, single-cell suspensions of tissues were prepared, and the percentages of calcein- and PKH26-labeled cells were determined by flow cytometric analysis. A minimum of 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 previously described (4, 8).
Statistical analysis
All data are shown as the mean ± SEM, unless otherwise indicated. Paired comparisons between groups were conducted using Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role of L-selectin in the migration of T and B cells from the
circulation into lymphoid tissues was examined in short term (1-h) in
vivo migration assays. Calcein-labeled splenocytes from
L-selectin-/- or wild-type mice were injected into
the tail veins of individual wild-type mice. Although the spleens of
L-selectin-deficient mice are larger than the spleens of wild-type
mice, the distribution of lymphocyte subsets remains relatively
unchanged in spleens of 2-mo-old L-selectin-deficient mice (4, 8, 33).
Since the relative number of T and B cells varied in each donor mouse,
the effect of injecting varying numbers of splenocytes (1–4 x
107) on T and B cell migration was assessed. Increasing the
number of injected splenocytes beyond 
2 x 107 did
not have a significant effect on the number or the relative frequency
of T and B cells migrating into tissues in 1-h migration assays (data
not shown). Therefore, the number of splenocytes injected into mice was
adjusted whenever possible to provide similar frequencies of T and B
cells within samples of cells that were being compared directly.
The greatest numbers of injected wild-type splenocytes were recovered
from the spleen (8.8 ± 1.1% of injected cells) after 1 h,
with lower numbers in PLN (0.38 ± 0.08%), MLN (0.41 ±
0.07%), and Peyer’s patches (0.03 ± 0.01%; Table I and Fig. 1A). T cells were the
predominant lymphocyte population migrating into PLN, MLN, Peyer’s
patches, and spleen (Fig. 1, B and C).
Similar relative percentages of injected T and B cells were found in
the blood. The relative differences in numbers of T and B cells
migrating into tissues compared with the numbers of T and B cells
injected indicated that T cells were 17 ± 2, 9 ± 2, and
4 ± 1 times more likely than B cells to migrate into PLN, MLN,
and Peyer’s patches, respectively.
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A and Table I). The percentages of injected
L-selectin-/- T cells that migrated into PLN, MLN, and
Peyer’s patches were reduced by 98, 97, and 77%, respectively,
compared with those of wild-type cells (Fig. 1B).
L-selectin-/- B cell migrations into PLN, MLN, and
Peyer’s patches were reduced by 95, 86, and 80%, respectively,
compared with those of wild-type B cells (Fig. 1C and Table I). Thus, the migrations of both T and B cells into PLN, MLN, and
Peyer’s patches at 1 h are predominantly dependent on L-selectin
expression.
The rate of lymphocyte entry into tissues was also assessed by
determining the proportion of calcein-labeled cells in each tissue
after 1-h migration assays. These determinations also compensated for
any size differences between tissues, since they are a function of the
number of cells recovered from each tissue sample. T cells migrated
into PLN, MLN, Peyer’s patches, and spleen at 22-, 10-, 4-, and 2-fold
higher frequencies than B cells, respectively (Table I). The rate of T
lymphocyte migration into PLN and MLN was almost double the rate of
entry into Peyer’s patches. By contrast, the rate of B lymphocyte
migration into PLN was almost half the rate of entry into MLN and
Peyer’s patches. Nonetheless, the loss of L-selectin expression
reduced the rates of T cell entry into PLN, MLN, and Peyer’s patches
by 98, 96, and 73%, respectively. Similarly, the loss of L-selectin
expression reduced the rates of B cell entry into PLN, MLN, and
Peyer’s patches by 98, 90, and 81%, respectively. Therefore,
migration of both lymphocyte subsets was dependent on L-selectin
expression.
Long term migration of T and B cells from L-selectin-/- mice
The role of L-selectin in the accumulation of calcein-labeled
splenocytes in lymphoid tissues was examined in long term (48 h) in
vivo migration assays. The greatest numbers of injected wild-type
lymphocytes were recovered from the spleen (7.2 ± 1.0%), with
smaller numbers from PLN (1.2 ± 0.2%), MLN (1.7 ± 0.4%),
and Peyer’s patches (0.12 ± 0.04%; Fig. 1D and Table I). The relative differences in numbers of T and B cells migrating into
tissues compared with the numbers of T and B cells injected indicated
that T cells localized 9 ± 1 and 6 ± 1 times more
efficiently than B cells in PLN and MLN, respectively (Table I). By
contrast, more B cells accumulated in Peyer’s patches than T cells
(T/B ratio = 0.5 ± 0.1; Table I). Similar frequencies of
injected T and B cells were found in the blood, but more T cells than B
cells accumulated in the spleen (Fig. 1, E and
F). Therefore, T cells localized predominantly in
PLN, MLN, and spleen, while B cells were preferentially retained in
Peyer’s patches relative to T cells.
The loss of L-selectin dramatically impaired both T and B cell (99%
decrease) localization in PLN at 48 h compared with that of
wild-type splenocytes (Fig. 1, E and F, and Table I). The percentage of injected L-selectin-/- lymphocytes
that localized in MLN was reduced by approximately 93%
(p < 0.01) for T cells and by approximately
77% (p < 0.01) for B cells relative to that
of their wild-type counterparts (Fig. 1, E and
F). There was only a 38% reduction in the percentage
of injected L-selectin-/- T cells recovered from Peyer’s
patches, while localization of L-selectin-/- and that of
wild-type B cells were equivalent (Fig. 1, E and
F). Localization of both T cells and B cells in the
spleen was increased by loss of L-selectin, although significantly
(p < 0.01) more L-selectin-/- T
cells migrated to the spleen than B cells. Therefore, loss of
L-selectin expression severely altered both T cell and B cell
localization, with T cells being affected to a greater extent than B
cells.
Migration of CD4+ and CD8+ T cells into secondary lymphoid tissues
The role of L-selectin in CD4+ and
CD8+ T cell migration to lymphoid tissues was examined 1
and 48 h after injection of L-selectin-/- or
wild-type splenocytes. There was no difference in the migration of
wild-type CD4+ and CD8+ cells at either time
point, except CD4+ cells migrated approximately twofold
faster than CD8+ cells to Peyer’s patches in 1-h migration
assays (p < 0.05; Table II). PLN and MLN migrations were markedly
reduced (>96%) for both CD4+ and CD8+ T cells
by the loss of L-selectin in 1-h migration assays (Table II). Peyer’s
patch migration of CD4+ and CD8+ T cells was
also significantly reduced (76–81%) by L-selectin loss. After 48
h, the lack of L-selectin expression reduced both CD4+ and
CD8+ cell localization in PLN and MLN by about 98 and 92%,
respectively (Table II). L-selectin loss only reduced CD4+
and CD8+ cell migration into Peyer’s patches by about
39%. The loss of L-selectin expression resulted in increased
localization of both CD4+ and CD8+ cells in the
spleen (Table II). Therefore, CD4+ and CD8+ T
cells were similarly dependent on L-selectin expression for migration
into lymphoid tissues.
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Since the preferential migration of T cells to lymphoid tissues
relative to B cells could result from differences in adhesion molecule
expression between these lymphocyte subsets, L-selectin and
4ß7 expression were assessed by
two-color immunofluorescence flow cytometry. Almost all the T and B
cells from blood expressed L-selectin and
4ß7 (Table III). Most of the T and B cells from PLN,
MLN, Peyer’s patches, and spleen also expressed L-selectin and
4ß7 (Table III). However, fewer
CD4+ cells expressed L-selectin than CD8+ cells
(Table III). CD4+ cells from Peyer’s patches expressed
L-selectin at particularly low frequencies. Similarly, fewer
CD4+ cells expressed 4ß7 than
B cells or CD8+ cells.
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4ß7 on individual lymphocytes in secondary
lymphoid tissues was examined using three-color fluorescence cytometric
analysis. The majority of T and B cells (80–94%) in blood, PLN, and
MLN were L-selectin/4ß7 double positive
(Fig. 2). Although most T and B cells in
Peyer’s patches were L-selectin/4ß7
double positive, the single positive T and B cells were generally
4ß7+. In the spleen, similar
proportions (
55–60%) of T and B cells were
L-selectin/4ß7 double positive, although
there was a slightly higher frequency of
L-selectin+/4ß7-
T cells than B cells (15 vs 7%). Therefore, the majority of
circulating T and B cells express both L-selectin and
4ß7, although variability in expression
occurs for lymphocytes already in lymphoid tissues.
T cells always expressed L-selectin at significantly higher cell
surface densities than B cells (Fig. 3,
A and C, and Table III). Relative mean
fluorescence staining levels for L-selectin expression by T cells were
consistently 1.5 to 2.0 times those of B cells, regardless of the
tissue of origin (p < 0.001; Fig. 3C). By contrast, mean
4ß7 expression levels on T cells were 50
to 71% those of B cells in all tissues (p <
0.001; Fig. 3, B and C, and Table III). This
difference in 4ß7 expression levels was
also found for T and B lymphocytes from L-selectin-/-
mice (data not shown). Regardless of the tissue source, the mean linear
fluorescence staining intensity of CD8+ cells for
L-selectin was 29 to 40% greater than that for CD4+ cells
(Table III). However, 4ß7 expression
levels were equivalent for CD4+ and CD8+ cells.
These findings demonstrate intrinsic subset-specific differences
between T and B cell regulation of L-selectin and
4ß7 cell surface expression that are
independent of the secondary lymphoid tissue of origin.
L-selectin expression levels regulate lymphocyte migration
L-selectin expression levels on leukocytes from hemizygous
L-selectin+/- mice are approximately 50% of
wild-type levels (4). This difference is consistent for
CD4+ and CD8+ T cell and B220+ B
cell populations (Fig. 4A). Therefore,
migration of splenocytes from L-selectin+/- mice was
examined in short term in vivo migration assays to determine whether
levels of L-selectin expression significantly affect lymphocyte
migration. Calcein-labeled L-selectin+/- or wild-type
splenocytes were mixed with equal numbers of internal control
PKH26-labeled wild-type splenocytes and injected into the tail veins of
recipient wild-type mice. After 1 h, single-cell suspensions of
tissues from recipient mice were prepared. The ratio of calcein-labeled
test cells (L-selectin+/- or wild-type) to PKH26-labeled
control cells was determined for each tissue (Ro) and
normalized by dividing by the injected ratio of calcein-labeled test
cells to PKH26-labeled control cells (Ri). Migrations of
L-selectin+/- splenocytes to PLN, MLN, and Peyer’s
patches at 1 h were reduced by 69, 68, and 47%, respectively,
compared with those of wild-type splenocytes (Fig. 4B). Therefore, migrations of lymphocytes to PLN,
MLN, and Peyer’s patches correlated directly with expression levels of
L-selectin.
The cellularity of lymphoid tissues in hemizygous L-selectin+/- mice was assessed to determine whether reduced L-selectin expression results in long term migration defects. The numbers of lymphocytes in the spleens (86 ± 6 x 106), PLN (4.9 ± 0.8 x 106), MLN (6.8 ± 1.2 x 106), Peyer’s patches (1.0 ± 0.2 x 106), and blood (4.4 ± 1.1 x 106/ml) of four L-selectin+/- mice were similar to those of their wild-type littermates. The frequencies of CD4+ (29 ± 4%), CD8+ (15 ± 1%), and B220+ (47 ± 4%) cells in blood were also similar to those of wild-type littermates. Therefore, although reduced L-selectin levels delayed significantly the kinetics of lymphocyte entry into lymphoid tissues, this did not cause an obvious redistribution of lymphocyte subsets.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Table I). Nonetheless, both T and B cell migrations into PLN
and MLN were reduced severely in the absence of L-selectin. T and B
cell migration into Peyer’s patches was also predominantly L-selectin
dependent, although approximately 30% of L-selectin-/-
splenocytes still entered this tissue (Table I). By contrast, the loss
of L-selectin resulted in significantly increased T and B cell
migration into the spleen. Migration of CD4+ and
CD8+ cells was similar, and both subsets required
L-selectin expression for migration (Table II). Therefore, although T
cells migrate into all lymphoid tissues at a faster tempo than B cells,
both cell populations are predominantly dependent on L-selectin
expression for entry into these tissues.
The more efficient migration of T cells into peripheral lymphoid
tissues relative to that of B cells is partly explained by the finding
that T cells consistently expressed L-selectin at 50 to 100% higher
levels than B cells regardless of their tissue source (Fig. 3 and Table III). Conversely, T cells expressed 4ß7
integrin at significantly lower levels than B cells regardless of their
tissue source (Fig. 3 and Table III). These results contrast markedly
with previous studies showing variability in L-selectin expression
among lymphocyte subsets (34, 35). Nonetheless, intrinsic differences
in L-selectin expression levels observed on T and B cells in the
current studies correlated directly with lymphocyte migration across
HEV. A 50% reduction in L-selectin expression by lymphocytes from
hemizygous L-selectin+/- mice resulted in a 50 to 70%
decrease in short term lymphocyte migration into peripheral lymphoid
tissues relative to wild-type lymphocytes (Fig. 4). These results
demonstrate that the preferential migration of T lymphocytes into
secondary lymphoid tissues may be directly regulated by the higher
levels of endogenous L-selectin expression. Factors in addition to
L-selectin expression levels are also likely to influence
subset-specific lymphocyte migration, since a twofold reduction in
L-selectin on T cells from L-selectin+/- mice did not
reduce the level of T cell migration to that of normal B
lymphocytes.
Most circulating lymphocytes simultaneously expressed both
L-selectin and 4ß7 integrin (Fig. 2).
However, lymphocyte utilization of 4ß7 for
entry into lymphoid tissues is relatively inefficient (4, 8). In the
absence of L-selectin expression, T and B cells migrated preferentially
to the spleen rather than to MLN or Peyer’s patches (Fig. 1). In
addition, despite having lower levels of
4ß7 expression (Fig. 3 and Table III), T
cells demonstrated a fivefold higher rate of migration into Peyer’s
patches than B cells in the absence of L-selectin expression
(p < 0.004; Table I). However, higher levels
of 4ß7 expression by B cells may
facilitate B cell retention in Peyer’s patches relative to that of T
cells during 48-h migration assays (Fig. 1 and Table I). Since
lymphocyte entry into Peyer’s patches is entirely dependent upon
lymphocyte-HEV interactions (36) and L-selectin loss significantly
inhibits lymphocyte migration into Peyer’s patches in short term
assays (Fig. 1 and Table I), increased retention or utilization of
L-selectin-independent adhesion pathways must account for the
accumulation of Peyer’s patch lymphocytes in long term assays (Fig. 1)
and in L-selectin-/- mice (4, 8). Specific lymphocyte
subsets do not appear to selectively enter Peyer’s patches in the
absence of L-selectin, since Peyer’s patch lymphocytes from wild-type
and L-selectin-deficient mice predominantly have the phenotypic
characteristics of naive lymphocytes, and increased numbers of
4ß7high, CD44high,
or CD18high memory cells are not observed (our unpublished
observations) (8). Therefore, L-selectin expression is required for the
efficient migration of T and B cells into peripheral lymphoid tissues,
although other factors influence their long term localization in
Peyer’s patches.
Our recent in vivo studies examining leukocyte rolling in Peyer’s
patches of L-selectin-deficient mice and ß7
integrin-deficient mice (37) support the conclusion that L-selectin is
a dominant factor in this process. Although wild-type and
ß7 integrin-deficient mice have similar frequencies of
rolling leukocytes in Peyer’s patches, rolling flux is reduced by
about 80% in L-selectin-deficient mice. ß7 integrins
presumably mediate the rolling of the remaining cells at characteristic
slow velocities. Consistent with this, 4ß7
and 4ß1 integrins can each mediate rolling
and firm adhesion during in vitro assays (38, 39, 40). In situ studies have
also identified distinct roles for L-selectin and the
4ß7 and LFA-1 integrins in lymphocyte
binding to Peyer’s patch HEV (41). Collectively, these studies
indicate that although 4ß7 ligands are
present on Peyer’s patch HEV, these ligands are unable to support
efficient attachment and firm adhesion of most T and B cells when they
lack L-selectin. This inability probably reflects a requirement for the
simultaneous engagement of multiple adhesion receptors to support
efficient leukocyte rolling on vascular endothelium in lymphoid
tissues, as occurs at sites of inflammation (42, 50). Whether the
requirement for L-selectin expression in this process relates to its
role as a signal transduction molecule remains to be determined
experimentally (30, 43, 44).
Different patterns of B cell and CD4+ and CD8+ T cell migration have been reported for sheep, humans, rats, and mice. Many of these differences can be attributed to differences in experimental approach, differences in the assay systems used, or differences in the source of cells being assayed. However, the results of this study suggest that these variances may also relate to species-specific differences in L-selectin expression levels. There is remarkable heterogeneity in the levels and patterns of L-selectin expression by lymphocytes among different animal species (12, 30, 45, 46). Therefore, variability in published experimental results is expected given that small differences in L-selectin expression are likely to significantly affect lymphocyte migration patterns. Future studies of lymphocyte migration must take these considerations into account.
The current findings demonstrate that lymphocyte subset-specific migration into secondary lymphoid tissues is regulated in part by expression levels of L-selectin. Moreover, higher L-selectin expression by T cells is likely to be a major factor in their preferential migration to secondary lymphoid tissues relative to that of B cells. Indeed, the finding that L-selectin deficiency affects most significantly the migration of CD4+ T cells and memory T cells (8) is consistent with the findings of the current study. Regulation of L-selectin expression is clearly a complex and dynamic process, as L-selectin expression levels change rapidly in response to numerous stimuli, including cellular activation (47, 48). In addition, naive lymphocytes predominantly express high levels of L-selectin, while memory cells are more heterogeneous in L-selectin expression (8, 49). The finding that a twofold difference in L-selectin expression can dramatically alter lymphocyte migration in vivo establishes an important role for adhesion receptor expression levels in the regulation of lymphocyte migration. Consequently, our current results suggest that even relatively small changes in L-selectin expression levels may have dramatic effects on the efficiency of adhesion molecule-ligand interactions and leukocyte entry into tissues. This paradigm is likely to apply to other adhesion molecules as well.
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Footnotes |
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2 Both authors contributed equally to this study and share first authorship.
3 Address correspondence and reprint requests to Dr. Thomas F. Tedder, Department of Immunology, Box 3010, Duke University Medical Center, Durham, NC 27710.
4 Abbreviations used in this paper: PLN, peripheral lymph node; MLN, mesenteric lymph node; HEV, high endothelial venule; L-selectin-/-, L-selectin deficient; PE, phycoerythrin; 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 September 12, 1997. Accepted for publication January 21, 1998.
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References |
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+T19+ lymphocytes through peripheral lymph nodes. Immunology 70:235.[Medline]
4ß7-mediated adhesion to vascular cell adhesion molecule-1, mucosal addressin-1, fibronectin, and lymphocyte aggregation. J. Immunol. 153:3847.[Abstract]
4 integrins mediate lymphocyte attachment and rolling under physiologic flow. Cell 80:413.[Medline]
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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