By
¶
From the * Leukocyte Adhesion Laboratory and the Lymphocyte Molecular Biology Laboratory,
Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom; the § Center of
Anatomy, Medical School of Hannover, D-30623 Hannover, Germany; the
Department of
Immunology, Medical Clinic, University Hospital Eppendorf, D-20246 Hamburg, Germany; and ¶ Charité Clinic, Humboldt University, D-10117 Berlin, Germany
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Abstract |
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Using lymphocyte function-associated antigen (LFA)-1/
mice, we have examined the role of
LFA-1 and other integrins in the recirculation of lymphocytes. LFA-1 has a key role in migration to peripheral lymph nodes (pLNs), and influences migration into other LNs. Second, the
4 integrins,
4
7 and
4
1, have a hitherto unrecognized ability to compensate for the lack
of LFA-1 in migration to pLNs. These findings are confirmed using normal mice and blocking
LFA-1 and
4 monoclonal antibodies. Unexpectedly, vascular cell adhesion molecule (VCAM)-1,
which is essential in inflammatory responses, serves as the ligand for the
4 integrins on pLN
high endothelial venules. VCAM-1 also participates in trafficking into mesenteric LNs and
Peyer's patch nodes where mucosal addressin cell adhesion molecule 1 (MAdCAM-1), the
4
7-specific ligand, dominates. Both
4
1, interacting with ligand VCAM-1, and also LFA-1
participate in substantial lymphocyte recirculation through bone marrow. These observations
suggest that organ-specific adhesion receptor usage in mature lymphocyte recirculation is not as
rigidly adhered to as previously considered, and that the same basic sets of adhesion receptors
are used in both lymphocyte homing and inflammatory responses.
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Introduction |
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The integrin LFA-1 (CD11a/CD18), which belongs to
the 2 family of integrin receptors, is expressed by all
leukocytes and has a central role in the functions of these
cells. When leukocytes respond to inflammatory signals,
LFA-1 acts together with other adhesion receptors to direct
the cells into injured tissues (see references 1). Thus, the
selectin adhesion receptors allow transient leukocyte contact
with stimulated endothelium, causing a rolling movement
which is further arrested by the action of the
4 integrins.
This stage is followed by a signaling event that activates
LFA-1 to promote firm adhesion of the leukocytes and
their migration across the endothelium towards the area of injury.
In contrast, mature lymphocytes, but not myeloid cells,
circulate continually through secondary lymphoid tissue
of the peripheral lymph nodes (pLNs),1 mesenteric LNs
(mLNs), and Peyer's patches (PPs), thereby increasing the
opportunity for an encounter with antigen. To migrate
across the specialized high endothelial venules (HEVs) of
these LNs, lymphocytes have been proposed to use organ-specific sets of adhesion receptors (4, 5). Migration into
mucosal tissue such as PPs is greatly dependent on the 4
integrin,
4
7, which has no apparent role in homing to
the pLNs (6, 7). On the other hand, homing of naive lymphocytes to pLNs has been considered to be dependent
upon L-selectin (CD62L) but not to involve the
4 integrins (8, 9). However, parallels are emerging between the
processes of lymphocyte recirculation and the response to inflammatory signals. LFA-1 influences migration into mucosal tissue as well as the pLNs (10), and there is now evidence
that recirculating lymphocytes also undergo an activation
step when they contact the HEVs. LFA-1 can be activated
by signals arising from the binding of lymphocyte L-selectin
to HEV ligand, GlyCAM-1 (11). HEV-expressed chemokines B lymphocyte chemoattractant (BLC) and secondary
lymphoid tissue chemokine (SLC) cause adhesion of naive
B cells (12) and T cells (13), respectively, and SLC has been
demonstrated to activate LFA-1.
Ligands for most of the lymphocyte adhesion receptors
have been identified on the HEVs. L-selectin recognizes
peripheral node addressin (PNAd), a group of fucosylated
and sialylated LewisX-expressing proteins on pLN HEVs
(see reference 14). LFA-1 functions through recognition of
its ligands intercellular adhesion molecule (ICAM)-1 or
ICAM-2 (see reference 15). The 4
7 integrin recognizes
MAdCAM-1, which is expressed on HEVs in PPs and
mLNs (16, 17), and VCAM-1 and fibronectin. The alternative
4 integrin,
4
1, which has a major role in inflammation (18), also recognizes ligands VCAM-1 and
fibronectin. VCAM-1 is expressed by endothelia under inflammatory conditions (21, 22) and is thought to be absent
on normal LN endothelium (22, 23). Fibronectin is generally associated only with the subendothelial matrix,
but there is a report that a spliced variant is expressed in
rheumatoid arthritis tissue (24). Thus, the ligand binding
activities of the two
4 integrins are distinctive, in that
MAdCAM-1 is involved in
4
7-mediated naive lymphocyte homing to mucosal sites whereas VCAM-1 primarily serves as the ligand for
4
1 at inflammatory sites.
Recently, two studies using LFA-1-deficient mice have
confirmed many of the previous assignments of LFA-1
function, for example, in assays for T cell function such as
MLR and delayed-type hypersensitivity responses (25, 26).
In this study, we have used LFA-1/
mice to reexamine
the role of LFA-1 in the recirculation of lymphocytes and
to investigate alternative mechanisms used in its absence. We confirm an important role for LFA-1 in LN localization, identify an unexpected role for
4 integrins,
4
7
and
4
1, in pLN homing that is mediated by HEV-
expressed VCAM-1, and finally demonstrate that
4
1/
VCAM-1 together with LFA-1 is involved in migration of
lymphocytes to bone marrow.
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Materials and Methods |
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Generation of LFA-1 Mutant Mice.
A 7-kb PCR product amplified from murine 129/Sv genomic DNA and containing exon 2 of the Lfa-1 gene was subcloned into pSP72. A selection cassette containing stop codons in all three frames, an independent ribosomal entry sequence (IRES) followed by the LacZ gene with an SV40 polyadenylation signal, and a neomycin phosphotransferase gene was introduced into exon 2. Culture and transfection of GK129 embryonic stem (ES) cells was done as described previously (27). EcoRI-digested ES cell DNA was analyzed by Southern blot analysis using probe A, and homologous recombination was confirmed using probe B (see Fig. 1 a). Blastocyst injection and breeding have been described previously (27). Mice were genotyped from EcoRI-digested tail DNA by Southern blot (data not shown) or PCR (Fig. 1 b). A multiplex PCR using primers on either side of the selection cassette and one within the neomycin resistance gene yielded fragments of 90 bp (wild-type) and 660 bp (LFA-1-deficient), respectively.
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Animal Husbandry.
All mice were kept under specific pathogen-free conditions and in accordance with United Kingdom Home Office regulations. The mice were monitored on a six-monthly basis for a wide selection of parasites, bacteria, and fungi and were regularly found to be pathogen free. LN sections from sentinel mice belonging to litters used in the reported experiments were inspected for the presence of germinal centers by hematoxylin and eosin staining and for positive MAdCAM-1 staining in pLNs (28). These latter indicators of an inflammatory response were routinely negative for mice housed in London and Hamburg.mAbs and Other Reagents.
The following purified rat mAbs were used in this study: LFA-1Preparation of Cells for Flow Cytometry.
Single cell suspensions from 8-12-wk-old mice were prepared by mincing or rubbing between glass slides with frequent rinsing with 5% FCS/RPMI or 0.2% BSA/PBS. Bone marrow cells were harvested by flushing with 5% FCS/RPMI via cut ends of tibias and femurs, followed by disaggregation and filtering through nylon gauze. Cells were washed and incubated at 5 × 106 cells/ml with specific primary mAbs, then FITC-conjugated goat anti-rat Ig (1:200; Jackson ImmunoResearch Laboratories) at 4°C for 30 min. Cells were fixed in 1% HCHO/PBS before analysis on a FACScanTM instrument (Becton Dickinson).In Vivo Homing Experiments Using CellTrackerTM Fluorescent Dye-labeled Lymphocytes.
Lymphocytes from pLNs and mLNs of LFA-1+/+ and LFA-1In Vivo Homing Experiments Using 51Cr-labeled Lymphocytes.
Lymphocytes obtained as above were labeled with 20 µCi 51Cr/ml as described previously (29), and dead cells removed by centrifugation on a 17% Nycodenz cushion (Nycomed). 5 × 106 lymphocytes in 200 µl were injected into the tail vein of recipient 8-12-wk-old C57BL/6 mice. mAbs were coinjected as Fab fragments and at 300 µg/200 µl. Sample groups of four mice were used in each condition. The mice were killed after 1 h, and the distribution of radioactivity was determined for the different organs and for the residual body mass. PBLs were calculated for a blood volume of 1.5 ml.Labeling, Injection, and Detection of Donor Cells in the Host pLNs.
LN cells were labeled by dissolving 1 mg digoxigenin in 0.5 ml DMSO and incubating the lymphocytes for 15 min at a final concentration of 5 × 107 cells/ml with 60 µg digoxigenin. After washing, 0.8 × 107 cells were injected intravenously, and pLNs were removed 30 min later and frozen in liquid nitrogen. To localize the donor cells in the HEVs, two antigens were revealed simultaneously as described (31). In brief, cryostat sections were incubated with the anti-HEV mAb MECA-325, which was revealed by the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique (blue [31]). The donor cells were identified by a peroxidase-conjugated antidigoxigenin antibody (brown) as described previously (31). The area of the HEVs (excluding the lumen) together with the area within 4-cell diameters surrounding the vessel was then determined (31), and the number of injected cells adhering to and within the endothelium, and within the tissue was analyzed.Immunohistochemistry.
Sections of 6 µm were cut from snap-frozen tissues of pLN, mLN, and PP and placed on silane-coated slides which were fixed in acetone for 10 min and allowed to air dry. Slides were washed in standard Tris-buffered saline (TBS), pH 7.6, and primary mAb was added for 1 h at room temperature. mAbs were used at optimal dilution: 10 µg/ml for anti- VCAM-1 mAb MK2.7 (IgG1) and IgG1 isotype control mAb PyLT-1, MECA-79 at 4 µg/ml, and MECA-367 as tissue culture supernatant. Sections were also tested with null IgG1 and IgG2a mAbs (PharMingen; data not shown). Biotinylated rabbit anti-rat Ig (1:100; Vector Laboratories) was added for 35 min. A tertiary stage was carried out using the StreptABComplex/HRP kit following the manufacturer's instructions (K377; Dako), and the slides were incubated again for 35 min before exposure to 1 mg/ml 3,3'diaminobenzidine. ![]() |
Results |
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Lfa-1, the gene for the
LFA-1 subunit, was mutated in ES cells using a replacement-type targeting vector and the strategy shown in Fig. 1 a.
G418-resistant colonies were identified by Southern blot
analysis of EcoR1-digested ES cell genomic DNA using the probe shown in Fig. 1 a. Of 400 G418-resistant ES cell
colonies, 10 correctly targeted colonies were identified.
Three different homologously targeted ES cell clones were
injected into C57BL/6 blastocysts, and all of them were
transmitted through the germline. Southern blot analysis of
mouse tail DNA enabled identification of LFA-1 wild-type
(+/+), heterozygous (+/
), and gene-deleted status (
/
)
(data not shown). This was confirmed by PCR analysis (Fig. 1 b).
To
establish that the capacity for LFA-1 synthesis had been
completely ablated, leukocyte populations were examined
for LFA-1 expression and function. Thymocytes from
LFA-1/
mice were compared with LFA-1+/+ littermates
and shown to be devoid of LFA-1 expression (Fig. 1 c).
The lack of LFA-1 surface expression was also demonstrated for leukocytes from other lymphoid tissue, including pLN, mLN, PP, spleen, bone marrow, and blood (data
not shown). To test for absence of LFA-1 function, control
phorbol ester-treated LFA-1+/+ thymocytes were adhered
to murine ICAM-1-transfected COS-7 cells, and this adhesion could be blocked with anti-LFA-1 mAb H68 (data not shown). In contrast, no adhesion was evident with
LFA-1
/
thymocytes. Therefore, testing for both expression and function of the LFA-1 receptor provided further
proof that the LFA-1
/
mice were totally deficient in this
2 integrin.
Analysis of LFA-1/
compared with LFA-1+/+ mice revealed an increase in spleen
size and a decrease in size of the pLNs, as observed previously (25). For 30-g male mice (n = 29), the LFA-1
/
spleens were 1.7 times larger than those of LFA-1+/+ mice
(182.0 ± 56.4 compared with 107.8 ± 30.0 mg). For 25-g
female mice (n = 37), the same comparison yielded a
1.2-fold increase in weight (137.6 ± 33.0 compared with
118.5 ± 42.3 mg). Second, the pLNs from LFA-1
/
mice
were smaller than those from LFA-1+/+ mice, with an average decreased lymphocyte number for LFA-1
/
mice of
~30% that of the LFA-1+/+ littermates (3.54 ± 0.74 × 106 compared with 12.15 ± 2.90 × 106; n = 9).
To discover whether alteration had occurred in lymphocyte numbers in general or in a particular subtype, we next
analyzed CD4 and CD8 T cell subsets and used mAb B220
to detect B cells. In the pLNs, substantial loss in numbers of
CD4 and CD8 T cells as well as a deficiency in B cells was
observed (Fig. 2). Furthermore, there was no difference in
naive versus memory phenotype in LFA-1/
and LFA-1+/+
mice as indicated by expression levels of L-selectin, CD44,
and CD45RB antigens (data not shown). In the other LNs,
wild-type and LFA-1-deficient mice were similar in terms
of lymphocyte numbers and subsets, as was expected from
the fact that these LNs were comparable in size between
the two types of mice. In spleen, there was a significant increase in CD4 T cells and CD8 cells (Fig. 2).
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To gain
further information about the diminished cellularity of the
pLNs and to investigate trafficking capabilities of LFA-1-deficient lymphocytes, short-term migration studies were
performed. The approach taken was to label LFA-1/
and
LFA-1+/+ lymphocytes with two distinguishable CT orange
and green fluorescent dyes and inject equivalent numbers
into the tail vein of C57BL/6 recipients. This allowed direct comparison of the homing activity of the lymphocytes
within each LN setting (29). An example of the methodology is illustrated in Fig. 3 a. When the pLN lymphocytes were examined for proportions of CT orange to green after
1 h of homing, it was observed that ~13% of LFA-1
/
cells compared with LFA-1+/+ cells had migrated into the
pLNs (Fig. 3 a). We then examined the relative ability of
LFA-1
/
cells to gain entry into other secondary lymphoid tissues (Fig. 3 b). When compared with LFA-1+/+,
the LFA-1
/
cells were most poorly represented in pLNs
(0.21 ± 0.01), followed by mLNs (0.51 ± 0.02) and PPs
(0.68 ± 0.03). In contrast, there was a marked increase in
the LFA-1
/
cells in the spleen (1.29 ± 0.04) which is
likely due to redistribution of lymphocytes from LN to
spleen. These findings on the effect of LFA-1 absence on
the trafficking of lymphocytes were confirmed using the
technique of tracking 51Cr-labeled lymphocytes to compare
homing in LFA-1
/
and LFA-1+/+ mice (data not shown).
When CD4, CD8, and B220+ lymphocyte subsets were
analyzed separately, the ratio of migrated to injected lymphocytes was identical among the subsets whether derived
from wild-type or LFA-1
/
mice, indicating that the lack
of LFA-1 caused equal difficulty for all types of lymphocytes to gain entry into the LNs (data not shown).
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The above findings showed that LFA-1/
cells
migrated less effectively, particularly to the pLNs, within
the 1-h period but did not reveal the stage at which the
lack of LFA-1 had its effect. To discover where LFA-1 deficiency caused difficulty, we turned to histochemistry and
examined pLN tissue sections from mice injected with
LFA-1
/
and LFA-1+/+ lymphocytes for 30 min. As
shown in Fig. 4, the LFA-1-deficient lymphocytes bound
less well to HEVs than the LFA-1-expressing lymphocytes.
The total numbers of lymphocytes at the HEV level and surrounding 4-cell diameters were 285 ± 70 cells/mm2 for
LFA-1+/+ (Fig. 4, a and b) and 85 ± 10 cells/mm2 for LFA-1
/
lymphocytes (Fig. 4, c and d) (n = 3). These data suggest that loss of LFA-1 expression reduces lymphocyte adherence to and transmigration across HEVs by 70%, thereby
causing substantially diminished recruitment into pLNs.
When the cells were divided between those adhering to and
within the HEVs and those found within 4-cell diameters beyond HEVs, the following proportions were observed: for
LFA-1+/+ lymphocytes, 73 ± 7 and 27± 2%; for LFA-1
/
lymphocytes, 78 ± 1 and 22 ± 1%, respectively. These data
suggest that the major block is at the level of the HEVs. If
the deficiency had been acting selectively at the level of the
transmigration step, a larger accumulation of lymphocytes at
the HEV level would have been expected.
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Although LFA-1 was obviously playing a critical role at the
HEV level, a proportion of lymphocytes remained capable
of migrating into lymphoid tissue (Fig. 3 b, and Fig. 4). Of
other adhesion receptors that might substitute for LFA-1,
the 4 integrins were attractive candidates, as they were active
in migration in other situations. In the next series of experiments, the migratory behavior of CT-labeled LFA-1
/
and
LFA-1+/+ cells was compared in host animals simultaneously injected with Fab fragments of
4 mAb or
4
7-specific mAb. The
4 mAb completely prevented the migration of the residual numbers of LFA-1
/
lymphocytes
homing to pLNs, mLNs, and PPs (Fig. 5). In addition, the
4
7-specific mAb blocked lymphocyte entry into pLNs
to ~22% and completely prevented migration into mLNs
and PPs. There was no significant effect of
4 or
4
7
mAbs on migration into spleen.
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These results were duplicated in a 51Cr lymphocyte labeling experiment in which 4 mAb abolished all entry
into the pLNs, mLNs, and PPs of LFA-1
/
lymphocytes
(data not shown). The
4
7 mAb blocked entry into mLNs and PPs and inhibited entry into pLNs to ~35%
(data not shown). It was possible that the
4-dependent
LFA-1
/
cells migrating into pLNs were a specific subset
of lymphocytes expanded in the LFA-1-deficient environment. However, phenotyping of these cells using a third
fluorescent tag of Tricolor-conjugated anti-rat Ig showed
the migrated LFA-1
/
and LFA-1+/+ lymphocytes to have
identical phenotypic profiles with regard to their levels of
4 and
4
7 integrins and L-selectin (data not shown).
The suggestion that 4 integrins,
4
7 acting together
with
4
1, have a role in migration to the pLNs has not
previously been recognized. To further confirm the findings and to gain information about tissues other than secondary LNs, 51Cr-labeled lymphocytes from normal BALB/c
mice were coinjected with 300 µg of Fab fragments from
either
4 mAb, LFA-1 mAb, or both into host BALB/c
mice. The findings with the single mAbs were as reported
previously (7, 10; Fig. 6), but the combination of
4 and
LFA-1 mAbs totally prevented migration into pLNs as well
as mLNs and PPs (Fig. 6). There was also an appreciable
decrease in migration into intestinal tissue and into the
"body" (see below). This mAb blockade provoked an increase in circulating blood cells as well as an increase in migration into the spleen. Put together, these observations
provide evidence that the
4 integrins operating together
with LFA-1 have an essential role in migration to pLNs, as
found previously for other secondary LNs.
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The identification
of the ligand(s) recognized by the 4 integrins, particularly
in pLNs, was next explored. Using a MAdCAM-1-specific mAb, homing to both PPs and mLNs was substantially prevented, as described previously (16, 17; Fig. 5). However,
the mAb had no significant effect on migration of LFA-1
/
lymphocytes into pLNs and spleen. VCAM-1 is another
ligand recognized by both
4
1 and
4
7 integrins (32, 33),
and in the present experiments, the anti-VCAM-1 mAb
MK2.7 completely eliminated homing of LFA-1
/
lymphocytes to pLNs and also substantially blocked entry into both mLNs and PPs although not into spleen (Fig. 5).
The foregoing experiments showed that
VCAM-1 has a role in the trafficking into LNs, suggesting
that this ligand is indeed expressed on the HEVs. To further
address this question, immunohistochemical staining was
performed using fresh frozen tissue sections of pLNs, mLNs,
and PPs from LFA-1/
and LFA-1+/+ mice and normal
C57BL/6 mice. In pLNs, anti-VCAM-1 mAb MK2.7 (IgG1) was identified to label the same HEVs as PNAd-specific mAb MECA-79 (Fig. 7, a and b). Of interest was
the observation that the MAdCAM-1 mAb MECA-367
was negative, as was an IgG1 isotype control mAb, PyLT-1
(Fig. 7, c and d). In mLNs where both MAdCAM-1 and
PNAd are chiefly coexpressed on HEVs (34), VCAM-1 is also present, whereas the isotype control mAb was negative (Fig. 7, e-h). In a preliminary quantitative study of mLNs, complete
overlap was observed in HEV staining of VCAM-1 with one
other HEV ligand, MAdCAM-1 (data not shown). The level
of VCAM-1 expression was comparable on HEVs from all
LNs and from LFA-1+/+, LFA-1
/
, and C57BL/6 mice
(data not shown). Therefore the absence of LFA-1 did not induce a compensatory increase in expression of VCAM-1.
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To confirm that VCAM-1 is expressed on the luminal surface of the endothelium and therefore accessible to recirculating lymphocytes, C57BL/6 mice were intravenously injected for 10 min with rat anti-VCAM-1 mAb MK2.7 or anti-HEV mAb MECA-325. On tissue sections stained with anti-rat Ig, both the pLN and mLN HEVs scored positive to a similar degree for both the anti- VCAM-1 and the anti-HEV mAbs (data not shown). This experiment provided a second type of histochemical proof that VCAM-1 is luminally expressed on the HEVs of the LNs.
A Role forIn the migration experiments with 51Cr-labeled lymphocytes, significantly fewer lymphocytes distributed into the
mouse carcass after treatment with a combination of LFA-1
and 4 mAbs (see Fig. 6). To discover the identity of the
relevant tissue compartment, individual body parts of muscle, heart, kidney, thymus, and bone were isolated and
counted separately. The target tissue with diminished lymphocyte migration was determined to be the limb bones, indicating that lymphocyte recirculation was occurring in the
bone marrow (data not shown). Within 1 h, a single femur
recruited ~1% of 51Cr-labeled lymphocytes, suggesting that
the total bone marrow compartment attracted lymphocytes
at a rate comparable to the 5-10% lymphocyte entry into
the combined LNs. Which lymphocytes were migrating
into bone marrow was tested by using CT-labeled lymphocytes and subsequent staining with subset-specific mAbs detected with Tricolor-conjugated anti-rat Ig. The ratio of
migrated to injected CD4/CD8/B220 subsets was ~0.5/
1.0/2.0. This advantage for B cell and disadvantage for CD4
T cells were independent of LFA-1 expression status.
LFA-1+/+ and LFA-1/
cells did not significantly differ in
their trafficking to bone marrow (ratio of LFA-1
/
to LFA-1+/+ was 1.1; data not shown), suggesting that the migration
was not solely reliant on LFA-1. However, when LFA-1
/
lymphocytes were coinjected with
4 mAb, the trafficking to
bone marrow was completely abolished (Fig. 8). These results
suggest that migration into bone marrow can be accomplished either by LFA-1 or in its absence by an
4 integrin.
As the
4
7 mAb had a partial effect on migration, whereas
4 mAb blocked migration completely, we conclude that in
bone marrow, in contrast to the migration into LNs,
4
1
dominates
4
7. Finally, the VCAM-1 mAb, but not MAdCAM-1 mAb, had an inhibitory effect. Considered together, these results demonstrate that migration of recirculating lymphocytes into bone marrow can be mediated by either LFA-1
or the
4 integrins, and, as in pLNs, that VCAM-1 serves as
the chief ligand for the latter receptors.
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Discussion |
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This study shows that LFA-1 has a key role in migration to
the pLNs, other LNs, and bone marrow but not into the
spleen. Also revealed is a hitherto unrecognized ability of the
4 integrins,
4
7 and
4
1, to compensate for the lack of
LFA-1 in lymphocyte trafficking to pLNs and other lymphoid
tissues, including bone marrow. In general, these findings
highlight common features between lymphocyte homing and
the response to inflammatory stimuli and extend the validity of
the multistep model of adhesion and transmigration.
The LFA-1/
mouse described in this report has, as its key
phenotypic characteristic, pLNs that contain ~30% normal
lymphocyte numbers, as also noted previously (25). The decreased trafficking of LFA-1
/
lymphocytes to the pLNs to
~20-30% of wild-type lymphocytes suggested that LFA-1
/
lymphocytes, irrespective of which subset, had difficulty
gaining entry to the LNs. This was confirmed by microscopic
studies showing that circulating LFA-1
/
lymphocytes
bound poorly to HEVs. Migration into mLNs and PPs was
also depressed, but LN cell counts were normal, suggesting compensatory measures were in operation in these tissues
but not in pLNs. The decrease in migration of lymphocytes
to pLNs is consistent with previous work using function-blocking LFA-1 mAbs (10) and has recently been reported in
another study using LFA-1-deficient mice (35). Thus, the
general importance of LFA-1 in mature lymphocyte trafficking to secondary lymphoid tissue is confirmed.
In our studies, LFA-1/
lymphocytes were able to gain
entry into the pLNs, albeit at a lower level, suggesting involvement of further receptors. We here demonstrate these
additional receptors to be the
4 integrins. Skewed receptor usage towards increased expression of
4 integrins in
LFA-1
/
mice as a possible cause of experimental bias was
excluded (data not shown). The fact that the presence of
mAbs to both LFA-1 and
4 integrins caused complete
blockade of lymphocyte entry into normal pLNs and other
LNs strongly implies that
4 has a critical role in migration
of normal lymphocytes into pLNs. That this role for
4 integrin has not previously been observed might be because LFA-1 has a larger role than
4 integrin in adherence to
pLN HEVs than HEVs of other organs. This is in good
agreement with intravital microscopy studies which show
LFA-1 and L-selectin are the essential codependent pLN-specific adhesion pair for lymphocyte adherence to pLN
vessels (36). Put together, the data suggest that
4 integrins
have a less prominent role in adherence to pLN HEVs, but
potentially a larger role in the transmigration step.
Our data also suggest unexpectedly that 4
7 acts as the
major
4 receptor in conjunction with
4
1 in lymphocyte recirculation into pLNs. There has previously been no
evidence to link
4
7 with pLN migration, although its
dominant role in trafficking into mucosal tissue of the
mLNs and PPs is well documented (3, 17) and is confirmed
here. The integrin
4
1 has usually been associated with
inflammatory responses (18) rather than with lymphocyte recirculation. However, as these two
4 integrins might be acting either in synergy or in sequence, the extent of the contribution of
4
1 must await a potent murine CD29
blocking mAb. The results imply that the succession of adhesive events in lymphocyte recirculation is similar to that
in inflammatory responses, and that the
4 integrins in cooperation with L-selectin and LFA-1 have a more specific
and necessary role than previously perceived.
The data presented here identify VCAM-1 and not
MAdCAM-1 as ligand on pLN HEVs for 4 integrins in
spite of
4
7 involvement. The result was unexpected, as
4
1 has been identified as the major VCAM-1 binding integrin in inflammatory responses (37) in studies using transfectant (38) and cell lines (17). VCAM-1 also made a significant contribution to trafficking into mLNs and, to a lesser
extent, PPs. This cooperative activity with MAdCAM-1 was
also unexpected, as it has previously been considered that entry into these LNs was regulated only by MAdCAM-1. The
findings presented here suggest that potential roles for
4
7
in addition to
4
1 should be sought in other circumstances where VCAM-1 is the major ligand.
One explanation for having overlooked the importance
of VCAM-1 as an HEV ligand is its reported absence from
normal lymphoid tissue (22, 23, 39), with the exception of
a report on its expression on rat HEVs (40). In the present
study, expression occurred at comparable levels on HEVs
in pLNs, mLNs, and PP LNs and without obvious difference between LFA-1/
mice, their wild-type littermates,
C57BL/6, or BALB/c mice. Such broad and constant presence of VCAM-1 implies it is constitutively expressed
rather than as a consequence of an inflammatory signal or a
compensatory mechanism in LFA-1
/
mice.
The involvement of VCAM-1 raises the issue as to
whether lymphocytes can use this ligand for migration
across HEVs into the LN as well as adhesion to the luminal
surface of HEVs. In this study, the lack of LFA-1 decreased
migration across HEVs by ~25% only, suggesting involvement of other receptor/ligand pair(s) (data not shown). For
HUVECs, VCAM-1 is reported to be restricted to an apical location and therefore available for adhesion but not for
transendothelial migration (41). However, monocytes can
transmigrate HUVECs, making use of 4/VCAM-1 independently of
2 integrins (42). In mice, two major forms of
VCAM-1 have been identified, the common seven-domain
form and also a three-domain glycosylphosphatidylinositol
(GPI)-linked splice variant which is induced by inflammation (43, 44). It is of interest that in polarized epithelial cells,
the GPI-linked VCAM-1 was found apically, whereas the
seven-domain VCAM-1 was localized to the basolateral surface, where it could theoretically serve as a ligand for transmigration (45). The issue of where VCAM-1 is expressed, particularly in murine HEVs, warrants further investigation.
Migration of recirculating lymphocytes to bone marrow, a primary lymphoid tissue, has been noted in the past (46). Bone marrow serves as a site of hematopoiesis and for B cell maturation in the mammal. It can also act as a reservoir for a primary immune response in the absence of a spleen and presence of L-selectin mAb (47). In this study, we find an unexpectedly high degree of mature lymphocyte recirculation in which B cells dominate into bone marrow. We suggest here that bone marrow, but not the thymus (data not shown), should be considered as a major part of the lymphocyte recirculation network.
Migration to bone marrow is restricted by adhesion
mechanisms. Both human (48) and murine (49) hematopoietic progenitors make use of 4 integrin to lodge in the
bone marrow. The
4 integrins are also reported to be
necessary for correct lymphocyte development (50). We
show that normal lymphocyte recirculation through the bone
marrow is regulated by LFA-1 and the
4 integrins and, in contrast to the LNs,
4
1 substantially dominates
4
7. In
this context, it is again VCAM-1 and not MAdCAM-1
which acts as ligand for the
4 integrins. VCAM-1 is expressed constitutively by bone marrow sinusoidal vasculature (51, 52), and as progenitor cells have been demonstrated to roll on bone marrow endothelial VCAM-1 (48),
it can be speculated that other lymphocytes might behave
similarly. However VCAM-1 is also expressed constitutively by stromal reticular cells (53, 54), and it is possible
that lymphocytes may be retarded not only at the level of
the endothelium but also within the bone marrow matrix.
Factors produced by the stromal cells might be beneficial
for the maintenance of the recirculating lymphocytes. For
example, the interaction with VCAM-1 might suppress lymphocyte apoptosis (55). On the other hand, the incoming cells might contribute to the regulation of hematopoiesis.
In summary, the observations presented here extend and
validate the concept of a multistage adhesion response requiring selectins and integrins. Rather than exclusive LN-specific
use of receptors such as LFA-1 and L-selectin in trafficking to
pLNs or 4
7 to mucosal sites, we have presented evidence
that LFA-1 and the
4 integrins can operate in migration of
unstimulated lymphocytes to the pLNs as well as other secondary LNs and bone marrow. These general features, including the use of VCAM-1 as an
4 integrin ligand, resemble the response to an inflammatory signal. Our findings suggest a unifying hypothesis for migration of lymphocytes
across HEVs into secondary lymphoid tissue and also establish
further parallels between the activities of adhesion receptors
in the "homing" context and the mechanisms used in the
more sporadic responses to tissue injury and inflammation.
![]() |
Footnotes |
---|
Address correspondence to Nancy Hogg, Leukocyte Adhesion Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, UK. Phone: 44-171-269-3255; Fax: 44-171-269-3093; E-mail: hogg{at}icrf.icnet.uk
Received for publication 17 September 1998 and in revised form 25 February 1999.
C. Berlin-Rufenach was supported by an EMBO Fellowship. F. Otto was supported by Deutsche Forschungsgemeinschaft fellowship Ot 134/1-1. M. Mathies was supported by The Claremont Colleges, Claremont, CA. This work was funded by the Deutsche Forschungsgemeinschaft (A. Hamann) and the Imperial Cancer Research Fund.We thank Ian Rosewell for blastocyst injection, and Tracy Crafton and Peter Hagger for mouse breeding. For carrying out the histochemical staining procedures, we are indebted to Phillipa Munson and Keith Miller, Department of Histopathology, University College London Medical School, and George Elia and Richard Poulsom, Histopathology Laboratory, Imperial Cancer Research Fund. We also gratefully acknowledge the statistical help of Henry Potts, Imperial Cancer Research Fund Medical Statistics Group, Oxford. We thank Britta Engelhardt for the murine ICAM-1 construct; and Mark Singer, Steve Rosen, Fumio Takei, and Michel Pierres for mAbs.
Abbreviations used in this paper CT, CellTrackerTM; ES, embryonic stem; HEV, high endothelial venule; HUVEC, human umbilical vein endothelial cell; ICAM, intercellular adhesion molecule; MAdCAM-1, mucosal addressin cell adhesion molecule 1; pLN, peripheral lymph node; PNAd, peripheral node addressin; PP, Peyer's patch; VCAM, vascular cell adhesion molecule.
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