By
From the * Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford
University, Stanford, California 94305, and the Veterans Affairs Palo Alto Health Care Systems,
Palo Alto, California 94304; and The Center for Blood Research and the Department of Pathology,
Harvard Medical School, Boston, Massachusetts 02115
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Abstract |
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To characterize the adhesion cascade that directs lymphocyte homing to peripheral lymph
nodes (PLNs), we investigated the molecular mechanisms of lymphocyte interactions with the
microvasculature of subiliac lymph nodes. We found that endogenous white blood cells and
adoptively transferred lymph node lymphocytes (LNCs) tethered and rolled in postcapillary
high endothelial venules (HEVs) and to a lesser extent in collecting venules. Similarly, firm arrest occurred nearly exclusively in the paracortical HEVs. Endogenous polymorphonuclear
(PMNs) and mononuclear leukocytes (MNLs) attached and rolled in HEVs at similar frequencies, but only MNLs arrested suggesting that the events downstream of primary rolling interactions critically determine the specificity of lymphocyte recruitment. Antibody inhibition studies revealed that L-selectin was responsible for attachment and rolling of LNCs, and that LFA-1 was essential for sticking. LFA-1-dependent arrest was also abolished by pertussis toxin, implicating a requirement for Gi
-protein-linked signaling.
4 integrins, which play a critical role
in lymphocyte homing to Peyer's Patches, made no significant contribution to attachment,
rolling, or sticking in resting PLNs. Velocity analysis of interacting LNCs revealed no detectable contribution by LFA-1 to rolling. Taken together, our results suggest that lymphocyte- HEV interactions within PLNs are almost exclusively initiated by L-selectin followed by a G
protein-coupled lymphocyte-specific activation event and activation-induced engagement of
LFA-1. These events constitute a unique adhesion cascade that dictates the specificity of lymphocyte homing to PLNs.
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Introduction |
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The surveillance of the body for foreign antigens is a critical function of the immune system. Lymphocytes migrate from the blood into tissues and secondary lymphoid organs, and return back to the blood via lymph vessels and the thoracic duct (1). The majority of lymphocytes are capable of tissue selective trafficking (termed "homing"), recognizing organ-specific adhesion molecules on specialized endothelial cells (2). Tissue-specific homing is thought to control the extent and scope of immune responses, and to account for the regional compartmentalization of immune system functions (3).
A multistep model for leukocyte homing has been proposed (4), and aspects of it have been confirmed by various in vitro and in vivo models (7). Leukocytes are specifically recruited by combinatorial molecular events: blood-borne cells use primary adhesion molecules to tether and roll on the luminal wall of postcapillary venules, and to encounter and rapidly transduce signals to upregulate secondary adhesion molecules, which then mediate firm arrest. Thus, a variety of adhesive and/or signaling events are used in unique combinations to achieve tissue and subset specificity of homing.
In situ microscopy experiments have demonstrated that
such a multistep model applies to lymphocyte homing to
Peyer's patches (PPs)1. Bargatze et al. found that initial attachment in PPs is L-selectin-dependent, and that established rolling involves both L-selectin and 4
7 integrins
that interact with the mucosal addressin, MAdCAM-1 (11). Recent studies in gene-targeted mice have confirmed these
distinct but overlapping functions for L-selectin and
4
7
(12, 13). Subsequent arrest of rolling cells is mediated by
4
7 and LFA-1 integrins, and requires a G protein-linked
signal that is inhibited by lymphocyte treatment with pertussis toxin (PTX; reference 9). Thus, in PPs, lymphocyte
homing requires a cascade of four distinct molecular steps.
A similar multistep adhesion cascade may govern lymphocyte homing to peripheral lymph nodes (PLNs). For example, L-selectin deficiency in mice or treatment of wild-type animals with mAbs to L-selectin or peripheral node addressin (PNAd), the high endothelial venule (HEV)-specific ligand for L-selectin, resulted in impaired lymphocyte homing to PLNs (12, 14). Anti-LFA-1 mAbs also reduced homing to PLNs, and PLNs in LFA-1-deficient mice were reportedly smaller than those in heterozygous littermates (19, 20). Finally, treatment of lymphocytes with PTX inhibited migration to PLNs (21), and phenotypically mature thymocytes expressing transgenic PTX homed poorly in adoptive transfer experiments (22). Thus, lymphocyte homing to PLNs appears to require at least three molecular events: L-selectin binding to PNAd; G protein- mediated signaling; and engagement of LFA-1. The spatial and temporal coordination of these events during lymphocyte-HEV interactions has not been examined in vivo.
We have used a novel technique to visualize and dissect interactions of lymphocytes with HEVs in subiliac PLNs of mice. Our findings confirm that lymphocyte homing to PLN-HEVs involves a coordinated multistep process, define important differences from previously described adhesion cascades that mediate homing to other tissues, and help explain the unique recruitment of lymphocyte subsets to PLNs.
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Materials and Methods |
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Reagents.
FITC-dextran (10 mg/ml; 150 kD mol mass; Sigma Chemical Co., St. Louis, MO) and rhodamine 6G (2 mg/ ml; Molecular Probes, Inc., Eugene, OR) were diluted in saline and undissolved particles were removed by centrifugation; both reagents were freshly prepared for each experiment. 2Monoclonal Antibodies.
mAbs Mel-14 (rat IgG2a, anti-murine L-selectin; reference 14) and MECA 79 (rat IgM, anti-PNAd; reference 15) were generated in our laboratories. Hybridoma cells for these mAbs as well as mAbs Tib 213 (rat IgG2b, anti-murine CD11a; reference 24) and PS/2 (rat IgG2b, anti-murineLymphocyte Isolation and Treatment.
Lymph node cells (LNCs) were freshly isolated from PLNs (axillary, brachial, cervical, and subiliac) and mesenteric (M) LNs. Cells were washed and resuspended to 1-2 × 107 cells/ml in warm cRPMI. Representative samples were stained with FITC-conjugated mAbs to T and B cell markers (CD3 and B220, respectively) and determined by flow cytometry to be ~95% lymphocytes. For fluorescent labeling, 10 ml of LNCs was incubated with 5 µl BCECF for 20 min at 37°C. Lymphocytes were centrifuged through 5 ml of warm bovine calf serum and supernatant was discarded. The pellet was resuspended to 1-2 × 107 cells/ml in warm cRPMI. Aliquots of LNCs were pretreated with one or more of the following reagents: various mAbs (see above); PTX, which inactivatesAnimals.
We used adult BALB/c mice (Charles River Laboratories, Wilmington, MA) of both sexes (20-30 g body wt). Mice were housed in a viral antigen-free barrier facility, and kept on sterilized lab chow and water. All experimental procedures comply with National Institutes of Health (Bethesda, MD) guidelines for the care and use of laboratory animals and were approved by the Standing Committees on Animals of Harvard Medical School and The Center for Blood Research.Subiliac LN Preparation and Intravital Microscopy.
Mice were anesthetized by intraperitoneal injection (10 ml/kg) of physiologic saline containing xylazine (1 mg/ml) and ketamine (5 mg/ml). The left subiliac LN was prepared for intravital microscopy as previously described (25). In brief, polyethylene catheters were inserted in the right jugular vein and the left carotid artery. A catheter in the right femoral artery was used for injection of cell suspensions and mAbs. The left subiliac LN was exposed and dissected free of fat tissue. The node was positioned over a microscope slide on a plexiglass stage and kept moist with sterile saline. Sterile vacuum grease and a glass cover slip were applied to enclose the preparation for microscopic viewing. The stage was then placed on an intravital microscope (IV-500; Mikron Instruments, San Diego, CA) equipped with a silicon-intensified target camera (VE1000SIT; Dage mti, Michigan City, IN). The node was visualized by bright field transillumination or epifluorescence from a video-triggered Xenon arc stroboscope (Chadwick Helmuth, El Monte, CA). Video images were recorded using a time base generator (For-A, Montvale, NJ) and a Hi8 VCR (Sony, Boston, MA). Some experiments included injection of FITC-dextran solution (2 mg/ml, 10 ml/kg body wt) after recordings of cell behavior to determine vessel structure, luminal diameter, and venular order. The latter was determined by counting successive generations of branches upstream from the epigastric vein as previously described (25).Analysis of Endogenous White Blood Cell Behavior.
Circulating white blood cells (WBCs) were labeled by intravenous injection of rhodamine 6G (20 mg/kg body wt). This fluorophor stains nuclei and mitochondria. Thus, it allows visualization of myeloid and lymphoid cells as well as (the more faintly labeled) platelets in the circulation, whereas RBCs remain unstained. The distinct nuclear staining patterns of polymorphonuclear (PMNs) and mononuclear leukocytes (MNLs) made it possible to distinguish between these two cell types. An ×100 oil immersion objective with adjustable numerical aperture (Carl Zeiss, Inc., Thornwood, NY) was focused on the upper luminal surface of superficially located HEVs. Using this approach, we determined the nuclear shape of rolling and sticking cells in the plane of focus. Video analyses performed by different blinded observers yielded essentially identical results, indicating that this method is feasible and reproducible. Between 16 and 83 consecutive rolling cells per venule were categorized as PMNs and MNLs, respectively. Results are expressed as frequency of PMNs and MNLs per 100 identifiable cells that rolled or were stuck (i.e., cells remaining stationary forAnalysis of BCECF-labeled LNC in PLN-HEVs.
Small boluses (10-50 µl) of BCECF-labeled LNCs (1-2 × 107 cells/ml) were retrogradely injected via the femoral artery catheter. LNC behavior in PLNs was recorded through ×10 or ×20 water immersion objectives (Achroplan, Carl Zeiss Inc.). Cell behavior in individual vessel segments was determined by playback of video tapes. Cells were considered noninteracting when they moved at the velocity of RBCs, whereas detectably lower velocities were defined as rolling. Cells were considered interacting whether they rolled, arrested, or both. The rolling fraction is the percentage of cells that interact with a given venule in the total number of cells that enter that venule during the same period. The sticking fraction is the percentage of interacting cells that become immobile forStatistical Methods.
Data in graphs and tables are shown as mean ± SD, unless otherwise indicated. For statistical comparison of two samples, a two-tailed Student's t test was used when applicable. Multiple comparisons were performed employing the Kruskal-Wallis test and Bonferoni correction of P. Velocity histograms were compared using the Mann-Whitney U-test and the Kolmogorov-Smirnov test. Differences were considered statistically significant when P <0.05. ![]() |
Results |
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Nuclei of endogenous WBCs were labeled with rhodamine 6G to study the behavior of PMNs and MNLs in subiliac PLNs (Fig. 1). In agreement with studies of other organs, we observed heterogeneous adhesive behavior in venules, whereas arterioles or capillaries did not support interactions. High-power microscopy of superficial HEV segments allowed us to distinguish rolling and/or sticking MNLs and PMNs in the plane of focus. Thus, although some cells could not be categorized when they were out of focus, it was possible to determine the relative frequency of subset behavior. Of all the identifiable rolling WBCs in HEVs (300 cells in 7 HEVs of 3 animals) the frequency of MNLs was 30%; the remainder (70%) were PMNs. The proportion of PMNs in the rolling population was somewhat higher than their frequency in systemic blood (Fig. 2); differential blood counts showed that 44 and 56% of circulating WBCs were PMNs and MNLs, respectively (the frequency of PMNs is slightly elevated over normal levels for mice, and most likely reflects release of neutrophils from the marginating pool due to surgical trauma). We conclude that the ability to tether and roll within HEVs is not specific for lymphocytes, but is shared by other leukocyte subsets, including neutrophils.
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A subpopulation of initially rolling cells eventually arrested, adhering firmly. This behavior was almost exclusively limited to MNLs, whereas PMNs continued to roll and, eventually, detached from the vessel wall. Indeed, 23.2 ± 8.9% (mean ± SD) of rolling MNLs became stuck during their observable passage through HEVs, whereas only 0.5 ± 0.6% of PMNs displayed this behavior (data not shown). Similarly, a survey of firmly adherent cells (i.e., cells that were already stuck when the recording was started and did not move for at least 30 s thereafter) in HEVs revealed that nearly all of these cells were MNLs as well (10 HEVs in 3 animals).
Behavior of Adoptively Transferred LNCs in PLN-HEVs.Although in situ staining of WBCs permitted the differentiation of the nuclear shape of rolling cells in segments of some HEVs, rhodamine 6G labeling did not allow us to perform a selective and quantitative analysis of lymphocyte behavior in the entire PLN microcirculation. Therefore, we assessed the adhesive behavior of pooled lymphocytes that were isolated from peripheral and mesenteric nodes and stained in vitro with the intracellular fluorophor BCECF. Labeled LNCs were retrogradely injected through the right femoral artery and observed during their passage through the left subiliac PLN.
LNC interactions mirrored the behavior of the endogenous MNLs described above. A fraction (~30% on average) of LNCs did not interact with the venular walls, but the majority of cells rolled, especially in higher order venules. A small fraction of cells exhibited either multiple interactive behaviors (e.g., momentary stops or hesitations, skipping, variable rolling speeds) or transient arrest and release (preceded and/or followed by rolling). A significant number of rolling LNCs (up to 30% in some HEVs) subsequently became firmly adherent. Less than 1% became stationary immediately upon entry into HEVs, a site where microvascular hemodynamics and endothelial cell properties may change abruptly.
In contrast to rhodamine 6G staining of WBCs, which requires high magnification for reliable detection of cells, BCECF-labeling confers significant brightness and, therefore, allows visualization of labeled cells at lower magnification (×10 objective) and within a larger field of view. This permits parallel evaluation of LNC behavior within different segments of the nodal venular tree. A recent analysis of the microvascular anatomy of murine subiliac PLNs has shown that up to five branching orders can be identified in a typical venular tree (25). One (or more) order I venule is located in the hilus of subiliac PLNs and drains into a branch of the extralymphoid superficial epigastric vein. Order I venules are formed by the merging of smaller collecting venules (order II) in the nodal medulla that collect blood from higher order postcapillary venules (orders III-V) in the sub- and paracortex. To determine if there are segmental differences in lymphocyte recruitment, we correlated rolling and sticking fractions, as well as the accumulation of sticking LNCs, with the venular order (Fig. 3 A). In agreement with previous studies using L-selectin- transfected lymphoma cells (25), we found that LNCs were able to attach and roll in all venules draining the node proper, but rolling fractions were significantly larger in higher order venules (Fig. 3 B). Likewise, arresting cells were much more frequently observed in the highest order venules (Fig. 3 C). The paracortical orders III-V HEVs also supported the majority of long-term sticking, resulting in the preferential accumulation of LNCs in these venular segments (Fig. 3 D). A significant number of HEVs were noted whose lumen widened abruptly where several capillaries converged in the paracortex. This transition results in an immediate slowing of cells, and may lead to a local disruption of smooth blood flow, which in turn may aid in promoting lymphocyte-HEV interactions. It was primarily at these sites that rare lymphocytes became stuck without discernible rolling preceding the arrest.
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To investigate the contribution of L-selectin to localization of lymphocytes in PLNs, we pretreated LNCs
with Mel-14, a function-blocking anti-murine L-selectin
mAb. The antibody effect was strikingly complete, inhibiting nearly all interactions of labeled LNCs with the PLN
vasculature (Fig. 4). This differs significantly from previous
intravital microscopy studies in murine PPs, in which anti-
L-selectin only partially reduced interacting fractions (11,
13). In this site, the integrin 4
7 can also contribute to
lymphocyte tethering and rolling. Thus, we tested whether
integrins were similarly able to support LNC rolling in
PLNs. No significant effect on the frequency of rolling was
found with neutralizing antibodies to LFA-1 or
4 integrins.
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The 4
7 integrin component of
LNC rolling in PPs was found to strengthen rolling interactions and to reduce lymphocyte rolling velocity (11, 13).
We asked whether there might be a similarly significant
contribution by
4 or LFA-1 integrins to the strength of
LNC rolling interactions in PLNs. To address this question, we compared microvascular hemodynamics and Vroll
histograms by measuring velocities of at least 35 consecutive (
15 rolling and
20 noninteracting) cells in typical
order III HEV before and after antibody treatment (Table
1). Video analysis allowed us to make precise comparisons
of Vroll within the same vessels before and after mAb treatment (Fig. 5, A and B). No significant mAb effect could be
ascertained.
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Since Gi-linked signaling pathways are known to be involved in physiologic integrin activation and lymphocyte
homing to PLNs, we also measured the Vroll of LNCs in
which these signaling molecules had been blocked by pretreatment with PTX. Excluding those cells that arrested,
there was no significant difference between Vroll profiles of
LNCs treated with functionally active PTX or the inactive mutant form, MTX, or with untreated cells (Fig. 5 C and
data not shown).
Isolated LNCs and recipient mice were pretreated
with anti-CD11a antibody to block LFA-1-dependent adhesion, and cell behavior was observed within subiliac PLNs.
The mAb treatment essentially abrogated LNC sticking in
all venular orders (Fig. 6). Importantly, sticking was also
abolished when LNC rolling was blocked by treatment with anti-L-selectin mAb Mel-14, even when LFA-1 function was not blocked. Treatment with either anti-L-selectin or anti-LFA-1 also eliminated the behavior of the small
subset of LNCs that seemed to arrest immediately upon entry into HEVs, without discernible rolling (data not shown).
In contrast, anti-4 integrin antibody had no statistically
significant effect on LNC sticking.
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Our findings strongly suggest that LFA-1 is responsible for firm LNC arrest, but not
for initial rolling interactions. This integrin is known to require cell activation signals to become functional (27). Thus,
it seemed likely that a selective activation event was operating to mediate the transition from L-selectin- to LFA-1-
dependent adhesion in rolling LNCs. We tested the hypothesis that this activation step involved PTX-sensitive
signaling pathway(s). The effect of PTX treatment was
similar to that of anti-CD11a, in that LNCs were able to
attach and roll normally in PLN-HEVs (Fig. 7 A), but were
unable to arrest (Fig. 7 B). In contrast, mutant inactive PTX
(MTX) did not inhibit LNC sticking. The mutant form of
PTX contains two amino acid substitutions (Arg9 Lys and
Glu129
Gly) and retains the ability to interact with cell
membranes, but lacks the enzymatic ability to ADP-ribosylate
i subunits of heterotrimeric G proteins (23). Thus, it
seems likely that the blockade of sticking by PTX was due
to inactivation of G protein-linked signaling, and not due
to intrinsic surface modifications and/or steric hindrance of
requisite surface molecules.
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Discussion |
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We have examined the interactions of blood-borne leukocytes with PLN-HEVs in order to dissect the molecular mechanisms of lymphocyte homing to PLNs. The experimental data suggest that an organ-specific adhesion cascade operates within resting PLNs that is selective for and efficient in recruiting naive lymphocytes, while excluding myeloid cells or lymphocyte populations that express little or no L-selectin (Fig. 8). Although L-selectin mediates attachment and rolling interactions in PLN-HEVs, LFA-1 was required for subsequent lymphocyte sticking. Adhesion via LFA-1, but not L-selectin, was inhibited by PTX, indicating that heterotrimeric G protein signaling is required for affinity upregulation of LFA-1. This cascade of molecular-adhesive events in PLNs is similar to, but molecularly distinct from, those previously characterized for lymphocyte homing to PPs and intestinal lamina propria, and for granulocyte accumulation in other organs (11, 28, 29).
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Previous ultrastructural and histologic studies of the PLN subcortex and medulla have suggested that lymphocyte extravasation may occur preferentially within paracortical venules that feature specialized luminal morphology. These post-capillary vessels (corresponding to order IV and V venules in subiliac LNs) are lined by high cuboidal endothelial cells. Further downstream, endothelial cells are arranged in an overlapping plate pattern followed by a smooth layer of flat endothelial cells that is indistinguishable from nonlymphoid microvessels (corresponding to the lining of order I and II venules; references 1, 30, 31). The counter-receptors for L-selectin and LFA-1 are found in abundance on the luminal surface of postcapillary HEVs, and in particular on ridgelike structures that may further facilitate or reinforce cellular interactions (32, 33). Our results provide a functional correlate for these morphologic observations: leukocyte rolling and lymphocyte sticking were spatially regulated, occurring preferentially in high order subcortical venules. Thus, our studies confirm those of Marchesi and Gowans who proposed over 30 years ago that lymphocyte homing is a unique feature of HEV in the nodal paracortex (31).
Early experiments in mice demonstrated the inhibition
of lymphocyte homing to PLNs by treatment with mAb
Mel-14 to murine L-selectin and thus L-selectin has been
dubbed the lymphocyte homing receptor (14). In addition
to its well-known function as a mediator of leukocyte rolling, recent studies have shown that L-selectin ligation can
induce intracellular signaling events (34, 35) and that extensive antibody- or ligand-induced cross-linking of L-selectin
can trigger functional upregulation of 2 integrins in neutrophils and lymphocytes (36, 37). Our analysis of LNCs and
endogenous leukocyte behavior demonstrates that L-selectin is both necessary and sufficient for tethering and rolling
of LNCs in all venular orders, but suggests that L-selectin
engagement alone is insufficient to induce sticking.
On rolling cells, L-selectin engagement is presumably
brief and restricted to the small fraction of total L-selectin
molecules within the area of contact with the vessel wall.
Our results show that L-selectin-mediated rolling of PMNs
in HEVs was rarely followed by sticking, and a detailed
analysis of Vrolls suggests that the strength of LNC rolling
interactions was not influenced by LFA-1 or 4 integrins.
Moreover, L-selectin signaling has been reported to be insensitive to PTX (34), whereas LNC sticking in HEVs was
essentially abrogated by PTX pretreatment. Thus, L-selectin signaling, if it occurs during lymphocyte-HEV interactions, does not appear sufficient to mediate arrest or alter
rolling through integrin activation. However, our studies
do not exclude the possibility that L-selectin signaling may
synergize with other activation pathways that involve PTX-sensitive G proteins.
Lymphocytes from noninflamed lymph nodes are primarily
naive T and B cells that are characteristically L-selectinhi,
LFA-1+, and 4Io (38, 39). Consistent with this specialized
array of surface adhesion molecules, naive lymphocytes are
the predominant subset that enters PLNs via HEVs (39,
40). The finding that LNC accumulation in murine PLNs
is dependent on L-selectin implies that lymphocytes that do
not express L-selectin, or express only low levels, may be
excluded from homing to PLNs because they cannot tether
and roll along the vessel wall. L-selectinlow lymphocyte
populations may include many recently activated cells as
well as a sizable fraction (~30%) of circulating memory cells (38, 39). On the other hand, these results suggest that, unless precluded by the existence of naive cell-specific activating factors, memory cell subsets that express L-selectin and
LFA-1 should be capable of homing to PLNs via the HEV
route. Indeed, recent short-term homing studies of splenic
memory T cell subsets indicate that a significant number of
memory cells retain a significant capacity to home via PLN-HEVs in mice (39).
The concept that L-selectin is essential for lymphocyte traffic to PLNs is also in agreement with the phenotype of L-selectin-deficient mice, which display an ~90% reduction in the size and cellularity of PLNs due to a greatly reduced influx of lymphocytes via HEV (12, 16). The majority of resident lymphocytes in the PLNs of these animals display a memory phenotype, and it has been proposed that these cells arrive in PLNs predominantly via afferent lymph vessels (18). However, it is possible that some L-selectin-deficient cells may reach PLNs after adhering to HEVs through an alternative pathway. We have previously shown that circulating activated platelets can bridge between L-selectin deficient lymphocytes and HEVs because platelet P-selectin mediates platelet tethering to both P-selectin glycoprotein ligand 1 (PSGL-1) on lymphocytes and PNAd on HEVs (41, 42). Consistent with this idea, we have previously shown that the rolling of endogenous WBCs in wild-type mice was not completely inhibitable by anti- L-selectin mAb (41). Moreover, ~10% of the WBCs that passed through PLNs in L-selectin-deficient mice rolled in HEVs (42). The fact that LNCs did not display L-selectin- independent rolling in our experiments does not contradict this platelet delivery concept. It is unlikely that LNCs had significant numbers of platelets on their surface because they were directly isolated from LNs and washed extensively before injection, and LNC behavior was only analyzed for a short time (seconds to minutes) after intraarterial injection. This period may not have been sufficient to allow significant association with activated platelets which may be infrequently encountered in the blood stream.
Lymphocyte homing to PLNs in LFA-1-deficient mice has not been directly examined as yet. However, PLNs in these mice are reportedly smaller than those of wild-type littermates (20). Moreover, it has been reported that although systemically-induced delayed type hypersensitivity responses are apparently normal in the absence of LFA-1 (43), recall responses to cutaneously applied antigen are severely impaired in LFA-1-deficient mice (20). This phenotype is similar to that of L-selectin-deficient mice in which an inability to mount a cutaneous hypersensitivity response was shown to be due to the lack of naive lymphocyte homing to the draining PLNs where cutaneous antigens are presented (16, 17). Our findings demonstrating LFA-1 involvement in lymphocyte arrest in PLN-HEVs may provide an explanation for these observations, suggesting that the inefficient cutaneous response in LFA-1-deficient mice may reflect a defect in normal sensitization of lymphocytes within PLNs.
LFA-1 exists in an inactive state on resting LNCs, but its
ligand binding activity may be rapidly modulated by a variety of mechanisms (27, 44). In addition to L-selectin cross-linking (discussed above), several distinct signaling pathways
have been implicated in 2 integrin activation, including ligation of lymphocyte antigen receptors and chemoattractant
receptors that signal through heterotrimeric G proteins.
The latter include receptors for physiologic chemoattractants such as chemokines, a class of receptors whose signaling is both rapid and sensitive to inactivation by PTX (46, 47).
We found that LFA-1-dependent firm adhesion of
LNCs in PLN-HEVs was PTX-sensitive, analogous to prior
studies of LNC homing to PPs (9, 11). However, the
PTX-induced inhibition of LNC sticking in PPs was reversible by LNC activation with PMA, a phorbol ester that
directly activates protein kinase C (9). We attempted but
were unable to circumvent the effects of PTX inhibition
on LNC sticking in PLN-HEVs by PMA pretreatment of
LNCs before injection. PMA has been shown to upregulate the affinity of leukocyte integrins, including LFA-1 (48)
and 4
7 (49), but PMA treatment also causes rapid shedding of L-selectin and resulted in a dramatic reduction in
LNC rolling in PLN-HEVs (reference 50 and data not
shown). We performed additional in vitro studies using a
parallel plate flow chamber (41) and affinity-isolated PNAd
and intercellular adhesion molecule (ICAM)-1, endothelial
ligands for L-selectin and LFA-1, respectively. PMA induced equivalent sticking of both PTX and mutant toxin-
treated LNCs to ICAM-1 under static conditions or at low
shear (1.6 dyne/cm2) within a narrow temporal window
(4.5-6 min after activation, our unpublished observation).
Surface-expressed L-selectin was concomitantly downregulated, resulting in a failure of most cells to tether to the coated
substrate at higher shear rates or at later time points. In contrast to PLNs, primary adhesion of LNCs in PPs can be alternatively mediated by either L-selectin or
4
7 (11). Thus,
the ability of PMA-activated, PTX-treated LNCs to stick
in PP-HEVs, but not PLN-HEVs, was most likely due to
the differential contribution by
4
7 to primary adhesion
and may also have resulted in more efficient sticking in PP-HEVs afforded by the synergy of activated
4
7 and LFA-1.
Taken together, our results suggest that rolling lymphocytes are activated in PLN-HEVs by one or more potent
adhesion-triggering chemoattractant(s) that exert their effect
via Gi-coupled lymphocyte surface receptor(s). Our observations suggest that unless one postulates signals occurring before lymphocyte entry into HEVs, the signaling event
must be capable of triggering LFA-1 in less than 1 s. Interestingly, we noted LNCs to be extraordinarily sensitive to
temperature effects: LNC preparations that were not carefully kept at 37°C before intraarterial injection rolled normally in HEVs, but were incapable of sticking during their
initial passage through HEVs (our unpublished observation).
This indicates that the efficient detection and processing of
the chemoattractant signal and/or the subsequent integrin
response are critically dependent on unperturbed cellular
metabolism. While the identity of the lymphocyte chemoattractant(s) in PLN-HEVs remain(s) to be determined, our
findings do not exclude the possibility that signals which
upregulate LFA-1 affinity may be sufficient for arrest, but
insufficient for subsequent diapedesis.
Our results imply that the recruitment of (mostly naive) lymphocytes to PLNs is also controlled in part by a selective ability to recognize specific chemoattractant(s). Nearly all granulocytes and 70-80% of all circulating lymphocytes (constituting the majority of MNLs) express L-selectin (51). The observation that rolling PMNs in PLN-HEVs were slightly more frequent than MNLs when compared to their respective frequency in the blood stream is consistent with the differential prevalence of L-selectin+ cells in the two subsets. However, as originally noted by Lewinsohn et al., the distinction between granulocyte and lymphocyte recruitment to PLNs is not based on differential engagement of the homing receptor L-selectin (51). Although both PMNs and MNLs are known to express LFA-1 in an inactive or low-affinity conformation (27), we observed that endogenous MNLs, but not PMNs, could firmly arrest in PLN-HEVs. This implies that the HEV-specific activation signal was selectively detected by rolling lymphocytes, but not by myeloid cells. Though variations in homing receptor expression reflect differentiation of lymphocyte subpopulations (and critically impact recirculatory routes), the possible role that chemoattractants may play in regulating subpopulation homing (e.g., naive versus memory lymphocytes) remains to be determined.
The data presented here describe lymphocyte homing to
PLNs as similar to, but distinct from, the multi-step adhesion cascade in PPs, which requires lymphocyte 4
7 interaction with endothelial MAdCAM-1. In this site,
4
7
plays a critical bridging role, slowing the L-selectin-initiated
rolling of cells sufficiently to allow LFA-1 engagement. In
addition,
4
7 can also mediate tethering (albeit less efficiently than L-selectin), thus allowing some lymphocytes to
home to PPs in an L-selectin-independent manner. Indeed,
although anti-L-selectin greatly reduced the total leukocyte rolling flux in PP-HEVs (65-90%), the fraction of lymphocytes that entered PP-HEVs and subsequently became stuck
was reduced by only 35% (11, 13). Although lymphocyte
homing to rat PLNs has been reported to involve
4 integrins, and expression of the
4 integrin ligand VCAM-1
has been found in rat PLN-HEVs (52), most HEVs in uninflamed adult murine and human PLNs were reported to
express little, if any, MAdCAM-1 or vascular cell adhesion molecule 1 (VCAM-1; 53-55). This is consistent with our
present findings and with earlier homing studies using anti-
4 mAbs or
7-deficient animals (13, 56, 57).
The apparent lack of a requisite bridging adhesive interaction between L-selectin and 2 integrin engagement in
PLNs may reflect the much higher density of L-selectin
ligands on PLN-HEVs, which alone may allow sufficiently
strong and slow rolling interactions (15). Our analysis of
hemodynamic parameters presented here and in an earlier
report (25) indicate that, on average, Vblood (1.1 ± 0.5 mm/s
versus 3.2 ± 0.2 mm/s) and WSR (228 ± 75 s
1 versus
418 ± 39 s
1) are significantly lower in PLN venules than
those encountered in PP-HEVs (58), yet the Vroll is similar
(11, 58). Although the requirement for
4
7 in PP homing may reflect the greater need for strengthened rolling
adhesions to allow sufficient slowing for LFA-1 engagement, the dissimilar requirements of PLN and PP homing
clearly provide a mechanism for the differential recruitment of compartmentalized lymphocyte subsets (39).
However, our results do not formally exclude the existence of additional intermediate adhesive interactions in PLNs. In addition to the platelet delivery mechanism (discussed above), a number of other receptors have been implicated in PLN homing, such as the hyaluronate receptor CD44 and the mucin CD43 on lymphocytes, and vascular adhesion protein 1 (VAP-1) expressed on human PLN-HEVs (59). On the other hand, the strikingly complete inhibition of LNC rolling and sticking by mAbs to L-selectin and LFA-1, respectively, suggests that any additional molecular adhesive pathways (other than platelet P-selectin) are unable to confer detectable mechanical stability to either of these events under the experimental conditions employed here.
In conclusion, we have demonstrated a unique adhesion cascade mediating lymphocyte homing to PLN-HEVs. We have confirmed the site of lymphocyte adhesion in PLNs as high-order subcortical post-capillary venules, and have refined the understanding of leukocyte adhesive behavior therein. The critical roles of L-selectin for attachment and rolling and affinity-upregulated LFA-1 for firm adhesion in normal lymph nodes have profound implications for the recirculatory routes of naive and memory lymphocyte populations, and illustrate the singular role that chemoattractant signals play in recruiting or excluding leukocyte subsets from specialized tissues.
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Footnotes |
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Address correspondence to Dr. Ulrich H. von Andrian, The Center for Blood Research, 200 Longwood Ave., Boston, MA 02115. Phone: 617-278-3130; Fax: 617-278-3190; E-mail: uva{at}cbr.med.harvard.edu
Received for publication 18 August 1997 and in revised form 19 November 1997.
1 Abbreviations used in this paper: BCECF, 2We wish to thank Guiying Cheng and Brian Fors for outstanding technical support, Dr. Rino Rappuoli for providing MTX, and Jay Mizgerd for critical reading of the manuscript.
This study was supported by National Institutes of Health grants HL-54936 and HL-56949 (to U.H. von Andrian), and GM-37734 and AI-37832 (to E.C. Butcher).
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