Molecular Mechanisms of Lymphocyte Homing to Peripheral ...

Molecular Mechanisms of Lymphocyte Homing to Peripheral
Lymph Nodes

By R.Aaron Warnock,* Sanaz Askari,‡ Eugene C. Butcher,*
and Ulrich H. von Andrian‡

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

Summary

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 ar-
rest occurred nearly exclusively in the paracortical HEVs. Endogenous polymorphonuclear
(PMNs) and mononuclear leukocytes (MNLs) attached and rolled in HEVs at similar frequen-
cies, but only MNLs arrested suggesting that the events downstream of primary rolling interac-
tions critically determine the specificity of lymphocyte recruitment. Antibody inhibition stud-
ies 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, impli-
cating a requirement for G␣iϪ-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 detect-
able 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 lym-
phocyte homing to PLNs.

The surveillance of the body for foreign antigens is a riety of adhesive and/or signaling events are used in unique
critical function of the immune system. Lymphocytes combinations to achieve tissue and subset specificity of
migrate from the blood into tissues and secondary lym- homing.
phoid organs, and return back to the blood via lymph ves-
sels and the thoracic duct (1). The majority of lymphocytes In situ microscopy experiments have demonstrated that
are capable of tissue selective trafficking (termed “hom- such a multistep model applies to lymphocyte homing to
ing”), recognizing organ-specific adhesion molecules on Peyer’s patches (PPs)1. Bargatze et al. found that initial at-
specialized endothelial cells (2). Tissue-specific homing is tachment in PPs is L-selectin–dependent, and that estab-
thought to control the extent and scope of immune re- lished rolling involves both L-selectin and ␣4␤7 integrins
sponses, and to account for the regional compartmentaliza- that interact with the mucosal addressin, MAdCAM-1 (11).
tion of immune system functions (3). Recent studies in gene-targeted mice have confirmed these
distinct but overlapping functions for L-selectin and ␣4␤7
A multistep model for leukocyte homing has been proposed
(4–6), and aspects of it have been confirmed by various in 1Abbreviations used in this paper: BCECF, 2Ј7Ј,-bis-(2-carboxyethyl)-5(and
vitro and in vivo models (7–11). Leukocytes are specifically 6) carboxyfluorescein; cRPMI, RPMI 1640 ϩ 10% bovine calf serum;
recruited by combinatorial molecular events: blood-borne HEV, high endothelial venule; LNC, lymph node lymphocyte; MLN,
cells use primary adhesion molecules to tether and roll on mesenteric lymph node; MNL, mononuclear lymphocyte; MTX, mutant
the luminal wall of postcapillary venules, and to encounter pertussis toxin; PP, Peyer’s patch; PLN, peripheral lymph nodes; PNAd,
and rapidly transduce signals to upregulate secondary adhe- peripheral node addressin; PTX, pertussis toxin; Vblood, mean blood flow
sion molecules, which then mediate firm arrest. Thus, a va- velocity; Vfast, mean fast cell velocity; Vrel, relative rolling velocity; Vroll,
rolling velocity; WBC, white blood cell.

205 The Journal of Experimental Medicine • Volume 187, Number 2, January 19, 1998 205–216
http://www.jem.org

(12, 13). Subsequent arrest of rolling cells is mediated by Lymphocyte Isolation and Treatment. Lymph node cells (LNCs)
␣4␤7 and LFA-1 integrins, and requires a G protein–linked were freshly isolated from PLNs (axillary, brachial, cervical, and
signal that is inhibited by lymphocyte treatment with per- subiliac) and mesenteric (M) LNs. Cells were washed and resus-
tussis toxin (PTX; reference 9). Thus, in PPs, lymphocyte pended to 1–2 ϫ 107 cells/ml in warm cRPMI. Representative
homing requires a cascade of four distinct molecular steps. samples were stained with FITC-conjugated mAbs to T and B
cell markers (CD3 and B220, respectively) and determined by
A similar multistep adhesion cascade may govern lym- flow cytometry to be ‫ف‬95% lymphocytes.
phocyte homing to peripheral lymph nodes (PLNs). For
example, L-selectin deficiency in mice or treatment of wild- For fluorescent labeling, 10 ml of LNCs was incubated with 5 ␮l
type animals with mAbs to L-selectin or peripheral node BCECF for 20 min at 37ЊC. Lymphocytes were centrifuged through
addressin (PNAd), the high endothelial venule (HEV)–spe- 5 ml of warm bovine calf serum and supernatant was discarded. The
cific ligand for L-selectin, resulted in impaired lymphocyte pellet was resuspended to 1–2 ϫ 107 cells/ml in warm cRPMI.
homing to PLNs (12, 14–18). Anti–LFA-1 mAbs also re- Aliquots of LNCs were pretreated with one or more of the fol-
duced homing to PLNs, and PLNs in LFA-1–deficient lowing reagents: various mAbs (see above); PTX, which inacti-
mice were reportedly smaller than those in heterozygous vates ␣i-linked G proteins; or MTX, which lacks the enzymatic
littermates (19, 20). Finally, treatment of lymphocytes with activity of native PTX (23). PTX and MTX treatment (100 ng/ml)
PTX inhibited migration to PLNs (21), and phenotypically was for 2 h at 37ЊC and was combined with BCECF-labeling
mature thymocytes expressing transgenic PTX homed during the final 20 min. Treated cells were washed and used im-
poorly in adoptive transfer experiments (22). Thus, lym- mediately.
phocyte homing to PLNs appears to require at least three
molecular events: L-selectin binding to PNAd; G protein– Animals. We used adult BALB/c mice (Charles River Labo-
mediated signaling; and engagement of LFA-1. The spatial ratories, Wilmington, MA) of both sexes (20–30 g body wt).
and temporal coordination of these events during lympho- Mice were housed in a viral antigen–free barrier facility, and kept
cyte–HEV interactions has not been examined in vivo. on sterilized lab chow and water. All experimental procedures
comply with National Institutes of Health (Bethesda, MD) guide-
We have used a novel technique to visualize and dissect lines for the care and use of laboratory animals and were approved
interactions of lymphocytes with HEVs in subiliac PLNs of by the Standing Committees on Animals of Harvard Medical
mice. Our findings confirm that lymphocyte homing to School and The Center for Blood Research.
PLN-HEVs involves a coordinated multistep process, de-
fine important differences from previously described adhe- Subiliac LN Preparation and Intravital Microscopy. Mice were anes-
sion cascades that mediate homing to other tissues, and thetized by intraperitoneal injection (10 ml/kg) of physiologic sa-
help explain the unique recruitment of lymphocyte subsets line containing xylazine (1 mg/ml) and ketamine (5 mg/ml). The
to PLNs. left subiliac LN was prepared for intravital microscopy as previ-
ously described (25). In brief, polyethylene catheters were in-
Materials and Methods serted in the right jugular vein and the left carotid artery. A cath-
eter in the right femoral artery was used for injection of cell
Reagents. FITC-dextran (10 mg/ml; 150 kD mol mass; suspensions and mAbs. The left subiliac LN was exposed and dis-
Sigma Chemical Co., St. Louis, MO) and rhodamine 6G (2 mg/ sected free of fat tissue. The node was positioned over a micro-
ml; Molecular Probes, Inc., Eugene, OR) were diluted in saline scope slide on a plexiglass stage and kept moist with sterile saline.
and undissolved particles were removed by centrifugation; both Sterile vacuum grease and a glass cover slip were applied to en-
reagents were freshly prepared for each experiment. 2Ј7Ј,-bis-(2- close the preparation for microscopic viewing. The stage was
carboxyethyl)-5(and 6) carboxyfluorescein (BCECF; Molecular then placed on an intravital microscope (IV-500; Mikron Instru-
Probes, Inc.) was prepared as a 0.5 mg/ml stock solution in DMSO ments, San Diego, CA) equipped with a silicon-intensified target
and used within 1 wk. EDTA, PMA, and PTX were all from camera (VE1000SIT; Dage mti, Michigan City, IN). The node
Sigma Chemical Co. Mutant PTX (MTX) with a two amino acid was visualized by bright field transillumination or epifluorescence
substitution (PT9K129G; reference 23) was a gift of Dr. Rino from a video-triggered Xenon arc stroboscope (Chadwick Hel-
Rappuoli (IRIS, Chiron Vaccines Immunobiological Research In- muth, El Monte, CA). Video images were recorded using a time
stitute, Sienna, Italy). base generator (For-A, Montvale, NJ) and a Hi8 VCR (Sony,
Boston, MA). Some experiments included injection of FITC-
Monoclonal Antibodies. mAbs Mel-14 (rat IgG2a, anti–murine dextran solution (2 mg/ml, 10 ml/kg body wt) after recordings of
L-selectin; reference 14) and MECA 79 (rat IgM, anti-PNAd; cell behavior to determine vessel structure, luminal diameter, and
reference 15) were generated in our laboratories. Hybridoma cells venular order. The latter was determined by counting successive
for these mAbs as well as mAbs Tib 213 (rat IgG2b, anti–murine generations of branches upstream from the epigastric vein as pre-
CD11a; reference 24) and PS/2 (rat IgG2b, anti–murine ␣4, viously described (25).
from American Type Culture Collection, Rockville, MD) were
purified from culture supernatant. mAbs were stored at Ϫ70ЊC in Analysis of Endogenous White Blood Cell Behavior. Circulating
endotoxin-free saline at 1 mg/ml. For inhibition studies, 50 ␮g of white blood cells (WBCs) were labeled by intravenous injection
antibody was used to preincubate lymphocytes (5–10 ϫ 106 cells) of rhodamine 6G (20 mg/kg body wt). This fluorophor stains nu-
in 0.3–0.5 ml RPMI 1640 (Biowhittaker, Walkersville, MD), ϩ clei and mitochondria. Thus, it allows visualization of myeloid and
10% bovine calf serum (cRPMI) for 10 min. Animals were given lymphoid cells as well as (the more faintly labeled) platelets in the
an additional 50 ␮g intravenously 5 min before cell injection to circulation, whereas RBCs remain unstained. The distinct nuclear
ensure saturating circulating mAb levels. FITC-conjugated mAbs staining patterns of polymorphonuclear (PMNs) and mononu-
to murine CD3 and B220 were from PharMingen (San Diego, CA). clear leukocytes (MNLs) made it possible to distinguish between
these two cell types. An ϫ100 oil immersion objective with ad-
justable 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

206 Peripheral Lymph Node Homing

rolling and sticking cells in the plane of focus. Video analyses per- (Vroll) as previously described (25). To correct for the influence of
formed by different blinded observers yielded essentially identical varying Vblood on rolling, the relative rolling velocity (Vrel) was
results, indicating that this method is feasible and reproducible. Be- also calculated according to the following formula: Vrel ϭ Vroll/
tween 16 and 83 consecutive rolling cells per venule were catego- Vblood ϫ 100%. Vroll data from different experiments were pooled
rized as PMNs and MNLs, respectively. Results are expressed as to compare treatment effects on Vroll histograms, a sensitive indi-
frequency of PMNs and MNLs per 100 identifiable cells that cator of the strength of rolling interactions.
rolled or were stuck (i.e., cells remaining stationary for у30 s) in
the field of view. Cells that could not be clearly identified (e.g., Statistical Methods. Data in graphs and tables are shown as
because they were out of focus) were excluded. mean Ϯ SD, unless otherwise indicated. For statistical compari-
son of two samples, a two-tailed Student’s t test was used when
Parallel to the video recordings, 20 ␮l blood samples were applicable. Multiple comparisons were performed employing the
taken from the retroorbital plexus and diluted in Turk’s solution Kruskal-Wallis test and Bonferoni correction of P. Velocity his-
for differential WBC counts on a hemocytometer. tograms were compared using the Mann-Whitney U-test and the
Kolmogorov-Smirnov test. Differences were considered statisti-
Analysis of BCECF-labeled LNC in PLN-HEVs. Small boluses cally significant when P Ͻ0.05.
(10–50 ␮l) of BCECF-labeled LNCs (1–2 ϫ 107 cells/ml) were
retrogradely injected via the femoral artery catheter. LNC behav- Results
ior in PLNs was recorded through ϫ10 or ϫ20 water immersion
objectives (Achroplan, Carl Zeiss Inc.). Cell behavior in individ- Endogenous Leukocyte Behavior in Subiliac PLNs. Nuclei of
ual vessel segments was determined by playback of video tapes. endogenous WBCs were labeled with rhodamine 6G to
Cells were considered noninteracting when they moved at the study the behavior of PMNs and MNLs in subiliac PLNs
velocity of RBCs, whereas detectably lower velocities were de- (Fig. 1). In agreement with studies of other organs, we ob-
fined as rolling. Cells were considered interacting whether they served heterogeneous adhesive behavior in venules, whereas
rolled, arrested, or both. The rolling fraction is the percentage of arterioles or capillaries did not support interactions. High-
cells that interact with a given venule in the total number of cells power microscopy of superficial HEV segments allowed us
that enter that venule during the same period. The sticking frac- to distinguish rolling and/or sticking MNLs and PMNs in the
tion is the percentage of interacting cells that become immobile plane of focus. Thus, although some cells could not be cat-
for у30 s. In some experiments, sticking LNCs that accumulated egorized when they were out of focus, it was possible to
in PLN venules during the 10-min period after intraarterial bolus determine the relative frequency of subset behavior. Of all
injection of 107 LNCs were counted. LNC accumulation was ex- the identifiable rolling WBCs in HEVs (300 cells in 7 HEVs
pressed as number of sticking cells per mm2 of lumenal surface of 3 animals) the frequency of MNLs was 30%; the remain-
area (calculated from venular diameter and length assuming cylin- der (70%) were PMNs. The proportion of PMNs in the
drical geometry). A PC-based image analysis system (26) was used rolling population was somewhat higher than their fre-
to measure velocities of у15 consecutive rolling and 20 nonin- quency in systemic blood (Fig. 2); differential blood counts
teracting LNCs before and after treatment with mAbs, PTX, or showed that 44 and 56% of circulating WBCs were PMNs
MTX in one representative venule per animal. Data were used to
determine mean fast cell velocity (Vfast), centerline velocity, mean
blood flow velocity (Vblood), wall shear rate, and rolling velocity

Figure 1. Both PMNs and MNLs roll, but only MNLs stick in PLN-HEVs. Endogenous circulating WBCs were labeled in situ with the nuclear dye
rhodamine 6G. Cells were recorded by fluorescence microscopy through an ϫ100 objective during their passage through HEVs. The micrographs show
typical scenes in a segment of an order III venule (50-␮m diameter, blood flow from top to bottom). Lines were drawn to demarcate lateral borders of the
lumenal compartment. WBCs in the plane of focus show two distinct nuclear staining patterns allowing the identification of PMNs (irregular shape) and
MNLs (homogenous round or oval staining pattern). (A) Two rolling PMNs (arrows) and several sticking MNLs (stars) can be seen. (B) 3.7 s later, one of the
PMNs (A, lower left) has rolled out of the field of view, whereas the other PMN (arrowhead) has continued to roll slowly through the vascular segment.
Sticking MNLs did not change their position. In the meantime, several additional rolling PMNs and MNLs entered the field of view.

207 Warnock et al.

Figure 2. Frequency of rolling and sticking MNLs and PMNs in non- age) of LNCs did not interact with the venular walls, but
inflamed PLN-HEVs. Nuclei of circulating WBCs were fluorescently la- the majority of cells rolled, especially in higher order ven-
beled by intravenous injection of rhodamine 6G. Differential counts of ules. A small fraction of cells exhibited either multiple in-
PMNs and MNLs in systemic blood were determined in parallel to video teractive behaviors (e.g., momentary stops or hesitations,
recordings of WBC behavior in PLN-HEVs. The frequency of PMNs and skipping, variable rolling speeds) or transient arrest and re-
MNLs in the total flux of identifiable rolling cells (16–83 consecutive roll- lease (preceded and/or followed by rolling). A significant
ing cells in 7 HEVs of 3 separate preparations) was determined. 10 HEVs number of rolling LNCs (up to 30% in some HEVs) subse-
in 3 preparations were scanned to determine the frequency of sticking cell quently became firmly adherent. Less than 1% became sta-
subsets (i.e., cells that did not move during a 30 s time period). *P Ͻ0.01; tionary immediately upon entry into HEVs, a site where
**P Ͻ0.001 versus MNL (Student’s t test). microvascular hemodynamics and endothelial cell proper-
ties may change abruptly.
and MNLs, respectively (the frequency of PMNs is slightly
elevated over normal levels for mice, and most likely re- In contrast to rhodamine 6G staining of WBCs, which
flects release of neutrophils from the marginating pool due requires high magnification for reliable detection of cells,
to surgical trauma). We conclude that the ability to tether BCECF-labeling confers significant brightness and, there-
and roll within HEVs is not specific for lymphocytes, but is fore, allows visualization of labeled cells at lower magnifi-
shared by other leukocyte subsets, including neutrophils. cation (ϫ10 objective) and within a larger field of view.
This permits parallel evaluation of LNC behavior within
A subpopulation of initially rolling cells eventually ar- different segments of the nodal venular tree. A recent anal-
rested, adhering firmly. This behavior was almost exclusively ysis of the microvascular anatomy of murine subiliac PLNs
limited to MNLs, whereas PMNs continued to roll and, has shown that up to five branching orders can be identi-
eventually, detached from the vessel wall. Indeed, 23.2 Ϯ fied in a typical venular tree (25). One (or more) order I
8.9% (mean Ϯ SD) of rolling MNLs became stuck during venule is located in the hilus of subiliac PLNs and drains
their observable passage through HEVs, whereas only 0.5 Ϯ into a branch of the extralymphoid superficial epigastric
0.6% of PMNs displayed this behavior (data not shown). Sim- vein. Order I venules are formed by the merging of smaller
ilarly, a survey of firmly adherent cells (i.e., cells that were collecting venules (order II) in the nodal medulla that col-
already stuck when the recording was started and did not lect blood from higher order postcapillary venules (orders
move for at least 30 s thereafter) in HEVs revealed that nearly III–V) in the sub- and paracortex. To determine if there
all of these cells were MNLs as well (10 HEVs in 3 animals). are segmental differences in lymphocyte recruitment, we
correlated rolling and sticking fractions, as well as the accu-
Behavior of Adoptively Transferred LNCs in PLN-HEVs. mulation of sticking LNCs, with the venular order (Fig. 3
Although in situ staining of WBCs permitted the differen- A). In agreement with previous studies using L-selectin–
tiation of the nuclear shape of rolling cells in segments of transfected lymphoma cells (25), we found that LNCs were
some HEVs, rhodamine 6G labeling did not allow us to able to attach and roll in all venules draining the node
perform a selective and quantitative analysis of lymphocyte proper, but rolling fractions were significantly larger in
behavior in the entire PLN microcirculation. Therefore, we higher order venules (Fig. 3 B). Likewise, arresting cells
assessed the adhesive behavior of pooled lymphocytes that were much more frequently observed in the highest order
were isolated from peripheral and mesenteric nodes and venules (Fig. 3 C). The paracortical orders III–V HEVs also
stained in vitro with the intracellular fluorophor BCECF. supported the majority of long-term sticking, resulting in
Labeled LNCs were retrogradely injected through the right the preferential accumulation of LNCs in these venular
femoral artery and observed during their passage through segments (Fig. 3 D). A significant number of HEVs were
the left subiliac PLN. noted whose lumen widened abruptly where several capil-
laries converged in the paracortex. This transition results in
LNC interactions mirrored the behavior of the endoge- an immediate slowing of cells, and may lead to a local dis-
nous MNLs described above. A fraction (‫ف‬30% on aver- ruption 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.

Lymphocyte Adhesion in PLN Venules Is L-selectin–depen-
dent. To investigate the contribution of L-selectin to lo-
calization of lymphocytes in PLNs, we pretreated LNCs
with Mel-14, a function-blocking anti–murine L-selectin
mAb. The antibody effect was strikingly complete, inhibit-
ing 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

208 Peripheral Lymph Node Homing

Figure 3. Lymphocyte interac-
tions in the nodal venular tree are
spatially restricted. (A) Micro-
graphs show a typical subiliac LN
preparation at low (ϫ4 objective,
left) and high (ϫ20 objective, right)
magnification. The panel on the
left shows the LN embedded in
fatty tissue and partially covered by
the superficial epigastric artery
(SEA) and vein (SEV). Venous
blood from the LN drains into an
extralymphoid side branch of the
SEV via a large order I venule in
the nodal medulla (I in left panel).
An area of the PLN (rectangle on
left) containing some of the tribu-
taries (orders II–V) to this collect-
ing venule is shown on the right
30 min after injection of BCECF-
labeled LNCs (some cells appear
larger because of bleeding of the
bright fluorescence signal and/or
because they are out of focus). In-
teracting LNCs are preferentially
found in high-order HEV, and
rarely stick in order I or II venules.
(B) Rolling fractions and (C) stick-
ing fractions of BCECF-labeled
LNCs in PLN venules were correlated with venular order. LNCs were retrogradely injected into the right femoral artery, and their rolling and sticking
behavior during their initial passage through the LN was analyzed as described in Materials and Methods. Data shown are means Ϯ SEM of 9 experi-
ments (4–10 venules each). (D) LNC accumulation (10 min after injection of 107 cells) in a representative subiliac LN preparation. Bars depict mean Ϯ
SD of accumulated LNCs in a total of 67 venules.

integrins were similarly able to support LNC rolling in of Vroll within the same vessels before and after mAb treat-
PLNs. No significant effect on the frequency of rolling was ment (Fig. 5, A and B). No significant mAb effect could be
found with neutralizing antibodies to LFA-1 or ␣4 inte- ascertained.
grins.
Since G␣i-linked signaling pathways are known to be in-
The Strength of LNC Rolling Interactions Is Integrin- and G volved in physiologic integrin activation and lymphocyte
Protein–independent. The ␣4␤7 integrin component of homing to PLNs, we also measured the Vroll of LNCs in
LNC rolling in PPs was found to strengthen rolling inter- which these signaling molecules had been blocked by pre-
actions and to reduce lymphocyte rolling velocity (11, 13). treatment with PTX. Excluding those cells that arrested,
We asked whether there might be a similarly significant there was no significant difference between Vroll profiles of
contribution by ␣4 or LFA-1 integrins to the strength of LNCs treated with functionally active PTX or the inactive
LNC rolling interactions in PLNs. To address this ques- mutant form, MTX, or with untreated cells (Fig. 5 C and
tion, we compared microvascular hemodynamics and Vroll data not shown).
histograms by measuring velocities of at least 35 consecu-
tive (у15 rolling and у20 noninteracting) cells in typical LFA-1 Mediates Firm Adherence of Lymphocytes in PLN-
order III HEV before and after antibody treatment (Table HEVs. Isolated LNCs and recipient mice were pretreated
1). Video analysis allowed us to make precise comparisons with anti-CD11a antibody to block LFA-1–dependent ad-
hesion, and cell behavior was observed within subiliac PLNs.
Figure 4. Lymphocyte rolling The mAb treatment essentially abrogated LNC sticking in
in PLNs is L-selectin–dependent. all venular orders (Fig. 6). Importantly, sticking was also
LNCs rolling fractions were ana- abolished when LNC rolling was blocked by treatment
lyzed before (control) and after with anti–L-selectin mAb Mel-14, even when LFA-1 func-
treatment of cells and animals tion was not blocked. Treatment with either anti–L-selec-
with blocking mAbs (␣4, mAb tin or anti–LFA-1 also eliminated the behavior of the small
PS/2; LFA-1, mAb Tib 213; subset of LNCs that seemed to arrest immediately upon en-
L-selectin, mAb Mel-14). Bars try into HEVs, without discernible rolling (data not shown).
depict rolling fractions as percent- In contrast, anti-␣4 integrin antibody had no statistically
age of control cell rolling in the significant effect on LNC sticking.
same venule; data shown are mean Ϯ SEM of rolling fractions from 3–5
experiments of 4–10 vessels each. **P Ͻ0.001 versus all groups (Kruskal Rolling Lymphocytes Require Rapid G␣i-linked Signaling to
Wallis test with Bonferoni correction). Trigger Sticking in PLN-HEVs. Our findings strongly sug-
gest that LFA-1 is responsible for firm LNC arrest, but not
209 Warnock et al.

Table 1. Venular Microhemodynamics and Rolling Velocities of LNCs

mAb No. Venules/

inhibition No. animals Diameter Vfast Vblood WSR (WSS) Vroll (Median) Vrel (Median)

␮m ␮m/s ␮m/s sϪ1 (dyne/cm2) ␮m/s %

Control 3/3 38.4 Ϯ 5.9 1236 Ϯ 603 1105 Ϯ 456 228 Ϯ 75 (5.6 Ϯ 1.9) 38.6 Ϯ 21.7 (14.7) 3.4 Ϯ 2.8 (2.0)
L-selectin
3/3 38.4 Ϯ 5.9 1099 Ϯ 266 1168 Ϯ 486 250 Ϯ 59 (6.3 Ϯ 1.5) N/A N/A

Control 4/4 36.2 Ϯ 8.5 999 Ϯ 521 1150 Ϯ 309 267 Ϯ 82 (6.7 Ϯ 2.1) 24.2 Ϯ 18.6 (10.0) 2.7 Ϯ 1.8 (0.9)

␣4 integrin 4/4 26.2 Ϯ 8.5 1456 Ϯ 673 1297 Ϯ 475 289 Ϯ 70 (7.2 Ϯ 1.7) 34.1 Ϯ 29.5 (15.8) 2.7 Ϯ 2.7 (1.3)

Control 3/3 32.7 Ϯ 4.8 1103 Ϯ 482 1021 Ϯ 386 248 Ϯ 71 (6.2 Ϯ 1.8) 43.0 Ϯ 34.9 (32.1) 4.5 Ϯ 4.0 (3.5)
LFA-1 3/3 32.7 Ϯ 4.8 1109 Ϯ 462 1149 Ϯ 492 273 Ϯ 81 (6.8 Ϯ 2.0) 44.9 Ϯ 37.6 (30.8) 4.4 Ϯ 3.8 (2.6)

MTX 3/3 36.9 Ϯ 5.0 1592 Ϯ 815 1392 Ϯ 949 319 Ϯ 91 (8.0 Ϯ 2.3) 64.1 Ϯ 40.4 (23.2) 4.9 Ϯ 3.7 (1.4)
PTX 3/3 36.9 Ϯ 5.0 1792 Ϯ 809 1569 Ϯ 662 332 Ϯ 99 (8.3 Ϯ 2.5) 66.4 Ϯ 48.6 (33.9) 5.2 Ϯ 5.0 (1.9)

Mean velocities of noninteracting (Vfast) and rolling (Vroll) LNCs in one representative order III venule per animal were measured. These data to-
gether with the venular diameter were used to calculate Vblood and wall shear rate (WSR) as described in Materials and Methods. The wall shear stress
(WSS) was estimated based on the assumption that the blood viscosity in HEVs is 0.025 Poise. To correct for hemodynamic differences between

control and teatment periods in individual venules that may have mechanically affected LNC rolling behavior, Vroll was also expressed as percentage
of Vfast (Vrel). Anti–L-selectin treatment essentially eliminated LNC rolling in HEVs. Thus, no data for Vroll and Vrel were obtainable. Data shown are
mean Ϯ SD. Since velocity histograms of Vroll indicate that data were not normally distributed (see Fig. 5), median values are shown as well.

for initial rolling interactions. This integrin is known to re- pothesis that this activation step involved PTX-sensitive
quire cell activation signals to become functional (27). Thus, signaling pathway(s). The effect of PTX treatment was
it seemed likely that a selective activation event was operat- similar to that of anti-CD11a, in that LNCs were able to
ing to mediate the transition from L-selectin– to LFA-1– attach and roll normally in PLN-HEVs (Fig. 7 A), but were
dependent adhesion in rolling LNCs. We tested the hy- unable to arrest (Fig. 7 B). In contrast, mutant inactive PTX

Figure 5. The strength of LNC rolling in HEVs is not augmented by ␣4 or LFA-1 integrins, and is not PTX sensitive. Frequency histograms were
generated by measuring rolling velocities of individual LNCs in order III venules before (control) and after treatment with (A) anti–LFA-1, (B) anti-␣4,
or (C) PTX or MTX. Vroll, Vfast, and Vblood were determined in the same vessels before and after mAb, MTX, or PTX treatment as described in Materials
and Methods (results of hemodynamic measurements are given in Table 1). To account for hemodynamic differences between control and treatment pe-
riod, Vrel was calculated as percentage of Vblood. Frequency distributions were calculated after cells were assigned to velocity classes with Vrel Ͼ0% to Ͻ1%,
1% to Ͻ2%, and so on. Statistical comparison of population means and distributions revealed no significant differences between rolling velocities (P
Ͼ0.05).

210 Peripheral Lymph Node Homing

Figure 6. Lymphocyte sticking in PLN-HEVs is mediated by LFA-1. Figure 7. Sticking of LNCs in PLN-HEV, but not rolling, is blocked
The fraction of rolling LNCs that became stuck for у30 s was determined by PTX. To test the role of G␣i-linked proteins, LNCs were treated with
before (control) and after treatment of cells and animals with blocking PTX or the inactive mutant form, MTX, as a control. Cells were labeled
mAbs (␣4, mAb PS/2; LFA-1, mAb Tib 213; L-selectin, mAb Mel-14). with BCECF and injected into the feeding artery of a subiliac PLN. (A)
Bars depict sticking fractions as percentage of control cell sticking in the Rolling and (B) sticking fractions of treated LNCs in subiliac LN venules
same venule; data shown are mean Ϯ SEM of sticking fractions from 3–5 were determined. Data are shown as mean Ϯ SEM of 3 experiments (5–10
experiments (4–10 venules each). **P Ͻ0.001 versus ␣4 and control venules each). **P Ͻ0.001 (Student’s t test).
group (Kruskal Wallis test with Bonferoni correction).
overlapping plate pattern followed by a smooth layer of flat
(MTX) did not inhibit LNC sticking. The mutant form of endothelial cells that is indistinguishable from nonlymphoid
PTX contains two amino acid substitutions (Arg9→Lys and microvessels (corresponding to the lining of order I and II
Glu129→Gly) and retains the ability to interact with cell venules; references 1, 30, 31). The counter-receptors for
membranes, but lacks the enzymatic ability to ADP-ribosy- L-selectin and LFA-1 are found in abundance on the lumi-
late ␣i subunits of heterotrimeric G proteins (23). Thus, it nal surface of postcapillary HEVs, and in particular on
seems likely that the blockade of sticking by PTX was due ridgelike structures that may further facilitate or reinforce
to inactivation of G protein–linked signaling, and not due cellular interactions (32, 33). Our results provide a func-
to intrinsic surface modifications and/or steric hindrance of tional correlate for these morphologic observations: leuko-
requisite surface molecules.

Discussion Figure 8. Homing to PLN is mediated by a unique cascade of molecu-
lar adhesive and signaling events. At least three distinct steps must occur
We have examined the interactions of blood-borne leu- for lymphocyte homing via HEVs. Only cells that express L-selectin at
kocytes with PLN-HEVs in order to dissect the molecular sufficient density can tether and roll via binding to PNAd (Step 1). Most
mechanisms of lymphocyte homing to PLNs. The experi- lymphocytes (such as subsets of activated/memory cells) that express little
mental data suggest that an organ-specific adhesion cascade or no L-selectin pass HEVs without interacting. Subsequently, rolling
operates within resting PLNs that is selective for and effi- cells must encounter a G␣i-linked activating stimulus (Step 2) which trig-
cient in recruiting naive lymphocytes, while excluding my- gers functional upregulation of LFA-1 (Step 3). Rolling PMNs cannot re-
eloid cells or lymphocyte populations that express little or spond to this lymphocyte-specific chemoattractant signal. The unique
no L-selectin (Fig. 8). Although L-selectin mediates attach- combination of these consecutive steps results in the highly efficient and
ment and rolling interactions in PLN-HEVs, LFA-1 was selective recruitment of preferentially naive lymphocytes.
required for subsequent lymphocyte sticking. Adhesion via
LFA-1, but not L-selectin, was inhibited by PTX, indicat-
ing 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 dis-
tinct from, those previously characterized for lymphocyte
homing to PPs and intestinal lamina propria, and for granu-
locyte accumulation in other organs (11, 28, 29).

Previous ultrastructural and histologic studies of the PLN
subcortex and medulla have suggested that lymphocyte ex-
travasation may occur preferentially within paracortical ven-
ules 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

211 Warnock et al.

cyte rolling and lymphocyte sticking were spatially regu- L-selectin–deficient mice, which display an ‫ف‬90% reduc-
lated, occurring preferentially in high order subcortical tion in the size and cellularity of PLNs due to a greatly re-
venules. Thus, our studies confirm those of Marchesi and duced influx of lymphocytes via HEV (12, 16–18). The
Gowans who proposed over 30 years ago that lymphocyte majority of resident lymphocytes in the PLNs of these ani-
homing is a unique feature of HEV in the nodal paracortex mals display a memory phenotype, and it has been pro-
(31). posed that these cells arrive in PLNs predominantly via af-
ferent lymph vessels (18). However, it is possible that some
Early experiments in mice demonstrated the inhibition L-selectin–deficient cells may reach PLNs after adhering to
of lymphocyte homing to PLNs by treatment with mAb HEVs through an alternative pathway. We have previously
Mel-14 to murine L-selectin and thus L-selectin has been shown that circulating activated platelets can bridge be-
dubbed the lymphocyte homing receptor (14). In addition tween L-selectin deficient lymphocytes and HEVs because
to its well-known function as a mediator of leukocyte roll- platelet P-selectin mediates platelet tethering to both P-selec-
ing, recent studies have shown that L-selectin ligation can tin glycoprotein ligand 1 (PSGL-1) on lymphocytes and
induce intracellular signaling events (34, 35) and that exten- PNAd on HEVs (41, 42). Consistent with this idea, we
sive antibody- or ligand-induced cross-linking of L-selectin have previously shown that the rolling of endogenous WBCs
can trigger functional upregulation of ␤2 integrins in neutro- in wild-type mice was not completely inhibitable by anti–
phils and lymphocytes (36, 37). Our analysis of LNCs and L-selectin mAb (41). Moreover, ‫ف‬10% of the WBCs that
endogenous leukocyte behavior demonstrates that L-selec- passed through PLNs in L-selectin–deficient mice rolled in
tin is both necessary and sufficient for tethering and rolling HEVs (42). The fact that LNCs did not display L-selectin–
of LNCs in all venular orders, but suggests that L-selectin independent rolling in our experiments does not contradict
engagement alone is insufficient to induce sticking. this platelet delivery concept. It is unlikely that LNCs had
significant numbers of platelets on their surface because
On rolling cells, L-selectin engagement is presumably they were directly isolated from LNs and washed exten-
brief and restricted to the small fraction of total L-selectin sively before injection, and LNC behavior was only ana-
molecules within the area of contact with the vessel wall. lyzed for a short time (seconds to minutes) after intraarterial
Our results show that L-selectin–mediated rolling of PMNs injection. This period may not have been sufficient to al-
in HEVs was rarely followed by sticking, and a detailed low significant association with activated platelets which
analysis of Vrolls suggests that the strength of LNC rolling may be infrequently encountered in the blood stream.
interactions was not influenced by LFA-1 or ␣4 integrins.
Moreover, L-selectin signaling has been reported to be in- Lymphocyte homing to PLNs in LFA-1–deficient mice
sensitive to PTX (34), whereas LNC sticking in HEVs was has not been directly examined as yet. However, PLNs in
essentially abrogated by PTX pretreatment. Thus, L-selec- these mice are reportedly smaller than those of wild-type
tin signaling, if it occurs during lymphocyte–HEV interac- littermates (20). Moreover, it has been reported that al-
tions, does not appear sufficient to mediate arrest or alter though systemically-induced delayed type hypersensitivity
rolling through integrin activation. However, our studies responses are apparently normal in the absence of LFA-1
do not exclude the possibility that L-selectin signaling may (43), recall responses to cutaneously applied antigen are se-
synergize with other activation pathways that involve PTX- verely impaired in LFA-1–deficient mice (20). This pheno-
sensitive G proteins. type is similar to that of L-selectin–deficient mice in which
an inability to mount a cutaneous hypersensitivity response
Lymphocytes from noninflamed lymph nodes are primarily was shown to be due to the lack of naive lymphocyte hom-
naive T and B cells that are characteristically L-selectinhi, ing to the draining PLNs where cutaneous antigens are
LFA-1ϩ, and ␣4Io (38, 39). Consistent with this specialized presented (16, 17). Our findings demonstrating LFA-1 in-
array of surface adhesion molecules, naive lymphocytes are volvement in lymphocyte arrest in PLN–HEVs may pro-
the predominant subset that enters PLNs via HEVs (39, vide an explanation for these observations, suggesting that
40). The finding that LNC accumulation in murine PLNs the inefficient cutaneous response in LFA-1–deficient mice
is dependent on L-selectin implies that lymphocytes that do may reflect a defect in normal sensitization of lymphocytes
not express L-selectin, or express only low levels, may be within PLNs.
excluded from homing to PLNs because they cannot tether
and roll along the vessel wall. L-selectinlow lymphocyte LFA-1 exists in an inactive state on resting LNCs, but its
populations may include many recently activated cells as ligand binding activity may be rapidly modulated by a vari-
well as a sizable fraction (‫ف‬30%) of circulating memory cells ety of mechanisms (27, 44–46). In addition to L-selectin cross-
(38, 39). On the other hand, these results suggest that, un- linking (discussed above), several distinct signaling pathways
less precluded by the existence of naive cell-specific activat- have been implicated in ␤2 integrin activation, including li-
ing factors, memory cell subsets that express L-selectin and gation of lymphocyte antigen receptors and chemoattractant
LFA-1 should be capable of homing to PLNs via the HEV receptors that signal through heterotrimeric G proteins.
route. Indeed, recent short-term homing studies of splenic The latter include receptors for physiologic chemoattract-
memory T cell subsets indicate that a significant number of ants such as chemokines, a class of receptors whose signal-
memory cells retain a significant capacity to home via PLN- ing is both rapid and sensitive to inactivation by PTX (46, 47).
HEVs in mice (39).
We found that LFA-1–dependent firm adhesion of
The concept that L-selectin is essential for lymphocyte LNCs in PLN-HEVs was PTX-sensitive, analogous to prior
traffic to PLNs is also in agreement with the phenotype of

212 Peripheral Lymph Node Homing

studies of LNC homing to PPs (9, 11). However, the subsets. However, as originally noted by Lewinsohn et al.,
PTX-induced inhibition of LNC sticking in PPs was re- the distinction between granulocyte and lymphocyte re-
versible by LNC activation with PMA, a phorbol ester that cruitment to PLNs is not based on differential engagement
directly activates protein kinase C (9). We attempted but of the homing receptor L-selectin (51). Although both
were unable to circumvent the effects of PTX inhibition PMNs and MNLs are known to express LFA-1 in an inac-
on LNC sticking in PLN-HEVs by PMA pretreatment of tive or low-affinity conformation (27), we observed that
LNCs before injection. PMA has been shown to upregu- endogenous MNLs, but not PMNs, could firmly arrest in
late the affinity of leukocyte integrins, including LFA-1 (48) PLN-HEVs. This implies that the HEV-specific activation
and ␣4␤7 (49), but PMA treatment also causes rapid shed- signal was selectively detected by rolling lymphocytes, but
ding of L-selectin and resulted in a dramatic reduction in not by myeloid cells. Though variations in homing recep-
LNC rolling in PLN-HEVs (reference 50 and data not tor expression reflect differentiation of lymphocyte sub-
shown). We performed additional in vitro studies using a populations (and critically impact recirculatory routes), the
parallel plate flow chamber (41) and affinity-isolated PNAd possible role that chemoattractants may play in regulating
and intercellular adhesion molecule (ICAM)–1, endothelial subpopulation homing (e.g., naive versus memory lympho-
ligands for L-selectin and LFA-1, respectively. PMA in- cytes) remains to be determined.
duced equivalent sticking of both PTX and mutant toxin–
treated LNCs to ICAM-1 under static conditions or at low The data presented here describe lymphocyte homing to
shear (1.6 dyne/cm2) within a narrow temporal window PLNs as similar to, but distinct from, the multi-step adhe-
(4.5–6 min after activation, our unpublished observation). sion cascade in PPs, which requires lymphocyte ␣4␤7 in-
Surface-expressed L-selectin was concomitantly downregu- teraction with endothelial MAdCAM-1. In this site, ␣4␤7
lated, resulting in a failure of most cells to tether to the coated plays a critical bridging role, slowing the L-selectin–initiated
substrate at higher shear rates or at later time points. In con- rolling of cells sufficiently to allow LFA-1 engagement. In
trast to PLNs, primary adhesion of LNCs in PPs can be al- addition, ␣4␤7 can also mediate tethering (albeit less effi-
ternatively mediated by either L-selectin or ␣4␤7 (11). Thus, ciently than L-selectin), thus allowing some lymphocytes to
the ability of PMA-activated, PTX-treated LNCs to stick home to PPs in an L-selectin–independent manner. Indeed,
in PP-HEVs, but not PLN-HEVs, was most likely due to although anti–L-selectin greatly reduced the total leukocyte
the differential contribution by ␣4␤7 to primary adhesion rolling flux in PP-HEVs (65–90%), the fraction of lympho-
and may also have resulted in more efficient sticking in PP- cytes that entered PP-HEVs and subsequently became stuck
HEVs afforded by the synergy of activated ␣4␤7 and LFA-1. was reduced by only 35% (11, 13). Although lymphocyte
homing to rat PLNs has been reported to involve ␣4 inte-
Taken together, our results suggest that rolling lympho- grins, and expression of the ␣4 integrin ligand VCAM-1
cytes are activated in PLN-HEVs by one or more potent has been found in rat PLN-HEVs (52), most HEVs in un-
adhesion-triggering chemoattractant(s) that exert their effect inflamed adult murine and human PLNs were reported to
via G␣i-coupled lymphocyte surface receptor(s). Our ob- express little, if any, MAdCAM-1 or vascular cell adhesion
servations suggest that unless one postulates signals occur- molecule 1 (VCAM-1; 53–55). This is consistent with our
ring before lymphocyte entry into HEVs, the signaling event present findings and with earlier homing studies using anti-
must be capable of triggering LFA-1 in less than 1 s. Inter- ␣4 mAbs or ␤7-deficient animals (13, 56, 57).
estingly, we noted LNCs to be extraordinarily sensitive to
temperature effects: LNC preparations that were not care- The apparent lack of a requisite bridging adhesive inter-
fully kept at 37ЊC before intraarterial injection rolled nor- action between L-selectin and ␤2 integrin engagement in
mally in HEVs, but were incapable of sticking during their PLNs may reflect the much higher density of L-selectin
initial passage through HEVs (our unpublished observation). ligands on PLN-HEVs, which alone may allow sufficiently
This indicates that the efficient detection and processing of strong and slow rolling interactions (15). Our analysis of
the chemoattractant signal and/or the subsequent integrin hemodynamic parameters presented here and in an earlier
response are critically dependent on unperturbed cellular report (25) indicate that, on average, Vblood (1.1 Ϯ 0.5 mm/s
metabolism. While the identity of the lymphocyte chemo- versus 3.2 Ϯ 0.2 mm/s) and WSR (228 Ϯ 75 sϪ1 versus
attractant(s) in PLN-HEVs remain(s) to be determined, our 418 Ϯ 39 sϪ1) are significantly lower in PLN venules than
findings do not exclude the possibility that signals which those encountered in PP-HEVs (58), yet the Vroll is similar
upregulate LFA-1 affinity may be sufficient for arrest, but (11, 58). Although the requirement for ␣4␤7 in PP hom-
insufficient for subsequent diapedesis. ing may reflect the greater need for strengthened rolling
adhesions to allow sufficient slowing for LFA-1 engage-
Our results imply that the recruitment of (mostly naive) ment, the dissimilar requirements of PLN and PP homing
lymphocytes to PLNs is also controlled in part by a selec- clearly provide a mechanism for the differential recruitment
tive ability to recognize specific chemoattractant(s). Nearly of compartmentalized lymphocyte subsets (39).
all granulocytes and 70–80% of all circulating lymphocytes
(constituting the majority of MNLs) express L-selectin (51). However, our results do not formally exclude the exis-
The observation that rolling PMNs in PLN-HEVs were tence of additional intermediate adhesive interactions in PLNs.
slightly more frequent than MNLs when compared to their In addition to the platelet delivery mechanism (discussed
respective frequency in the blood stream is consistent with above), a number of other receptors have been implicated
the differential prevalence of L-selectinϩ cells in the two in PLN homing, such as the hyaluronate receptor CD44
and the mucin CD43 on lymphocytes, and vascular adhe-
213 Warnock et al.

sion protein 1 (VAP-1) expressed on human PLN-HEVs high-order subcortical post-capillary venules, and have re-
(59–61). On the other hand, the strikingly complete inhibi- fined the understanding of leukocyte adhesive behavior
tion of LNC rolling and sticking by mAbs to L-selectin and therein. The critical roles of L-selectin for attachment and
LFA-1, respectively, suggests that any additional molecular rolling and affinity-upregulated LFA-1 for firm adhesion in
adhesive pathways (other than platelet P-selectin) are un- normal lymph nodes have profound implications for the re-
able to confer detectable mechanical stability to either of these circulatory routes of naive and memory lymphocyte popu-
events under the experimental conditions employed here. lations, and illustrate the singular role that chemoattractant
signals play in recruiting or excluding leukocyte subsets
In conclusion, we have demonstrated a unique adhesion from specialized tissues.
cascade mediating lymphocyte homing to PLN-HEVs. We
have confirmed the site of lymphocyte adhesion in PLNs as

We 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).

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: [email protected]

Received for publication 18 August 1997 and in revised form 19 November 1997.

References model confirmed and refined. Immunity. 3:99–108.
12. Arbonès, M.L., D.C. Ord, K. Ley, H. Ratech, C. Maynard-
1. Gowans, J.L., and E.J. Knight. 1964. The route of re-circula-
tion of lymphocytes in the rat. Proc. R. Soc. Lond. B. Biol. Curry, G. Otten, D.J. Capon, and T.F. Tedder. 1994. Lym-
159:257–282. phocyte homing and leukocyte rolling and migration are im-
paired in L-selectin–deficient mice. Immunity. 1:247–260.
2. Butcher, E.C., R.G. Scollay, and I.L. Weissman. 1980. Or- 13. Wagner, N., J. Lohler, E.J. Kunkel, K. Ley, E. Leung, G.
gan specificity of lymphocyte migration: mediation by highly Krissansen, K. Rajewsky, and W. Müller. 1996. Critical role
selective lymphocyte interaction with organ-specific determi- for ␤7 integrins in formation of the gut-associated lymphoid
nants on high endothelial venules. Eur. J. Immunol. 10:556–561. tissue. Nature. 382:366–370.
14. Gallatin, W.M., I.L. Weissman, and E.C. Butcher. 1983. A
3. Butcher, E.C., and L.J. Picker. 1996. Lymphocyte homing cell-surface molecule involved in organ-specific homing of
and homeostasis. Science. 272:60–66. lymphocytes. Nature. 304:30–34.
15. Streeter, P.R., B.T.N. Rouse, and E.C. Butcher. 1988. Im-
4. Butcher, E.C. 1991. Leukocyte-endothelial cell recognition: munohistologic and functional characterization of a vascular
three (or more) steps to specificity and diversity. Cell. 67: addressin involved in lymphocyte homing into peripheral
1033–1036. lymph nodes. J. Cell Biol. 107:1853–1862.
16. Tedder, T.F., D.A. Steeber, and P. Pizcueta. 1995. L-selec-
5. Shimizu, Y., W. Newman, Y. Tanaka, and S. Shaw. 1992. tin–deficient mice have impaired leukocyte recruitment into
Lymphocyte interactions with endothelial cells. Immunol. To- inflammatory sites. J. Exp. Med. 181:2259–2264.
day. 13:106–112. 17. Catalina, M.D., M.C. Carroll, H. Arizpe, A. Takashima, P.
Estess, and M.H. Siegelman. 1996. The route of antigen en-
6. Springer, T.A. 1994. Traffic signals for lymphocyte recircula- try determines the requirement for L-selectin during immune
tion and leukocyte emigration: the multi-step paradigm. Cell. responses. J. Exp. Med. 184:2341–2351.
76:301–314. 18. Xu, J., I.S. Grewal, G.P. Geba, and R.A. Flavell. 1996. Im-
paired primary T cell responses in L-selectin–deficient mice.
7. von Andrian, U.H., J.D. Chambers, L.M. McEvoy, R.F. J. Exp. Med. 183:589–598.
Bargatze, K.E. Arfors, and E.C. Butcher. 1991. Two-step 19. Hamann, A., D.J. Westrich, A. Duijevstijn, E.C. Butcher, H.
model of leukocyte–endothelial cell interaction in inflamma- Baisch, R. Harder, and H.G. Thiele. 1988. Evidence for an
tion: distinct roles for LECAM-1 and the leukocyte ␤2 inte- accessory role of LFA-1 in lymphocyte–high endothelium in-
grins in vivo. Proc. Natl. Acad. Sci. USA. 88:7538–7542. teraction during homing. J. Immunol. 140:693–699.
20. Schmits, R., T.M. Kundig, D.M. Baker, G. Shumaker, J.J.
8. Lawrence, M.B., and T.A. Springer. 1991. Leukocytes roll Simard, G. Duncan, A. Wakeham, A. Shahinian, A. van der
on a selectin at physiologic flow rates: distinction from and Heiden, M.F. Bachmann, et al. 1996. LFA-1–deficient mice
prerequisite for adhesion through integrins. Cell. 65:859–873. show normal CTL responses to virus but fail to reject immu-
nogenic tumor. J. Exp. Med. 183:1415–1426.
9. Bargatze, R.F., and E.C. Butcher. 1993. Rapid G protein– 21. Spangrude, G.J., B.A. Braaten, and R.A. Daynes. 1984. Mo-
regulated activation event involved in lymphocyte binding to
high endothelial venules. J. Exp. Med. 178:367–372.

10. Lawrence, M.B., E.L. Berg, E.C. Butcher, and T.A. Springer.
1995. Rolling of lymphocytes and neutrophils on peripheral
node addressin and subsequent arrest on ICAM-1 in shear
flow. Eur. J. Immunol. 25:1025–1031.

11. Bargatze, R.F., M.A. Jutila, and E.C. Butcher. 1995. Distinct
roles of L-selectin and integrins ␣4␤7 and LFA-1 in lympho-
cyte homing to Peyer’s patch–HEV in situ: the multistep

214 Peripheral Lymph Node Homing

lecular mechanisms of lymphocyte extravasation. I. Studies of 37. Hwang, S.T., M.S. Singer, P.A. Giblin, T.A. Yednock, K.B.
two selective inhibitors of lymphocyte recirculation. J. Immu- Bacon, S.I. Simon, and S.D. Rosen. 1996. GlyCAM-1, a
nol. 132:354–362. physiologic ligand for L-selectin, activates ␤ 2 integrins on
22. Chaffin, K.E., C.R. Beals, T.M. Wilkie, K.A. Forbush, M.I. naive peripheral lymphocytes. J. Exp. Med. 184:1343–1348.
Simon, and R.M. Perlmutter. 1990. Dissection of thymocyte
signaling pathways by in vivo expression of pertussis toxin 38. Picker, L.J., and E.C. Butcher. 1992. Physiological and mo-
ADP-ribosyltransferase. EMBO (Eur. Mol. Biol. Organ.) J. 9: lecular mechanisms of lymphocyte homing. Annu. Rev. Im-
3821–3829. munol. 10:561–591.
23. Pizza, M., A. Covacci, A. Bartoloni, M. Perugini, L. Nenci-
oni, M.T. De Magistris, L. Villa, D. Nucci, R. Manetti, and 39. Williams, M.B., and E.C. Butcher. 1997. Homing of naïve
M. Bugnoli. 1989. Mutants of pertussis toxin suitable for vac- and memory T lymphocyte subsets to Peyer’s patches, lymph
cine development. Science. 246:497–500. nodes, and spleen. J. Immunol. 159:1746–1752.
24. Sanchez-Madrid, F., P. Simon, S. Thompson, and T.A.
Springer. 1983. Mapping of antigenic and functional epitopes 40. Mackay, C. 1993. Homing of naive, memory and effector
on the ␣ and ␤ subunits of two related glycoproteins in- lymphocytes. Curr. Opin. Immunol. 5:423–427.
volved in cell interactions, LFA-1 and Mac-1. J. Exp. Med.
158:586–602. 41. Diacovo, T.G., K.D. Puri, R.A. Warnock, T.A. Springer,
25. von Andrian, U.H. 1996. Intravital microscopy of the pe- and U.H. von Andrian. 1996. Platelet-mediated lymphocyte
ripheral lymph node microcirculation in mice. Microcircula- delivery to high endothelial venules. Science. 273:252–255.
tion. 3:287–300.
26. Pries, A.R. 1988. A versatile video image analysis system for 42. Diacovo, T.G., M.D. Catalina, M.H. Siegelman, and U.H.
microcirculatory research. Int. J. Microcirc. Clin. Exp. 7:327–345. von Andrian. 1998. Circulating activated platelets reconsti-
27. Stewart, M., and N. Hogg. 1996. Regulation of leukocyte tute lymphocyte homing and peripheral immunity in L-selec-
integrin function: Affinity vs. avidity. J. Cell. Biochem. 61: tin–deficient mice. J. Exp. Med. 187:197–204.
554–561.
28. von Andrian, U.H., P. Hansell, J.D. Chambers, E.M. Berger, 43. Shier, P., G. Otulakowski, K. Ngo, J. Panakos, E. Chour-
I.T. Filho, E.C. Butcher, and K.E. Arfors. 1992. L-selectin mouzis, L. Christjansen, C.Y. Lau, and W.P. Fung-Leung.
function is required for ␤2-integrin–mediated neutrophil ad- 1996. Impaired immune responses toward alloantigens and
hesion at physiological shear rates in vivo. Am. J. Physiol. tumor cells but normal thymic selection in mice deficient in
263:H1034–H1044. the ␤2 integrin leukocyte function-associated antigen–1. J.
29. Kanwar, S., D.C. Bullard, M.J. Hickey, C. Wayne Smith, Immunol. 157:5375–5386.
A.L. Beaudet, B.A. Wolitzky, and P. Kubes. 1997. The asso-
ciation between ␣4-integrin, P-selectin, and E-selectin in an 44. Dustin, M.L., and T.A. Springer. 1989. T cell receptor cross-
allergic model of inflammation. J. Exp. Med. 185:1077–1087. linking transiently stimulates adhesiveness through LFA-1.
30. De Bruyn, P.P., and Y. Cho. 1990. Structure and function of Nature. 341:619–624.
high endothelial postcapillary venules in lymphocyte circula-
tion. Nature. 84:85–101. 45. van Kooyk, Y., P. van de Wiel-van Kemenade, P. Weder,
31. Marchesi, V.T., and J.L. Gowans. 1963. The migration of T.W. Kuijpers, and C.G. Figdor. 1989. Enhancement of LFA-
lymphocytes through the endothelium of venules in lymph 1–mediated cell adhesion by triggering through CD2 or CD3
nodes: an electron microscope study. Proc. R. Soc. Lond. B. on T lymphocytes. Nature. 342:811–813.
Biol. 159:283–290.
32. Kikuta, A., and S.D. Rosen. 1994. Localization of ligands for 46. Laudanna, C., J.J. Campbell, and E.C. Butcher. 1996. Role
L-selectin in mouse peripheral lymph node high endothelial of Rho in chemoattractant-activated leukocyte adhesion
cells by colloidal gold conjugates. Blood. 84:3766–3775. through integrins. Science. 271:981–983.
33. Sasaki, K., Y. Okouchi, H.-J. Rothkötter, and R. Pabst.
1996. Ultrastructural localization of the intercellular adhesion 47. Campbell, J.J., J. Hedrick, A. Zlotnik, M.A. Siani, D.A.
molecule (ICAM-1) on the cell surface of high endothelial Thompson, and E.C. Butcher. 1998. Novel chemokines trig-
venules in lymph nodes. Anat. Rec. 244:105–111. ger rapid arrest of rolling lymphocytes under physiologic
34. Laudanna, C., G. Constantin, P. Baron, E. Scarpini, G. Scar- shear. Science. In press.
lato, G. Cabrini, C. Dechecchi, F. Rossi, M.A. Cassatella,
and G. Berton. 1994. Sulfatides trigger increase of cytosolic 48. Nickoloff, B.J., and R.S. Mitra. 1988. Phorbol ester treat-
free calcium and enhanced expression of tumor necrosis fac- ment enhances binding of the mononuclear leukocytes to
tor-␣ and interleukin-8 mRNA in human neutrophils. J. autologous and allogenic gamma-interferon–treated keratino-
Biol. Chem. 269:4021–4026. cytes, which are blocked by anti–LFA-1 monoclonal anti-
35. Brenner, B., E. Gulbins, K. Schlottmann, U. Koppenhoefer, body. J. Investig. Dermatol. 90:684–689.
G.L. Busch, B. Walzog, M. Steinhausen, K.M. Coggeshall,
O. Linderkamp, and F. Lang. 1997. L-selectin activates the 49. Andrew, D.P., C. Berlin, S. Honda, T. Yoshino, A. Ha-
Ras pathway via the tyrosine kinase p56lck. Proc. Natl. Acad. mann, B. Holzmann, P.J. Kilshaw, and E.C. Butcher. 1994.
Sci. USA. 93:15376–15381. Distinct but overlapping epitopes are involved in ␣4␤7-
36. Sikorski, M.A., D.E. Staunton, and J.W. Mier. 1996. L-selec- mediated adhesion to vascular cell adhesion molecule-1, mu-
tin crosslinking induces integrin-dependent adhesion: evi- cosal addressin-1, fibronectin, and lymphocyte aggregation. J.
dence for a signaling pathway involving PTK but not PKC. Immunol. 153:3847–3861.
Cell Adhes. Commun. 4:355–367.
50. Jung, T.M., W.M. Gallatin, I.L. Weissman, and M.O. Dai-
ley. 1988. Down-regulation of homing receptors after T cell
activation. J. Immunol. 141:4110–4117.

51. Lewinsohn, D.M., R.F. Bargatze, and E.C. Butcher. 1987.
Leukocyte-endothelial cell recognition: evidence of a com-
mon molecular mechanism shared by neutrophils, lympho-
cytes, and other leukocytes. J. Immunol. 138:4313–4321.

52. May, M.J., G. Entwistle, M.J. Humphries, and A. Ager.
1993. VCAM-1 is a CS1 peptide-inhibitable adhesion mole-
cule expressed by lymph node high endothelium. J. Cell. Sci.
106:109–119.

53. Szabo, M.C., E.C. Butcher, and L.M. McEvoy. 1997. Spe-
cialization of mucosal follicular dendritic cells revealed by

215 Warnock et al.

mucosal addressin-cell adhesion molecule-1 display. J. Immu- 2929.
nol. 158:5584–5588. 58. Miura, S., Y. Tsuzuki, I. Kurose, M. Suematsu, T. Shige-
54. Hahne, M., M. Lenter, U. Jäger, S. Isenmann, and D. Vest-
weber. 1993. VCAM-1 is not involved in LPAM-1 (␣4␤p/ matsu, H. Kimura, H. Higuchi, H. Serizawa, H. Yagita, K.
␣4␤7) mediated binding of lymphoma cells to high endothe- Okumura, et al. 1996. Endotoxin stimulates lymphocyte-
lial venules of mucosa-associated lymph nodes. Eur. J. Cell. endothelial interactions in rat intestinal Peyer’s patches and
Biol. 61:290–298. villus mucosa. Am. J. Physiol. 271:G282–G292.
55. Nakache, M., E.L. Berg, P.R. Streeter, and E.C. Butcher. 59. Jalkanen, S., R.F. Bargatze, J. de los Toyos, and E.C.
1989. The mucosal vascular addressin is a tissue-specific en- Butcher. 1987. Lymphocyte recognition of high endothe-
dothelial cell adhesion molecule for circulating lymphocytes. lium: antibodies to distinct epitopes of an 85–95 kD glyco-
Nature. 337:179–181. protein antigen differentially inhibit lymphocyte binding to
56. Hamann, A., D.P. Andrew, D. Jablonski-Westrich, B. Holz- lymph node, mucosal and synovial endothelial cells. J. Cell
mann, and E.C. Butcher. 1994. Role of ␣4-integrins in lym- Biol. 105:983–993.
phocyte homing to mucosal tissues in vivo. J. Immunol. 152: 60. McEvoy, L.M., H. Sun, J.G. Frelinger, and E.C. Butcher.
3282–3293. 1997. Anti-CD43 Inhibition of T Cell Homing. J. Exp. Med.
57. Hamann, A., D. Jablonski-Westrich, P. Jonas, and H.-G. 185:1493–1498.
Thiele. 1991. Homing receptors reexamined: mouse LECAM-1 61. Salmi, M., and S. Jalkanen. 1996. Human vascular adhesion
(MEL-14 antigen) is involved in lymphocyte migration into protein 1 (VAP-1) is a unique sialoglycoprotein that mediates
gut-associated lymphoid tissue. Eur. J. Immunol. 21:2925– carbohydrate-dependent binding of lymphocytes to endothe-
lial cells. J. Exp. Med. 183:569–579.

216 Peripheral Lymph Node Homing