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*
Department of Anatomy and Program in Immunology and
Cardiovascular Research Institute, University of California, San Francisco, CA 94143
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lß2 (LFA-1) binding to ICAM-1 or ICAM-2 on
the endothelium (7, 8), after which the lymphocytes eventually
transmigrate across the endothelium into the underlying tissue. A key
step in this multistep process is the rapid activation of this
integrin. Until recently, the nature of the signals that induce up-regulation of integrins and lead to firm arrest of rolling lymphocytes has been obscure. Intravital microscopy studies have demonstrated that the ß2 integrin-mediated arrest of rolling lymphocytes in HEV of peripheral lymph nodes and Peyer’s patches is PTX sensitive (7, 9), indicating that a G protein-linked signal-transduction mechanism is a component of the recruitment cascade. Chemokines that bind to G protein-coupled receptors have emerged as candidates for these integrin activation signals (2, 3, 10). They constitute a large family of structurally related polypeptides that are primarily characterized by their ability to direct migration of selective subsets of leukocytes (11, 12).
Chemokines and other chemoattractants induce strong and rapid
ß2 integrin-mediated adhesion of neutrophils to ICAM-1
(13) and have been implicated in the recruitment of neutrophils to
inflammatory sites (12, 14). Many chemokines that exhibit
chemoattractant activity for lymphocytes are not effective in
triggering adhesion of normal lymphocytes to endothelial ligands for
integrins, e.g., ICAM-1 (15), in a manner that is sufficiently fast or
robust to be physiologically relevant to lymphocyte homing. However, we
showed recently that the newly described C-C chemokine SLC (16), also
referred to as Exodus-2 (17), thymus-derived chemotactic agent 4 (18),
and 6Ckine (19), satisfies many of the requirements for such a homing
chemokine (20). In chemotaxis assays, SLC attracts T and B lymphocytes,
exhibiting the strongest activity toward naive T lymphocytes, but is
not active toward monocytes or neutrophils. Strikingly, one of the
major sites of expression of SLC mRNA in lymph nodes and Peyer’s
patches is HEC (20). When SLC is added in soluble form to T cells, it
induces shear-resistant ß2 integrin-mediated binding to
ICAM-1. This activity is exerted primarily on naive T lymphocytes, the
predominant lymphocyte subset that enters peripheral lymph nodes
through HEV (21, 22, 23). In recent studies, Campbell et al. (24) confirmed
the ability of SLC (referred to as 6Ckine in their study) to induce
adhesion of T cells to ICAM-1 and showed in addition that two other
chemokines (SDF-1 and MIP-3ß) also exhibited pronounced activity.
Furthermore, it was demonstrated that these three chemokines can induce
the rapid arrest of unfractionated lymph node cells on a mixed
substrate of PNAd and ICAM-1.
Because of the demonstrated expression of SLC mRNA in HEC, we have chosen to extend this preliminary characterization to evaluate the potential role of SLC in lymphocyte homing more thoroughly. We have generated an artificial HEV substrate consisting of coimmobilized PNAd, ICAM-1, and SLC in a parallel plate flow chamber. Under conditions of physiologic shear, we have examined the ability of SLC to induce the arrest of rolling lymphocytes with respect to kinetics, stability of the resulting adhesive interactions, lymphocyte subset selectivity, leukocyte specificity, and overall efficiency of the process. We show below that most of the features of lymphocyte homing in vivo are recapitulated in our artificial HEV. Our results strongly point to the critical participation of SLC in the homing of lymphocytes to secondary lymphoid organs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The anti-ß2 integrin mAb R15/7 (IgG1) was a
gift from Dr. R. Rothlein (Boehringer Ingelheim Pharmaceuticals,
Ridgefield, CT). Anti-ß2 integrin mAb IB4 was provided by
Dr. S. Simon (Baylor College of Medicine, Houston, TX). mAb24 was a
kind gift of Dr. N. Hogg (Imperial Cancer Research Fund, London, U.K.).
Purified anti-L-selectin mAb LAM1.3 was obtained from Dr. T. Tedder
(Duke University, Durham, NC). Anti-CD45RA and anti-CD45RO mAbs
were obtained from K. Bacon (DNAX, Palo Alto, CA). Anti-CD3 mAb OKT3
was provided by Dr. A. Weiss (University of California, San Francisco,
CA). Recombinant RANTES, SDF-1, and MIP-1ß were provided by Dr. T.
Schall; rIL-8 was obtained from R&D Systems (Minneapolis, MN). The
anti-4 integrin mAb HP2/1 (IgG1) was purchased from
Immunotech (Westbrook, ME); anti-CD14 mAb and anti-CD19 mAb
were from Caltag Laboratories (South San Francisco, CA).
Human PNAd was purified from detergent lysates from human tonsil with MECA-79 mAb-Sepharose (25, 26). Soluble rICAM-1-Fc chimera was produced using the plasmid provided by Dr. D. L. Simmons (University of Oxford, Headington Oxford, U.K.). SLC was expressed as previously described (20). The SLC-tail peptide, containing amino acids 76–110 of mature mouse SLC, was prepared on an Applied Biosystems (Foster City, CA) 433A peptide synthesizer and purified by HPLC.
T lymphocytes, neutrophils, and B lymphocytes
Human T lymphocytes and naive/memory T lymphocyte subsets were isolated from venous blood samples, as previously described (27, 28). T lymphocytes or naive and memory subsets were obtained by negative immunomagnetic selection with anti-CD14 (monocyte marker), anti-CD19 (B cell marker), and either anti-CD45RA (naive T lymphocytes) or anti-CD45RO mAbs (memory T lymphocytes). Neutrophils were isolated using a one-step Histopaque gradient. The cells were resuspended in HBSS without Ca+ or Mg+ plus 0.2% BSA (buffer A) at 5 x 106 cells/ml and kept at room temperature for a maximum of 2 h. B lymphocytes were isolated from fresh human tonsils and purified on a Histopaque gradient, followed by negative selection using anti-CD3 (T cell marker) and anti-CD14 mAbs. Typically, the resulting B cells were >96% pure.
mAb24 binding assay
mAb24 binding assays were performed as previously described
(27). Briefly, isolated naive T lymphocytes were resuspended in buffer
B at 1 x 105 cells/50 µl buffer containing either
0.5 µg/ml FITC-mAb24 or nonimmune class-matched 0.5 µg/ml FITC-IgG1
(Caltag). The lymphocytes were exposed to SLC or SDF-1 for varying
periods of time at 37°C, washed, fixed in paraformaldehyde, and
analyzed by flow cytometry. As a positive control, cells were treated
with 1 mM Mn.
Preparation of adhesion substrates
Adhesion substrates for controlled shear-detachment assays were
generated by coating 10 µg/ml ICAM-1-Fc in Tris-buffered saline
(TBS), pH 9, onto bacteriologic petri dishes (Corning, San Mateo, CA)
for 2 h at room temperature. After washing with PBS, chemokine
(SLC, SDF-1) was coated at a concentration that induced maximal
activation effects (
10 µg/ml, see Fig. 4
A) in TBS, pH 9,
for 1 h. The substrates were washed and blocked with 3% BSA. For
rolling and arrest assays, 1 µg/ml PNAd, diluted in TBS, pH 8.5, was
coated at 4°C overnight. The plates were washed, exposed to 5–10
µg/ml ICAM-1-Fc for 2 h, washed, treated with chemokine (10
µg/ml of SLC, MIP-1ß, or SDF-1) for 1 h, and blocked with
BSA.
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The substrate-coated slides were incorporated as the lower wall
of a parallel plate flow chamber and mounted on the stage of an
inverted phase-contrast microscope (Diaphot TMD; Nikon, Garden City,
NY). For convenience, all flow experiments were performed at room
temperature. A variety of chemokines can robustly up-regulate integrin
function on T lymphocytes at this temperature (15, 24). For
quantification of bound cells and velocities, NIH Image 1.61 was used.
T lymphocytes were perfused through the flow chamber at 1–2 x
106 cells/ml in HBSS with Ca+ or
Mg+ supplemented with 0.2% BSA (buffer B); neutrophils
were used at 0.25–0.5 x 106 cells/ml. For inhibition
studies, cells (107/ml) were treated with 15 or 20 µg/ml
anti-ß2 integrin mAb R15/7 (IgG1) or isotype-matched
anti-4 integrin Ab HP2/1 (IgG1) for 20 min at room
temperature and diluted 1/10 before injection into the flow chamber. T
lymphocytes were preincubated with 100 ng/ml PTX or its B oligomer
(List Laboratories, Campbell, CA) at 37°C for 1.5 h. In some
experiments, cells were pretreated with 250 nM basic SLC C-terminal
peptide for 20–30 min.
For detachment assays, T lymphocytes were perfused into the flow chamber and allowed to settle onto the substrate for 6 min under static conditions. Flow was then initiated and increased 2- to 2.5-fold in steps every 10 s up to a maximum of 35 dynes/cm2. The number of cells remaining bound at each time interval was determined and was expressed as the percentage of input cells remaining bound.
For rolling and arrest assays, cells were perfused at 1 dyne/cm2 into the flow chamber, and a single field of view (4x objective) was recorded for the first 4 min, after which three additional fields were taped for 15 s each. The number of interacting cells was determined by averaging the total number of arrested and rolling cells in each of the four fields of view. Arrested cells were defined as those that remained stationary during the 15-s interval. These cells were verified as stably arrested because they assumed a phase dark spread morphology and by overlaying the succession of captured images in the computer analysis. The accumulation of arrested cells within a field of view over 4 min of flow was expressed as the percentage of the interacting (rolling plus arrested) cells that were arrested in a field. Cell displacement was followed for 2–4 s to determine rolling velocities. The kinetics of SLC-induced arrest was evaluated by tabulating the time intervals between the initiation of rolling and cell arrest over 4 min of flow. Only those cells that tethered and were arrested within the field of view were included in the analysis.
Statistical analysis
Data were analyzed where indicated using a paired two-tailed Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lß2 through
a G protein-coupled receptor
We previously demonstrated that treatment of naive T lymphocytes
with soluble SLC resulted in increased shear-resistant binding to
immobilized ICAM-1 (20). It is believed that chemokines that act on
leukocytes within blood vessels must be immobilized on the endothelium
for optimal efficacy (29). Otherwise, the chemokine would be dissipated
in the blood flow. Evidence for such an immobilization of IL-8 has been
recently presented (30). It is noteworthy that SLC has the
distinguishing characteristic of a highly basic C-terminal extension of
about 35 amino acids (16), which is likely to serve as an
immobilization domain. Because of these considerations, we wanted to
determine whether immobilized SLC would also induce shear-resistant
binding of naive T lymphocytes to ICAM-1, perhaps even more efficiently
than SLC in solution. We purified naive T lymphocytes based on the
expression of CD45RA and CD45RO (31). In a parallel plate flow chamber,
cells were allowed to settle down under static conditions on a
substrate of ICAM-1 with or without coimmobilized SLC. Our first
observation was that SLC induced the spreading of cells on ICAM-1/SLC
within 3–5 min of contact, while lymphocytes on ICAM-1 alone retained
a rounded shape. When flow was initiated, and shear stress was
increased in a stepwise fashion, naive T lymphocytes bound very weakly
to an ICAM-1 substrate alone, but showed dramatically increased
shear-resistant binding on a substrate consisting of ICAM-1 with
coimmobilized SLC. SLC induced binding over a broad range of shear
stresses (7- to 12-fold enhancement), with the strongest effect at 0.5
dynes/cm2 (Fig. 1A). At low and medium shear
stresses, immobilized SLC induced shear-resistant binding to ICAM-1
twofold more efficiently than SLC in solution (20).
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B). This residual binding was most
likely due to the interaction of the lymphocytes with the immobilized
chemokine. PTX inhibits signaling of many G protein-coupled chemokine
receptors by ADP-ribosylating G proteins (32). Treatment of naive T
lymphocytes with PTX reduced binding to SLC/ICAM-1 to background levels
at high shear stress, while at intermediate and low shear stresses the
effects were partial (Fig. 1B). Treatment of lymphocytes
with the B oligomer of PTX (no ADP-ribosylating activity) did not
affect SLC-induced binding.
CC chemokines such as MIP-1ß and RANTES that did not up-regulate
ß2 integrin function on T lymphocytes when used in
solution (15) also did not induce an effect after immobilization (data
not shown). Immobilized SDF-1, which is a highly efficacious lymphocyte
chemoattractant for both naive and memory T cells (24, 33), promoted
the binding of naive T lymphocytes to ICAM-1 to a comparable extent as
SLC (Fig. 1B).
SLC activates Lß2 on B lymphocytes
B lymphocytes, like T lymphocytes, can enter peripheral lymph
nodes through HEV (34, 35). To determine whether SLC might play a role
in the interaction of B lymphocytes with HEV, we performed
shear-detachment assays with purified tonsillar B cells.
Coimmobilization of SLC with ICAM-1 resulted in increased
shear-resistant binding of B lymphocytes to the substrate over a broad
range of shear stresses. The enhanced adhesion was blocked
substantially by an anti-ß2 integrin Ab (Fig. 2).
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Integrin-mediated adhesion of cells can be enhanced by two general
mechanisms: 1) increase in integrin affinity for a ligand; and/or 2)
increase in the overall avidity of cell contact through integrin
clustering or cell spreading (36, 37, 38). mAb24 binds to an activation
epitope on ß2 integrins that is indicative of increased
affinity of the receptor for ICAM-1 (37). To test whether SLC or
SDF-1 induced the mAb24 epitope, naive T lymphocytes were treated
with each chemokine (10 µg/ml) for 3 min at 37°C and then stained
with fluoresceinated mAb24 (Fig. 3). The
epitope was not induced in either case. Treatment with lower
concentration of chemokines (0.1 or 1 µg/ml) or for longer periods of
incubation (10 and 25 min) also failed to induce the mAb24 epitope
(data not shown). In contrast, manganese that directly induces high
affinity conformation states of integrins (39) produced a marked
up-regulation of mAb24 binding.
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Chemokines must act very quickly in the homing cascade to arrest
rolling lymphocytes, or the lymphocytes will move out of HEV before
being arrested. To test whether immobilized SLC can act on rolling
cells rapidly and efficiently under physiologic flow conditions, we
attempted to reconstitute an artificial HEV substrate in the flow
chamber by coating the bottom surface with PNAd and ICAM-1 with or
without SLC. T lymphocytes were injected into the chamber at a shear
stress of 1 dyne/cm2, which resulted in a rolling velocity
of 48 ± 6 µm/s. Tethering and rolling of lymphocytes were
completely inhibited with Abs against L-selectin (data not shown). On a
substrate consisting of PNAd and ICAM-1, only 6% of the interacting
cells (combined rolling and arrested cells) were arrested (Fig. 4A). However, coimmobilization
of SLC induced a dramatic increase in the proportion of arrested cells
in a dose-dependent manner (Fig. 4A). At 10 µg/ml of SLC,
91% of the cells became arrested. Treatment of T lymphocytes with an
anti-ß2 integrin-blocking Ab or PTX did not perturb
the tethering and rolling of cells on the substrate, but greatly
reduced the number of arrested cells (Fig. 4B). Cells
incubated with the B oligomer of PTX behaved similarly to untreated
cells. As observed in static adhesion assays, SLC induced spreading of
arrested cells on the substrate within 3–5 min. In contrast, the few
arrested cells that were observed in the absence of SLC did not spread
on the substrate (data not shown). Cells that accumulated during flow
under the influence of SLC were resistant to detachment up to a shear
stress of 35 dynes/cm2 (data not shown).
Intravital microscopy studies have shown that the arrest of rolling
lymphocytes within HEV occurs very rapidly, within less than 1 s
(7). To evaluate how long rolling T lymphocytes had to be exposed to
immobilized SLC before being arrested, cells that tethered and were
arrested within the field of view were analyzed. The intervals between
initial contact (tethering) and arrest were measured. A histogram of
the intervals reveals a broad distribution of times, with 35% of cells
arresting in less than 1 s and a median time of arrest of 2.5
s (Fig. 5).
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Naive T lymphocytes, as determined by expression of cell surface
markers, are the predominant subset of T lymphocytes that enters
peripheral lymph nodes via HEV (21, 22, 23, 40). In short-term trafficking
experiments, naive T lymphocytes were 5–10-fold more efficient than
memory T cells in entering peripheral lymph nodes through HEV (22, 23).
On a SLC/PNAd/ICAM-1 substrate, SLC exerted a more pronounced effect on
the accumulation of arrested naive cells as compared with memory cells
over a 4-min period of observation (Fig. 6). Treatment with the
anti-ß2 integrin Ab confirmed that the observed
effect was due to ß2 integrin function (Fig. 6). Neither
naive T lymphocytes nor memory T cells responded to MIP-1ß in this
assay. However, immobilized SDF-1 induced similar effects to those
seen with SLC (Fig. 6).
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). Naive and
memory T lymphocytes rolled with the same velocity (35 ± 7
µm/s) at a shear stress of 1 dyne/cm2. On a per rolling
cell basis, naive T lymphocytes were arrested three times more
efficiently, resulting in an overall sixfold enhanced recruitment of
the naive cells (Table I).
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Neutrophils express L-selectin and ß2 integrins (3),
but normally they do not enter peripheral lymph nodes through HEV (7).
To test the leukocyte cell-type specificity of SLC, neutrophils were
infused into the flow chamber and were allowed to interact with a
substrate consisting of PNAd/ICAM-1 with or without SLC. In initial
experiments, we observed a strong background adhesion of neutrophils
that was not inhibited by an anti-ß2 integrin Ab. We
suspected that this binding might be due to an interaction of the
negatively charged surface of neutrophils with the very basic
C-terminal sequence of SLC. When neutrophils were pretreated with a
synthetic peptide consisting of the 35 C-terminal amino acids of SLC,
the background binding was eliminated. As observed previously,
neutrophils tethered and rolled on a substrate of PNAd/ICAM-1 (41).
After 4 min of flow, we observed that 32% of the interacting
neutrophils were arrested on this substrate through a ß2
integrin-mediated interaction (an anti-ß2 integrin
mAb blocks the interaction; data not shown), indicating a high level of
spontaneous activation of the neutrophils (Fig. 7). Coimmobilization of SLC together with
PNAd and ICAM-1 resulted in an actual decrease in the number of
arrested cells (11%). However, coimmobilization of IL-8 (a chemokine
specific for neutrophils) induced dramatic arrest of the neutrophils
(in the presence of the synthetic peptide) (Fig. 7). The peptide did
not alter the response of T lymphocytes, since immobilized SLC still
induced the arrest of a high proportion of these cells. In striking
contrast, there was virtually no response of the T lymphocytes to IL-8
(Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SLC became the focus of our attention, because we found that SLC mRNA was strongly expressed in HEV of lymph nodes (20). If this chemokine were present on the luminal surface of the endothelial cells, it would have an ideal localization to serve in the intravascular activation of ß2 integrins on lymphocytes. A distinguishing feature of SLC is that it possesses a highly basic C-terminal extension of 35 amino acids not found in other chemokines (16), providing a potential mechanism to anchor SLC in the lumen of HEV through interactions with anionic molecules on the apical aspect of the endothelial cells. In this way, SLC would be prevented from being washed away by the blood flow and would be available at the appropriate site to interact with rolling lymphocytes.
Our first characterization of SLC employed a static adhesion assay, in which the addition of soluble SLC to T lymphocytes resulted in increased ß2 integrin-dependent adhesion to immobilized ICAM-1 (20). This response was measured as increased resistance of the treated cells to detachment by physiologic shear stresses in a parallel plate flow chamber. In the present study, we found that SLC, when coimmobilized with ICAM-1, induced shear-resistant adhesion to ICAM-1 for both T cells and B cells. Interestingly, the response of T cells to immobilized SLC was significantly greater than that to soluble SLC (20). Future studies should be directed at determining whether localized stimulation of SLC receptors by immobilized SLC is essential for optimal activation of lymphocyte integrins.
The present study has examined the actions of SLC in a dynamic setting that recapitulated critical events occurring in HEV. We have determined that immobilized SLC induces a dramatic arrest of T lymphocytes rolling on a mixed surface of PNAd and ICAM-1. A large number of T cells were rapidly arrested, with the predicted characteristics deduced from in situ observations of homing (7): tethering and rolling required L-selectin, and arrest was mediated by ß2 integrins and dependent on PTX-sensitive events. We further observed that SLC induced the spreading of arrested lymphocytes within 3–5 min of exposure. The arrested lymphocytes were very firmly attached, resisting detachment at shear stresses up to 35 dynes/cm2, well in excess of the shear stresses observed in vivo. Having established the basic phenomenon of SLC-induced arrest of rolling T cells, we wanted to thoroughly characterize the action of SLC with respect to kinetics of arrest, efficiency of arrest, lymphocyte subset selectivity of the response (naive versus memory), and leukocyte class specificity (lymphocyte versus neutrophil). As reviewed below, there is a remarkable degree of correspondence with in situ observations.
Von Andrian and coworkers (7) found shear stresses between 5.6 and 8.3 dynes/cm2 in HEV in vivo and a range of lymphocyte-rolling velocities from 24–66 µm/s. Twenty-three percent of the rolling mononuclear leukocytes were arrested, the majority responding within less than 1 s. Similarly, Bjerknes et al. (42) estimated that approximately 20% of the lymphocyte-tethering events on HEV in Peyer’s patches resulted in persistent arrest of the cells. At a shear stress of 1 dyne/cm2, in which maximum tethering of lymphocytes onto PNAd occurred, we observed that T lymphocytes rolled with a velocity of 48 ± 6 µm/s, with 18.8 ± 3.5% of the rolling cells becoming arrested. In our system, 35% of the arrested cells responded to SLC in less than 1 s, with the median time between tethering and arrest of 2.5 s. It is possible that SLC, PNAd, and ICAM-1 might be more efficiently presented on the HEV endothelium in vivo than in the flow chamber, for example, displayed on microvilli as it has been observed for L-selectin ligands such as CD34 and for chemokines (30, 43, 44). In addition, the use of 22°C in our flow chamber rather than 37°C might have resulted in slower kinetics of activation. In contrast to our results, Campbell et al. (24), who also performed their experiments at 22°C, found a more rapid response to immobilized SLC, with a mean rolling time before arrest of 510 ms. The discrepancy between these results might be attributed to species difference of the rolling cells (mouse versus human) or to the fact that this group used a mixture of lymphocytes isolated from peripheral lymph nodes, whereas we employed lymphocytes purified from peripheral blood. Since many of the lymph node cells would have been recruited recently across HEV and exposed to chemokines, there may have been residual effects of activation in this population.
Also pertinent to these issues is that L-selectin not only mediates
rolling of cells, but also can function as a signal-transduction
molecule in triggering the activation of leukocyte integrins (27, 28, 45). Ligation of L-selectin by GlyCAM-1 (a soluble, secreted
HEV-derived ligand for L-selectin) or by artificial Ab cross-linking
induces the rapid (within seconds) activation of both ß1
and ß2 integrins on lymphocytes. L-selectin cross-linking
elicits the mAb24 activation epitope on naive T lymphocytes (27),
indicative of an affinity change in LFA-1 (37), but does not increase
the epitope on memory T lymphocytes. The activation of ß1
integrin through the L-selectin pathway also appears to involve
affinity changes, as shown by direct binding of soluble fibronectin
(28). In this study, we demonstrate that neither SLC nor SDF-1
elicits the mAb24 epitope on naive T lymphocytes (20, 24). A related
observation was made by Jakubowski et al. (46), who reported that
MIP-1ß-treated peripheral T cells do not bind a soluble VCAM-1/Ig
chimera, but exhibit stimulated adhesion to substrates coated with this
ligand. In contrast to the SLC-induced responses (Figs. 1B
and 4B), signaling through L-selectin was insensitive to PTX
treatment (P. A. Giblin and S. D. Rosen, unpublished
observations). In addition, unlike SLC (see above), L-selectin
cross-linking did not induce cell spreading on ICAM-1 substrates (K.T.,
unpublished), suggesting that SLC may stimulate integrin-dependent
adhesion through avidity modulation, whereas the L-selectin pathway
primarily affects the affinity state of the integrins. Interesting
parallels were found by Stewart and coworkers, who compared the effects
of magnesium and phorbol ester on ß2 integrin function in
T cells (37, 47). While magnesium induces a high affinity state of
LFA-1 for binding soluble ICAM-1, the enhanced cell adhesion to ICAM-1
substrates after phorbol ester treatment occurs by means of low
affinity receptors and is facilitated through cell spreading. Taken
together, our data suggest that the mechanisms of ß2
integrin activation through the L-selectin and SLC pathways differ
fundamentally from each other and must involve distinct signaling steps
(47). While L-selectin signaling may not be sufficient by itself to
mediate arrest of rolling lymphocytes, there may be synergy with the
chemokine signaling pathway. Such synergy may reinforce the selective
recruitment of naive versus memory T cells (see below) and may
facilitate the speed and overall efficiency of cell arrest. In fact,
L-selectin cross-linking and IL-8 have been shown to induce
ß2 integrin-mediated adhesion of neutrophils in a
synergistic manner (45). Additional experiments are required to examine
the combined action of L-selectin-mediated signaling and SLC in
integrin activation during lymphocyte homing.
A fundamental issue in lymphocyte homing concerns selectivity in
recruitment of naive versus memory lymphocytes. Naive T lymphocytes are
the predominant lymphocyte subset that is recruited to lymph nodes via
HEV (21, 22, 23). Memory T lymphocytes also cross lymph node HEV, although
in significantly smaller numbers than naive T lymphocytes (21, 22, 23). The
majority of human naive T lymphocytes (
80%) and a substantial
fraction of memory T lymphocytes (50%) express L-selectin at a
comparable level per cell (27, 28). Consistent with these cell surface
levels, we observed that naive cells tethered on PNAd with a twofold
higher efficiency than memory cells, but rolled with similar
velocities. However, the difference in L-selectin expression does not
explain the 5–10-fold less efficient recruitment of memory T
lymphocytes across HEV. Our present results can account for this
discrepancy, in that the efficiency of SLC-induced arrest was threefold
higher for naive T lymphocytes than for memory cells in the
reconstituted HEV. Taking into consideration the twofold enhanced
tethering of naive cells, we would predict a sixfold higher recruitment
efficiency for naive T lymphocytes, which is essentially in agreement
with findings in in vivo homing studies (22, 23).
A further striking aspect of homing specificity is that neutrophils are normally not recruited across HEV of lymph nodes (7). The observations of Warnock et al. (7) suggest that the exclusion of neutrophils is imposed at the level of integrin activation, since neutrophils are able to roll via L-selectin on HEV, but do not become arrested even though they possess activatable ß2 integrins. In the reconstituted HEV, we showed that SLC did not induce the arrest of neutrophils, whereas IL-8, a known chemoattractant for these leukocytes, induced efficient arrest. Conversely, IL-8 had no significant effect on the arrest of lymphocytes. This lack of responsiveness of neutrophils to SLC was also observed in chemotaxis assays (16, 17, 18, 19, 20).
In conclusion, we have generated an artificial in vitro HEV system that has allowed us to recapitulate most of the critical features of the early events in lymphocyte homing to peripheral lymph nodes. A role for chemokines in homing of T lymphocytes to secondary lymphoid organs has long been suggested (2, 3), but heretofore no chemokine has been found that showed the required expression pattern and activities. We have now shown SLC fulfills these requirements. Further studies are needed to define the receptor for SLC and to elucidate the intracellular signaling responses elicited by SLC.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Steven D. Rosen, Lung Biology Center, Box 0854, University of California, San Francisco, CA 94143-0854. E-mail address:
3 Abbreviations used in this paper: HEC, high endothelial cell; HEV, high endothelial venule; MIP, macrophage-inflammatory protein; PNAd, peripheral lymph node addressin; PTX, pertussis toxin; SLC, secondary lymphoid-tissue chemokine.
Received for publication May 26, 1998. Accepted for publication August 7, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A. Matussek, J. Lauber, A. Bergau, W. Hansen, M. Rohde, K. E. J. Dittmar, M. Gunzer, M. Mengel, P. Gatzlaff, M. Hartmann, et al. Molecular and functional analysis of Shiga toxin-induced response patterns in human vascular endothelial cells Blood, August 15, 2003; 102(4): 1323 - 1332. [Abstract] [Full Text] [PDF]
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J. V. Stein, S. F. Soriano, C. M'rini, C. Nombela-Arrieta, G. G. de Buitrago, J. M. Rodriguez-Frade, M. Mellado, J.-P. Girard, and C. Martinez-A. CCR7-mediated physiological lymphocyte homing involves activation of a tyrosine kinase pathway Blood, January 1, 2003; 101(1): 38 - 44. [Abstract] [Full Text] [PDF]
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 W. Maki, R. E. Morales, V. A. Carroll, W. G. Telford, R. N. Knibbs, L. M. Stoolman, and S. T. Hwang CCR6 Colocalizes with CD18 and Enhances Adhesion to Activated Endothelial Cells in CCR6-Transduced Jurkat T Cells J. Immunol., September 1, 2002; 169(5): 2346 - 2353. [Abstract] [Full Text] [PDF]
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T. Okada, V. N. Ngo, E. H. Ekland, R. Forster, M. Lipp, D. R. Littman, and J. G. Cyster Chemokine Requirements for B Cell Entry to Lymph Nodes and Peyer's Patches J. Exp. Med., July 1, 2002; 196(1): 65 - 75. [Abstract] [Full Text] [PDF]
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S.-C. Chen, G. Vassileva, D. Kinsley, S. Holzmann, D. Manfra, M. T. Wiekowski, N. Romani, and S. A. Lira Ectopic Expression of the Murine Chemokines CCL21a and CCL21b Induces the Formation of Lymph Node-Like Structures in Pancreas, But Not Skin, of Transgenic Mice J. Immunol., February 1, 2002; 168(3): 1001 - 1008. [Abstract] [Full Text] [PDF]
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K. W. Christopherson II, J. J. Campbell, and R. A. Hromas Transgenic overexpression of the CC chemokine CCL21 disrupts T-cell migration Blood, December 15, 2001; 98(13): 3562 - 3568. [Abstract] [Full Text] [PDF]
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 S. W. Chensue Molecular Machinations: Chemokine Signals in Host-Pathogen Interactions Clin. Microbiol. Rev., October 1, 2001; 14(4): 821 - 835. [Abstract] [Full Text] [PDF]
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J. A. DiVietro, M. J. Smith, B. R. E. Smith, L. Petruzzelli, R. S. Larson, and M. B. Lawrence Immobilized IL-8 Triggers Progressive Activation of Neutrophils Rolling In Vitro on P-Selectin and Intercellular Adhesion Molecule-1 J. Immunol., October 1, 2001; 167(7): 4017 - 4025. [Abstract] [Full Text] [PDF]
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J. Lieberman, P. Shankar, N. Manjunath, and J. Andersson Dressed to kill? A review of why antiviral CD8 T lymphocytes fail to prevent progressive immunodeficiency in HIV-1 infection Blood, September 15, 2001; 98(6): 1667 - 1677. [Abstract] [Full Text] [PDF]
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 K. Christopherson II and R. Hromas Chemokine Regulation of Normal and Pathologic Immune Responses Stem Cells, September 1, 2001; 19(5): 388 - 396. [Abstract] [Full Text] [PDF]
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G. Chen, P. Shankar, C. Lange, H. Valdez, P. R. Skolnik, L. Wu, N. Manjunath, and J. Lieberman CD8 T cells specific for human immunodeficiency virus, Epstein-Barr virus, and cytomegalovirus lack molecules for homing to lymphoid sites of infection Blood, July 1, 2001; 98(1): 156 - 164. [Abstract] [Full Text] [PDF]
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J. R. Chan, S. J. Hyduk, and M. I. Cybulsky Chemoattractants Induce a Rapid and Transient Upregulation of Monocyte {alpha}4 Integrin Affinity for Vascular Cell Adhesion Molecule 1 Which Mediates Arrest: An Early Step in the Process of Emigration J. Exp. Med., May 14, 2001; 193(10): 1149 - 1158. [Abstract] [Full Text] [PDF]
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A. Hafezi-Moghadam, K. L. Thomas, A. J. Prorock, Y. Huo, and K. Ley L-Selectin Shedding Regulates Leukocyte Recruitment J. Exp. Med., April 2, 2001; 193(7): 863 - 872. [Abstract] [Full Text] [PDF]
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Z. Ding, K. Xiong, and T. B. Issekutz Chemokines stimulate human T lymphocyte transendothelial migration to utilize VLA-4 in addition to LFA-1 J. Leukoc. Biol., March 1, 2001; 69(3): 458 - 466. [Abstract] [Full Text]
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R. Gerli, P. Gresele, O. Bistoni, C. Paolucci, L. Lanfrancone, S. Fiorucci, C. Muscat, and V. Costantini Salicylates Inhibit T Cell Adhesion on Endothelium Under Nonstatic Conditions: Induction of L-Selectin Shedding by a Tyrosine Kinase-Dependent Mechanism J. Immunol., January 15, 2001; 166(2): 832 - 840. [Abstract] [Full Text] [PDF]
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H. Nakano and M. D. Gunn Gene Duplications at the Chemokine Locus on Mouse Chromosome 4: Multiple Strain-Specific Haplotypes and the Deletion of Secondary Lymphoid-Organ Chemokine and EBI-1 Ligand Chemokine Genes in the plt Mutation J. Immunol., January 1, 2001; 166(1): 361 - 369. [Abstract] [Full Text] [PDF]
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D. J. Fitzhugh, S. Naik, S. W. Caughman, and S. T. Hwang Cutting Edge: C-C Chemokine Receptor 6 Is Essential for Arrest of a Subset of Memory T Cells on Activated Dermal Microvascular Endothelial Cells Under Physiologic Flow Conditions In Vitro J. Immunol., December 15, 2000; 165(12): 6677 - 6681. [Abstract] [Full Text] [PDF]
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J. V. Stein, A. Rot, Y. Luo, M. Narasimhaswamy, H. Nakano, M. D. Gunn, A. Matsuzawa, E. J. Quackenbush, M. E. Dorf, and U. H. von Andrian The CC Chemokine Thymus-derived Chemotactic Agent 4 (TCA-4, Secondary Lymphoid Tissue Chemokine, 6Ckine, Exodus-2) Triggers Lymphocyte Function-associated Antigen 1-mediated Arrest of Rolling T Lymphocytes in Peripheral Lymph Node High Endothelial Venules J. Exp. Med., January 3, 2000; 191(1): 61 - 76. [Abstract] [Full Text] [PDF]
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H. Hasegawa, T. Nomura, M. Kohno, N. Tateishi, Y. Suzuki, N. Maeda, R. Fujisawa, O. Yoshie, and S. Fujita Increased chemokine receptor CCR7/EBI1 expression enhances the infiltration of lymphoid organs by adult T-cell leukemia cells Blood, January 1, 2000; 95(1): 30 - 38. [Abstract] [Full Text] [PDF]
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 J. G. Cyster Chemokines and Cell Migration in Secondary Lymphoid Organs Science, December 10, 1999; 286(5447): 2098 - 2102. [Abstract] [Full Text]
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K. Tangemann, A. Bistrup, S. Hemmerich, and S. D. Rosen Sulfation of a High Endothelial Venule–expressed Ligand for L-Selectin: Effects on Tethering and Rolling of Lymphocytes J. Exp. Med., October 4, 1999; 190(7): 935 - 942. [Abstract] [Full Text] [PDF]
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M. D. Gunn, S. Kyuwa, C. Tam, T. Kakiuchi, A. Matsuzawa, L. T. Williams, and H. Nakano Mice Lacking Expression of Secondary Lymphoid Organ Chemokine Have Defects in Lymphocyte Homing and Dendritic Cell Localization J. Exp. Med., February 1, 1999; 189(3): 451 - 460. [Abstract] [Full Text] [PDF]
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