Uncovering Quantitative Aspects of Chemokine-Inducible and IntegrinDependent Adhesion of Human T Helper Cells Under Flow Conditions
By Daniele D’Ambrosio*, Francesco Sinigaglia* and Carlo Laudanna‡
* Roche Milano Ricerche, 20132 Milano Italy.
‡ Section of General Pathology, Department of Pathology, University of Verona,
37134 Verona, Italy
Running Title: Chemokine-Inducible and Integrin-Dependent Adhesion of T Helper
Cells under flow
Keywords: Th1/Th2 cells, lymphocyte homing, chemokines, integrins, signal
transduction.
Word count: 5982
Address correspondence to Dr. Daniele D’Ambrosio, Roche Milano Ricerche, Via
Olgettina 58, Milano, Italy. I-20132.
Phone: +3902-2884803. FAX: +3902-2153203.
E-mail address: daniele.dambrosio@roche.com
Abstract
Vascular recognition leading to extravasation of circulating T cells is critically
dependent on the coordinated action of chemokines and adhesion receptors. Although
several molecules potentially involved in vascular recognition by T helper (Th) cells
have been identified, how distinct molecular signals are integrated at the cellular level
is unknown. Here, we utilized human Th1 and Th2 cells as a model system to explore
quantitative aspects of chemokine-inducible and integrin-dependent adhesion under
physiologic flow conditions. We show that distinct chemokines promote subsecond
induction of tethering, rolling and firm adhesion of Th1 and Th2 cells on immobilized
integrin 4 ligands MAdCAM-1 and VCAM-1. Efficiency of CCL17 and CCL22
(ligands of CCR4) and CXCL9 and CXCL11 (ligands of CXCR3) in inducing rolling
or rolling followed by firm adhesion of Th1 and Th2 cells correlated with the level of
chemokine receptor expression, with affinity of the chemokine for the receptor and
ultimately with intensity of receptor signaling, as revealed by evaluation of
intracellular calcium mobilization. Our data indicate that vascular recognition by
distinct subsets of circulating T cells is accomplished by quantitative integration of
multiple qualitatively different signals displayed by endothelial cells.
Introduction
Recruitment of blood borne leukocytes into tissues requires the activation of integrindependent arrest on endothelial cell surface as a prerequisite for subsequent diapedesis
(1). Very rapid integrin activation is mandatory to efficient leukocyte arrest under
flow and previous data have shown that intracellular signaling pathways generated by
heterotrimeric Gi protein-coupled receptors leads to rapid integrin triggering in vivo
(2-4). Chemokines, which generate heterotrimeric Gi protein-dependent signaling
pathways, have been shown to be physiological activators of rapid lymphocyte arrest
along high endothelial venules (HEV) in secondary lymphoid organs, and play a
central role in lymphocyte tissue selective homing (5-7). Transient tethering and
rolling precede firm adhesion of circulating leukocytes and are essential to slow
leukocyte motion, thus facilitating microenviromental sampling and subsequent
interaction with proadhesive chemokines presented by the endothelial cells (8, 9).
Tethering and rolling of leukocytes on vessel walls are primarily mediated by
specialized selectins and mucins (10, 11), although 4 integrins, namely 4 1 (very
late antigen-4, VLA-4) and the mucosal homing receptor 4 7 have been shown to
support tethering and slow rolling (inflammatory rolling) (12, 13). These relatively
loose adhesive interactions are rapidly converted into integrin-dependent firm
adhesion upon chemokine receptor engagement and generation of intracellular signals.
These molecular events have been proposed to act in a sequential and combinatorial
digital fashion to dictate the exquisite specificity of leukocyte recruitment (1, 14, 15).
However, several lines of evidence suggest that selectins, chemokines and integrins
do not simply act in a linear and sequential fashion, but may instead functionally
overlap (16-18), fostering the need to revise the current multistep extravasation
model. Nevertheless, the coordinated activity of chemokines and adhesion receptors
dictates the specificity of leukocyte’s vascular recognition (5). In the case of
lymphocytes, distinct subsets of T cells have been shown to possess specific patterns
of vascular recognition and tissue-homing abilities likely resulting from their
characteristic repertoire of homing receptor’s expression (19, 20).
Functionally distinct subsets of CD4+ T helper cells constitute a useful
paradigm to understand how the concerted action of chemokines and adhesion
receptors regulates the specificity of vascular recognition. T helper 1 (Th1) and Th2
cells home to distinct inflammatory sites and differentially express P- and E-selectin
ligands and a large repertoire of chemokine receptors that have been proposed to
mediate recruitment of Th1 and Th2 cells to inflamed tissues (21-26). However, there
is a poor understanding of how the interplay between chemokine and adhesion
receptors regulates the specificity of vascular recognition by distinct subsets of T
cells. As a first attempt to investigate how distinct molecular signals are quantitatively
integrated at the cellular level to achieve the specificity of vascular recognition, we
have analyzed the requirements for chemokine-induced and integrin-dependent
adhesion of human Th1 and Th2 cells under physiologic flow conditions. We
document that distinct chemokines promote subsecond induction of tethering, rolling
and firm adhesion of Th1 and Th2 cells on immobilized integrin 4 ligands
MAdCAM-1 and VCAM-1. The efficiency of chemokine-induced rolling or rolling
followed by firm adhesion correlates with the level of chemokine receptor expression
and affinity of the ligand for the receptor and ultimately with intensity of receptortriggered signaling events.
Materials and Methods
Generation of polarized human helper T lymphocytes. Human neonatal leukocytes
were isolated from freshly collected, heparinized, neonatal blood by Ficoll-paque
(Pharmacia biotech AB, Uppsala, Sweden) density gradient centrifugation. Polarized
helper T cell lines were generated as previously described by stimulation with 2 g/ml
phytoemoagglutinin (PHA) (Wellcome, Beckenham, UK) in the presence of various
combinations of cytokines and anti-cytokine antibodies (27). Th1 cells were generated
by the addition of 5 ng/ml IL-12 (Hoffmann La Roche Inc., Nutley, NJ) and 200 ng/ml
neutralising anti-IL-4 antibody (Pharmingen, San Diego, CA). Th2 cells were
generated by the addition of 10 ng/ml IL-4 (Pharmingen, San Diego, CA) and 2 g/ml
neutralising anti-IL-12 antibodies 17F7 and 20C2. The cells were cultured in complete
medium (RPMI 1640 (Sigma Chemical Co., St. Louis, MO) supplemented with 5%
FetalClone (Hyclone, Logan UT), 2 mM L-glutamine, 1 mM sodium pyruvate, 100
U/ml penicillin-streptomycin). On day 3 the cultures were washed and expanded in
complete medium with addition of 100 U/ml IL-2 (Hoffman-La Roche Inc., Nutley,
NJ).
Cell surface and intracellular cytokine staining. Cells were washed in FACS buffer
(50 mM phosphate, 150 mM NaCl, pH 7.4; 1% FetalClone; 0.05% sodium azide) and
incubated with anti-human CCR4, CXCR3, integrin  1,  7, 4 or an isotype-matched
control (Pharmingen, San Diego, CA) for 30 minutes on ice, washed and analyzed by
FACScan flow cytometry (Becton Dickinson, Mountain View, CA). Single cell
analysis of IFN- and IL-4 production was performed as previously described (28).
Briefly, T cells were collected 10 days after priming, washed, and 106 cells were
restimulated with PMA (50 ng/ml) and ionomycin (1 g/ml) (Sigma Chemicals, St.
Louis, MO) for 4 h at 37 C in complete medium. Brefeldin A (10 g/ml) (Sigma) was
added during the last two hours of incubation. Fixed cells were permeabilized with
saponin, stained with anti-huIFN--FITC (Pharmingen, San Diego, CA) and anti-huIL4-PE mAbs (Pharmingen), following a protocol provided by the manufacturer and
subsequently analyzed by FACScan flow cytometry (Becton Dickinson, Mountain
View, CA).
Analysis of intracellular calcium mobilization. Fluo-3AM loading was performed as
previously described by incubating the cells (5
X
106/ml) in buffer A (HBSS with 10
mM Hepes) with 2 M Fluo-3AM (Molecular Probes, Eugene, OR) at 37° C for 30
min (27). The incubation was prolonged for 30 min after addition of an equal volume
of buffer B (HBSS with 10 mM Hepes and 5% FCS). Cells were washed twice in
buffer B, resuspended at 2 X 106/ml and analyzed by FACS. Emissions at 525 and 613
nm were measured on a log scale before and after stimulation with the chemokines
(CCL17, CCL22, CXCL9 and CXCL11 were purchased from R&D Systems Inc.,
Minneapolis, MN or Dictagene, Epalinges CH).
Adhesion under flow. Recombinant human MAdCAM-1 and VCAM-1 IgG fusion
proteins were a kind gift of Drs. Ueli Gubler and Louis Renzetti (Roche Nutley, New
Jersey). MAdCAM-1 and VCAM-1 were engineered as IgG fusion proteins using
human IgG1 CH2-CH3 domains onto which the extracellular domains of MAdCAM-1
and VCAM-1 were fused. MAdCAM-1 sequence was from aa. 1 to 331 ending before
the transmembrane domain. VCAM-1 sequence was from amino acid 1 to 696 ending
two residues before the transmembrane domain. Both constructs were expressed in
Drosophila cells, purified by affinity chromatography from lysates of Drosophila cells
and stored at –80 0C. Before use, MAdCAM-1 and VCAM-1 IgG fusion proteins (0.5
mg/ml) were dialyzed against PBS containing 1%  -octyl glucoside. 100 l microcap
glass capillary tubes (Drummond Scientific Company, USA) were coated for 16 hours
at 4°C with 20 l of human MAdCAM-1 and VCAM-1 at 2000 sites/m2. Site
densities per square micrometer of immobilized MAdCAM-1 and VCAM-1 were
calculated by using a
125
I-anti human IgG1 heavy chain monoclonal antibody, as
previously described (29). Before use, tubes were washed and co-coated with 20 l of
2 M chemokines for 60 min. After washing with PBS, the behavior of interacting Th1
and Th2 lymphocytes was recorded on S-VHS videotape and analyzed frame by frame,
as described (30). Single areas of 0.2 mm2 were recorded for at least 30 seconds.
Interactions (rolling, arrest or both) of > 1s were considered significant and were
scored. Lymphocytes that remained firmly adherent for > 10 s were considered fully
adherent (4).
Quantification of chemokine immobilization. 10 mm long sections of 100 l microcap
glass capillary tubes were coated with human MAdCAM-1 or VCAM-1 at 2000
sites/m2, as described above.
(specific
activity
Human recombinant
2000Ci/mmol;
125
I-CCL17 and
Amersham-Pharmacia
Biotech,
125
I-CCL22,
UK),
were
reconstituted at 100 Ci/ml in PBS. A labeled/unlabeled (1:100) mixture of CCL17
and CCL22 was made containing 5 pmoles of 125I-chemokines in 100 l of PBS. 10 l
of chemokine mixture (corresponding to 50 pmoles of chemokine) were added to the
capillary tubes to co-coat a 10 mm long section. After variable incubation times at
room temperature, the tubes were then washed with 10 ml of PBS at a flow rate of 10
dyne/cm2. Radioactivity bound to the tubes was measured with a gamma counter and
transformed in number of molecules/m2. Background binding to glass in absence of
MAdCAM-1 or VCAM-1 was calculated for both chemokines and was subtracted
from the binding in the presence of immobilized MadCAM-1 or VCAM-1. The
number of molecules of each chemokine specifically immobilized by one molecule of
MAdCAM-1 or VCAM-1 was finally calculated.
Results
4 integrins support tethering and rolling of human Th1 and Th2 cells
Polarized CD4+ T helper (Th) cell lines were generated from leukocytes isolated from
human cord blood as previously described (27). The Th1 and Th2 phenotype of CD4+
T cell lines was determined by restimulation and intracellular staining for IFN- and
IL-4 production (Fig. 1A). Surface expression of integrins 4,  1 and  7 that dimerize
to make 4 1 (very late antigen-4, VLA-4) and mucosal homing receptor 4 7 was
investigated and found to be similarly elevated in both Th1 and Th2 cell cultures (Fig.
1B). Since integrins 4 have been shown to support primary adhesive interactions,
namely tethering and rolling due to their microvillous distribution (12, 13), we
investigated real time interactions of Th1 and Th2 cells with the immobilized 4
integrin ligands VCAM-1 and MAdCAM-1 under conditions of physiologic flow.
MAdCAM-1 was found to support, in a site density-dependent linear fashion, a high
number of primary adhesive interactions of Th1 and Th2 cells, few of which
converted into firm adhesions independently of agonist engagement. By contrast,
VCAM-1 poorly supported primary adhesion of Th1 or Th2 cells even at higher site
densities, suggesting differences in 4 1 versus 4 7 integrin activation states (Fig.
1C). These data for the first time demonstrate that the expression of 4 integrins on
Th1 and Th2 cells is functional and promotes tethering and rolling under conditions of
flow, indicating a potential mechanism for rolling of Th2 cells that reportedly lack
expression of P- and E-selectin ligands (21, 22).
Chemokine-induced integrin-dependent adhesion of human Th1 and Th2 cells in flow
conditions
To assess the effect of chemokines on integrin-dependent adhesion of Th1 and Th2
cell in conditions of physiologic flow, chemokines were co-immobilized with
MAdCAM-1 or with VCAM-1. At the density of 2000 sites/mm2, MAdCAM-1 but
not VCAM-1 effectively supported tethering and rolling of both Th1 and Th2 cells.
Co-immobilization of MAdCAM-1 with CCL17 (formerly TARC) or CCL22
(formerly MDC) that engage the chemokine receptor CCR4 (preferentially expressed
on Th2 cells, Fig. 4A), led to a marked up-regulation in the number of firmly adherent
Th2 cells (Fig. 2A). The same chemokines co-immobilized with VCAM-1 induced a
powerful subsecond up-regulation of tethering and rolling, rapidly followed by firm
adhesion in Th2 cells (Fig. 2B). Interestingly, CCL22 was consistently more efficient
than CCL17 in triggering conversion from rolling to firm adhesion of Th2 cells on
both integrin ligands VCAM-1 and MAdCAM-1 (Fig. 2A and B). Surprisingly,
CCL17 and CCL22 were able to trigger lower but relevant interactions also of Th1
cells on both integrin ligands (Fig. 2A and B). CCL17 induced a moderate upregulation of Th1 cell rolling on VCAM-1 (Fig. 2B), but was ineffective in triggering
the complete transition from rolling to firm adhesion and this was particularly evident
on VCAM-1 (Fig. 2B). In contrast, CCL22 consistently triggered a remarkable level
of firm adhesion also in Th1 cells on MAdCAM-1 as well as on VCAM-1. Our data
indicate that although CCR4 expression is low on Th1 cells (Fig. 4A), it is able to
trigger a significant 4-integrin activation leading to moderate levels of tethering,
rolling and firm adhesion. These findings clearly document chemokine-inducible and
integrin-dependent adhesion of Th1 and Th2 cells in conditions of physiologic flow
and confirm the recently documented rapid chemokine-inducible and 4 1-integrindependent lymphocyte tethering and rolling (18). They also indicate subtle
quantitative and/or qualitative differences between CCR4-sharing chemokines CCL17
and CCL22 in triggering integrin-dependent adhesion under flow.
Presentation of chemokines by immobilized VCAM-1 and MAdCAM-1
The differences observed between CCL17 and CCL22 could be due to their reported
different affinities for CCR4 (31, 32), and consequently to the efficiency of receptor
triggered signaling events. Alternatively, differences could be simply due to a
different degree of chemokine immobilization. Initial analysis showed that
chemokines bind to glass (background binding, data not shown). We next quantified
the number of molecules of CCL17 and CCL22 immobilized in presence of
MAdCAM-1 or VCAM-1. The presence of MAdCAM-1 or VCAM-1 highly
increased the amount of chemokine immobilized. The experiment revealed an
extraordinary high number of CCL17 and CCL22 molecules that are specifically
bound to one molecule of integrin ligand (Fig. 3). Binding was rather rapid as it was
clearly detectable within 15 min. and almost reached the plateau within 30 min.
MAdCAM-1 was consistently able to adsorb more chemokine and even more
interestingly both VCAM-1 and MAdCAM-1 were capable to adsorb CCL17 more
efficiently than CCL22 (Fig. 3). These data show for the first time that both
MAdCAM-1 and VCAM-1 are able to directly bind chemokines and suggest that
integrin ligands can act as surprisingly efficacious chemokine presenting molecules.
Importantly, CCL17 is presented more efficiently than CCL22 and this indicates that
the lower efficiency of CCL17 to trigger rapid arrest of cells under flow is not due to a
reduced surface presentation of the chemokine.
Potency of receptor-triggered calcium mobilization by CCL17 and CCL22 correlates
with the efficiency of induction of integrin dependent adhesion under flow conditions
Next, we compared the ability of CCL17 and CCL22 to trigger chemokine receptortransduced early signaling events by analyzing intracellular calcium mobilization on
Th2 cells. Stimulation of Th2 cells with CCL17 followed by stimulation with CCL22
and viceversa demonstrated that CCL22 was able to fully desensitize the cells to
subsequent stimulation with CCL17 (Fig. 4A). By contrast, CCL17 failed to fully
desensitize Th2 cells to CCL22. As expected, stimulation of Th1 cells with CCL17 or
CCL22 induced a much lower calcium mobilization response (data not shown). Since
it is formally possible, and it has been suggested in the literature that CCL22 may bind
to an unidentified receptor in addition to CCR4 (33), we have extended our analysis to
mouse L1.2 pre-B cells transfected with human CCR4 receptor. L1.2 parental cells
failed to mobilize calcium in response to either CCL17 or CCL22 (data not shown)
and as seen with Th2 cells, CCL22 was able to fully desensitize to CCL17 but not the
reverse (Fig. 4B). CCL17 and CCL22 have been reported to bind to CCR4 with
different affinities (31, 32), CCL22 exhibiting higher affinity than CCL17. Thus, it
seems likely that the higher affinity resulting in higher signaling potency of CCL22
relative to CCL17 may help establish a functional hierarchy between these
chemokines. Collectively, our data suggest that the lower efficiency of CCL17 in
triggering a complete integrin-dependent adhesive transition from rolling to firm
adhesion, particularly on cells expressing low levels of CCR4 such as Th1 cells, is
dependent on its reduced signaling potency. These findings also suggest that the
induction of integrin-dependent tethering and rolling versus firm adhesion by
chemokines may depend upon fulfilling the requirements for distinct signaling
thresholds achieved by the specific signaling output of chemokine receptors.
Efficiency of chemokine-induced integrin dependent adhesion in flow conditions
correlates with level of chemokine receptor’ expression and ligand potency
We next wished to show whether the differences observed between CCL17 and
CCL22 in triggering tethering/rolling and firm adhesion of Th1 and Th2 cells in flow
conditions reflect a general phenomenon linked to the quantitative integration of
receptor expression level and ligand potency. To this end, we analyzed the adhesive
interactions of Th1 and Th2 cells on immobilized VCAM-1 and MAdCAM-1 in
response to CXCL9 and CXCL11 (formerly Mig and I-TAC), which bind specifically
to the CXCR3 receptor that is preferentially expressed on Th1 cells (Fig. 5A).
Analysis of intracellular calcium mobilization in response to CXCL9 and CXCL11
showed that CXCL11 was able to fully desensitize Th1 cells to subsequent
stimulation with CXCL9, but not viceversa (Fig. 5B). In flow assays, both
chemokines induced a marked up-regulation of the number of firmly adherent Th1
cells on MAdCAM-1 and VCAM-1 and of rolling Th1 cells on VCAM-1 (Fig. 5C and
5D). Notably, CXCL11 was consistently more efficient than CXCL9 in the conversion
of rolling into firmly adherent Th1 cells on both integrin ligands (Fig. 5C and 5D).
CXCL11 was also able to trigger a significant number of Th2 cell interactions on
VCAM-1 and MAdCAM-1, a fraction of which consisted of firmly adherent cells
(Fig. 5C and 5D). By contrast, CXCL9 was able to induce modest rolling but no arrest
of Th2 cells on VCAM-1 or MAdCAM-1 (Fig. 5C and D). Overall, these data depict a
pattern of interactions induced by CXCL9 and CXCL11 that mirrors that seen with
CCL17 and CCL22. Thus, the quantity and quality of chemokine-inducible integrindependent adhesion appears to be quantitatively dependent on receptor-triggered
signaling events, which result from the integration at the cellular level of both
chemokine receptor expression and ligand affinity.
Discussion
In this study we have explored the molecular basis regulating the specificity of
vascular recognition by functionally distinct subsets of Th cells. We have employed in
vitro derived human Th1 and Th2 cells as a model system to investigate quantitative
aspects of chemokine-inducible and integrin-dependent adhesion of lymphocytes in
conditions of physiologic flow. Previous studies have demonstrated that distinct
subsets of T helper cells possess a unique repertoire of homing receptors, which have
been proposed to control the specificity of vascular recognition (21-23, 25, 26, 28).
However, several issues remained still poorly defined. For instance, selective
expression of selectin ligands on Th1 cells has been proposed to mediate rolling of
these cells, but the mechanisms mediating rolling of Th2 cells are still undefined.
Furthermore, it is unclear whether and which chemokines and chemokine receptors
are able to induce the arrest of Th1 or Th2 cells on vascular ligands under flow
conditions. Here, we have provided the first evidence that endothelial vascular ligands
VCAM-1 and MAdCAM-1 can support rolling and adhesion of Th1 and Th2 cells
under flow conditions and that a defined set of immobilized chemokines can promote
the selective arrest of Th1 or Th2 cells on these vascular ligands. Although rolling and
adhesion on VCAM-1 and MAdCAM-1 is not a Th2 selective phenomenon, several
lines of evidence suggest that VCAM-1 may function primarily to mediate Th2 cell
adhesion. First, expression of VCAM-1 on endothelial cells is highly inducible by the
Th2-signature cytokine IL-4, suggesting involvement of VCAM-1 in an amplification
loop sustaining the recruitment of Th2 cells to local inflammatory sites (34, 35).
Second, VCAM-1 and integrin 4 inhibitors have been proven efficacious in
preventing allergic airway inflammatory responses (36, 37). Overall, these and our
data suggest that integrin 4 1-mediated recognition of VCAM-1 on endothelial cells
may play a critical role in the vascular recognition and inflammatory tissue
recruitment of Th2 cells.
In the course of our experiments, we have confirmed the recently documented
ability of chemokines to promote transient tethering and rolling of T cells on VCAM1 mediated by 4 integrins (18). Most importantly, here we have uncovered several
quantitative aspects that contribute to the regulation of integrin-dependent adhesion
under flow conditions. We have shown that distinct chemokines acting through the
same receptor can promote qualitatively distinct adhesive interactions (rolling versus
firm adhesion) depending on their relative affinity and agonistic potency. Less potent
chemokines were found to be less efficient in inducing firm adhesion, particularly on
cells that expressed lower levels of receptor. A previous study by Campbell et al. (38),
showed that the level of chemokine receptor expression constitutes a critical
threshold-sensitive parameter for induction of lymphocyte arrest under flow
conditions. In that study, only receptors that were expressed above a certain level were
able to trigger arrest under flow, whereas chemotaxis was a much less threshold-
sensitive functional response. It was argued that the different efficiency with which
chemokines were able to trigger chemotaxis versus integrin-dependent arrest relied on
differential receptor occupancy requirements for triggering of the two phenomena. In
such a model, differential affinities between ligands for their binding to the same
receptor could actually be translated into differential receptor occupancies, thus
resulting in distinct signaling potencies. Indeed, our data illustrate that chemokine
ligand affinity and agonistic potency represent additional parameters regulating the
efficiency of induction of lymphocyte arrest under flow conditions and therefore
influence the specificity of vascular recognition. In our study, CCL17 appears to be a
more selective agonist for vascular recognition by cells expressing high levels of
CCR4 such as Th2 cells. In contrast, CCL22 was able to induce significant arrest of
rolling Th1 cells that express lower levels of CCR4. Thus, CCL17 although less
potent than CCL22 may be a more selective clue for vascular recognition. In this
scenario, CCL17 could act to arrest circulating cells expressing high levels of CCR4
such as Th2 but not Th1 cells, while CCL22 could guide the same CCR4-expressing
cells within the underlying tissues. Interestingly, two lines of evidence support this
hierarchy of action between CCL17 and CCL22. First, CCL17 but not CCL22
expression has been documented on vascular endothelium in vitro and in vivo (39-41).
Second, CCL22 but not CCL17 is sensitive to CD26 proteolytic degradation
indicating that CCL17 may be a more stable ligand adept at presentation by
endothelial cells (33, 41, 42). At this regard, it is noteworthy that we have documented
more efficient immobilization of CCL17 than CCL22 on VCAM-1 and MAdCAM-1.
Overall, these findings support the contention that CCL17 may be the most
appropriate trigger for vascular recognition of CCR4 expressing cells. Taken together,
these findings underscore the potential general significance of having multiple
chemokines engaging the same receptor with different affinities and agonistic
potency. Immobilization of CCL17, CCL22, CXCL9, CXCL11, CXCL10 and
CXCL12 (Figs. 2, 5 and data not shown) invariably stimulated tethering/rolling of
Th1 and Th2 cells on VCAM-1. This phenomenon has recently been described by
Grabovsky et al. (18), and has been causally linked to subsecond induction of 4 1
integrin clustering induced by chemokine receptor signaling. Consistent with this
study, we have found that PTX treatment completely abolished chemokine-induced
tethering/rolling as well as firm adhesion of Th1 and Th2 cells without affecting basal
tethering/rolling observed on MAdCAM-1 (data not shown). However, it remains
unclear whether this phenomenon pertains only to VCAM-1 or can also occur with
4 7 ligand MAdCAM-1. While chemokine-inducible tethering and rolling on
VCAM-1 was always observed, it was difficult to see on MAdCAM-1 presumably
due to the high number of spontaneous interactions. Our attempts to further clarify
this issue by analyzing chemokine-inducible adhesion of Th1 and Th2 cells on
decreasing site densities of immobilized MAdCAM-1 were hampered by the fact that
the efficiency of chemokine immobilization diminished proportionally, thus
undermining a meaningful interpretation of the results.
Overall, our data indicate that chemokine receptor expression, chemokine
affinity/potency and integrin expression are not independent threshold-sensitive
parameters, but they are instead quantitatively and functionally integrated at the
cellular level to achieve a global threshold of signals required to trigger integrindependent vascular recognition. These findings indicate that the finely tuned
specificity of vascular recognition by lymphocytes is achieved by the quantitative
integration of signals delivered by chemokines and integrins rather than by their linear
and sequential involvement.
Figure Legends
Figure 1.
(A) Single cell analysis of intracellular IFN- and IL-4 production by human Th1 and
Th2 cells generated in vitro. Cord blood derived Th1 and Th2 cells were harvested
and restimulated with PMA and ionomycin as described. The intracellular production
of IL-4 and IFN- was analyzed by flow cytometry. (B) Surface expression of
integrins4,  1 and  7 on human Th1 and Th2 cells. Shown are representative FACS
profiles of Th1 and Th2 cells stained with antibodies specific for integrins 4,  1 and
 7 (thick lines) or isotype control antibodies (thin lines). (C) Adhesive interactions of
Th1 and Th2 cells with MAdCAM-1 and VCAM-1 under flow in the absence of
chemokine triggering. Increasing number of sites/m2 of immobilized MAdCAM-1
support tethering and rolling of Th1 and Th2 cells with increasing efficiency. Firm
adhesion is only minimally supported. In contrast, VCAM-1 does not support either
tethering, rolling as well as firm adhesion even at much higher number of sites/m2.
Values are mean ± SD of rolling or firm adherent cells during 30 s from at least three
separate 0.2 m2 areas of the same capillary tube. Values are from a representative
experiment of three.
Figure 2. CCL17 and CCL22 trigger rapid adhesive interactions of Th1 and Th2 cells
on MAdCAM-1 and VCAM-1 under flow. (A) 2000 sites/m2 of immobilized
MAdCAM-1 support tethering and rolling and, upon chemokine triggering, firm
adhesion of Th1 and Th2 cells. CCL17 and particularly CCL22 triggered robust
sticking of Th2 cells. On Th1 cells, CCL17 induced minimal sticking, whereas
CCL22 triggered a remarkable level of firm adhesion. (B) 2000 sites/m2 of
immobilized VCAM-1 support, upon chemokine triggering, tethering, rolling as well
as firm adhesion in Th1 and Th2 cells. CCL17 and CCL22 triggered marked upregulation of tethering/rolling and robust sticking of Th2 cells. On Th1 cells, CCL17
and CCL22 induced tethering/rolling. However, CCL17 failed to induce sticking,
whereas CCL22 triggered a significant level of firm adhesion. Values are mean ± SD
of rolling or firm adherent cells during 30 s from at least three separate 0.2 m2 areas
of the same capillary tube. The number of Th1 and Th2 cells tethering/rolling (empty
bars) or sticking (black bars) is indicated. Values are from a representative experiment
of three.
Figure 3. Immobilized MAdCAM-1 and VCAM-1 bind and present chemokines.
MAdCAM-1 is more efficient than VCAM-1 in binding CCL17 and CCL22. Both
MAdCAM-1 and VCAM-1 bind much more efficiently CCL17 than CCL22. Shown
is a time-course of specific chemokine binding to VCAM-1 or MAdCAM-1
immobilized at 2000 sites/m2 on glass capillaries. Values depicted indicate the
number of molecule of chemokine bound to one molecule of immobilized MAdCAM1 or VCAM-1 calculated as described in Materials and Methods. A representative
experiment of two is shown.
Figure 4. Th1 and Th2 cell surface CCR4 expression and differential signaling
potencies of CCR4 ligands CCL17 and CCL22. (A) Surface expression of chemokine
receptor CCR4 on human Th1 and Th2 cells. Shown are representative FACS profiles
of Th1 and Th2 cells stained with antibody specific for chemokine receptor CCR4
(thick lines) or isotype control antibody (thin lines). (B) Hierarchical homologous
desensitization of intracellular calcium mobilization in Th2 cells in response to
CCL17 and CCL22. Th2 cells and L1.2 cells expressing hCCR4 were loaded with the
fluorescent Ca2+ indicator Fluo-3 AM. Real time intracellular calcium mobilization in
Th2 cells or CCR4-expressing L1.2 cells was monitored before and after addition of
CCL17 or CCL22 (200 ng/ml) by FACS analysis. Time of addition of CCL17 or
CCL22 is indicated by the arrow. CCL22 fully desensitizes the response to CCL17 in
both Th2 and CCR4-expressing L1.2 cells, but not viceversa. Shown is the
intracellular calcium mobilization response profile recorded for emissions at 525 nm.
One representative experiment of three performed is shown
Figure 5.
The efficiency of CXCL9 and CXCL11 triggering of rapid adhesion of Th1 and Th2
cells on MAdCAM-1 and VCAM-1 under flow correlates with agonistic potency and
chemokine receptor CXCR3 expression level. (A) Surface expression of chemokine
receptor CXCR3 on human Th1 and Th2 cells. Shown are representative FACS
profiles of Th1 and Th2 cells stained with antibody specific for chemokine receptor
CXCR3 (thick lines) or isotype control antibody (thin lines). (B) Hierarchical
desensitization of intracellular calcium mobilization in response to CXCL9 and
CXCL11 in Th1 cells. Th1 cells were loaded with the fluorescent Ca2+ indicator Fluo3 AM. Real time intracellular calcium mobilization in Th1 cells was monitored before
and after addition of CXCL9 or CXCL11 (200 ng/ml) by FACS analysis. Time of
addition of CXCL9 or CCL11 is indicated by the arrow. CXCL11 fully desensitizes
the response to CXCL9 in Th1 cells but not viceversa. Shown is the intracellular
calcium mobilization response profile recorded for emissions at 525 nm. One
representative experiment of three performed is shown. (C) 2000 sites/m2 of
immobilized MAdCAM-1 support tethering and rolling and, upon chemokine
triggering, firm adhesion of Th1 and Th2 cells. CXCL9 and particularly CXCL11
triggered robust sticking of Th1 cells. On Th2 cells, CXCL9 failed to induce l
sticking, whereas CXCL11 triggered a modest level of firm adhesion. (D) 2000
sites/m2 of immobilized VCAM-1 support, upon chemokine triggering, tethering,
rolling as well as firm adhesion in Th1 and Th2 cells. CXCL9 and CXCL11 triggered
marked up-regulation of tethering/rolling and robust sticking of Th1 cells. On Th2
cells, CXCL9 and CXCL11 induced tethering/rolling. However, CXCL9 failed to
induce sticking, whereas CXCL1 triggered a significant level of firm adhesion. Values
are mean ± SD of rolling or firm adherent cells during 30 s from at least three separate
0.2 m2 areas of the same capillary tube. The number of Th1 and Th2 cells
tethering/rolling (empty bars) or sticking (black bars) is indicated. Values are from a
representative experiment of three.
Acknowledgements
This work was partially supported by cofinanziamento MURST and University of
Verona, Progetto Sanità 1996/97, Fondazione Cassa di Risparmio, Ministero della
Sanità (ricerca finalizzata), CNR. We thank members of Roche Milano Ricerche for
critical comments and discussions.
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