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Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3904-3911
Eosinophil Tethering to Interleukin-4-Activated Endothelial Cells
Requires Both P-Selectin and Vascular Cell Adhesion Molecule-1
By
Kamala D. Patel
From the Department of Physiology and Biophysics, University of
Calgary, Calgary, Alberta, Canada.
 |
ABSTRACT |
We examined the mechanisms used by eosinophils to tether and
accumulate on interleukin-4 (IL-4)-stimulated human umbilical vein
endothelial cells (HUVECs) under flow conditions. As previously reported, HUVECs treated for 24 hours with 20 ng/mL IL-4 had increased expression of P-selectin and vascular cell adhesion molecule-1 (VCAM-1)
but not E-selectin. We found that eosinophils tethered and rolled on
IL-4-stimulated HUVECs at physiologic shear stresses. Eosinophil
rolling was quickly followed by firm adhesion. Treatment with either an
anti-P-selectin monoclonal antibody (MoAb) or an anti-VCAM-1 MoAb
decreased both eosinophil tethering and accumulation at 2 dyn/cm2. VCAM-1 interacts with  4-integrins expressed on
eosinophils. We found that an anti-4-integrin MoAb also blocked
eosinophil tethering and accumulation at 2 dyn/cm2. None of
these MoAbs alone had an impact on eosinophil accumulation at lower
shear stresses, but when either an anti-VCAM-1 or an anti-4-integrin MoAb was used in combination with an
anti-P-selectin MoAb, all eosinophil tethering and accumulation on
IL-4-stimulated HUVECs were blocked. This was true at both high and
low shear stresses. These data show that both P-selectin and VCAM-1 are required to tether eosinophils at high shear stresses, but at low shear
stresses these adhesion proteins can act independently to recruit
eosinophils to IL-4-stimulated HUVECs.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
EOSINOPHILS REPRESENT a minor fraction of
circulating leukocytes; however, they are a major component of the
cellular infiltrate found in allergic diseases such as bronchial
asthma,1,2 allergic rhinitis, and atopic
dermatitis.3 Eosinophil cytotoxic mediators are believed to
participate in epithelial cell shedding, mucous hypersecretion,
microvascular leakage, and smooth muscle contraction associated with
bronchiolar asthma.4 Because of the central role
eosinophils play in these events, investigators are now trying to
understand the molecular mechanisms responsible for selective
recruitment of eosinophils into sites of allergic inflammation
(reviewed by Wardlaw5). Because eosinophils must exit the
vasculature and migrate into tissue to exert their effects, recent
studies have focused on the adhesive interactions between vascular
endothelial cells and eosinophils.
Leukocyte tissue infiltration is a multistep process that is initiated
by leukocyte tethering and rolling along activated endothelium under
flow conditions.6,7 Leukocyte tethering and rolling is
mediated predominately through the selectin family of adhesion
proteins.8 Selectin expression is restricted to the
vasculature, with L-selectin expressed on most circulating leukocytes,
E-selectin expressed on cytokine-activated endothelial cells, and
P-selectin expressed on both activated endothelial cells and
platelets.9 Eosinophils can use all of the selectins to
form attachments under shear conditions10,11; however,
P-selectin is much better at mediating eosinophil recruitment than
E-selectin both in vivo12 and in vitro.10
The 4-integrins (4 1 and/or 47) can also support
leukocyte tethering and rolling by interacting with their ligands
vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell
adhesion molecule-1 (MadCAM-1).13,14 Although
4-integrins can participate in primary tethering to VCAM-1, several
studies have demonstrated that leukocyte subclasses interact
differently with VCAM-1. 4-integrins on T cells participate in both
initial tethering and rolling on VCAM-1,13-15 whereas
4-integrins on monocytes do not participate in initial tethering but
instead act to stabilize rolling adhesions and support firm
adhesion.16 Cytokines can also alter leukocyte interactions
with VCAM-1. VCAM-1 expressed on interleukin-1 (IL-1)- or
IL-4-stimulated human umbilical vein endothelial cells (HUVECs) support T-cell rolling, but VCAM-1 expressed on HUVECs stimulated with
both IL-1 and IL-4 does not support T-cell rolling.17 Thus, the role that VCAM-1 plays in selective recruitment of eosinophils will
depend not only on expression and activation of 4-integrins on
eosinophils, but also on the mechanism of endothelial cell activation.
Finally, some studies suggest that shear forces will also govern
leukocyte-VCAM-1 interactions.13,14,18
This study examines the interactions of eosinophils with HUVECs
stimulated for 24 hours with IL-4, a cytokine purported to play a
critical role in eosinophil recruitment in allergic
inflammation.19 We show that eosinophils tether and adhere
to IL-4-stimulated HUVECs under shear conditions. Surprisingly, we
have found that eosinophil tethering and accumulation are dependent on
both P-selectin and VCAM-1/4-integrin interactions. Blocking any of
these proteins decreases both eosinophil tethering and accumulation at
higher shear stresses. As the shear stress is lowered, P-selectin and VCAM-1/4-integrins act independently to accumulate eosinophils. This
study clearly demonstrates both overlapping and cooperative roles for
these adhesion proteins in eosinophil recruitment.
| |
MATERIALS AND METHODS |
Reagents and antibodies.
Human recombinant IL-4 and human recombinant tumor necrosis factor-
(TNF-) were from R & D Systems, Inc (Minneapolis, MN). Hanks'
balanced salt solution with Ca2+ and Mg2+
(HBSS), lymphoprep 1077, Dulbecco's modified Eagle's medium
(DMEM), and Media 199 (M199) were from GIBCO BRL, Life
Technologies (Grand Island, NY). Thirty-five-millimeter dishes were
from Corning (Corning, NY) and all other plasticware was from Becton
Dickinson (Franklin Lakes, NJ). Human serum albumin (HSA) was from
Immuno US (Rochester, MI). Enhanced chemiluminescence reagents were
from Amersham (Buckinghamshire, UK). Histamine was from Sigma (St
Louis, MO). All other chemicals were from BDH, Inc (Toronto, Ontario,
Canada).
The antihuman P-selectin monoclonal antibodies (MoAbs) S12 and G1 (both
IgG1- ) were prepared and characterized as
described.20,21 G1, but not S12, blocks binding of
P-selectin to leukocytes.22 The blocking antihuman
E-selectin MoAbs ES1 (IgG1-) was prepared and characterized as
described.23 The blocking antihuman VCAM-1 MoAb 1.G11B1
(IgG1), the antihuman 4-integrin MoAb H2/1 (IgG1), and the antihuman
2-integrin MoAb (IgG2b) were purchased from Serotec (Oxford, UK).
Anti-CD16 MoAb conjugated to paramagnetic beads was purchased from
Miltenyi (Auburn, CA).
Cell culture and isolation.
HUVECs were isolated from individual umbilical cords and grown in M199
with 20% human serum as primary cultures in either 35-mm dishes or in
48-well plates, as described.24 Only monolayers of primary
cultures that were tightly confluent were used for these studies.
Granulocytes were isolated from normal human donors by dextran
sedimentation, hypotonic lysis, and density centrifugation on
lymphoprep 1077, as described.24 Eosinophils were isolated from granulocytes by negative selection with CD16 microbeads using the
Magnetic Cell Separation System (MACS), as described.10 Eosinophils were greater than 94% pure as assessed by Kimura staining and/or Wright-Geimsa staining. L cells stably expressing human VCAM-1 were kindly provided by Dr John Elliott (University of Alberta,
Edmonton, Alberta, Canada) and were maintained in DMEM with 10% fetal
calf serum (FCS), as described.25
Adhesion under flow conditions.
Fluid shear stresses present in the microvasculature were simulated in
a dual chamber parallel-plate flow chamber, as previously described.23,26 Confluent monolayers of primary HUVECs were treated with 20 ng/mL of IL-4 in M199 with 0.5% HSA (M199/A) for 24 hours and then washed once with HBSS. In other experiments, L cells
stably transfected with human VCAM-1 were grown to confluence and used
in the flow chamber. Eosinophils (5 × 105/mL) in
HBSS/0.5% HSA (HBSS/A) were perfused through the chamber at the
desired wall shear stresses. Eosinophil accumulation was determined
after 4 minutes of rolling. These interactions were visualized with a
×10 objective using phase-contrast video microscopy. Interactions
were quantified using a computer imaging system (NIH Image, Bethesda,
MD). The number of adherent or rolling leukocytes was measured by
digitizing six random image frames per condition. Tethering events were
determined using frame-by-frame analysis during the first 60 seconds of
perfusion, as described.10 Only cells that directly
tethered to the surface (primary tethers) were counted. All experiments
were performed at 37°C, unless otherwise indicated. In certain
experiments, eosinophils or HUVECs were preincubated for 10 minutes
with the specified MoAb and rolling was assayed in the continued
presence of the MoAb.
Determination of adhesion protein expression.
Surface expression of adhesion proteins on control and IL-4-stimulated
HUVECs was determined using a modified enzyme-linked immunosorbent
assay (ELISA), as described.25 Total protein expression in
control and IL-4-stimulated HUVECs was determined by Western blotting,
as described.27
Statistics.
All experiments were performed at least three times and the data are
presented as the mean and SEM of those replicates. Statistical differences between experimental groups were evaluated using the unpaired Student's t-test. P values  .05 were
considered significant.
| |
RESULTS |
Eosinophils accumulate on IL-4-stimulated HUVECs under shear
conditions.
We found that eosinophils accumulated on IL-4-stimulated HUVECs at
shear stresses as high as 4 dyn/cm2
(Fig 1A); however, because there were many
more interactions occurring at 2 dyn/cm2, this shear stress
was the highest used in later experiments. Unlike eosinophil rolling on
purified E-selectin or P-selectin,10 most eosinophils
rolled for less than 1 cell diameter before becoming firmly adherent
(Fig 1B). Thus, eosinophils do not participate in rolling interactions
on IL-4-stimulated HUVECs, but instead become firmly adherent. Over a
period of minutes, these eosinophils become activated, changing shape
and spreading on the endothelial cell monolayer
(Fig 2A and B). These data demonstrate that
IL-4-stimulated HUVECs can tether, bind, and activate eosinophils
under shear conditions.
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| Fig 1.
Eosinophil accumulation on IL-4-stimulated HUVECs.
Confluent monolayers of primary HUVECs were washed once with HBSS and
then stimulated with either M199/A alone (control) or M199/A containing
20 ng/mL IL-4 (IL-4). After 24 hours at 37°C, the monolayers were
washed with HBSS and assembled in the flow chamber. Purified
eosinophils (5 × 105/mL) were perfused over the
monolayers at the specified shear stresses and eosinophil accumulation
(A), rolling, and adhesion (B) were determined. The data represent the
mean and SEM of at least three independent experiments. *P .05.
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| Fig 2.
Eosinophils bound to IL-4-stimulated HUVECs.
Eosinophils were perfused over (A) control or (B) IL-4-stimulated
HUVECs as described in Fig 1. After attachment under shear conditions,
video images were obtained using a ×40 objective. These images were
captured as described in Materials and Methods and photographs were
made. Eosinophils bound to control HUVECs (A) remain phase-bright,
whereas eosinophils bound to IL-4-stimulated HUVECs (B) change
shape and spread on the monolayer.
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P-selectin mediates eosinophil tethering to IL-4-stimulated HUVECs
at high shear stresses.
IL-4-stimulated HUVECs have increased expression of P-selectin, both
intracellularly and on the cell surface (Yao et al28 and
data not shown). Further treatment of these endothelial cells with
histamine dramatically increases the surface expression of P-selectin
and eosinophil attachment (Yao et al28 and data not shown);
however, because eosinophils bind to P-selectin at very low site
densities,29 we chose to explore the role of
P-selectin on IL-4-activated endothelial cells in the absence of
histamine in these experiments. We treated IL-4-stimulated HUVECs with
a blocking MoAb directed against P-selectin to determine if the elevated levels of P-selectin present on the surface of
IL-4-stimulated HUVECs were sufficient to participate in eosinophil
recruitment. We found that blocking P-selectin significantly inhibited
eosinophil tethering (Fig 3A). This
inhibition of tethering translated to a 65% reduction in accumulation
(Fig 3B). A nonblocking P-selectin MoAb had no effect on either
tethering (Fig 3A) or accumulation (Fig 3B). As we decreased the shear
stress below physiological levels, eosinophils began to accumulate on
these HUVECs in a P-selectin-independent manner (Fig 3B). Thus,
P-selectin participates in tethering and rolling of eosinophils at high
shear stresses, but other mechanisms are sufficient to mediate these
interactions at lower shear stresses.
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| Fig 3.
P-selectin participates in eosinophil accumulation and
tethering to IL-4-stimulated HUVECs. Monolayers of HUVECs were treated
as described in Fig 1. Before assembly of the flow chamber, HUVECs were
treated for 10 minutes at 37°C with HBSS/A alone or HBSS/A
containing 5 µg/mL of either a nonblocking (S12) or blocking (G1)
MoAb directed against P-selectin. The flow chamber was assembled and
eosinophils also containing the specified MoAb were perfused through
the chamber. (A) Tethering of eosinophils was determined at 2 dyn/cm2, as described in Materials and Methods. (B)
Accumulation of eosinophils was determined at the specified wall shear
stresses as described in Materials and Methods. The data represent the
mean and SEM of at least three independent experiments. *P .05; **P .0001.
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VCAM-1 and 4-integrins participate in eosinophil tethering and
accumulation on IL-4-stimulated HUVECs.
VCAM-1 expressed on L cells is capable of mediating tethering, rolling,
and adhesion of eosinophils under shear conditions (Fig 4A and B). These interactions only
occurred at shear stresses less than 2 dyn/cm2 (Fig 4A).
Almost half of the eosinophils that tethered to VCAM-1 also
participated in rolling interactions on this adhesion protein (Fig 4B).
We found that an antibody directed against the 4-integrins was able
to completely block these interactions (Fig 4A), confirming that
4-integrins on eosinophils interact with VCAM-1 under shear conditions.
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| Fig 4.
Eosinophils tether and roll on VCAM-1-transfected L
cells. Confluent monolayers of nontransfected and VCAM-1-transfected L
cells were washed once with HBSS and then placed in flow assembly.
Eosinophils (5 × 105/mL) were perfused over these cells
at the specified wall shear stresses. (A) Accumulation, (B) rolling,
and adherence were determined as described in Materials and Methods. In
some experiments, eosinophils were pretreated for 10 minutes at
37°C with 2 µg/mL of H2/1, an anti-4-integrin MoAb, before
perfusion over VCAM-1-transfected cells. The data represent the mean
and SEM of at least three independent experiments.
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To determine if VCAM-1 or 4-integrins were participating in
eosinophil tethering and accumulation on IL-4-stimulated HUVECs, we
used MoAbs to block these adhesion proteins. Surprisingly, these MoAbs
dramatically decreased eosinophil tethering at 2 dyn/cm2
(Fig 5A). This inhibition of tethering led
to decreased eosinophil accumulation on IL-4-stimulated HUVECs at 2 dyn/cm2 (Fig 5B). As with P-selectin, as the shear stress
was decreased below physiological shear, the ability of these
antibodies alone to block accumulation also diminished (Fig 5B). Taken
together, these data show that VCAM-1/4-integrins can mediate
eosinophil tethering and accumulation under shear conditions both in a
purified system and in the context of IL-4-stimulated HUVECs.
Strikingly, VCAM-1 and 4-integrins can mediate significant tethering
at 2 dyn/cm2, a value above any described for other
leukocytes.
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| Fig 5.
VCAM-1 and 4-integrins participate in eosinophil
accumulation and tethering to IL-4-stimulated HUVECs. Monolayers of
HUVECs were treated as described in Fig 1. Before assembly of the flow
chamber, HUVECs were treated for 10 minutes at 37°C with HBSS/A
alone or HBSS/A containing 5 µg/mL of 1.G11B1, an MoAb directed
against VCAM-1. Alternatively, eosinophils were pretreated 10 minutes
at 37°C with 2 µg/mL of H2/1, an anti-4-integrin MoAb, before
perfusion over IL-4-stimulated HUVECs. The flow chamber was assembled
and eosinophils also containing the specified MoAb were perfused
through the chamber. (A) Tethering of eosinophils was determined at 2 dyn/cm2, as described in Materials and Methods. (B)
Accumulation of eosinophils was determined at the specified wall shear
stresses as described in Materials and Methods. The data represent the
mean and SEM of at least three independent experiments. *P .05; **P .0001.
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P-selectin, VCAM-1, and 4-integrins act together to recruit
eosinophils to IL-4-stimulated HUVECs.
We next determined if P-selectin and VCAM-1/4-integrins were acting
together to optimally recruit eosinophils to IL-4-stimulated HUVECs.
In these experiments, MoAbs directed against P-selectin, VCAM-1, and
4-integrins were used alone or in combination and eosinophil
tethering and accumulation on IL-4-stimulated HUVECs were
determined. We found that blockade of P-selectin and either VCAM-1
or 4-integrins completely blocked eosinophil tethering at 2 dyn/cm2 (Fig 6). Inhibition of
tethering lead to ablation of accumulation at 2 dyn/cm2
(Fig 7A). As the shear was lowered, either
P-selectin or VCAM-1/4-integrins could independently recruit
eosinophils; however, inhibition of both of the adhesive pathways
abolished accumulation at low shears as well (Fig 7B). Using a
P-selectin MoAb in conjunction with either a VCAM-1 MoAb or an
4-integrin MoAb was nearly as effective as using all three MoAbs
together (Figs 6 and 7A and B). Taken together, these data demonstrate
that P-selectin and VCAM-1 expressed on the surface of IL-4-stimulated
HUVECs act in concert to recruit eosinophils under shear conditions, in
part through interactions with 4-integrins on eosinophils.
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| Fig 6.
P-selectin, VCAM-1, and 4-integrins together mediate
eosinophil tethering on IL-4-stimulated HUVECs. Monolayers of HUVECs
were treated as described in Fig 1. Before assembly of the flow
chamber, HUVECs were treated for 10 minutes at 37°C with HBSS/A
alone; HBSS/A containing 5 µg/mL of 1.G11B1, an MoAb directed against
VCAM-1; 5 µg/mL of either a nonblocking (S12) or blocking (G1) MoAb
directed against P-selectin; or 5 µg/mL of both anti-VCAM-1 and G1
MoAbs. In some experiments, eosinophils were also pretreated for 10 minutes at 37°C with 2 µg/mL of H2/1, an anti-4-integrin
MoAb, before perfusion over IL-4-stimulated HUVECs. The flow chamber
was assembled and eosinophils also containing the specified MoAbs were
perfused through the chamber. Tethering of eosinophils was determined
at 2 dyn/cm2, as described in Materials and Methods. The
data represent the mean and SEM of at least three independent
experiments. *P .05; **P .01.
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| Fig 7.
P-selectin, VCAM-1, and 4-integrins together mediate
eosinophil accumulation on IL-4-stimulated HUVECs. These experiments
were performed exactly as described in Fig 6. Accumulation of
eosinophils was determined at (A) 2 dyn/cm2 and (B) 1 dyn/cm2, as described in Materials and methods. The data
represent the mean and SEM of at least three independent experiments.
*P .05; **P .0001;  P .001 with
respect to P-selectin MoAb alone.
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-4-integrins and 2-integrins both participate in firm adhesion
of eosinophils to IL-4-stimulated endothelial cells.
As stated earlier, eosinophils do not roll on IL-4-stimulated
endothelial cells; instead, they immediately arrest and eventually activate and spread on the endothelial cell monolayer (Figs 1B and 2).
In all of the experiments performed in this study using P-selectin,
VCAM-1, and 4-integrin MoAbs either alone or in concert, all the
cells remaining on the endothelial cell surface were firmly adherent,
not participating in rolling adhesion
(Table 1). This suggested that another
adhesive mechanism was participating in the firm adhesion of
eosinophils to IL-4-stimulated endothelial cells. Like other
leukocytes, eosinophils have 2-integrins on their surface. When
activated, 2-integrins can interact with ICAM-1 on the surface of
endothelial cells supporting firm adhesion. We treated eosinophils with
an anti-2-integrin MoAb to evaluate the role of this adhesion
molecule in eosinophil firm adhesion in this system. We found that an
anti-2-integrin MoAb alone had no effect on either accumulation
(data not shown) or firm adhesion (Table 1). When we used this MoAb
together with an anti-P-selectin MoAb, we found no further effect on
accumulation (data not shown) or firm adhesion (Table 1). However, when
an anti-2-integrin MoAb was used with an anti-4-integrin MoAb,
the percentage of firmly adherent cells decreased dramatically (Table
1). Instead, eosinophils were participating in rolling interactions
similar to those observed during eosinophil interactions with purified P-selectin. These data suggest that both 4- and 2-integrins act
together to firmly adhere eosinophils to IL-4-stimulated endothelial cells.
| |
DISCUSSION |
Eosinophils may represent a minor fraction of the circulating
leukocytes; however, they are a major component of the cellular infiltrate found in diseases such as atopic dermatitis, allergic rhinitis, and bronchial asthma.1,30 Selective infiltration of these cells may be achieved at several levels, including the initial
interaction between eosinophils and activated endothelium at sites of
allergic inflammation.
We examined eosinophil accumulation on IL-4-stimulated HUVECs, because
IL-4 has been shown to be an important cytokine in allergic
inflammation.19 We found that HUVECs stimulated 24 hours
with IL-4 tethered and accumulated eosinophils at shear stresses as
high as 4 dyn/cm2 (Fig 1A). At 2 dyn/cm2, this
response was quite robust. We initially hypothesized that tethering at
2 dyn/cm2 was due exclusively to the expression of
P-selectin on IL-4-activated endothelial cells, because low site
densities of P-selectin have been shown to support eosinophil
rolling.29 Yet, when we used an antibody directed against
P-selectin, there was still residual tethering of eosinophils at 2 dyn/cm2 (Fig 3A). As a result of this residual tethering,
some eosinophils were still able to accumulate on IL-4-stimulated
HUVECs in the presence of an anti-P-selectin MoAb. These data
suggested that another adhesion protein was mediating eosinophil
tethering to these endothelial cells.
Although the selectins mediate leukocyte recruitment under shear
conditions both in vitro and in vivo,8 there are now
several in vivo models of leukocyte recruitment in which selectin
blockade does not prevent leukocyte recruitment and tissue
infiltration. Sriramarao et al12,31 showed that inhibition
of either L-selectin or 4-integrins blocked eosinophil infiltration
into the rabbit mesentery after IL-1 stimulation, suggesting a role for
the 4-integrins in eosinophil recruitment in this model. Johnston et
al32 went on to show that 4-integrins could mediate both
rolling and adhesion in chronically inflamed rat mesentery and that
rolling could occur independent of selectins. These data suggest that,
under some conditions, leukocyte recruitment can occur independent of
selectins.
4-integrins can mediate leukocyte tethering to purified
VCAM-1.13-15 T cells13,15 and
eosinophils18 tether to purified VCAM-1 at shear stresses
between 0.5 and 1 dyn/cm2, but, as the shear stress is
increased to 2 dyn/cm2, few cells tether and accumulate on
VCAM-1. In contrast, monocytes expressing 4-integrins do not tether
to VCAM-1 at either high or low shear stresses.33 To date,
VCAM-1 expressed on cytokine-activated HUVECs has not been shown to
participate in tethering of peripheral blood
leukocytes,16,18,33,34 although data using lymphocyte cell
lines suggest that the VCAM-1 expressed is capable of mediating attachment in the absence of a selectin.17 Instead, data
suggest that VCAM-1 acts to stabilize the adhesion of both peripheral blood T cells and monocytes after these cells have been tethered by a
selectin.
We first examined the ability of freshly isolated eosinophils to
interact with VCAM-1 transfectants and found that eosinophils tethered
and accumulated at low shear stresses in a manner similar to T cells.
At increased site densities, VCAM-1 may be able to mediate eosinophil
tethering at higher shear stresses; however, we only examined
eosinophil interactions at a single site density. We then addressed the
role of VCAM-1/4-integrins interactions in eosinophil recruitment on
IL-4-stimulated HUVECs. In contrast to other leukocytes, we found that
eosinophils tethered to VCAM-1 expressed on IL-4-activated HUVECs, and
this tethering was occurring at physiologic shear stresses.
P-selectin and VCAM-1/4-integrins work cooperatively to optimize
eosinophil interactions at physiologic shear stresses, because MoAbs
directed against either VCAM-1 or 4-integrins used together with an
anti-P-selectin MoAb blocked all eosinophil tethering and accumulation
on IL-4-stimulated HUVECs. As the shear stress was lowered, P-selectin
and VCAM-1 acted independently to recruit eosinophils. This difference
in adhesion molecule use is likely due to the forces exerted on the
flowing eosinophil at high versus low shear stresses. The loss of one
receptor ligand pair at high shear stresses is sufficient to impair
eosinophil tethering and accumulation, whereas the decreased forces
exerted on the eosinophil at lower shear stresses allow for loss of
some interactions without affecting eosinophil accumulation.
A striking observation was that eosinophils did not roll on
IL-4-stimulated endothelial cells. Instead, eosinophils tethered and
rapidly became firmly adherent. MoAbs directed against P-selectin, VCAM-1, and/or 4-integrins alone or in concert did not alter the nature of these eosinophil interactions, yet eosinophils do roll on
purified surfaces expressing P-selectin10 or VCAM-1 (this
study). When we explored a role for the 2-integrins in firm
adhesion, we found that an anti-2-integrin MoAb alone did not alter
the percentage of adherent cells (Table 1). However, an
anti-2-integrin MoAb together with an anti-4 MoAb not only decreased attachment, but also led to a dramatic increase in the percentage of rolling eosinophils (Table 1). Thus, just as P-selectin and VCAM-1/4-integrins act together to tether eosinophils,
2-integrins and VCAM-1/4-integrins act together to support firm
adhesion.
Activation of 2-integrins on eosinophils along with the data in Fig
2 showing the dramatic shape change and transmigration of eosinophils
on IL-4-stimulated endothelial cells suggest that these endothelial
cells express or release an eosinophil activator(s). Ligation of
adhesion receptors may prime eosinophils to respond optimally to these
activation signals. Ligation of P-selectin glycoprotein
ligand-1 on neutrophils leads to increased tyrosine phosphorylation on several target proteins and activates members of the
MAP kinase family.35 Similar activation occurs in
eosinophils upon ligation of PSGL-1 (manuscript in
preparation). Ligation of the 4-integrins has also been
shown to activate MAP kinase family proteins in monocytic
cells36 and may also play a role in eosinophil activation.
Thus, both outside-in signaling through PSGL-1 or 4-integrins and
release of eosinophil activators from IL-4-stimulated endothelial
cells may be responsible for the abrupt adhesion of eosinophils in this
system.
Our results show that eosinophils use both P-selectin and VCAM-1 to
tether and accumulate on IL-4-stimulated HUVECs. Both of these
molecules are critical at high shear stresses, but either can
independently meditate accumulation at lower shear stresses. At all
shear stresses, eosinophils rapidly become firmly adherent, an
interaction that uses both 4- and 2-integrins. The ability of
eosinophils to effectively use multiple adhesion molecules both for
tethering and firm adhesion may be particularly important in
understanding the mechanisms of eosinophil trafficking in vivo where
vascular flow can regulate the efficacy of interactions. MoAbs directed
against both the selectins and VCAM-1/4-integrins may be required to
prevent eosinophil recruitment at lower shear stresses, whereas in
faster flowing vessels, blocking the selectins would be sufficient to
inhibit recruitment. This is the first time that endothelial VCAM-1,
through interactions with 4-integrins, has been shown to support
significant tethering of eosinophils at shear stresses greater than 1 dyn/cm2. Thus, these experiments may shed some light on the
mechanisms used for the selective accumulation of eosinophils at sites
of allergic inflammation.
| |
ACKNOWLEDGMENT |
The author thanks Dr Rodger McEver and Dr John Elliot for their
generous gifts of reagents; Dr Paul Kubes for critical reading of this
manuscript; Evelyn Lailey for her technical assistance; and the Labor
and Delivery unit at the Foothills Hospital in Calgary for their
assistance in providing umbilical cords.
| |
FOOTNOTES |
Submitted February 26, 1998;
accepted July 8, 1998.
Supported by grants from the Medical Research Council of Canada
(MT-14180) and the Alberta Heritage Foundation for Medical Research
(970234).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Kamala D. Patel, PhD, Department of
Physiology and Biophysics, University of Calgary, 3330 Hospital Dr NW,
Calgary, Alberta, Canada T2N 4N1.
| |
REFERENCES |
1.
Gleich GJ:
The eosinophil and bronchial asthma: Current understanding.
J Allergy Clin Immunol
85:422, 1990[Medline]
[Order article via Infotrieve]
2.
Sedgwick JB, Calhoun WJ, Gleich GJ, Kita H, Abrams JS, Schwartz LB, Volovitz B, Ben-Yaakov M, Busse WW:
Immediate and late airway response of allergic rhinitis patients to segmental antigen challenge. Characterization of eosinophil and mast cell mediators.
Am Rev Respir Dis
144:1274, 1991[Medline]
[Order article via Infotrieve]
3.
Wardlaw AJ, Symon FS, Walsh GM:
Eosinophil adhesion in allergic inflammation.
J Allergy Clin Immunol
94:1163, 1994[Medline]
[Order article via Infotrieve]
4.
Frigas E, Motojima S, Gleich GJ:
The eosinophilic injury to the mucosa of the airways in the pathogenesis of bronchial asthma.
Eur Respir J Suppl
13:123s, 1991[Medline]
[Order article via Infotrieve]
5.
Wardlaw A:
The eosinophil: New insights into its function in human health and disease [editorial].
J Pathol
179:355, 1996[Medline]
[Order article via Infotrieve]
6.
Zimmerman GA, Prescott SM, McIntyre TM:
Endothelial cell interactions with granulocytes: Tethering and signaling molecules.
Immunol Today
13:93, 1992[Medline]
[Order article via Infotrieve]
7.
Springer TA:
Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration.
Annu Rev Physiol
57:827, 1995[Medline]
[Order article via Infotrieve]
8.
McEver RP, Moore KL, Cummings RD:
Leukocyte trafficking mediated by selectin-carbohydrate interactions.
J Biol Chem
270:11025, 1995[Abstract/Free Full Text]
9.
McEver RP:
Selectins.
Curr Opin Immunol
6:75, 1994[Medline]
[Order article via Infotrieve]
10.
Patel KD, McEver RP:
Comparison of tethering and rolling of eosinophils and neutrophils through selectins and P-selectin glycoprotein ligand-1.
J Immunol
159:4555, 1997[Abstract]
11.
Fuhlbrigge RC, Alon R, Puri KD, Lowe JB, Springer TA:
Sialylated, fucosylated ligands for L-selectin expressed on leukocytes mediate tethering and rolling adhesions in physiologic flow conditions.
J Cell Biol
135:837, 1996[Abstract/Free Full Text]
12.
Sriramarao P, Broide DH:
Differential regulation of eosinophil adhesion under conditions of flow in vivo.
Ann NY Acad Sci
796:218, 1996[Medline]
[Order article via Infotrieve]
13.
Alon R, Kassner PD, Carr MW, Finger EB, Hemler ME, Springer TA:
The integrin VLA-4 supports tethering and rolling in flow on VCAM-1.
J Cell Biol
128:1243, 1995[Abstract/Free Full Text]
14.
Berlin C, Bargatze RF, Campbell JJ, von Andrian UH, Szabo MC, Hasslen SR, Nelson RD, Berg EL, Erlandsen SL, Butcher EC:
Alpha 4 integrins mediate lymphocyte attachment and rolling under physiologic flow.
Cell
80:413, 1995[Medline]
[Order article via Infotrieve]
15.
Lichtman AH, Ding H, Henault L, Vachino G, Camphausen R, Cumming D, Luscinskas FW:
CD45RA-RO+ (memory) but not CD45RA+RO (naive) T cells roll efficiently on E- and P-selectin and vascular cell adhesion molecule-1 under flow.
J Immunol
158:3640, 1997[Abstract]
16.
Luscinskas FW, Ding H, Tan P, Cumming D, Tedder TF, Gerritsen ME:
L- and P-selectins, but not CD49d (VLA-4) integrins, mediate monocyte initial attachment to TNF-alpha-activated vascular endothelium under flow in vitro.
J Immunol
157:326, 1996[Abstract]
17.
Abe Y, Ballantyne CM, Smith CW:
Functions of domain 1 and 4 of vascular cell adhesion molecule-1 in alpha4 integrin-dependent adhesion under static and flow conditions are differentially regulated.
J Immunol
157:5061, 1996[Abstract]
18.
Kitayama J, Fuhlbrigge RC, Puri KD, Springer TA:
P-selectin, L-selectin, and alpha 4 integrin have distinct roles in eosinophil tethering and arrest on vascular endothelial cells under physiological flow conditions.
J Immunol
159:3929, 1997[Abstract]
19. Borish L, Rosenwasser LJ: Update on cytokines. J Allergy Clin
Immunol 97:719 (quiz 734), 1996
20.
McEver RP, Martin MN:
A monoclonal antibody to a membrane glycoprotein binds only to activated platelets.
J Biol Chem
259:9799, 1984[Abstract/Free Full Text]
21.
Geng JG, Bevilacqua MP, Moore KL, McIntyre TM, Prescott SM, Kim JM, Bliss GA, Zimmerman GA, McEver RP:
Rapid neutrophil adhesion to activated endothelium mediated by GMP-140.
Nature
343:757, 1990[Medline]
[Order article via Infotrieve]
22.
Moore KL, Eaton SF, Lyons DE, Lichenstein HS, Cummings RD, McEver RP:
The P-selectin glycoprotein ligand from human neutrophils displays sialylated, fucosylated, O-linked poly-N-acetyllactosamine.
J Biol Chem
269:23318, 1994[Abstract/Free Full Text]
23.
Patel KD, Moore KL, Nollert MU, McEver RP:
Neutrophils use both shared and distinct mechanisms to adhere to selectins under static and flow conditions.
J Clin Invest
96:1887, 1995
24.
Zimmerman GA, McIntyre TM, Prescott SM:
Thrombin stimulates the adherence of neutrophils to human endothelial cells in vitro.
J Clin Invest
76:2235, 1985
25.
Reinhardt PH, Elliott JF, Kubes P:
Neutrophils can adhere via alpha4beta1-integrin under flow conditions.
Blood
89:3837, 1997[Abstract/Free Full Text]
26.
Moore KL, Patel KD, Bruehl RE, Li F, Johnson DA, Lichenstein HS, Cummings RD, Bainton DF, McEver RP:
P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin.
J Cell Biol
128:661, 1995[Abstract/Free Full Text]
27.
Patel KD, Nollert MU, McEver RP:
P-selectin must extend a sufficient length from the plasma membrane to mediate rolling of neutrophils.
J Cell Biol
131:1893, 1995[Abstract/Free Full Text]
28.
Yao L, Pan J, Setiadi H, Patel KD, McEver RP:
Interleukin 4 or oncostatinMinduces a prolonged increase in P-selectin mRNA and protein in human endothelial cells.
J Exp Med
184:81, 1996[Abstract/Free Full Text]
29.
Symon FA, Lawrence MB, Williamson ML, Walsh GM, Watson SR, Wardlaw AJ:
Functional and structural characterization of the eosinophil P-selectin ligand.
J Immunol
157:1711, 1996[Abstract]
30.
Wardlaw AJ, Moqbel R, Kay AB:
Eosinophils: Biology and role in disease.
Adv Immunol
60:151, 1995[Medline]
[Order article via Infotrieve]
31.
Sriramarao P, von Andrian UH, Butcher EC, Bourdon MA, Broide DH:
L-selectin and very late antigen-4 integrin promote eosinophil rolling at physiological shear rates in vivo.
J Immunol
153:4238, 1994[Abstract]
32.
Johnston B, Issekutz TB, Kubes P:
The alpha 4-integrin supports leukocyte rolling and adhesion in chronically inflamed postcapillary venules in vivo.
J Exp Med
183:1995, 1996[Abstract/Free Full Text]
33.
Luscinskas FW, Kansas GS, Ding H, Pizcueta P, Schleiffenbaum BE, Tedder TF, Gimbrone MA Jr:
Monocyte rolling, arrest and spreading on IL-4-activated vascular endothelium under flow is mediated via sequential action of L-selectin, beta 1-integrins, and beta 2-integrins.
J Cell Biol
125:1417, 1994[Abstract/Free Full Text]
34.
Luscinskas FW, Ding H, Lichtman AH:
P-selectin and vascular cell adhesion molecule 1 mediate rolling and arrest, respectively, of CD4+ T lymphocytes on tumor necrosis factor alpha-activated vascular endothelium under flow.
J Exp Med
181:1179, 1995[Abstract/Free Full Text]
35.
Hidari KI, Weyrich AS, Zimmerman GA, McEver RP:
Engagement of P-selectin glycoprotein ligand-1 enhances tyrosine phosphorylation and activates mitogen-activated protein kinases in human neutrophils.
J Biol Chem
272:28750, 1997[Abstract/Free Full Text]
36.
McGilvray ID, Lu Z, Bitar R, Dackiw APB, Davreux CJ, Rotstein OD:
VLA-4 integrin cross-linking on human monocytic THP-1 cells induces tissue factor expression by a mechanism involving mitogen-activated protein kinase.
J Biol Chem
272:10287, 1997[Abstract/Free Full Text]
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Abstract
Introduction