|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 592-601
IMMUNOBIOLOGY
VCAM-1 is more effective than MAdCAM-1 in supporting eosinophil
rolling under conditions of shear flow
P. Sriramarao,
Richard G. DiScipio,
Ronald R. Cobb,
Myron Cybulsky,
Greg Stachnick,
Diego Castaneda,
Mariano Elices, and
David H. Broide
From the Laboratory of Immunology and Vascular Biology, La Jolla
Institute for Experimental Medicine La Jolla, CA; Cytel Corporation, La
Jolla, CA; Department of Biology, Tanabe Research Laboratories, San
Diego, CA; Department of Laboratory Medicine and Pathobiology,
University of Toronto, Toronto, Ontario; and Department of
Medicine, University of California San Diego, La Jolla, CA.
 |
Abstract |
The ability of the  4 integrin counterligands vascular cell
adhesion molecule (VCAM)-1 or mucosal addressin (MAd)CAM-1 to support eosinophil rolling or firm adhesion under conditions of physiologic flow has not been delineated. Using a parallel plate flow
chamber in vitro and intravital microscopy in vivo, we demonstrate that
eosinophil rolling and adhesion on VCAM-1 is mediated by both 4 1
and 47 integrins. Eosinophils rolled equally efficiently on both
VCAM-1 2 domain and VCAM-1 7 domain, suggesting that the N-terminal 2 domains of VCAM-1 are sufficient to support eosinophil rolling under
conditions of flow. Furthermore, activation of the eosinophil 1
integrin with monoclonal antibody (mAb) 8A2 resulted in both resistance
to shear stress-induced detachment from VCAM-1 in vitro and in stable
arrest of rolling eosinophils on interleukin (IL)-1-stimulated
venules in vivo. Eosinophils rolled less efficiently on MAdCAM-1- than
on VCAM-1-coated coverslips under conditions of flow. However,
eosinophils firmly adhered as efficiently to MAdCAM-1 as to VCAM-1.
Overall, these results demonstrate that both VCAM-1 and MAdCAM-1 can
support eosinophil firm adhesion under conditions of flow. In contrast,
VCAM-1 is significantly more efficient than MAdCAM-1 in supporting
eosinophil rolling under conditions of flow.
(Blood. 2000;95:592-601)
© 2000 by The American Society of Hematology.
| |
Introduction |
The recruitment of eosinophils to extravascular tissue
sites during episodes of asthma and allergic inflammation is mediated by the adhesive interactions between circulating eosinophils and vascular endothelial cells.1-4 In vitro, the adherence of
eosinophils to cytokine-stimulated cultured endothelial cells is
mediated by several endothelial-expressed adhesion molecules, including E-selectin (CD62E),5 P-selectin
(CD62P),6 intercellular adhesion molecule (ICAM)-1
(CD54),7 and vascular cell adhesion molecule (VCAM)-1
(CD106).8 The interaction of eosinophils with these
vascular adhesion molecules is mediated by eosinophil cell surface
receptors, including L-selectin (CD62L),9 P-selectin glycoprotein ligand (PSGL)-1,10 and 2
(CD18)11 and  4 (CD49d) integrins.12
Eosinophils express both 41 and 47
integrins,13,14 both of which bind to VCAM-1.15
In contrast, only 47 integrins mediate eosinophil adhesion to
mucosal addressin MAdCAM-1.16 The importance of 4
integrins to eosinophil recruitment has been supported by several
studies demonstrating that anti-4 monoclonal antibodies (mAbs) block
the accumulation of eosinophils at sites of allergic inflammation in
vivo.17-19
We have recently demonstrated that the sequential events of eosinophil
adhesion to vascular endothelial cells in vivo (ie, rolling, adhesion,
and transmigration) are mediated by distinct adhesion
receptors.3,20,62 The initial interaction between eosinophils and vascular endothelial cells is weak and transient under
conditions of flow and is characterized by rolling in postcapillary venules. Eosinophil rolling in inflamed postcapillary venules is
mediated by 41 integrins and L-selectin20 as well as
by vascular P-selectin21,22 but not
E-selectin.23,24 The presence of integrin activating
factors released at inflamed tissue sites could then trigger stable
adhesion of rolling eosinophils followed by their emigration into
extravascular tissue sites.62 Although the relative
contribution of individual adhesion molecules and cytokines to the
sequential steps of eosinophil adhesion to endothelium is only
partially understood, it is clear that the regulation of eosinophil
adhesion at any of these sequential steps could be critical to the
sequestration of eosinophils at sites of allergic inflammation.
Recent studies have revealed that 4 integrins expressed by
eosinophils are capable of existing in different activation states characterized by either low or high affinity binding to counterligands such as VCAM-1.25,26 In a single-cell adhesion assay,
activation of eosinophils with granulocyte-macrophage
colony-stimulating factor (GM-CSF) resulted in enhancement of the
binding strength of eosinophil-expressed 4 integrins for
VCAM-1-coated surfaces.25 Similarly, the ability
of activating agents such as phorbol myristate acetate (PMA), manganese
(Mn)2+, and 1 integrin-activating mAbs to alter the
activation state of 4 integrins on lymphocytes has also been
demonstrated.27-29 Activation of the lymphocyte 41 or
47 integrin results in transformation of either of these
lymphocyte integrin rolling receptors into firm adhesion receptors in
vitro.27,28 However, the regulation of 4 integrin
activation in vivo (as opposed to flow chamber studies in vitro) and
the relative contribution of the two 4 integrins, 41 and
47, in mediating the initial events of eosinophil adhesion in
vivo (ie, rolling in comparison to firm adhesion) during inflammation
has not been clearly delineated. In the present investigation, we have
made the novel observation that (a) eosinophils roll more efficiently
on VCAM-1 than on MAdCAM-1 and (b) the eosinophil 1 integrin
activation state can rapidly upregulate eosinophil firm adhesion and
resistance to detachment from VCAM-1 in vitro and in vivo. In addition,
we demonstrate that eosinophils roll equally efficiently on both VCAM-1
2d (VCAM-1 2 domain) as well as on VCAM-1 7d (VCAM-1 7 domain),
suggesting that the N-terminal 2 domains of VCAM-1 are sufficient to
support eosinophil rolling under conditions of flow.
| |
Materials and methods |
Isolation and labeling of eosinophils
Eosinophils were purified from the peripheral blood of patients
suffering from mild asthma/allergic rhinitis as previously described in
this laboratory20 in a protocol approved by the University
of California, San Diego, Human Subjects Committee. Eosinophils with
more than 95% purity and more than 95% viability were recovered by
negative selection using a Magnetic Assembly Cell Separator (Miltenyi
Biotec, Burlingame, CA) and magnetized anti-CD16
Ab.30 These eosinophils were used in the in vitro laminar flow chamber adhesion assay and in the in vivo
adhesion experiments. For the in vivo experiments, eosinophils
were fluorescently labeled with carboxy fluorescein diacetate (CFDA;
Molecular Probes, Eugene, OR) as previously described.20
Eosinophils were resuspended in Dulbecco's phosphate buffered saline
(DPBS) containing 0.01% glucose at a concentration of 1 × 107 cells/mL and kept at room temperature in the dark until use.
Antibodies
Adhesion-blocking mAbs directed against human 4 (mAb P4G9 and mAb
P4C2),31 anti-human 1 (mAb P4C10),32
anti-human 2 mAb IB4,33 and anti-rabbit VCAM-1 (mAb Rb
1/9)34 were used. A 1 integrin-activating antibody,
mAb 8A2,35 was obtained from Dr John Harlan (University of
Washington, Seattle, WA), and a rat anti-mouse 7 mAb (F1B504) with
cross-reactivity to human 736 was obtained from Dr
Eugene Butcher (Stanford University, Stanford, CA) and used in the
adhesion experiments in vitro and in vivo.
Recombinant VCAM-1
The soluble VCAM-1 used in the in vitro flow chamber studies is a
truncated form of VCAM-1 containing all 7 extracellular immunoglobulin
(Ig) domains, including the VLA-4 binding sites on domains 1 and 4. The
soluble VCAM-1 was produced by Dr Carl Perez (Cytel Corporation, La
Jolla, CA) in a mammalian expression vector and purified by
immunoaffinity chromatography on the anti-VLA-4 mAb P3H12 as previously
described.25
Recombinant MAdCAM-1
Soluble MAdCAM-1 used in these studies contains the entire
extracellular domain of MAdCAM-1.16 The soluble MAdCAM-1
was expressed using a baculovirus expression vector containing the human Fc sequence and Sf9 cells grown in SF90011
serum-free media (Life Technologies, Gaithersburg, MD).
Recombinant MAdCAM-1 protein was purified from the supernatants using
protein A affinity purification membranes using the procedures
recommended by the manufacturer (Nygene, Golden Bridges, NY).
In vitro laminar flow eosinophil VCAM-1 and MAdCAM-1 adhesion assay
The ability of eosinophils to roll on soluble VCAM-1 or MAdCAM-1 was
assessed using an in vitro parallel plate laminar flow chamber as
previously described in this laboratory.23 Briefly, glass
coverslips were coated with soluble VCAM-1 or MAdCAM-1 (200 µL at 10 µg/mL) for 1 hour at 37°C. Unbound sites on the coverslips were
then blocked with BSA for 15 minutes. The glass coverslip with
immobilized VCAM-1 or MAdCAM-1 was then positioned in the bottom of a
parallel plate flow chamber (100 µm thickness), where the coverslip
was exposed to different flow conditions. Defined levels of flow were
applied to the coverslips in the flow chamber by perfusing warm media
(RPMI containing 0.75 mM calcium (Ca)2+ and magnesium
(Mg)2+ and 0.2% HSA) through a syringe pump (Harvard
Apparatus, South Natick, MA). Different wall shear stress levels were
achieved by varying the flow rates as previously
described.23 Care was taken to eliminate air bubbles in the
channel during loading of the coverslip and assembly of the flow
chamber. The flow apparatus with the immobilized VCAM-1 or MAdCAM-1 was
mounted onto the stage of an inverted phase contrast microscope (Nikkon
Inc, Garden City, NY). The flow chamber was then perfused with
eosinophils (2 × 105 cells) for 2 minutes, and
interaction of the injected cells with VCAM-1 or MAdCAM-1 was observed
and videorecorded. Rolling or adherent eosinophils were identified by
qualitative assessment of their interaction with VCAM-1 or MAdCAM-1.
Rolling eosinophils in contact with VCAM-1 or MAdCAM-1 demonstrated
multiple discrete interruptions and flowed slowly, while adherent
eosinophils remained stationary at a given point for extended times
(more than 30 seconds). Recorded images were subjected to offline video
analysis to manually enumerate the number of interacting eosinophils.
All results are expressed as the number of rolling or adherent
eosinophils per field (average of 4 fields; 20× field) per 2 × 105 eosinophils during a 2-minute observation period. Data
represent mean ± SEM. In some experiments, eosinophils were
preincubated with a function-blocking anti-4, anti-1, or
anti-7 integrin mAbs (50 µg/mL), or isotype-matched control
antibody, for 20 minutes at room temperature prior to infusion of
eosinophils into the flow chamber.
Influence of a 1 integrin-activating mAb on the resistance
of eosinophils to detachment from VCAM-1
To evaluate the influence of a 1 integrin-activating antibody on
the resistance of eosinophils to detachment from VCAM-1, stepwise
increases in shear stress were applied for 15 seconds at each shear
force (2-20 dyn/cm2) to eosinophils adherent to VCAM-1 in
the flow chamber. The number of eosinophils firmly adherent to VCAM-1
per field was recorded before and after each stepwise increase in shear
stress. In these experiments, eosinophils were incubated with 2 µg/mL
of a 1 integrin-activating mAb 8A2, or control antibody, for 15 minutes prior to infusion of eosinophils into the flow chamber.
Eosinophil static adhesion and resistance to detachment from
MAdCAM-1
Because few eosinophils that were perfused into the flow chamber
rolled or adhered to MAdCAM-1, additional experiments were performed to
demonstrate that eosinophils were able to adhere to MAdCAM-1 in a
static adhesion assay as previously reported.14 In these
experiments 2 × 105 eosinophils were directly placed on
the MAdCAM-1-coated coverslip used in the flow chamber experiments and
allowed to adhere for 15 to 30 minutes at room temperature in a static
adhesion assay. At the end of the incubation period, the coverslip was
washed with media to remove nonadherent eosinophils. The coverslip was then positioned in the bottom of the parallel plate flow chamber and
mounted on the stage of the inverted microscope as described above. The
number of eosinophils firmly adherent to MAdCAM-1 was videorecorded in
the absence of flow conditions. To evaluate the resistance of
eosinophils to detachment from MAdCAM-1, stepwise increases in shear
stress were then applied for 15 seconds at each shear force (2-20 dyn/cm2) to eosinophils adherent to MAdCAM-1 in the flow chamber.
In selected experiments, the specificity of the eosinophil-expressed
47 interaction with MAdCAM-1 was determined by preincubating the eosinophils with an anti-7 mAb prior to adding
eosinophils to MAdCAM-1-coated coverslips used in the static adhesion assay.
VCAM-1 2d and VCAM-1 7d eosinophil flow chamber adhesion assay
To explore the relative importance of the N-terminal 2 domains of
VCAM-1 (VCAM-1 2d) as compared to the 7-domain form of VCAM-1 (VCAM-1
7d) in subserving an eosinophil rolling or adhesive interaction under
conditions of flow, we used varying concentrations (0.5-10 µg/mL) of
VCAM-1 2d and VCAM-1 7d in our flow chamber adhesion assay as described
above. VCAM-1 2d and VCAM-1 7d (expressed using a baculovirus
expression vector), were kindly provided by Tanabe Research
Laboratories, San Diego, CA, and have been used in static eosinophil
adhesion assays as previously described.37
Rabbit preparation for local mesenteric vascular bed instillation of
an anti-VCAM-1 mAb and infusion of fluorescently labeled
eosinophils in vivo
Rolling of human eosinophils in the postcapillary venules of the
interleukin [IL]-1-stimulated mesenteric circulation of New
Zealand White rabbits in vivo was visualized by intravital microscopy
as previously described.20,23 The ability of local mesenteric vascular bed instillation of an anti-rabbit VCAM-1 mAb Rb
1/9 to block eosinophil adhesion in vivo was assessed using a balloon
catheter method previously described to administer anti-E-selectin mAbs.23,38 In brief, the rabbit mesentery was exteriorized and a side branch of the superior mesenteric artery cannulated with
PE-10 tubing.23 The collateral mesenteric circulation to the ileum was occluded using occluder clamps. A balloon catheter was
then gently wrapped and tied around the main stem of the superior mesenteric artery, upstream of the cannulated side branch, to allow
temporary but complete occlusion of the blood flow through the
mesenteric artery. Initially, video recordings were obtained to
determine the baseline rolling of fluorescently labeled human eosinophils in the mesenteric venules. Subsequently, the mesenteric blood flow was briefly interrupted by inflation of the balloon catheter
with an air-filled syringe. Immediately after complete cessation of
blood flow, 3 mL of a neutralizing anti-rabbit VCAM-1 (Rb 1/9), or an
isotype-matched antibody, at a concentration of 50 µg/mL was slowly
infused through the cannulated tubing as previously described.23 The injected mAb Rb 1/9 or isotype-matched
control antibodies were allowed to interact with the rabbit endothelium for a maximum of 10 minutes. Thereafter, the balloon catheter was
deflated, and previous levels of blood flow resumed immediately. CFDA-labeled eosinophils were then injected and the interaction of
eosinophils with antibody-treated vessel walls was determined over a
15-minute observation period.
In some experiments, eosinophils were incubated ex vivo with either
function-blocking anti-4, anti-1, anti-7, or control mAbs (50 µg/mL) for 20 minutes at room temperature and then injected into the
rabbit mesentery as previously described.20 In other experiments, eosinophils were incubated with 20 µg/mL of a 1 integrin-activating mAb 8A2 for 5 to 15 minutes prior to their injection into the mesenteric circulation.
Intravital microscopy and image analysis
CFDA-labeled eosinophils (0.2 × 107 to
0.5 × 107) were infused through the cannulated artery
approximately 6 to 10 hours after IL-1 stimulation. The passage of
eosinophils in the inflamed venules was made visible by stroboscopic
epi-illumination as previously described20,23 and recorded
using an SVHS video recorder. The video recordings were
analyzed offline by manually counting the total number of CFDA-labeled
eosinophils passing through a venular segment (total flux). The tapes
were rewound, and only those cells found to be visibly rolling along
the venular wall were counted (rolling flux). The rolling fraction
(Rf ) was calculated as the percentage of rolling cells in the total
flux of eosinophils passing through a venule during a given injection.
Eosinophils were considered adherent if they remained stationary for
more than 30 seconds in the mesenteric vessels.
Analysis of eosinophil rolling velocities
The mean rolling velocity of injected eosinophils in
IL-1-stimulated mesenteric venules before and after mAb treatment
was manually determined by frame-by-frame analysis of recorded video images as previously described.39 The mean rolling velocity of eosinophils are expressed as mm/sec ± SD.
Statistics
The interaction between eosinophils and venular endothelium in vivo
before and after mAb treatment was analyzed by the Student t test using
a statistical software package (SigmaStat, Jandel Scientific, San Rafael, CA). The rolling or adhesion of eosinophils to
VCAM-1 or MAdCAM-1 in vitro in the laminar flow chamber was compared by
the Student t test using a statistical software package (In Stat, San
Diego, CA). Results are given as mean ± SD (unless otherwise
indicated) and P values of <.05 were considered statistically significant.
| |
Results |
Eosinophils roll and adhere on VCAM-1 under conditions of
physiologic shear stress in vitro: comparison with rolling and
adhesion on MAdCAM-1
The ability of eosinophils to interact with VCAM-1 at physiologic
conditions of shear force was examined using a parallel plate flow
chamber in vitro. Significant number of eosinophils rolled on VCAM-1
(35.7 ± 6.8) but not on control BSA-coated cover slips
(2.3 ± 1.5) (P = .01) (n = 3) (shear stress 0.7 dyn/cm2) (Figure 1A). There was significant firm adhesion
of flowing eosinophils to VCAM-1 (45.4 ± 9.1) but not to control
BSA-coated cover slips (0.3 ± 0.1) (P = .01) (n = 3)
(shear stress 0.7 dyn/cm2) (Figure 1B). The increase in
level of shear stress from 0.7 to 1.4 dyn/cm2 was
associated with a decrease in the number of eosinophils rolling on, or
adhering to, VCAM-1 (Figure 1).
View larger version (22K):
[in this window]
[in a new window]
| Fig 1.
Eosinophil rolling on VCAM-1 in vitro.
Eosinophils were infused at a flow rate of 0.7 and 1.4 dyn/cm2 into a parallel plate flow chamber containing
VCAM-1- or BSA-coated coverslips. The number of rolling eosinophils
(A) and adherent eosinophils (B) during continuous flow periods of 2 minutes were recorded and subjected to offline analysis. Results of
experiments performed are presented as the mean ± SEM (n = 4
experiments). At flow rates of 0.7 dyn/cm2, significant
numbers of eosinophils rolled on VCAM-1 (P = .001 vs BSA) and
adhered to VCAM-1 (P = .001 vs BSA). At flow rates of 1.4 dyn/cm2, significant numbers of eosinophils rolled on
VCAM-1 (P = .001 vs BSA), but the number of eosinophils
adherent to VCAM-1 was significantly reduced. Solid bar, BSA; hatched
bar, VCAM-1.
|
|
Eosinophils also rolled on MAdCAM-1 (7.1 ± 0.9) (shear stress 0.7 dyn/cm2), but this was less efficient than on VCAM-1
(35.7 ± 6.8) (P = .001) (n = 3) (Figure
2A). The eosinophil rolling on MAdCAM-1 (7.1 ± 0.9) did not lead to sustained eosinophil firm adhesion to
MAdCAM-1 of the rolling eosinophils. In contrast, eosinophil rolling on
VCAM-1 (35.7 ± 6.8) frequently led to subsequent eosinophil firm
adhesion to VCAM-1 (45.4 ± 9.1) (Figure 2A).
View larger version (2335K):
[in this window]
[in a new window]
| Fig 2.
Eosinophil flow and static adhesion to MAdCAM-1.
(A) Flow chamber study. Eosinophils were infused at a flow rate of 0.7 dyn/cm2 into a parallel plate flow chamber containing
MAdCAM-1-, VCAM-1-, or BSA-coated coverslips. The number of rolling
eosinophils and adherent eosinophils during continuous flow periods of
2 minutes was recorded and subjected to offline analysis. Results of
experiments performed are presented as the mean ± SEM (n = 4
experiments). At flow rates of 0.7 dyn/cm2, significant
numbers of eosinophils rolled on VCAM-1 (P = .001 vs BSA) and
adhered to VCAM-1 (P = .001 vs BSA). At the same flow rate,
significantly fewer eosinophils rolled on MAdCAM-1 compared with VCAM-1
(P = .001) with few rolling eosinophils remaining firmly
adherent to MAdCAM-1. (B) Static adhesion assay. Eosinophils were
allowed to adhere for 30 minutes to a MAdCAM-1- or BSA-coated
coverslip that had been subjected either to no shear stress (preflow
panel) or to 1.4 dyn/cm2 shear stress (to simulate whether
flow would strip MAdCAM-1 from the coverslip) (postflow panel).
Nonadherent cells were then washed from the coverslip, and the
coverslip was placed in the flow chamber. The number of adherent
eosinophils was recorded on videotape. In the detachment panel, the
eosinophils that had adhered to MAdCAM-1 (preflow panel) were subjected
to shear stress (20 dyn/cm2) to determine whether the
firmly adherent eosinophils were resistant to detachment from MAdCAM-1.
There was no significant difference in the number of eosinophils
adherent to MAdCAM-1 before and after application of shear force to the
coverslip (preflow vs postflow) or after application of shear force to
eosinophils adherent to MAdCAM-1 (P = not significant)
(n = 3).
|
|
The reduced rolling and adhesion of eosinophils to MAdCAM-1
was not due to an inability of eosinophils to bind in sufficient numbers to MAdCAM-1-coated coverslips as evidenced by the following static adhesion experiments. In a static adhesion assay, using the same
MAdCAM-1-coated coverslips as in the flow chamber experiments, significant numbers of eosinophils firmly adhered to MAdCAM-1 (103.3 ± 17.1) (n = 3) as compared to BSA-coated coverslips
(2.1 ± 1.3) (P = .001). These firmly adherent eosinophils
were as resistant to detachment from MAdCAM-1 as from
VCAM-1-coated coverslips (less than 1% of eosinophils detached from
either MAdCAM-1 or VCAM-1 when shear stress was increased from 2 to 20 dyn/cm2). Preincubation of eosinophils with a
neutralizing antibody to 7 integrins significantly inhibited
eosinophil binding to MAdCAM-1 by 94.2 ± 5.7% in the static adhesion
assay, whereas a control anti-1 antibody inhibited eosinophil
binding by less than 5%. To determine whether the reduced ability of
eosinophils to adhere to MAdCAM-1 in the flow chamber experiments could
be due to stripping of MAdCAM-1 off the coverslip during the flow
chamber experiments, we performed experiments in which we subjected the
MAdCAM-1-coated coverslip to the maximal shear stress (1.4 dyn/cm2) used in the eosinophil MAdCAM-1 flow adhesion
studies. After the MAdCAM-1-coated coverslip was subjected to 1.4 dyn/cm2 shear stress, eosinophils were added to the
coverslip. The number of eosinophils adherent to MAdCAM-1-coated
coverslips that had been subjected to shear stress (105.5 ± 13.9
eosinophils) determined under static adhesion assay conditions did not
differ significantly from the number of eosinophils adherent to
MAdCAM-1-coated coverslips before they were subjected to 1.4 dyn/cm2 shear stress (103.3 ± 17.1 eosinophils) (Figure
2B).
Effect of anti-4, anti-1, and anti-7 mAbs on eosinophil
rolling and adhesion to VCAM-1 in vitro
Eosinophils preincubated with an anti-4 mAb P4C2 did not exhibit
significant rolling on VCAM-1 (4.3 ± 1.6 eosinophils treated with
an anti-4 mAb rolling on VCAM-1 vs 23.5 ± 9.0 eosinophils treated with an isotype-matched mAb rolling on VCAM-1)
(P = .01) (Figure 3A). To
determine whether 41 or 47 integrins subserved the
eosinophil rolling function on VCAM-1, eosinophils were preincubated with neutralizing antibod ies to individual integrins (1 or 7) or to both integrins. The combination of anti-1 (P4C10) and anti-7 (FIB504) integrin mAbs almost completely inhibited eosinophil rolling on VCAM-1 (P = .01) (n = 3 experiments) (Figure
3B). When used individually, the anti-1 Ab and the anti-7 Ab were
each able to inhibit eosinophil rolling on VCAM-1 but did so less
efficiently than the combination of the anti-1 and anti-7 Abs
(Figure 3B).
View larger version (2735K):
[in this window]
[in a new window]
| Fig 3.
Effect of anti-4, anti-1, and anti-7 integrin
mAbs on eosinophil rolling on VCAM-1 in vitro.
Eosinophils (preincubated with either an anti-4, anti-1,
anti-7, anti-1 and anti-7 in combination, or control mAb) were
infused at a flow rate of 0.7 dyn/cm2 into a parallel plate
flow chamber containing VCAM-1- or BSA-coated coverslip. The number of
rolling eosinophils during continuous flow periods of 2 minutes was
recorded and subjected to offline analysis. Results of experiments
performed are presented as the mean ± SEM (n = 3 experiments). (A)
The anti-4 mAb significantly inhibited eosinophil rolling on VCAM-1
(P = .01 vs control). (B) The anti-1 and anti-7 mAbs in
combination (P = .01 vs control) as well as the individual
anti-1 mAb (P = .05 vs control) and anti-7 mAb
(P = .05 vs control) inhibited eosinophil rolling on
VCAM-1.
|
|
Comparison of eosinophil rolling and adhesion on VCAM-1 2d and
VCAM-1 7d under conditions of flow
Previous studies have demonstrated the importance of an IDSPL
sequence in domains 1 and 4 of VCAM-1 to the binding of 4 integrins to VCAM-1 in static adhesion assays40-42 but have not
investigated their role in eosinophil/VCAM-1 interactions under
conditions of flow. We have compared the ability of eosinophils to roll
and adhere to a 2-domain VCAM-1 (VCAM-1 2d containing 4 integrin binding domain 1 but not domain 4) and a 7-domain VCAM-1 (VCAM-1 7d
containing 4 integrin binding domain 1 and domain 4) under conditions of flow. In these studies, we noted that the VCAM-1 2d was
as efficient as the VCAM-1 7d in mediating eosinophil rolling under
conditions of flow (Figure 4A). Although
there was a trend for eosinophils to firmly adhere in greater numbers
to VCAM-1 7d compared with VCAM-1 2d at shear stress of 0.7 dyn/cm2 (14.8 ± 7.1 adherent eosinophils vs
12.6 ± 9.2 adherent eosinophils) (n = 4) (P = .59) and
at shear stress of 1.4 dyn/cm2 (4.8 ± 3.3 adherent
eosinophils vs 3.8 ± 3.9 adherent eosinophils) (n = 4)
(P = .39), this was not statistically significant (Figure 4B). These studies suggest that the N-terminal 2 domains of VCAM-1 (containing the IDSPL sequence recognized by 4 integrins in domain 1 of VCAM-1)40-42 are sufficient to subserve an eosinophil
rolling function under conditions of flow. These studies do not exclude a role for VCAM-1 domain 4 (or other VCAM-1 domains 3-7) in
strengthening the adhesive interaction of eosinophils that have rolled
or adhered on the N-terminal 2 domains of VCAM-1.
View larger version (1514K):
[in this window]
[in a new window]
| Fig 4.
Eosinophil rolling and firm adhesion on VCAM-1 2d and
VCAM-1 7d.
Eosinophils were infused at a flow rate of 0.7 and 1.4 dyn/cm2 into a parallel plate flow chamber containing
VCAM-1 2d-, VCAM-1 7d-, or BSA-coated coverslip. The number of
rolling eosinophils (A) and adherent eosinophils (B) during continuous
flow periods of 2 minutes was recorded and subjected to offline
analysis. Results of experiments performed are presented as the mean ± SEM (n = 4 experiments). At flow rates of 0.7 dyn/cm2, significant numbers of eosinophils rolled on
VCAM-1 2d (P = .01 vs BSA) and VCAM-1 7d (P = .01
vs BSA) as well as adhered to VCAM-1 2d (P = .01 vs BSA) and
VCAM-7d (P = .001 vs BSA). There was no significant
difference in the number of eosinophils rolling or adhering to VCAM-1
2d vs VCAM-1 7d (P = not significant).
|
|
Effect of 1 integrin-activating mAb on eosinophil rolling
and detachment from VCAM-1 in vitro
The 1 integrin-activating mAb 8A2 decreased the number of
eosinophils rolling on VCAM-1 (Figure 5A)
and resulted in firmly adherent eosinophils that were resistant to
detachment from VCAM-1 when exposed to stepwise increases in shear
stress from 2 to 20 dyn/cm2 (Figure 5B). To investigate the
specificity of the eosinophil 1 integrin interaction with VCAM-1, we
examined the ability of the anti-1 integrin mAb P4C10 to block the
1 integrin-activating mAb 8A2-induced adhesion of eosinophils to
VCAM-1-coated coverslips. These studies demonstrated that the
anti-1 mAb P4C10 inhibited the 1-activating mAb 8A2-induced
adhesion of eosinophils to VCAM-1 by 92 ± 7%. These experiments
demonstrate the specificity of the 1 integrin/VCAM-1 interaction
studied and exclude significant integrin cross talk as being
responsible for the results observed.
View larger version (29K):
[in this window]
[in a new window]
| Fig 5.
Effect of 1 integrin activation on eosinophil rolling
and detachment from VCAM-1 in vitro.
Eosinophils (preincubated with a 1 integrin-activating Ab or
control Ab) were infused into a parallel plate flow chamber containing
VCAM-1- or BSA-coated coverslip. The number of (A) rolling eosinophils
during continuous flow periods of 2 minutes was recorded and subjected
to offline analysis. To evaluate the influence of the 1-activating
antibody on the resistance of eosinophils to detachment from VCAM-1
(B), stepwise increases in shear stress were applied for 15 seconds at
each shear force (2 to 20 dyn/cm2) to eosinophils adherent
to VCAM-1 in the flow chamber. The number of eosinophils firmly
adherent to VCAM-1 per field was recorded before and after each
stepwise increase in shear stress. The number of detached eosinophils
is expressed as a percentage of the total number of eosinophils
adherent to VCAM-1 before stepwise increases in shear stress were
applied to the coverslip in the flow chamber. Results of experiments
performed at a flow rate of 0.7 dyn/cm2 are presented as
the mean ± SEM (n = 3 experiments). Solid bar, mAb 8A2 treated;
hatched bar, control.
|
|
Demonstration of 41- and 47-dependent eosinophil rolling
in inflamed mesenteric venules in vivo
To determine the relative in vivo contribution of 41 and
47 integrins to eosinophil rolling, we visualized the interaction of CFDA-labeled eosinophils in the IL-1-stimulated mesenteric circulation by intravital microscopy. Although donor-to-donor variation
was observed, eosinophils were found to roll avidly (Rf:
42.2 ± 22.2%) in postcapillary venules (Figure
6) as previously reported.20,23
Because we previously demonstrated 4-dependent eosinophil rolling in
mesenteric venules,20 we next compared the relative ability
of (4)1 versus (4)7 integrins to mediate the 4 component
of eosinophil rolling in vivo. CFDA-labeled eosinophils were first
preincubated with 50 µg/mL of either the anti-1 mAb P4C10, the
anti-7 mAb FIB504, a combination of the anti-1 and anti-7
mAbs, or anti-2 mAb IB4 for 20 minutes prior to intravascular administration of eosinophils in vivo. Pretreatment of eosinophils with
either the anti-1 or the anti-7 mAbs resulted in a significant inhibition of eosinophil rolling (anti-1: 29.6 ± 8.5%
inhibition, P = .001 vs control; anti-7: 21.5 ± 15.9%
inhibition, P = .004 vs control) (Figure 6). Preincubation of
eosinophils with the anti-1 and anti-7 mAb in combination
resulted in a greater reduction in eosinophil rolling than that induced
by the individual mAbs alone (46.9 ± 12.5% inhibition,
P = .0001 vs control) and was comparable to the effect of the
anti-4 mAb treatment (43.2 ± 14.1% inhibition,
P = .0001 vs control) (Figure 6). These results suggest that
eosinophil rolling in cytokine-stimulated mesenteric venules is
mediated by both 41 and 47 integrins.
View larger version (30K):
[in this window]
[in a new window]
| Fig 6.
41 and 47 integrins support eosinophil
rolling in postcapillary venules in vivo.
CFDA-labeled eosinophils were preincubated with either
function-blocking anti-4, anti-1, anti-7, or anti-1 plus
anti-7 mAb in combination prior to the administration of eosinophils
into the mesenteric microcirculation. The fraction of rolling
eosinophils (Rf ) was determined in IL-1-stimulated rabbit
mesenteric venules (n = 5 to 12 rabbits). The ability of the
different mAbs to block eosinophil rolling (% inhibition) was
determined. Data represent mean ± SD. There was significant
inhibition of eosinophil rolling induced by the anti-4 mAb
(P = .0001 vs control), the anti-1 mAb (P = .001
vs control), the anti-7 mAb (P = .004 vs control), and the
combination of anti-1 and anti-7 mAbs (P = .0001 vs
control) but not by the anti-2 mAb (P = not significant vs
control).
|
|
Anti-rabbit VCAM-1 mAbs blocks eosinophil rolling in vivo
Because VCAM-1 is one of the endothelial-expressed ligands for both
41 and 47, we investigated whether inducible rolling of
human eosinophils in the rabbit mesentery was mediated by VCAM-1. Blood
vessels pretreated with an anti-VCAM-1 mAb Rb 1/9 exhibited a 27% to
53% inhibition in rolling at the different time points studied as
compared to rolling observed in the control period (ie, prior to mAb
administration) (Figure 7). Similar treatment of blood vessels with an
isotype-matched control antibody (IgG1) had minimal effect on the flux
of rolling eosinophils (Figure 7). As
previously reported,23,38 the local intravascular
administration of anti-adhesion molecule mAbs in the rabbit mesentery
allows for the analysis of the function of the mAb for a brief time
until the mAb is presumably removed from the endothelial cell surface by blood flow or other mechanisms. The effect of the anti-VCAM-1 mAb
blockade induced by local instillation of the mAb was observed to last
up to about 30 minutes, after which time eosinophils rolled at the same
flux as observed during the control period (Figure 7). As described
previously,23 temporary occlusion of the mesenteric artery
neither altered the rolling flux of injected eosinophils nor induced
any spontaneous adhesion of injected eosinophils.
View larger version (22K):
[in this window]
[in a new window]
| Fig 7.
Anti-VCAM-1 mAb Rb 1/9 blocks eosinophil rolling in
venular endothelium in vivo.
Fluorescently labeled eosinophils were injected into the superior
mesenteric artery and their baseline rolling on IL-1-stimulated
venular endothelium determined. The flow of the mesenteric circulation
was temporarily occluded and an anti-VCAM-1 (Rb 1/9) or control (mouse
IgG1) mAb infused. After a 10-minute incubation of the mAb with the
endothelial surface, the blood flow was restored and the CFDA-labeled
eosinophils injected. The flux of rolling eosinophils was determined at
different time points (up to 1 hour postinfusion of eosinophils into
the antibody treated mesenteric venules) by frame-by-frame analysis of
recorded video images. The effect of mAb blockade lasted for up to
about 30 minutes after resumption of flow. The results are expressed as
percent rolling of eosinophils compared with rolling observed before
mAb treatment (% of average control). The values represent mean ± SD. There was significant inhibition of eosinophil rolling 10 to 15 minutes posttreatment with mAb Rb 1/9 (P = .05 vs
control) but not with mouse IgG (P = not significant vs
control).  , mAb Rb 1/9;  , mouse IgG1.
|
|
Effect of 41 integrin activation on eosinophil firm
adhesion in vivo
Because we25 and others26 have demonstrated
that 41 integrins on eosinophils can exist in different
functional states in vitro, we examined whether altering the functional
state of 41 in vivo would convert it from an eosinophil rolling
receptor to a firm adhesion receptor. Spontaneous adhesion of
eosinophils infused into mesenteric venules is not frequently
encountered and has been observed in less than 10% of the donors we
have tested thus far. CFDA-labeled eosinophils were therefore activated
with mAb 8A2 (1 integrin-activating mAb) or a control
function-blocking mAb P4C10 for 3 to 5 minutes prior to administration
of eosinophils into the IL-1-stimulated mesenteric circulation.
Similar to our in vitro observations demonstrating a resistance to
detachment from VCAM-1 of 1 integrin activated eosinophils (Figure
5B), activation of the eosinophil 1 integrin resulted in an increase in the number of firmly adherent eosinophils (2 to 6 adherent eosinophils/250 µm venular length) in the mesenteric venules in vivo
(P = .02 vs control) (Figure 8).
In contrast to the 1-activating mAb 8A2, treatment of eosinophils
with a control mAb P4C10 did not result in the increased adhesion of
rolling eosinophils. The 1-activated adherent eosinophils were
observed to be stationary for up to 15 to 30 minutes, after which time
some of the adherent eosinophils (about 25%) were observed to detach
from the endothelial surface. No firm adhesion of eosinophils was
observed in the arterioles.
View larger version (40K):
[in this window]
[in a new window]
| Fig 8.
Stimulation with 1-activating mAb 8A2 results in
stable arrest of rolling eosinophils in IL-1-stimulated mesenteric
venules.
Eosinophils were incubated ex vivo with anti-1 integrin-activating
mAb 8A2 (20 µg/mL) for 3 to 5 minutes prior to administration of
eosinophils into the rabbit mesentery. The ability of the rolling
eosinophils to adhere firmly in postcapillary venules (treated with
anti-VCAM-1 mAb Rb 1/9 or control antibody (IgG1) (as described in
Figure 7) was determined. The results represent the number of adherent
eosinophils per 250 µm length of venule (mean ± SD) during the 5 minutes of eosinophil infusion after resumption of blood flow.
Eosinophil control vs eosinophil and 1-activating mAb 8A2
(P = .02); eosinophil and 1-activating mAb 8A2 vs
anti-VCAM-1 mAb Rb 1/9 (P = .04): anti-VCAM-1 vs mouse IgG1
control (P = .05).
|
|
The activation-dependent stable adhesion of eosinophils induced by the
1 integrin-activating mAb 8A2 was significantly inhibited (58.8 ± 8.9% inhibition) (P = .04) when the mesenteric
venules were pretreated with an anti-VCAM-1 mAb Rb 1/9 (Figure 8). In contrast, mouse IgG1 (Figure 8) or adhesion blocking anti-1, anti-7, or anti-4 mAbs (data not shown) failed to stimulate the
adhesion of rolling eosinophils. These studies suggest that the
41 integrin can subserve multiple functions of
eosinophil-endothelial cell interactions in vivo, ie, rolling as well
as activation-dependent firm adhesion in vivo.
Effect of antibody blockade on the velocity distribution profiles of
rolling eosinophils
In addition to evaluating the influence of the anti-4, anti-1,
and anti-7 mAbs on eosinophil rolling in vivo, we also examined the
effect of these mAbs on the velocity distribution profiles of rolling
eosinophils in venular segments in vivo. The mean velocities (mm/sec)
of rolling eosinophils prior to and after ex vivo mAb treatment were
determined in each of the representative venules (n = 7 rabbits, 2 to
3 venules/rabbit) (Figure 9). Fluorescently labeled eosinophils were pretreated with 50 µg/mL of either
anti-1, anti-7, anti-1 and anti-7, or anti-2 integrin
mAbs prior to their injection into the cannulated mesentery artery. The
mean velocity of each eosinophil in a representative venule was
determined by frame-by-frame analysis. The mean rolling velocity of
control eosinophils was observed to be 0.26 ± 0.07 mm/sec. In
contrast, eosinophils rolled at significantly higher velocities when
pretreated with either the anti-4 mAb P4G9 (0.44 ± 0.09 mm/sec;
P = .003 vs control), the anti-1 mAb P4C10
(0.43 ± 0.11 mm/sec; P = .004 vs control), or the
anti-7 mAb FIB504 (0.42 ± 0.09 mm/sec; P = .005 vs
control). In contrast, treatment of eosinophils with a control
anti-2 integrin mAb IB4 (which does not block eosinophil rolling)20 had no significant effect on mean rolling
velocity (0.28 ± 0.06 mm/sec).
View larger version (51K):
[in this window]
[in a new window]
| Fig 9.
Effect of anti-integrin mAb treatment on the velocity
distribution profiles of rolling eosinophil in mesenteric venules.
The passage of rolling eosinophils in IL-1-stimulated mesenteric
venules was recorded. The velocity of consecutive rolling eosinophils
was determined before and after eosinophil treatment with either
anti-1, anti-7, anti-1 plus anti-7, anti-4, or anti-2
mAbs (n = 7 rabbits; 2 to 3 representative venules per rabbit). The
velocity of rolling eosinophils (mm/sec) was manually determined by
frame-by-frame analysis of recorded video images and represented as
mean ± SD. The rolling velocity of eosinophils was increased by
pretreatment of eosinophils with either anti-1 (P = .004
vs control), anti-7 (P = .005 vs control), anti-1 plus
anti-7 (P = .003 vs control), and anti-4 mAbs
(P = .003 vs control) but not by pretreatment with anti-2
mAbs.
|
|
| |
Discussion |
In contrast to the well-established role of
selectins43,44 in mediating the initial rolling of
leukocytes on vascular endothelium, the potential importance of 4
integrins to this rolling interaction has only recently been
appreciated.20,28,45 Our initial identification of
4-integrin dependent rolling of eosinophils on endothelium in
vivo20 did not explore the relative contribution of
41 compared with 47 integrins to eosinophil rolling; nor
did it characterize whether 4 integrin counterligands VCAM-1 and
MAdCAM-1 could both support eosinophil rolling and firm adhesion under conditions of flow. The novel observations in this study include the
observations that (a) eosinophils roll more efficiently on VCAM-1 than
on MAdCAM-1, (b) both VCAM-1 and MAdCAM-1 contribute to eosinophil firm
adhesion under conditions of flow, (c) eosinophils roll equally
efficiently on both VCAM-1 2d and VCAM-1 7d, suggesting that the
N-terminal 2 domains of VCAM-1 are sufficient to support eosinophil
rolling under conditions of flow, and (d) the functional status of the
eosinophil 1 integrin determines the number of eosinophils firmly
adherent to endothelium in vivo.
Prior studies have demonstrated by fluorescence-activated cell sorting,
immunostaining, and immunoprecipitation that eosinophils from mild
atopics, a similar study population to that used in our study, express
both 41 and 47.13,14 Under static conditions, eosinophils are able to use both 41 or 47 integrins to bind to VCAM-1,14 while only 47 mediates eosinophil
binding MAdCAM-1.46 The reduced rolling of eosinophils on
MAdCAM-1 as compared to VCAM-1 noted in our flow chamber studies may be
due to the weaker single receptor interaction of eosinophils with
MAdCAM-1 (ie, only 47 interacts with MAdCAM-1) compared with the
stronger dual eosinophil receptor interaction with VCAM-1 (ie, 47
and 41 both interact with VCAM-1). The reduced ability of
eosinophils to roll on MAdCAM-1 was not due to technical factors
related to eosinophil adhesion to MAdCAM-1 because eosinophils bound
firmly to MAdCAM-1 and VCAM-1 in static adhesion assays and resisted detachment from both of these ligands. In addition, stripping of
MAdCAM-1 from the coverslip during the flow chamber experiment is
unlikely because eosinophils adhered in static adhesion assays to
MAdCAM-1-coated coverslips that had previously been subjected to flow.
Based on these observations, MAdCAM-1 expression by endothelial cells
is more likely to contribute to eosinophil firm adhesion as opposed to
eosinophil rolling on endothelial cells. In the context of eosinophils
and asthma, VCAM-1 is more likely than MAdCAM-1 to play a significant
role in eosinophil recruitment to the lung because VCAM-1 is expressed
in the lung47 but MAdCAM-1 is predominantly expressed in
the gastrointestinal tract and is absent or expressed at very low
levels in nongastrointestinal sites including the lung.48
Nevertheless, MAdCAM-1 may play a more significant role in eosinophil
recruitment to the gastrointestinal tract.
Because MAdCAM-1 binds only to 47 but VCAM-1 binds to both
47 and 41, research has focused on understanding the
different integrin binding sites in VCAM-1 and MAdCAM-1. Domain
swapping and construction of chimeric soluble forms of MAdCAM-1 have
shown that the N-terminal 2 domains of MAdCAM-1 are both required and sufficient for efficient 47 binding.49,50 A GLTDSL
amino acid sequence in domain 1 of MAdCAM-1 is essential for binding of
MAdCAM-1 to 47.49,50 Another unique feature of
MAdCAM-1 is present in domain 2, which contains a negatively charged
ribbon loop of 11 amino acids.49,50 This negatively
charged "antenna," extending outward from domain 2 and reaching
close to the GLTDSL motif in domain 1, may contribute to integrin
binding. The interaction of 47 with MAdCAM-1 or VCAM-1 is likely
to involve both unique as well as overlapping adhesion contact sites
based on studies demonstrating differential inhibition of adhesion
induced by mAb directed against different epitopes of
47.51
Our intravital microscopy studies extend our understanding of the
adhesion receptors that subserve a rolling function on the eosinophil
cell surface in vivo to include 41 and 47 as well as
previously described L-selectin.20 In addition to 41,
47, and L-selectin, flow chamber studies in vitro have
demonstrated that eosinophils also use PSGL-1 to roll on the
endothelial ligand P-selectin.10 Thus, eosinophils express
at least four receptors (41, 47, L-selectin, and PSGL-1)
capable of mediating eosinophil rolling on endothelial cells expressing
the appropriate counterligand at sites of allergic inflammation.
This study also extends our knowledge of the endothelial cell surface
adhesion counterreceptors that subserve a rolling function for
eosinophils in vivo to include VCAM-1 as well as previously reported
P-selectin.21,22 The importance of extending results obtained with eosinophils in static adhesion assays to in vivo physiologic evaluation is underscored by studies of eosinophil adhesion
to E-selectin. While E-selectin is readily able to support eosinophil
adhesion under static conditions in vitro,5 studies performed with eosinophils in flow chambers and eosinophils in vivo23,24 demonstrate that E-selectin preferentially
supports neutrophil but not eosinophil rolling. As tissue infiltration by eosinophils but not neutrophils is a hallmark of allergic
inflammation, whereas tissue infiltration with neutrophils but not
eosinophils is a hallmark of bacterial infection, the eosinophil
41/47 rolling interaction with endothelial-expressed
VCAM-1, and the neutrophil rolling interaction with endothelial
expressed E-selectin, may allow for preferential eosinophil or
neutrophil recruitment pathways. In contrast, both eosinophils and
neutrophils use L-selectin and P-selectin as rolling receptors in vivo,
suggesting that neither of these pathways would account for selective
eosinophil or neutrophil tissue recruitment. The selective recruitment
of eosinophils as opposed to neutrophils to sites of allergic
inflammation will not only be influenced by the above-mentioned
adhesion receptors but also by the tissue expression of chemokines that
preferentially attract eosinophils (ie, eotaxin, RANTES, MIP-1,
MCP-4)52,53 as opposed to neutrophils.
Recent studies have assisted in defining the ligand binding sites on
4 integrins and its counterreceptor VCAM-1.40-42,54 The
N-terminal portion of integrin subunits (about 440 amino acids)
contains 7 sequence repeats.54 The regions of 4 integrin critical for ligand binding contained in this N-terminal portion are
not adjacent in the primary 4 integrin structure.54
Rather, the 7 N-terminal sequence repeats of 4 integrins are
proposed to fold into a -propeller domain with the integrin binding
site for VCAM-1 on the upper face of the propeller.55
The predominant form of VCAM-1 in vivo has an amino-terminal
extracellular region comprising 7 immunoglobulin-like
domains.54 Functional studies of VCAM-1 have identified a
conserved integrin-binding motif in VCAM-1 domains 1 and
4.40-42 The crystal structure of the first 2 domains of
VCAM-1 has been elucidated56 and demonstrates that the
integrin binding motif (IDSPL) of VCAM-1 domain 1 is highly exposed and
available for integrin binding.56 At present, the crystal
structure of VCAM-1 domains 3 through 7 has not been reported. Our
studies demonstrate that eosinophils roll equally efficiently on both
VCAM-1 2d as well as on VCAM-1 7d, suggesting that the N-terminal 2 domains of VCAM-1 are sufficient to support eosinophil rolling under
conditions of flow. These studies do not exclude a role for VCAM-1
domain 4 (containing a known integrin binding site) or other sequences
in VCAM-1 domains 3-7 in strengthening the adhesive interaction of
eosinophils that have rolled or adhered on the N-terminal 2 domains of
VCAM-1. Interestingly, VCAM-1 domain 1 would project furthest from the
endothelial cell surface into the lumen of the blood vessel, allowing
for ease of initial eosinophil 4-integrin rolling interaction with
this VCAM-1 domain as opposed to other VCAM-1 domains not projecting as
far into the blood vessel lumen. Studies using Ramos cells (a
Burkitt's lymphoma cell line), cytokine-stimulated human umbilical
vein endothelial cells, and Abs against domain 1 and domain 4 of VCAM-1
also demonstrate that VCAM-1 domain 1 is solely responsible for
4-integrin-dependent primary capture of Ramos cells under
conditions of flow, whereas both VCAM-1 domains 1 and 4 are used in
4-integrin-dependent Ramos cell adhesion under static
conditions.57
Several laboratories,27-29 including ours,25
have demonstrated that integrins such as 41 (VLA-4) expressed by
eosinophils can change their affinity for counterligands such as VCAM-1
without changing their level of integrin expression, presumably by
changing the conformation of the integrin from a low- to a
high-affinity state. This change in integrin affinity can be induced by
several stimuli including cytokines,25 extracellular
divalent cations such as manganese,26 and a 1
integrin-activating Ab.26 We were interested to determine
how upregulating the functional state of 41 on eosinophils with a
1 integrin-activating Ab might influence the ability of 41 to
function either as a rolling or firm adhesion receptor under conditions
of blood flow in vivo. Our studies of eosinophils activated with the
1-activating Ab suggest that upregulating 1 integrin function
increases the number of eosinophils adherent to VCAM-1 in vivo. This
1 integrin effect on eosinophil accumulation on VCAM-1 in vivo is
probably mediated by increasing the strength of eosinophil adhesion to
VCAM-1 in vivo with resultant fewer eosinophils detaching from VCAM-1
once adherent. This conclusion is also supported by our in vitro
studies in which 1 integrin-activated eosinophils were resistant to
detachment from VCAM-1 in a flow chamber at levels of shear stress
considerably higher than those observed in the microcirculation in
vivo. Thus, locally released cytokines such as GM-CSF,25
chemokines such as eotaxin,58 or other integrin-activating
mediators, released at sites of allergic inflammation in vivo, have the
potential to upregulate 41 function as well as the number of
endothelial adherent eosinophils by promoting their resistance to detachment.
The importance of 4 integrins to eosinophil recruitment has been
suggested from several studies of animal models of allergic inflammation, in which pretreatment with anti-4 mAbs resulted in the
reduction in the accumulation of eosinophils in the airways as well as
reduced bronchial hyperreactivity.17-19 However, not all
studies have demonstrated a correlation between the
anti-4-Ab-mediated inhibition of eosinophil recruitment, and the
anti-4-mediated inhibition of bronchial
hyperreactivity,59 suggesting that the anti-4 Ab may not
only be influencing eosinophil recruitment but also eosinophil
activation in tissue sites. In this regard, it is of interest that
eosinophil 41 receptors can be activated with either the CS-1
region of fibronectin (induces eosinophils to release
GM-CSF),60 or with VCAM-1 (induces eosinophils to release
superoxide).61 While targeting the 4/VCAM-1 eosinophil endothelial cell adhesion pathway in allergic inflammation is attractive in terms of its selective effect on eosinophil but not
neutrophil rolling and adhesion, eosinophils can bypass this pathway
using alternate endothelial-expressed rolling receptors (P-selectin)
and firm adhesion receptors (ICAM-1). Indeed, our studies of
ragweed-induced peritoneal eosinophil recruitment in P-selectin/ICAM-1-deficient mice treated with an anti-VCAM-1 mAb (near
complete inhibition of eosinophil recruitment) suggest that the VCAM-1
pathway contributes approximately 25% to 38% to eosinophil peritoneal
recruitment.21 However, the degree of inhibition of
eosinophil recruitment may vary with the vascular bed studied, the
animal species studied, or the route of administration of the anti-4
Ab.59
Overall, this study demonstrates that eosinophils roll more efficiently
on VCAM-1 versus MAdCAM-1, whereas eosinophils firmly adhere as
efficiently to MAdCAM-1 as to VCAM-1. The 4 integrin VCAM-1 pathway
may therefore be able to support both of the first 2 sequential steps
of eosinophil adhesion to endothelium (ie, rolling and firm adhesion)
independent of the requirement for alternate adhesion receptors (ie,
selectins, 2 integrin/ICAM-1). In contrast, the 4 integrin
MAdCAM-1 pathway would be more efficient in subserving eosinophil firm
adhesion to endothelium as opposed to eosinophil rolling on
endothelium, especially in the gastrointestinal tract where MAdCAM-1 is
expressed. In addition, because 41 can rapidly modulate its
function in vivo to resist eosinophil detachment from endothelium under
conditions of flow in vivo, this modulation of receptor function may
promote eosinophil accumulation at sites of inflammation.
| |
Acknowledgments |
We thank Greg Hughes and Tim Gifford for technical assistance and
Lanesha Hill for assistance in the preparation of the manuscript. This
study complies with National Institutes of Health guidelines for the
care and use of laboratory animals.
| |
Footnotes |
Submitted September 9, 1998; accepted August 25, 1999.
Supported by the UCSD General Clinical Research Center grant
M01 RR0827 from the National Institutes of Health and by National Institutes of Health grants AI 35796, AI 29974, AI 33977, AI 38425, and
AI 22415.
Reprints: Pragada Sriramarao, Laboratory of Immunology and
Vascular Biology, La Jolla Institute of Experimental Medicine, 505 Coast Blvd South, La Jolla, CA 92037; email: rao{at}ljiem.org.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Weller PF, Lim K, Wan HC, et al.
Role of the eosinophil in allergic reactions.
Eur Respir J Suppl.
1996;22:109s-115s[Medline]
[Order article via Infotrieve].
2.
Wardlaw AJ, Moqbel R, Kay AB.
Eosinophils: biology and role in disease.
Adv Immunol.
1995;60:151-266[Medline]
[Order article via Infotrieve].
3.
Sriramarao P, Broide DH.
Differential regulation of eosinophil adhesion under conditions of flow in vivo.
Ann N Y Acad Sci.
1996;796:218-225[Medline]
[Order article via Infotrieve].
4.
Bochner BS.
Cellular adhesion and its antagonism.
J Allergy Clin Immunol.
1997;100:581-585[Medline]
[Order article via Infotrieve].
5.
Kyan-Aung U, Haskard DO, Poston RN, Thornhill MH, Lee TH.
Endothelial leukocyte adhesion molecule-1 and intercellular adhesion molecule-1 mediate the adhesion of eosinophils to endothelial cells in vitro and are expressed by endothelium in allergic cutaneous inflammation in vivo.
J Immunol.
1991;146:521-528[Abstract].
6.
Wein M, Sterbinsky SA, Bickel CA, Schleimer RP, Bochner BS.
Comparison of human eosinophil and neutrophil ligands for P-selectin: ligands for P-selectin differ from those from E-selectin.
Am J Respir Cell Mol Biol.
1995;12:315-319[Abstract].
7.
Bochner BS, Lucinskas FW, Gimbrone MA Jr, et al.
Adhesion of human basophils, eosinophils, and neutrophils to IL-1 activated human vascular endothelial cells: contributions of endothelial cell adhesion molecules.
J Exp Med.
1991;173:1553-1557[Abstract/Free Full Text].
8.
Dobrina A, Menegazzi R, Carlos TM, et al.
Mechanisms of eosinophil adherence to cultured vascular endothelial cells: eosinophils bind to cytokine-induced endothelial ligand vascular cell adhesion molecule-1 via the very late activation antigen-4 integrin receptor.
J Clin Invest.
1991;88:20-26.
9.
Knol EF, Tackey F, Tedder TF, et al.
Comparison of human eosinophil and neutrophil adhesion to endothelial cells under nonstatic conditions. Role of L-selectin.
J Immunol.
1994;153:2161-2167[Abstract].
10.
Symon FA, Walsh GM, Watson SR, Wardlaw AJ.
Eosinophil adhesion to nasal polyp endothelium is P-selectin-dependent.
J Exp Med.
1994;180:371-376[Abstract/Free Full Text].
11.
Nakajima H, Sano H, Nishimura T, Yoshida S, Iwamoto I.
Role of vascular cell adhesion molecule 1/very late activation antigen 4 and intercellular adhesion molecule 1/lymphocyte function-associated antigen 1 interactions in antigen-induced eosinophil and T cell recruitment into the tissue.
J Exp Med.
1994;179:1145-1154[Abstract/Free Full Text].
12.
Walsh GM, Mermod JJ, Hartnell A, Kay AB, Wardlaw AJ.
Human eosinophil, but not neutrophil, adherence to IL-1 stimulated human umbilical vascular endothelial cells is 41 (very late antigen-4) dependent.
J Immunol.
1991;146:3419-3423[Abstract].
13.
Erle DJ, Briskin MJ, Butcher EC, Garcia-Pardo A, Lazarovits AI, Tidswell M.
Expression and function of the MAdCAM-1 receptor, integrin alpha 4 beta 7 on human leukocytes.
J Immunol.
1994;153:517-528[Abstract].
14.
Walsh GM, Symon FA, Lazarovils AL, Wardlaw AJ.
Integrin alpha 4 beta 7 mediates human eosinophil interaction with MAdCAM-1, VCAM-1 and fibronectin.
Immunology.
1996;89:112-119[Medline]
[Order article via Infotrieve].
15.
Elices MJ, Osborn L, Takada Y, et al.
VCAM-1 on activated endothelial cells interacts with leukocyte integrin VLA-4 at a site distinct from VLA-4/fibronectin binding site.
Cell.
1990;60:577-584[Medline]
[Order article via Infotrieve].
16.
Briskin MJ, McEvoy LM, Butcher EC.
MAdCAM-1 has homology to immunoglobulin and mucin like adhesion receptors and to IgA1.
Nature.
1993;363:461-464[Medline]
[Order article via Infotrieve].
17.
Abraham WM, Sielczak MW, Ahmed A, et al.
4-integrins mediate antigen-induced late bronchial responses and prolonged airway hyperresponsiveness in sheep.
J Clin Invest.
1994;93:776-787.
18.
Weg VB, Williams TJ, Lobb RR, Noushargh S.
A monoclonal antibody recognizing very late activation antigen-4 inhibits eosinophil accumulation in vivo.
J Exp Med.
1993;177:561-566[Abstract/Free Full Text].
19.
Pretolani M, Ruffie C, Lapa e Silva JR, Joseph D, Lobb RR, Vargaftig BB.
Antibody to very late activation antigen 4 prevents antigen-induced bronchial hyperreactivity and cellular infiltration in the guinea pig airways.
J Exp Med.
1994;180:795-805[Abstract/Free Full Text].
20.
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.
1994;153:4238-4246[Abstract].
21.
Broide DH, Humber D, Sriramarao P.
Inhibition of eosinophil rolling and recruitment in P-selectin and ICAM-1 deficient mice. [authorship erratum appears in Blood 1998;92:343]
Blood.
1998;91:2847-2856[Abstract/Free Full Text].
22.
Symon FA, Lawrence MB, Williamson ML, Walsh GM, Watson SR, Wardlaw AJ.
Functional and structural characterization of the eosinophil P-selectin ligand.
J Immunol.
1996;157:1711-1719[Abstract].
23.
Sriramarao P, Norton CR, Borgström P, DiScipio RG, Wolitzky BA, Broide DH.
E-selectin preferentially supports neutrophil but not eosinophil rolling under conditions of flow in vitro and in vivo.
J Immunol.
1996;157:4672-4680[Abstract].
24.
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.
1997;159:3929-3939[Abstract].
25.
Sung L-L, Yang PL, Elices M, Jin G, Sriramarao P, Broide DH.
Granulocyte-macrophage colony-stimulating factor regulates the functional adhesive state of very late antigen-4 expressed by eosinophils.
J Immunol.
1997;158:919-927[Abstract].
26.
Matsumoto K, Sterbinsky SA, Bickel CA, Zhou DF, Kovach NL, Bochner BS.
Regulation of alpha 4 integrin-mediated adhesion of human eosinophils to fibronectin and vascular cell adhesion molecule-1.
J Allergy Clin Immunol.
1997;99:648-656[Medline]
[Order article via Infotrieve].
27.
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.
1995;128:1243-1253[Abstract/Free Full Text].
28.
Berlin C, Bargatze RF, Campbell JJ, et al.
4 integrins mediate lymphocyte attachment and rolling under physiologic flow.
Cell.
1995;80:413-422[Medline]
[Order article via Infotrieve].
29.
Yednock T, Cannon C, Vandevert C, et al.
Alpha 4 beta 1 integrin-dependent cell adhesion is regulated by a low affinity receptor pool that is conformationally responsive to ligand.
J Biol Chem.
1995;270:28,740-28,750[Abstract/Free Full Text].
30.
Hansel TT, Pound PD, Thompson RA.
Isolation of eosinophils from human blood.
J Immunol Methods.
1990;127:153-164[Medline]
[Order article via Infotrieve].
31.
Wayner EA, Garcia-Pardo A, Humphries MJ, McDonald JA, Carter WG.
Identification of characterization of the T lymphocyte adhesion receptor for an alternative cell attachment domain (CS-1) in plasma fibronectin.
J Cell Biol.
1989;109:1321-1330[Abstract/Free Full Text].
32.
Carter WG, Wayner EA, Bouchard TS, Kaur P.
The role of integrins alpha 2 beta 1 and alpha 3 beta 1 in cell-cell and cell-substrate adhesion of human epidermal cells.
J Cell Biol.
1990;110:1387-1404[Abstract/Free Full Text].
33.
Wright SD, Rao PE, van Voorhis WC, et al.
Identification of the C3bi receptor of human monocytes and macrophages by using monoclonal antibodies.
Proc Natl Acad Sci U S A.
1983;80:5699-5703[Abstract/Free Full Text].
34.
Cybulsky M, Gimbrone MA.
Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis.
Science.
1991;251:788-791[Abstract/Free Full Text].
35.
Kovach NL, Carlos TM, Yee E, Harlan JM.
A monoclonal antibody to beta 1 integrin (CD29) stimulates VLA-dependent adherence of leukocytes to human umbilical vein endothelial cells and matrix components.
J Cell Biol.
1992;116:499-509[Abstract/Free Full Text].
36.
Andrew DP, Berlin C, Honda S, Yoshimo T, Butcher EC.
Distinct but overlapping epitopes are involved in 47 mediated adhesion to vascular cell adhesion molecule-1, mucosal addressin-1, fibronectin, and lymphocyte aggregation.
J Immunol.
1994;153:3847-3861[Abstract].
37.
Tsuruta R, Cobb RR, Mastrangelo M, Lazarides E, Cardarelli PM.
Soluble vascular cell adhesion molecule (VCAM)-Fc fusion protein induces leukotriene C4 secretion in platelet-activating factor-stimulated eosinophils.
J Leuk Biol.
1999;65:71-79[Abstract].
38.
Olofsson A, Arfors K, Ramezani L, Wolitzky B, Butcher E, von Andrian U.
E-selectin mediates leukocyte rolling in interleukin-1-treated rabbit mesentery venules.
Blood.
1994;84:2749[Abstract/Free Full Text].
39.
von Andrian UH, Hansell P, Chambers JD, et al.
L-selectin function is required for 2 integrin-mediated neutrophil adhesion at physiological shear rates in vivo.
Am J Physiol.
1992;263:H1034-H1044[Abstract/Free Full Text].
40.
Osborn L, Vassallo C, Browning BG, et al.
Arrangement of domains, and amino acid residues required for binding of vascular cell adhesion molecule-1 to its counter-receptor VLA-4 (41).
J Cell Biol.
1994;124:601-608[Abstract/Free Full Text].
41.
Vonderheide RH, Tedder TF, Springer TA, Staunton DE.
Residues within a conserved amino acid motif of domains 1 and 4 of VCAM-1 are required for binding to VLA-4.
J Cell Biol.
1994;125:215-222[Abstract/Free Full Text].
42.
Renz ME, Chiu HH, Jones S, et al.
Structural requirements for adhesion of soluble recombinant murine vascular cell adhesion molecule-1 to 41.
J Cell Biol.
1994;125:1395-1406[Abstract/Free Full Text].
43.
Butcher EC.
Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity.
Cell.
1991;67:1033-1036[Medline]
[Order article via Infotrieve].
44.
Springer TA.
Traffic signals for lymphocyte recirculation and leukocyte emigration: the multi-step paradigm.
Cell.
1994;76:301-314[Medline]
[Order article via Infotrieve].
45.
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.
1996;183:1995-2006[Abstract/Free Full Text].
46.
Tidswell M, Pachynski R, Wu SW, et al.
Structure-function analysis of the integrin 7 subunit: identification of domains involved in adhesion to MAdCAM-1.
J Immunol.
1997;159:1497-1505[Abstract].
47.
Fukuda T, Fukushima Y, Numao T, et al.
Role of interleukin-4 and vascular cell adhesion molecule-1 in selective eosinophil migration into the airways in allergic asthma.
Am J Respir Cell Mol Biol.
1996;14:84-94[Abstract].
48.
Briskin M, Winsor-Hines D, Shyjan A, et al.
Human mucosal addressin cell adhesion molecule-1 is preferentially expressed in intestinal tract and associated lymphoid tissue.
Am J Pathol.
1997;51:97-110.
49.
Briskin MJ, Rott L, Butcher EC.
Structural requirements for mucosal vascular addressin binding to its lymphocyte receptor 47: Common themes among integrin-Ig family interactions.
J Immunol.
1996;156:719-726[Abstract].
50.
Tan K, Casasnovas J, Liu J-H, Briskin M, Springer T, Wang J.
The structure of immunoglobulin domains 1 and 2 of MAdCAM-1 reveals novel features important for integrin recognition.
Structure.
1998;6:793-801[Medline]
[Order article via Infotrieve].
51.
Andrew DP, Berlin C, Honda S, et al.
Distinct but overlapping epitopes are involved in 47-mediated adhesion to vascular cell adhesion molecule-1, mucosal addressin-1, fibronectin, and lymphocyte aggregation.
J Immunol.
1994;153:3847-3861.
52.
Lukacs NW, Strieter RM, Chensue SW, Kunkel SL.
Activation and regulation of chemokines in allergic airway inflammation.
J Leukoc Biol.
1996;59:13-17[Abstract].
53.
Luster AD, Rothenberg ME.
Role of the monocyte chemoattractant protein and eotaxin subfamily of chemokines in allergic inflammation.
J Leukoc Biol.
1997;62:620-633[Abstract].
54.
Irie A, Kamata T, Takada Y.
Multiple loop structures critical for ligand binding of the integrin 4 subunit in the upper face of the -propeller model.
Proc Natl Acad Sci USA.
1997;94:7198-7203[Abstract/Free Full Text].
55.
Springer TA.
Folding of the N-terminal ligand binding region of integrin alpha subunits into a beta-propeller domain.
Proc Natl Acad Sci U S A.
1997;94:65-72[Abstract/Free Full Text].
56.
Jones EY, Harlos K, Bottomley MJ, et al.
Crystal structure of an integrin-binding fragment of vascular cell adhesion molecule-1 at 1.8A resolution.
Nature.
1995;373:539-544[Medline]
[Order article via Infotrieve].
57.
Abe Y, Ballantyne CM, Smith CW.
Functions of domain 1 and domain 4 of vascular cell adhesion molecule 1 in 4 integrin dependent adhesion under static and flow conditions are differentially regulated.
J Immunol.
1996;157:5061-5069[Abstract].
58.
Weber C, Kitayama J, Springer TA.
Differential regulation of 1-2-integrin avidity by chemoattractants in eosinophils.
Proc Natl Acad Sci U S A.
1996;93:10,939-10,944[Abstract/Free Full Text].
59.
Henderson WR Jr, Chi EY, Albert RK, et al.
Blockade of CD49d (4 integrin) on intrapulmonary but not circulating leukocytes inhibits airway inflammation and hyperresponsiveness in a mouse model of asthma.
J Clin Invest.
1997;100:3083-3092[Medline]
[Order article via Infotrieve].
60.
Anwar AR, Moqbel R, Walsh GM, Kay AB, Wardlaw AJ.
Adhesion to fibronectin prolongs eosinophil survival.
J Exp Med.
1993;177:839-843[Abstract/Free Full Text].
61.
Nagata M, Sedgwick JB, Bates ME, Kita H, Busse WW.
Eosinophil adhesion to vascular cell adhesion molecule-1 activates superoxide anion generation.
J Immunol.
1995;155:2194-2202[Abstract].
62.
DiScipio RG, Daffern PJ, Jagels MA, Broide DH, Sriramarao P.
A comparison of C3a and C5a-mediated stable adhesion of rolling eosinophils in postcapillary venules and transendothelial migration in vitro and in vivo.
J Immunol.
1999;162:1127-1136[Abstract/Free Full Text].
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
D. J. Song, J. Y. Cho, S. Y. Lee, M. Miller, P. Rosenthal, P. Soroosh, M. Croft, M. Zhang, A. Varki, and D. H. Broide
Anti-Siglec-F Antibody Reduces Allergen-Induced Eosinophilic Inflammation and Airway Remodeling
J. Immunol.,
October 15, 2009;
183(8):
5333 - 5341.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. I. Zuberi, X. N. Ge, S. Jiang, N. S. Bahaie, B. N. Kang, R. M. Hosseinkhani, E. M. Frenzel, M. M. Fuster, J. D. Esko, S. P. Rao, et al.
Deficiency of Endothelial Heparan Sulfates Attenuates Allergic Airway Inflammation
J. Immunol.,
September 15, 2009;
183(6):
3971 - 3979.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. J. Tumes, A. Connolly, and L. A. Dent
Expression of survivin in lung eosinophils is associated with pathology in a mouse model of allergic asthma
Int. Immunol.,
June 1, 2009;
21(6):
633 - 644.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. R. Barthel, M. W. Johansson, D. M. McNamee, and D. F. Mosher
Roles of integrin activation in eosinophil function and the eosinophilic inflammation of asthma
J. Leukoc. Biol.,
January 1, 2008;
83(1):
1 - 12.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. K. Khaldoyanidi, V. V. Glinsky, L. Sikora, A. B. Glinskii, V. V. Mossine, T. P. Quinn, G. V. Glinsky, and P. Sriramarao
MDA-MB-435 Human Breast Carcinoma Cell Homo- and Heterotypic Adhesion under Flow Conditions Is Mediated in Part by Thomsen-Friedenreich Antigen-Galectin-3 Interactions
J. Biol. Chem.,
January 31, 2003;
278(6):
4127 - 4134.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Y. Larbi, J. P. Dangerfield, F. J. Culley, D. Marshall, D. O. Haskard, P. J. Jose, T. J. Williams, and S. Nourshargh
P-selectin mediates IL-13-induced eosinophil transmigration but not eotaxin generation in vivo: a comparative study with IL-4-elicited responses
J. Leukoc. Biol.,
January 1, 2003;
73(1):
65 - 73.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Broide
Fast Flowing Eosinophils . Signals for Stopping and Stepping Out of Blood Vessels
Am. J. Respir. Cell Mol. Biol.,
June 1, 2002;
26(6):
637 - 640.
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Miller, K.-L. P. Sung, W. A. Muller, J. Y. Cho, M. Roman, D. Castaneda, J. Nayar, T. Condon, J. Kim, P. Sriramarao, et al.
Eosinophil Tissue Recruitment to Sites of Allergic Inflammation in the Lung Is Platelet Endothelial Cell Adhesion Molecule Independent
J. Immunol.,
August 15, 2001;
167(4):
2292 - 2297.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Y. Larbi, A. R. Allen, F. W. K. Tam, D. O. Haskard, R. R. Lobb, P. M. R. Silva, and S. Nourshargh
VCAM-1 has a tissue-specific role in mediating interleukin-4-induced eosinophil accumulation in rat models: evidence for a dissociation between endothelial-cell VCAM-1 expression and a functional role in eosinophil migration
Blood,
November 15, 2000;
96(10):
3601 - 3609.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Tachimoto, M. M. Burdick, S. A. Hudson, M. Kikuchi, K. Konstantopoulos, and B. S. Bochner
CCR3-Active Chemokines Promote Rapid Detachment of Eosinophils from VCAM-1 In Vitro
J. Immunol.,
September 1, 2000;
165(5):
2748 - 2754.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction