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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2847-2856
Inhibition of Eosinophil Rolling and Recruitment in P-Selectin- and
Intracellular Adhesion Molecule-1-Deficient Mice
By
David H. Broide,
David Humber, and
P. Sriramarao
From the Department of Medicine, University of California, San Diego,
San Diego, CA; and the Laboratory of Immunology and Vascular Biology,
La Jolla Institute for Experimental Medicine, La Jolla, CA.
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ABSTRACT |
To determine the relative in vivo importance of endothelial
expressed adhesion molecules to eosinophil rolling, adhesion, and
transmigration, we have induced eosinophilic peritonitis using ragweed
allergen in P-selectin-deficient, intracellular adhesion molecule-1 (ICAM-1)-deficient and control wild-type mice.
Circulating leukocytes visualized by intravital microscopy exhibited
reduced rolling and firm adhesion in P-selectin-deficient mice and
reduced firm adhesion in ICAM-1-deficient mice. Eosinophils exhibited reduced rolling and firm adhesion to endothelium in
P-selectin-deficient mice. Eosinophil recruitment in
P-selectin-deficient mice (~75% inhibition of eosinophil
recruitment) and ICAM-1-deficient mice (~67% inhibition of
eosinophil recruitment) was significantly reduced compared with
wild-type mice. Eosinophil recruitment was not completely inhibited in
P-selectin/ICAM-1 double-mutant mice (eosinophil recruitment inhibited
~62%). However, pretreatment of P-selectin/ICAM-1-deficient mice
with an anti-vascular cell adhesion molecule (VCAM)
antibody induced near complete inhibition of eosinophil recruitment.
Overall, these studies show that eosinophil rolling and firm adhesion
is significantly reduced in P-selectin-deficient mice and that
P-selectin, ICAM-1, and VCAM are important to eosinophil peritoneal
recruitment after ragweed challenge.
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INTRODUCTION |
THE RECRUITMENT OF eosinophils to sites
of allergic inflammation in the peritoneal microcirculation in vivo is
a multistep process characterized by initial eosinophil intravascular
rolling and firm adhesion to endothelium, followed by sequential
eosinophil diapedesis between endothelial cells and chemotaxis into
tissues.1,2 In vitro studies of eosinophils using a
rotational assay to simulate shear stress associated with blood flow
have demonstrated a role for L-selectin in eosinophil adhesion to human
umbilical vein endothelial cells placed on a horizontal
rotator.3 Using intravital videomicroscopy, we have
identified that the adhesion molecules used by eosinophils in vivo in
the initial steps of rolling on endothelium (L-selectin- and
VLA-4-dependent)2 differ from that used by other
circulating leukocytes such as neutrophils (L-selectin-dependent and
VLA-4-independent)2,4 under conditions of blood flow in
vivo. These in vivo experiments with eosinophils identified that the
paradigm of the strict separation of leukocyte selectins as rolling
receptors, and leukocyte integrins as firm adhesion receptors under
conditions of blood flow did not hold for all leukocyte integrins. The
observation that an eosinophil expressed integrin, namely VLA-4, can
function as a rolling receptor in vivo2 has subsequently
been confirmed by other investigators studying the rolling function of
VLA-4 expressed by T lymphocytes in vitro and in vivo.5,6
Whereas the above-noted studies have attempted to address the role of
eosinophil expressed adhesion molecules (ie, L-selectin and VLA-4),
this study focuses on the relative importance of adhesion molecules
(P-selectin, intracellular adhesion molecule-1 [ICAM-1]) expressed by endothelial cells to eosinophil rolling, adhesion, and
transmigration in vivo. Endothelial cells store P-selectin preformed in
Weibel-Palade bodies.7 Upon in vitro stimulation with
histamine or thrombin, Weibel-Palade bodies fuse with the endothelial
cell surface membrane and expose P-selectin rapidly (within a few
minutes of stimulation) and transiently (peak expression 20 to 30 minutes) to the luminal cell surface.7 In contrast to
histamine and thrombin stimulation, cytokines such as tumor necrosis
factor induce sustained P-selectin expression by cultured endothelial cells.8 The important in vivo functional role
of P-selectin as a neutrophil adhesion counter-receptor has previously been shown in P-selectin-deficient mice.9-11 Additional
studies demonstrate that neutrophils use P-selectin glycoprotein
ligand-1 (PSGL-1) to roll on P-selectin12 and that
neutralizing antibodies to P-selectin significantly attenuate
neutrophil influx in animal models of inflammation.13 In
limited studies of eosinophils and P-selectin, eosinophils have been
shown to bind to both purified P-selectin14 and to
P-selectin expressed by nasal polyp endothelium using static in vitro
adhesion assays.15 Eosinophils also roll on P-selectin in
vitro, as demonstrated in a flow chamber assay.16
Murine ICAM-1 has been molecularly cloned17 and, like human
ICAM-1, has five extracellular Ig-like domains. Amino acid
substitutions in the ICAM-1 extracellular domains have indicated that
the primary binding site for the eosinophil counter receptor CD11a/CD18
(LFA-1) is located in the NH2-terminal first domain of
ICAM-1.17 In vitro studies have shown that eosinophils bind
to ICAM-118 and that levels of ICAM-1 expression are
increased in the nasal mucosa of patients with perennial allergic
rhinitis.19 In vivo studies using neutralizing antibodies
to ICAM-1 in animal models of eosinophilic inflammation have produced
conflicting results concerning the role of ICAM-1 in eosinophil
recruitment, with some studies demonstrating inhibition of eosinophil
recruitment,20 whereas other studies show no inhibition of
eosinophil recruitment.21,22 To obviate methodologic
concerns about studies using neutralizing antibodies to ICAM-1 (dose,
route, and timing of administration of antibody, affinity of antibody,
nonspecific binding of antibody to Fc receptors on inflammatory cells
and not to ICAM-1 on endothelial cells), we have used ICAM-1-deficient
mice to study the role of ICAM-1 in eosinophil adhesion and
recruitment. Studies of neutrophil recruitment in P-selectin- and
ICAM-1-deficient mice infected in the peritoneal cavity with
streptococcus pneumoniae demonstrate an approximately 60% to 70%
reduction in acute neutrophil migration into the peritoneal cavity in
mice with either mutation alone and complete inhibition of neutrophil
recruitment in P-selectin/ICAM-1 double-mutant mice.10 We
have used the same P-selectin- and ICAM-1-deficient
mice,10 as well as P-selectin/ICAM-1 double-mutant mice,10 to determine the relative importance of P-selectin
and ICAM-1 to an eosinophilic inflammatory response in the peritoneum in response to ragweed allergen challenge (as opposed to streptococcus pneumoniae infection as the inflammatory stimulus for neutrophils in
the above-mentioned studies).10
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MATERIALS AND METHODS |
Adhesion molecule knockout mice.
P-selectin-deficient, ICAM-1-deficient, P-selectin/ICAM-1
double-mutant, as well as C57BL/6 background control wild-type female mice aged 8 to 10 weeks were purchased from Jackson Laboratories (Bar
Harbor, ME). The above-noted mice were developed by Dr
Arthur Beaudet (Department of Molecular Genetics, Baylor College of
Medicine, Houston, TX) and used previously in studies of neutrophil
adhesion in a model of bacterial infection in mice.10
Mouse model of peritoneal eosinophilic inflammation: Ragweed
allergen immunization and peritoneal allergen challenge.
The techniques used for ragweed immunization and challenge are similar
to those previously described by other investigators.23-25 Previous studies of neutrophil recruitment into the peritoneal cavity
in these adhesion molecule-deficient mice were performed 2 to 4 hours
after bacterial infection by Bullard et al,10 whereas the
studies of eosinophil peritoneal recruitment we describe were performed
at a later time point optimal for evaluation of eosinophil recruitment
(24 to 48 hours after allergen challenge). Mice are immunized by a
series of five injections of a 1:1,000 dilution of a ragweed pollen
extract (Miles Inc, Spokane, WA): 0.1 mL is injected subcutaneously on
days 0 and 1, and 0.2 mL is injected subcutaneously on days 6, 8, and
14. A control group of ragweed immunized mice (challenged with
phosphate-buffered saline [PBS] diluent) and nonimmunized mice
(prepared by subcutaneous injections of isotonic saline instead of the
ragweed pollen extract) follow the same immunization schedule. Three to
five mice are included in each group of mice studied. The mice are
challenged on day 20 by the intraperitoneal injection of 0.2 mL of the
ragweed allergen (or control PBS diluent).
To determine whether wild-type and adhesion molecule-deficient mice
were equivalently sensitized to allergen, immediate hypersensitivity skin tests were performed. Wild-type and adhesion molecule-deficient mice were sensitized to ragweed as described. On day 20, 50 µL of
ragweed antigen or diluent control was injected into the shaved backs
of the different groups of mice. Immediately after antigen administration, 200 µL of 1% Evans blue dye was injected into the
tail vein of the mice. Blueing of the skin at the antigen challenged
(but not diluent challenged) sites occurred within 10 minutes of
antigen challenge. The size of the blueing reaction (15 to 20 mm
diameter) was not significantly different in wild-type, P-selectin-deficient, or ICAM-1-deficient mice.
In selected experiments to evaluate the contribution of vascular cell
adhesion molecule (VCAM) expression to eosinophil
recruitment, two groups of P-selectin/ICAM-1 double-mutant mice were
immunized over a 14-day period with ragweed allergen as described
above. On day 20 of the ragweed sensitization protocol, either a rat IgG1 antimouse VCAM monoclonal antibody (MoAb) mk 2.7 (1 mg/kg body
weight; kindly provided by Dr E. Butcher, Stanford, CA)26 or a species- and isotype-matched control MoAb was injected
intravenously. Two hours after the antibody was administered, the mice
were challenged by the intraperitoneal injection of allergen and the
number of peritoneal eosinophils was enumerated 48 hours later.
Assessment of cells in the peritoneal cavity.
At time points before (day 0) and after immunization, as well as before
and 48 hours after intraperitoneal allergen challenge (day 22), the
mice were killed by cervical dislocation. Two milliliters of PBS
containing 6 U/mL of heparin was injected intraperitoneally, the
abdomen was massaged, and the peritoneal infusion was collected after
the peritoneum was opened. An appropriate PBS dilution of the recovered
peritoneal fluid was added to trypan blue, and the viability and total
number of white blood cells were counted with a hemocytometer.
Differential leukocyte counts were performed after brief acetone
fixation and staining of the peritoneal cells with
May-Grünwald-Giemsa stains. The percentage of eosinophils present
on each slide was assessed by counting a minimum of 300 cells in random
high-power fields using a light microscope (40× magnification) to
display the slide image on a TV monitor (Videometric 150 image analysis
program; American Innovision, San Diego, CA). In addition, because mast
cells participate in allergen-induced inflammation, the number of
resident peritoneal mast cells was compared in wild-type and adhesion
molecule-deficient mice.
Assay for eosinophil peroxidase (EPO).
In addition to enumerating the number of eosinophils in the peritoneal
cavity, EPO, an eosinophil cytoplasmic granule protein, was assayed
using the substrate solution O-phenylenediamine dihydrochloride (OPD)
and a calorimetric assay.27 Peritoneal cell pellets
(105 cells) were lysed in 0.02% CTAB, 0.05% triton and
added to 2 mL of assay buffer (0.1 mol/L phosphate buffer, pH 6.8, 8 mmol/L OPD, 0.01% H2O2). Reaction volumes were
incubated in duplicate for 10 minutes at room temperature and read in a
spectrophotometer at 492 nm wavelength (Shimadza UV160U, Tokyo, Japan).
Preparation of mice for detection of leukocyte rolling in the
peritoneal microcirculation.
Adhesion molecule-deficient or control wild-type mice (25 to 35 g body
weight) were anesthetized with a subcutaneous injection of saline
solution containing a cocktail of ketamine hydrochloride and Xylazine
(7.5 mg and 2.5 mg, respectively, per 100 mg body weight). The mice
were then placed on a heating pad maintained at 37°C. A midline
incision was made and the mesentery was gently exteriorized and spread
on a heated glass window (37.5°C) of the stage of a Leitz
intravital microscope (Wetzlar, Germany). The exteriorized portion of
mouse mesentery was kept continuously moist with endotoxin-free
isotonic saline solution (pH 7.4). Other parts of the intestine that
were exposed but not microscopically observed were kept moist with
isotonic saline-soaked cotton pads and the mesentery was covered with
Saran Wrap. To minimize endotoxin contamination, Saran
Wrap (Dow Brands LP, Indianapolis, IN) was presoaked with 1%
E-Toxa-Clean (Sigma Chemical Co, St Louis, MO) overnight, followed by
rinsing in 70% ethanol and endotoxin-free distilled water and a final
wash with sterile isotonic saline solution.
Intravital microscopy and image analysis.
The passage of circulating leukocytes in the peritoneal
microcirculation was made visible by transillumination using a Nikon 10× (numerical aperture [NA], 0.30), 20× (NA, 0.40), or
40× (NA, 0.55) water immersion objective (Melville, NY), as
previously described.2,28 All microscopic images were
recorded through a silicon-intensified tube camera (SIT68; Dage-MTI,
Michigan City, IN) attached to the microscope and connected to a Sony
monitor (Tokyo, Japan) and an SVHS video recorder (JVC
HR-S66004) for off-line analysis of eosinophil rolling.
Video recordings of different postcapillary and collecting venules
(range, 20 to 65 µm) were analyzed for assessment of rolling and
adhesion of leukocytes as previously described.2,28 Rolling
leukocytes were quantitated by counting the number of cells interacting
with the vessel wall in 1 minute in a plane perpendicular to a vessel
axis, whereas those cells that were found to be stationary for at least
1 minute were considered as adherent or sticking cells. All studies
were conducted between 0 to 1 hour after exteriorization of the mouse mesentery.
Isolation of murine eosinophils from interleukin-5 (IL-5) transgenic
mice.
In selected experiments, mouse eosinophils of greater than 90% purity
and greater than 98% viability were purified from IL-5 transgenic
mice, kindly provided by Dr Colin Sanderson (Perth, Australia).29 IL-5 transgenic mice (age, 10 weeks old) have peripheral blood leukocyte differential cell counts exhibiting 42% ± 12% peripheral blood eosinophilia (n = 3). The contaminating white blood cells comprise 44% T lymphocytes, 2% mononuclear cells, and 10% neutrophils. To purify the eosinophils, we diluted blood drawn
from six IL-5 transgenic mice 1:1 in PBS, 0.1 mmol/L EDTA and layered
it onto a discontinuous density gradient of percoll (1.075, 1.080, 1.085, 1.090, and 1.095). Eosinophils (85% to 95% pure; 98% viable)
banded between the 1.085 and 1.090 layers. Fluorescence-activated cell
sorting analysis showed that the purified eosinophils
expressed L-selectin, as well as  4 and  2 integrin cell surface
receptors (data not shown).
Eosinophils with at least 98% viability and greater than 90% purity
were selected and labeled with carboxy fluorescein diacetate (CFDA;
Molecular Probes, Eugene, OR) as previously described for human
eosinophils and murine mast cells.2,28 CFDA-labeled eosinophils were resuspended at a concentration of 0.5 × 107 cells/200 µL of PBS containing 0.01% glucose and
kept at room temperature in the dark until used. Eosinophils were then
injected into the tail vein of mice previously sensitized with ragweed allergen and challenged 24 hours before surgery with either ragweed or
saline and observed by intravital fluorescence microscopy. All studies
were conducted between 0 and 1 hour after exteriorization of the mouse
mesentery.
Visualization of eosinophils in mouse mesentery.
The rolling of mouse eosinophils in mesenteric venules was made visible
by stroboscopic epi-illumination using a video-triggered Xenon lamp
(Chadwick Helmuth, El Monte, CA) and Leitz Ploemopak epi-illuminator
employing an I2 filter block (Wetzlar, Germany). All images were
recorded through a silicon-intensified tube camera (SIT68; Dage MTI,
Michigan City, IN) using a 10× or 20× water immersion
objective (Nikon), as described previously.2 The rolling
fraction (Rf) and the rolling velocity of CFDA-labeled mouse
eosinophils in ragweed and diluent PBS challenged mice (wild-type control and P-selectin-deficient mice) was determined by
frame-by-frame analysis, as previously described.2,28
Statistics.
The number of eosinophils and mast cells, as well as EPO levels in
peritoneal fluid, were compared by multiple comparisons of paired data
by Student's t-test using a statistical software package (In
Stat, San Diego, CA). P values of less than .05 were considered
to be statistically significant. All results are given as the mean ± standard error of the mean (SEM). Rolling fractions of murine
leukocytes and injected eosinophils were compared by multiple
comparisons of paired data by Student's t-test using a
statistical software package (SigmaStat, Jandel Scientific). P
values of less than .05 were considered statistically significant. All
results are given as the mean ± standard error (SE).
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RESULTS |
Mouse model of eosinophilic peritonitis: P-selectin-deficient mice.
Control wild-type mice (n = 9 mice; 3 separate experiments), when
immunized and challenged with ragweed allergen, developed a significant
peritoneal cavity eosinophilia (17.3% ± 3.5% eosinophils; Fig 1) compared with wild-type mice that
were not challenged with ragweed (0.9% ± 0.4% eosinophils;
P = .03) or compared with wild-type mice that were immunized
with ragweed and challenged with PBS diluent (2.5% ± 0.8%
eosinophils; P = .04). In contrast to control wild-type mice,
P-selectin-deficient mice immunized with ragweed developed less
peritoneal eosinophilia when challenged with an intraperitoneal
injection of ragweed allergen (P-selectin-deficient mice 4.3% ± 2.0% peritoneal eosinophils, ~75% inhibition compared with
ragweed-challenged wild-type mice, P = .005; Fig 1).
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| Fig 1.
Comparison of eosinophil recruitment and EPO levels in
P-selectin-deficient and wild-type mice. Ragweed-sensitized mice
(P-selectin-deficient or control wild-type mice; n = 9 mice; 3 separate experiments) were challenged with an intraperitoneal injection
of ragweed allergen. Forty-eight hours later, the percentage of
transmigrated peritoneal eosinophils (A) and EPO levels (B) were
assessed. P-selectin-deficient mice developed significantly less
peritoneal eosinophilia (P = .005) and lower EPO levels
(P = .05) compared with control wild-type mice after
allergen.
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There was a trend to an increase in the total leukocyte count in the
peritoneal cavity of wild-type mice after, compared with before,
allergen challenge (89.4 ± 53.6 × 105 peritoneal
cells after allergen; 44.0 ± 14.8 × 105
peritoneal cells before allergen; n = 3 experiments; P = .19) and P-selectin-deficient mice (94.8 ± 41.4 × 105
peritoneal cells after allergen; 76.6 ± 13.6 × 105 peritoneal cells before allergen; n = 3 experiments;
P = .48), but this did not reach statistical significance.
Mouse model of eosinophilic peritonitis: ICAM-1-deficient mice.
Control wild-type mice (n = 10 mice; 3 separate experiments), when
immunized and challenged with ragweed allergen, developed a significant
peritoneal cavity eosinophilia (11.6% ± 2.9% eosinophils) compared with wild-type mice that were not challenged with ragweed (0.2% ± 0.1% eosinophils; P = .05) or compared
with wild-type mice that were immunized with ragweed and challenged
with PBS diluent (1.4% ± 0.6% eosinophils). In contrast to
control wild-type mice, ICAM-1-deficient mice immunized with ragweed
developed less peritoneal eosinophilia when challenged with an
intraperitoneal injection of ragweed allergen (ICAM-1-deficient mice
3.8% ± 1.1% peritoneal eosinophils, ~67% inhibition compared
with ragweed challenged wild-type mice; P = .03;
Fig 2).
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| Fig 2.
Comparison of eosinophil recruitment and EPO levels in
ICAM-1-deficient and wild-type mice. Ragweed-sensitized mice
(ICAM-1-deficient or control wild-type mice; n = 10 mice; 3 separate
experiments) were challenged with an intraperitoneal injection of
ragweed allergen. Forty-eight hours later, the percentage of
transmigrated peritoneal eosinophils (A) and EPO levels (B) were
assessed. ICAM-1-deficient mice developed significantly less
peritoneal eosinophilia (P = .03) and lower EPO levels
(P = .05) compared with control wild-type mice after
allergen.
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As with studies of P-selectin-deficient mice, there was a trend to an
increase in the total leukocyte count in the peritoneal cavity of
wild-type mice after versus before allergen challenge (73.3 ± 50.5 × 105 peritoneal cells after allergen; 34.2 ± 10.2 × 105 peritoneal cells before
allergen; n = 3 experiments; P = .22) and ICAM-1-deficient
mice (48.9 ± 20.6 × 105 peritoneal cells after
allergen; 37.1 ± 7.1 × 105 peritoneal cells
before allergen; n = 3 experiments; P = .37), but this did not
reach statistical significance.
Kinetic studies of eosinophilic peritonitis.
Because peak eosinophil recruitment in this model of eosinophilic
peritonitis occurs from 24 to 72 hours after allergen challenge, our
initial studies (reported above) focused on the 48-hour post-allergen challenge time point. We also performed studies of eosinophil recruitment at earlier time points. Control wild-type mice, when immunized and challenged with ragweed allergen, developed a significant peritoneal eosinophilia (18.3% ± 1.7 % eosinophils) 24 hours
after allergen challenge compared with ICAM-1-deficient mice (6.3% ± 1.8% eosinophils; P = .01) or compared with
P-selectin-deficient mice (5.3% ± 0.6% eosinophils; P = .001). These studies show that there was no significant difference in
the degree of inhibition of eosinophil recruitment in ICAM-1-deficient
mice at 24 hours after allergen challenge (66% inhibition v
wild-type mice) compared with 48 hours after allergen challenge (67%
inhibition v wild-type mice). Similarly, our studies of
P-selectin-deficient mice showed that there was no significant
difference in the degree of inhibition of eosinophil recruitment in
P-selectin-deficient mice at 24 hours after allergen challenge (72%
inhibition v wild-type mice) compared with 48 hours after
allergen challenge (75% inhibition v wild-type mice).
We also performed studies at 12 hours after allergen challenge that are
more difficult to interpret because of the very low level of
allergen-induced eosinophil recruitment in wild-type mice at 12 hours
(3% eosinophils) compared with 24 hours (18% eosinophils) or 48 hours
(17% eosinophils) after allergen challenge. Nevertheless, there is a
similar degree of inhibition of eosinophil recruitment 12 hours after
allergen challenge in ICAM-1-deficient mice (62% inhibition compared
with wild-type mice) and P-selectin-deficient mice (63% inhibition
compared with wild-type mice). These studies clearly demonstrate the
importance of P-selectin and ICAM-1 at the 24-hour and 48-hour
post-allergen challenge time points and also suggest a role for
P-selectin and ICAM-1 as early as the 12-hour time point (the caveat
being the low numbers of eosinophils recruited in wild-type mice).
Peritoneal EPO levels.
Control wild-type mice (n = 9 mice; 3 separate experiments), when
immunized and challenged with ragweed allergen, developed a significant
increase in peritoneal cavity EPO levels (1.35 ± 0.35 EPO units)
compared with wild-type mice that were not challenged with ragweed
(0.08 ± 0.05 EPO units; P = .01) or compared with wild-type
mice that were immunized with ragweed and challenged with PBS diluent
(0.14 ± 0.06 EPO units; P = .01). In contrast, P-selectin-deficient and ICAM-1-deficient mice immunized with ragweed
developed lower levels of peritoneal EPO when challenged with an
intraperitoneal injection of ragweed allergen (P-selectin-deficient mice 0.54 ± 0.16 EPO units, ~60% inhibition compared with
ragweed-challenged wild-type mice 1.35 ± 0.35 EPO units; P = .05; Fig 1; ICAM-1-deficient mice 0.30 ± 0.07 EPO units, ~63%
inhibition compared with ragweed challenged wild-type mice 0.81 ± 0.24 EPO units; P = .05; Fig 2).
Peritoneal mast cells and mononuclear cells.
In contrast to the changes noted in the number of recruited eosinophils
in control wild-type and adhesion molecule-deficient mice after
allergen challenge, there were no significant differences in the number
of resident peritoneal mast cells in wild-type and ICAM-1-deficient
mice before (ICAM-1 wild-type 4.1% ± 1.3% v
ICAM-1-deficient 4.7% ± 0.9%) or after allergen challenge
(ICAM-1 wild-type 2.8% ± 0.4% v ICAM-1-deficient 3.2% ± 0.3%).
There was a slight, but statistically insignificant, reduction in
peritoneal mast cells in P-selectin-deficient compared with control
wild-type mice before (P-selectin wild-type 3.2% ± 0.5% v
P-selectin-deficient 2.0% ± 0.3%) and after allergen challenge (P-selectin wild-type 1.5% ± 0.7% v P-selectin-deficient
1.1% ± 0.5%). There was also a trend for antigen-challenged
P-selectin-deficient, ICAM-1-deficient, as well as wild-type mice to
have a lower percentage of peritoneal mast cells compared with their
respective pre-antigen control. This is probably due to the fact that
antigen challenge induces mast cell degranulation, rendering some
post-antigen degranulated peritoneal mast cells invisible with the
granule-based stain we have used to enumerate mast cells.
Mononuclear cells comprised the majority of peritoneal cells in
wild-type (96.1 ± 0.1% before allergen v 83.3% ± 3.7% after allergen), P-selectin-deficient (97.6% ± 0.3% before
allergen v 91.5% ± 2.1% after allergen), and
ICAM-1-deficient mice (94.9% ± 0.8% before allergen v
93.3% ± 1.2% after allergen). Rare neutrophils that comprised
less than 1% to 2% of peritoneal cells were occasionally noted in
wild-type mice challenged with allergen.
Peripheral blood leukocytes.
There was no significant difference in the number of circulating
eosinophils in wild-type (2.1%) compared with P-selectin-deficient (2.0%) or ICAM-1-deficient mice (2.2%). P-selectin/ICAM-1-deficient mice had a slight increase in circulating eosinophils (3.1%) compared with wild-type mice (2.1%). As previously noted,10 there
was a mild increase in the total peripheral blood leukocyte cell count as well as the percentage of neutrophils in
P-selectin/ICAM-1-deficient, ICAM-1-deficient, and
P-selectin-deficient mice compared with wild-type mice (wild-type mice
6.4 ± 0.8 × 103 total leukocytes/µL; 2.1% ± 0.2% eosinophils, 14.8% ± 6.3% neutrophils, 83.1% ± 7.4% mononuclear cells; P-selectin-deficient mice 8.1 ± 0.7 × 103 total leukocytes/µL; 2.0% ± 0.3%
eosinophils, 22.4% ± 10.6% neutrophils, 75.6% ± 8.4%
mononuclear cells; ICAM-1-deficient mice 11.5 ± 0.6 × 103 total leukocytes/µL; 2.2% ± 0.1% eosinophils,
27.1% ± 10.3% neutrophils, 70.7% ± 12.6% mononuclear cells;
and P-selectin/ICAM-1-deficient mice 11.8 ± 0.6 × 103 total leukocytes/µL; 3.1% ± 0.2% eosinophils,
29.6% ± 9.8% neutrophils, 67.3% ± 12.7% mononuclear cells).
Intravital microscopy and leukocyte rolling.
The effect of ragweed challenge on leukocyte rolling and firm adhesion
in mesenteric venules of wild-type (n = 11 mice, 34 venules) as well as
P-selectin (n = 6 mice, 19 venules) and ICAM-1-deficient mice (n = 5 mice, 18 venules) was examined by intravital microscopy (Table 1). There was no significant
difference in hemodynamic parameters measured (venular diameter, mean
blood flow velocity, shear rate, and shear stress;
Table 2) in wild-type compared with
P-selectin-deficient or ICAM-1-deficient mice. Leukocyte rolling was
observed predominantly in postcapillary and collecting venules and was
rarely observed in arterioles. In wild-type mice, challenge with
ragweed allergen resulted in a fourfold increase in leukocyte rolling
compared with challenge with PBS diluent (ragweed challenge 68.1 ± 11.1 rolling leukocytes/minute v PBS diluent challenge 16.4 ± 5.3 rolling leukocytes/minute; P = .004; Table 1).
However, leukocyte rolling in ragweed-challenged P-selectin-deficient mice (4.2 ± 0.3 rolling leukocytes/minute) was significantly less than that observed in control ragweed-challenged wild-type mice (68.1 ± 11.1 rolling leukocytes/minute; P = .0001). PBS diluent challenge failed to induce detectable leukocyte rolling in
P-selectin-deficient mice during the 1-hour observation period.
Allergen challenge of ICAM-1-deficient mice with ragweed resulted in a
threefold increase in leukocyte rolling (45.1 ± 4.5 rolling
leukocytes/minute) compared with PBS diluent-challenged ICAM-1-deficient mice (13.9 ± 2.2 rolling leukocytes/minute;
P = .002). Furthermore, no significant differences in
leukocyte rolling was observed between ragweed-challenged wild-type
mice and ragweed challenged ICAM-1-deficient mice.
Intravital microscopy and leukocyte firm adhesion.
We next examined the effect of allergen challenge on leukocyte firm
adhesion (adherent leukocytes/100 µm venule length) in the mesenteric
venules (Table 1). In wild-type mice, intraperitoneal challenge with
ragweed resulted in a fourfold increase in leukocyte adhesion compared
with PBS diluent challenge (ragweed challenge 10.6 ± 2.2 adherent
leukocytes v PBS diluent challenge 2.3 ± 0.9 adherent
leukocytes; P = .02). Leukocyte adhesion in ragweed-challenged P-selectin-deficient mice was found to be significantly reduced compared with ragweed-challenged wild-type mice (P-selectin-deficient 2.8 ± 1.2 adherent leukocytes v wild-type mice 10.6 ± 2.2 adherent leukocytes; P = .0001). Minimal adhesion of murine
leukocytes was observed in P-selectin-deficient mice that were
challenged with PBS diluent (0.2 ± 0.4 adherent leukocytes).
Although allergen challenge had induced significant leukocyte rolling
in ICAM-1-deficient mice, it failed to induce significant leukocyte
adhesion in the ICAM-1-deficient mice (1.1 ± 0.4 adherent
leukocytes in ragweed-challenged ICAM-1-deficient mice; v 10.6 ± 2.2 adherent leukocytes in ragweed-challenged wild-type mice;
P = .007). As observed with P-selectin-deficient mice, PBS
diluent administration failed to induce significant firm adhesion of
rolling leukocytes in ICAM-1-deficient mice (0.6 ± 0.3 adherent
leukocytes).
Intravital microscopy and eosinophil rolling and adhesion in
P-selectin-deficient mice.
Because the above-noted studies characterized total leukocyte, but not
eosinophil, rolling and adhesion, we examined the ability of purified
murine eosinophils to roll and adhere to vascular endothelium in vivo
(Table 3). Eosinophils were purified from IL-5 transgenic mice and labeled with CFDA before injection into the
tail vein. The passage of the fluorescently labeled eosinophils in the
mesenteric circulation was made visible by stroboscopic epi-illumination (Fig 3). Similar to
studies of total leukocyte rolling, allergen challenge with ragweed
induced a 1.8-fold increase in eosinophil rolling in the mesenteric
venules of wild-type mice challenged with ragweed (eosinophil Rf:
26.7% ± 2.6%) compared with wild-type mice challenged
with PBS diluent (eosinophil Rf: 9.1% ± 1.5%; P = .002;
Table 3). The rolling of eosinophils in venules of ragweed challenged
P-selectin-deficient mice was found to be dramatically reduced
(eosinophil Rf: 1.2% ± 0.8%; P = .0002 v
ragweed-challenged wild-type mice). PBS diluent challenge failed to
induce detectable eosinophil rolling in P-selectin-deficient mice.
View this table:
[in this window]
[in a new window]
|
Table 3.
Effect of Ragweed Challenge on Eosinophil Rolling and
Adhesion to Endothelium in Mesenteric Venules of Wild-Type and
P-Selectin-Deficient Mice
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|
View larger version (49K):
[in this window]
[in a new window]
| Fig 3.
Eosinophil rolling and firm adhesion visualized by
intravital videomicroscopy. Ragweed-sensitized wild-type mice were
challenged with an intraperitoneal injection of ragweed. Twenty-four
hours after intraperitoneal ragweed allergen challenge, fluorescently labeled eosinophils in the peritoneal microcirculation were visualized in vivo using intravital videomicroscopy. (A) through (F) are sequential videotape images of the same venule (V) and arteriole (A).
Blood flow in the venule is from left to right. Three firmly adherent
eosinophils ( ; A) are visualized in the same relative position in
(A) through (F), whereas two different eosinophils (eosinophil #1 =  ; and eosinophil #2 = ) rolling along the venular endothelium
are noted in different positions in (A) through (D) (rolling eosinophil
#1 = ) and (C) through (E) (rolling eosinophil #2 = ).
|
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The ability of eosinophils to firmly adhere to mesenteric endothelium
in P-selectin-deficient and wild-type mice after allergen challenge
was also determined. In wild-type mice ragweed challenge resulted in an
eightfold increase in eosinophil adhesion (ragweed challenge 3.3 ± 1.1 adherent eosinophils v 0.4 ± 0.3 adherent eosinophils
after PBS diluent challenge; P = .001; Table 3). As observed
with murine leukocytes, reduced levels of eosinophil adhesion was
observed in P-selectin-deficient compared with wild-type mice that
were challenged with ragweed (0.8 ± 0.5 adherent eosinophils in
ragweed challenged P-selectin-deficient mice v 3.3 ± 0.5 adherent eosinophils in ragweed challenged wild-type mice,
P = .001), whereas no detectable adhesion of
eosinophils was observed in PBS diluent-challenged P-selectin-deficient mice. A comparison of velocity distribution profiles of rolling eosinophils showed that the eosinophil rolling velocity in ragweed-challenged P-selectin-deficient mice (216.3 ± 31.9 µm/s) was significantly greater than the eosinophil rolling velocity in ragweed-challenged wild-type mice (35.7 ± 8.3 µm/s; P < .001).
P-selectin/ICAM-1 double-mutant mice.
Because studies with neutrophil emigration into the peritoneum during
streptococcus pneumoniae inducing peritonitis demonstrated complete
inhibition of neutrophil emigration into the peritoneum of
P-selectin/ICAM-1 double-mutant mice,10 we performed
similar experiments evaluating eosinophil recruitment after ragweed
allergen challenge. In contrast to studies with neutrophils in which
there was complete inhibition of neutrophil emigration,10
eosinophil recruitment in P-selectin/ICAM-1 double-mutant mice
challenged with allergen was not completely inhibited (eosinophil
recruitment inhibited ~62%; P = .01;
Fig 4) and was similar to that noted with
either P-selectin-deficient (eosinophil recruitment inhibited ~75%)
or ICAM-1-deficient mice (eosinophil recruitment inhibited ~67%)
challenged with allergen.
View larger version (34K):
[in this window]
[in a new window]
| Fig 4.
Evaluation of eosinophil recruitment in
P-selectin/ICAM-1-deficient double-mutant mice. Ragweed-sensitized
mice (P-selectin/ICAM-1-deficient or control wild-type mice) were
challenged with an intraperitoneal injection of ragweed. Forty-eight
hours later, the percentage of transmigrated peritoneal eosinophils was
assessed by light microscopy. P-selectin/ICAM-1-deficient
double-mutant mice developed significantly less peritoneal eosinophilia
compared with control wild-type mice after allergen (P = .01).
|
|
Experiments were performed with an anti-VCAM antibody to determine the
contribution of VCAM to eosinophil recruitment in
P-selectin/ICAM-1-deficient mice. Pretreatment of P-selectin/ICAM-1
double-mutant mice with either an anti-VCAM or control antibody before
the day-20 peritoneal allergen challenge resulted in near complete
inhibition of eosinophil recruitment into the peritoneal cavity 48 hours later only in the mice receiving the anti-VCAM antibody (~93%
inhibition of eosinophil recruitment). In contrast, the species- and
isotype-matched antibody had no additional effect on eosinophil
recruitment in the control group of P-selectin/ICAM double-mutant mice
(~53% inhibition of eosinophil recruitment; wild-type mice 16.6% ± 8.4% peritoneal eosinophils; P-selectin/ICAM-1-deficient mice
pretreated with control antibody 7.8% ± 1.2% peritoneal
eosinophils; P-selectin/ICAM-1-deficient mice pretreated with an
anti-VCAM antibody 1.1% ± 0.2% peritoneal eosinophils;
Fig 5).
View larger version (18K):
[in this window]
[in a new window]
| Fig 5.
Inhibition of eosinophil recruitment in
P-selectin/ICAM-1-deficient double-mutant mice treated with an
anti-VCAM antibody. Ragweed-sensitized P-selectin/ICAM-1-deficient
mice were pretreated with either a rat IgG1 antimouse VCAM MoAb or a
species- and isotype-matched control antibody. Two hours after the
antibody was administered intravenously, the mice were challenged by
the intraperitoneal injection of allergen and the number of peritoneal
eosinophils enumerated 48 hours later.
|
|
| |
DISCUSSION |
In this study, we demonstrate that endothelial-expressed P-selectin and
ICAM-1 subserve important functions in the recruitment of eosinophils
to sites of allergic inflammation. In addition, we show that eosinophil
rolling is significantly reduced in P-selectin-deficient mice at sites
of allergen challenge 24 hours after challenge. Because significant
peritoneal eosinophilia (but not neutrophilia) is present 48 hours
after intraperitoneal allergen challenge,23-25 our
recruitment studies focused on analyzing the influx of eosinophils at
48 hours.23-25 Our study confirmed previous studies in
several laboratories that have shown that intraperitoneal allergen
challenge induces a peritoneal eosinophilia that is present at 8 hours
and peaks between 24 and 72 hours.23-25 A small influx of
neutrophils that peaks at 8 hours and is no longer present after 24 hours has also previously been noted in this mouse
model.23-25 Our studies using intravital microscopy have
demonstrated that total leukocyte rolling in mesenteric venules is
significantly induced in ragweed-challenged wild-type and
ICAM-1-deficient mice. In contrast, total leukocyte rolling is
significantly inhibited in ragweed-challenged P-selectin-deficient mice. Studies with fluorescently labeled eosinophils confirm that not
only total leukocyte, but also eosinophil rolling is significantly reduced in P-selectin-deficient mice. In addition, eosinophils roll
appreciably faster in P-selectin-deficient compared with wild-type
mice, suggesting that the absence of P-selectin weakens the strength of
the initial eosinophil endothelial cell interaction, resulting in
eosinophils rolling at higher velocities. Intravital microscopy studies
also demonstrated that total leukocyte firm adhesion was significantly
reduced in ragweed-challenged ICAM-1-deficient and
P-selectin-deficient mice as compared with ragweed-challenged wild-type mice. These observations underscore the importance of ICAM-1
as a firm adhesion receptor in vivo. The reduced firm adhesion of
leukocytes in P-selectin-deficient mice challenged with allergen also
suggests that leukocyte rolling and adhesion are sequential at sites of
allergen challenge. A model of sequential leukocyte adhesion would
require initial leukocyte rolling before firm adhesion. Thus, the
absence of significant leukocyte rolling in the mesenteric venules of
P-selectin-deficient mice would not allow for the subsequent firm
adhesion of leukocytes to firm adhesion receptors such as ICAM-1.
Indeed, studies with fluorescently labeled eosinophils confirmed that
not only total leukocytes, but also eosinophils, exhibited reduced
rolling and firm adhesion in P-selectin-deficient as compared with
wild-type mice challenged with allergen.
Our studies with ICAM-1-deficient mice (~67% inhibition of
eosinophil recruitment) also demonstrated an important role for this
endothelial-expressed adhesion molecule in recruitment of eosinophils
after allergen challenge. Studies with antibodies to ICAM-1 in animal
models of asthma have previously shown conflicting results regarding
the role of ICAM-1 in eosinophil recruitment into the
allergen-challenged lung.20-22 In a primate model of
asthma, antibodies against ICAM-1 decreased BAL eosinophil infiltration (~65%) and attenuated bronchial hyperresponsiveness,20
whereas in a mouse model antibodies to ICAM-1 had no effect on
eosinophil recruitment.21,22 The conflicting results in
these studies may relate to methodologic differences in the studies in
which different animal models (mouse v primate), different
antibodies (rat mouse ICAM-1 MoAb YN1/1.7 v MoAb R6.5),
different route of antibody administration (intraperitoneal v
intravenous), and different antigens (ascaris v ovalbumin) were
used.20-22 Our study using ICAM-1-deficient mice would
suggest that ICAM-1 is important to eosinophil recruitment to the
peritoneal cavity in mice, as has previously been demonstrated for
eosinophil recruitment to the lung in primates.20 A
potential confounding variable in the studies of the ICAM-1-deficient
mice we have used in this study is the recent demonstration that these
mice are not completely deficient in ICAM and do express small amounts
of alternatively spliced ICAM (as assessed by
immunohistochemistry).30 At present, the in vivo biologic
and functional significance of these alternative spliced forms of ICAM
are not known. The demonstration in this study that eosinophil
recruitment was inhibited approximately 67% in ICAM-1-deficient mice
suggests that ICAM-1 plays a significant role in eosinophil
recruitment. Although it is possible that alternative isoforms of
ICAM-1 could contribute to the approximately 33% of eosinophil
recruitment not inhibited in ICAM-1 mutant mice, our studies using
neutralizing antibodies to VCAM in P-selectin/ICAM-1 double-mutant mice
suggest that VCAM is more likely than these alternate isoforms of ICAM
to participate in eosinophil recruitment into the peritoneal cavity.
Previous studies by Gonzalo et al31 and our
group32 have shown that eosinophil recruitment into the
lungs of ovalbumin-challenged P-selectin-deficient mice is inhibited
70% to 80% compared with wild-type mice 3 hours after allergen
challenge. In contrast, Gonzalo et al31 showed that
eosinophil recruitment into the lungs of ovalbumin-challenged mice was
increased 2.5-fold 7 hours after allergen inhalation challenge. The
difference in eosinophil requirements for P-selectin at 7 hours
compared with other time points after allergen challenge may depend on
the vascular bed studied (pulmonary v peritoneal), the antigen
used (ovalbumin by Gonzalo et al and ragweed in our model), or the
method of sensitization and challenge (single v multiple
repetitive challenge). It is difficult to directly compare kinetic
results in the two studies (pulmonary versus peritoneal), because the
time 0 hours in the Gonzalo et al pulmonary study was preceded by 7 consecutive days of daily inhaled allergen, whereas in our peritoneal
study the single day-20 intraperitoneal injection of allergen was not
preceded by daily allergen challenge for 7 days. Further studies will
need to define whether repetitive allergen challenge compared with single allergen challenge alters the kinetic requirements of
eosinophils for P-selectin.
Overall, these studies demonstrate that P-selectin and ICAM-1 play an
important role in eosinophil recruitment to sites of allergic
inflammation. However, neither of these adhesion molecules is
completely able to inhibit eosinophil recruitment, and neither adhesion
molecule is specific for eosinophils as P-selectin and ICAM-1 are also
important to neutrophil10 and T
lymphocyte11,33,34 adhesion. Studies with P-selectin/ICAM-1
double-mutant mice suggest that approximately 25% to 40% of
eosinophil recruitment at sites of allergen challenge in the peritoneal
cavity occurs through a P-selectin/ICAM-1-independent pathway. In
terms of P-selectin-independent endothelial mechanisms of eosinophil
rolling along vascular endothelium, E-selectin and VCAM-1 are important
candidate endothelial expressed rolling receptors. In addition, VCAM-1
can function as an ICAM-1-independent eosinophil firm adhesion
pathway. Our studies in which we were able to nearly completely inhibit
eosinophil recruitment in P-selectin/ICAM-1 double-mutant mice
pretreated with neutralizing antibodies to VCAM, suggest that VCAM
contributes most significantly to P-selectin/ICAM-1-independent eosinophil recruitment. VCAM-1 expressed by endothelial cells can
function both as a firm adhesion and a rolling receptor.6 Studies using either T cells or 4 integrin transfected cells have
shown that cells expressing an 4 integrin can roll on purified VCAM-1, but not on ICAM-1 or fibronectin.6 Studies using
eosinophils have also demonstrated that neutralizing antibodies to
4, but not to 2 integrins can inhibit
eosinophil rolling in vivo,2 presumably through an
interaction between eosinophil expressed 4 and endothelial expressed
VCAM-1. However, at present, in vitro flow chamber or in vivo studies
with eosinophils and neutralizing antibodies to VCAM-1 have not been
published. The importance of P-selectin and VCAM-1, but not E-selectin
to eosinophil rolling is also suggested from in vitro flow chamber
studies and in vivo studies of the rabbit microcirculation in which
E-selectin was shown to preferentially support
neutrophil35,36 but not eosinophil rolling.37
However, in this study, we have not directly evaluated the role of
E-selectin in supporting eosinophil rolling. Nevertheless, overall,
these studies suggest that P-selectin supports both eosinophil and
neutrophil rolling,10,12 whereas VCAM-1 supports eosinophil but not neutrophil rolling and adhesion and E-selectin predominantly supports neutrophil but not eosinophil rolling.35-38
In summary, we report the first demonstration of murine eosinophil
rolling and firm adhesion in mouse mesenteric venules at sites of
allergen challenge using intravital microscopy. These studies also
provide evidence for the importance of vascular P-selectin, ICAM-1, and
VCAM-1 in the rolling, adhesion, and recruitment of murine eosinophils
and leukocytes in a model of allergic eosinophilic peritonitis.
| |
FOOTNOTES |
Submitted May 13, 1997;
accepted December 9, 1997.
Supported by National Institutes of Health Grants No. AI 33977 and AI
38425 (to D.H.B.) and AI 35796 (to P.S.).
Address reprint requests to David H. Broide, MBCHB, University of
California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0635.
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.
| |
ACKNOWLEDGMENT |
The authors thank Lauri Doval for expert secretarial support during the
preparation of the manuscript, Dr Colin Sanderson for providing IL-5
transgenic mice, and Gregory K. Hughes for technical support.
| |
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K. Y. Larbi, J. P. Dangerfield, F. J. Culley, D. Marshall, D. O. Haskard, P. J. Jose, T. J. Williams, and S. Nourshargh
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N. W. Lukacs, A. John, A. Berlin, D. C. Bullard, R. Knibbs, and L. M. Stoolman
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B. S. Edwards, M. S. Curry, H. Tsuji, D. Brown, R. S. Larson, and L. A. Sklar
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M. A. Giembycz and M. A. Lindsay
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R. G. DiScipio, P. J. Daffern, M. A. Jagels, D. H. Broide, and P. Sriramarao
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Abstract
Introduction
Abstract