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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2345-2352
Overlapping Functions of E- and P-Selectin in Neutrophil
Recruitment During Acute Inflammation
By
Jonathon W. Homeister,
Mengkun Zhang,
Paul S. Frenette,
Richard O. Hynes,
Denisa D. Wagner,
John B. Lowe, and
Rory M. Marks
From the Departments of Pathology and Internal Medicine and the
Howard Hughes Medical Institute at The University of Michigan Medical
Center, Ann Arbor, MI; the Center for Cancer Research, the Department
of Biology, and the Howard Hughes Medical Institute, Massachusetts
Institute of Technology, Cambridge, MA; and The Center for Blood
Research, and the Department of Pathology, Harvard Medical School,
Boston, MA.
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ABSTRACT |
Selectin adhesion molecules mediate leukocyte rolling on activated
endothelium, a prerequisite to leukocyte accumulation at sites of
inflammation. The precise role of each selectin (E-, P-, and L-) in
this process is unclear and may vary depending on the particular
inflammatory stimulus, vascular bed, leukocyte subset, and species;
most data suggest discrete functional roles for each selectin. To
define the relative roles of E- and P-selectin in mediating neutrophil
accumulation in acute dermal inflammation, mice genetically deficient
in E-selectin, P-selectin, or both E- and P-selectin were injected
intradermally with zymosan. Luminal endothelial expression of E- and
P-selectin in response to zymosan was documented in wild-type mice by
intravenous administration of fluorochrome-labeled anti-E- and
anti-P-selectin antibodies. In mice deficient in E- or P-selectin,
neutrophil accumulation was unchanged or only subtly reduced relative
to wild-type control mice. In mice deficient in both E- and P-selectin,
neutrophil accumulation was significantly reduced (87% at 4 hours and
79% at 8 hours). These data demonstrate that, in this model of acute inflammation, there is considerable overlap in the functions of E- and
P-selectin; loss of both selectins was required to impair neutrophil
accumulation.
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INTRODUCTION |
LEUKOCYTE EMIGRATION from the bloodstream
into the tissues in response to an inflammatory stimulus involves
multiple sequential events. These include a low-affinity adhesive
rolling interaction of leukocytes on activated endothelium, subsequent
leukocyte activation, immobilization, and endothelial
transmigration.1 The initial rolling interaction along
venular endothelium at physiologic shear stress is principally mediated
by the selectin family of adhesion molecules consisting of L-, P-, and
E-selectins. L-selectin is constitutively expressed on all leukocytes,
whereas E- and P-selectin are expressed on activated endothelial cells
(P-selectin is also expressed on activated platelets). E-selectin
expression requires de novo synthesis, whereas P-selectin is rapidly
translocated to the cell membrane from storage sites in Weibel-Palade
bodies (endothelium) or  granules (platelets).2
The precise roles of the selectins in vivo, individually and in
concert, remain undetermined. The close structural similarity of the
selectins suggests they may have overlapping functions, and this may be
especially true for E- and P-selectin, which are both expressed on
activated endothelium. However, many studies have demonstrated discrete
functional differences between these two molecules. Such functional
specificity may be dependent on a particular inflammatory stimulus,
adherent leukocyte subset, vascular bed, and species, causing
difficulty in assigning selectin function. In in vivo murine models,
the degree of functional overlap between E- and P-selectin is unclear
from published studies. Early (<8 hours) in a thioglycollate-induced
model of peritonitis, polymorphonuclear leukocyte (PMN)
accumulation is essentially normal in E-selectin-deficient mice,3 but is moderately reduced in P-selectin-deficient
mice and severely reduced in mice deficient in both E- and P-selectin (E-/P-selectin deficient),4,5 demonstrating discrete
functional differences for the two selectins. Anti-selectin antibody
studies have also generally demonstrated functional differences for the selectins in the chemical peritonitis model.6 Functional
differences for E- and P-selectin are likewise evident in
Streptococcal-induced peritonitis7 and cytokine-induced
meningitis.8 In these models, there is a moderate reduction
in PMN accumulation in P-selectin-deficient mice, but a marked
reduction in E-/P-selectin double-deficient mice.
However, other studies suggest similar functions for the selectins. All
three selectins contribute to PMN accumulation in a murine model of
lipopolysaccharide-induced pleurisy, and E- and P-selectin functions
are similar.9 Six hours after the initiation of a chemical
peritonitis, E- and P-selectin functions appear similar, although at 2 hours their functions appear to be discrete.3 Also, in
delayed-type contact hypersensitivity, a T-cell-dependent inflammatory
lesion, PMN accumulation is dependent on overlapping E- and P-selectin
functions.3
An acute inflammatory lesion can be induced by zymosan, a constituent
of Saccharomyces cerevisiae cell wall. Zymosan-induced inflammation does not require previous sensitization,10-12
is complement-dependent, is associated with cytokine expression, and is
characterized acutely by rapid influx of PMNs. Zymosan directly
activates the alternative pathway of complement, leading to formation
of the anaphylatoxins C3a and C5a and the membrane attack complex
C5b-9. The anaphylatoxins are chemotactic for PMNs, and C5a directly
and indirectly induces expression of P-selectin on endothelial
cells.13-16 The membrane attack complex induces and
enhances P- and E-selectin expression, respectively.14,17
Zymosan also induces production of cytokines such as interleukin-1
(IL-1) and tumor necrosis factor (TNF)18-20 that
then cause endothelial selectin expression.21-24 Because
zymosan induces multiple interdependent molecular mechanisms, it is an attractive model for studying inflammation in vivo.
Gene targeting techniques have been used to generate mice genetically
deficient in one or more selectins. These mice have been extremely
useful in determining the roles of adhesion molecules in physiologic
and pathophysiologic conditions, including inflammation. This study
uses mice genetically deficient in E-, P-, and both E- and P-selectin
to determine the roles of these molecules in PMN accumulation in
zymosan-induced acute dermal inflammation. The results indicate that
zymosan causes both E- and P-selectin expression in the dermal
microvasculature, either of which is sufficient for PMN accumulation.
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MATERIALS AND METHODS |
Mice.
Mice deficient in E-, P-, or both E- and P-selectin were generated as
previously described4,5 and referred to as P+/E , P/E+, and P/E, respectively. Each individual
strain was tested with its own genetically matched control strain. All
experiments were performed (by J.W.H.) blinded to animal genotype. All
mice were male, 8 to 9 weeks old, and weighing 18 to 28 g. Genomic DNA
was prepared from tail samples at the completion of the experiment, and
polymerase chain reaction (PCR) amplification using
appropriate probes was performed on each DNA sample to ensure correct
assignment of genotype.
Eight-week-old B6D2F1/J male mice (Jackson Laboratory, Bar Harbor, ME)
were used for in vivo immunofluorescent studies of E-and P-selectin
expression in wild-type mice and for time- and dose-effect studies.
Experiments were performed in accordance with the guidelines of The
University of Michigan Committee on the Use and Care of Animals.
Veterinary care was provided by The University of Michigan Unit for
Laboratory Animal Medicine.
Zymosan-induced dermal inflammation.
Zymosan A from Saccharomyces cerevisiae (Sigma, St Louis, MO)
was suspended in phosphate-buffered saline (PBS) at a
concentration of 20 mg/mL. Mice were anesthetized with ether and an
intradermal injection of 20 µL PBS (control) or zymosan suspension
was placed in the dorsum of each ear using a microsyringe (Hamilton Co,
Reno, NV) and 30-gauge needle. At defined times after injection, the mice were killed and the entire ears were removed at their base. Specimens were snap frozen in liquid nitrogen and stored at
80°C until assayed.
Dermal myeloperoxidase content.
Tissue myeloperoxidase (MPO) content was used to quantitate tissue PMN
accumulation.25 Frozen specimens (80°C) were
transferred into liquid nitrogen and shattered in a pulverizing device
(Biospec Products, Bartlesville, OK). Tissue fragments were suspended
in 0.5% hexadecyltrimethylammonium bromide in 50 mmol/L potassium phosphate buffer, pH 6.0 (HTAB buffer), cooled on ice, homogenized, sonicated on ice for 30 seconds, freeze thawed three times, and resonicated. The resulting suspension was centrifuged at
40,000g for 20 minutes at 4°C. Thirty microliters of
supernatant were assayed for MPO activity in a microtiter plate in HTAB
buffer at room temperature with (final concentration) hydrogen peroxide (0.056 g/L) and o-dianisidine (0.139 g/L), by monitoring OD 450 nm
(Titertek Multiskan Plus; Flow Laboratories, McLean, VA). The reaction
was stopped with 0.1% sodium azide. Human PMN MPO standards were
included in each assay to quantitate and standardize MPO content.
Tissue histology and immunofluorescent microscopy.
Ears were harvested from euthanized mice 8 hours after injection with
zymosan suspension or diluent alone, fixed overnight in 10% buffered
formalin, and processed routinely for paraffin block production.
Four-micron sections were cut and stained with hematoxylin and eosin.
To assess luminal endothelial expression of P- and E-selectin, in vivo
immunofluorescence studies were performed. Immediately after injecting
ears with PBS or zymosan, mice received intravenously via tail vein 20 µg of fluorescein-conjugated rat antimouse P-selectin (PharMingen,
San Diego, CA) and 60 µg of R-phycoerythrin-conjugated rat antimouse
E-selectin (PharMingen) in a total volume of 350 µL PBS. Control
animals received 80 µg of fluorescein-conjugated rat IgG (Sigma).
Three hours after ear injection, an additional 30 µg of
R-phycoerythrin-conjugated antimouse E-selectin or rat IgG (control)
was administered in 300 µL PBS. Doses of antibodies administered were
calculated based on target molecule saturating concentrations and
half-lives of these antibodies reported in a model of
thioglycollate-induced murine peritonitis.6 Six hours after
ear injection, mice were euthanized, the ears were harvested, and a
portion was embedded and frozen in Tissue-Tek O.C.T. Compound (Miles,
Elkhart, IN). Ten-micrometer sections were cut with a cryostat,
mounted, and examined for tissue fluorescence by epi-illumination. The
number of fluorescent microvascular elements in a total of six sections
(sectioned at least 30 µm apart and from 2 different ears) were
counted in six consecutive high power fields. Examination of undiluted
antibody solutions showed that there was no cross-detection of the two
fluorochromes.
Statistical analyses.
The data presented represent the arithmetic mean ± SEM, unless
otherwise noted. There was substantial variability between the
wild-type strains (ie, control strains for each selectin-deficient genotype) in the absolute values of zymosan-stimulated MPO activity at
4 hours (see Fig 4A). Therefore, at this time point, for the purpose of
comparison between groups, zymosan-stimulated MPO activity was also
expressed as the percentage relative to PBS-stimulated MPO activity in
the contralateral ear of the same animal. MPO values (absolute or
percentage of control) for each group of selectin-specific knock-out
mice at all time points were compared using the Mann-Whitney nonparametric ranking test. Other multigroup comparisons were performed
using analysis of variance with a post-hoc Scheffe' F test.
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| Fig 4.
Zymosan-induced neutrophil accumulation dependence on
selectin genotype at (A) 4 hours and (B) 8 hours after injection.
Tissue MPO content, measured by colorimetric enzymatic reaction and
quantitated as units of myeloperoxidase per ear, was determined for
each selectin-deficient genotype, and their genetically matched
wild-type control (normal expression of both E- and P-selectin) 4 hours
and 8 hours after intradermal injection of vehicle (PBS) or 400 µg
zymosan. See text for normalized data and statistical comparisons. The
bracketed number indicates the number of animals assessed per group.
*P  .05 for zymosan-injected E-/P-selectin-deficient mice
versus zymosan-injected genetically matched wild-type control and
versus zymosan-injected E-selectin-deficient or P-selectin-deficient
mice.
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RESULTS |
Time- and dose-dependency of zymosan-induced dermal inflammation in
wild-type mice.
PMN accumulation in response to intradermal injection of zymosan was
determined at 8 hours after administration to wild-type mice. Results
show a zymosan dose-dependent increase in accumulation of MPO
(Fig 1A) that increased to the maximum 800 µg dose tested. A dose of 400 µg (near-maximal PMN accumulation)
was used for all subsequent studies. Analysis of the time course of
tissue MPO showed that PMN accumulation increased from 2 hours to a
maximum at 12 hours and then decreased slightly by 18 hours (Fig 1B). In experiments using selectin-deficient mice, tissue MPO content was
determined both early (4 hours) and at a later time point (8 hours)
near maximal PMN accumulation.
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| Fig 1.
(A) Dose-dependent zymosan-induced dermal neutrophil
accumulation. Colorimetric determination of tissue myeloperoxidase
content (OD 450 nm) of mouse ears was determined 8 hours after
subcutaneous injection of the indicated amount of zymosan. n = 2 animals per group. (B) Time-dependent zymosan-induced dermal neutrophil
accumulation. Tissue myeloperoxidase content of mouse ears, measured as
described above and quantitated as units of myeloperoxidase per ear,
was determined at the indicated time after the intradermal injection of
400 µg zymosan. n = 4 animals per group.
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Selectin expression in zymosan inflammation.
In vivo immunofluorescence using intravenously administered
monospecific anti-selectin antibodies was used to detect selectin expression at the luminal surface of microvascular endothelium (Fig 2A and B). These studies demonstrated
both P- and E-selectin expression in vascular structures in ears
injected with either PBS or zymosan, with minimal expression in
unmanipulated ears. The number of fluorescent-associated vessels in
zymosan-injected ears was greater than in PBS-injected ears, which was,
in turn, greater than in unmanipulated ears. In animals that received
control fluorescein-labeled normal IgG, no significant binding to ear vasculature was identified. The fluorescence associated with the anti-P-selectin antibody (Fig 2B) was focally granular in nature, possibly representing internalized P-selectin being recycled into storage granules, as demonstrated by Subramaniam et al.26
Figure 3 is a quantitative comparison of
the number of microvessels expressing P-selectin in each of the
treatment groups. Qualitatively similar results were observed for
E-selectin. Quantitation of E-selectin expression was not performed,
because we used different fluorochromes for the active and control
antibodies, and significant fluorochrome quenching was encountered.
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| Fig 2.
Immunofluorescence photomicrographs of vascular selectin
expression in zymosan-injected ears in wild-type mice. Tissue was
prepared for immunofluorescent microscopy 6 hours after subcutaneous
zymosan injection and intravenous administration of
R-phycoerythrin-conjugated anti-E-selectin antibody and
fluorescein-conjugated anti-P-selectin antibody. Photomicrographs
(original magnification × 400) were taken adjacent to a subcutaneous
zymosan deposit and represent the fluorescence associated with a single
tubular, branching vascular structure as viewed with appropriate
filters for (A) R-phycoerythrin (anti-E-selectin) or (B) fluorescein
(anti-P-selectin).
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| Fig 3.
Vascular P-selectin expression. The number of
microvascular structures associated with green
(fluorescein-anti-P-selectin) fluorescence was determined in tissue
sections (n = 6 per group) of ears from wild-type mice receiving
intravenous fluorescein-conjugated control IgG (control Ab) or
fluorescein-conjugated anti-P-selectin antibody (selectin Ab). The
ears were uninjected, injected with vehicle (phosphate-buffered saline
[PBS]), or injected with 400 µg zymosan. *P = .05 versus
all other groups.
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Zymosan-induced dermal inflammation in selectin-deficient mice.
Zymosan-induced ear inflammation was assessed in P-selectin,
E-selectin, and E-/P-selectin double-deficient mice as well as in
control animals for each strain with normal selectin expression. PMN
accumulation in PBS or zymosan-injected ears was determined by
measuring tissue MPO content at 4 and 8 hours after injection. The MPO
content of ears injected with PBS was similar for all genotypes and
time points and was similar to that measured in other strains of mice
(data not shown). Therefore, at each time point, the MPO contents of
PBS-injected ears from all groups were pooled for the purpose of data
presentation (Fig 4A and B).
Four hours after injection (Fig 4A), zymosan-stimulated MPO activity
(v genetically matched wild-type controls) was slightly decreased in P-selectin-deficient mice (6.7 ± 0.6 v
9.9 ± 2.0 U/ear) and slightly increased in
E-selectin-deficient mice (10.6 ± 2.1 v 6.8 ± 0.4 U/ear), but greatly decreased in E-/P-selectin double-deficient mice
(3.5 ± 0.5 v 8.6 ± 1.3 U/ear) to an amount near that
measured in PBS-injected ears. A multigroup comparison using analysis
of variance with a post-hoc Scheffe' F test confirmed that the only
significant reduction in MPO (v matched wild-type genetic
control) was associated with the E-/P-selectin double-deficient mice.
However, there was significant variation in absolute MPO values among
the three zymosan-injected wild-type genetic control groups. Therefore,
to facilitate intergroup comparison, the zymosan-stimulated MPO
activity in all animals was also expressed as a percentage of the
PBS-stimulated MPO activity in the contralateral ear of the same
animal. Normalized values were then compared between each knockout and
its genetically matched wild-type control group (Mann-Whitney test). A
subtle but not statistically significant attenuation of
zymosan-stimulated MPO activity was detected in the
P-selectin-deficient mice (267% ± 39% v 368% ± 42%
in wild-type genetic controls; P = .055). In
E-selectin-deficient mice, zymosan-stimulated MPO activity was similar
to that in matched wild-type genetic controls (355% ± 36%
v 295% ± 72%, respectively; P = .15). However, in
the E-/P-selectin double-deficient mice, the zymosan-stimulated MPO
activity was only 137% ± 7% of PBS-stimulated activity (v 335% ± 57% in wild-type genetic controls; P = .004).
Eight hours after injection (Fig 4B), the range of absolute MPO values
among the three wild-type genetic control groups was small, abrogating
the necessity for normalizing the data; the zymosan-stimulated MPO
activity in the three wild-type genetic control groups ranged from 3.8 to 4.5 times the PBS-stimulated activity. Zymosan-stimulated MPO
activity did not vary between the E- or P-selectin-deficient mice and
their matched wild-type genetic controls (E-selectin knockout, 16.5 ± 1.3 U/ear v E-selectin wild-type, 15.0 ± 1.2 U/ear,
P = .273; P-selectin knockout, 15.8 ±1.1 U/ear v
P-selectin wild-type, 14.9 ± 1.2 U/ear, P = .465). In
contrast, the zymosan-stimulated MPO activity in
E-/P-selectin-deficient mice was 6.9 ± 0.6 U/ear (v 17.8 ± 1.6 U/ear in wild-type genetic controls, P = .004), only
1.7 times the PBS-stimulated activity. The zymosan-stimulated MPO
activity in E-/P-selectin-deficient mice was also significantly
reduced when compared with that in E-selectin-deficient mice
(P = .003) and P-selectin-deficient mice (P = .003).
Similar results were also obtained at 15 hours after injection (data
not shown).
Histology of zymosan-induced dermal inflammation.
Photomicrographs of hematoxylin and eosin-stained sections of ears 8 hours after zymosan injection, in wild-type and E-/P-selectin double-deficient mice, are shown in Fig 5.
The ear is composed of a strip of cartilage covered on each side with
loose connective tissue and skin. Bundles of skeletal muscle are
present within the connective tissue on the dorsal aspect of the ear.
PBS-injected ears were characterized by disruption of the loose
subcutaneous connective tissue. However, no significant inflammatory
infiltrate or edema fluid was observed. Zymosan-injected ears from both
wild-type (Fig 5A) and selectin-deficient mice (Fig 5B) are
characterized by deposits of zymosan particles within edematous tissue.
The deposits were rimmed and effectively walled off by a layer of phagocytic cells, which are predominately PMNs. These cells had phagocytized zymosan particles that pushed their nucleus to the outer
region of cytoplasm, distorting their morphology. The tissue peripheral
to these micro-abscesses contained an inflammatory infiltrate of
varying intensity composed primarily of PMNs, with occasional
mononuclear cells, mast cells, and eosinophils in similar proportions
among genotypes. Blood vessels were readily identified that contained
marginated PMNs (Fig 5C). Qualitatively, the inflammatory response was similar between wild-type and selectin-deficient mice.
Figure 5B shows that the PMNs that were able to complete vascular
transmigration into the interstitial tissue in the
selectin-deficient mice were fully capable of migrating through the
tissue to the inflammatory focus, phagocytizing the zymosan
particles, and participating in walling off of the inflammatory
material. The inflammatory infiltrate was less intense overall in the
E-/P-selectin double-deficient mice, but there was spatial variability
in the intensity of the infiltrate within tissue sections, and
morphologic assessment of differences between genotypes was more
accurately determined by measuring whole tissue MPO content.
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| Fig 5.
Photomicrographs of zymosan-injected tissue in wild-type
and E- and P-selectin-deficient mice. Ear tissue was removed 8 hours
after zymosan injection and sections were stained with hematoxylin and
eosin. (A) Wild-type mouse (original magnification × 400).
Micro-abscess consisting of aggregates of zymosan (Z) surrounded and
walled off by a rim of leukocytes (between arrowheads). The leukocytes
are predominantly neutrophils, many of which have distorted morphology
due to having phagocytized zymosan particles. Adjacent connective
tissue also contains a neutrophilic infiltrate. (B) E- and
P-selectin-deficient mouse (original magnification × 400). General
features are similar to those described in (A). However, the overall
intensity of the leukocyte infiltrate is markedly reduced in comparison
with the wild-type mouse tissue. (C) Higher magnification of inset from
(A) (original magnification × 1,000). Neutrophils (arrows) adherent
to endothelial cells lining a venule in the vicinity of a
micro-abscess.
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DISCUSSION |
Previous studies have indicated that leukocyte adhesion to
microvascular endothelium and accumulation of PMNs at a site of inflammation depend on expression of selectin adhesion
molecules.1,2 Most studies comparing the function of the
endothelial selectins in these processes suggest discrete functional
differences for E- and P-selectin. We expected that zymosan-induced
acute dermal inflammation would result in endothelial expression of E-
and P-selectin and that their function in PMN accumulation would
resemble those previously demonstrated.
Immunofluorescent detection of selectins at the luminal endothelium of
dermal micro-vessels demonstrated both E- and P-selectin expression,
with a basal level of P-selectin detected in unmanipulated ears.
Expression increased after treatment with PBS or zymosan. Because only
selectin expression at the endothelial luminal surface is relevant to
leukocyte-endothelial adhesion, intravascular administration of
fluorochrome-labeled antibodies was used to identify surface-expressed selectins. The anti-selectin antibodies were administered
simultaneously with initiation of dermal inflammation to ensure that
they would be present in vivo for the entire experimental protocol. The
vessel-associated fluorescence detected at the end of the 6-hour
experiment demonstrates that the selectins were expressed at the
endothelial cell luminal surface at some time during the treatment
period. However, the experiment as performed could not indicate the
time course of expression of E- and P-selectin during the experiment;
both cell surface as well as subsequently internalized
antibody-selectin complexes26 would have been detected with
the technique used.
The source of the basal levels of luminal P-selectin detected is
unclear. Possibilities include low-level constitutive P-selectin expression resulting from trafficking of the molecule after
biosynthesis through the plasma membrane en route to Weibel-Palade
bodies or a basal level of constitutive Weibel-Palade body fusion with
the plasma membrane. The moderate increase in P-selectin expression after PBS injection shows that the minor inflammatory reaction caused
by raising a subcutaneous blister of PBS is sufficient to cause
Weibel-Palade body degranulation. Similar results with P-selectin
expression have been obtained in in vivo models of leukocyte rolling,
in which P-selectin-dependent rolling has been demonstrated merely
after exteriorization of a vascular bed in the absence of other
inflammatory stimuli.5,27,28 Increased P-selectin detection
after PBS or zymosan injection might also in part be due to activated
platelets adherent to activated endothelium. This platelet-expressed
P-selectin can mediate lymphocyte delivery to high endothelial
venules,29 but does not mediate PMN delivery to sites of
inflammation.30 Although PBS and zymosan injection both
resulted in increased selectin expression, marked increases in MPO
content were only seen in zymosan-injected ears. These findings
indicate that PMN extravasation depends on additional inflammatory
mediators formed only in the presence of zymosan. The results
demonstrate that selectin expression is necessary, but not sufficient,
for acute dermal inflammation.
The MPO data demonstrate a clear dependence on either E- or P-selectin
expression for PMN accumulation in acute dermal inflammation. At 4 and
8 hours after injection, PMN accumulation is largely prevented in mice
deficient in both E- and P-selectin. However, whereas selectin
dependence is evident at all time points, the precise function of E- or
P-selectin varies among the early (4 hours) and late (8 hours) time
points. At 4 hours, the overlap in E- and P-selectin function is not
complete. The 4-hour data suggest that P-selectin plays a greater role
in PMN accumulation at this early time point. This is consistent with
P-selectin being the predominant selectin expressed early after
endothelial cell activation, because it is preformed and stored in
Weibel-Palade bodies, requiring only translocation to the cell surface
for expression, whereas E-selectin expression requires de novo
synthesis after cell activation.31,32 Several studies have
demonstrated a particular dependence on P-selectin for PMN accumulation
early in an inflammatory response.5,7
At 8 hours after injection, either E- or P-selectin alone can fully
support PMN accumulation. Mice deficient in either E- or P-selectin
have PMN accumulation comparable to wild-type controls, and only
E-/P-selectin double deletion significantly blocks PMN accumulation.
Thus, there is overlap in E- and P-selectin function, with respect to
PMN accumulation, in acute dermal inflammation. It is possible that
discrete functions for each selectin may relate to accumulation of
inflammatory cells other than PMNs that may express only ligands for
one or the other selectin. Alternatively, inflammatory stimuli other
than zymosan may be associated with expression of only one of the
selectins. Doerschuk et al33 have demonstrated that
inflammatory mechanisms, including adhesion molecule expression, vary
among different organs and with different inflammogens. Thus, organ to
organ differences may account for the varied functional roles of the
selectins observed in this study.
The mechanism of PMN recruitment that accounts for the low level of
residual zymosan-stimulated MPO activity (above PBS-stimulated MPO
activity) in the E-/P-selectin double-deficient mice is unclear. The
relation between rolling flux or velocity determinations and extravascular PMN accumulation over time is unknown. In addition, although the number of rolling or adherent leukocytes in inflamed (exteriorized or TNF-treated) mesenteric or cremasteric venules of
E-/P-selectin double-deficient mice is extremely low, small numbers of
leukocytes can nevertheless extravasate over time in response to a
bacterial or thioglycollate-induced peritonitis in these
animals.4,7 Possible residual mechanisms of leukocyte rolling include an L-selectin-mediated interaction with a putative inducible ligand on nonlymphoid venular endothelium.34-36
The overlapping functions of E- and P-selectin in mediating PMN
recruitment to sites of acute dermal inflammation were compatible with
some prior studies. In a dermal delayed-type contact hypersensitivity (DTH) model in mice, tissue edema and PMN accumulation were decreased in E-selectin-deficient mice treated with anti-P-selectin antibody, but not in untreated E-selectin-deficient mice or in P-selectin antibody-treated wild-type mice (ie, functional loss of both E- and
P-selectin was required to decrease PMN accumulation), suggesting an
overlap of E- and P-selectin functions.3 However, these results differ from DTH studies in P-selectin-deficient mice, in which
mononuclear cell and PMN accumulation was decreased, and this was
attributed solely to the absence of only P-selectin.37 However, extrapolation of results from DTH to the zymosan model may not
be appropriate because of potential differences in mechanisms responsible for leukocyte accumulation.
This and other studies have determined the selectin-dependent nature of
an array of inflammatory responses. Although differences in the roles
of each individual selectin exist among models, organs, and disease
processes, the significance of the selectins in mediating these
responses is clear. Manipulation of the interaction between selectins
and their ligands will likely be important in modulating inflammatory
processes such as dermal inflammation.
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FOOTNOTES |
Submitted February 26, 1998;
accepted May 18, 1998.
Supported by National Institutes of Health Grants No. HL41484 (R.O.H.),
HL53756 (D.D.W.), AI33189, CA71932 (J.B.L.), AI33189 (R.M.M.); the
Howard Hughes Medical Institute (J.B.L. and R.O.H.); and the Pew
Scholars Program (R.M.M.).
Address reprint requests to Rory M. Marks, MD, Department of Internal
Medicine, 5520 MSRB I, The University of Michigan Medical Center, Ann
Arbor, MI 48109-0680; e-mail: rmarks{at}umich.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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ACKNOWLEDGMENT |
The authors gratefully acknowledge Faye Silverstein and John Barks for
help with the statistical analyses and Mollie Ullman-Cullere for animal
care and transport.
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Abstract
Introduction
Abstract