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1 Department of Biomedical Engineering, University of Virginia School of Medicine, Charlottesville, Virginia 22908; and 2 Department of Comparative Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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It has not been determined whether
L-selectin-mediated rolling can promote leukocyte adhesion in vivo
independent of P- and E-selectin. We used intravital microscopy of E-
and P-selectin double-mutant mice (E
/P) stimulated with
tumor necrosis factor-
for 6-8 h to investigate the importance
of L-selectin-dependent rolling in cremaster muscle venules. Rolling
leukocyte flux in E/P mice was 9 ± 2 cells/min compared with 77 ± 17 cells/min in wild-type (WT) mice.
Pretreatment with the L-selectin monoclonal antibody
MEL-14 significantly reduced rolling in both
E/P (by 89%) and WT mice (by 79%). L-selectin-dependent
rolling in E/P mice resulted in leukocyte adhesion
comparable to that seen in WT mice. MEL-14 pretreatment of
E/P mice reduced leukocyte adhesion by 50%. The majority
(~80%) of intravascular leukocytes in both WT and E/P
mice were neutrophils. We conclude that L-selectin can mediate rolling
that results in sufficient leukocyte recruitment to account for the
robust inflammatory response seen in E/P mice at later
times.
inflammation; leukocyte adhesion; knockout mice; intravital microscopy
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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LEUKOCYTE-ENDOTHELIAL CELL interactions mediated by
several classes of adhesion molecules, including selectins,
immunoglobulin-like molecules, and integrins, are critical for the
inflammatory response. The selectins are specialized to mediate initial
leukocyte capture and rolling under flow conditions (34).
L-selectin is constitutively expressed on circulating
leukocytes and is shed after activation. E-selectin and
P-selectin are expressed on cytokine-activated endothelium.
P-selectin is also stored in Weibel-Palade bodies of
endothelial cells and -granules of platelets, allowing rapid surface
expression after stimulation by secretagogues like histamine or
thrombin. The importance of the selectins for a normal inflammatory response in humans is most dramatically illustrated by a rare defect in
fucose metabolism termed leukocyte adhesion deficiency type II. These
patients suffer from infections and are unable to recruit adequate
numbers of leukocytes (8) because the carbohydrate groups necessary for
selectin binding cannot be synthesized.
Recently, gene-targeted mice lacking P-selectin (7, 26), L-selectin (2,
36), E-selectin (6, 20), and a combination of E- and P-selectin
(E/P) (6, 10) have been produced by gene targeting and
homologous recombination. In L-selectin-deficient mice, neutrophil
recruitment into peritonitis 4, 24, and 48 h after challenge with
thioglycollate is reduced but not eliminated (2, 33). In addition, both
trauma-induced (22) and tumor necrosis factor (TNF)--induced
leukocyte rolling (19) are impaired in these mice. P-selectin-deficient
mice show reduced neutrophil recruitment into thioglycollate- or
Streptococcus pneumoniae-induced peritonitis at early time points (7, 26) but a normal level of
neutrophil recruitment at later time points (24-48 h).
Concomitantly, leukocyte rolling is absent immediately after tissue
exteriorization in cremaster muscle venules (22) and mesenteric venules
(26), but significant rolling is seen after treatment with TNF-
(22). E-selectin-deficient mice show normal neutrophil
recruitment to thioglycollate-induced peritonitis (20) and an elevated
level of leukocyte rolling at increased velocity in TNF--stimulated cremaster muscle venules (19). E-selectin function appears to overlap
with P-selectin because monoclonal antibodies (mAb) to P-selectin in
E-selectin-deficient mice produce a severe defect in neutrophil
recruitment into the peritoneal cavity after 6 h (20). Similarly, mAb
to P-selectin in E-selectin-deficient mice (19) or mAb to E-selectin in
P-selectin-deficient mice (18) almost completely block leukocyte
rolling in cremaster venules treated with TNF- for 3 h. These
results are consistent with impaired leukocyte rolling and recruitment
seen in mice deficient in both E- and P-selectin (6, 10). These mice
develop a phenotype of leukocyte adhesion deficiency syndrome and
exhibit drastically elevated leukocyte counts and serum immunoglobulin
(Ig) G levels. In E/P mice, neutrophil accumulation in
response to S. pneumoniae peritonitis
is completely absent at 4 h but not reduced at 24 h (6). The
discrepancy observed between leukocyte recruitment at early and later
time points suggested that E- and P-selectin-independent adhesion
mechanisms may mediate leukocyte rolling and adhesion at later time
points during inflammation. L-selectin, the only selectin present in
the E/P mice, is a primary candidate that might be
responsible for neutrophil recruitment at later time points. Hence,
this study was conducted to investigate the role of
L-selectin in leukocyte rolling in venules of the cremaster muscle of E/P mice.
L-selectin-dependent rolling in vivo has previously been shown using
transfected cells (23, 35), but purely L-selectin-dependent rolling of
endogenous neutrophils in vivo has never directly been demonstrated.
Reduced leukocyte rolling in L-selectin-deficient mice suggests that
L-selectin function is necessary for normal rolling (2, 22), but under
the conditions tested previously (no intentional stimulation or 3-h
TNF- treatment), all leukocyte rolling was eliminated when both E-
and P-selectin were blocked (18, 19) or absent (6). Although venular
endothelium in vivo expresses L-selectin ligand activity as seen by
intravital microscopy without intentional stimulation (23, 35), several reports indicate that optimal L-selectin ligand activity is expressed on inflamed endothelium at later times after cytokine stimulation. L-selectin-dependent adhesion was found to be maximal on human umbilical vein endothelial cells treated with TNF-,
interleukin-1
, or lipopolysaccharide for 6-24 h (30) and on
microvascular endothelial cells treated with TNF- for 24-48 h
(5), and optimal L-selectin-dependent rolling and adhesion under flow
occurs 6-24 h after TNF- treatment of human microvascular
endothelial cells (37). Therefore, we hypothesized that long-term
TNF- treatment of mice may also induce expression of more or better
L-selectin ligands on venular endothelium in vivo. We reasoned that the
more robust L-selectin ligand activity induced by prolonged cytokine
treatment may facilitate the detection of L-selectin-dependent
neutrophil rolling. In this study, we show that 6-8 h after
TNF- stimulation neutrophils roll in cremaster venules of
E/P mice, and this rolling appears to be sufficient to
produce levels of leukocyte adhesion comparable to those seen in
wild-type mice under the same conditions. This observation correlates
with and suggests a mechanism for the substantial neutrophil recruitment seen in E/P mice at late time points.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Animals. Male E-selectin and P-selectin double-null mutant mice (6) and L-selectin null mutant mice (2) were obtained from established colonies derived from gene-targeted founders. The mutant mice were backcrossed into a C57BL/6 background for at least five generations. Control mice were of matching age and strain. All mice were free of overt cutaneous infection. All experiments were conducted at the University of Virginia under a protocol approved by the institutional animal care and use committee.
Materials. A hybridoma cell line
producing a mAb against murine L-selectin, MEL-14 (rat IgG2a) (12), was
obtained from American Type Culture Collection (Rockville, MD), and the
mAb was purified from hybridoma supernatant by affinity chromatography
on a protein G column. Hybridomas were grown in endotoxin-free
Iscove's modified Dulbecco's medium (GIBCO, Grand Island, NY) with
10% fetal calf serum with low endotoxin (<2 endotoxin units/ml by
Limulus amoebocyte assay). Recombinant murine TNF-
(Genzyme, Cambridge, MA) was injected at a dose of 500 ng/mouse in a
volume of 0.3 ml saline containing 30 U of heparin intrascrotally 6 h
before the beginning of the intravital microscopic experiment. In some
animals, MEL-14 (100 µg/mouse) was administered as an intraperitoneal
injection at the time of TNF- injection (MEL-14 pretreatment group).
Flow cytometry. Expression of L-selectin on mouse neutrophils obtained both from peripheral blood and bone marrow was determined by direct immunofluorescence. Briefly, mouse whole blood was obtained from a plastic catheter inserted into the carotid artery. Bone marrow was obtained by flushing both femurs and tibia with sterile phosphate-buffered saline (PBS). The whole blood or bone marrow was incubated with phycoerythrin (PE)-labeled mAb MEL-14 to L-selectin (Pharmingen, San Diego, CA, 0.5 µg/106 cells), followed by incubation with fluorescein isothiocyanate (FITC)-labeled mAb GR-1 (Pharmingen, 0.5 µg/106 cells) to identify granulocytes. After lysis of red blood cells (150 mM NH4Cl, 10 mM NaHCO3, 1 mM Na2EDTA in deionized, distilled water), the cells were analyzed for forward scatter, side scatter, FITC fluorescence, and PE fluorescence using a laser flow cytometer (Becton Dickinson, FACScan, San Jose, CA). Neutrophils were gated by expression of GR-1 antigen. Data are presented as fluorescence histograms of L-selectin expression of GR-1 positive cells on a four-decade log scale.
Intravital microscopy. For intravital microscopy, mice were anesthetized with an intraperitoneal injection of ketamine hydrochloride (Ketalar, Parke-Davis, 100 mg/kg, Morris Plains, NJ) after pretreatment with pentobarbital sodium (30 mg/kg ip, Nembutal, Abbott Laboratories, North Chicago, IL) and atropine (0.1 mg/kg ip, Elkins-Sinn, Cherry Hill, NJ). The trachea, one jugular vein, and one carotid artery were cannulated. Body temperature was kept at 37°C with a thermocontrolled heating pad. The mice received ~0.2 ml/h diluted pentobarbital in saline intravenously to maintain anesthesia and a neutral fluid balance. The cremaster muscle was prepared for intravital microscopy as described (19). The epididymis and testis were gently pinned to the side, exposing the well-perfused cremaster microcirculation. Time 0 was set at the beginning of the cremaster surgery. The cremaster muscle was superfused with thermocontrolled (36°C) bicarbonate-buffered saline.
Microscopic observations were made on a Zeiss intravital microscope
(Axioskop, Thornwood, NY) with a saline immersion objective (SW 40/0.75
NA). Each venule was observed for ~90 s. Venules with diameters
between 25 and 80 µm were observed, and video recordings were made
through a charge-coupled device camera system (Dage-MTI, model
VE-1000CD, Michigan City, IN) on a Panasonic S-VHS recorder. Microvascular center line red blood cell velocity was measured using a
dual photodiode and a digital on-line cross-correlation program (27).
Center line velocities were converted to mean blood flow velocities by
multiplying with an empirical factor of 0.625 (25). Wall shear rates
(
w) were estimated as
w = 2.12(8Vb /d),
where Vb is the
mean blood flow velocity, d is the diameter of the vessel, and 2.12 is a median empirical correction factor obtained from actual velocity profiles measured in microvessels in vivo (28). Microvessel diameters and rolling leukocyte
velocities were measured using a digital image-processing system (27). Each rolling leukocyte passing a line perpendicular to the vessel axis
was counted, and the leukocyte rolling flux was expressed as rolling
cells per minute. All rolling leukocytes measured were interacting with
the wall of endothelium, not with other leukocytes present in the
vessel. For determination of rolling velocities, rolling leukocytes
were followed for ~150 µm downstream, and velocity was calculated
by dividing this distance by the elapsed time period. Firmly adherent
cells were defined to be any leukocytes not moving for at least 30 s.
Adherent cells for each venular segment were counted and quantified as
number of cells per area of the venular segment, which was calculated
by assuming cylindrical geometry of the venular segment. Blood samples
(10 µl each) were taken throughout the experiment from the carotid
catheter at ~45-min intervals to analyze systemic leukocyte
concentrations. Differential leukocyte counts were obtained by Kimura
stain of the blood samples. Rolling leukocyte flux fraction was
determined by dividing the rolling flux by total leukocyte flux given
by the product of
Vb, venule
cross-sectional area
(d/2)2,
and systemic leukocyte count.
Histology. To obtain intravascular differential leukocyte counts, we fixed the entire cremaster muscle by dripping 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) onto the tissue. The cremaster was removed, mounted flat on a gelatinized glass slide, and air dried for 5-10 min, followed by fixation in 4% paraformaldehyde in 0.1 M phosphate buffer for 24 h at 4°C. After fixation, the tissue was washed three times in phosphate buffer with 5% ethanol, stained in Giemsa (Sigma, St. Louis, MO) at room temperature for 5-10 min, and differentiated in 0.01% acetic acid for contrast. The differentiated slides were sequentially washed in water, in 75, 95, and 100% ethanol, and in xylene, followed by mounting in Permount. Observations were made on a Zeiss microscope with a ×100, 1.4-NA oil immersion objective.
Statistics. Average leukocyte rolling flux fractions, rolling velocities, systemic leukocyte counts, and differentials between genotypes were compared using an ANOVA followed by a Student-Newman-Keuls multiple-comparison procedure. Statistical significance was set at P < 0.05.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Systemic leukocyte counts. The
systemic leukocyte counts in mice deficient in both E- and P-selectin
were found to be highly elevated (~9-fold increase) compared with the
leukocyte counts in wild-type mice (Table
1). After 6-8 h of TNF-, the
majority of circulating leukocytes in wild-type and E/P
mice were neutrophils (Table 1). The leukocyte counts after MEL-14
pretreatment at the time of TNF- injection were not significantly
different from mice without mAb pretreatment for both wild-type and
E/P mice (data not shown).
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Hemodynamics in mouse cremaster venules.
We observed leukocyte rolling and adhesion in a total of 161 venules in
five wild-type, four L-selectin null mutant, and eight E/P mice. The average diameter was 41 ± 1 µm,
the average center line blood flow velocity was 1.7 ± 0.1 mm/s, and the calculated mean wall shear rate was 472 ± 17 s
1. These values
were not significantly different between the investigated groups (Table
1).
Leukocyte rolling flux.
In venules of the cremaster muscle after 6-8 h of TNF-
stimulation, an average of 77 cells/min rolled in the wild-type mice (Fig. 1), more than in wild-type mice after
2-3 h of TNF- (37 cells/min) (19). The leukocyte rolling flux
was significantly lower in E/P mice (9 cells/min). This
residual rolling was almost completely inhibited (89% reduction) by
pretreatment with the L-selectin mAb MEL-14 at the time of TNF-
injection. A similar inhibition of leukocyte rolling flux was observed
when MEL-14 was injected acutely during intravital microscopy (67%
reduction). Isotype-matched control antibody had no effect on leukocyte
rolling (data not shown). These findings indicate that the majority of residual rolling seen in cremaster venules of E- and P-selectin double-mutant mice is L-selectin dependent.
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injection. The leukocyte rolling flux observed 6-8 h after
this treatment was reduced to 16 ± 2 cells/min, which is 79% below
the level seen in wild-type mice without MEL-14 pretreatment. A similar
low level of leukocyte rolling flux was found in L-selectin-deficient mice (14 ± 2 cells/min). Hence, eliminating L-selectin function by
gene targeting or by a blocking mAb each significantly impairs leukocyte rolling after 6-8 h of TNF- stimulation in vivo.
Because the leukocyte counts are much higher in the E/P
mice than in the wild-type or L-selectin-deficient mice, the leukocyte rolling flux seen in E/P mice at 6-8 h after TNF-
stimulation corresponds to a very small leukocyte rolling flux fraction
(<1%), defined as the number of rolling leukocytes divided by the
total number of leukocytes passing during the same time interval.
Therefore, the L-selectin-dependent leukocyte rolling in
E/P mice appears to be much less efficient than the
leukocyte rolling in the wild-type mice, because the number of rolling
cells represents a much smaller percentage of circulating leukocytes.
A complicating factor in these studies is that L-selectin can
be shed from the surface of neutrophils after stimulation with chemoattractants (16). At least one neutrophil chemoattractant, granulocyte colony-stimulating factor, has previously been shown to be
elevated in the circulation of E/P mice (10).
L-selectin expression in E/P mice has
previously been reported to be decreased on most granulocytes in one
study (6) and on 50% of the circulating granulocytes in the other
(10). We determined L-selectin expression in our mice and found only a
small percentage (7.6%) of peripheral blood neutrophils expressing
L-selectin at normal levels. The remaining majority of neutrophils
expressed L-selectin at 20-fold lower levels (Fig.
2). This was paralleled by a small fraction of rolling leukocytes in E/P mice as shown above. As
noted previously (6), the expression of L-selectin on bone
marrow-derived neutrophils from E/P mice was similar to
that found in wild-type mice, suggesting that reduced L-selectin
expression is due to shedding of L-selectin in peripheral blood
neutrophils.
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/P, or L-selectin-deficient
mice occurred between individual leukocytes and the
endothelium.
Leukocyte rolling velocity. Because
L-selectin-dependent leukocyte rolling after prolonged cytokine
stimulation was not investigated previously, we sought to more fully
characterize the properties of this type of rolling. Previous work has
shown that, at the site densities prevailing in venules in vivo,
E-selectin mediates the slowest rolling (<10 µm/s) (19), P-selectin
mediates rolling at intermediate velocities (20-50 µm/s) (15,
18, 19), and the L-selectin ligand activity expressed in mildly
activated endothelium after tissue trauma supports rolling only at
higher velocities (>100 µm/s) (15). The mean leukocyte rolling
velocity observed after 6-8 h of TNF- stimulation (12 µm/s)
was less than trauma-induced leukocyte rolling velocity (41 µm/s)
(15) but higher than leukocyte rolling velocity after 2-3 h of
TNF- stimulation (5 µm/s) (19). The present data show that
L-selectin-dependent rolling in E/P mice receiving
prolonged cytokine stimulation proceeds at velocities only slightly
higher than those seen in wild-type mice (19 vs. 12 µm/s,
respectively, P < 0.05, Fig.
3). This finding suggests that 6-8 h
after TNF- treatment, L-selectin ligands are expressed on the
endothelium of venules that can support rolling at much lower
velocities than those seen in L-selectin-dependent rolling without
TNF- treatment (15), although not quite at the low velocities
typical of E-selectin-mediated leukocyte rolling (19). The remaining
difference in rolling velocities is further emphasized by experiments
in L-selectin null mice (Fig. 3C),
which show lower rolling velocities than E/P mice under
the same conditions (8 vs. 19 µm/s,
P < 0.05). Treating
E/P mice with a function-blocking L-selectin mAb (MEL-14)
leaves very few rolling leukocytes, which roll at slightly reduced
velocities (Fig. 3D, 15 ± 1 µm/s, P < 0.05 vs. Fig.
3B and vs. Fig.
3C).
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/P mice
is sufficient to promote significant adhesion of neutrophils in venules
of the cremaster muscle. In contrast to impaired leukocyte rolling
observed in E/P mice, the number of adherent leukocytes
was similar in wild-type and E/P mice (Fig.
4). Furthermore, pretreatment of wild-type
mice with MEL-14 did not significantly alter the level of leukocyte adherence in cremaster muscle venules. Similarly, mice deficient in
L-selectin showed the same level of leukocyte adherence in cremaster
muscle venules as wild-type mice (Fig. 4). These data suggest that
L-selectin is not required for a normal level of leukocyte adhesion in
response to TNF- when both E- and P-selectin are present. However,
when E/P mice were pretreated with MEL-14, leukocyte
adhesion was significantly reduced (by 50%), indicating that
L-selectin becomes critical for neutrophil adhesion in the simultaneous
absence of both E- and P-selectin. Acute treatment with MEL-14 had no
effect on the number of adherent leukocytes in E/P mice
(data not shown), suggesting that the majority of adherent leukocytes
accumulates during the course of inflammation induced by TNF- given
6 h before the preparation for intravital microscopy.
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stimulation (Fig. 5A
and Table 2). Similarly, neutrophils make
up the majority of leukocytes present within venules of
E/P mouse cremaster muscles (Fig.
5B and Table 2). These data show that
most cells being recruited in both wild-type and E/P mice
are neutrophils. In E/P mice, we saw significant numbers
of eosinophils inside the cremaster venules, which were much less
frequent in wild-type mice. Eosinophils can use selectin-independent
mechanisms for rolling, in particular
41-integrin
(31). The apparent increase of the prevalence of eosinophils in venules
of E/P mice may be caused by normal eosinophil rolling in
the presence of a smaller number of rolling neutrophils. In addition,
the number of circulating eosinophils is increased approximately
sixfold in E/P compared with wild-type mice (10).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Our data show that L-selectin mediates leukocyte rolling, leading to
significant adhesion in E/P mice receiving prolonged cytokine treatment. The residual leukocyte rolling in E/P
mice amounted to ~12% of the level seen in wild-type mice under the same conditions. The leukocyte rolling seen in E/P mice
corresponds to a very low (<1%) rolling leukocyte fraction because
the systemic neutrophil counts in these mice are very high. Thus only a
small fraction of neutrophils is recruited from a very large pool of circulating leukocytes in E/P mice, suggesting that
leukocyte rolling is relatively inefficient in
E/P compared with wild type mice. However,
the elevated circulating counts appear to compensate for the
inefficiency of the rolling process, leading to near-normal levels of
neutrophils becoming firmly adherent under physiological conditions.
The low fraction of rolling neutrophils is paralleled by a small number
of circulating neutrophils expressing L-selectin in these mice (6, 10).
In wild-type mice, all neutrophils express L-selectin (21). The reason
for deficient L-selectin expression in E/P mice is not
known but is thought to be secondary to inflammatory neutrophil
activators inducing L-selectin shedding (6).
Our functional data show that L-selectin ligand(s) are present in
venules of the mouse cremaster muscle after 6-8 h of TNF- stimulation, which appear to be responsible for mediating the majority
of leukocyte rolling observed in E/P mice and a
substantial portion of rolling in wild-type mice. Because these
L-selectin ligand(s) on inflamed venules have not been identified at
the molecular level, it is currently not possible to directly measure the expression of L-selectin ligand(s) in vivo. Previous in
vivo studies have shown that functional L-selectin ligand activity is
expressed in venules even without TNF- stimulation (23, 24, 35), but
the rolling velocity data suggest that the L-selectin ligand activity
induced in mouse cremaster venules by prolonged cytokine treatment may
be enhanced or qualitatively distinct from that expressed in mildly
stimulated endothelium (15). Because rolling is not seen in venules of
untreated E/P mice or E/P mice treated with
TNF- for only 2-3 h (6), it is tempting to speculate that the
low levels of L-selectin expression in these mice may be responsible
for the inability of leukocytes to roll on more moderately stimulated
endothelium.
Because a defect in leukocyte rolling was seen in L-selectin-deficient
mice both with and without intentional stimulation (2, 22) and after
short-term TNF- treatment (19), the endothelial L-selectin ligands
expressed after mild stimulation may require normal expression of
L-selectin on circulating leukocytes for function, whereas the
L-selectin ligands induced by TNF- treatment for 8 h appear to be
able to function at the lower levels of L-selectin seen in
E/P mice. This conclusion is also supported by the
finding that antibody blockade of both E- and P-selectin in wild-type
mice (19) in which L-selectin expression is normal produces a
substantial but incomplete inhibition of leukocyte rolling after
2-3 h of TNF-, whereas rolling is completely absent in
E/P mice in which L-selectin expression is reduced (6). In addition, shedding of L-selectin in E/P mice is likely
to cause elevated levels of soluble L-selectin, which may partially inhibit L-selectin-dependent interactions (29). Previous studies have
shown that circulating L-selectin in mice treated with complete Freund's adjuvant is elevated three- to fourfold at 6-12 h (32).
After 6-8 h of TNF-, the slowest rolling was seen in
L-selectin-deficient mice (rolling dominated by
E-selectin), followed by wild-type mice (rolling through all
3 selectins), and the highest rolling velocity was found in
E/P mice (rolling mainly through L-selectin). Interestingly, the few leukocytes that continued to roll after blocking L-selectin function in E/P mice
through a selectin-independent pathway did so at velocities lower than those seen in E/P mice. This residual rolling seen even
after blocking L-selectin in E/P mice suggests that a
selectin-independent mechanism, e.g., through
4-integrin (13), can mediate
some rolling. Nevertheless, our data show that L-selectin function is
necessary for efficient rolling in both wild-type and E/P mice.
Although MEL-14 pretreatment in wild-type mice did not affect the level
of adherent leukocytes, E/P mice showed significantly reduced leukocyte adherence after MEL-14 pretreatment. This indicates that L-selectin is critical for leukocyte recruitment when
E- and P-selectin are not available. Our observation may
explain an earlier finding showing that both P- and
L-selectin must be blocked to substantially inhibit neutrophil
migration into the inflamed peritoneal cavity (4). Of note, our
findings correlate with the pattern of inflammatory defects seen in
E/P mice, in which inflammation is severely impaired at
early time points but normalizes at later times (6).
As was the case for rolling, the inhibition of neutrophil adhesion in
E/P mice by pretreatment with L-selectin mAb was
substantial but not complete. Even after pretreatment with L-selectin
antibody, we still observed a significant number of adherent cells.
Selectin-independent mechanisms of leukocyte recruitment under shear
flow have been described and may involve
2-integrins (11) and
1-integrins (13, 31). These
mechanisms may account for the remaining neutrophil adhesion. In
addition, novel selectin-like adhesion molecules may be involved in
binding neutrophils to long-term cytokine-treated endothelial cells
(14).
Taken together, this study shows that during cytokine-induced
inflammation adhesion mechanisms independent of endothelial E- and
P-selectins mediate leukocyte rolling and adhesion in mouse cremaster
muscle venules. L-selectin is responsible for the majority of residual
leukocyte rolling and is necessary for near-normal neutrophil
accumulation in E/P mice at later time points. The direct
demonstration of a significant role of L-selectin for leukocyte rolling
and adhesion after prolonged cytokine treatment helps explain the
inflammatory phenotype seen in various selectin-deficient mice and
implies L-selectin as a key inflammatory adhesion molecule in health
and disease.
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ACKNOWLEDGEMENTS |
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We thank Dr. T. F. Tedder, Duke University, for providing the L-selectin-deficient mice and William Ross for assistance with flow cytometry. This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grant R-01 HL-54136 to K. Ley. U. Jung is supported by a predoctoral fellowship (NHLBI Training Grant T-32 HL-07284 to B. R. Duling). C. L. Ramos is supported by NHLBI National Research Service Award postdoctoral fellowship HL09578-02.
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FOOTNOTES |
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Address for reprint requests: K. Ley, Univ. of Virginia School of Medicine, Dept. of Biomedical Engineering, Health Sciences Center, Box 377, Charlottesville, VA 22908.
Received 24 September 1997; accepted in final form 28 January 1998.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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