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Department of Biomedical Engineering, University of Virginia School of Medicine, Charlottesville, Virginia 22908
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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To study
selectin-independent leukocyte recruitment and the role of
intercellular adhesion molecule-1 (ICAM-1), we generated mice lacking
all three selectins and ICAM-1 (E/P/L/I
/) by bone marrow
transplantation. These mice were viable and appeared healthy under
vivarium conditions, although they showed a 97% reduction in leukocyte
rolling, a 63% reduction in leukocyte firm adhesion, and a 99%
reduction of neutrophil recruitment in a thioglycollate-induced model
of peritonitis at 4 and 24 h. Mononuclear cell recruitment was
almost unaffected. All residual leukocyte rolling and most leukocyte
adhesion in these mice depended on 
4-integrins, but a
small number of leukocytes (6% of wild-type control) still became adherent in the absence of all known rolling mechanisms (E-, P-, L-selectin and 4-integrins). A striking similarity of
leukocyte adhesion efficiency in E/P/L/ and E/P/I/ mice
suggests a pathway in which leukocyte rolling through L-selectin
requires ICAM-1 for adhesion and recruitment. Comparison of our data
with mice lacking individual or other combinations of adhesion
molecules reveal that elimination of more adhesion molecules further
reduces leukocyte recruitment but the effect is less than additive.
neutrophil adhesion; thioglycollate-induced peritonitis; intravital
microscopy; knockout mice; 4-integrins
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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EFFICIENT LEUKOCYTE
RECRUITMENT to sites of inflammation requires the selectin family
of adhesion molecules. E-, P-, and L-selectin mediate leukocyte capture
and rolling on the inflamed vessel wall (4, 12). The
generation of mice lacking one, two, or three selectins has provided
valuable information in the overlapping and unique functions of each
selectin in efficient leukocyte recruitment in inflammation (1,
2, 7, 9, 18, 22, 27). E- and P-selectin double knockout mice
(E/P/) treated with an L-selectin-blocking antibody or mice made
deficient in E-, P-, and L-selectin by transplanting L/ bone marrow
into E/P/ mice showed drastically reduced leukocyte rolling
(9, 11). Residual rolling in selectin-deficient mice was
blocked with an 4-integrin monoclonal antibody (MAb)
(9). A similar role of 4-integrins
in leukocyte recruitment was also shown in a model of allergic
inflammation (13). Therefore, selectins mediate the
majority of leukocyte capture and rolling, and
4-integrins mediate the capture and rolling of a small
fraction of circulating leukocytes.
Selectin-mediated leukocyte capture and rolling has been a generally accepted prerequisite for firm leukocyte adhesion and subsequent transmigration (4, 12). However, despite very few rolling cells in selectin-deficient mice (9, 11), a significant amount of leukocyte adhesion remained. Kubes et al. (15) estimated that rolling must be reduced by >90% to significantly affect firm adhesion.
Firm leukocyte adhesion is mediated primarily through 
2
(CD18)-integrins. 2-Integrins are a family of four
heterodimers, CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), CD11c/CD18
(p150,95), and CD11d/CD18. LFA-1 is the predominant
2-integrin on lymphocytes and neutrophils
(14). Neutrophils also express Mac-1 (30). Both LFA-1 and Mac-1 can interact with intercelular cell adhesion-1 (ICAM-1) expressed on resting (8) and inflamed (5,
6, 8, 21, 29) endothelial cells.
ICAM-1 was recently shown to be important in stabilizing P- and
L-selectin-mediated leukocyte rolling. Mice deficient in P-selectin and
ICAM-1 did not display leukocyte rolling after trauma-induced inflammation (16), had significantly reduced leukocyte
rolling and increased rolling velocities after tumor necrosis
factor- (TNF-) treatment compared with ICAM-1/ and wild-type
mice (16), and showed almost a complete absence of
neutrophil recruitment in a peritonitis model (3).
L-selectin/ICAM-1 double-mutant mice also showed increased rolling
velocities and decreased neutrophil recruitment in various experimental
models of inflammation (31, 32). These data suggest ICAM-1
is synergistic with P- and L-selectin in mediating efficient leukocyte recruitment.
To address the possible role of ICAM-1 in firm leukocyte adhesion and
recruitment in selectin-deficient mice and to further investigate
selectin-independent mechanisms of leukocyte rolling, firm adhesion,
and recruitment, we generated novel mice deficient in four adhesion
molecules (E-, P-, and L-selectin and ICAM-1). We tested the impact of
the absence of these adhesion molecules in two models of inflammation,
a TNF--induced model of inflammation of the mouse cremaster muscle
and thioglycollate-induced peritonitis.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals.
L-selectin null (L/) (1) and E- and P-selectin and
ICAM-1 triple null (E/P/I/) (24) mice were obtained
from established colonies maintained at the University of Virginia
Health Sciences Center vivarium under specific pathogen-free conditions
in a barrier facility. All mice used in these studies were back-crossed
into the C57BL/6 background for at least six generations. C57BL/6
wild-type mice (Hilltop Lab Animals, Scottdale, PA) were maintained at
the University of Virginia Health Sciences Center vivarium until needed for experiments.
Models of inflammation.
Leukocyte recruitment was studied in a TNF--induced model of
inflammation of the mouse cremaster muscle and in a
thioglycollate-induced peritonitis. The long-term (6 h) TNF- model
of inflammation of the mouse cremaster muscle has been described
previously (9, 11). TNF- enhances the expression of
P-selectin and ICAM-1 on the endothelial surface. The expression of
E-selectin and ligands for L-selectin and 4-integrins
are also induced by treatment with TNF- (11). This
model is accessible to intravital microscopy and allows direct
observation of the mechanisms underlying leukocyte recruitment.
Antibodies and cytokines.
The rat anti-mouse MAb to 4-integrin PS/2 (rat IgG2b)
(23), purified from hybridoma culture supernatant, has
been previously shown to specifically block 4-integrin
function in vivo (9). In some experiments, PS/2 (100 µg/mouse) was administered intraperitoneally at the time of TNF-
injection. In the 6-h TNF--induced model of inflammation,
intravascular coagulation has been described as a consequence of the
severe inflammation (9). Therefore, E/P/I/ and
E/P/L/I/ mice were injected with 10 units of hirudin (Sigma
Chemical; St. Louis, MO) at the time of TNF- administration and 10 units of hirudin 2.5 h before cremaster exteriorization to prevent
intravascular coagulation.
Bone marrow transplantation.
Mice deficient in E-, P-, and L-selectin and ICAM-1 (E/P/L/I/) were
generated by transplanting L-selectin-deficient (L/) bone marrow
into E/P/I/ mice. Bone marrow transplant recipient mice were
2-3 mo of age.
/ mice were transplanted with wild-type
bone marrow or L/ bone marrow to generate the E/P/ and
E/P/L/ groups (9). E/P/I/ mice were transplanted
with wild-type bone marrow to generate E/P/I/ control mice.
Intravital microscopy.
For the model of TNF--induced acute inflammation, mice were
anesthetized with an intraperitoneal injection of ketamine
hydrochloride (Ketalar, 125 mg/kg, Parke-Davis; Morris Plains, NJ),
xylazine (12.5 mg/kg, Vedco; St. Joseph, MO), and atropine (0.25 mg/kg, Elkins-Sinn; Cherry Hill, NJ). The trachea was intubated and anesthetic (diluted pentobarbital sodium in saline) was administered throughout the intravital experiment through one cannulated jugular vein. Blood
pressure was monitored, and blood samples were obtained through a
cannulated carotid artery. Mice were kept at a constant temperature of
37°C with a thermo-controlled heating pad (Physitemp Instruments;
Clifton, NJ) during the intravital microscopic experiment. The
cremaster muscle was prepared for intravital microscopy as described (17). Recombinant murine TNF- (Genzyme;
Cambridge, MA) was injected intrascrotally at a dose of 500 ng per
mouse in a volume of 0.3 ml of sterile saline 6 h before
exteriorization of the cremaster muscle. The cremaster muscle was
superfused with thermo-controlled (35°C) bicarbonate-buffered saline.
Throughout the experiment, blood samples were taken from the carotid
catheter to analyze systemic leukocyte concentrations. Kimura-stained
blood samples were analyzed by using a hemocytometer to obtain
leukocyte counts. Microscope observations were made with a Zeiss
intravital microscope (Axioskop; Thornwood, NY) by using a saline
immersion objective (SW 40/0.75 numerical aperture). Venules with
diameters between 20 and 80 µm were observed and recorded via a CCD
camera system (model VE-1000CD, Dage-MTI; Michigan City, IN) on a
Panasonic S-VHS recorder. Centerline red blood cell velocity was
measured by using a dual photodiode and a digital on-line
cross-correlation program (CircuSoft Instrumentation; Hockessin, DE).
Mean blood flow velocities were obtained by multiplying the centerline
velocity by an empirical factor of 0.625 (20). Wall shear
rates (
w) were estimated as 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 velocity profiles measured in
microvessels in vivo (26).
Rolling and adhesion parameters.
A digital image processing system was used to measure microvessel
diameters, lengths, and leukocyte rolling velocities (25). Leukocyte rolling flux, expressed as leukocytes per minute, was calculated by counting leukocytes rolling past a line perpendicular to
the vessel axis (19). Rolling flux fraction was calculated as described (19) by dividing leukocyte rolling flux by
total leukocyte flux estimated as
[WBC]Vb
(d/2)2, where
[WBC] is the systemic leukocyte count. Adherent leukocytes were
defined as leukocytes that did not move for at least 30 s. The
total number of adherent leukocytes was determined for each venule
segment (~200 µm) and expressed per unit area of inside surface
area of the venule.
Flow cytometry. Expression of L-selectin on mouse neutrophils obtained from both peripheral blood and bone marrow was determined by direct immunofluorescence. Bone marrow cells were harvested as described above using PBS (GIBCO) with 0.01% azide. Whole blood or bone marrow was incubated with fluorescein isothiocyanate (FITC)-labeled MAb Gr-1 (0.5 µg/106 cells, Pharmingen; San Diego, CA) to identify granulocytes, and phycoerythrin-labeled MAb MEL-14 to label L-selectin (0.5 µg/106 cells, Pharmingen) or isotype control (rat IgG2a, 0.5 µg/106 cells, Pharmingen). Samples were incubated for 30 min on ice. Unlabeled antibody was removed by aspiration after centrifugation. Bone marrow cells were resuspended in PBS with 0.01% azide. Peripheral blood was resuspended in 150 mM NH4Cl, 10 mM NaHCO3, and 1 mM Na2EDTA in deionized, distilled water to lyse red blood cells. Cells were analyzed by forward scatter, side scatter, FITC fluorescence, and phycoerythrin fluorescence using a laser flow cytometer (FACScan, Becton-Dickinson; San Jose, CA). Neutrophils were identified and gated by expression of Gr-1 antigen measured by incubation with Gr-1-FITC (0.5 µg/106 cells, Pharmingen). Data are presented as fluorescence histograms of MEL-14 expression of Gr-1-positive cells on a four-decade log scale.
Histology. To differentiate intravascular and interstitial leukocytes, cremaster muscle whole mounts were prepared. While the cremaster muscle was still mounted on the stage for intravital microscopy, the tissue was fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The cremaster muscle was removed and mounted flat on a gelatanized-treated glass slide, air dried for 5-10 min, and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 24 h at 4°C. After fixation, the tissue was washed three times in 0.1 M phosphate buffer with 5% ethanol, stained with Giemsa (Sigma) at room temperature for 5 min, and differentiated in 0.01% acetic acid for contrast. The differentiated slides were washed in water, 75% ethanol, 95% ethanol, 100% ethanol, and fresh xylene, followed by mounting in mounting media (Sigma). The Giemsa-stained cremaster muscles were observed using a Zeiss microscope with a ×100, 1.4 numerical aperture oil immersion objective (Zeiss, Germany). Intravascular and interstitial leukocytes were counted and differentiated into neutrophils, eosinophils, and mononuclear cells. The interstitial tissue observed was a circular area (diameter of 183 µm) bisected by each venule.
Thioglycollate-induced peritonitis.
Wild-type, E/P/I/, and E/P/L/I/ mice were injected
intraperitoneally with 1 ml of 4% thioglycollate (Sigma) in deionized water at time 0. At 4 or 24 h mice were killed with a
lethal injection of pentobarbital sodium and injected intraperitoneally
with 5 ml ice-cold PBS (without Ca2+, Mg2+)
containing 2 mM EDTA, their abdomens were massaged, and total lavage
fluid was collected through a small slit cut into the peritoneal cavity. Collected cells were pelleted by centrifugation and resuspended in 5 ml of 150 mM NH4Cl, 10 mM NaHCO3, and 1 mM
Na2EDTA in deionized, distilled water to lyse red blood
cells. The number of recruited leukocytes was counted, and leukocyte
differentials were obtained from Kimura-stained samples and verified
using stained cytospins. Systemic leukocyte concentrations were
determined at 0 and 4 or 24 h from Kimura-stained blood samples
collected from the tail vein. To obtain a leukocyte recruitment
efficiency (10), the leukocyte concentration in the
peritoneal lavage was divided by the leukocyte concentration in the
circulation. Data obtained from E/P/I/ mice transplanted with
wild-type bone marrow were not significantly different from data
obtained in E/P/I/ mice; therefore, nontransplanted wild-type and
E/P/I/ mice were also used in the thioglycollate-induced
peritonitis studies.
Statistics. Average leukocyte rolling velocities, leukocyte adhesion, systemic leukocyte counts, and differentials between groups were compared using one-way ANOVA and Kruskal-Wallis multiple comparison test. Statistical significance was set at P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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E/P/L/I/ chimeric mice.
E/P/L/I/ mice were generated by transplanting lethally irradiated
E/P/I/ mice with L/ bone marrow. E/P/I/ control mice were
also generated through bone marrow transplantation (reconstituted with
wild-type bone marrow). At 4 wk after reconstitution, peripheral blood
(data not shown) and bone marrow of chimeric mice were analyzed for
L-selectin expression (Fig. 1).
L-selectin expression was undetectable on bone marrow and blood cells
in mice transplanted with L/ bone marrow, confirming that
reconstitution was efficient and complete. E/P/I/ and E/P/L/I/
mice appeared healthy and did not show ulcerative dermatitis as seen in
E/P/ mice (2).
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TNF--induced inflammation of the cremaster muscle: systemic
leukocyte counts and hemodynamics in mouse cremaster venules.
The systemic leukocyte counts in E/P/I/ and E/P/L/I/ mice were
significantly elevated (approximately a 4-fold increase; Table
1) after TNF- treatment compared with
wild-type mice (4,390 ± 750/µl; see Ref. 11). We
investigated leukocyte rolling and adhesion in 81 venules in six
E/P/I/ and seven E/P/L/I/ mice. The average vessel diameter was
51.8 ± 2.8 µm. The severe inflammatory model using long-term
TNF- treatment (6 h) resulted in reduced blood flow velocities
(Table 1) and, therefore, lower wall shear rates than in untreated or
short-term TNF--treated mice. The average centerline velocity was
1.1 ± 0.1 mm/s, and the calculated wall shear rate was 240 ±20
s
1.
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Leukocyte rolling.
TNF- (6 h) induces the expression of E-selectin, enhances the
expression of P-selectin, and induces L-selectin- and
4-integrin-dependent rolling (8, 11). We
have previously shown that leukocyte rolling in E/P/
(2) and E/P/L/ mice is drastically reduced (9) compared with wild-type mice. Residual rolling in mice lacking all three selectins was shown to be 4-integrin
dependent (9).
/ mice (2.7 ± 0.5 cells/min; Fig. 2A) was
similar to E/P/L/ mice (2.0 ± 0.4 cells/min) and lower than
in E/P/ mice (5.2 ± 0.8 cells/min). E/P/L/I/ mice yielded
a leukocyte rolling flux (2.1 ± 0.3 cells/min; Fig. 2A) similar to E/P/I/ and E/P/L/ mice, which was
approximately a 60% reduction from the level of leukocyte rolling in
E/P/ mice and a more than 97% reduction compared with wild-type
mice (11).
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/ mice (Fig.
2B). Removing L-selectin and/or ICAM-1 resulted in a further
reduction in the leukocyte rolling flux fraction in E/P/L/
(0.4 ± 0.6%), E/P/I/ (0.3 ± 0.9%), and E/P/L/I/
(0.3 ± 0.6%) mice (Fig. 2B).
Leukocyte rolling in E/P/L/, E/P/I/, and E/P/L/I/ mice was
completely blocked by a MAb to 4-integrin, showing that 4-integrins are capable of capturing a very small
fraction of circulating leukocytes (Fig. 2, A and
B). These data also show that the L-selectin-mediated
rolling present in E/P/ mice (11) does not occur in
the absence of ICAM-1 because the leukocyte rolling flux and flux
fraction are the same for E/P/I/ and E/P/L/ mice.
Leukocyte rolling velocities after 6 h of TNF- treatment in
wild-type, E/P/, and E/P/L/ mice were previously determined to
be 13.3 ± 1.0, 15.7 ± 1.2, and 13.6 ± 1.2 µm/s,
respectively (9). E/P/I/ and E/P/L/I/ mice showed
average leukocyte rolling velocities of 19.9 ± 1.8 and 19.7 ± 2.5 µm/s, respectively (Fig. 3). All
leukocyte rolling in E/P/L/, E/P/I/, and E/P/L/I/ mice was
4-integrin dependent (Fig. 2B). The
marginally elevated leukocyte rolling velocities seen in E/P/I/
mice and the significantly elevated leukocyte rolling velocities seen
in E/P/L/I/ mice, therefore, suggest a role of ICAM-1 in leukocyte
rolling for the subset of leukocytes captured by
4-integrins.
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Leukocyte adhesion and transmigration.
Although there were dramatic reductions of leukocyte rolling in
E/P/, E/P/L/, E/P/I/, and E/P/L/I/ mice (Fig. 2,
A and B), TNF- treatment for 6 h still
resulted in a substantial amount of leukocyte adhesion in all the
mutants (400-600 adherent leukocytes/mm2) (Fig.
2C). Leukocyte adhesion was reduced by ~40-50% in
mutant mice compared with wild-type mice (1,080 ± 170 adherent
leukocytes/mm2) (Fig. 2C). Blocking all
leukocyte rolling in E/P/I/ and E/P/L/I/ mice by pretreatment
with a MAb to 4-integrin at the time of induction of
inflammation almost completely abolished leukocyte adhesion (Fig.
2C).
/ mice showed a
significant reduction in neutrophil recruitment (63%) (9)
compared with that observed in wild-type mice (81%) (9). Neutrophil recruitment was further reduced in the absence of ICAM-1 (43% in E/P/I/ and 36% in E/P/L/I/ mice; Table
2). To appreciate the impact of these
adhesion molecules on neutrophil and mononuclear cell recruitment, the
number of adherent neutrophils and mononuclear cells per squared
millimeter of vessel surface area was estimated by multiplying the
intravascular differential counts obtained by histology (Table 2) with
the leukocyte adhesion density obtained by intravital microscopy (Fig.
2C). These data show that despite similar leukocyte adhesion
levels (cells/mm2) in E/P/ compared with E/P/I/ and
in E/P/L/ compared with E/P/L/I/ mice, the elimination of
ICAM-1 significantly impairs neutrophil recruitment (Fig.
4).
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Thioglycollate-induced peritonitis.
A thioglycollate-induced peritonitis model of inflammation was used to
further investigate selectin-independent leukocyte recruitment.
Peritoneal lavage fluid was collected at 4 or 24 h following
thioglycollate injection, and the number of neutrophils and mononuclear
cells recruited was counted (Fig.
5A). Neutrophil recruitment
was severely impaired in E/P/I/ and E/P/L/I/ mice compared with
that in wild-type mice at 4 and 24 h (Fig. 5A). Mononuclear cell recruitment in E/P/I/ and E/P/L/I/ mice was unaffected (Fig. 5A).
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/ mice, and from 17,200 to 8,600 cells/µl in E/P/L/I/
mice. At 24 h, circulating mononuclear cells counts had
largely recovered to baseline levels.
To obtain an overall leukocyte recruitment efficiency, the
concentration of leukocytes recruited to the peritoneum was divided by
the concentration of available circulating leukocytes and normalized to
a wild-type efficiency of 1.0 (equalling 100%) (Fig. 5C).
E/P/I/ and E/P/L/I/ mutants had drastically reduced neutrophil
recruitment efficiency at 4 h (2.2% and 1.1% of wild-type,
respectively). The reduction was even more severe at 24 h (0.8%
and 0.5% of wild-type, respectively) (Fig. 5C). The
progressive reduction in the percentage of neutrophil recruitment
relative to the wild-type controls for E/P/I/ and E/P/L/I/ mice
shows a cumulative effect of the absence of adhesion molecules on
effective neutrophil recruitment.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Quadruple adhesion molecule-deficient E/P/L/I/ mice are viable
and, without challenge, apparently healthy. However, they display a
remarkable impairment in leukocyte capture, rolling, adhesion, and
recruitment. Mononuclear cell recruitment appeared largely unaffected
in the absence of selectins and ICAM-1, indicating a greater role of
selectin- and ICAM-1-mediated recruitment for neutrophils than for
mononuclear cells.
Our previous work (9) and that of others (27)
have shown the role of selectins in mediating efficient neutrophil
recruitment to sites of inflammation. Leukocyte capture and rolling,
largely mediated by selectins, is the generally accepted prerequisite for firm leukocyte adhesion. Surprisingly, a substantial amount of
leukocyte adhesion occurred in E/P/L/ or E/P/L/I/ mice (~40%
of wild-type) despite very few rolling leukocytes (~3% of wild-type). This is consistent with previous studies using antibody blockade of selectin function. Kubes et al. (15) showed
that rolling must be blocked by at least 90% to significantly affect recruitment. Mice lacking all three selectins by repeated gene targeting (27) or by bone marrow chimerism
(9) also show very low rolling but still significant
recruitment. The E/P/I/ and E/P/L/I/ mice studied here,
however, reveal much more severely compromised adhesion and recruitment
of neutrophils in the cremaster muscle. This represents a very severe
impairment of neutrophil recruitment.
Interestingly, some leukocyte adhesion remained in E/P/L/I/ mice
when leukocyte rolling was completely removed with a MAb to
4-integrins. These data suggest the existence of both
neutrophil and mononuclear cell recruitment mechanisms that completely
bypass all known rolling mechanisms through selectins and
4-integrins.
The number of adherent leukocytes found in cremaster venules reflects
the steady-state balance between new leukocyte adhesion from the
rolling pool and leukocyte transmigration. We have investigated the
mechanisms leading to leukocyte adhesion and found a reduction of
leukocyte rolling that was more severe than the reduction in the number
of adherent leukocytes. The substantial amount of selectin- and
ICAM-1-independent leukocyte adhesion could therefore result from a
defect in cell transmigration in these mice. Such an additional effect
on transmigration is suggested by the data obtained in the peritonitis
model. Recruitment of neutrophils in E/P/L/I/ mice in the
peritonitis model was reduced by more than 95% compared with wild-type
controls despite only a 83% reduction in the number of firmly adhered
cells in the TNF- model of inflammation. The mechanisms
underlying these differential effects remain to be explored by detailed
studies of the transmigration process.
Leukocyte adhesion in E/P/L/I/ mice was similar to the level of
leukocyte adhesion in selectin-deficient mice (E/P/L/). This
suggests that ICAM-1 is not important in mediating firm adhesion of
leukocytes recruited through selectin-independent mechanisms. However,
the leukocyte differentials reveal a preferential reduction in
neutrophil recruitment in the absence of ICAM-1.
The leukocyte rolling data reveal a unique cooperative function between
ICAM-1 and L-selectin in mediating efficient leukocyte recruitment. The
majority of leukocyte rolling in E/P/ mice was previously shown to
be L-selectin dependent (11). Here, E/P/I/ mice showed
leukocyte rolling similar to E/P/L/ mice, and this level was not
further reduced in E/P/L/I/ mice. These data suggest that the
L-selectin-mediated rolling seen in E/P/ mice requires the presence
of ICAM-1. ICAM-1 appears necessary for stabilizing rolling of
leukocytes captured through L-selectin. This is consistent with earlier
findings in a different model. Mice lacking both P-selectin and ICAM-1
show almost no trauma-induced rolling (16) and severely
reduced neutrophil recruitment in a peritonitis model (3).
The disruption of leukocyte rolling in the absence of ICAM-1 is further
substantiated in the present study by the elevated leukocyte rolling
velocities in E/P/I/ and E/P/L/I/ mice compared with E/P/
and E/P/L/ mice. Taken together, these data suggest a model of
neutrophil recruitment in which preferential pathways may exist.
Leukocytes initially captured by and rolling on L-selectin appear to
require ICAM-1 for adhesion and ultimately recruitment. This
requirement for ICAM-1 is much less stringent when P- and/or E-selectin
are also present, as can be seen for the mild inflammatory defect in
ICAM-1/ mice when all selectins are present (28, 33).
In conclusion, novel E/P/L/I/ mice were used in two models of
inflammation to investigate selectin- and rolling-independent mechanisms of leukocyte recruitment. The data show that selectins and
ICAM-1 are all involved in efficient neutrophil recruitment. Eliminating more adhesion molecules further reduces neutrophil recruitment efficiency, but the effect is less than additive. These
mice clearly show and highlight leukocyte adhesion and recruitment through 4-integrin, which may allow E/P/L/I/ mice to
be alive and viable. The similar phenotype seen in E/P/L/ mice and
E/P/I/ mice without a further defect in E/P/L/I/ mice suggests
that ICAM-1 is necessary for recruitment of leukocytes that use
L-selectin to roll.
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ACKNOWLEDGEMENTS |
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We thank Dr. Daniel C. Bullard, University of Alabama at
Birmingham, for providing E/P/I/ breeding pairs. We thank Jennifer Bryant for mouse husbandry.
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FOOTNOTES |
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Funding for the study was provided by National Heart, Lung, and Blood Institute Grant HL-54136 to K. Ley. S. B. Forlow was supported by an institutional training grant (T-32HL-07284 to B. R. Duling) and a Special Opportunity Award to K. Ley by the Whitaker Foundation.
Address for reprint requests and other correspondence: K. Ley, Dept. of Biomedical Engineering, Health Sciences Center Box 800759, Univ. of Virginia, Charlottesville, Virginia 22908 (E-mail: klausley{at}virginia.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 28 April 2000; accepted in final form 15 September 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Significantly different from wild-type and E/P