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Department of Biomedical Engineering, University of Virginia Health Sciences Center, Charlottesville, VA 22908
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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90%) in inflammation induced by TNF-
. Residual
rolling in L/P-/- mice was very slow (3.6 ± 0.2
µm/s after TNF-). L/E-/- mice (only P-selectin
present) showed rolling similar to that of L-/- at
increased velocities (15.1 ± 0.3 µm/s). The number of adherent
leukocytes after 2 or 6 h of TNF- treatment was not
significantly reduced in L/E-/- or L/P-/-
mice. E/L/P-/- mice showed very little rolling after
TNF-, all of which was blocked by mAb to 4 integrin.
Adherent and emigrated neutrophils were significantly reduced at 6
h after TNF-. We conclude that any one of the selectins can support
some neutrophil recruitment but eliminating all three selectins
significantly impairs neutrophil recruitment. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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treatment (6), and
significantly reduced after long term (>6 h) TNF- treatment of
L-/- mice (7). P-selectin-deficient mice
(P-/-) show reduced neutrophil recruitment in models of
peritonitis at 2 and 4 h but normal neutrophil recruitment at
later times (2, 8). This corresponds to a striking absence of leukocyte
rolling in P-/- mice at early time points (5), which is
largely restored after treatment with TNF- (5, 9).
E-selectin-deficient mice (E-/-) have an inflammatory
defect when P-selectin is also blocked (3), do not show slow leukocyte
rolling (6), have less efficient adhesion in response to
chemoattractant (10), and succumb to i.p. infection with
Streptococcus pneumoniae at a dose that is cleared by
wild-type (WT) mice (11). Taken together, the current evidence suggests
that mice with null mutations at individual selectin loci have
mild to moderate inflammatory defects, with the severity of defects
being L-/- > P-/- > E-/-.
Mice with concomitant null mutations at the E- and P-selectin loci (8, 12) have at least 10-fold elevated neutrophil counts, high levels of
circulating inflammatory cytokines, reduced L-selectin expression due
to chronic inflammation, and severely impaired neutrophil recruitment
into chemically induced peritonitis lesions at 2 and 8 h and into
bacterial peritonitis at 4 h, but not at 24 h (8, 12). In
addition, these mice suffer from spontaneous skin erosions and mucosal
infections, which limit their life span. E/P-/- mice show
no rolling after trauma or short term TNF- treatment (8) and little
rolling after prolonged TNF- (>6 h) (7). A study based on Ab
blockade showed reduced neutrophil recruitment to peritonitis when both
L- and P-selectin were blocked, suggesting that L- and P-selectin
may also have overlapping functions (13). Consistent with this, it was
shown that rolling is severely reduced in short term TNF--treated
P-/- mice receiving an L-selectin Ab (5) or in
L-/- mice receiving a P-selectin Ab (6). By
contrast, blocking E-selectin in addition to L-selectin had no effect
in this same model (6).
Each selectin mediates rolling at a characteristic velocity. Under physiological conditions of wall shear stress and selectin site density in vivo, the velocity of L-selectin-mediated rolling is highest (14) and that of E-selectin-mediated rolling is lowest (6). In vitro studies have suggested that L-selectin is most efficient at capturing leukocytes from the flow (15, 16), whereas E-selectin mediates stable rolling. P-selectin appears to be able to both initiate (17) and maintain (18) leukocyte rolling.
On the basis of these findings, we hypothesized that mice lacking both
L- and P-selectin would have a substantial defect in leukocyte rolling
beyond the defect seen in mice lacking P-selectin only or L-selectin
only. We further predicted that L/E-/- mice should not
have a severe rolling defect beyond that seen in the single mutants,
because P-selectin can support both leukocyte capture and rolling. To
directly test these hypotheses, we have generated mice lacking both L-
and endothelial P-selectin, L- and E-selectin, or all three selectins.
The chimeric mice were generated by transplanting L-/-
bone marrow into lethally irradiated P-/-,
E-/- or E/P-/- mice. The phenotype of these
mice was investigated under conditions of mild trauma caused by
exteriorizing the cremaster muscle for intravital microscopy (5)
and in two models of TNF--induced inflammation with different
selectin requirements (6, 7).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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E-selectin null (E-/-) (8), P-selectin null (P-/-) (19), L-selectin null (L-/-) (1), E- and P-selectin double null (8), and C57BL/6 WT mice were obtained from established colonies maintained at the University of Virginia Health Sciences Center vivarium. All mutant mice used in this study were backcrossed into the C57BL/6 strain for at least six generations.
Abs and cytokines
The rat anti-mouse mAb to E-selectin, 9A9 (rat IgG1), has
been previously shown to specifically block E-selectin function in
vitro (20) and in vivo (9). mAb 9A9 was a generous gift of Dr. B.
Wolitzky (Hoffman-La Roche, Nutley, NJ). mAb to P-selectin, RB40.34
(rat IgG1), is a function-blocking mAb against murine P-selectin (13).
mAb R1/2 to 4 integrin (21) was purified from hybridoma
culture supernatant. All Abs were injected i.v. at a dose of 30
µg/mouse. For the two models of TNF--induced inflammation,
recombinant murine TNF- (Genzyme, Cambridge, MA) was injected
intrascrotally at a dose of 500 ng/mouse in a volume of 0.3 ml of
sterile saline either 2 or 6 h before the beginning of the
intravital microscopic experiments as described previously (7). In the
6-h group, 30 U of heparin (Elkins-Sinn, Cherry Hill, NJ) were injected
at the time of TNF- administration to prevent intravascular
coagulation (7).
Bone marrow transplantation
Recipient mice were irradiated in two doses of 550–600 rads
each, for a total of 1100–1200 rads, 4 h apart. Donor mice were
killed by lethal injection of sodium pentobarbital (Nembutal, Abbott
Laboratories, North Chicago, IL), and bone marrow cells from both
femurs and tibias were harvested under sterile conditions.
Approximately 50 million nucleated bone marrow cells were obtained from
each donor mouse. Bones were flushed with RPMI (Life Technologies,
Grand Island, NY) (without phenol red) with 10% FCS (Atlanta
Biologicals, Norcross, GA). Suspended bone marrow cells were washed and
lysed in 1.5 mM NH4Cl lysing solution. The bone marrow
cells were washed twice and counted using a standard hemocytometer.
Approximately 1–2 million unfractionated bone marrow cells in 200 µl
of media were delivered i.v. through the tail vein of each recipient
mouse. For E/P-/- recipients, 10 million cells were
infused per mouse, because some E/P-/- mice died when
reconstituted with 1–2 million unfractionated bone marrow cells.
Recipient mice were housed in a barrier facility (individually
ventilated cages, high energy particulate arresting filter-filtered
air), under pathogen-free conditions before and after bone marrow
transplantation. After bone marrow transplantation, the mice were
maintained on autoclaved water with antibiotics (0.7 mM neomycin
sulfate, 60 µM tetracycline, and 0.37 mM trimethoprim) (Sigma, St.
Louis, MO) and fed autoclaved food. These conditions were maintained
for 4–5 wk. At this point, peripheral leukocyte counts and
differentials had returned to normal, and the mice were ready for
intravital microscopic experiments.
Intravital microscopy
Mice were anesthetized with an i.p. injection of ketamine
hydrochloride (100 mg/kg, Ketalar, Parke-Davis, Morris Plains, NJ)
after pretreatment with sodium pentobarbital (30 mg/kg i.p., Nembutal,
Abbott Laboratories, North Chicago, IL) and atropine (0.1 mg/kg i.p.,
Elkins-Sinn). The trachea was intubated, and one jugular vein was
cannulated for administration of anesthetic throughout the intravital
microscopic experiment. One carotid artery was cannulated for blood
pressure monitoring, blood samples, and systemic mAb injections. Mice
were kept at a constant temperature of 37°C with a thermo-controlled
heating lamp (Physitemp, Clifton, NJ) and received 0.2 ml/h diluted
pentobarbital in saline i.v. to maintain anesthesia and a neutral fluid
balance. The cremaster muscle was prepared for intravital microscopy as
described (5). The epididymis and testis were gently pinned to the
side, exposing the cremaster microcirculation. Time 0 was set at the
beginning of the cremaster muscle exteriorization. The cremaster muscle
was superfused with thermo-controlled (35°C) bicarbonate-buffered
saline. 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 evaluating Kimura-stained blood samples in a hemocytometer.
Microscopic observations were made on a Zeiss intravital microscope
(Axioskop, Carl Zeiss, Thornwood, NY) with 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
(Panasonic, Osaka, Japan). Centerline RBC velocity was measured using a
dual photodiode and a digital on-line cross-correlation program (22).
Centerline velocities were converted to mean blood flow
velocities by multiplying with an empirical factor of 0.625 (23). 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 (24).
Rolling and adhesion parameters
Microvessel diameters, lengths, and rolling leukocyte velocities
were measured with a digital image processing system (22). Each rolling
leukocyte passing a line perpendicular to the vessel axis was counted,
and leukocyte rolling flux was expressed as leukocytes per minute.
Rolling flux fraction was calculated as described (5) by dividing
leukocyte rolling flux by total leukocyte flux estimated as [WBC]
vb 
(d/2)2, where
[WBC] is the systemic leukocyte count, vb is
mean blood flow velocity, and d is the venule diameter.
Leukocyte rolling velocities were measured over a constant 2-s time
window. The rolling velocities of 10 leukocytes were measured in each
venule. Adherent cells were defined to be the leukocytes that were not
moving for at least 30 s. The total number of adherent cells were
measured for each segment of venule (200 µm long) and expressed
per unit area of inside surface area of the venule. The surface area
was calculated from diameter and length, assuming cylindrical geometry
of the venule.
Flow cytometry
Flow cytometry was used to detect L-selectin expression on peripheral blood leukocytes as well as bone marrow cells of the recipient mice. Peripheral blood was obtained from the carotid catheter. Bone marrow cells were harvested as described above using PBS (Life Technologies) with 0.01% azide. For each sample tube, a 200-µl volume of peripheral blood or bone marrow cell suspension was incubated with either MEL-14 conjugated with PE or rat IgG2a conjugated with PE (both PharMingen, San Diego, CA) for 30 min (0.5 µg/106 cells). Cells were centrifuged and aspirated. Bone marrow cells were resuspended in 500 µl of PBS with 0.01% azide. Peripheral blood leukocytes were lysed in 1.5 ml of 1.5 mM NH4Cl solution. Flow cytometry data were analyzed using a four-decade FACScan and Cell Quest software package (Becton Dickinson, San Jose, CA). For each sample, 10,000 cells were analyzed.
Histology
To differentiate intravascular and interstitial leukocytes, whole mounts of cremaster muscle were prepared by dropping 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) onto the tissue while tissue was still mounted on the cremaster stage for intravital microscopy. Each cremaster muscle was removed, mounted flat on a poly-L-lysine (Sigma)-treated glass slide, and air dried for 5–10 min, followed by fixation 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 stain (Sigma) at room temperature for 5–10 min, and differentiated in 0.01% acetic acid for contrast. The differentiated slides were sequentially 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 on a Zeiss microscope with a 100x, 1.4 numerical aperture oil immersion objective (Carl Zeiss). Intravascular and interstitial leukocytes were counted and differentiated into neutrophils, eosinophils, and mononuclear cells. The interstitial tissue observed was a circular area (with a diameter of 141 µm) bisected by each venule.
Statistics
Average leukocyte rolling flux fractions, leukocyte adhesion, leukocyte rolling velocities, leukocyte counts and differentials between groups were compared with the one-way ANOVA Kruskal-Wallis multiple comparison test. Statistical significance was set at p < 0.05, indicated by *.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At the selected dose of irradiation (1100 rads, fractionated), all
nontreated mice died within 2 wk of the irradiation, whereas all mice
injected with donor bone marrow survived (data not shown). At 4 wk
after reconstitution, we investigated the degree of chimerism in our
mice by measuring L-selectin expression on nucleated cells in the bone
marrow and peripheral blood (Fig. 1
).
After reconstitution with L-/- bone marrow, L-selectin
expression was undetectable on at least 99% of the bone marrow and
blood cells. P-/- or E-/- mice could
readily be reconstituted with L-/- bone marrow cells at a
dose of 1–2 million per mouse. By contrast, E/P-/- mice
required larger numbers of bone marrow cells (10 million per mouse) for
efficient reconstitution. This finding suggests that a combined absence
of P- and E-selectin may impair homing of hemopoietic stem cells, but
this issue was not investigated further in the present study. Recently,
another group has shown that absence of E- and P-selectin indeed
impairs engraftment of bone marrow stem cells (25).
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Mild trauma rapidly induces P-selectin-dependent leukocyte rolling
(2, 5, 26), followed by a second phase which is both L-selectin and
P-selectin dependent (5, 14, 27). E-selectin is not or very faintly
expressed in cremaster muscle vessels under these conditions (28).
Here, we assessed leukocyte rolling in 126 venules of 12 mice with
velocities, diameters, and shear rates (Table I). Rolling is expressed as leukocyte
rolling flux fraction, which represents the number of rolling
leukocytes divided by the total number of leukocytes passing through
the same venule (29). L/P-/- mice showed a complete
absence of leukocyte rolling at >30 min after exteriorization,
compared with normal rolling in WT mice (15 ± 1% (5)) and to
residual rolling in P-/- mice reported earlier (rolling
flux fraction, 4 ± 1% (5)). There were too few rolling
leukocytes in L/P-/- mice to measure leukocyte rolling
velocity. However, in control mice in which WT bone marrow was injected
into lethally irradiated C57BL/6 WT mice, the rolling velocity at >30
min after exteriorization was 48 ± 6 µm/s, similar to the 49
µm/s reported earlier for WT mice under these conditions (14).
Rolling velocity in WT mice reconstituted with L-/- bone
marrow was slightly reduced to 40 ± 1 µm/s, similar to previous
findings in L-/- mice (14, 30). These findings confirm
that P-selectin mediates slower leukocyte rolling than L-selectin under
these conditions and show that trauma-induced rolling is eliminated
when both P- and L-selectin are absent (Table I).
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treatment
Short term treatment with TNF- is sufficient to induce
expression of E-selectin and enhance the expression of P-selectin on
the venular endothelium in this model (28). Here, we report leukocyte
rolling in 248 venules of 25 TNF--treated mice lacking various
selectins. Hemodynamic parameters were similar in these mice (Table II). The leukocyte rolling flux fraction
after short term TNF- treatment was slightly reduced in WT mice
reconstituted with L-/- bone marrow (Fig. 2), similar to previous findings in
L-/- mice (6). E-/- mice reconstituted with
WT bone marrow showed increased levels of leukocyte rolling, consistent
with previous findings in E-/- mice (6) and in mice
pretreated with an E-selectin Ab (6, 31). All rolling in these mice was
blocked by a P-selectin Ab (Fig. 2).
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). Mice lacking endothelial
P-selectin were generated by reconstituting P-selectin
-/- mice with WT bone marrow. Rolling flux fraction in
these mice was similar to that previously reported for
P-/- mice (5), suggesting that platelet P-selectin does
not play an important role in this model of inflammation.
In marked contrast, novel mice lacking both P- and L-selectin
(L/P-/-) showed very low levels of leukocyte rolling
after 2 h of TNF- treatment (Fig. 2). These findings suggest
that L- or P-selectin are required to initiate rolling and that
E-selectin cannot serve that particular function. This provides clear
evidence of a nonoverlapping function of P- and L-selectin compared
with E-selectin. Under these conditions, all residual rolling in
L/P-/- mice was blocked by injecting a blocking
E-selectin Ab, mAb 9A9 (Fig. 2).
Next, we investigated the rolling velocities in these mice. WT mice
reconstituted with L-/- bone marrow showed rolling at
5.0 ± 0.3 µm/s (Fig. 3), similar
to rolling in L-/- mice (6.4 ± 0.5 µm/s (6)).
Similarly, rolling velocity in P-/- reconstituted with WT
bone marrow was 5.9 ± 0.3 µm/s, comparable to 6.4 ± 1.9
µm/s found in P-/- mice (6). However, rolling velocity
was dramatically increased in E-/- mice reconstituted
with WT bone marrow, reaching an average of 31.1 ± 0.9 µm/s.
Again, this is similar to rolling velocities found in
E-/- mice (6).
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) and has an average rolling velocity of 14.8 ± 0.4 µm/s.
Because this is a novel mouse, it can be compared only indirectly with
previous data, which showed an average rolling velocity of 14.5 ±
1.4 µm/s in L-selectin-deficient mice treated with an E-selectin Ab
(6).
The most interesting result in this model is the finding that residual
rolling in L/P-/- mice proceeds at very low velocities,
averaging 3.5 ± 0.2 µm/s (Fig. 3). These mice have very low
numbers of rolling leukocytes, which use E-selectin for rolling (Fig. 2). This represents the first isolation of purely E-selectin-dependent
rolling in vivo.
Long term (6 h) TNF- treatment
Long term TNF- treatment has previously been shown to induce
L-selectin and 4 integrin-dependent rolling in
E/P-/- mice (7). Now, we use L-/- bone
marrow transplantation into E-/-, P-/- and
E/P-/- mice to investigate how rolling is altered when
only P-selectin (L/E-/-), only E-selectin
(L/P-/-), and no selectin (E/L/P-/-) is
present (Fig. 4). These investigations
were made in 161 hemodynamically comparable venules of 26 mice (Table III).
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treatment,
1.2 ± 0.3% (Fig. 4) compared with 10.6 ± 2.7% in WT mice
treated in the same way (7). Rolling was also reduced when
L-/- bone marrow was transplanted into E-/-
mice, but much less so than in WT mice reconstituted with
L-/- bone marrow. This probably reflects the increase in
leukocyte rolling flux produced by absence of E-selectin (6). Rolling
was very low in L/P-/- mice, showing that E-selectin
alone is not very efficient at supporting leukocyte rolling under these
conditions. The rolling flux fraction seen in these
L/P-/- mice, 0.9 ± 0.3% (Fig. 4), is similar to
that found in E/P-/- mice reconstituted with WT bone
marrow (0.9 ± 0.2%, Fig. 4). This rolling flux fraction was
further reduced to 0.4 ± 0.06% in E/L/P-/- mice
lacking all three selectins. These data show that L-selectin plays an
important role in leukocyte rolling after long term TNF- treatment,
because E/L/P-/- mice show less rolling than
E/P-/- mice (7). In contrast to the findings obtained
after TNF- treatment for 2 h, rolling was not totally
abolished even in the absence of all three selectins. Rolling was
completely blocked only after a blocking 4 integrin Ab,
mAb PS/2, was injected in these mice.
The average rolling velocity in WT mice reconstituted with
L-/- bone marrow and treated with TNF- for 6 h
before surgery was 10.7 ± 1.2 µm/s (Fig. 5), similar to values seen in
L-/- mice under the same conditions (8 ± 0.8 µm/s
(7)). L/E-/- mice showed elevated rolling velocities of
15.2 ± 0.9 µm/s, suggesting that this may be the characteristic
velocity of P-selectin-mediated rolling under long term TNF-
treatment. Isolated P-selectin-dependent rolling has not previously
been investigated in this model. The L/P-/- mouse is also
novel, showing a rolling velocity for E-selectin of 4.5 ± 0.3
µm/s (Fig. 5). This is not quite as low as the 3.6 µm/s seen in
short term TNF- model (see Fig. 3), but significantly lower than
rolling in WT mice after 6 h of TNF- treatment (Fig. 5).
Transplanting E/P-/- mice with WT bone marrow yields
rolling at an average velocity of 15.7 ± 1.2 µm/s, not
different from the velocity seen in E/P-/- mice (19
± 2 µm/s (7)). This finding suggests that the presence of P-selectin
on platelets in the bone marrow-transplanted mice shown here has no
major impact on leukocyte rolling velocity (Fig. 5) or flux (see Fig. 4) in this model.
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4 integrin-mediated
rolling, because all rolling in E/L/P-/- mice was blocked
by a mAb to 4 (Fig. 4). Leukocyte adhesion and transmigration
In spite of the massive changes in leukocyte rolling parameters,
the number of adherent neutrophils inside microvessels was similar in
all mutants after 2 h of TNF- (data not shown). This finding
suggests that sufficient overlap exists among the functions of the
selectins to ensure neutrophil delivery in acute inflammation even when
one or two of three selectins are absent. After 6 h of TNF-
treatment, neutrophil adhesion was slightly but not significantly
reduced in each of the single and double mutants (Fig. 6). A significant reduction was seen only
in E/L/P-/- mice lacking all three selectins. Remarkably,
the composition of adherent leukocytes was drastically altered in
E/L/P-/- mice. In E/L/P-/- mice,
neutrophils accounted for only 63% of all intravascular leukocytes,
with a balance of 25% mononuclear cells and 8% eosinophils. This
shows that absence of selectins specifically impairs neutrophil
recruitment but leaves mononuclear cell and eosinophil recruitment
relatively intact. These findings were also reflected in the number of
neutrophils found in the tissue surrounding the venules (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Under more severe inflammatory conditions, modeled here by treatment
with TNF- for >6 h, the rolling defects seen in both single- and
double-mutant mice are insufficient to significantly block leukocyte
recruitment to cremaster muscle venules or into the tissue. This is
remarkable and surprising in view of the significant rolling defects
seen in these mice. Apparently, under inflammatory conditions, even a
small number of rolling leukocytes can suffice to deliver
neutrophils to sites of inflammation. This is reminiscent of findings
obtained in a study exploring the therapeutic potential of inhibiting
P- and L-selectin (32). In that study, Kubes at al. reported that
rolling must be inhibited by at least 90% to see therapeutic effects
in terms of reduced neutrophil recruitment to inflammatory lesions.
With TNF- treatment for 6 h, leukocyte rolling is reduced by
95% in mice lacking all three selectins (E/L/P-/-).
In E/L/P-/- mice, neutrophil recruitment is significantly
impaired (Fig. 6).
The L/P-/- and L/E-/- isolate the function of E-selectin and P-selectin in vivo, respectively. Clearly, E-selectin alone is not nearly as good at mediating capture, rolling, and recruitment as P-selectin is. The rolling defect described here for L/P-/- mice appears to be of similar severity to that seen in E/P-/- mice described elsewhere (7, 8). By contrast, L/E-/- mice show only a moderate reduction of leukocyte rolling. The present data indicate that P-selectin is the most versatile of the selectins, because it alone can mediate reasonable levels of neutrophil rolling in all three models. This is achieved neither by L-selectin alone (as seen in E/P-/- mice) nor by E-selectin alone (as seen in L/P-/- mice), which show significant restrictions in the number of rolling leukocytes.
In E/L/P-/- mice, neutrophil recruitment is significantly
impaired (Fig. 6). This suggests that the multiple inflammatory defects
in the human disease, leukocyte adhesion deficiency II (33), must
likely be attributed to defects in all selectins contributing to
neutrophil recruitment. A previous report has shown that neutrophils
obtained from these patients have impaired ligand function for E- and
P-selectin and show impaired rolling in a rabbit model of inflammation
(34). The present data suggest that in addition to having defective E-
and P-selectin ligands, L-selectin ligand function may also be impaired
in these patients.
The generation of E/L/P-/- mice for the first time
isolates 4-dependent leukocyte rolling in vivo. This
rolling occurs at a reduced efficiency, as seen by a low leukocyte
rolling flux, and at an intermediate velocity of 13–14 µm/s.
Previous studies have shown that 4 integrins can mediate
capture and rolling on activated endothelial cells (35) and on isolated
VCAM-1 (36) in flow chamber-based in vitro assays. In vivo, a role for
4 integrins in leukocyte rolling has previously been
shown in a model of Mycobacterium butyricum-induced
vasculitis (37) and in lymphocyte attachment to high endothelial
venules in Peyer’s patches (38, 39). In these studies, the role of
4 integrins in leukocyte rolling was not clearly
separated from the contribution of the selectins to rolling. Here, we
show that 4 integrins mediate leukocyte rolling in
E/L/P-/- mice, although at a drastically reduced
efficiency compared with selectin-competent mice.
Although our experiments were not specifically designed to address the
role of P-selectin expressed on platelets for leukocyte rolling and
recruitment, the present data suggest that platelet P-selectin may not
be very important under the conditions tested. In the trauma-induced
and the short term TNF--induced models, P-/- mice
reconstituted with WT bone marrow, which express P-selectin on
platelets, show rolling and adhesion similar to that seen in
P-/- mice (5), which lack P-selectin on both platelets
and endothelial cells. In the long term (6 h) TNF- model,
P-/- mice have not been tested; therefore the current
data cannot be compared. Our findings are consistent with a recent
study (40) in which reconstitution of E/P-/- mice with WT
bone marrow failed to correct the elevated neutrophil counts,
development of spontaneous skin lesions, and reduced neutrophil
recruitment into peritonitis. However, negative findings with respect
to platelet P-selectin in Ref. 40 and in the present study do not rule
out the possibility that platelet P-selectin may be involved in
leukocyte recruitment in other models, as has recently been shown for
lymphocyte homing to peripheral lymph nodes (41).
Based on our new findings, best estimates can be given of the
quantitative parameters describing rolling in vivo (Table IV). A previous version of this table (6)
contained estimates based on data from gene-targeted mice combined with
Ab blockade. Table IV now lists the best estimates of rolling fluxes
and rolling velocities as mediated by individual selectins entirely
based on gene-targeted mice. The data listed are valid for the
indicated adhesion molecules at the expression levels seen under three
conditions, mild trauma, short term acute inflammation, and long term
acute inflammation. This table shows strong synergistic effects for the
selectins, because, possibly with the exception of trauma-induced
rolling, the sum of the leukocyte rolling fluxes produced by each
selectin is significantly less than the rolling flux observed in intact
mice. The new estimates are entirely consistent with the previous ones
(6), but the data are more complete and now for the first time include
estimates for 4 integrin-mediated leukocyte rolling.
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Acknowledgments |
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Footnotes |
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2 Current address: Parke-Davis, Inc., Pharmaceutical Research, Vascular and Cardiac Diseases Department, 2800 Plymouth Road, Ann Arbor, MI 48105.
3 Address correspondence and reprint requests to Dr. Klaus Ley, Department of Biomedical Engineering, University of Virginia, Box 377, Health Sciences Center, Charlottesville, VA 22908. E-mail address:
4 Abbreviations used in this paper: E-/-, E-selectin null; P-/-, P-selectin null; L-/-, L-selectin null; E/P-/-, E- and P-selectin null; L/P-/-, L and P-selectin null; L/E-/-, L- and E-selectin null; E/L/P-/-, L-, E-, and P-selectin null; WT, wild-type.
Received for publication February 8, 1999. Accepted for publication March 5, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-activated vascular endothelium under flow in vitro. J. Immunol. 157:326.[Abstract]
4-integrins in lymphocyte homing to mucosal tissues in vivo. J. Immunol. 152:3282.[Abstract]
4 integrin supports leukocyte rolling and adhesion in chronically inflamed postcapillary venules in vivo. J. Exp. Med. 183:1995.This article has been cited by other articles:
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  A. Shigeta, M. Matsumoto, T. F. Tedder, J. B. Lowe, M. Miyasaka, and T. Hirata An L-selectin ligand distinct from P-selectin glycoprotein ligand-1 is expressed on endothelial cells and promotes neutrophil rolling in inflammation Blood, December 15, 2008; 112(13): 4915 - 4923. [Abstract] [Full Text] [PDF]
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 A. Ramachandran, S. Jha, and D. J. Lefer REVIEW Paper: Pathophysiology of Myocardial Reperfusion Injury: The Role of Genetically Engineered Mouse Models Vet. Pathol., September 1, 2008; 45(5): 698 - 706. [Abstract] [Full Text] [PDF]
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S. Yakubenia, D. Frommhold, D. Scholch, C. C. Hellbusch, C. Korner, B. Petri, C. Jones, U. Ipe, M. G. Bixel, R. Krempien, et al. Leukocyte trafficking in a mouse model for leukocyte adhesion deficiency II/congenital disorder of glycosylation IIc Blood, August 15, 2008; 112(4): 1472 - 1481. [Abstract] [Full Text] [PDF]
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J. Chang, P. A. Shi, E. Y. Chiang, and P. S. Frenette Intravenous immunoglobulins reverse acute vaso-occlusive crises in sickle cell mice through rapid inhibition of neutrophil adhesion Blood, January 15, 2008; 111(2): 915 - 923. [Abstract] [Full Text] [PDF]
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 R. Sumagin and I. H. Sarelius A role for ICAM-1 in maintenance of leukocyte-endothelial cell rolling interactions in inflamed arterioles Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H2786 - H2798. [Abstract] [Full Text] [PDF]
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M. Osaka, S. Hagita, M. Haraguchi, M. Kajimura, M. Suematsu, and M. Yoshida Real-time imaging of mechanically injured femoral artery in mice reveals a biphasic pattern of leukocyte accumulation Am J Physiol Heart Circ Physiol, April 1, 2007; 292(4): H1876 - H1882. [Abstract] [Full Text] [PDF]
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 M. Phillipson, B. Heit, P. Colarusso, L. Liu, C. M. Ballantyne, and P. Kubes Intraluminal crawling of neutrophils to emigration sites: a molecularly distinct process from adhesion in the recruitment cascade J. Exp. Med., November 27, 2006; 203(12): 2569 - 2575. [Abstract] [Full Text] [PDF]
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S. R. McMeekin, I. Dransfield, A. G. Rossi, C. Haslett, and T. R. Walker E-selectin permits communication between PAF receptors and TRPC channels in human neutrophils Blood, June 15, 2006; 107(12): 4938 - 4945. [Abstract] [Full Text] [PDF]
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 D. Stengel, K. Bauwens, D. Keh, H. Gerlach, A. Ekkernkamp, R. Tauber, and T. Kerner Prognostic Value of an Early Soluble L-Selectin (sCD62L) Assay for Risk Assessment in Blunt Multiple Trauma: A Metaanalysis Clin. Chem., January 1, 2005; 51(1): 16 - 24. [Abstract] [Full Text] [PDF]
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R. C. O. Zanardo, C. S. Bonder, J. M. Hwang, G. Andonegui, L. Liu, D. Vestweber, L. Zbytnuik, and P. Kubes A down-regulatable E-selectin ligand is functionally important for PSGL-1-independent leukocyte-endothelial cell interactions Blood, December 1, 2004; 104(12): 3766 - 3773. [Abstract] [Full Text] [PDF]
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S.-C. Chen, C.-C. Huang, C.-L. Chien, C.-J. Jeng, H.-T. Su, E. Chiang, M.-R. Liu, C. H. H. Wu, C.-N. Chang, and R.-H. Lin Cross-linking of P-selectin glycoprotein ligand-1 induces death of activated T cells Blood, November 15, 2004; 104(10): 3233 - 3242. [Abstract] [Full Text] [PDF]
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 M. L. Smith, M. Sperandio, E. V. Galkina, and K. Ley Autoperfused mouse flow chamber reveals synergistic neutrophil accumulation through P-selectin and E-selectin J. Leukoc. Biol., November 1, 2004; 76(5): 985 - 993. [Abstract] [Full Text] [PDF]
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 Y. Huo, L. Zhao, M. C. Hyman, P. Shashkin, B. L. Harry, T. Burcin, S. B. Forlow, M. A. Stark, D. F. Smith, S. Clarke, et al. Critical Role of Macrophage 12/15-Lipoxygenase for Atherosclerosis in Apolipoprotein E-Deficient Mice Circulation, October 5, 2004; 110(14): 2024 - 2031. [Abstract] [Full Text] [PDF]
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 E. M. Rijcken, M. G. Laukoetter, C. Anthoni, S. Meier, R. Mennigen, H.-U. Spiegel, M. Bruewer, N. Senninger, D. Vestweber, and C. F. Krieglstein Immunoblockade of PSGL-1 attenuates established experimental murine colitis by reduction of leukocyte rolling Am J Physiol Gastrointest Liver Physiol, July 1, 2004; 287(1): G115 - G124. [Abstract] [Full Text] [PDF]
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 S. Szanto, I. Gal, A. Gonda, T. T. Glant, and K. Mikecz Expression of L-Selectin, but Not CD44, Is Required for Early Neutrophil Extravasation in Antigen-Induced Arthritis J. Immunol., June 1, 2004; 172(11): 6723 - 6734. [Abstract] [Full Text] [PDF]
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 D. Manka, S. B. Forlow, J. M. Sanders, D. Hurwitz, D. K. Bennett, S. A. Green, K. Ley, and I. J. Sarembock Critical Role of Platelet P-Selectin in the Response to Arterial Injury in Apolipoprotein-E-Deficient Mice Arterioscler Thromb Vasc Biol, June 1, 2004; 24(6): 1124 - 1129. [Abstract] [Full Text] [PDF]
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 I. E. Hoefer, N. van Royen, J. E. Rectenwald, E. Deindl, J. Hua, M. Jost, S. Grundmann, M. Voskuil, C. K. Ozaki, J. J. Piek, et al. Arteriogenesis Proceeds via ICAM-1/Mac-1- Mediated Mechanisms Circ. Res., May 14, 2004; 94(9): 1179 - 1185. [Abstract] [Full Text] [PDF]
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 A. D. Blann, S. K. Nadar, and G. Y.H. Lip The adhesion molecule P-selectin and cardiovascular disease Eur. Heart J., December 2, 2003; 24(24): 2166 - 2179. [Abstract] [Full Text] [PDF]
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 J. Frenette, N. Chbinou, C. Godbout, D. Marsolais, and P. S. Frenette Macrophages, not neutrophils, infiltrate skeletal muscle in mice deficient in P/E selectins after mechanical reloading Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2003; 285(4): R727 - R732. [Abstract] [Full Text] [PDF]
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M. Sperandio, M. L. Smith, S. B. Forlow, T. S. Olson, L. Xia, R. P. McEver, and K. Ley P-selectin Glycoprotein Ligand-1 Mediates L-Selectin-dependent Leukocyte Rolling in Venules J. Exp. Med., May 19, 2003; 197(10): 1355 - 1363. [Abstract] [Full Text] [PDF]
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A. E. R. Hicks, S. L. Nolan, V. C. Ridger, P. G. Hellewell, and K. E. Norman Recombinant P-selectin glycoprotein ligand-1 directly inhibits leukocyte rolling by all 3 selectins in vivo: complete inhibition of rolling is not required for anti-inflammatory effect Blood, April 15, 2003; 101(8): 3249 - 3256. [Abstract] [Full Text] [PDF]
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C. A. St. Hill, S. R. Alexander, and B. Walcheck Indirect capture augments leukocyte accumulation on P-selectin in flowing whole blood J. Leukoc. Biol., April 1, 2003; 73(4): 464 - 471. [Abstract] [Full Text] [PDF]
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K. E. Norman, M. J. Cotter, J. B. Stewart, K. B. Abbitt, M. Ali, B. E. Wagner, W. A. H. Wallace, S. B. Forlow, and P. G. Hellewell Combined anticoagulant and antiselectin treatments prevent lethal intravascular coagulation Blood, February 1, 2003; 101(3): 921 - 928. [Abstract] [Full Text] [PDF]
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S. B. Forlow, E. J. White, K. L. Thomas, G. J. Bagby, P. L. Foley, and K. Ley T Cell Requirement for Development of Chronic Ulcerative Dermatitis in E- and P-Selectin-Deficient Mice J. Immunol., November 1, 2002; 169(9): 4797 - 4804. [Abstract] [Full Text] [PDF]
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T. Hirata, B. C. Furie, and B. Furie P-, E-, and L-Selectin Mediate Migration of Activated CD8+ T Lymphocytes into Inflamed Skin J. Immunol., October 15, 2002; 169(8): 4307 - 4313. [Abstract] [Full Text] [PDF]
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 S. B. FORLOW, P. L. FOLEY, and K. LEY Severely reduced neutrophil adhesion and impaired host defense against fecal and commensal bacteria in CD18-/-P-selectin-/- double null mice FASEB J, October 1, 2002; 16(12): 1488 - 1496. [Abstract] [Full Text] [PDF]
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J. Kirveskari, M. Helinto, J. A. O. Moilanen, T. Paavonen, T. M. T. Tervo, and R. Renkonen Hydrocortisone reduced in vivo, inflammation-induced slow rolling of leukocytes and their extravasation into human conjunctiva Blood, August 28, 2002; 100(6): 2203 - 2207. [Abstract] [Full Text] [PDF]
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S. Hudak, M. Hagen, Y. Liu, D. Catron, E. Oldham, L. M. McEvoy, and E. P. Bowman Immune Surveillance and Effector Functions of CCR10+ Skin Homing T Cells J. Immunol., August 1, 2002; 169(3): 1189 - 1196. [Abstract] [Full Text] [PDF]
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M. Gironella, M. Molla, A. Salas, A. Soriano, M. Sans, D. Closa, P. Engel, A. Salas, J. M. Pique, and J. Panes The role of P-selectin in experimental colitis as determined by antibody immunoblockade and genetically deficient mice J. Leukoc. Biol., July 1, 2002; 72(1): 56 - 64. [Abstract] [Full Text] [PDF]
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D. A. Carlow, S. Y. Corbel, M. J. Williams, and H. J. Ziltener IL-2, -4, and -15 Differentially Regulate O-Glycan Branching and P-Selectin Ligand Formation in Activated CD8 T Cells J. Immunol., December 15, 2001; 167(12): 6841 - 6848. [Abstract] [Full Text] [PDF]
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S. B. Forlow, J. R. Schurr, J. K. Kolls, G. J. Bagby, P. O. Schwarzenberger, and K. Ley Increased granulopoiesis through interleukin-17 and granulocyte colony-stimulating factor in leukocyte adhesion molecule-deficient mice Blood, December 1, 2001; 98(12): 3309 - 3314. [Abstract] [Full Text] [PDF]
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K. SINGBARTL, S. B. FORLOW, and K. LEY Platelet, but not endothelial, P-selectin is critical for neutrophil-mediated acute postischemic renal failure FASEB J, November 1, 2001; 15(13): 2337 - 2344. [Abstract] [Full Text] [PDF]
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R. G. Collins, U. Jung, M. Ramirez, D. C. Bullard, M. J. Hicks, C. W. Smith, K. Ley, and A. L. Beaudet Dermal and pulmonary inflammatory disease in E-selectin and P-selectin double-null mice is reduced in triple-selectin-null mice Blood, August 1, 2001; 98(3): 727 - 735. [Abstract] [Full Text] [PDF]
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D. Bouvard, C. Brakebusch, E. Gustafsson, A. Aszodi, T. Bengtsson, A. Berna, and R. Fassler Functional Consequences of Integrin Gene Mutations in Mice Circ. Res., July 30, 2001; 89(3): 211 - 223. [Abstract] [Full Text] [PDF]
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E. E. Eriksson, X. Xie, J. Werr, P. Thoren, and L. Lindbom Importance of Primary Capture and L-Selectin-Dependent Secondary Capture in Leukocyte Accumulation in Inflammation and Atherosclerosis in Vivo J. Exp. Med., July 16, 2001; 194(2): 205 - 218. [Abstract] [Full Text] [PDF]
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M. Sperandio, A. Thatte, D. Foy, L. G. Ellies, J. D. Marth, and K. Ley Severe impairment of leukocyte rolling in venules of core 2 glucosaminyltransferase-deficient mice Blood, June 15, 2001; 97(12): 3812 - 3819. [Abstract] [Full Text] [PDF]
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S. B. Forlow and K. Ley Selectin-independent leukocyte rolling and adhesion in mice deficient in E-, P-, and L-selectin and ICAM-1 Am J Physiol Heart Circ Physiol, February 1, 2001; 280(2): H634 - H641. [Abstract] [Full Text] [PDF]
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J. Carvalho-Tavares, M. J. Hickey, J. Hutchison, J. Michaud, I. T. Sutcliffe, and P. Kubes A Role for Platelets and Endothelial Selectins in Tumor Necrosis Factor-{alpha}-Induced Leukocyte Recruitment in the Brain Microvasculature Circ. Res., December 8, 2000; 87(12): 1141 - 1148. [Abstract] [Full Text] [PDF]
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T. Hirata, G. Merrill-Skoloff, M. Aab, J. Yang, B. C. Furie, and B. Furie P-Selectin Glycoprotein Ligand 1 (Psgl-1) Is a Physiological Ligand for E-Selectin in Mediating T Helper 1 Lymphocyte Migration J. Exp. Med., December 4, 2000; 192(11): 1669 - 1676. [Abstract] [Full Text] [PDF]
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P. S. Frenette and L. Weiss Sulfated glycans induce rapid hematopoietic progenitor cell mobilization: evidence for selectin-dependent and independent mechanisms Blood, October 1, 2000; 96(7): 2460 - 2468. [Abstract] [Full Text] [PDF]
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P. Bauer, C. W. Lush, P. R. Kvietys, J. M. Russell, and D. N. Granger Role of endotoxin in the expression of endothelial selectins after cecal ligation and perforation Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2000; 278(5): R1140 - R1147. [Abstract] [Full Text] [PDF]
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E. J. Kunkel, J. L. Dunne, and K. Ley Leukocyte Arrest During Cytokine-Dependent Inflammation In Vivo J. Immunol., March 15, 2000; 164(6): 3301 - 3308. [Abstract] [Full Text] [PDF]
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 S. D. Robinson, P. S. Frenette, H. Rayburn, M. Cummiskey, M. Ullman-Cullere, D. D. Wagner, and R. O. Hynes Multiple, targeted deficiencies in selectins reveal a predominant role for P-selectin in leukocyte recruitment PNAS, September 28, 1999; 96(20): 11452 - 11457. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
WT) transplantation with L-/- bone
marrow (C, D), in P-/- mice before
(P-/-) and after
(L-/-
)
was significantly reduced to 63% compared with 90–95% in all
other groups (*, p < 0.05) and WT mice (dotted
lines) (7). Fraction of mononuclear cells (
) and
eosinophils (
) significantly increased (*,
p < 0.05).