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PHAGOCYTES
From the Department of Biomedical Engineering and
Cardiovascular Research Center, University of Virginia Health Sciences
Center, Charlottesville, VA; and the Departments of Medicine and
Molecular and Human Genetics, Baylor College of Medicine, Houston, TX.
Previously it was shown that During an inflammatory response, neutrophils roll
along the wall of inflamed venules before they come to a stop, adhere,
and transmigrate. The original paradigm of leukocyte adhesion held that
rolling and adhesion were separate, sequential steps.1 However, recent work has shown that the Although it is known that the Both LFA-1 and Mac-1 are known to bind to a number of ligands. One
LFA-1 ligand on endothelial cells is intracellular adhesion molecule-1
(ICAM-1).15 In addition, LFA-1 also binds to ICAM-2 on
endothelial cells and platelets and ICAM-3 on
lymphocytes.16 Mac-1 binds to ICAM-1, ICAM-2, and a number
of other ligands, including iC3b, factor X, fibrinogen, and many
denatured proteins.17,18 Although ICAM-1 is necessary for
neutrophil adhesion to unstimulated endothelium, it is probably not
significantly involved in either slow rolling3,19 or
chemoattractant-induced firm adhesion of leukocytes in inflamed
venules.20
Here, we test the hypothesis that LFA-1 and Mac-1 cooperate to slow
down rolling leukocytes. We examined leukocyte rolling during an
inflammatory response induced by TNF- Mice
Gene-targeted mice lacking CD1823 were obtained from Dr
Arthur L. Beaudet (Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX). Gene-targeted mice lacking LFA-121 or Mac-122 were obtained from Dr
Christie M. Ballantyne (Department of Medicine, Baylor College of
Medicine, Houston, TX). All mice were back-crossed into the C57BL/6
strain for at least 7 generations. Wild-type C57BL/6 mice were from
Hilltop Labs (Scottsdale, PA) or Jackson Laboratories (C57BL/6J) (Bar Harbor, ME). We used male mice with a mean age of 17 weeks and a mean
weight of 27 g. Six groups of mice were studied: wild-type, CD18 Reagents Murine recombinant TNF- (0.5 µg/mouse) was obtained from R
& D Systems (Minneapolis, MN). The blocking mAb M1/70, specific for the
M subunit of Mac-1 (rat IgG2b, 30 µg/mouse) was obtained from Pharmingen (San Diego,
CA).24 The LFA-1 mAb TIB-217 (rat IgG2a , 30 µg/mouse) was purified at the Lymphocyte Culture Center at the
University of Virginia from hybridoma supernatant (American Type
Culture Collection, Rockville, MD).25
Intravital microscopy Two hours before exteriorization of the cremaster muscle, all mice were injected intrascrotally with 0.5 µg TNF- in 0.30 mL
isotonic saline. Mice were anesthetized with ketamine hydrochloride (125 mg/kg; Sanofi Winthrop Pharmaceuticals, New York, NY), xylazine (12.5 mg/kg TranquiVed; Phoenix Scientific, St Joseph, MO), and atropine sulfate (0.025 mg/kg; Fujisawa USA, Deerfield, IL)
intraperitoneally. Mice were kept at 37°C; trachea, jugular vein (for
additional anesthetic), and one carotid artery (for blood sampling and
mAb injections) were cannulated with polyethylene tubing (Becton
Dickinson, Sparks, MD). After surgery, the cremaster muscle was
prepared for intravital microscopy, the epididymis and testis were
pinned to the side, and the cremaster was superfused with a
thermocontrolled (37°C) bicarbonate-buffered saline (131.9 mM NaCl,
18 mM NaHCO3, 4.7 mM KCl, 2.0 mM
CaCl2 · 2H2O, and 1.2 mM MgCl2)
equilibrated with 5% CO2 in N2. All
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 between 20 and 90 µm were videotaped through a CCD camera system (model VE-1000CD,
Dage-MTI, Michigan City, IN) for approximately 90 s/venule on a VHS
recorder (Panasonic AG-W1) for off-line analysis of leukocyte rolling
velocity and adhesion data. The vessel centerline blood velocity was
measured using a dual photodiode and a digital on-line cross
correlation program as previously described.26 Mean blood
flow velocity, Vb, was approximated by multiplying the
centerline blood velocity by a factor of 0.625.17 Wall
shear rate, w, was estimated as
w = 2.12 × 8 × [Vb/d],
where 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.28 Systemic leukocyte counts were
taken from 10 µL carotid blood samples throughout the experiment and
stained in 90 mL Kimura stain (11 mL toludine blue, 0.8 mL 0.03% light
green SF yellowish [Sigma, St Louis, MO], 0.5 mL saturated saponin
[Sigma] in 50% ethanol, and 5 mL 1/15 M phosphate buffer, pH 6.4) in
a hemocytometer to obtain absolute numbers of leukocytes per microliter
and a 2-part differential count (Table
2).
Flow cytometry Heparinized whole mouse blood was incubated with either LFA-1 mAb (M1/70) or Mac-1 mAb (TIB217) followed by a fluorescein isothiocyanate (FITC)-labeled secondary antibody (polyclonal antirat Ig, Pharmingen). Appropriate FITC-labeled isotype controls were used for each mAb (R35-95 for M1/70 and G15-337 for TIB217, both from Pharmingen).Data analysis Vessel diameter and length were measured using electronic calipers. Adherent leukocytes were defined being stationary for more than 30 seconds. Adhesion numbers are expressed per unit surface area of the vessel, assuming cylindrical geometry. In vessels larger than 40 µm, only one half of the vessel is in sharp focus, and the sampled surface area was approximated as a half-cylinder. Rolling velocities were measured for 10 leukocytes per vessel picked at random by viewing the translation during 2 seconds.For each of the groups studied, mean leukocyte rolling velocity and the
distribution of rolling velocities were determined. Quartile averages
were calculated and compared. To study the effects of shear rate on
rolling velocity and adhesion, individual venules for each group were
stratified into 2 shear rate ranges, low (200-500 s
To confirm that LFA-1 and Mac-1 proteins were absent in
LFA-1
Rolling velocity distribution To determine whether LFA-1 and/or Mac-1 modulated leukocyte rolling velocity, rolling velocity was measured in each of the experimental groups after 2 hours of stimulation with TNF- (Figure 2). Leukocytes in wild-type mouse venules
treated with TNF- rolled at an average velocity of 4.8 ± 0.3
µm/s. Leukocytes in CD18 / venules rolled
significantly faster than wild-type, with an average rolling velocity
of 28.5 ± 2.1 µm/s, confirming a key role for 2-integrins in slow rolling.2,5
To discern which of the Influence of wall shear rate To determine if shear rate had an effect on rolling velocity, rolling velocity data were stratified by shear rate into 2 groups, low shear rate (200-500 s 1) and high shear rate (500-1000 s 1) (Figure 3). Cumulative
frequency histograms for all experimental groups were created to
observe the dependence of rolling velocity on wall shear rate. In
wild-type mice, the median rolling velocity did not differ from the low
shear rate range (3.9 µm/s) to the high shear rate range (3.5 µm/s), suggesting that there is little dependence of rolling velocity
on shear rate in these mice. In CD18 / mice, rolling
velocity increased significantly (P < .05) from the low
shear rate range to the high shear rate range, almost doubling the
median rolling velocity from 17.3 µm/s to 33.4 µm/s. The median
rolling velocity in Mac-1 / mice increased from 4.7 µm/s in the low shear rate range to 8.5 µm/s in the high shear rate
range. In LFA-1 / mice, rolling velocities increased
from 5.7 µm/s in the low shear rate range to 6.5 µm/s in the high
shear rate range. At low shear rates (200-500 s 1),
rolling velocities in LFA-1 / mice averaged 9.3 ± 0.9
µm/s (Figure 3E) and were significantly higher than the rolling
velocity in Mac-1 / mice (6.8 ± 0.8 µm/s,
Figure 3C).
To investigate the cooperative effect of Mac-1 and LFA-1 on shear
rate-dependent rolling velocity, the rolling velocity was measured in
venules of Mac-1 To statistically analyze rolling velocities in more detail, averages
were calculated for the lowest, second, third, and highest quartiles
for both the low and higher shear rate venules (Figure 4). In all quartiles and both shear rate
ranges, the rolling velocity was highest in CD18
Role of LFA-1 and Mac-1 in leukocyte adhesion To assess the role of the 2-integrins on adhesion,
the number of adherent cells (stationary > 30 seconds) was determined in all venules, stratified by shear rate into low (200-500 s 1) and high (500-1000 s 1) groups. In
CD18 / mice, adhesion was influenced by shear rate,
decreasing from 1009 ± 311 adherent leukocytes/mm2 in
the low shear group to 422 ± 86 adherent leukocytes/mm2
in the high shear group. In wild-type mice, adhesion tended to decrease
from 770 ± 45 adherent leukocytes/mm2 in the low shear
group to 486 ± 104 adherent leukocytes/mm2 in the high
shear group, but the difference was not statistically significant.
Interestingly, the absolute numbers of adherent leukocytes were not
different among the groups tested, including CD18 /
mice. This is consistent with previous findings in that
mouse2 and suggests that mice lacking
2-integrins compensate for the severe adhesion defect by
increased systemic leukocyte counts.
Leukocyte adhesion efficiency The efficiency of neutrophil adhesion can be expressed as a ratio of the number of leukocytes available to adhere and those that do adhere.2 Consistent with previous reports,21-23 CD18 / mice showed a nearly
13-fold increase in circulating neutrophils compared to wild-type,
LFA-1 / mice showed almost a 5-fold increase, and
Mac-1 / showed a 4-fold increase (Figure
5B). Overall adhesion efficiency was
calculated by dividing the number of adherent by the number of
circulating neutrophils.2 We found that adhesion
efficiency was markedly and significantly decreased in
CD18 / mice as compared to wild-type mice. A similar
significant decrease is seen in the LFA-1 / mice. In
Mac-1 / mice, adhesion efficiency was significantly
decreased in venules with low wall shear rate only (Figure 5C). Taken
together, these data show that the adhesion impairment is more severe
in LFA-1 / than in Mac-1 / mice,
suggesting that LFA-1 is more important for leukocyte arrest than
Mac-1.
This study shows that removing either Mac-1 or LFA-1 from
mice leads to significantly increased average leukocyte rolling velocities in inflamed venules, but these velocities were still significantly lower than in CD18 Obviously, both Mac-1 and LFA-1 are important to slow down rolling
leukocytes. However, in the low shear rate range, LFA-1 is more
important to slow down the fastest rolling leukocytes, as additionally
blocking Mac-1 in LFA-1 These results show that Mac-1 and LFA-1 work cooperatively to slow down rolling leukocytes. Although many studies have examined the relative roles of these molecules in the later stages of inflammation, namely, firm adhesion and transmigration, this is the first study to elucidate the roles of Mac-1 and LFA-1 in leukocyte rolling. In previous work from our laboratory, we have shown that CD18 integrins are important for slow rolling2 and the conversion of leukocytes from rolling to adhesion.5 To address this conversion, we determined the impact of the absence of LFA-1 or Mac-1 on firm leukocyte adhesion. These data suggest that LFA-1 is more important than Mac-1 for inducing firm adhesion, although both LFA-1 and Mac-1 are involved in slow rolling. This is consistent with a recent study that determined the relative roles of Mac-1 and LFA-1 in adhesion to ICAM-1. Neutrophils were preincubated with saturating concentrations of anti-LFA-1 or anti-Mac-1 mAbs or both and then allowed to interact with ICAM-1-expressing cells in a cone-plate viscometer. LFA-1 accounted for the majority of cell adhesion capture under shear conditions, whereas Mac-1 supported stable adhesion over several minutes of chemotactic stimulation, suggesting that LFA-1 and Mac-1 may serve sequential rather than parallel functions.29 During an inflammatory response, mAbs to LFA-1 effectively
inhibit adhesion of PMNs to endothelial cells in culture that express ligands for LFA-1.30 In an in vitro static adhesion study
with neutrophils from Mac-1 Previous studies have suggested that adhesion via LFA-1 and Mac-1
proceed through independent pathways. Chimeras with extracellular and
transmembrane regions of the In conclusion, either Mac-1 alone or LFA-1 alone is sufficient to slow
down rolling leukocytes in TNF-
We thank Nick Douris, Jennifer Bryant, and Michele Kirkpatrick for animal husbandry. We would also like to thank Jim White for genotyping.
Submitted May 11, 2001; accepted September 4, 2001.
Supported by National Institutes of Health grants HL-54136 (K.L.), HL-42550 and HL-62243 (C.M.B.), and AI-32177 (A.L.B.). J.L.D. is supported by National Institutes of Health Training Grant T32 HL-07284 to B. R. Duling.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Klaus Ley, Department of Biomedical Engineering, University of Virginia, PO Box 800759, Charlottesville, VA 22908; e-mail: klausley{at}virginia.edu.
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