Departments of 1Genomics and Pathobiology and 3Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294; 2Department of Pathology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130; and4 Research Service, Birmingham Department of Veterans Affairs Medical Center, Birmingham, Alabama 35233
Submitted 18 July 2002 ; accepted in final form 3 March 2003
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ABSTRACT |
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intercellular adhesion molecule-1; cell adhesion; leukocytes; vascular endothelium; videomicroscopy
It is therefore interesting that in the ICAM-1 / mouse, leukocyte rolling is faster than in the wild type (22). This is despite the fact that purified ICAM-1 does not support rolling in vitro, except when ICAM-1 receptors are transfected into rolling cells that do not normally express them (14, 19). Normal T cells, expressing native lymphocyte function-associated antigen (LFA)-1 as an ICAM-1 receptor, do not roll at all when perfused over a purified ICAM-1 substrate under shear but immediately arrest (19). This apparent discrepancy can be reconciled if ICAM-1, rather than acting as an independent rolling receptor, cooperates with selectins to optimize the rolling process. In fact, in vivo experiments using mice deficient in multiple adhesion molecules demonstrated that ICAM-1 expression is required to optimize selectin-mediated rolling (22). However, the nature of this functional synergism between ICAM-1 and selectins (what ICAM-1 did to influence rolling velocity) was not determined in that system.
To understand how ICAM-1 might influence rolling, we used an in vitro flow system capable of high-temporal-resolution and high-spatial-resolution imaging with computerized motion tracking and analysis. Using the WEHI 274.1 monocytic cell line and cultured aortic endothelium from wild-type and ICAM-1 / mice, we found that average rolling velocity was increased in the absence of ICAM-1. High-temporal-resolution analysis demonstrated that these differences in rolling velocity were due to transient events with durations on a time scale of tens of milliseconds. Thus, although ICAM-1 is not necessary for rolling interactions, it modulates these interactions by stabilizing rolling. This function may be important for the efficient endothelial cell sampling that is necessary to initiate and regulate an inflammatory response.
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MATERIALS AND METHODS |
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In vitro rolling and adhesion assay. Cells of the WEHI 274.1 monocyte-like cell line were loaded with fluorescent dye by 20-min incubation at room temperature with 1 µM 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF; Molecular Probes, Eugene OR) in HBSS, resuspended in HBSS at 106 cells/ml, and loaded into a 25-ml syringe. A GlycoTech (Rockville, MD) flow chamber insert and gasket were inserted into the dish to form a laminar flow chamber that could be viewed on a microscope. Cells were injected into the flow chamber in HBSS at controlled physiological shear rates with a programmable syringe pump (KD Scientific, New Hope, PA). Cells were viewed on an Axiovert 100 microscope (Zeiss, Thornwood, NY) equipped with a charge-coupled device (CCD) camera (model 300T-RC; Dage-MTI, Michigan City, IN) and viewed as either phase-contrast or fluorescent images. Video was recorded onto sVHS videotape, and selected sequences were digitized to TIF files with the Perception video editing package (Perception PVR-2500; Digital Processing Systems, Markham, ON, Canada). Video images were analyzed to yield position measurements every 1/30 s with Metamorph software (Universal Imaging, West Chester, PA). Position measurements were processed by programs written for this purpose in D. F. Kucik's laboratory to determine velocities, accelerations, and arrest durations. Counts of rolling and firmly adherent cells were determined by visual review of video sequences and confirmed by computer analysis.
Calculation of critical rolling velocity. To distinguish cells
rolling on the endothelium via adhesive interactions from those freely flowing
in the perfusion buffer close to the endothelium, a critical rolling velocity
(Vcrit; Ref.
17) was calculated according
to Vcrit =r
, where
is shear
rate, r is the radius of the rolling cell, and
is a
dimensionless drag coefficient (in our case, 0.5) derived from theoretical
analysis of a sphere flowing close to a wall
(5). Any cell traveling at a
velocity below Vcrit is considered a rolling cell, as it
is assumed to be retarded by an adhesive interaction with the endothelium.
Western blot analysis. Western blotting analysis was performed as previously described (11, 12). Briefly, cell monolayers were lysed in 200 µl of RIPA buffer containing protease inhibitors (1 mM PMSF, 25 µg/ml aprotinin, 2 µg/ml leupeptin). Cell lysate protein concentrations were determined with the Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA). Forty micrograms of total protein from each sample was separated on an 8% SDS polyacrylamide gel, transferred to polyvinylidene difluoride (PVDF) membrane, and blocked at room temperature for 4 h. Anti-mouse-P-selectin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the membrane at a dilution of 1:200 and incubated at 4°C overnight. The membrane was washed and incubated with horseradish peroxidase (HRP)-linked donkey anti-goat IgG at a dilution of 1:2,000 for 2 h. The membrane was then washed and developed with the ECL chemiluminescence detection system from Amersham (Amersham Biosciences, Little Chalfont, UK).
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RESULTS |
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Imaging of rolling leukocytes on cultured endothelium. To relate adhesive behavior to biophysical events occurring on a millisecond time scale, we used high-speed digital image acquisition to capture large numbers of high-resolution images of rolling monocytic cells. Typically, 2,700 uncompressed, full-screen, 720 x 486-pixel images were captured at 30 images/s for each data set. The high-speed image acquisition revealed transient events, with durations of <100 ms, that would have been missed with a lower measurement frequency. High spatial resolution made possible accurate velocity and acceleration measurements, because of more accurate position measurements for each monocyte in each image. We combined this with computer analysis of thousands of images, making it practical to analyze a sufficient number of events to accurately calculate mean velocities, quantify acceleration events, and determine statistical significance. This system allowed us to detect and characterize transient events that provide insights into ICAM-1's role in leukocyte rolling, both on stimulated and unstimulated endothelium.
Images were obtained with both phase-contrast and fluorescent optics with a cooled CCD camera and digitized at high resolution (Fig. 1). Phase-contrast imaging was used to assess the integrity of the endothelial cell monolayer. Fluorescent imaging then enabled automated computer tracking of dye-loaded WEHI 274.1 cells. To accurately calculate velocities and accelerations, positions were determined for each cell 30 times/s for a 90-s video sequence. These position measurements were then converted from pixels to micrometers with a known size standard.
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ICAM-1 modulates monocyte rolling velocity on unstimulated aortic endothelium. Although monocyte interactions with endothelium are less frequent and weaker in the absence of endothelial cell stimulation, a number of adhesive interactions do occur. To determine the role of ICAM-1 in monocyte rolling on unstimulated endothelium, we calculated average rolling velocities for WEHI 274.1 cells rolling on either wild-type or ICAM-1-deficient endothelium. In our system, as in vivo (22), average rolling velocity was significantly greater on ICAM-1-deficient endothelium compared with wild-type endothelium (Fig. 2, A and B; P < 0.05 by Mann-Whitney test). A close examination of the velocity distribution revealed that the difference between wild-type and ICAM-1 / endothelium was not caused by a simple shift in the mean rolling velocity of the population but by an absence of slowly rolling cells on ICAM-1-deficient endothelium. To examine this difference in greater detail, we plotted instantaneous velocity (the velocity in each 0.033-s measurement interval) vs. time for individual cells (Fig. 2, C and D). Figure 2C illustrates transient arrests, typically of <1-s duration, that occur on wild-type endothelium. These transient arrests result in decreased average rolling velocity. Between these arrests, the cells roll at the same velocity on ICAM-1-sufficient and ICAM-1-deficient endothelium. Transient arrests occurred regularly on wild-type endothelium, as reflected by the number of cells with decreased rolling velocity in Fig. 2A, but were not observed on the ICAM-1-deficient endothelial monolayers.
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ICAM-1 modulates monocyte rolling velocity on
TNF--stimulated aortic endothelium. Stimulation of
endothelium by inflammatory cytokines results in a number of changes
including, but not limited to, increased expression of adhesion molecules. The
net effect is slower leukocyte rolling
(2). To understand the role of
ICAM-1 in monocyte interaction with inflamed endothelium, we measured average
velocities for WEHI 274.1 cells rolling on either wild-type or
ICAM-1-deficient endothelium after treatment with TNF-
, a potent
stimulator of endothelial cells (10 ng/ml for 6 h). As expected, average
rolling velocity was markedly decreased on both wild-type and ICAM-1-deficient
endothelium compared with unstimulated endothelium
(Fig. 3, A and
B; compare Fig. 2,
A and B). However, the median velocity on
ICAM-1-deficient endothelial cells was significantly greater than on wild-type
cells (P < 0.01 by Mann-Whitney test). To begin to understand the
underlying mechanism for this difference, we plotted instantaneous rolling
velocity profiles for individual cells
(Fig. 3, C and
D). Importantly, there were intervals in which rolling
velocity in the absence of ICAM-1 was similar to that on wild-type endothelium
(marked by gray bars in Fig.
3D). However, rolling on ICAM-1-deficient endothelium was
characterized by rapid acceleration events (RAEs) with durations averaging
0.1 s. During these RAEs, the rolling cells transiently accelerated to
velocities as high as 10-fold greater than the average on wild-type
endothelium.
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Quantitation of cell "jerkiness." RAEs represented
sudden accelerations that caused the motion of the rolling cells to appear
jerky when observed by video. To quantify this jerkiness, we calculated
instantaneous accelerations, i.e., the change in velocity between each 0.033-s
interval, for each rolling cell. Figure 4,
A and B, are representative instantaneous
acceleration plots for individual cells rolling on ICAM-1-sufficient and
-deficient endothelium, respectively. Although rolling cells exhibited
occasional RAEs even on wild-type endothelium, these events were much more
frequent on ICAM-1-deficient endothelium. Using the criterion that an
acceleration spike >1,000 µm/s2 (2 standard deviations
greater than the median acceleration on wild-type endothelium) constitutes an
RAE, we determined the number of events per cell
(Fig. 4C). Cells
rolling on ICAM-1-deficient endothelium had a significantly greater number of
RAEs than those rolling on wild-type endothelium (mean ± SE = 1.74
± 0.31 and 5.87 ± 0.75 for wild type and ICAM-1 deficient,
respectively; P < 0.01 by Mann-Whitney test).
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P-selectin expression is equivalent on wild-type and ICAM-1-deficient
endothelium. Work with gene-targeted mice showed that ICAM-1 and
P-selectin cooperate in rolling and leukocyte recruitment in vivo
(15). To confirm that
P-selectin expression was equivalent in wild-type and ICAM-1-deficient mouse
endothelium, we measured P-selectin levels by Western blot. P-selectin
expression was equivalent both before and after TNF- stimulation
(Fig. 5).
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ICAM-1 modulates frequency of firm adhesion, but not capture, of WEHI
274.1 monocytic cells on TNF--stimulated endothelium. To
determine the role of ICAM-1 in initial attachment and in firm adhesion of
monocytes to endothelium under flow, the number of cells perfused into the
flow chamber that rolled and/or adhered firmly was determined
(Fig. 6). Cells with average
velocities below the critical rolling velocity (see MATERIALS AND
METHODS) for >1 s were scored as rolling. Cells that moved <1 cell
diameter in 5 s were scored as adherent. Because, in all cases, cells were
perfused at a concentration of 0.5 x 106 cells/ml, and the
area of observation was the same for all measurements, the numbers of cells
counted reflect the fraction of cells flowing through the chamber that rolled
or arrested. Figure 6
demonstrates that expression of ICAM-1 by endothelial cells had no effect on
the likelihood that perfused WEHI cells would be captured and roll. However,
significantly fewer cells progressed to firm arrest on ICAM-1-deficient
endothelium (P < 0.01).
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DISCUSSION |
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We found that, even on unstimulated endothelium, monocytic cells displayed transient adhesion events, lasting 12 s each. These transient arrests, averaged over time, contributed to a slowing of rolling velocity. Genetic deletion of ICAM-1 led to loss of these transient events. It has been suggested that the amount of time required to traverse a local area may influence the likelihood that firm adhesion and subsequent transmigration will occur (8). We suggest that these momentary pauses may allow the cells more time to sample the local endothelial cell surface, which could be important early in the development of inflammation.
However, ICAM-1 modulated rolling to an even greater extent on
TNF--stimulated endothelium. As expected, on both wild-type and ICAM-1
/ endothelium, TNF-
treatment resulted in slower rolling.
Our results confirm that ICAM-1 is not itself essential to rolling adhesions,
because just as many cells rolled on the ICAM-1 / endothelium as
on the wild type. However, our high-resolution analysis demonstrated that
ICAM-1 changes the nature of the rolling interaction. That is, slow rolling is
stable on wild-type endothelium but punctuated by transient accelerations on
ICAM-1 / endothelial cells. These sudden accelerations account
for the differences in the average rolling velocity of the leukocytes on
wild-type vs. ICAM-1 / endothelium.
Importantly, between acceleration spikes, the monocytes on ICAM-1
/ endothelium rolled at the same velocity as on the wild-type
endothelium. In fact, for 30% of each individual cell rolling profile,
rolling on ICAM-1 / endothelium was indistinguishable from
rolling on wild-type endothelium. This is to be expected if the role of ICAM-1
is not to independently support rolling but to modulate it.
We suggest that the transient acceleration events represent escapes from the constraints on motion imposed by rolling adhesions. That is, in the absence of ICAM-1, selectins are insufficient to maintain close, uninterrupted contact between monocytes and endothelial cells, so the monocytes occasionally "jump." Similar jumps have been reported for mixed leukocytes rolling in vivo in the presence of L-selectin shedding (6).
Although several receptors bind simultaneously during cell rolling,
previous work using gene-targeted mice lacking multiple adhesion receptors
showed that the ICAM-1 effect on rolling velocity depends primarily on
cooperation with P-selectin
(15) and L-selectin
(22). The ICAM-1
/ mouse has been studied extensively, and this gene deletion has
not been shown to influence expression of other adhesion molecules. It is
formally possible, however, that the differences we observed in this study
between wild-type and ICAM-1 / mouse endothelium were partially
due to differences in expression of either P-selectin or endothelial ligands
for L-selectin (which is expressed on leukocytes). A number of molecules have
been shown to serve as L-selectin ligands on high endothelial vein (HEV)
endothelium, but L-selectin ligands on non-HEV endothelium have not been
defined (9). Therefore, it is
not possible to determine whether differential expression of L-selectin
ligands might play a role on the aortic endothelium used here. Expression of
P-selectin, however, both before and after TNF- stimulation, was
roughly equivalent on the wild-type and ICAM-1 / endothelium, as
determined by Western blot (Fig.
5).
Our findings are consistent with a recent study (7) examining the mechanism of increased leukocyte rolling velocity in mice deficient in the integrin LFA-1, a major receptor for ICAM-1. Because capture rates (number of cells engaging the endothelium and rolling) were similar to those in wild-type mice, those authors proposed that LFA-1 had no effect on the net "on" rate of cells binding to the endothelium. However, because LFA-1 influenced rolling velocity, they suggested that LFA-1 expression might affect the "off" rate, providing stability to transient selectin tethers slowing the velocity of rolling leukocytes. Similarly, we found that deficiency of ICAM-1, one of the ligands for LFA-1, had no effect on the attachment phase of the rolling interaction. However, our results demonstrate directly that ICAM-1 expression stabilizes leukocyte rolling on a time scale of tens of milliseconds. This effectively modulates the leukocyte off rate.
In summary, our results provide a direct demonstration that ICAM-1 stabilizes rolling interactions. Without ICAM-1 the endothelial surface remains capable of mediating monocyte rolling, but this rolling is perturbed. It has been suggested that slower rolling allows leukocytes to integrate signals and is therefore causally related to firm adhesion (8). Indeed, we found that firm adhesion was less frequent on ICAM-1-deficient endothelium, where rolling contact was more likely to be interrupted. Thus we propose that an important role for ICAM-1 in regulation of monocyte adhesion is to stabilize rolling interactions, damping out accelerations and decreasing rolling velocity. This cooperative function of ICAM-1 may have consequences for development of firm adhesion and eventual transmigration into tissues.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
Present address of C. G. Kevil: Department of Pathology, Louisiana State University Health Sciences Center, Shreveport, LA 71130.
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