High-temporal-resolution analysis demonstrates that ICAM-1 stabilizes WEHI 274.1 monocytic cell rolling on endothelium

Christopher G. Kevil,1 John H. Chidlow,2 Daniel C. Bullard,1 and Dennis F. Kucik3,4

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leukocyte rolling, adhesion, and migration on vascular endothelium involve several sets of adhesion molecules that interact simultaneously. Each of these receptor-ligand pairs may play multiple roles. We examined the role of ICAM-1 in adhesive interactions with mouse aortic endothelial cells (MAECs) in an in vitro flow system. Average rolling velocity of the monocytic cell line WEHI 274.1 was increased on ICAM-1-deficient MAECs compared with wild-type MAECs, both with and without TNF-{alpha} stimulation. High-temporal-resolution analysis provided insights into the underlying basis for these differences. Without TNF-{alpha} stimulation, average rolling velocity was slower on wild-type than on ICAM-1-deficient endothelium because of brief (<1 s) pauses. On TNF-{alpha}-stimulated ICAM-1-deficient endothelium, cells rolled faster because of transient accelerations, producing "jerky" rolling. Firm adhesion to ICAM-1-deficient MAECs was significantly reduced compared with wild-type MAECs, although the number of rolling cells was similar. These results demonstrate directly that ICAM-1 affects rolling velocity by stabilizing leukocyte rolling.

intercellular adhesion molecule-1; cell adhesion; leukocytes; vascular endothelium; videomicroscopy


LEUKOCYTE-ENDOTHELIAL CELL interactions during an inflammatory response are thought to occur through a series of steps, progressing from rolling to firm adhesion and finally migration into tissues (1). Previous work suggested that rolling and firm adhesion each use a specific set of adhesion molecules. Although there are differences among leukocyte types, the primary role of each adhesion molecule is similar in most systems. In neutrophils, for example, rolling is primarily mediated by the interaction of selectins with their carbohydrate ligands, whereas ICAM-1, a ligand for {beta}2-integrins (3, 4, 21, 24), is thought to be important for firm adhesion and migration (1, 18, 20).

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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Isolation and culture of endothelial cells. Endothelial cells purified from mouse aorta were isolated as previously described (10) in the laboratory of D. C. Bullard, according to a protocol approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee, and cultured in dishes precoated with 1% gelatin (Sigma, St. Louis, MO). The cells were maintained for 7 days in MCDB-131 medium supplemented with 10% FBS, bovine brain extract, heparin, hydrocortisone, and antibiotics to obtain confluent endothelial cell monolayers.

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 ={gamma}r{beta}, where {gamma} is shear rate, r is the radius of the rolling cell, and {beta} 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).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell system. In this study, we used WEHI 274.1, a well-established monocyte-like cell line (23) that has been used to study monocyte adhesion (23, 25). In previous work (13), we demonstrated that the adhesive properties of this cell line on TNF-{alpha}-stimulated endothelium were similar to those of whole blood monocytes. We used this cell line to investigate leukocyte interactions with cultured mouse aortic endothelial cells (MAECs) from both wild-type and ICAM-1-deficient mouse aortic endothelium. A major advantage of this genetic approach is that it avoids the possible nonspecific effects of antibodies or other inhibitors.

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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Fig. 1. Imaging of WEHI 274.1 cells rolling in vitro on TNF-{alpha}-treated mouse aortic endothelial cell (MAEC) monolayers. A and B: phase-contrast images of WEHI 274.1 cell adhesion (arrows) to 10 ng/ml TNF-{alpha}-stimulated wild-type and ICAM-1 –/– MAECs, respectively. C and D: a similar preparation imaged by fluorescence in wild-type (C) and ICAM-1 –/– (D) MAECs. Each preparation was imaged first by phase contrast to ensure that the endothelial cell monolayer was intact and then by fluorescence for computer tracking of rolling cells. Elongated, blurred images represent cells rapidly flowing in the perfusion buffer, not interacting with the endothelium.

 

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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Fig. 2. ICAM-1 modulates WEHI rolling velocity on unstimulated MAECs. A: scatterplot of WEHI 274.1 average rolling velocities on unstimulated wild-type and ICAM-1 –/– MAECs. There is a wide distribution of rolling velocities on the wild-type endothelium, whereas all cells roll rapidly on the ICAM-1-deficient endothelium (P < 0.05 by rank-sum test). B: relative frequency histograms of WEHI average rolling velocities on unstimulated MAEC. Open bars, wild-type MAECs; filled bars, ICAM-1 –/– MAECs. C: rolling velocity profile showing a transient arrest on wild-type MAECs. The slower cells on wild-type endothelium exhibit pauses, typically of <1-s duration, that dramatically decrease their average rolling velocities. D: rolling profile on unstimulated ICAM-1 –/– MAECs. Although there was some variability in rolling velocity, pauses were not observed.

 

ICAM-1 modulates monocyte rolling velocity on TNF-{alpha}-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-{alpha}, 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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Fig. 3. ICAM-1 modulates WEHI rolling velocity on wild-type MAECs. A: scatterplot of WEHI average rolling velocities on TNF-{alpha}-stimulated wild-type and ICAM-1 –/– MAECs. TNF-{alpha} treatment results in slower WEHI rolling compared with unstimulated MAECs (cf. Fig. 3). The effect is more pronounced with wild-type MAECs. B: relative frequency histograms of average rolling velocities on TNF-{alpha}-treated MAECs. Open bars, wild-type MAECs; filled bars, WEHI ICAM-1 –/– MAECs. C: sample WEHI cell rolling velocity profile on TNF-{alpha}-stimulated wild-type MAECs. D: sample rolling profile of WEHI cell on ICAM-1 –/– endothelium. Note that on ICAM-1-deficient endothelium, rolling has characteristic "jumps," i.e., short-duration (~0.1 s) periods of increased velocity. Rolling velocities between these jumps (gray bars), however, are similar to those on wild-type endothelium.

 

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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Fig. 4. WEHI acceleration profiles on TNF-{alpha} wild-type and ICAM-1 –/– MAECs. Acceleration profiles were generated to quantify "jerkiness" of WEHI rolling on TNF-{alpha}-stimulated wild-type and ICAM-1 –/– MAEC. A and B: typical acceleration profiles for WEHI cells rolling on wild-type and ICAM-1 –/– MAECs, respectively. C: quantitation of rapid acceleration events (RAEs) for WEHI cells on wild-type and ICAM-1 –/– endothelium. Acceleration >1,000 µm/s2 was considered an RAE. The difference between wild-type and ICAM-1 –/– endothelium is significant by the Mann-Whitney test (P < 0.05).

 

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-{alpha} stimulation (Fig. 5).



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Fig. 5. Equivalent expression of P-selectin on wild-type and ICAM-1 –/– endothelial cells. Expression of P-selectin (indicated by arrow) was measured both before and after TNF-{alpha} stimulation. Very little P-selectin was present on either wild-type or ICAM-1 –/– MAECs before stimulation. TNF-{alpha} treatment resulted in roughly equivalent increased expression of P-selectin for both cell types. Numbers on left indicate appropriate molecular weights.

 

ICAM-1 modulates frequency of firm adhesion, but not capture, of WEHI 274.1 monocytic cells on TNF-{alpha}-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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Fig. 6. Number of rolling and firmly adherent WEHI monocytes on TNF-{alpha} treated MAECs. A: number of rolling cells on wild-type and ICAM-1 –/– endothelium. The presence or absence of ICAM-1 on MAECs does not influence the number of monocytes that attach and roll. B: Firmly adherent cells. WEHI firm adhesion to ICAM-1 –/– MAEC is significantly decreased compared with wild-type MAEC, indicating that ICAM-1 is important for monocyte firm adhesion.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we used high-temporal-resolution imaging and analysis to demonstrate that ICAM-1 effects on cell rolling are caused by transient events. The ICAM-1-{beta}2-integrin interaction has long been thought to be important for firm adhesion and migration and not directly involved in rolling (1, 16, 18, 20). Recent experiments with mice deficient in each of these adhesion molecules provided cause to question this dichotomy, suggesting a role for ICAM-1 in rolling in vivo. In the current study, we provide direct evidence that, although ICAM-1 is not necessary for leukocyte rolling, it affects rolling velocity by stabilizing rolling interactions.

We found that, even on unstimulated endothelium, monocytic cells displayed transient adhesion events, lasting 1–2 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-{alpha}-stimulated endothelium. As expected, on both wild-type and ICAM-1 –/– endothelium, TNF-{alpha} 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-{alpha} 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.


    ACKNOWLEDGMENTS
 
This work was supported by National Institutes of Health Grants HL-10312 to C. G. Kevil and AR-46404 to D. C. Bullard and a Department of Veterans Affairs Merit Review Award to D. F. Kucik.


    FOOTNOTES
 

Address for reprint requests and other correspondence: D. F. Kucik, Univ. of Alabama at Birmingham, VHG019 Dept. of Pathology, 1670 University Blvd., Birmingham, AL 35294 (E-mail: kucik{at}uab.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.

Present address of C. G. Kevil: Department of Pathology, Louisiana State University Health Sciences Center, Shreveport, LA 71130.


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