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(Circulation Research. 1996;79:1196-1204.)
© 1996 American Heart Association, Inc.

Articles

Distinct Phenotype of E-Selectin–Deficient Mice

E-Selectin Is Required for Slow Leukocyte Rolling In Vivo

Eric J. Kunkel, Klaus Ley

the Department of Biomedical Engineering, University of Virginia School of Medicine, Charlottesville.

Correspondence to Klaus Ley, MD, University of Virginia School of Medicine, Department of Biomedical Engineering, Box 377, Health Sciences Center, Charlottesville, VA 22908. E-mail kfl3f@virginia.edu.

	Abstract

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Leukocyte capture and rolling are mediated by calcium-dependent lectins expressed on most leukocytes (L-selectin) and the vascular endothelium (P- and E-selectin). To study the role of the selectins during inflammation, we have investigated leukocyte rolling in venules of tumor necrosis factor- (TNF-)–treated mouse cremaster muscles in wild-type mice and gene-targeted mice with homozygous deficiency for L-, P-, or E-selectin (L-/-, P-/-, or E-/-, respectively). TNF- treatment induces expression of E-selectin and increases expression of P-selectin on endothelial cells. Consistent with previous reports of redundant P- and E-selectin function, a combination of monoclonal antibodies (mAbs) against P- and E-selectin (RB40.34 and 9A9, respectively) was necessary to block rolling in wild-type mice. The rolling leukocyte flux fraction (percent rolling cells) in L-/- mice was similar to that in wild-type mice, but rolling in these mice was blocked by a P-selectin mAb. The velocity of rolling leukocytes in TNF-–treated wild-type, P-/-, or L-/- mice was 5 to 10 times slower (3 to 7 µm/s) than during trauma-induced rolling (20 to 50 µm/s). In contrast, leukocytes in venules of TNF-–treated E-/- mice rolled significantly faster (12 to 20 µm/s); the rolling leukocyte flux fraction was more than doubled compared with wild-type, L-/-, or P-/- mice; and the number of adherent leukocytes was reduced. Addition of an E-selectin mAb, but not a P-selectin mAb, increased rolling flux fraction and rolling velocity in wild-type mice. Histological analysis revealed that 90% to 95% of all leukocytes interacting (rolling and adherent) with the venular endothelium in TNF-–treated wild-type, L-/-, P-/-, and E-/- mice were granulocytes. These results identify a previously unrecognized phenotype of E-/- mice by establishing that at the site densities prevailing in vivo, E-selectin is responsible for slow (5 µm/s) granulocyte rolling. E-selectin–dependent slow rolling drastically increases the transit time of leukocytes rolling through an inflamed tissue and thus aids in targeting leukocytes activated by chemoattractants to the inflammatory microenvironment.

Key Words: venule • microcirculation • inflammation • gene targeting • shear rate

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
During normal trafficking, and in order to reach inflammatory loci, free-flowing blood leukocytes must exit the vasculature and enter perivascular tissue, where they can then proceed to carry out their effector functions. In this process, leukocytes are slowed from the free-stream blood flow velocity (on the order of 1 to 10 mm/s in the microvasculature) before becoming fully stationary on the endothelium. The movement of leukocytes from the vasculature into extravascular tissue occurs in several clear sequential steps: morphological and rheological margination, capture, rolling, activation, firm adhesion, diapedesis, and tissue migration.^{1 2 3}

Leukocyte rolling is thought to serve two major functions. On one hand, rolling increases the transit time of leukocytes passing through venules, prolonging exposure to locally produced chemoattractants and thus promoting leukocyte activation and adhesion within a short distance after encountering these activators. On the other hand, rolling appears to be necessary to slow leukocytes down from the free-stream velocity before integrin-mediated firm adhesion can occur.⁴

Leukocyte capture and rolling in most tissues are mediated by a family of calcium-dependent mammalian lectins (L-, P-, and E-selectin)^{5 6 7 8} and their respective carbohydrate-rich ligands,^{9 10} although ₄ integrins can also mediate rolling.^{11 12 13} L-Selectin is constitutively expressed at the tips of microfolds on granulocytes,¹⁴ monocytes, and most lymphocytes and is enzymatically shed after leukocyte activation.¹⁵ P-Selectin, stored preformed in Weibel-Palade bodies, is rapidly expressed at the luminal surface of endothelial cells after stimulation with mediators such as histamine or thrombin.¹⁶ Stimulation of endothelial cells with the inflammatory cytokines TNF- or interleukin-1 induces de novo expression of E-selectin^{17 18} as well as increased P-selectin expression.^{18 19} The importance of the selectins in a normal inflammatory response is reflected in the moderate inflammatory deficiencies observed in mice lacking individual selectins^{20 21 22 23 24} and the severe inflammatory defects in mice with combined deficiencies.^{25 26 27} Many observations show that the selectins tend to function redundantly in mediating rolling in many tissues.^{8 22 28}

Several functional differences between the selectins regarding their ability to mediate rolling in vivo have been observed. In P-selectin–deficient mice, trauma-induced leukocyte rolling is initially absent,²⁰ and when rolling appears after 1 to 2 hours, the rolling flux fraction is very low (between 10% and 15% of the value seen in wild-type mice).^{8 28} Trauma-induced rolling in these mice is almost exclusively L-selectin dependent,⁸ with an average leukocyte rolling velocity that is three to five times faster (averaging 130 µm/s) than rolling in wild-type mice.²⁹ L-Selectin–transfected cells also roll less effectively than neutrophils when injected into rat mesenteric venules.³⁰ These data suggest that L-selectin alone is not sufficient to mediate leukocyte rolling at the typical velocities seen in vivo. Nevertheless, L-selectin is required for a normal inflammatory response, since mice deficient in L-selectin have impaired leukocyte recruitment in a peritonitis model of inflammation²¹ and reduced edema and leukocyte infiltration in a delayed-type hypersensitivity response.²⁴

In L-selectin–deficient mice, trauma-induced leukocyte rolling is normal initially^{8 21} but decreases significantly as time progresses.⁸ Trauma-induced rolling in these mice is exclusively P-selectin dependent,⁸ with a rolling velocity (averaging 40 µm/s) identical to that seen in wild-type mice.²⁹ P-Selectin–deficient mice have normal long-term neutrophil recruitment into inflammatory sites and show only a small reduction in mononuclear cell recruitment during a delayed-type hypersensitivity response.²³ These data indicate that although P-selectin is necessary for trauma-induced rolling, it is not critical for successful granulocyte recruitment during inflammation.

The functional role of E-selectin in mediating rolling during cytokine-dependent inflammation is less well understood, and E-selectin–deficient mice show no obvious phenotypic difference from wild-type mice.²² It has been reported that the velocity of rolling leukocytes is much lower in mesenteric venules of P-selectin–deficient mice with peritonitis,³¹ where E-selectin expression is likely. The reason for the apparent redundancy between P- and E-selectin in mediating leukocyte rolling during inflammation is unclear, and the possibility exists that each molecule has distinct functions in addition to these overlapping functions.

In order to explore the functional role of each of the selectins in mediating leukocyte rolling during inflammation, we have used TNF-–treated mouse cremaster muscles in wild-type and L-, P-, and E-selectin–deficient mice. TNF- treatment is known to increase the expression of P-selectin and induce the expression of E-selectin throughout the venular tree of the cremaster muscle (U. Jung and K. Ley, unpublished data). Predominantly granulocyte rolling and adhesion are induced in this model of acute inflammation. In the present study, we examine which of the selectins mediates the residual rolling in each genotype through mAb treatments and measure the rolling leukocyte flux fraction and leukocyte rolling velocity over the physiological shear rate range from 90 to 1400 s^-1. Our data show that E-selectin is required to mediate leukocyte-endothelial rolling interactions at velocities below 5 µm/s. This identifies a previously unrecognized phenotype of E-selectin–deficient mice and suggests that E-selectin serves to target leukocyte recruitment locally.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
mAbs and Cytokines
The following two mAbs were used in the present study: RB40.34 (rat IgG1, 30 µg per mouse), a mAb against murine P-selectin that completely blocks adhesion of HL-60 promyelocytes to immobilized P-selectin,³² and 9A9 (rat IgG1, 30 µg per mouse), a mAb against murine E-selectin that blocks HL-60 promyelocyte adhesion to transfected COS monolayers.³³ Murine recombinant TNF- (0.5 µg per mouse) was obtained from Genzyme Corp.

Animals
A total of 29 male mice >8 weeks in age and weighing between 21 and 47 g were used in the present study. Mice genetically engineered to be deficient in P-selectin,²⁵ L-selectin,²¹ and E-selectin²⁷ were prepared as described earlier by targeted gene disruption. The P-selectin– and L-selectin–deficient mice used in the present study were at least a fifth generation backcross onto a C57BL/6 background. The E-selectin–deficient mice were of a mixed C57BL/6x129Sv background. Control experiments were performed in age- and strain-matched wild-type mice. Rolling in C57BL/6 and C57BL/6x129Sv wild-type mice was indistinguishable under all conditions; therefore, the data from both strains were combined.

Intravital Microscopy
After premedication (given intraperitoneally) with 30 mg/kg sodium pentobarbital (Nembutal, Abbott Laboratories) and 0.1 mg/kg atropine (Elkins-Sinn, Inc), mice were anesthetized with an intraperitoneal injection of 100 mg/kg ketamine hydrochloride (Ketalar, Parke-Davis). All mice were pretreated 2 to 2.5 hours before surgery with an intrascrotal injection of 0.5 µg murine TNF- in 0.30 mL isotonic saline. Mice were prepared for the ensuing cremaster exteriorization surgery and data collection by cannulation of the trachea to facilitate spontaneous respiration, cannulation of the jugular vein for injection of anesthetic, antibodies, and saline, and cannulation of the carotid artery for blood pressure monitoring and blood sampling. During the experiment, mice were thermocontrolled at 36°C using an oral thermistor and monitoring unit (Thermalert TH-5 and TCAT-1A controller, Physitemp Instruments, Inc) and an infrared heat lamp. Blood pressure was monitored (model BPMT-2, Stemtech, Inc) and maintained in the range of 60 to 100 mm Hg.

The cremaster was prepared for intravital microscopy as described previously.^{28 34} If the artery-vein pair connecting the epididymis to the cremaster was a major feed-drainage pair, then the epididymis and testis were pinned to the side; otherwise, these vessels were pinched off and severed, and the testis was gently pushed back into the peritoneal cavity or pinned laterally. With either method, the cremaster was well perfused. The surgical procedure was completed in 6 to 10 minutes. During and after this procedure, the cremaster was superfused with thermocontrolled (36°C) bicarbonate-buffered saline as described previously.²⁸

An intravital microscope (Axioskop, Carl Zeiss, Inc) with a saline immersion objective (SW 40, 0.75 numerical aperture) was used to make microscopic observations. Venules with diameters between 15 and 80 µm were observed and recorded through a CCD camera system (model VE-1000CD, Dage-MTI, Inc) for 60 seconds each (S-VHS recorder, Panasonic). The centerline erythrocyte velocity in recorded microvessels was measured using a dual photodiode and a digital on-line cross-correlation program³⁵ running on an IBM-compatible computer system. Centerline velocities were converted to mean blood flow velocities by dividing the centerline velocity by an empirical factor of 1.6.³⁶ Throughout the experiment, 10-µL blood samples were withdrawn at 45-minute intervals from the carotid catheter and analyzed for leukocyte concentration (expressed as number of leukocytes per microliter of whole blood). Blood smears were stained with a three-step stain (LeukoStat, Fisher Scientific Co), from which a differential leukocyte count was obtained. Additional blood samples were taken, using the above procedure, before and after administration of mAbs and at the termination of the experiment.

Data Analysis
Microvessel diameter was measured from video recordings using a digital image–processing system.³⁵ The rolling leukocyte flux fraction was determined as described previously.²⁸ Briefly, the rolling leukocyte flux fraction was determined from video recordings by counting all visible cells passing through a plane perpendicular to the vessel axis and dividing this number by the total leukocyte flux through the vessel, which can be estimated by the product of the systemic leukocyte count, mean blood flow velocity, and estimated vessel cross-sectional area. Wall shear rate was estimated from mean blood flow velocity as described previously.³⁷ The rolling flux fraction represents the fraction of rolling leukocytes as a percentage of all leukocytes passing through the microvessel per unit time. Individual leukocyte rolling velocities were measured from video recordings by randomly choosing 5 to 15 leukocytes per venule and measuring the time necessary to travel a fixed distance (30 to 50 µm in TNF-–treated venules) using a digital image–processing system.³⁵ In vessels with large amounts of adhesion, leukocytes having rapid transient interactions with other adherent leukocytes were not chosen for analysis.

Histology
Whole-mount mouse cremaster muscles were Giemsa-stained as described previously.²⁹ Briefly, cremaster muscles prepared for intravital observation were laid flat on polylysine-coated glass slides and fixed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer (7.4 pH) for 24 hours at 4°C. Fixed tissues were then washed in 5% ethanol in 0.1 mol/L phosphate buffer and stained with Giemsa stain (Sigma Chemical Co) for 5 to 10 minutes. Staining contrast was enhanced with a solution of 0.01% acetic acid in 0.1 mol/L phosphate buffer. Tissues were then washed in water, dehydrated with ethanol, washed in xylene, and mounted using Permount (Fisher Scientific). Two-part leukocyte differentials (percent granulocytes and percent mononuclear leukocytes) were obtained in 250-µm-long segments of venules with diameters between 25 and 65 µm. As reported earlier,^{29 38} the cells present in each segment of venule represent both rolling and firmly adherent leukocytes.

Statistical Analysis
The dependence of leukocyte rolling flux fraction on hemodynamic parameters was analyzed using a multiple linear regression after hyperbolic transformation and an ANCOVA. Average leukocyte rolling flux fractions in control and treatment groups, systemic leukocyte counts and differentials between genotypes, and histological vessel leukocyte differentials were compared using an ANOVA followed by a Student-Newman-Keuls multiple comparison procedure. Comparison of rolling leukocyte velocity between groups was done using a nonparametric Kruskal-Wallis one-way ANOVA to determine significant differences, followed by Dunn's test for multiple comparisons. SPSS software (SPSS, Inc) was used for the above statistical analyses. Statistical significance was set at a value of P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
All mice appeared healthy and active and had no obvious abnormalities. During the 2- to 2.5-hour TNF- incubation period, mice exhibited no signs of pain or discomfort. Systemic leukocyte counts were lower than previously reported in untreated mice of the same genotype^{20 22 24} but not significantly different between genotypes (Table 1). This reduction is probably due to system-wide leukocyte adherence and diapedesis as well as possible bone marrow effects caused by the injected TNF-. Because of the difficulty in differentiating monocytes from lymphocytes by morphology alone, monocytes (constituting 2% to 3% of all circulating leukocytes in each genotype, P=NS between genotypes) and lymphocytes were combined and designated mononuclear leukocytes. The systemic percentages of neutrophils and mononuclear leukocytes remained similar (Table 1) to those seen in untreated mice, although the neutrophil percentage was consistently higher than previously reported.^{20 22 24 25} This may be due to recruitment of the sequestered granulocyte pool in these mice caused by the stress of anesthesia and surgery. The percentage of circulating neutrophils in E-selectin–deficient mice was significantly higher than in the other genotypes (Table 1). Differentiation of leukocytes in venules of fixed and Giemsa-stained tissues showed that granulocytes were the predominant (>90%, P=NS between genotypes) class of leukocytes interacting with the TNF-–stimulated venular endothelium in all investigated genotypes (Table 2).

View this table: [in this window] [in a new window]   Table 1. Systemic Leukocyte Counts and Two-Part Differentials After TNF- Treatment by Genotype

View this table: [in this window] [in a new window]   Table 2. Differentiation of Leukocytes Interacting With the Endothelium of TNF-–Stimulated Venules by Genotype

Hemodynamic Variation in Leukocyte Rolling Flux
Leukocyte rolling flux fraction was investigated in 124 venules of 7 TNF-–treated wild-type mice, 99 venules in 12 TNF-–treated L-selectin–deficient mice, 35 venules in 4 TNF-–treated P-selectin–deficient mice, and 60 venules in 6 TNF-–treated E-selectin–deficient mice. Venular diameters ranged from 22 to 70 µm, with an overall mean±SEM of 34±5 µm. The centerline erythrocyte velocity in these venules ranged from 0.3 to 6 mm/s and averaged 1.2±0.8 mm/s. Mean wall shear rates were similar in all investigated mouse genotypes (Table 3). The multiple linear regression correlation for TNF-–treated wild-type mice was used to correct flux fractions in all mice, as described previously.⁸ About 30% of the observed variation in the rolling leukocyte flux fraction in TNF-–treated wild-type mice was due to variation in microvascular parameters (venular surface-to-volume ratio and wall shear rate, data not shown).

View this table: [in this window] [in a new window]   Table 3. Average Hemodynamic Parameters in Venules of Different Mouse Genotypes

Selectin-Mediated Rolling in TNF-–Treated Cremaster Venules
Consistent with the fact that it is necessary to block P-selectin function in E-selectin–deficient mice in order to significantly reduce peritoneal leukocyte infiltration²² and that a mAb to E-selectin (9A9) ablates leukocyte rolling in mice lacking P-selectin expression,²⁸ we found that it was necessary to block both P- and E-selectin function in venules of TNF-–treated wild-type mice (Fig 1A) in order to significantly decrease leukocyte rolling (from 23.0±1.5% to 5.5±1.4%, P<.01). This finding is consistent with the lack of leukocyte rolling seen in TNF-–treated E-selectin/P-selectin double-mutant mice.^{26 27} The rolling flux fraction in TNF-–treated wild-type mice (25%) was consistent with previously published results^{8 28} and increased slightly (to 34.1±4.7%, P<.05) with the addition of a blocking E-selectin mAb, whereas no significant increase was seen with the addition of a blocking P-selectin mAb (28.0±2.6%, P=NS) (Fig 1A). The increase in the rolling leukocyte flux fraction after mAb blockade of E-selectin suggests that E-selectin may be involved in leukocyte arrest or adhesion. In E-selectin–deficient mice, the rolling flux fraction after TNF- treatment was found to be significantly higher than in TNF-–treated wild-type mice (58.3±6.2%, P<.05) and was almost totally P-selectin dependent (dropped to 0.7±0.4% after addition of the P-selectin mAb, P<.01) (Fig 1C).

View larger version (32K): [in this window] [in a new window]   Figure 1. Leukocyte rolling flux fraction in TNF-–treated wild-type and L-selectin– and E-selectin–deficient mice. Mice were pretreated with an intrascrotal injection of TNF- (0.5 µg) 2 to 2.5 hours before cremaster exteriorization. A, Leukocyte rolling flux fraction in wild-type mice (62 venules) was not affected by the addition of the blocking P-selectin mAb RB40.34 (19 venules, P=NS) but increased with the addition of the blocking E-selectin mAb 9A9 (22 venules, *P<.05). Consistent with redundant function of P- and E-selectin during cytokine-induced inflammation, both mAbs (RB40.34 and 9A9) were necessary to reduce the rolling flux fraction (21 venules, **P<.01) by 80% to 90%. Residual rolling may be due to L-selectin or selectin-independent mechanisms. B, The leukocyte rolling flux fraction in L-selectin–deficient mice was slightly lower than in wild-type mice (58 venules, P<.05) and was highly P-selectin dependent (23 venules, **P<.01). The addition of 9A9 did not affect the rolling flux fraction (10 venules, P=NS). The addition of both mAbs (RB40.34 and 9A9) had no additional effect on rolling flux fraction compared with the addition of RB40.34 alone (8 venules). C, The rolling flux fraction in E-selectin–deficient mice was almost three times higher than in wild-type mice (40 venules, #P<.05), possibly because of reduced firm adhesion, and was entirely P-selectin dependent (13 venules, **P<.01). Data are shown as mean±SEM.

L-Selectin plays a role in mediating rolling after surgical trauma^{8 29} and is necessary for neutrophil recruitment during inflammation.²⁴ Consistent with this, we found that the leukocyte rolling flux fraction in L-selectin–deficient mice was slightly lower than that in wild-type mice (15.7±1.2% versus 23.0±1.5%, P<.05.) (Fig 1B). Interestingly, leukocyte rolling in these mice was clearly more dependent on P-selectin than in wild-type mice, as the addition of a P-selectin mAb alone significantly reduced leukocyte rolling (to 4.4±0.6%, P<.01). In contrast, a blocking E-selectin mAb did not reduce rolling but increased it slightly (Fig 1B). Administration of the blocking P- and E-selectin mAbs together lowered the flux to the same level as the P-selectin mAb alone (3.2±1.0%, P<.01) (Fig 1B). These data suggest that lack of L-selectin renders TNF-–induced rolling more P-selectin dependent.

With these data, we show unequivocally that P- and E-selectin mediate rolling in TNF-–treated mice in a redundant manner. The higher rolling flux fraction seen in wild-type mice treated with a blocking E-selectin mAb (9A9) and in E-selectin–deficient mice is surprising and suggests a possible role for E-selectin other than mediating rolling, possibly in the initiation of firm adhesion (C.L. Ramos, E.J. Kunkel, M.B. Lawrence, U. Jung, D. Vestweber, R. Bosse, K.W. McIntyre, K.M. Gillooly, C.R. Norton, B.A. Wolitzky, and K. Ley, unpublished data). Similar to the reduction in leukocyte rolling seen in the absence of P- and E-selectin,^{26 27} lack of L-selectin in conjunction with blockade of P-selectin function also has a deleterious effect on leukocyte rolling in TNF-–treated venules, possibly by affecting leukocyte capture. Moreover, the lower leukocyte rolling flux fraction seen in L-selectin–deficient mice (Fig 1B) suggests that a step preceding rolling, such as capture, may be predominantly L-selectin dependent.

Velocity of Rolling Leukocytes in TNF-–Treated Cremaster Venules
We have analyzed the velocities of rolling leukocytes in venules of TNF-–treated wild-type and L-, P-, and E-selectin–deficient mice with similar hemodynamic conditions (Table 3). The velocity tracings of three representative leukocytes in a wild-type mouse (Fig 2A) show the slow steady rolling observed after TNF- treatment. The median rolling velocity in TNF-–treated wild-type mice was 3.2 µm/s (Fig 3), and the rolling leukocyte velocity distribution (Fig 4A) was highly right-skewed.

View larger version (29K): [in this window] [in a new window]   Figure 2. Velocity tracing of representative leukocytes from TNF-–treated wild-type and L-selectin–, P-selectin–, and E-selectin–deficient mice. TNF-–treated venules with similar critical velocities (Vcrit, 130 to 154 µm/s) were selected for each genotype, and the velocity of three rolling leukocytes was measured at discrete time intervals (0.5 seconds for panel D and 2.0 seconds for panels A, B, and C). A, Slow steady leukocyte rolling is shown in a venule of a wild-type mouse. B, Leukocytes rolling in venule of an L-selectin–deficient mouse show a slightly higher rolling velocity and more fluctuation. C, Leukocytes rolled in a wild-type mouse treated with RB40.34 () in a manner similar to that of leukocytes in a P-selectin–deficient mouse (). D, Leukocytes in an E-selectin–deficient mouse () and a wild-type mouse treated with 9A9 () rolled at five to six times higher velocities and with more fluctuation.

View larger version (20K): [in this window] [in a new window]   Figure 3. Median rolling leukocyte velocity in TNF-–treated wild-type and L-selectin–, P-selectin–, and E-selectin–deficient mice. Box plot of data from Fig 4 shows median (heavy line), 25th, and 75th percentiles (top and bottom of box) and the range of measured rolling velocities, excluding calculated outliers and extremes (whiskers). P-Selectin function was not involved in mediating slow rolling, since P-selectin–deficient mice and wild-type mice treated with RB40.34 were shown to have median velocities that were not different from those of wild-type mice. E-Selectin–deficient mice had a greatly increased median velocity (*P<.05 vs all others), as did wild-type mice treated with 9A9 (#P<.05 vs all others except E-selectin–deficient mice).

View larger version (44K): [in this window] [in a new window]   Figure 4. Rolling leukocyte velocity distributions in TNF-–treated wild-type and L-selectin–, P-selectin–, and E-selectin–deficient mice. Five to 15 rolling leukocytes were selected from each observed venule at random, and the time necessary for the cells to roll at fixed distance (30 to 50 µm) was measured. The distribution of rolling leukocyte velocity in L-selectin–deficient mice (D), P-selectin–deficient mice (E), and wild-type mice treated with RB40.34 (B) was not significantly different from the distribution in wild-type mice (A). E-Selectin–deficient mice (F) had a rolling velocity distribution that is shifted to higher velocities. Wild-type mice treated with 9A9 (C), like E-selectin–deficient mice, showed a shift to higher velocities but not to the same extent. At least 20 venules in four different mice were examined for each genotype and treatment. The last bar in all histograms represents all cells with velocities >50 µm/s. The total number of cells analyzed (n) is shown.

In order to understand whether a particular selectin was responsible for this slow rolling velocity, mice deficient in each selectin as well as mAb-treated wild-type mice were investigated. P-Selectin–deficient mice, as well as wild-type mice treated with the blocking P-selectin mAb RB40.34, exhibited the same slow rolling (Fig 2C) and rolling velocity distribution (Fig 4B and 4E). The median velocity was 3.5 µm/s in P-selectin–deficient mice and 3.7 µm/s in wild-type mice treated with RB40.34 (Fig 3). Interestingly, the velocity of typical leukocytes in E-selectin–deficient mice was five to six times higher than in wild-type mice (Fig 2D). The distribution of rolling velocities (Fig 4F) was much wider in these mice, with the median velocity being 17.1 µm/s (P<.05 versus wild-type and P- and L-selectin–deficient mice) (Fig 3). The addition of a blocking E-selectin mAb (9A9) to wild-type mice caused a similar, though less pronounced, increase in rolling velocity (Fig 2D), shifting the velocity distribution to higher velocities (Fig 4C) and increasing the median velocity (to 8.8 µm/s, P<.05 versus wild-type and L- and P-selectin–deficient mice) (Fig 3). These data show that E-selectin is responsible for the slow rolling present in mouse cremaster venules after treatment with TNF-.

The velocity of representative rolling leukocytes in L-selectin–deficient mice (Fig 2B) was only slightly higher than that in wild-type mice. Generally, these leukocytes also exhibited less steady rolling interactions and more velocity fluctuations. The distribution of individual leukocyte rolling velocities in L-selectin–deficient mice, however, is not different from that in wild-type mice (Fig 4D), and the median is only slightly higher (4.3 µm/s) (Fig 3).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The role of E-selectin in mediating leukocyte rolling and recruitment during inflammation has been difficult to ascertain because of its partial redundancy with P- and L-selectin.^{7 22 28} The TNF-–stimulated mouse cremaster muscle model provides evidence for a unique role for E-selectin in mediating rolling. We show clearly that although P- and E-selectin together are redundant in mediating rolling in TNF-–stimulated venules, E-selectin is required to mediate slow granulocyte rolling. Additionally, lack of L-selectin appears to reduce the efficiency of E-selectin function, as shown by the sensitivity of rolling to P-selectin antibodies in these mice. A summary of changes in the leukocyte rolling flux fraction and rolling leukocyte velocity when individual selectins or pairs of selectins are blocked can be seen in Table 4.

View this table: [in this window] [in a new window]   Table 4. Summary of Leukocyte Rolling Flux Fraction and Rolling Velocity After TNF- Treatment With Respect to Selectin Function Blocked

Similar to what is observed after acute trauma,^{29 38} the large majority (>90%) of leukocytes interacting with the venular endothelium in all genotypes (wild-type and L-, P-, and E-selectin–deficient mice) are granulocytes. Myeloid cells (including granulocytes) express functional ligands for both P- and E-selectin,^{39 40} and neutrophils, which make up the vast majority of granulocytes in mice, can interact readily with P- and/or E-selectin in vitro.⁴¹ These data would suggest that in the TNF-–treated mouse cremaster model of inflammation, E-selectin is mediating slow granulocyte rolling and participating in the transition from rolling to adhesion largely for granulocytes. Although limited subsets of lymphocytes express functional ligands for P- and/or E-selectin and can roll on these adhesion molecules in vitro,^{42 43 44} it is not possible to determine whether E-selectin mediates slow lymphocyte rolling in this in vivo model because of the low percentage of interacting mononuclear leukocytes. Lymphocytes are also known to be able to use ₄ß₁–VCAM-1 interactions to roll slowly and then adhere firmly in vitro.^{11 12 13} VCAM-1 can be induced in vitro as early as 2 hours after TNF- stimulation of endothelial cells⁴⁵ and may be expressed on venular endothelium of TNF-–stimulated mouse cremaster muscles as well.

Because the predominant type of leukocytes observed in this model is granulocytes, it remains unclear what role E-selectin plays in lymphocyte recruitment in vivo. It is possible that E-selectin may mediate slow rolling and the transition from rolling to adhesion for granulocytes while ₄ß₁–VCAM-1 interactions may be responsible for the same steps when dealing with lymphocyte recruitment. It is also possible that E-selectin may mediate slow rolling for all leukocytes able to bind to this molecule under physiological conditions. Further studies using different inflammatory models in which lymphocyte interactions are easily observed are necessary to clarify the role of E-selectin in lymphocyte recruitment.

Surprisingly, the rolling flux fraction in TNF-–treated E-selectin–deficient mice is almost threefold higher than in similarly treated wild-type mice and similar to the rolling flux fraction seen in wild-type mice after acute trauma. Interestingly, in both instances, rolling is mediated mostly by L- and P-selectin.^{8 28} The increase in rolling flux fraction in the absence of E-selectin suggests that the presence of E-selectin contributes in some way to leukocyte arrest and adhesion, which would remove leukocytes from the rolling pool. This is consistent with previous reports suggesting that E-selectin may have a role in leukocyte adhesion in vitro^{46 47} and in vivo (C.L. Ramos, E.J. Kunkel, M.B. Lawrence, U. Jung, D. Vestweber, R. Bosse, K.W. McIntyre, K.M. Gillooly, C.R. Norton, B.A. Wolitzky, and K. Ley, unpublished data, and Reference 48).

The leukocyte rolling velocity in venules of acutely exteriorized tissues in vivo is known to decrease at low wall shear rates.^{49 50 51 52} Throughout the upper physiological range, however, rolling velocity varies little with shear rate.^{49 50} In the present study, we used groups of venules with similar wall shear rates to reduce the influence of hemodynamic parameters (Table 2). In the group in which the highest rolling velocities were observed (E-selectin–deficient mice), the wall shear rate was not higher than in the other groups. This excludes the possibility that higher wall shear rates may have caused faster rolling in E-selectin–deficient mice.

An in vitro reconstitution assay has previously shown that neutrophils roll more slowly on immobilized E-selectin than P-selectin at similar site densities.⁴¹ In contrast, P-selectin expressed on CHO cells appears to support slower, more efficient rolling than E-selectin expressed on CHO cells at similar site densities.⁵³ Although we show here that slow leukocyte rolling is mediated by E-selectin, the question of unknown selectin and ligand site densities remains to be addressed in vivo. Data on selectin site densities in vivo are not currently available. Pretreatment with TNF- induces expression of both E-selectin and P-selectin on endothelial cells of wild-type mice,^{17 18 19} including endothelial cells in the cremaster muscle (U. Jung and K. Ley, unpublished data). Under these conditions, we observed slow leukocyte rolling. Similarly, leukocytes in TNF-–treated P-selectin–deficient mice, in which E-selectin is the only vascular selectin, roll slowly as well. Conversely, leukocytes in TNF-–treated E-selectin–deficient mice, in which only P-selectin is expressed on the vascular endothelium, roll much more rapidly. Therefore, it is reasonable to conclude that at the site densities prevailing in vivo, E-selectin mediates slow leukocyte rolling.

The reduced rolling velocity of leukocytes could be due to the binding characteristics of E-selectin. The selectins are thought to mediate leukocyte capture and rolling because of their rapid binding kinetics. A selectin molecule must bind its ligand rapidly, before the blood flow carries the cell out of range, and then release the bond at the trailing edge of the rolling cell. Slower leukocyte rolling could be due to a slower bond dissociation rate. Although data on the bond formation and dissociation rates for E-selectin are not available, the estimated bond dissociation rate for P-selectin is on the order of 3 s^-1,⁵⁴ and P-selectin mediates leukocyte rolling in vivo at velocities in the range of 30 to 50 µm/s.²⁹ The characteristic rolling velocities for each of the selectins are probably a function of both the selectin site density and the rate at which the receptor-ligand bonds are broken, which depends on bond kinetics and mechanical stresses. E-Selectin may have a slower bond dissociation rate than P-selectin, and/or it may be expressed at higher site densities than P-selectin. Greatly increased expression of P-selectin after stimulation with histamine⁵⁵ or leukotriene C₄⁵⁶ indeed causes a moderate reduction of leukocyte rolling velocity (from 40 to 20 µm/s) but is unable to produce slow rolling at 3 to 5 µm/s, as observed in the present study. This suggests that a slower bond dissociation rate for E-selectin is likely to be primarily responsible for the slow leukocyte rolling.

We also show that the rolling flux fraction in TNF-–treated L-selectin–deficient mice is highly dependent on P-selectin function. It is known that L-selectin alone is not sufficient to mediate leukocyte rolling at the velocities normally observed in vivo^{8 29} but that L-selectin function is necessary in order to have cells attach to and roll on E-selectin.⁵⁷ Our data suggest that when both L- and P-selectin function are blocked, cells may not be able to attach to and roll on E-selectin. The rapid expression of P-selectin¹⁶ and its sustained presence during an inflammatory response^{18 19} as well as its length⁵⁸ and the location of its ligand (P-selectin glycoprotein ligand-1) on leukocyte microfolds⁵⁹ support the concept that P-selectin may, like L-selectin, serve as a capturing molecule.² However, the slightly decreased rolling flux fraction in the absence of L-selectin also shows that both L- and P-selectin functions are necessary for optimal leukocyte-endothelium interactions. In the absence of P-selectin, L-selectin apparently is required to initiate leukocyte interaction, which can then result in sustained slow rolling mediated by E-selectin.

In summary, we show that E-selectin, at the site densities prevailing in vivo, mediates granulocyte rolling at a significantly lower velocity than either L- or P-selectin. Also, we show that either L- or P-selectin must be present to mediate efficient capture of free-flowing leukocytes before they can then roll on E-selectin. In the present study, we highlight a differential role for E-selectin in vivo and identify a unique phenotype of E-selectin–deficient mice. Slow leukocyte rolling (3 to 5 µm/s) as mediated by E-selectin greatly increases the transit time of leukocytes through microvascular networks. Leukocyte transit time is increased by a factor of 5 to 10 over trauma-induced leukocyte rolling (30 to 50 µm/s) and by a factor of 100 to 200 over noninteracting leukocytes (>400 µm/s). Because leukocyte activation in the presence of chemoattractant is thought to require a fixed amount of time, leukocyte adhesion and extravasation will occur nearer to the original site of exposure when leukocytes are rolling slowly. Slow E-selectin–mediated leukocyte rolling may serve to locally target an inflammatory response and may promote the transition from rolling to firm adhesion more rapidly after leukocytes encounter chemoattractant.

	Selected Abbreviations and Acronyms

 

CHO = Chinese hamster ovary mAb = monoclonal antibody TNF- = tumor necrosis factor- VCAM = vascular cell adhesion molecule

	Acknowledgments

 
This study was supported by National Institutes of Health grant HL-54136 to Dr Ley. Mr Kunkel was supported by National Heart, Lung, and Blood Institute grant T-32 HL-07284 (to B.R. Duling). We thank T.F. Tedder at Duke University for providing the L-selectin–deficient mice and D.C. Bullard and A.L. Beaudet at Baylor College of Medicine for providing the P- and E-selectin–deficient mice. We also thank D. Vestweber at the University of Munster for providing the mAb RB40.34 and B.A. Wolitzky at Hoffmann-La Roche, Inc, for providing the mAb 9A9. We also thank U. Jung for assistance in performing the histology.

Received July 16, 1996; accepted October 3, 1996.

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				Y. Yu, K. S. Moulton, M. K. Khan, S. Vineberg, E. Boye, V. M. Davis, P. E. O'Donnell, J. Bischoff, and D. S. Milstone E-selectin is required for the antiangiogenic activity of endostatin PNAS, May 25, 2004; 101(21): 8005 - 8010. [Abstract] [Full Text] [PDF]

				M. J. Smith, B. R. E. Smith, M. B. Lawrence, and K. R. Snapp Functional Analysis of the Combined Role of the O-Linked Branching Enzyme Core 2 {beta}1-6-N-Glucosaminyltransferase and Dimerization of P-selectin Glycoprotein Ligand-1 in Rolling on P-selectin J. Biol. Chem., May 21, 2004; 279(21): 21984 - 21991. [Abstract] [Full Text] [PDF]

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				H. S. Sakhalkar, M. K. Dalal, A. K. Salem, R. Ansari, J. Fu, M. F. Kiani, D. T. Kurjiaka, J. Hanes, K. M. Shakesheff, and D. J. Goetz Leukocyte-inspired biodegradable particles that selectively and avidly adhere to inflamed endothelium in vitro and in vivo PNAS, December 23, 2003; 100(26): 15895 - 15900. [Abstract] [Full Text] [PDF]

				E. S. Ong, X.-P. Gao, N. Xu, D. Predescu, A. Rahman, M. T. Broman, D. H. Jho, and A. B. Malik E. coli pneumonia induces CD18-independent airway neutrophil migration in the absence of increased lung vascular permeability Am J Physiol Lung Cell Mol Physiol, October 1, 2003; 285(4): L879 - L888. [Abstract] [Full Text] [PDF]

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				W. G. James, D. C. Bullard, and M. J. Hickey Critical Role of the {alpha}4 Integrin/VCAM-1 Pathway in Cerebral Leukocyte Trafficking in Lupus-Prone MRL/faslpr Mice J. Immunol., January 1, 2003; 170(1): 520 - 527. [Abstract] [Full Text] [PDF]

				L. G. Ellies, M. Sperandio, G. H. Underhill, J. Yousif, M. Smith, J. J. Priatel, G. S. Kansas, K. Ley, and J. D. Marth Sialyltransferase specificity in selectin ligand formation Blood, November 15, 2002; 100(10): 3618 - 3625. [Abstract] [Full Text] [PDF]

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				M.-C. Huang, O. Zollner, T. Moll, P. Maly, A. D. Thall, J. B. Lowe, and D. Vestweber P-selectin Glycoprotein Ligand-1 and E-selectin Ligand-1 Are Differentially Modified by Fucosyltransferases Fuc-TIV and Fuc-TVII in Mouse Neutrophils J. Biol. Chem., September 29, 2000; 275(40): 31353 - 31360. [Abstract] [Full Text] [PDF]

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