Importance of E-Selectin for Firm Leukocyte Adhesion In Vivo

From the Department of Biomedical Engineering, University of Virginia School of Medicine, Charlottesville (K.L., M.A., S.M.), and Department of Comparative Medicine, University of Alabama at Birmingham (D.C.B.).

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

	Abstract

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Abstract—Leukocyte adhesion under flow is preferentially mediated by the selectins. In this study we used intravital microscopy to investigate whether E-selectin may promote firm leukocyte adhesion in vivo. E-Selectin is expressed by endothelial cells activated with tumor necrosis factor- (TNF-) and causes slow leukocyte rolling. Microinjection of formyl-methionyl-leucyl-phenylalanine (fMLP) or macrophage inflammatory protein-2 (MIP-2) next to a venule of the TNF-–treated mouse cremaster muscle significantly increased the number of adherent leukocytes. In gene-targeted mice homozygous for a null mutation in the E-selectin gene or in wild-type mice treated with an E-selectin monoclonal antibody (mAb), this response was significantly attenuated (by >80%). No such defect was seen in intercellular adhesion molecule-1 (ICAM-1)–deficient mice. E-Selectin–null mice showed more rapid leukocyte rolling than wild-type or ICAM-1–deficient mice, resulting in significantly shortened leukocyte transit times through venules. Topical application of fMLP onto the whole cremaster muscle generated the same number of adherent leukocytes in wild-type and E-selectin–deficient mice. We conclude that slow leukocyte rolling through E-selectin results in long transit times, which are essential for efficient leukocyte adhesion in response to a local chemotactic stimulus.

Key Words: E-selectin • rolling • leukocyte transit • ICAM-1 • leukocyte adhesion

	Introduction

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The selectin family of adhesion molecules is critically involved in mediating leukocyte rolling, a prerequisite for leukocyte adhesion at sites of inflammation.^{1 2 3 4 5} Similarly, the selectins are thought to play a key role in postischemic injury.^{6 7} Previous studies have indicated that the selectins serve partially overlapping functions. This was most dramatically illustrated by the generation of mice homozygous for an induced null mutation in the E-selectin gene, which did not show obvious defects of inflammation unless P-selectin function was also blocked by a mAb⁸ or by an induced null mutation in the P-selectin gene.^{9 10} This overlap of E- and P-selectin function is also seen at the level of leukocyte rolling because most leukocyte rolling in inflamed venules of the cremaster muscle is blocked by injecting a mAb to P-selectin in E-selectin–deficient mice,¹¹ a mAb to E-selectin in P-selectin–deficient mice,¹² or a combination of P-selectin and E-selectin mAbs in wild-type mice.¹¹ These results are consistent with impaired leukocyte rolling and severely compromised inflammatory cell recruitment seen in mice deficient in both E- and P-selectin.^{9 10}

Although their functions are partially overlapping, distinct properties of E- and P-selectin exist. Recently, our group has shown that E-selectin supports leukocyte rolling at a typical velocity almost an order of magnitude lower than P-selectin at the site densities prevailing in vivo.¹¹ Injecting a function-blocking E-selectin antibody, mAb 9A9,¹³ increases the velocities of leukocytes rolling in venules of TNF-–treated wild-type mice. A different E-selectin antibody, mAb 10E9.6, has been shown to significantly reduce neutrophil recruitment to inflammatory sites¹⁴ but does not block E-selectin–dependent leukocyte rolling.^{12 15} Further study of this antibody revealed that it appears to inhibit a previously unknown function of E-selectin necessary for recruitment of leukocytes into some models of inflammation but not others.¹⁵ In addition, E-selectin has long been known to mediate adhesion of isolated neutrophils to endothelial cell monolayers in vitro in static assay systems.¹⁶ On the basis of these findings, we hypothesize that E-selectin may not only mediate leukocyte rolling but may also serve to promote firm leukocyte adhesion.

According to the multistep paradigm of leukocyte recruitment,^{2 3 4 5} firm leukocyte adhesion is mainly mediated through interactions between integrins expressed on leukocytes and their ligands expressed on the endothelial surface. Leukocyte integrins, including ß₂ and ₄, require conformational activation to efficiently support adhesive function.¹⁷ This conformational activation is thought to be induced by inside-out signaling processes secondary to leukocyte activation through chemoattractants. During inflammation, leukocytes can be activated by chemokines including MIP-2,^{18 19} formylated peptides,²⁰ and other peptide or lipid agonists. Both fMLP and MIP-2 activate neutrophils by binding to heptahelical, G-protein–coupled receptors on the neutrophil surface.¹⁸ After conformational activation, leukocyte integrins bind to various ligands on the endothelial surface, including ICAM-1 and ICAM-2 for ß₂ integrins and vascular cell adhesion molecule-1 for ₄ integrins.³ Treatment with TNF- is known to enhance expression of ICAM-1 and induce expression of E-selectin in venules of the mouse cremaster muscle.²¹ Locally targeted application of chemoattractants can be used to mimic the chemotactic stimulus. fMLP is a small, freely diffusible peptide²² and is therefore likely to enter the vascular space through the vessel wall. MIP-2 is a mouse homologue of interleukin-8²³ and is likely to be presented on the surface of endothelial cells after specific uptake and transport, as shown for human interleukin-8.²⁴ Micropipette injection of fMLP or MIP-2 into the interstitial tissue next to a venule induces local leukocyte activation and firm leukocyte adhesion.^{25 26} In the present study, we used gene-targeted mice to investigate (1) whether E-selectin may promote firm adhesion of leukocytes under in vivo conditions and (2) whether increased transit times secondary to reduced rolling velocities supported by E-selectin are critical for efficient conversion of leukocyte rolling to firm adhesion. We show that (1) functional blockade or absence of E-selectin reduces the recruitment of leukocytes in response to locally applied formyl peptide or MIP-2 and (2) leukocyte adhesion is restored when fMLP is superfused across the entire preparation, allowing prolonged exposure of leukocytes to chemoattractant.

	Materials and Methods

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Animals
In the present study, we used 28 male mice >8 weeks in age with a mean±SD weight of 29±5 g. Mice carrying a null mutation in the E-selectin gene were described earlier.¹⁰ Mice heterozygous for the E-selectin mutation were interbred, and viable and healthy homozygous E-selectin–deficient offspring were obtained. This was true both in a mixed 129Sv/C57BL/6 background and after back-crossing into the C57BL/6 background for five generations. The ICAM-1 mutant mice were described previously²⁷ and were back-crossed into the C57BL/6 background for 12 generations. Some wild-type mice were pretreated with an intravenous injection of 30 µg of mAb 9A9¹³ suspended in saline. This dose of mAb 9A9 has previously been shown to completely block E-selectin–mediated leukocyte rolling in mouse cremaster muscle venules.¹¹ All experiments were conducted under a protocol approved by the Institutional Animal Care and Use Committee.

Intravital Microscopy
Mice were anesthetized with 30 mg/kg sodium pentobarbital (Nembutal, Abbott Laboratories), 0.1 mg/kg atropine (Elkins-Sinn, Inc), and 100 mg/kg ketamine hydrochloride (Ketalar, Parke-Davis). All mice were pretreated 2 to 2.5 hours before surgery with an intrascrotal injection of 100 ng murine TNF- (Genzyme) in 0.30 mL isotonic saline. In preliminary experiments, this dose of TNF- was shown to be sufficient to induce E-selectin expression and slow leukocyte rolling (data not shown). This dose is five times lower than that used in previous studies of this same preparation.^{11 12 28} The trachea, one jugular vein, and one carotid artery were cannulated. Mice were thermocontrolled at 36°C with the use of an oral thermistor and monitoring unit (Thermalert TH-5 and TCAT-1A Controller, Physitemp Instruments) and an infrared heat lamp. Blood pressure was monitored with a carotid catheter (model BPMT-2, Stemtech, Inc) and ranged between 60 and 100 mm Hg.

The cremaster was prepared for intravital microscopy as described¹¹ without severing the major artery-vein pair connecting the epididymis to the cremaster. During and after surgery, the cremaster was superfused with thermocontrolled (36°C) bicarbonate-buffered saline as described.¹² An intravital microscope (Axioskop, Carl Zeiss, Inc) with a saline immersion objective (SW 40, 0.75 numerical aperture) was used to record venules with diameters between 26 and 75 µm through a CCD camera system (model VE-1000CD, Dage-MTI, Inc) on an S-VHS video recorder (S-VHS recorder, Panasonic). The centerline erythrocyte velocity in recorded microvessels was measured with the use of a dual photodiode and a digital on-line cross-correlation program²⁹ running on an IBM-compatible personal computer. Centerline velocities were converted to mean blood flow velocities, V_b, by dividing by an empirical factor of 1.6.³⁰ Wall shear rates, _w, were estimated as _w=2.12 (8V_b/d), where d is the in vivo diameter of the vessel and 2.12 is a median empirical correction factor obtained from velocity profiles measured in microvessels in vivo.³¹ Small blood samples (10 µL each) were obtained from the carotid catheter and analyzed for leukocyte concentration and two-part differentials in a hemocytometer with the use of Kimura stain.

Leukocyte rolling velocities and vessel diameters were measured from videotapes with the aid of a custom-designed digital imaging processing system.²⁹ To accurately observe the movements of rolling leukocytes, freeze frame advance was used. Since the time interval chosen to measure leukocyte rolling velocities can influence the shape of the observed distribution,³² we followed each leukocyte for a fixed time interval of 2 seconds to exclude potential bias introduced by different time intervals. The number of firmly adherent leukocytes was determined from video recordings and expressed as the number of leukocytes remaining stationary for 30 seconds in a 200-µm segment of venule. The number of adherent leukocytes was normalized for the systemic leukocyte counts in each mouse.

Local and Topical Application of Chemoattractant
Glass pipettes were drawn from standard borosilicate glass with an outer diameter of 1.0 mm (Stoelting) on a vertical pipette puller (Stoelting) as described.²⁶ The tip was beveled to a diameter of 10 to 14 µm with the use of a micropipette grinder (model EG-40, Narishige) with a 0.3-µm abrasive foil (No. 6775, AH Thomas). The pipettes were filled with 10 µL of either fMLP (10 µmol/L; Sigma) or MIP-2 (also known as KC¹⁹; 200 nmol/L, Austral Biologicals). These concentrations are 10-fold higher than the optimum concentration for either chemoattractant in vitro, allowing for 10-fold dilution of the injected fluid by tissue and superfusion fluids. A system composed of a 10-mL syringe connected to a three-way stopcock and tubing attached directly to the pipette in the pipette holder served as an air pressure reservoir. The pipette tip was placed within 30 µm of the vessel with a piezo-driven micromanipulator (model DC-3k, Märzhäuser-Wetzlar) so that the beveled tip penetrated the interstitial tissue. Once the pipette was in place, the vessel was recorded for 60 seconds. Air pressure was applied to the pipette via the tubing/syringe setup, causing injection of some of the pipette content (<1 µL), which was verified by observing visible swelling of the interstitial tissue surrounding the pipette tip. The vessel was recorded before, during, and after the 60-second infusion of chemoattractant for a total of 3 minutes. This mode of application results in exposure of a limited segment of venule to the chemoattractant diffusing away from the site of application (Figure 1A).

View larger version (75K): [in this window] [in a new window]   Figure 1. Experimental design for local application of chemoattractant via micropipette, exposing only a limited segment of venule as indicated by the black bar (A). Topical application (B) leads to exposure of the entire cremaster to a uniform concentration of chemoattractant (shaded area). At any given rolling velocity, the transit time during which the rolling leukocytes (light) are exposed to chemoattractant is much longer in panel B than in panel A. Leukocytes becoming adherent in response to chemoattractant are shown in black.

In separate experiments, fMLP was applied topically by replacing the superfusion buffer with buffered saline containing fMLP (1 µmol/L) for 1 minute on the entire cremaster preparation. In these experiments, the same four or five venules were investigated before and after application of chemoattractant, and the difference in the number of adherent cells was determined. This results in exposure of the whole cremaster muscle to a uniform concentration of chemoattractant (Figure 1B). Application of vehicle by either micropipette or topical exposure had no effect (data not shown).

Statistical Analysis
Comparison of numbers of adherent cells and of rolling leukocyte velocities between groups was done with the use of ANOVA to determine significant differences, followed by Newman-Keuls multiple comparison test. The velocity and transit time histograms were compared with the Kolmogorov-Smirnov test. To assess local differences in distributions, truncated distributions were analyzed (ie, all cells with velocities <10 µm/s or >40 µm/s, all cells with transit times >3 seconds). NCSS 6.0 software (Jerry Hintze, 1995) was used for statistical analyses.

	Results

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All mice used were clinically healthy and showed no obvious defects. In each group, we investigated between 12 and 21 venules in five to nine mice per group. The hemodynamic characteristics of the investigated venules were similar (Table 1). As described previously,¹¹ large numbers of slowly rolling leukocytes are observed in venules of the TNF-–treated cremaster muscle. In addition, some leukocytes remained stationary for 30 seconds (firmly adherent). The number of adherent leukocytes per venule was similar in E-selectin–deficient, ICAM-1–deficient, and wild-type mice with or without treatment with the blocking E-selectin mAb 9A9 (Figure 2). This suggests that the absence or blockade of either one of these adhesion molecules does not reduce firm leukocyte adhesion in response to TNF- in mouse cremaster venules in vivo.

View this table: [in this window] [in a new window]   Table 1. Systemic Leukocyte Counts After Intrascrotal Injection of 100 ng TNF-

View larger version (14K): [in this window] [in a new window]   Figure 2. The number of adherent leukocytes per 200-µm segment of venule in TNF-–treated cremaster muscle (before application of chemoattractant) is not affected by absence of E-selectin (E-sel.) or ICAM-1. Leukocyte adhesion was measured in 20 venules of 7 wild-type mice, 12 venules of 4 wild-type mice treated with the blocking E-selectin mAb 9A9, 21 venules of 5 E-selectin–deficient mice, and 18 venules of 6 ICAM-1–deficient mice.

Consistent with previous studies conducted in the same model,¹¹ 90% of all adherent and rolling leukocytes in mouse cremaster venules were polymorphonuclear granulocytes in wild-type, E-selectin–deficient, and ICAM-1–deficient mice. The systemic leukocyte counts and differentials measured in blood drawn from the carotid artery are shown in Table 1. As reported previously,^{27 33} ICAM-1–deficient mice show a mild leukocytosis. After TNF- treatment, the fraction of circulating polymorphonuclear granulocytes was found to be elevated in E-selectin–deficient mice, consistent with previous findings.¹¹

We analyzed the velocities of leukocytes rolling in hemodynamically similar venules of the cremaster muscle of wild-type, E-selectin–deficient, or ICAM-1–deficient mice treated with 100 ng of TNF- intrascrotally. The distribution of leukocyte rolling velocities is shown in Figure 3A. The median rolling velocity was 18 µm/s in wild-type mice, which was not quite as low as the rolling velocity prevailing in wild-type mice treated with 500 ng of TNF-¹¹ but significantly lower than the leukocyte rolling seen in this tissue without TNF- treatment.³⁴ A slight but significant (P<0.01) reduction in the fraction of slowly rolling leukocytes (<10 µm/s) was seen in ICAM-1–deficient mice (14%) compared with wild-type mice (33%), suggesting that ICAM-1 may contribute to supporting leukocyte rolling at very low velocities (Figure 3B). This lack of very slowly rolling cells had little impact on the median leukocyte rolling velocity, which was 24 µm/s in ICAM-1–deficient mice. More strikingly, the leukocyte rolling velocity was significantly (P<0.01) increased to a median of 40 µm/s in mice lacking E-selectin (Figure 3C). This is in agreement with an earlier study conducted in the same preparation at a higher dose of TNF-.¹¹ Figure 3D shows the cumulative histogram of the leukocyte rolling velocity distributions seen in the three groups to facilitate direct comparison between the groups. The E-selectin–deficient mice show >2-fold higher leukocyte rolling velocities than wild-type or ICAM-1–deficient mice. The elevation of mean rolling velocity was mainly due to a relative increase in the number of rapidly rolling leukocytes. In E-selectin–null mice, 50% of the leukocytes rolled at velocities >40 µm/s, whereas this fraction was only 21% in wild-type mice (P<0.01).

View larger version (28K): [in this window] [in a new window]   Figure 3. Rolling velocities measured in hemodynamically similar venules over a constant 2-second time window for each cell. A, Leukocytes (n=129) rolling in venules of wild-type mice; mean velocity, 27.5±2.6 µm/s. B, Similar rolling velocity (32.3±2.7 µm/s) in ICAM-1–deficient mice, but the fraction of cells rolling at <10 µm/s is reduced from 32% in wild-type mice to 14% in ICAM-1–deficient mice; n=121 leukocytes. C, Significant increase (P<0.01) of rolling velocity in E-selectin–deficient mice to 47.2±4.1 µm/s; n=105 rolling leukocytes. D, Cumulative histogram of rolling velocities allows direct comparison of distribution among groups.

In wild-type mice treated with TNF-, micropipette application of fMLP or MIP-2 induced a significant increase of leukocyte adhesion in the venule next to the micropipette beyond the level of adhesion induced by TNF- treatment. Leukocyte adhesion increased rapidly (within <1 minute) and was not significantly dependent on the number of leukocytes already adherent (data not shown). Chemoattractant application had no significant effect on blood flow velocity or wall shear rate (Table 2). Microinjection of vehicle did not induce any leukocyte adhesion (data not shown). Figure 4 shows the number of leukocytes becoming adherent in response to microinjection of fMLP (Figure 4A) or MIP-2 (Figure 4B), normalized by the systemic leukocyte count in each group, as shown in Table 1. The responses to MIP-2 and fMLP were qualitatively and quantitatively similar to each other. When the responses to fMLP were compared across genotypes, a 82% reduction of the adhesion response was seen in E-selectin–deficient mice (P<0.05) (Figure 4A). To ensure that this reduction was not limited to fMLP, we repeated this experiment using MIP-2, a chemoattractant that can be presented on the endothelial surface²⁴ and binds to a different receptor on the leukocyte.¹⁸ We found a similar reduction of induced leukocyte adhesion by 90% in E-selectin–null mice (P<0.01) (Figure 4B). The adhesion response in ICAM-1–deficient mice was not different from that in wild-type mice. We also tested leukocyte adhesion in response to fMLP microinjection in P-selectin–deficient mice¹⁰ treated with 100 ng TNF- intrascrotally and found similar numbers of leukocytes becoming adherent as in wild-type mice (30±4 leukocytes per venule, six venules in two P-selectin–deficient mice, not significantly different from 34±12 leukocytes in wild-type mice). These findings indicate that E-selectin is required to promote firm leukocyte adhesion in response to chemoattractant.

View larger version (25K): [in this window] [in a new window]   Figure 4. Number of leukocytes becoming adherent on microinjection of chemoattractants next to venules of the TNF-–treated mouse cremaster muscle. A, Microinjection of fMLP (10 µmol/L, 1 minute) induces adhesion of 32 leukocytes in a 200-µm segment of venule. This is significantly reduced by E-selectin mAb 9A9 and in E-selectin–deficient mice (*P<0.05). B, Microinjection of MIP-2 (200 nmol/L, 1 minute) causes a similar response, which is attenuated by 90% in E-selectin–deficient mice (**P<0.01).

To ensure that the deficit in leukocyte adhesion in response to chemoattractant was truly caused by the absence of E-selectin and not a consequence of gene targeting, we treated wild-type mice with mAb 9A9, a mAb to E-selectin known to block E-selectin–dependent leukocyte rolling.^{11 13} Similar to the observations made in E-selectin–deficient mice, intravenous injection of mAb 9A9 blocked 84% of the leukocyte adhesion response to fMLP (Figure 4A). Application of an isotype-matched control antibody had no effect (data not shown).

Since the rolling velocities in TNF-–treated venules are much higher in the absence of E-selectin (Figure 3 and Reference 11¹¹ ), the transit time of a leukocyte through any given segment of venule is correspondingly shorter in E-selectin–deficient mice than in wild-type mice. A small molecule like fMLP or MIP-2 applied by micropipette penetrates the interstitial tissue and percolates and diffuses away from the pipette in a radial fashion, exposing a limited segment of venule to chemoattractant (Figure 1). In the present set of experiments, all microinjections were performed in the same manner and at the same distance from the venule in each group, suggesting that the effective length of venule exposed to chemoattractant would be similar in both wild-type and E-selectin–deficient mice. Therefore, we reasoned that the shorter transit times of leukocytes rolling in E-selectin–deficient mice might be responsible for the diminished leukocyte recruitment in response to chemoattractant. To directly test this hypothesis, we exposed the whole cremaster muscle (rather than a single venule) to chemoattractant via superfusion. In response to superfused fMLP, the adhesion response was fully restored in E-selectin–deficient mice and not significantly different from the response seen in wild-type mice (Figure 5).

View larger version (17K): [in this window] [in a new window]   Figure 5. Leukocyte adhesion is restored in E-selectin–deficient mice by topical application of fMLP. The deficit in leukocyte adhesion in response to microinjection of fMLP (same data as in Figure 3A) (top) is overcome by exposing the whole cremaster muscle to fMLP (1 µmol/L) (bottom).

To quantitatively explore the impact of E-selectin on leukocyte transit through venules, we reanalyzed the rolling velocity data shown in Figure 3 and calculated leukocyte transit times through a 100-µm segment of venule (Figure 6). Seventy-two percent of leukocytes rolling in venules of wild-type mice took >3 seconds (dashed vertical line) to pass 100 µm. This fraction was similar (69.5%) in ICAM-1–deficient mice. Strikingly, this fraction was reduced to 44% in E-selectin–null mice (P<0.05). Correspondingly, the median transit time through a 100-µm segment of venule was significantly reduced to 2.4 seconds in E-selectin–null mice, less than half of the 5.4 seconds seen in wild-type mice (P<0.05). Although the median transit time of leukocytes rolling in venules of ICAM-1–deficient mice was only marginally reduced (to 4.1 seconds), very long transit times were absent in these mice. This corresponds to the lack of very slow rolling seen in ICAM-1–deficient mice (Figure 3).

View larger version (18K): [in this window] [in a new window]   Figure 6. Cumulative histogram of transit times of leukocytes rolling in venules of wild-type (solid line), E-selectin–deficient (thin line), and ICAM-1–deficient (broken line) mice. Note significant increase of the fraction of leukocytes with short (<3 seconds, dashed vertical line) transit times in E-selectin–deficient mice. The transit times are calculated per 100 µm of venule segment and are based on 129, 105, and 121 leukocytes in wild-type, E-selectin–deficient, and ICAM-1–deficient mice, respectively.

	Discussion

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Our data show that leukocyte adhesion in response to locally injected fMLP or MIP-2 is significantly reduced in E-selectin–deficient mice compared with wild-type mice. The response is restored by exposing a large area of cremaster muscle tissue to chemoattractant, suggesting that a reduced time of exposure of rolling leukocytes to chemoattractant underlies the deficit seen in E-selectin–deficient mice.

The striking defect of leukocyte adhesion in response to local chemoattractant in E-selectin–deficient mice shows that E-selectin is indeed required for efficient leukocyte delivery to a localized inflammatory stimulus, as speculated earlier.¹¹ This finding was reproduced by applying a function-blocking E-selectin antibody, suggesting that the leukocyte adhesion deficit was due to the lack of E-selectin function and not some developmental dysregulation in the gene-targeted mice. The fact that the deficient leukocyte recruitment could be overcome by exposing a larger area of the cremaster muscle (and hence a greater length of venule) to chemoattractant indicates that the transit time available for leukocyte activation becomes limiting when chemoattractant is available only locally. Therefore, E-selectin appears to be required for pinpointing the inflammatory response to a precise location. Interestingly, absence of P-selectin, which does not affect leukocyte rolling velocity after TNF- treatment,¹¹ has no impact on leukocyte accumulation in response to local chemoattractant under the conditions studied here.

In view of the normal recruitment of adherent cells in response to superfusion with fMLP, it is not surprising that E-selectin–deficient mice have no obvious defects in leukocyte recruitment to large, organ-size inflammatory lesions such as thioglycollate-induced peritonitis.⁸ Similarly, E-selectin antibodies have little impact on leukocyte recruitment in models of peritonitis in which C57BL/6 mice are used.¹⁵ Our data are consistent with the absence of defects in these models because chemoattractants are likely to be present at high concentrations in large areas, and leukocyte transit time through venules is probably not limiting under these conditions.

Interestingly, recent investigations have revealed that 93% of E-selectin–null mice succumb within 8 days to an intraperitoneal infection with Streptococcus pneumoniae, which is survived by 60% of wild-type mice.³⁵ This finding suggests that E-selectin can play a critical role in host defense, depending on the inflammatory stimulus used. Although the correlation between the inflammatory defect in the absence of E-selectin shown here and the findings in the peritonitis model is intriguing, a detailed mechanistic explanation of the susceptibility of E-selectin–null mice to S pneumoniae awaits more direct experimental verification.

ICAM-1 is a major ligand for leukocyte ß₂ integrins and is thought to be responsible for firm leukocyte adhesion in response to chemoattractants.^{2 3} Some in vivo studies have addressed the impact of blocking ICAM-1 function on leukocyte adhesion in venules. A recent study reports that a mAb to ICAM-1 inhibits 70% to 80% of the leukocyte adhesion response to fMLP superfused onto the rat mesentery for 20 to 40 minutes.³⁶ Similarly, the increase in leukocyte adhesion in response to the nitric oxide synthase inhibitor N^G-nitro-L-arginine methyl ester can partially be inhibited by antibodies to ICAM-1.^{37 38} In our model of TNF-–induced inflammation, we were unable to find a defect in leukocyte adhesion in ICAM-1–deficient mice. Under these conditions, other adhesion molecules that can serve as ligands for ß₂ integrins may be expressed on the inflamed endothelium, including ICAM-2³⁹ and possibly other as yet uncharacterized ligand molecules.⁴⁰ The ICAM-1–deficient mice prepared by homologous recombination^{27 33} are not true null mutants but express low levels of alternatively spliced variants of ICAM-1.⁴¹ Although the residual expression of ICAM-1 is low and probably insufficient to support significant leukocyte adhesion, we cannot formally rule out that alternatively spliced ICAM-1 may support leukocyte adhesion in our model. Interestingly, we did see a reduced number of very slowly rolling leukocytes (<10 µm/s) in ICAM-1–deficient mice, suggesting that ICAM-1 can reduce rolling velocity below the level achieved by selectin-mediated interactions alone. Interactions between ICAM-1 and ß₂ integrins have previously been shown to contribute to leukocyte rolling, particularly at low wall shear rates.^{42 43 44} Our data are consistent with a partial contribution of ICAM-1 to leukocyte rolling by modulating rolling velocities, although ICAM-1 alone is unable to support leukocyte adhesion in the presence of shear stress.⁴⁵ We have previously shown that the leukocyte rolling flux fraction (percentage of rolling leukocytes) is not reduced in ICAM-1–deficient mice treated with TNF-.¹²

Taken together, our data show that slow leukocyte rolling as mediated by E-selectin can be rate limiting for the delivery of leukocytes to small, circumscribed areas of inflammation without affecting the response to topically applied chemoattractant. The absence of a defect in leukocyte adhesion in ICAM-1–deficient mice suggests that alternative pathways can promote firm leukocyte adhesion and are sufficient for leukocyte recruitment in response to chemoattractant in this model.

	Selected Abbreviations and Acronyms

 

fMLP = formyl-methionyl-leucyl-phenylalanine ICAM = intercellular adhesion molecule mAb = monoclonal antibody MIP-2 = macrophage inflammatory protein-2 TNF- = tumor necrosis factor-

	Acknowledgments

 
This study was supported by US Public Health Service grant R01 HL-54136 to Dr Ley. We thank Arthur L. Beaudet, Howard Hughes Medical Institute and Department of Molecular Genetics, Baylor College of Medicine (Houston, Tex), for providing gene-targeted mice. Production and breeding of the mice were supported by US Public Health Service grant R01 AI-32177 to Dr Beaudet. S.J. Morgan received a graduate stipend of the Special Opportunity Award "Genetic Engineering Targeting Vascular Disease" awarded by the Whitaker Foundation to Dr Ley.

Received October 30, 1997; accepted April 6, 1998.

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