By
From the Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia 22908
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Abstract |
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The velocity of rolling leukocytes is thought to be determined by the expression of adhesion
molecules and the prevailing wall shear stress. Here, we investigate whether rapid cleavage of
L-selectin may be an additional physiologic regulatory parameter of leukocyte rolling. A
unique protease in the membrane of leukocytes cleaves L-selectin after activation, resulting in
L-selectin shedding. The hydroxamic acid-based metalloprotease inhibitor KD-IX-73-4 completely prevented L-selectin shedding in vitro and significantly decreased the rolling velocity of
leukocytes in untreated wild-type C57BL/6 mice from 55 to 35 µm/s in vivo. When E-selectin
was expressed on the endothelium (tumor necrosis factor [TNF]- treatment 2.5-3 h before
the experiment), rolling velocity was 4 µm/s and did not change after the application of KD-IX-73-4. However, KD-IX-73-4 decreased mean rolling velocity by 29% from 23 to 16 µm/s in
E-selectin-deficient mice treated with TNF-
. The reduction of velocity caused by KD-IX-73-4 was immediate (<5 s) after injection of KD-IX-73-4 as shown by a novel method using a
local catheter. These results establish a role for L-selectin shedding in regulating leukocyte rolling velocity in vivo.
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Introduction |
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Leukocyte rolling is the initial step in the recruitment of leukocytes to the site of acute inflammation (1, 2). L-, E-, and P-selectin are transmembrane glycoproteins known to mediate leukocyte rolling on microvascular endothelial cells (3). L-selectin (CD62L), a molecule expressed exclusively on the surface of leukocytes, interacts with a yet unidentified ligand on venular endothelial cells and participates in mediating leukocyte rolling under physiologic conditions (5, 6). It has been hypothesized that leukocyte rolling is accomplished by rapid formation and subsequent release of bonds between selectins and their ligand(s) (7, 8). A high rate of bond detachment of L-selectin under tension has been predicted (8) and is supported by experimental evidence (9, 10). The present study is designed to explore whether rapid cleavage of bound L-selectin may be responsible for releasing the trailing end of rolling leukocytes from the endothelial surface during rolling.
L-selectin is rapidly shed from the surface of the leukocytes upon their activation (11, 12). Stimuli inducing endoproteolytic release of L-selectin include chemokines like IL-8 and other chemoattractants (N-formylmethionyl leucylphenylalanine, platelet activating factor [12]). L-selectin shedding may also be one of the relevant factors in the recruitment of polymorphonuclear granulocytes (PMNs)1 from the bone marrow, since a reduction of L-selectin expression on polymorphonuclear leukocytes has been reported after their release from bone marrow (13).
L-selectin is cleaved at a membrane-proximal site between Lys321 and Ser322 in a region that links the second short consensus repeat (SCR) and the transmembrane domain (14). The protease releases a large soluble extracellular fragment (sL-selectin) of 68 kD and a 6-kD transmembrane peptide fragment (15). Soluble L-selectin is functionally active, and can inhibit leukocyte attachment to the endothelium (16). Although other cell markers (CD14, CD16, CD43, CD44, and CD50) also show a downregulation upon activation, the shedding of L-selectin is distinct, because it is unusually rapid and resistant to common protease inhibitors (12, 17), including the tissue inhibitor of metalloproteinases (TIMP [20]). An unidentified metalloprotease is believed to be responsible for the shedding of L-selectin (20). The activity of this metalloprotease can be effectively inhibited by a hydroxamic acid-based metalloprotease, KD-IX-73-4 (21). A similar hydroxamate-based metalloprotease inhibitor, N-(D,L-[2-(hydroxyaminocarbonyl)-methyl]-4-methylpentano)-L-3-(tert-butyl)-alanyl- L-alanine, 2-aminoethyl amide, blocks the release of TNF, TNF receptors (CD120a and CD120b), IL-6 receptor, and L-selectin release from leukocytes (22). The hydroxamic acid-based inhibitor of zinc-dependent matrix metalloproteinases, Ro 31-9790, has also been shown to inhibit L-selectin shedding from leukocytes (20).
Previous in vitro studies have shown that incubation of neutrophils with KD-IX-73-4 alters their rolling and causes increased neutrophil accumulation on immobilized MECA-79 antigen coated on the lower wall of a flow chamber (23). However, later in vitro results from Allport et al. with human neutrophils on activated human umbilical vein endothelial cells (HUVECs) did not show any regulatory function for L-selectin shedding in terms of neutrophil attachment, rolling, or transmigration (24). Motivated by the controversial role of L-selectin shedding, and the lack of any plausible explanation for the abundance of soluble L-selectin in vivo, we hypothesized a role for L-selectin shedding in the mechanism of physiologic leukocyte rolling.
To determine whether and to what extent L-selectin shedding affects leukocyte rolling, we designed intravital microscopic observations of rolling leukocytes before and after inhibition of L-selectin shedding. We also developed a new method for local application of compounds directly into the microcirculation of mouse cremaster muscle, which allows observations of rolling leukocytes during or briefly after their contact with the protease inhibitor KD-IX-73-4 or appropriate control peptide. We used this method to assess the contribution of rapid L-selectin cleavage to physiologic leukocyte rolling.
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Materials and Methods |
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Protease Inhibitor.
KD-IX-73-4 (HO-NH-CO-CH2-CH(CH2-CH(CH3)2)-CO-Nal-Ala-NH2) was a gift from Dr. T.K. Kishimoto (Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT). 0.5 mg of the protease inhibitor KD-IX-73-4 was solubilized in 50 µl DMSO and diluted with saline to a final concentration of 1 mg/ml. 150 µg of KD-IX-73-4 was used in in vivo experiments for an estimated initial serum concentration of 50 µg/ml. A related hydroxamic acid-based compound (slower isomer [SI]) HO-NH-CO-CH2-CH(CH2-CH(CH3)2)-CO-Nal-Ala-NH-CH2-CH2-NH2 was obtained from Peptides International. SI was solubilized, diluted, and aliquoted as described for KD-IX-73-4. SI was used in the experiments as a negative control. SI did not show any inhibitory effect on downregulation of L-selectin from neutrophils after activation with PMA (100 ng/ml; flow cytometry).mAbs.
mAb RB40.34 (rat IgG1, 30 µg/mouse) is a blocking mAb against murine P-selectin and was purified from hybridoma supernatant (25). mAb 9A9 (rat IgG1, 30 µg/mouse) is a blocking mAb against murine E-selectin (26, 27). TNF-Animals.
Experiments were performed on a total of 30 male mice 8-10 wk old and weighing 22-26 g. Mice included wild-type C57BL/6 (Hilltop), gene-targeted mice deficient in L-selectin (28), and gene-targeted mice deficient in E-selectin (29). Both L- and E-selectin knockout mice were backcrossed into a C57BL/6 background for at least seven generations.Intravital Microscopy.
For intravital microscopy, mice were anesthetized with an intraperitoneal injection of ketamine hydrochloride (100 mg/kg, Ketalar; Parke-Davis) after pretreatment with xylazine (0.05 mg/kg i.p.) and atropine (0.1 mg/kg i.p.; Elkins-Sinn). Animals were kept at 37°C with a thermo-controlled heating pad. Some mice were pretreated 2 or 6 h before surgery with an intrascrotal injection of 0.5 µg murine TNF-Local Catheter.
For local injection of the protease inhibitor KD-IX-73-4 and the negative control SI into the microcirculation of the cremaster muscle, a heparinized catheter was placed into the proximal part of the right femoral artery and advanced towards the branching section of the internal iliac artery from the common iliac artery, as shown in Fig. 1. The solutions (KD-IX-73-4 and SI) were injected as a 0.1-ml bolus containing 50 µg peptide. Upon injection of the bolus into the vasculature of the cremaster muscle, a brief hemodilution of the blood flowing through the observed venule indicated passage of the injected fluid. Centerline velocity and hematocrit returned to normal shortly after the passage of the bolus through the venule.
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Cremaster.
The cremaster muscle was prepared for intravital microscopy as described (31) and superfused with thermo-controlled 35°C bicarbonate-buffered saline saturated with 95% N2/ 5% CO2 (26). The exposed cremaster microcirculation remained well perfused. Time 0 was set at the beginning of the cremaster surgery, which took ~15 min. In the case of TNF-Flow Cytometry.
Mice were injected with KD-IX-73-4, or vehicle alone, and blood was obtained from the subclavian vein at 1 min after the injection. Mouse peripheral blood was stained with PE-conjugated anti-L-selectin mAb clone (cat. 01265B; PharMingen) or PE-conjugated isotype control mAb (rat IgG2a, cat. 11025A; PharMingen) 2 µl/ml at 4°C for 15 min. After lysis of the erythrocytes (lysing solution: 0.15 M NH4Cl, 0.01 M NaHCO3, and 0.001 M disodium EDTA), cells were washed once with BSA/PBS and fixed in 1% paraformaldehyde/PBS, and 0.5-1 × 104 cells/sample were analyzed by flow cytometry on a FACScan® (Becton Dickinson). The results reported are for granulocytes, gated by their characteristic forward and side scatter.Statistical Analysis.
Statistical comparisons were carried out using Kolmogorov-Smirnov two-sample t test. NCSS statistical software (http://www.icw.com/ncss) version 6.0.11 was used for the statistical analysis. Statistical significance was set at P < 0.05 or P < 0.01. ![]() |
Results |
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Fig. 2 shows the results of inhibition of L-selectin shedding in wild-type mice. Velocities of 750 rolling leukocytes in 15 vessels of 5 wild-type mice were obtained before and after the application of KD-IX-73-4. Observations were made 1-8 min after the injection of the protease inhibitor and within the first 1 h after starting the cremaster preparation. The mean velocity of all observed cells before the inhibition of L-selectin shedding was 59 ± 5.6 µm/s and after the inhibition was 39 ± 3.6 µm/s, which represents a reduction of the mean velocity by 33 ± 2%. As an example, Fig. 3 shows a composite photomicrograph illustrating the change in rolling velocity in one representative vessel.
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The related hydroxamic acid-based substance (SI) did not show any significant effect on the velocities of the rolling leukocytes. Interestingly, after the use of 150 µg SI (1 µg/ µl), the subsequent injection of 150 µg KD-IX-73-4 did not show any change in the velocity of rolling leukocytes, suggesting a possible competitive mechanism of interaction between the two hydroxamic acid-based substances and the L-selectin cleaving protease. Fig. 4 shows the results of the SI application followed by KD-IX-73-4 in wild-type mice.
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To determine the duration of the effect of the protease inhibitor in vivo, single venule recordings were made in two wild-type animals for 15-20 min after the application of KD-IX-73-4. Rolling velocities of 10 cells were obtained in 1-min steps after the injection of 150 µg KD-IX-73-4. Within the first 1 min after the injection of 150 µg KD-IX-73-4, the reduction of rolling velocity could be observed and persisted for ~9-10 min, after which the rolling velocity returned to control levels (Fig. 5).
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To assess whether the injected dose of KD-IX-73-4 resulted in complete inhibition of L-selectin shedding in vivo, we investigated the expression of L-selectin on neutrophils in peripheral blood by flow cytometry (Fig. 6). All neutrophils in peripheral blood of untreated mice expressed L-selectin, which was almost entirely downregulated after PMA activation. In contrast, PMNs obtained from mice 1 min after KD-IX-73-4 injection did not shed L-selectin in response to PMA activation. The inhibitory effect of KD-IX-73-4 in vivo was no longer detectable at 15 min after the injection. SI, the peptide used as a control, did not show any inhibitory effect on L-selectin shedding after PMA activation of the cells. Taken together, these data show that KD-IX-73-4 transiently inhibits L-selectin shedding in vivo. The time course of this inhibitory effect parallels the time course of reduction of rolling velocity by KD-IX-73-4.
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Systemic application of 150-300 µg KD-IX-73-4 did not cause an alteration of the systemic blood pressure, which remained between 80 and 95 mmHg throughout the experiments. Likewise, centerline velocities and diameters (and consequently the shear rates) of the observed venules were not affected by the systemic injections of KD-IX-73-4 or SI. Also, systemic white blood cell counts remained unchanged after systemic injection of KD-IX-73-4 (Table I).
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To determine whether the injected amount of KD-IX-73-4 (150 µg) in vivo was sufficient to cause maximal reduction of velocity, subsequent injections of KD-IX-73-4 (150-300 µg) were made in two wild-type animals to achieve an accumulation of the protease inhibitor in plasma. Such injections did not cause a further shift of velocities of the rolling cells compared with the cell rolling velocities after the first injection of 150 µg KD-IX-73-4 (data not shown). Therefore, an injection of 150 µg KD-IX-73-4 into a mouse produced complete inhibition of L-selectin shedding and maximal reduction of rolling velocity in vivo.
As another parameter relevant for leukocyte trafficking,
the flux of rolling leukocytes (number of rolling cells passing per unit of time) was measured in eight vessels of wild-type mice. Inhibition of L-selectin shedding caused a significant 22% increase of rolling flux compared with the
mean of the rolling flux of the same vessels before the KD-IX-73-4 treatment (118 cells/min). This finding suggests that
inhibition of L-selectin shedding not only reduces the velocity of rolling leukocytes, but also aids in promoting leukocyte capture from the free stream and/or stabilizes their
rolling along the venular tree. Furthermore, the number of
firmly adherent leukocytes was significantly increased after inhibition of L-selectin shedding in mice treated with
TNF- for 6 h (data not shown). In this model, rolling is
primarily L-selectin dependent (30). Concomitant with increased adhesion, inhibition of L-selectin shedding also increased the number of transmigrating cells, apparent by the
characteristic change of shape during their transendothelial
passage (data not shown). This suggests that, beyond its
prominent and immediate effect on the velocity of rolling leukocytes, L-selectin shedding significantly impacts leukocyte recruitment.
To investigate whether the effect of KD-IX-73-4 on the leukocyte velocities in vivo is specific to the inhibition of L-selectin shedding or whether other variables contribute to the shift of velocities, experiments with L-selectin-deficient mice were made. Fig. 7 shows leukocyte rolling velocities obtained in L-selectin-deficient mice before and after systemic application of KD-IX-73-4. There was no significant change of the mean rolling velocities measured after application of KD-IX-73-4 (37 ± 1.4 µm/s before and 38.2 ± 1.5 µm/s after the inhibition; 100 cells from 2 animals). The absence of the reduction of velocity in L-selectin-deficient mice shows that inhibition of L-selectin shedding with KD-IX-73-4 does not alter the rolling velocity of leukocytes unless L-selectin is expressed. Flux also remained unchanged after injection of KD-IX-73-4 in L-selectin-deficient mice (data not shown).
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The systemic application of the protease inhibitor into the jugular vein is likely to produce a homogenous serum concentration of the inhibitor throughout the vascular system. However, in order to be able to make a statement about the onset of the effect of KD-IX-73-4 on rolling velocity, it was necessary to observe rolling leukocytes from the moment of their first contact with the inhibitor. For this purpose, the protease inhibitor was injected as a small bolus (50 µg/0.1 ml) through a local catheter (Fig. 1) into the microcirculation of cremaster muscle. With the passage of the solution through the cremaster vasculature, the inhibitor washed over rolling leukocytes in the cremaster venules and might inhibit L-selectin shedding in these cells within the field of view. A brief hemodilution effect caused by injection of the bolus furnished evidence for the passage of the solution through the observed venules. Within the first 5 s after the passage of the bolus, velocities of 25 rolling leukocytes/venule were obtained. The mean velocity of 50 rolling leukocytes from 2 animals before contact with the protease inhibitor was 40.2 ± 3.1 µm/s, which was lowered to 29.9 ± 2.7 µm/s immediately after the injection of KD-IX-73-4. This equals a 26% reduction of leukocyte rolling velocity.
To explore the role of L-selectin shedding during cytokine-induced inflammation, we used intrascrotal injection
of TNF- 3 h before the observation period, as described
previously (6). Treatment of wild-type mice with intrascrotal injections of 0.5 µg TNF-
2 h before the experiments causes the expression of E-selectin and increased
expression of P-selectin on the endothelial cells (36). Presence of E-selectin on the endothelium of the cremaster venules lowers the mean leukocyte rolling velocity by almost one order of magnitude to under 5 µm/s (37). Fig. 8
A shows results of inhibition of L-selectin shedding with
KD-IX-73-4 in TNF-
-treated wild-type mice. The
mean velocity of 300 rolling leukocytes (4.5 µm/s) before
application of the protease inhibitor did not change significantly compared with the mean of 300 rolling leukocytes (4.1 µm/s) obtained between 1 and 6 min after the application of 150 µg KD-IX-73-4.
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We hypothesized that E-selectin-mediated leukocyte
rolling proceeded at such low velocities that inhibition of
L-selectin shedding did not influence the actual velocity
because the low off-rate of E-selectin (38) would determine and dominate the rolling velocity, no matter whether
L-selectin was being shed or not. Previous studies had shown
that rolling velocities were significantly higher in E-selectin-
deficient mice treated with TNF- than in wild-type mice
(37). To explore the reason for the apparent absence of an
effect of inhibiting L-selectin shedding in TNF-
-treated
mice, we used TNF-
-treated E-selectin-deficient mice. In
these mice, mean rolling velocity was 24 µm/s and was reduced to 17 µm/s after treatment with KD-IX-73-4 (a 29%
reduction of mean rolling velocity; Fig. 8 B).
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Discussion |
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Although proteolytic cleavage of L-selectin from the surface of leukocytes was described as early as 1989 (11, 12),
the physiologic relevance of L-selectin shedding in vivo has
remained unclear and the subject of controversial speculations. The results of this study establish L-selectin shedding
as a novel parameter, in addition to adhesion molecule expression and hemodynamic forces, relevant for regulation
of leukocytes rolling. Continuous L-selectin shedding appears necessary for physiologic leukocyte rolling at typical
velocities. Though Walcheck et al. have noted that inhibition of L-selectin shedding lowers the rolling velocity of
isolated neutrophils on immobilized MECA-79 antigen
(23), the results of other in vitro experiments using cultured endothelial cells seemed to contradict their conclusion (24). In experiments with TNF--treated HUVECs, a
role for L-selectin shedding in neutrophil attachment, rolling, or transmigration could not be shown. Our results in
untreated and TNF-
-treated wild-type mice can explain this discrepancy and establish the role of L-selectin shedding for leukocyte trafficking.
When E-selectin is expressed on endothelial cells, inhibition of L-selectin shedding has no effect on leukocyte rolling velocity. However, when E-selectin is not expressed,
either in wild-type mice not treated with an inflammatory
cytokine, or in E-selectin-deficient mice, the modulatory
role of L-selectin shedding is evident. In TNF--treated
cultured HUVECs (24), E-selectin is likely to be expressed.
This would explain why Allport et al. (24) did not find a velocity difference between human neutrophils with or without incubation with a zinc-dependent metalloproteinase inhibitor (Ro 31-9790). In the experiment of Walcheck et
al. (23), E-selectin is not present, because a purified L-selectin ligand (MECA-79) is used as the sole adhesive substrate.
We investigated the effect of inhibiting L-selectin on rolling in vivo both in the presence and absence of E-selectin.
In the absence of E-selectin, inhibition of L-selectin shedding causes a significant decrease of rolling velocity (as in reference 23), which is not detectable when E-selectin is involved in mediating leukocyte rolling (as in reference 24).
As presented in Fig. 2, a shift towards lower leukocyte rolling velocities can be seen with the inhibition of L-selectin shedding in wild-type mice without intentional induction of inflammation. The amount of protease inhibitor used in these animals provided a maximum inhibition of L-selectin shedding, since further injections of KD-IX-73-4 did not lower the rolling velocity of the cells further. The shift of velocity in these animals shows that normal leukocyte rolling requires continuous shedding of L-selectin. Consistent with this, Palecanda et al. detected soluble L-selectin in the plasma of healthy adults, whose peripheral blood leukocytes did not demonstrate any obvious signs of activation (39), and Schleiffenbaum et al. found high physiologic levels of soluble L-selectin in the serum of healthy volunteers (16). Since no soluble or secreted splice variants of L-selectin are known, we hypothesize that continuous L-selectin shedding during physiologic leukocyte rolling is a likely source for soluble L-selectin in the plasma of healthy individuals.
The fact that no change in the rolling velocity can be measured with the protease inhibitor KD-IX-73-4 in L-selectin- deficient mice shows that the observed shift of velocities in wild-type mice was specifically and exclusively caused by the inhibition of L-selectin shedding. Other molecules such as P- or E-selectin, which also mediate leukocyte rolling, do not contribute to the shift of velocity caused by the application of KD-IX-73-4.
As a negative control, we used the hydroxamic acid- based substance (SI), which is structurally closely related to the protease inhibitor KD-IX-73-4 but does not inhibit L-selectin shedding as shown by flow cytometry. Interestingly, after the use of SI, subsequent injections of KD-IX-73-4 did not show a shift of velocity. Therefore, SI may interact with the protease responsible for L-selectin shedding at the same binding site as KD-IX-73-4, thus competing with KD-IX-73-4 binding. PMNs from KD-IX-73-4- treated wild-type mice do not shed L-selectin in response to PMA activation. However, the inhibitory effect of KD-IX-73-4 in vivo is not permanent, as PMNs obtained 15 min after KD-IX-73-4 treatment responded to PMA activation by shedding L-selectin. This parallels our observations from the functional assay (Fig. 5) that the shift of velocity lasts for ~10 min after one injection. The inhibition of L-selectin shedding in vivo and the velocity shift in the functional assay can both be observed after the injection of the protease inhibitor KD-IX-73-4. These data are consistent with a causal relationship between the injection of KD-IX-73-4, the inhibition of the L-selectin shedding, and the shift of rolling velocity.
The current findings show that proteolytic cleavage of L-selectin shedding is relevant for physiologic leukocyte rolling. Since enzymatic processes often require metabolic energy, it may be worthwhile exploring whether L-selectin-mediated leukocyte rolling is modulated by cell metabolism or whether it is as independent of cell metabolism as formerly believed (40, 41). Furthermore, in light of the present study, a revision of the calculated dissociation rate of L-selectin appears necessary, which was made with the assumption that during stable rolling the average number of bonds that form and break will be equal (9). This assumption may prove erroneous, since inhibition of L-selectin shedding decreases rolling velocity. Therefore, we believe that the 7.5-11.5-fold faster L-selectin-mediated rolling does not necessarily require a 7-10-fold more rapid bond dissociation, as proposed by Alon et al. (9). Rather, continuous cleavage of L-selectin and breakage of bonds are likely to occur at the same time and thus cause short duration of L-selectin-mediated tethering events.
In conclusion, inhibition of L-selectin shedding decreases the leukocyte rolling velocity and increases the leukocyte rolling flux in vivo. A decreased rolling velocity increases the transit time of rolling leukocytes. Leukocyte transit times have recently been shown to be an important determinant of leukocyte recruitment in vivo (42). Modulation of L-selectin shedding is a novel and influential parameter in the leukocyte trafficking by virtue of its regulatory function on rolling velocity, rolling flux, and transit time of leukocytes.
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Footnotes |
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Address correspondence to Klaus Ley, Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22908. Phone: 804-924-1722; Fax: 804-982-3870; E-mail: kfl3f{at}virginia.edu
Received for publication 20 October 1998 and in revised form 28 January 1999.
We wish to thank Dr. T.K. Kishimoto for the gift of KD-IX-73-4 and for insightful discussion. We thank William Ross for help with flow cytometry experiments, and Nicholas Douris and Jennifer Bryant for mouse husbandry. L-selectin-deficient mice were from a colony based on breeders provided by Dr. T.F. Tedder, Duke University, Durham, NC. E-selectin-deficient mice were from a colony based on breeders provided by Dr. D.C. Bullard and Dr. A.L. Beaudet, Baylor College of Medicine, Houston, TX.
This work was supported by National Institutes of Health grant HL54136 to K. Ley.
Abbreviations used in this paper HUVEC, human umbilical vein endothelial cell; PMN, polymorphonuclear granulocyte; SI, slower isomer (negative control).
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References |
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1. | Ley, K., and T. Tedder. 1995. Leukocyte interactions with vascular endothelium. New insights into selectin-mediated attachment and rolling. J. Immunol. 155: 525-528 [Abstract]. |
2. | Springer, T.. 1995. Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu. Rev. Physiol. 57: 827-872 [Medline]. |
3. | Dore, M., R. Korthuis, D. Granger, M. Entman, and C. Smith. 1993. P-selectin mediates spontaneous leukocyte rolling in vivo. Blood. 82: 1308-1316 [Abstract]. |
4. |
Kansas, G..
1996.
Selectins and their ligands: current concepts
and controversies.
Blood.
88:
3259-3287
|
5. | Ley, K., P. Gaehtgens, C. Fennie, M. Singer, L. Lasky, and S. Rosen. 1991. Lectin-like cell adhesion molecule 1 mediates leukocyte rolling in mesenteric venules in vivo. Blood. 77: 2553-2555 [Abstract]. |
6. | Ley, K., D. Bullard, M. Arbones, R. Bosse, D. Vestweber, T. Tedder, and A. Beaudet. 1995. Sequential contribution of L-and P-selectin to leukocyte rolling in vivo. J. Exp. Med. 181: 669-675 [Abstract]. |
7. | Hammer, D., and S. Apte. 1992. Simulation of cell rolling and adhesion on surfaces in shear flow: general results and analysis of selectin-mediated neutrophil adhesion. Biophys. J. 63: 35-57 [Abstract]. |
8. | Tözeren, A., and K. Ley. 1992. How do selectins mediate leukocyte rolling in venules? Biophys. J. 63: 700-709 [Abstract]. |
9. |
Alon, R.,
S. Chen,
K. Puri,
E. Finger, and
T. Springer.
1997.
The kinetics of L-selectin tethers and the mechanics of selectin-mediated rolling.
J. Cell Biol.
138:
1169-1180
|
10. | Puri, K., S. Chen, and T. Springer. 1998. Modifying the mechanical property and shear threshold of L-selectin adhesion independently of equilibrium properties. Nature. 392: 930-933 [Medline]. |
11. |
Jutila, M.,
L. Rott,
E. Berg, and
E. Butcher.
1989.
Function
and regulation of the neutrophil MEL-14 antigen in vivo:
comparison with LFA-1 and MAC-1.
J. Immunol.
143:
3318-3324
|
12. | Kishimoto, T., M. Jutila, E. Berg, and E. Butcher. 1989. Neutrophil Mac-1 and MEL-14 adhesion proteins inversely regulated by chemotactic factors. Science. 245: 1238-1241 [Medline]. |
13. |
van Eeden, S.,
R. Miyagashima,
L. Haley, and
J. Hogg.
1997.
A possible role for L-selectin in the release of polymorphonuclear leukocytes from bone marrow.
Am. J. Physiol.
272:
H1717-H1724
|
14. | Chen, A., P. Engel, and T.F. Tedder. 1995. Structural requirements regulate endoproteolytic release of the L-selectin (CD62L) adhesion receptor from the cell surface of leukocytes. J. Exp. Med. 182: 519-530 [Abstract]. |
15. | Kahn, J., R. Ingraham, F. Shirley, G. Migaki, and T. Kishimoto. 1994. Membrane proximal cleavage of L-selectin: identification of the cleavage site and a 6-kD transmembrane peptide fragment of L-selectin. J. Cell Biol. 125: 461-470 [Abstract]. |
16. | Schleiffenbaum, B., O. Spertini, and T. Tedder. 1992. Soluble L-selectin is present in human plasma at high levels and retains functional activity. J. Cell Biol. 119: 229-238 [Abstract]. |
17. |
Bazil, V., and
J. Strominger.
1994.
Metalloprotease and serine
protease are involved in cleavage of CD43, CD44, and
CD16 from stimulated human granulocytes. Induction of
cleavage of L-selectin via CD16.
J. Immunol.
152:
1314-1322
|
18. | Kishimoto, T., J. Kahn, G. Migaki, E. Mainolfi, F. Shirley, R. Ingraham, and R. Rothlein. 1995. Regulation of L-selectin expression by membrane proximal proteolysis. Agents Actions Suppl. 47: 121-134 [Medline]. |
19. | Shipp, M., G. Stefano, S. Switzer, J. Griffin, and E. Reinherz. 1991. CD10 (CALLA)/neutral endopeptidase 24.11 modulates inflammatory peptide-induced changes in neutrophil morphology, migration, and adhesion proteins and is itself regulated by neutrophil activation. Blood. 78: 1834-1841 [Abstract]. |
20. |
Preece, G.,
G. Murphy, and
A. Ager.
1996.
Metalloproteinase-mediated regulation of L-selectin levels on leucocytes.
J.
Biol. Chem.
271:
11634-11640
|
21. |
Feehan, C.,
K. Darlak,
J. Kahn,
B. Walcheck,
A. Spatola, and
T. Kishimoto.
1996.
Shedding of the lymphocyte L-selectin
adhesion molecule is inhibited by a hydroxamic acid-based
protease inhibitor. Identification with an L-selectin-alkaline
phosphatase reporter.
J. Biol. Chem.
271:
7019-7024
|
22. | Bennett, T., E. Lynam, L. Sklar, and S. Rogelj. 1996. Hydroxamate-based metalloprotease inhibitor blocks shedding of L-selectin adhesion molecule from leukocytes: functional consequences for neutrophil aggregation. J. Immunol. 156: 3093-3097 [Abstract]. |
23. | Walcheck, B., J. Kahn, J. Fisher, B. Wang, R. Fisk, D. Payan, C. Feehan, R. Betageri, K. Darlak, A. Spatola, and T. Kishimoto. 1996. Neutrophil rolling altered by inhibition of L-selectin shedding in vitro. Nature. 380: 720-723 [Medline]. |
24. | Allport, J., H. Ding, A. Ager, D. Steeber, T. Tedder, and F. Luscinskas. 1997. L-selectin shedding does not regulate human neutrophil attachment, rolling, or transmigration across human vascular endothelium in vitro. J. Immunol. 158: 4365-4372 [Abstract]. |
25. | Bosse, R., and D. Vestweber. 1994. Only simultaneous blocking of the L- and P-selectin completely inhibits neutrophil migration into mouse peritoneum. Eur. J. Immunol. 24: 3019-3024 [Medline]. |
26. | Kunkel, E., U. Jung, D. Bullard, K. Norman, B. Wolitzky, D. Vestweber, A. Beaudet, and K. Ley. 1996. Absence of trauma-induced leukocyte rolling in mice deficient in both P-selectin and intercellular adhesion molecule 1. J. Exp. Med. 183: 57-65 [Abstract]. |
27. | Norton, C., J. Rumberger, D. Burns, and B. Wolitzky. 1993. Characterization of murine E-selectin expression in vitro using novel anti-mouse E-selectin monoclonal antibodies. Biochem. Biophys. Res. Commun. 195: 250-258 [Medline]. |
28. | Arbones, M., D. Ord, K. Ley, H. Ratech, C. Maynard-Curry, G. Otten, D. Capon, and T. Tedder. 1994. Lymphocyte homing and leukocyte rolling and migration are impaired in L-selectin-deficient mice. Immunity. 1: 247-260 [Medline]. |
29. | Bullard, D., E. Kunkel, H. Kubo, M. Hicks, I. Lorenzo, N. Doyle, C. Doerschuk, K. Ley, and A. Beaudet. 1996. Infectious susceptibility and severe deficiency of leukocyte rolling and recruitment in E-selectin and P-selectin double mutant mice. J. Exp. Med. 183: 2329-2336 [Abstract]. |
30. | Jung, U., C. Ramos, D. Bullard, and K. Ley. 1998. Gene-targeted mice reveal importance of L-selectin-dependent rolling for neutrophil adhesion. Am. J. Physiol. 43: H1785-H1791 . |
31. | Baez, S.. 1973. An open cremaster muscle preparation for the study of blood vessels by in vivo microscopy. Microvasc. Res. 5: 384-394 [Medline]. |
32. | Pries, A.. 1988. A versatile video image analysis system for microcirculatory research. Int. J. Microcirc. Clin. Exp. 7: 327-345 [Medline]. |
33. | Lipowsky, H., and B. Zweifach. 1978. Application of the "two-slit" photometric technique to the measurement of microvascular volumetric flow rates. Microvasc. Res. 15: 93-101 [Medline]. |
34. | Reneman, R., B. Woldhuis, M. oude Egbrink, D. Slaaf, and G. Tangelder. 1992. Concentration and velocity profiles of blood cells in the microcirculation. In Advances in Cardiovascular Engineering. N. Hwang, V. Turitto, and M. Yen, editors. Plenum Publishing Corp., New York. 25-40. |
35. | Ley, K., and P. Gaehtgens. 1991. Endothelial, not hemodynamic, differences are responsible for preferential leukocyte rolling in rat mesenteric venules. Circ. Res. 69: 1034-1041 [Abstract]. |
36. | Jung, U., and K. Ley. 1997. Regulation of E-selectin, P-selectin, and intercellular adhesion molecule 1 expression in mouse cremaster muscle vasculature. Microcirculation. 4: 311-319 [Medline]. |
37. |
Kunkel, E., and
K. Ley.
1996.
Distinct phenotype of E-selectin-deficient mice. E-selectin is required for slow leukocyte
rolling in vivo.
Circ. Res.
79:
1196-1204
|
38. | Kaplanski, G., C. Farnarier, O. Tissot, A. Pierres, A.M. Benoliel, M.C. Alessi, S. Kaplanski, and P. Bongrand. 1993. Granulocyte-endothelium initial adhesion. Analysis of transient binding events mediated by E-selectin in a laminar shear flow. Biophys. J. 64: 1922-1933 [Abstract]. |
39. | Palecanda, A., B. Walcheck, D. Bishop, and M. Jutila. 1992. Rapid activation-independent shedding of leukocyte L-selectin induced by crosslinking of the surface antigen. Eur. J. Immunol. 22: 1279-1286 [Medline]. |
40. | Lalor, P., J. Clements, R. Pigott, M. Humphries, J. Spragg, and G. Nash. 1997. Association between receptor density, cellular activation, and transformation of adhesive behavior of flowing lymphocytes binding to VCAM-1. Eur. J. Immunol. 27: 1422-1426 [Medline]. |
41. |
Lawrence, M., and
T. Springer.
1993.
Neutrophils roll on
E-selectin.
J. Immunol.
151:
6338-6346
|
42. |
Jung, U.,
K. Norman,
K. Scharffetter-Kochanek,
A. Beaudet, and
K. Ley.
1998.
Transit time of leukocytes rolling through
venules controls cytokine-induced inflammatory cell recruitment in vivo.
J. Clin. Invest.
102:
1526-1533
|