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Prepublished online as a Blood First Edition Paper on December 12, 2002; DOI 10.1182/blood-2002-07-2329.
PHAGOCYTES
From the Cardiovascular Research Group, University of
Sheffield, Sheffield, United Kingdom.
Selectin-dependent leukocyte rolling is one of the earliest steps
of an acute inflammatory response and, as such, contributes to many
inflammatory diseases. Although inhibiting leukocyte rolling with
selectin antagonists is a strategy that promises far-reaching clinical
benefit, the perceived value of this strategy has been limited by
studies using inactive, weak, or poorly characterized antagonists.
Recombinant P-selectin glycoprotein ligand-1-immunoglobulin (rPSGL-Ig) is a recombinant form of the best-characterized
selectin ligand (PSGL-1) fused to IgG, and is one of the best prospects in the search for effective selectin antagonists. We have used intravital microscopy to investigate the ability of rPSGL-Ig to influence leukocyte rolling in living blood vessels and find
that it can reduce rolling dependent on each of the selectins in vivo. Interestingly, doses of rPSGL-Ig required to reverse pre-existing leukocyte rolling are 30-fold higher than those required to limit inflammation, suggesting additional properties of this molecule. In
support of this, we find that rPSGL-Ig can bind the murine chemokine
KC and inhibit neutrophil migration toward this chemoattractant in vitro.
(Blood. 2003;101:3249-3256) Recruitment of leukocytes, although essential for
host defense, can also be deleterious, for example in
ischemia-reperfusion (I/R) injury,1
arthritis,2 and sepsis.3 As an early and necessary step in the leukocyte recruitment process, selectin-dependent rolling, therefore, presents an attractive drug target.
Although the selectins bind with moderate affinity to sialylated,
fucosylated carbohydrate ligands, typified by the tetrasaccharide sialyl Lewisx (sLex),4 preferred
natural ligands are more complex glycoconjugates.5 The
best-characterized selectin ligand is P-selectin glycoprotein ligand-1
(PSGL-1),6,7 a homodimeric sialomucin found on
leukocytes8,9 and platelets.10 PSGL-1, in its
correct glycoform, binds to P-, E- and L-selectins in
vitro5 and represents an important functional ligand for
all of these molecules.11-15
A recombinant fusion protein, recombinant PSGL-1-immunoglobulin
(rPSGL-Ig), consisting of 47 amino acids from the
NH2 terminal of human PSGL-1 linked to the Fc portion of
human IgG1 has been developed.16 To
preserve the function of the natural ligand, rPSGL-Ig is expressed in
cell lines containing enzymatic machinery for posttranslational
sulfation and glycosylation.17 Many investigators believe
that rPSGL-Ig will compete with cell surface selectin ligands to reduce
leukocyte rolling and thus limit inflammation.
Anti-inflammatory effects of rPSGL-Ig have been described. Treatment
with rPSGL-Ig accelerates thrombolysis and prevents reocclusion of injured porcine arteries18; protects against traumatic
shock,19 hemorrhage reinfusion injury,20 and
I/R injury in rats21; reduces myocardial I/R injury in
cats22; and limits acid-induced lung inflammation in
mice.23 Leukotriene C4
(LTC4)-induced rolling is also reduced by
rPSGL-Ig.17 In all of these studies, rPSGL-Ig was
administered prior to inflammatory stimulation, and direct effects on individual selectins were not investigated. We have used
well-characterized models of selectin-dependent leukocyte rolling to
demonstrate, for the first time, that rPSGL-Ig can directly inhibit
leukocyte rolling on all 3 selectins in vivo. We also report that
rPSGL-Ig inhibits inflammation at doses that only moderately alter
established leukocyte rolling and has supplementary activity
(inhibition of chemokine binding and function) not related to
inhibition of selectin-dependent rolling.
Animals
All procedures performed were approved by the University of Sheffield
ethics committee and by the Home Office Animals (Scientific Procedures)
Act 1986 of the United Kingdom.
rPSGL-Ig and controls
Cytokines and antibodies Murine recombinant tumor necrosis factor- (TNF),
purchased from R&D Systems (Abingdon, Oxon, United Kingdom), was
dissolved at 0.1 mg/mL in phosphate-buffered saline (PBS) containing
0.1% bovine serum albumin (BSA), sterile filtered, and stored in
500-ng aliquots at  20°C. Murine KC, purchased from
Peprotech (Rocky Hill, NJ), was reconstituted in PBS containing 0.1%
BSA at 10 4 M and stored in 10-µL aliquots at 20°C.
Rat antimouse antibodies against P-selectin (RB40.34,
IgG1), L-selectin (Mel-14, IgG2a), PSGL-1
(2PH1, IgG1), Ly-6G (RB6-8C5, IgG1), CD2
(rm2-5, IgG2b), CD5 (53-7.3, IgG2a), and CD45R
(RA3-6B2, IgG2a) were purchased from Pharmingen (Oxford,
United Kingdom). Rat antimouse intercellular adhesion molecule-1
(ICAM-1 [YN1/1]) was a gift from Dr C. Wegner (Abbott
Laboratories, Abbott Park, IL). Rat antimouse F4/80 antigen (CI:A3-1),
fluorescein isothiocyanate (FITC)-labeled rat antimouse CD18
(C71/16), and phycoerythrin (PE)-labeled rat IgG2a isotype negative control were purchased from Serotec (Kidlington, United Kingdom). Goat antirat IgG microbeads were obtained from Miltenyi Biotech (Bisley, United Kingdom). The isotype control for RB40.34 (8B9,
rat IgG1) was a kind gift from Dr B. A. Wolitzky (Hoffman-La Roche, Nutley, NJ). Rat antimouse E-selectin (10E6, rat IgG2b) used in
these experiments was also a kind gift from Dr B. A. Wolitzky. All
of these antibodies have been studied extensively by us and others.11,14,25-30 Antibodies were stored at 1 mg/mL in
10-µg aliquots at 20°C. Before injection into mice, cytokine and
antibody aliquot volumes were expanded to 200 µL by addition of
sterile saline.
Intravital microscopy Leukocyte rolling was observed in postcapillary venules of the murine cremaster muscle. For P-selectin-dependent leukocyte rolling, wild-type mice were used, and cremasters were stimulated merely by the surgery required for microscopic observation. For E-selectin-dependent/L-selectin-dependent leukocyte rolling, P-selectin/ mice were used, and cremasters were
stimulated with TNF (500 ng, intrascrotal) 2.5 hours prior to
observation. Leukocyte rolling with a strong requirement for L-selectin
has been described in E-selectin/P-selectin double-null mice stimulated
for 6 hours with TNF and treated with heparin to prevent
coagulation.31 Since these mice were not available in our
laboratory, E-selectin/ mice were pretreated with
P-selectin blocking antibody (30 µg), TNF (500 ng), and heparin
(50 U) 6 hours prior to observation. Immediately prior to
exteriorization of the cremaster for intravital microscopy, the
following cannulations were performed: the trachea to facilitate
respiration; the jugular vein to allow intravenous injection of
additional anesthesia and injection of rPSGL-Ig, vehicle, and
antibodies; and the carotid artery to permit blood sampling. Body
temperature was maintained at 37°C by means of a
thermocontrolled heat pad (PDtronics, Sheffield, United Kingdom).
The cremaster muscle was prepared for microscopic observation as described previously.27 Briefly, the testis, epididymis, and cremaster were extracted through a small incision in the scrotum. The tip of the cremaster was pinned so that the testis and cremaster were positioned across a 10-mm cover glass composing part of a specialized microscopy stage. Fat and connective tissue were carefully removed, and an excision was made through the cremaster allowing the muscle to be pinned out across the glass coverslip. Throughout this procedure and during intravital microscopy, the cremaster muscle was superfused with thermocontrolled (36°C) bicarbonate buffer solution (131.9 mM NaCl, 18 mM NaHCO3, 4.7 mM KCl, 2.0 mM CaCl2, and 2 mM MgSO4) through which a gas mixture of 5% CO2 in N2 was bubbled. Microscopic observations were made by means of a light microscope (Nikon Eclipse E600-FN; Nikon United Kingdom, Kingston, Surrey, United Kingdom) equipped with a water immersion objective (40 ×/0.80 W). Venules between 16- and 62-µm diameter were selected for observation. Images of rolling leukocytes were recorded by means of a charge-coupled device (CCD) camera (DC-330E; DAGE MTI, Michigan City, MI) onto S-VHS videocassettes. To investigate the instantaneous effects of rPSGL-Ig, venules were recorded continuously beginning 1 minute before treatment and continuing until 5 minutes after injection. In some experiments, attention was focused on areas immediately downstream of points where capillaries converged with small venules, allowing formation of new attachments to be visualized and quantified. In these experiments, attachment rates before and after treatments were determined for 10 minutes. In other experiments, leukocytes were tracked from the point of initial attachment through the network of vessels to points that could no longer be observed. Blood samples (10 µL) were drawn from the carotid artery at 10-minute intervals and at 2 minutes before and after treatments. These were analyzed for total systemic leukocyte concentrations. Venular blood flow velocity was measured as previously described,32 by means of a dual photodiode and digital online cross-correlation program (Microvessel Velocity OD-RT System; Circusoft Instrumentation LLC, Hockessin, DE). Leukocyte rolling induced by lower-body ischemia-reperfusion injury Wild-type C57BL/6 mice were anesthetized, and neck surgery was performed as described. The abdominal cavity was opened, and the intestines were moved carefully to one side. The aorta was uncovered in the abdominal cavity, gently detached from the vena cava, and occluded at a point immediately below the renal vasculature by means of a vessel clamp. Lower-body ischemia was maintained for 60 minutes, at which time the clamp was carefully removed.After clamp removal, the intestines were replaced, and the abdominal cavity was sutured. Vessels were reperfused for 30 minutes, after which time the cremaster muscle was exteriorized and leukocyte rolling observed by intravital microscopy. In some experiments, mice were given rPSGL-Ig (1 mg/kg) or vehicle as a pretreatment 55 minutes into the ischemic period (ie, just before reperfusion). Additional groups of animals were given rPSGL-Ig (1 mg/kg) or anti-P-selectin antibody (10 µg) 30 minutes after observation of rolling induced by I/R. Sham-treated mice received identical surgical manipulation followed by 60 minutes' sham ischemia and 30 minutes' sham reperfusion. Intravital microscopy data analysis After collection of intravital microscopy images, sequences of interest were digitized (Miromotion DC30; Pinnacle Systems, Mountain View, CA) and stored on a Macintosh (Seattle, WA) G3/400 computer prior to analysis. Rolling flux percentage was calculated as previously described.25 To allow easy comparison of rolling before and after application of inhibitors, some data are presented as the percentage of baseline rolling. Vessel geometry and leukocyte rolling velocities were measured by means of the public domain National Institutes of Health (NIH)-Image program (available at: http://rsb.info.nih.gov/nih-image) as described.33 Leukocyte tethering was quantified by counting the number of new attachments forming in a defined area of observation per unit of time. Tethering rate after inhibitor treatment is expressed as a percentage of the rate measured immediately prior to treatment. Some leukocytes were tracked through the network of cremasteric venules until they either detached, adhered, or rolled beyond the range of our imaging setup. These data are presented as percentages.Leukocyte accumulation in thioglycollate-induced peritonitis Negative controls (200 µL saline and 1 mg/kg CD4-Ig), rPSGL-Ig (1 mg/kg), anti-P-selectin antibody (10 µg RB40.34 per mouse), or anti-E-selectin antibody (10 µg 10E6 per mouse) were given as a pretreatment to wild-type mice. In these experiments, rPSGL-Ig was injected subcutaneously rather than intravenously. This allowed treatment of a large number of mice in close succession since preliminary experiments identified temporal variation as a major determinant of experimental error. At 15 minutes after pretreatment, 1-mL injections of 3% thioglycollate broth or 0.9% saline were administered intraperitoneally. Mice were killed 4 hours after thioglycollate by cervical dislocation. Heparinized saline (5 mL, 10 U/mL) was injected into the peritoneum; the abdomen was gently massaged; and lavage fluid was recovered via a small incision into the abdominal cavity. Samples of lavage fluid were taken for total leukocyte counts, which were performed by means of a modified Neubauer hemocytometer, and differential counts, which were performed on Giemsa-stained cytospin slides. Thioglycollate-induced peritonitis data are presented as the total number of neutrophils per peritoneum.Preparation and investigation of rPSGL-Ig-coated beads Nonfluorescent, protein A-coated 1-µm latex beads were purchased from Bangs Laboratories (Fishers, IN). Stock (1% wt/vol) beads were diluted 1/10 for labeling. Then, 200 µL of 0.1% (wt/vol) beads were incubated overnight at 4°C with 50 µg rPSGL-Ig or low-affinity rPSGL-Ig. Residual protein A sites on the beads were then blocked by incubation with an excess of Blockaid (Molecular Probes, Eugene, OR) for 15 minutes at room temperature. Beads were washed by centrifugation and resuspended at 0.1% wt/vol. Murine KC was fluorescently labeled by means of a kit according to the manufacturer's (Molecular Probes) instructions. Then, 10 µg fluorescent KC was incubated with 5 µL rPSGL-Ig- or low-affinity rPSGL-Ig-coated beads for 30 minutes at 4°C in a final volume of 100 µL. In some experiments, fluorescent KC was incubated with free rPSGL-Ig (10 µg/mL) or low-affinity rPSGL-Ig (10 µg/mL) prior to incubation with beads. Binding of fluorescent KC to nonfluorescent beads was assessed by means of flow cytometry.Purification of murine neutrophils Neutrophils were isolated from mouse blood as described.26 Briefly, blood was collected from the carotid artery into heparin (100 U/mL) and mixed with dextran (1.25% wt/vol in saline) to a final volume of 10 mL for each 1 mL blood collected. After erythrocyte sedimentation, leukocyte-rich plasma was collected and washed in PBS containing BSA (0.5% wt/vol). Antibodies against CD2, CD5, CD45R, F4/80, and ICAM-1 were added to target nonneutrophil cell types. Anti-ICAM-1 also recognizes neutrophils, but the dose of this antibody was titrated to preferentially target monocyes.26 Secondary antibody-coated magnetic microspheres were then added, and labeled cells removed by passing the cell suspension over a BS cell-separation column that was attached to a VarioMACS magnet (Miltenyi Biotech). After removal of residual erythrocytes by hypotonic lysis, neutrophils were washed once more in PBS plus BSA and resuspended at 2 × 106/mL.In vitro transmigration of murine neutrophils We used an in vitro model to investigate the influence of rPSGL-Ig on chemoattractant-induced migration of murine neutrophils. Neutrophils were treated with either CD4-Ig, low-affinity rPSGL-Ig, or rPSGL-Ig at different concentrations and then immediately dispensed (25 µL of 2 × 106/mL suspension) onto the filter membrane of a ChemoTx microplate (Neuro Probe, Gaithersburg, MD). The framed filter array was then positioned over a microplate loaded with different concentrations (108 to 106 M, 25 µL per well) of the murine chemoattractant KC or controls. Microplates were incubated for 3 hours at 37°C in 5%
CO2. After incubation, nonmigrated neutrophils were removed
from the top of the filter by wiping and washing 3 times with
RPMI. Migrated neutrophils were pelleted to the bottom of
microplate wells by centrifugation (300g, 10 minutes) and
then resuspended in 25 µL RPMI. Numbers of migrated neutrophils were
determined by means of a hemocytometer, and results expressed as the
percentage of neutrophils migrated or the percentage of control migration.
Statistics Differences between data sets were analyzed by means of GraphPad Prism software (San Diego, CA). Data were analyzed for deviation from a normal distribution, and tests for parametric or nonparametric data were used appropriately. Posttests (Dunn, Dunnet, or Tukey Kramer) for multiple comparisons were used when required.Online supplemental material Four QuickTime movies are included on the Blood website with this manuscript; see the Supplemental Videos link at the top of the online article. Movies were digitized from video archives and prepared for online viewing by means of Adobe Premiere version 5.1 (www.adobe.com/premiere) and QuickTime Pro version 5 (www.apple.com/quicktime) as described.33
rPSGL-Ig inhibits leukocyte rolling on all 3 selectins in vivo We used intravital microscopy to study the ability of rPSGL-Ig to prevent binding of natural selectin ligands and thereby disrupt leukocyte rolling in vivo.Surgical preparation of the mouse cremaster muscle for intravital
microscopy induces leukocyte rolling that is almost exclusively dependent on P-selectin.25 Baseline rolling was observed
30 minutes after such surgery and recorded for 1 minute. Indicated doses of rPSGL-Ig were injected intravenously, and the effects on
existing leukocyte rolling were observed. Low doses (1 mg/kg, 10 mg/kg)
of rPSGL-Ig did not alter the proportion of leukocytes rolling through
observed venules, whereas higher doses (30 mg/kg, 100 mg/kg) did
(Figure 1A). Interestingly, the
incomplete inhibition given by 30 mg/kg rPSGL-Ig was not exceeded by a
higher dose. Quicktime movies showing P-selectin-dependent leukocyte
rolling before (movie 1) and after (movie 2) 100 mg/kg rPSGL-Ig are
included with the online version of this manuscript. A PSGL-1 blocking antibody, 2PH1, inhibited leukocyte rolling to a level similar to that
of rPSGL-Ig, whereas P-selectin blocking antibody all but
abolished surgically induced rolling. A Quicktime movie comparing the
effect of P-selectin blocking antibody with that of rPSGL-Ig (movie 3)
is included with the online version. As well as reducing the proportion
of leukocytes rolling through observed venules, rPSGL-Ig also increased
the velocity of leukocytes continuing to roll (Figure 1B). Increased
rolling velocity was measurable with doses of rPSGL-Ig that did not
reduce the proportion of leukocytes rolling (ie, 1 mg/kg).
We have previously used TNF To investigate L-selectin-dependent leukocyte rolling, we treated
E-selectin By making these observations in the same vessels before and
after treatments, we controlled for vessel diameter as a possible influence on leukocyte rolling. Although vessels selected for observation in TNF
Low-dose rPSGL-Ig inhibits thioglycollate-induced peritonitis Low-dose (1 mg/kg) rPSGL-Ig failed to reduce rolling in any of these studies. We found this result surprising since others have described pronounced anti-inflammatory effects of this molecule at doses as low as 0.25 mg/kg. We therefore tested the anti-inflammatory activity of rPSGL-Ig in a peritonitis model.Few neutrophils were detected in peritoneal lavage samples of
unstimulated mice. Thioglycollate treatment caused approximately 5 million neutrophils to be recruited; a response that was not modified
by CD4-Ig (Figure 2). In contrast,
rPSGL-Ig (1 mg/kg) pretreatment inhibited thioglycollate-induced
recruitment of neutrophils to the peritoneum by 87%. The P-selectin
blocking antibody RB40.34 inhibited neutrophil recruitment by
approximately 77%, and a combination of RB40.34 and the E-selectin
blocking antibody 10E6 reduced recruitment by more than 90%. Thus, in
agreement with previous investigators, we find pronounced
anti-inflammatory activity of rPSGL-Ig at a relatively low
dose.
Low-dose rPSGL-Ig inhibits I/R-injury-induced leukocyte rolling We also tested the effects of low-dose rPSGL-Ig in a model of I/R injury. I/R injury was induced by clamping the aorta for 1 hour followed by 30 minutes of reperfusion. Leukocyte rolling was observed by means of intravital microscopy of cremaster muscles prepared at the end of the reperfusion period. Mice subjected to sham I/R injury followed by preparation of the cremaster muscle had levels of leukocyte rolling similar to those of mice receiving cremaster surgery alone, whereas I/R-injured mice had elevated levels of leukocyte rolling (Figure 3A). Treatment with rPSGL-Ig (1 mg/kg, intravenously) 5 minutes before reperfusion, inhibited the increased leukocyte rolling caused by I/R, although apparently not that caused by surgical exposure of the cremaster muscle 30 minutes later (Figure 3A). Systemic leukocyte concentrations, blood flow velocities, and diameters of vessels examined in I/R experiments are shown in Table 2. Mice subjected to the I/R procedure had significantly higher cremasteric blood flow but leukocyte concentrations similar to those of sham-treated mice. Pretreatment with rPSGL-Ig did not significantly influence any of the hemodynamic parameters studied in I/R experiments, and similarly sized vessels were selected.
Rolling in the cremaster muscle induced by I/R injury is known to be exclusively P-selectin dependent at early time points.35 Given that 1 mg/kg rPSGL-Ig failed to reduce surgically induced (P-selectin-dependent) rolling in earlier experiments (Figure 1A), we were surprised by its potent actions on I/R-induced rolling. To investigate the possibility that pretreatment with rPSGL-Ig confers some advantage to its activity, we tested the effects of 1 mg/kg rPSGL-Ig on pre-existing I/R-induced leukocyte rolling. Data in Figure 3B clearly demonstrate that 1 mg/kg rPSGL-Ig does not alter I/R-induced rolling if it is given after rolling has been established. This same rolling is all but abolished by a P-selectin blocking antibody, supporting earlier reports that such rolling is P-selectin dependent.35 Effect of rPSGL-Ig on leukocyte tethering Data in Figure 3 suggest considerable advantage to giving rPSGL-Ig as a pretreatment. Before leukocytes can begin rolling along vascular endothelium, they must first tether to it. Formation of the first molecular bond between a free-flowing leukocyte and endothelium may present more of a physical challenge than formation of subsequent bonds (which are facilitated by the existence of the first) and may therefore be more sensitive to inhibition. We designed experiments to test whether low-dose rPSGL-Ig could inhibit leukocyte tethering. Attention was focused on points where tethering was noted to preferentially occur and could be clearly demarked. This was most easily achieved by observing points where capillaries fed into venules (movie 4 in the online version of this article). Control tether formation was counted 30 minutes after surgical preparation of the cremaster. The tethering rate was not significantly altered by application of rPSGL-Ig at any of the doses studied. This suggests that the effect of 30 mg/kg rPSGL-Ig on rolling flux was not due to a reduction in the number of cells forming attachments to postcapillary venular endothelium. Tethering was abolished by P-selectin blocking antibody (Figure 4).
Effect of rPSGL-I on the fate of rolling leukocytes We hypothesized that rPSGL-Ig might destabilize rolling, allowing leukocytes to roll only short distances before detaching. Such an effect might explain the advantage of pretreatment and the potent anti-inflammatory activity of this molecule. To test this, we tracked leukocytes from their point of attachment in the smallest postcapillary venules of the cremaster to points where they could no longer be visualized (ie, as they entered very large vessels or left the cremaster). Velocities of leukocytes tracked through 4 different routes are shown in Figure 5A. Of the 4 leukocytes tracked prior to application of rPSGL-Ig (denoted by the black lines), 1 rolled for some distance and then firmly adhered (denoted by downward-pointing arrow), whereas the others rolled continuously from their point of attachment to points beyond observation (indicated by rightward-facing arrows). Mice were subsequently treated with 1 mg/kg rPSGL-Ig, and leukocyte velocities tracked through the same vessel networks (denoted by the gray lines). Although leukocytes rolled more quickly following 1 mg/kg rPSGL-Ig treatment, they were not more prone to detachment. The fate of a larger number of cells is shown in Figure 5B. Low-dose (1 mg/kg) rPSGL-Ig increased the percentage of cells rolling all the way out of the tissue by reducing the proportion that firmly adhered. Detachment of leukocytes from the endothelium was an infrequent event that was not appreciably altered by rPSGL-Ig at 1 mg/kg. In contrast, detachment was the most likely fate of leukocytes rolling in venules of mice treated with rPSGL-Ig at 30 mg/kg. Firm adhesion was rare in these mice, and no leukocytes rolled all the way out of the cremaster.
rPSGL-Ig binds the chemokine KC and inhibits its function PSGL-1 has certain functional requirements in common with chemokine receptors, including posttranslational sulfation and glycosylation.36 To investigate the possibility of chemokine binding to rPSGL-Ig, we coupled rPSGL-Ig to 1-µm beads and measured binding of fluorescent KC (a murine CXC chemokine) using flow cytometry. Fluorescent KC bound to beads coated with rPSGL-Ig (Figure 6A), but not to beads coated with a low-affinity form of rPSGL-Ig (Figure 6B). Preincubating fluorescent KC with soluble rPSGL-Ig (10 µg/mL) prevented subsequent binding to rPSGL-Ig-coated beads, whereas preincubation with low-affinity rPSGL-Ig (10 µg/mL) did not (Figure 6C).
We also used an in vitro neutrophil transmigration assay to investigate whether rPSGL-Ig could inhibit KC-induced responses. Mouse neutrophils (5 × 104/25 µL) placed onto filters of ChemoTx chambers migrated in a dose-dependent fashion toward the chemokine KC located in the wells of the microplate (Figure 6D). Interestingly, 10 µg/mL rPSGL-Ig inhibited migration toward KC. This concentration of rPSGL-Ig is roughly equivalent to the blood concentration given by the 1 mg/kg dose. Migration was not significantly affected by either CD4-Ig or low-affinity rPSGL-Ig. Inhibition of KC-induced neutrophil migration by rPSGL-Ig was dose dependent (Figure 6E).
It is proposed that selectin antagonists such as rPSGL-Ig function by competing with cell-bound selectin ligands to prevent leukocyte-endothelial cell interaction. Since leukocyte rolling depends on constant formation and rupture of selectin-ligand bonds, such competitive action should allow selectin antagonists to reverse established leukocyte rolling. In spite of extensive investigation of rPSGL-Ig, its selectin-binding properties16 and anti-inflammatory actions,17-22,37 ours is the first evidence that rPSGL-Ig does precisely this in vivo. We have shown that while high doses of rPSGL-Ig reduce pre-existing leukocyte rolling on all 3 selectins, lower doses (previously shown to be anti-inflammatory) have modest effects, unless given as a pretreatment. These observations support, but do not prove, the assumption that rPSGL-Ig is a competitive selectin inhibitor. Low-dose (1 mg/kg) rPSGL-Ig did not reduce established
P-selectin-dependent leukocyte rolling in venules of the mouse
cremaster, although we did detect moderately increased rolling
velocities following treatment at 1 mg/kg. We could consider leukocyte
rolling in simple terms, such as athletes on a running circuit and our point of observation as the start/finish line. According to such a
simple model, doubling velocity should also double the events at our
point of observation (providing the same number of runners show up,
none of them stop, and no one runs off the track). Studies with other
selectin inhibitors (particularly E-selectin inhibitors in
TNF Leukocyte rolling is not just a braking mechanism, allowing cells to decelerate as they near their target; it also enables them to sample their environment. Thus, rolling leukocytes are exposed to chemoattractants presented by vascular endothelial cells,39 and slow rolling enhances this exposure and promotes recruitment.38,40 Agents, such as rPSGL-Ig, that increase leukocyte rolling velocity may have profound anti-inflammatory effects by limiting the duration of contact between leukocytes and endothelium. The significance of such an effect is supported by the observation that 1 mg/kg rPSGL-Ig reduced the proportion of tracked leukocytes firmly adhering in venules of surgically stimulated mice without altering the number of leukocytes rolling through those venules. Although it is likely that the effect of rPSGL-Ig on leukocyte rolling velocity contributes to its impressive anti-inflammatory activity, we do not believe this to be the full explanation. There seems to be considerable advantage in giving this molecule as a pretreatment, which is not readily explained by this mechanism. Low-dose rPSGL-Ig (1 mg/kg or lower) given as a pretreatment is able to reduce leukocyte rolling in response to various stimuli (this and other17,19,20 studies), whereas the same dose given after rolling is established merely alters rolling velocity. An appealing explanation for the advantage of pretreatment is that low-dose rPSGL-Ig, rather than inhibiting leukocyte rolling per se, prevents initial attachment of leukocytes to the endothelium. Established rolling may be more resistant to inhibition, as one molecular bond between leukocyte and endothelial cell will promote formation of subsequent bonds between molecules that are held in proximity to one another. This explanation can be ruled out, however, by the observation that rPSGL-Ig does not alter tethering rate when given after stimulation of the cremaster. Since rPSGL-Ig is a large, complex molecule posttranslationally modified to express sialylated fucosylated glycans and sulfate, it may have the potential to interfere with multiple stages of the inflammatory response. Others have shown effects of rPSGL-Ig that are not readily related to competitive inhibition of selectin-selectin ligand binding, including changes in de novo synthesis of endogenous PSGL-119 and E-selectin mRNA.37 Our transmigration experiments suggest inhibition of chemoattractant-driven responses as an alternative or additional mechanism of action. A rationale for this observation may be that chemokine (and other chemoattractant) receptors require sulfation and posttranslational glycosylation similar to that of PSGL-1 in order to function36 and that various sulfated glycans (eg, heparan sulfate, chondroitin sulfate) are known to affect both chemokine41 and selectin42 binding. Large sulfated, glycosylated molecules such as rPSGL-Ig, and perhaps natural soluble PSGL-1, may present attractive surfaces for binding of chemokines. This binding may have proinflammatory or anti-inflammatory activities depending on the location of the molecule. Although the anti-inflammatory activity of low-dose rPSGL-Ig cannot be attributed entirely to direct inhibition of selectin-selectin ligand interaction (at least in mice), higher doses (30 to 100 mg/kg) of this molecule are able to reduce pre-existing rolling. Furthermore, this activity is apparent in models that favor either E-, L-, or P-selectin-dependent rolling. Although rPSGL-Ig can inhibit rolling by all 3 selectins in vivo, its activity appears to be incomplete, in that a significant proportion of rolling remains after rPSGL-Ig treatment in all experimental systems studied. In our model of surgically induced (P-selectin-dependent) rolling, for example, we see approximately 50% to 60% inhibition at 30 mg/kg and no further inhibition at 100 mg/kg. Treatment with P-selectin antibody, in contrast, abolishes leukocyte rolling in this model. Interestingly, antibodies against PSGL-1 are similarly unable to abolish P-selectin-dependent rolling,11,43,44 whereas gene-targeted mice lacking PSGL-1 have virtually no surgically induced rolling.45 We believe these data can be reconciled if we consider the partnership between the correctly modified N-terminus of PSGL-1 and P-selectin as a dominant, but not exclusive, interaction that can be inhibited by either PSGL-1 blocking antibodies or rPSGL-Ig. Data from PSGL-1 knockout mice suggest that residual rolling is still PSGL-1 dependent but may involve other parts of the molecule. There are other possible explanations for the observation that direct inhibition of leukocyte rolling by rPSGL-Ig is incomplete and requires such high doses. Firstly, rPSGL-Ig is based on the structure of human and not murine PSGL-1. Although human and murine selectins and selectin ligands have some sequence homology,5,7 we cannot rule out the possibility that species differences underlie the requirement for high doses of rPSGL-Ig and the incomplete effect. This argument does not explain the incomplete inhibition given by 2PH1, which is specifically directed against murine PSGL-1. Another antibody (4RA10) against murine PSGL-1 blocks more rolling than 2PH1,44 but is still not as effective as P-selectin blocking antibody. In summary, we have investigated the ability of rPSGL-Ig to influence leukocyte rolling. Although we find that rPSGL-Ig can inhibit leukocyte rolling via all 3 selectins in vivo if given in sufficient doses, the mechanism of its anti-inflammatory activity remains somewhat mysterious. Low-dose rPSGL-Ig has pronounced anti-inflammatory effects and can reduce leukocyte rolling if given as a pretreatment. The chemoattractant-driven responses that follow leukocyte rolling are also inhibited by rPSGL-Ig. The mechanism of this effect requires further investigation.
Submitted August 1, 2002; accepted November 12, 2002.
Prepublished online as Blood First Edition Paper, December 12, 2002; DOI 10.1182/blood-2002-07-2329.
Supported by grants FS/98051, FS/2001007, and FS/2002004 from the British Heart Foundation and grants 061305 and 057108 from the Wellcome Trust.
A.E.R.H. and S.L.N. contributed equally to this work.
The online version of the article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Keith Norman, Cardiovascular Research Group, University of Sheffield, Clinical Sciences Centre, Northern General Hospital, Herries Rd, Sheffield S5 7AU, United Kingdom; e-mail: k.norman{at}sheffield.ac.uk.
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Top
Abstract
Introduction
) or saline (
). Peritoneal lavage was
performed 4 hours after stimulation, and neutrophil numbers were
determined. The data represent the means ± SEMs of 7 mice in vehicle, CD4-Ig-, and rPSGL-Ig-pretreated groups, and 8 mice
in all other groups. Significant difference from vehicle control is
indicated by **P < .01.
; 30 mg/kg
rPSGL-Ig, 54 cells, 