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PHAGOCYTES
From the Cardiovascular Research Group and Section of
Surgery, Division of Clinical Sciences (NGH), University of Sheffield,
United Kingdom; and Novartis Pharma AG, Transplantation Preclinical
Research, Basel, Switzerland.
Selectin-dependent rolling is the earliest observable event in the
recruitment of leukocytes to inflamed tissues. Several glycoproteins
decorated with sialic acid, fucose, and/or sulfate have been shown to
bind the selectins. The best-characterized selectin ligand is
P-selectin glycoprotein-1 (PSGL-1) that supports P-selectin- dependent
rolling in vitro and in vivo. In vitro studies have suggested that
PSGL-1 may also be a ligand for E- and L-selectins. To study the in
vivo function of PSGL-1, without the influence of other leukocyte
proteins, the authors observed the interaction of PSGL-1-coated
microspheres in mouse venules stimulated to express P- and/or
E-selectin. Microspheres coated with functional recombinant PSGL-1
rolled in surgically stimulated and tumor necrosis factor alpha
(TNF Recruitment of leukocytes to inflamed tissues is
critical for defense against pathogens, but also contributes to host
injury in conditions such as ischemia-reperfusion injury.1
Leukocyte accumulation begins with attachment to and rolling along
postcapillary venules.2 Rolling is largely dependent on
the selectin family of adhesion molecules3 and is a
prerequisite for later arrest and diapedesis. Selectins are
differentially expressed with constitutive L-selectin on almost all
leukocytes, P-selectin released from storage granules to surfaces of
stimulated platelets and endothelial cells, and E-selectin appearing on
endothelial cells after cytokine stimulation.3
The selectins share a modular structure with a characteristic lectin
domain at the N-terminus, which is largely responsible for ligand
recognition. Selectins recognize certain carbohydrate structures such
as sialyl Lewisx (sLex) and its topoisomer
sialyl Lewisa (sLea).3 Sialic acid
and fucose are key elements for all selectins, whereas L-selectin also
requires sulfate on the sLex backbone.3,4
sLex and variants thereof can bind selectins in
vitro,5,6 and cells with proteins decorated by such
oligosaccharides roll on selectins under the conditions prevalent in
the living microcirculation.7 There is widespread
appreciation, however, that real selectin-ligand interaction requires
a specific protein backbone decorated with complex, branched
carbohydrates. The most characterized selectin ligand is P-selectin
glycoprotein ligand-1 (PSGL-1), which was initially identified as a
ligand for P-selectin,8,9 but is also reported to bind
E-10-13 and L-selectins.14 Gene-targeted mice, genetically deficient for PSGL-1 have been recently
described.15 Interestingly, these mice show defects in
P-selectin-mediated, but not E-selectin-mediated leukocyte rolling,
bringing the relevance of PSGL-1 as an E-selectin ligand into question.
The role of PSGL-1 as an L-selectin ligand was not addressed in detail
using these mice.
For binding to P-selectin, PSGL-1 requires a posttranslational
modification by enzymes to express sialic acid and fucose on polylactosamine extended core-2 O-glycans and sulfate on
certain N-terminal tyrosine residues.4 Cells lacking the
enzymes for modification of PSGL-1 (or other proteins) do not bind
selectins. Some studies suggest that covalent dimerization of PSGL-1 is
required for binding to P-selectin,16,17 although this has
been recently questioned.18 Physiologic binding of
leukocytes to P-selectin is almost entirely dependent on PSGL-1
because a monoclonal antibody (mAb) (PL-1) against PSGL-1 blocks the
rolling of neutrophils in vitro8 and in
vivo.9 Binding of L-selectin to PSGL-1 supports
leukocyte-leukocyte interaction in in vitro models and is blocked by
PL-1,14 although the importance of such interaction has
not been confirmed in vivo.19 Binding of PSGL-1 to
L-selectin requires sulfation of tyrosines in the amino-terminal region
and extended O-glycans containing fucose and sialic acid.
E-selectin interaction with PSGL-1 apparently differs from that of P-
or L-selectin in that sulfation is not required20 and PL-1
barely affects binding.4
Recent investigations revealed that microspheres coated with
sLex or sLea21,22 could roll on surfaces
expressing E-selectin, whereas microspheres coated with recombinant
PSGL-1 grown in COS cells expressing an Because conditions in the living microcirculation cannot be fully
reproduced using in vitro methods, our aim was to determine whether
PSGL-1 alone could support rolling in vivo. We previously used
recombinant PSGL-1 to define the biomechanics of P-selectin/PSGL-1 interaction.23 Here, we use PSGL-1-coated microspheres to
study the capacity of PSGL-1 to support rolling interaction with
endothelial selectins in vivo.
Reagents
Selectin ligand-coated microspheres
PSGL-1/IgG was purified and biotinylated as described previously.23 Biotinylated sLea-polylysine was also synthesized according to published methods.28 Yellow-green fluorescent microspheres (0.07 mL, 1.0-µm diameter) coated with avidin (Fluospheres, Molecular Probes, Eugene, OR) were washed twice by suspension in 5 mL phosphate-buffered saline (PBS), followed by centrifugation (2000g, 15 minutes). Microspheres were then incubated overnight at 4°C with 0.15 mg of biotinylated PSGL-1/IgG or sLea in 0.5 mL PBS. After incubation, microspheres were washed as previously described and resuspended in 0.35 mL PBS to make a final solution of 0.2% microspheres. Microspheres were stored at 4°C and used in rolling experiments within 10 days of coating. For injection into mice, the microsphere solution was diluted 1 to 10. Density of P-selectin glycoprotein ligand-1 on microspheres and on murine and human neutrophils Expression of PSGL-1 on mouse peripheral blood neutrophils was determined by whole blood flow cytometry. Peripheral blood was obtained by cardiac puncture from male C57BL/6 mice and 100 µL aliquots incubated for 30 minutes at 4°C with a saturating concentration of PE-conjugated antimouse-PSGL-1 monoclonal antibody (2PH1) or isotype-matched control antibody (R3-34). Cells were washed (2 mL CellWash [Pharmingen], 400g, 10 minutes) and erythrocytes lysed using PharmLyse (Pharmingen). After 2 further washes, cells were resuspended in 0.5 mL CellFix (Pharmingen) and stored in the dark at 4°C until analysis. Expression of PSGL-1 on human neutrophils was determined by the same method, substituting KPL1 (antihuman PSGL-1) for 2PH1.Because the PSGL-1 on our microspheres was recombinant human material, grown in a transfected cell line, we could not confidently assume that KPL1 would interact with our microspheres with the same efficiency as with native neutrophil PSGL-1. We therefore chose to determine the maximum possible PSGL-1 loading for our microspheres by measuring the number of biotin-binding sites. To do this we produced PE-labeled microspheres according to the method previously described for PSGL-1-coated microspheres, substituting an equimolar concentration of biotinylated PE (Molecular Probes) for biotinylated PSGL-1. The fluorescent intensity of PE microspheres and PE PSGL-1 antibody staining on peripheral blood neutrophils was determined using a FACScan flow cytometer and CellQuest data acquisition and analysis software (Becton-Dickinson Immunocytometry Systems, Mountain View, CA). Peripheral blood neutrophils were identified on the basis of forward (FSc) and side (SSc) light scatter characteristics and, for mouse cells, on the basis of positive staining with the granulocyte specific antibody RB6-8C5. The fluorescent intensity of gated cells was determined and the median channel of fluorescent intensity (MFI) recorded as a measure of antigen density. Cells incubated with the PE-conjugated control antibodies R3-34 (mouse) or F8-11-13 (human) were studied as controls. PE-conjugated microspheres were identified on the basis of FSc and SSc characteristics and a live analysis gate was placed around this population. The fluorescent intensity of the gated population was determined in logarithmic mode and the MFI recorded. Unstained micropheres acted as a negative control. The fluorescence intensities of the microspheres and peripheral blood neutrophils were converted into the number of molecules of equivalent fluorescence (MEF) by reference to a standard regression line generated using calibrated fluorescent microspheres (FluoroSpheres, Dako, Ely, UK). These comprise a mixture of 6 microsphere populations, each of which are labeled with a known number of fluorescent molecules. These microspheres contain a unique combination of fluorochromes, enabling calibration of a number of fluorescent markers, including PE. Microspheres were identified on the basis of light scatter, and the MFI of each population determined. A standard linear regression line of MFI versus the number of molecules of equivalent fluorescence was drawn, from which the MEF of PE-labeled neutrophils and microspheres were derived. Animals Male C57BL/6 mice (in-house colony), P-selectin knockout mice (in-house colony derived from C57BL/6J-Selptm1Bay, The Jackson Laboratory, Bar Harbor, ME) and E-selectin knockout mice (in-house colony derived from breeding pairs supplied by Dr B. A. Wolitzky)29 weighing between 25 and 35 g were used in these experiments. All procedures were approved by the University of Sheffield ethics committee and by the Home Office Animals (Scientific Procedures) Act 1986 of the UK. Mice were anesthetized with an intraperitoneal injection of 100 mg/kg ketamine hydrochloride after premedication with 30 mg/kg sodium pentobarbital and 0.1 mg/kg atropine sulfate. Some mice received an intrascrotal injection of 500 ng TNF 2.5 hours before
intravital microscopic observation. Other mice were depleted of
circulating neutrophils using RB6-8C5 (10 µg, intravenously
[iv]).
Intravital microscopy The cremaster was prepared for intravital microscopy and superfused as described by Ley et al.24 Venules were observed 10 to 30 minutes after surgical stimulation of tissue to study P-selectin-dependent rolling and 2.5 hours after TNF stimulation of
tissue to study combined E/P-selectin-dependent rolling.
Venules were observed using a Leitz diaplan (Leica
Microsystems, Cambridge, United Kingdom) equipped with a water
immersion objective (20x/0.5W). Images of rolling leukocytes were
recorded by a CCD camera (DC330EX, Dage MTI, Michigan City, MI)
onto sVHS videocassettes. After recording leukocyte rolling for 1 minute, passage of fluorescent microspheres was observed by dual flash stroboscopic (100-second Data analyses Sequences of interest were digitized (Miromotion DC30, Pinnacle Systems, Braunschweig, Germany) and analyzed using NIH-Image (National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/nih-image). To compare leukocyte rolling before and after different treatments, rolling flux percentage was calculated as described.24 Briefly, the number of leukocytes rolling past a fixed plane perpendicular to the vessel axis was determined. This value was expressed as a percentage of the total number of leukocytes passing through that vessel (calculated as the product of systemic leukocyte count and volumetric flow rate). Because all fluorescent microspheres (free-flowing and interacting) could be visualized, and because relatively small numbers of microspheres rolled continuously for long distances (rolling only short distances in surgically stimulated mice and rolling very slowly in TNF-stimulated
mice), we used a different method to quantify microsphere interaction.
Thus, microsphere rolling is quantified as percentage attachment rate per 100 µm vessel length. This value is calculated by dividing the
number of new attachments formed in a venule by the total number of
microspheres passing through that venule and then correcting for vessel
length. Attachment is defined as a rapid deceleration to a velocity
clearly lower than that of free-flowing microspheres. Although we
did not measure velocities of all observed microspheres, those that
were measured uniformly fell below critical velocity calculated as
described.9 Rolling of leukocytes and microspheres in surgically stimulated and TNF-stimulated wild-type mice and in
TNF-stimulated E / mice is also depicted in 2 video sequences on
the Blood website (see the Supplemental Videos link at the top of the online article). The vessels depicted in these sequences show leukocytes and beads rolling in surgically stimulated venules of
wild-type mice, TNF-stimulated venules of wild-type mice and TNF-stimulated venules of E-selectin / mice and are typical examples of those analyzed in Figures 2 to 5.
Statistics Differences between groups were compared by one-way analysis of variance (ANOVA), followed by Dunnett's test using INSTAT software (GraphPad Software, San Diego, CA).All measurements were compared with values given in similarly stimulated wild-type mice receiving no antibodies.
P-selectin glycoprotein ligand-1 expression on microspheres and on murine and human neutrophils We used flow cytometry to compare the maximum ligand coating density on our microspheres with that of PSGL-1 on murine neutrophils. The fluorescence histograms shown in Figure 1 show that microspheres coated with PE-biotin (at concentrations equivalent to concentrations of PSGL-1-biotin used to prepare microspheres for intravital microscopy studies) express fewer PE molecules than murine neutrophils labeled in whole blood with saturating concentrations of PE-conjugated anti-PSGL-1 antibody. Comparing the MFI of neutrophils and microspheres with the regression line generated from the MFI of calibrated microspheres (Figure 1), we calculate that murine neutrophils express approximately 75 000 PSGL-1 molecules per cell, whereas each of our coated microspheres expresses approximately 30 000 molecules. Because our measurement of PSGL-1 density on murine neutrophils is considerably higher than the number of P-selectin binding sites reported for human neutrophils,30 we decided to perform a direct comparison of PSGL-1 density on murine and human neutrophils. Human neutrophils stained with a saturating concentration of KPL1 had MFI of 179, whereas murine neutrophils stained with a saturating concentration of 2PH1 had MFI of 735. Converting the MFI of KPL1-stained neutrophils into molecules of equivalent fluorescence (by comparing with Dako fluorospheres), we estimate that there are approximately 18 000 PSGL-1 molecules on the surface of each human neutrophil. This value is in fairly close agreement with previously reported levels of P-selectin binding sites on neutrophils.30 Taken together, these results indicate that mouse neutrophils express considerably more PSGL-1 molecules on their surface than human neutrophils.
After correcting for the fact that surface area of a 1-µm microsphere is 1/49th that of a 7-µm neutrophil, it is clear that PSGL-1 density on our microspheres is considerably higher than that on mouse and human neutrophils. Although these differences may be reconciled by the fact that PSGL-1 on neutrophils is selectively targeted to dense clusters on the tips of microvilli, we also accept that the remarkable ability of these microspheres to mimic leukocyte rolling may be in part due to the increased density of PSGL-1. Hemodynamic variation in observed vessels Because hemodynamic variation can influence leukocyte rolling,24 diameters and centerline blood flow velocities were recorded for all observed vessels. These data are summarized in Table 1. Although there was some variation in average vessel diameters, no group differed significantly in this respect from equivalently stimulated wild-type mice receiving no antibodies. Surgically stimulated mice receiving P-selectin antibody, TNF-stimulated wild-type mice receiving combined E- and
P-selectin antibodies and TNF-stimulated P-selectin knockout mice
had somewhat lower centerline velocities than similarly stimulated
wild-type mice without antibodies. There is a negative relationship
between centerline velocity and rolling flux percentage24;
however, lower velocities do not explain the reduced
leukocyte/microsphere interaction seen.
Leukocyte rolling and microsphere interaction in surgically stimulated mice Initially, we investigated whether microspheres coated with PSGL-1 (293/FTIII) could roll in venules with P-selectin-dependent leukocyte rolling induced as described.24 Figure 2A and previous studies24,31 show that 10 to 30 minutes after surgical stimulation of tissue, more than 20% of leukocytes passing through observed venules were rolling, and that this rolling was abolished by treatment with the P-selectin antibody, RB40.34. Figure 2B shows interaction of coated microspheres in the same vessels. The profile of microsphere interaction in surgically stimulated venules largely mirrors that of leukocyte rolling. Thus, approximately 10% of PSGL-1(293/FTIII)-coated microspheres attached to and rolled for some time (0.5-2 seconds) along observed venules, and this rolling was abolished by treatment with RB40.34. 10A10, a nonblocking anti-P-selectin antibody, did not alter rolling of leukocytes or microspheres (data not shown). Microspheres coated with PSGL-1 (293) PSGL-1(CHO/FTIII) or sLea formed few interactions, emphasizing the importance of both protein backbone and correct glycosylation for function.
Because we were concerned that microspheres may be interacting with rolling leukocytes rather than with endothelial P-selectin, we also studied microsphere interaction in mice that had been previously depleted of neutrophils using antibody RB6-8C5. PMN were selectively and completely removed from the peripheral circulation for the duration of our experiments by RB6-8C5 treatment (10 µg, iv) (data not shown). RB6-8C5 also abolished leukocyte rolling in observed venules (Figure 2A), a result that was not unexpected because neutrophils comprise more than 90% of the rolling population under the conditions studied herein.32 Removing granulocytes from the circulation with RB6-8C5, and thus rolling neutrophils from observed venules had no effect on microsphere interaction (Figure 2B), suggesting direct binding between human PSGL-1 on the microsphere and murine P-selectin on the endothelium. Leukocyte rolling and microsphere interaction in
TNF produces leukocyte rolling that is dependent on
all 3 selectins. Thus, rolling in cremasteric venules of mice stimulated for 2 to 4 hours with TNF is abolished by a combination of P- and E-selectin antibodies,33 and substantially
inhibited when P-selectin deficiency is combined with L-selectin
antibody treatment.24 Blocking P-selectin
alone24 in TNF-stimulated mice produces little or no
effect, L-selectin deficiency results in a slight reduction of rolling
flux percentage,33 and blocking E-selectin alone produces
a characteristic increase in rolling velocity,33
emphasizing the importance of this molecule for maintenance of slow
leukocyte rolling. Figure 3 shows the
effect of antibody treatments on the percentage of leukocytes rolling in TNF stimulated venules of wild-type mice. As described
previously,34 blocking E-selectin caused a slight increase
in rolling flux percentage, whereas combined blockade of E- and
P-selectin markedly reduced leukocyte rolling in observed venules. As
seen in surgically stimulated vessels, treatment with RB6-8C5 caused a
substantial reduction of leukocyte rolling in TNF stimulated
venules. Figure 4 shows attachment rates
of differentially coated microspheres in venules of mice stimulated for
2.5 hours with TNF. As in surgically stimulated mice, control
microspheres (PSGL-1(CHO/FTIII), PSGL-1(293), or sLea
coating) formed few attachments (Figure 4). In contrast,
PSGL-1(293/FTIII)-coated microspheres formed a larger number of
attachments (Figure 4) and these attachments were generally long
lasting (more than 1 minute). Microsphere adhesion was not the result
of interaction with rolling leukocytes because their attachment was not
significantly altered by RB6-8C5. Blocking E-selectin did not
significantly alter PSGL-1(293/FTIII) microsphere attachment, whereas
blocking both E- and P-selectins reduced attachment to levels seen with PSGL-1(293) microspheres. A combination of control mAbs 10A10 and 14E4
did not alter microsphere interaction in TNF-stimulated venules
(data not shown). Interestingly, PSGL-1(293/FTIII)-coated microspheres
formed few interactions in P-selectin / mice (Figure 4) and those
that were seen were very brief. This indicates an absolute requirement
for P-selectin in the attachment of these beads.
Rolling velocities of leukocytes and microspheres in differentially stimulated mice A role for E-selectin-PSGL-1 interaction in the maintenance of leukocyte or microsphere rolling is not precluded by the observation that E-selectin inhibition or deficiency does not reduce rolling of PSGL-1(292/FTIII+ve)-coated microspheres (Figure 4). A role for E-selectin in the maintenance of slow leukocyte rolling has been described,33 and we have used leukocyte rolling velocity as a sensitive measure of selective E-selectin inhibition in vivo.31 Because E-selectin-PSGL-1 interaction might support slow rolling in vivo, we compared rolling velocities of leukocytes and PSGL-1(293/FTIII)-coated microspheres in surgically and TNF-stimulated wild-type mice and in TNF-stimulated E-selectin
/ mice. Rolling velocities of leukocytes and microspheres under
different conditions are compared in Figure
5. Leukocytes rolling through
TNF-stimulated venules of wild-type mice travel much more
slowly than those rolling through venules of surgically stimulated mice
(Figure 5A). In contrast, leukocytes rolling through TNF-stimulated
vessels of E-selectin / mice roll at velocities approaching those
measured in surgically stimulated mice. Figure 5B shows that, as is the case for leukocytes, the majority of microspheres interacting with
observed venules in TNF-stimulated wild-type mice travel at
considerably lower velocities than microspheres rolling in surgically
stimulated mice, whereas absence of E-selectin results in a
considerable increase in microsphere rolling velocity. Similar increases in leukocyte and microsphere velocities were also given by
E-selectin antibody (10E6) treatment (data not shown).
We have shown that P-selectin-PSGL-1 binding is the only molecular interaction required for rolling of microspheres in living blood vessels with physiologic blood flow. In addition, we find that although binding of E-selectin to PSGL-1 is not sufficient to initiate/support tangible rolling, this interaction can substantially limit rolling velocity when P-selectin is also present. The discovery that PSGL-1 sustains rolling on P-selectin is entirely consistent with previous in vitro work and with our own intravital microscopy studies using PSGL-1 blocking antibodies. Thus, microspheres coated with PSGL-1 roll on P-selectin in vitro, and blocking PSGL-1 prevents rolling of neutrophils on P-selectin in vitro8 and in vivo.9 The obligate role of PSGL-1 demonstrated by antibody-blocking studies, and reconstitution of rolling behavior by correctly glycosylated recombinant PSGL-1 support the concept that PSGL-1 is the only important P-selectin ligand on leukocytes, although it is possible that other leukocyte molecules might interact with P-selectin subsequent to PSGL-1-dependent adhesion. The PSGL-1 used in our assay was monomerically associated with one heavy chain of IgG, which will spontaneously dimerize with another PSGL-1-bearing heavy chain. Because we do not know about the distances between PSGL-1 molecules on these dimers, we cannot comment on whether they are relevant to dimerization of native PSGL-1. There is substantial in vitro evidence that PSGL-1 is also an
E-selectin ligand.10-13 Our discovery that
E-selectin-PSGL-1 interaction limits rolling velocity in
TNF Much of the earlier in vitro evidence for E-selectin-PSGL-1
interaction is based on static assays measuring, for example, binding
of CHO cells transfected with E-selectin (CHO-E) to purified immobilized PSGL-1.10 Recent work studying interaction of
selectin ligand-coated microspheres with CHO-E suggested that
PSGL-1,13 sLex,21,22 and
sLea22 are each sufficient to both initiate and support
rolling on E-selectin under simulated physiologic flow conditions. Our
findings contrast with those from in vitro flow assays on 2 points:
first, microspheres coated only with sLea did not form
substantial interactions under any of the conditions studied, and
secondly, PSGL-1(293/FTIII)-coated microspheres did not interact in
TNF Comparison of the velocity figures for leukocytes and microspheres
shown in Figure 5 reveals remarkable similarity between these 2 populations. The 2 populations not only show a similar profile (slow
rolling in TNF Our results appear to be in contrast to the findings of Yang et
al15 who studied the phenotype of PSGL-1 deficient mice and found no defect of E-selectin-mediated rolling. These results are
consistent, however, if one considers the possibility that the
genetically modified mice described by Yang et al15 might compensate for the lack of PSGL-1 by overexpressing other E-selectin ligands, or that overall density of E-selectin ligands is so great that
removing one of them has no consequence. Taken together, our results
and those of Yang et al15 suggest that, although PSGL-1 is
able to act as an E-selectin ligand in vivo, it is by no means the only
E-selectin ligand on leukocytes and may not even be the major one.
Although limiting molecular expression on the beads to PSGL-1 alone has
enabled us to reveal the function of this molecule as a ligand for both
P- and E-selectins in vivo, we have not addressed the importance of
other potential selectin ligands, of which there are a large number of
candidates, including ESL-1,35 L-selectin,36
and In summary, we have demonstrated that microspheres coated with
recombinant human PSGL-1-IgG chimera can attach to and roll along
murine venules differentially activated to express either P-selectin or
a combination of P- and E-selectins. In contrast to earlier in vitro
work,13,21,22 we find that, in the absence of P-selectin,
E-selectin cannot initiate rolling of PSGL-1-coated microspheres. We
do show, however, that the rolling velocity of PSGL-1-coated
microspheres in TNF
We are grateful to Dr Nicky Brown for helpful discussions and for the loan of the SIT camera.
Submitted December 7, 1999; accepted July 6, 2000.
Supported by British Heart Foundation studentships FS98051 and FS99040 (A.E.H. and M.J.C.). Grants awarded by the Royal Society (RSRG20061) and the Wellcome Trust (057108, 043571) funded purchase of the Dage 330EX camera, intravital microscopy equipment, and flow cytometer.
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 E. Norman, University of Sheffield, Clinical Sciences Centre, Northern General Hospital, Herries Road, Sheffield S5 7AU, UK; e-mail: k.norman{at}sheffield.ac.uk.
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Top
Abstract
Introduction
1,6
N-acetylglucosaminyltransferase (core 2GlcNAcT) that synthesizes the
core-2 structure and, as a result, cannot elaborate extended
O-glycans. PSGL-1/IgG from CHO/FTIII cells and from 293 cells without FTIII were used as inactive controls.
1 Strobex 11360, Chadwick
Helmuth, Mountain View, CA) epifluorescence illumination. Microspheres
injected into the carotid artery in 50 µL boluses could be observed
in the cremasteric circulation within 20 seconds and typically
circulated for 1 to 2 minutes. Activation of the Strobex lamp (Chadwick
Helmuth) was controlled by the video camera. Two flashes were produced
for each video frame, giving double images of fast free-flowing
microspheres. Distance between such images was used to calculate
centerline velocities of observed vessels. Images of fluorescent
microspheres were recorded using a silicon-intensified target camera
(C2400, Hammamatsu Photonics UK, Enfield, UK).
a mechanism that amplifies initial leukocyte accumulation on P-selectin in vitro.
J Clin Invest.
1996;98:1081-1087