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Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4691-4699
Differential Leukocyte Recruitment From Whole Blood Via Endothelial
Adhesion Molecules Under Shear Conditions
By
Paul H. Reinhardt and
Paul Kubes
From the Immunology Research Group, University of
Calgary, Calgary, Alberta, Canada.
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ABSTRACT |
The objective of this study was to determine if vascular cell
adhesion molecule (VCAM-1), E-selectin, and P-selectin could selectively recruit leukocyte subpopulations, and whether this was
affected by shear force or adhesion molecule concentration. Cover slips
coated with purified adhesion molecules were incorporated into laminar
flow chambers. Whole human blood was perfused for 5 minutes over these
cover slips at relative shear forces of 2 to 40 dynes/cm2.
Chasing the whole blood with buffer permitted visualization of
leukocyte-substratum interactions. Leukocytes were observed to roll on
and adhere to VCAM-1 at shears between 2 and 15 dynes/cm2.
As assessed by cover slip staining, the majority of these cells were
lymphocytes, but eosinophils, monocytes, and, surprisingly, neutrophils
were also recruited, events inhibitable by
anti- 4-integrin antibody (HP1/2). Neutrophils were
effectively recruited onto the selectins, with interactions occurring
at shears as high as 30 and 40 dynes/cm2 for E- and
P-selectin respectively. Eosinophils had high affinity for P- but not
E-selectin. Mononuclear cells did not have high affinity for either
selectin, but interacted avidly with VCAM-1. Antibodies against
P-selectin (G1) and E-selectin (ES-1) completely blocked interactions
on these substrates. Reducing the concentration of adhesion molecules
did not appreciably change recruitment patterns except for VCAM-1,
where neutrophils were no longer recruited. The novel use of whole
blood in flow chambers shows a partial selectivity of selectins and
VCAM-1 for certain subpopulations of leukocytes under varying
physiologic shear conditions.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
IT IS WELL APPRECIATED that the selective
recruitment of leukocytes occurs in various disease states and at
various time points during the progression of a single disease but
whether all leukocytes are indiscriminately recruited to the
endothelial surface before initiation of the selection process or
whether recruitment from the mainstream of blood is dependent on
adhesive topography of the endothelial surface remains unknown. The
process of leukocyte recruitment can be divided into four steps;
initial contact (tethering), rolling, firm adhesion, and emigration,
with each step being mediated by adhesion molecules expressed on the surface of activated endothelium. The leukocyte recruitment is designated as a cascade because interruption of the initial tethering and rolling prevents subsequent leukocyte adhesion and emigration. Therefore if selective, the initial capture of leukocytes would be a
rate-limiting event to subsequent recruitment of that particular cell
type and could potentially discriminate against other cell types.
This initial capture is known to be mediated by endothelial adhesion
molecules including P-selectin, E-selectin, and vascular cell adhesion
molecule (VCAM-1). P-selectin is found within membrane-bound vesicles
termed Weibel Palade bodies and is rapidly (within minutes) expressed
to the endothelial surface in response to histamine,1 thrombin,1,2 and reactive oxygen metabolites.3
Associated with the rapid expression of P-selectin is the early
recruitment of neutrophils,4 raising the possibility of
selective recruitment of neutrophils on this selectin. However, because
isolated lymphocytes, monocytes, and eosinophils can also interact with
P-selectin,5-7 the selectivity of this pathway, if any,
remains completely unappreciated. By contrast, E-selectin expression
requires de novo synthesis in response to inflammatory cytokines such
as interleukin-1 (IL-1), tumor necrosis factor (TNF), and
lipopolysaccharide (LPS), with maximal amounts occurring about 4 to 8 hours after stimulation.8 The expression of this selectin
coincides with further recruitment of neutrophils and monocyte
populations, however in vitro experiments show that this selectin can
support rolling of eosinophils and lymphocytes,7,9 again
providing little insight into selectivity for this adhesion molecule.
VCAM-1 expression also requires de novo synthesis and may reach peak
levels at 24 hours, slowly declining beyond that time
point.10 This adhesion molecule supports capture of
lymphocytes, monocytes, and eosinophils although these interactions typically occur at lower shear forces than observed for either of the
selectins.11-13 However, despite selectivity for
non-neutrophilic populations, there is some suggestion that even
neutrophils may adhere to VCAM-1 if sufficiently
activated.14
Generally, with in vitro approaches, the leukocytes are purified and in
the case of the more scarce population of cells, concentrated. The
adhesion molecules are coated onto the cover slip at concentrations that support adhesion and the shear forces are adjusted again so that
the cell type under study can interact with the substratum. This makes
direct comparison of efficiency of leukocyte recruitment between
studies almost impossible. Additionally, it is now well appreciated
that red blood cells enhance leukocyte-endothelial adhesion molecule
interactions by pushing the larger leukocytes to the
periphery,15,16 and therefore, certain interactions may be
missed. Therefore, at present it is unclear whether selection of
leukocytes for recruitment occurs after rolling is initiated or whether
the initial capture of leukocytes by selectins or VCAM-1 is a
discriminatory event favoring the recruitment of some but not other
cell types.
This study was designed to systematically assess which leukocytes would
be recruited from whole blood by a particular adhesion molecule. This
was accomplished by perfusing whole blood through a standard laminar
flow chamber for 5 minutes and allowing the cells to interact with a
given substratum. The whole blood is then chased with physiological
buffer for a brief period, which permits visualization of interacting
leukocytes while all red blood cells and noninteracting leukocytes are
rinsed away. At the end of the experiment the types of leukocytes
interacting with the cover slips are identified. We report herein, that
there is selectivity for different leukocyte populations dependent on the type of immobilized adhesion molecule on the cover slip as well as
the shear force at which the leukocytes are perfused.
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MATERIALS AND METHODS |
Soluble adhesion molecules and antibodies.
Recombinant soluble P-selectin,17 monoclonal antibodies
(MoAbs) G1 (blocking antibody for P-selectin 18), and S12
(nonblocking antibody for P-selectin 18) were generous
gifts from Dr R.P. McEver (University of Oklahoma, Oklahoma City,
OK). Soluble recombinant human VCAM-1 and MoAb HP1/2
(anti-4-integrin) were generously supplied by Dr R. Lobb, Biogen Inc (Cambridge, MA).19 MoAb ES-1 (blocking
antibody for E-selectin) was donated by Dr K.D. Patel, University of
Calgary (Calgary, Alberta, Canada). Soluble E-selectin was purchased
from R & D Systems (Minneapolis, MN).
Preparation of cover slips.
Glass cover slips (Fisher Scientific, Ottawa, Ontario, Canada) were
coated with soluble adhesion molecules at various concentrations (1 to
10 mg/mL) and incubated at 4°C for 18 hours. To inhibit nonspecific
interactions with the glass, cover slips were then incubated with 1%
bovine serum albumin (BSA; Sigma Chemical Co, St Louis, MO) at 37°C
for 2 hours. Whole blood for perfusion over protein-coated cover slips
was collected from healthy donors and heparinized (30 units/mL) to
prevent clotting.
Experimental protocol.
To study leukocyte recruitment from whole blood under shear conditions,
a flow chamber assay was established as previously described.14,20,21 This model allows for observations of
leukocyte interactions with various biologic substrata at defined shear forces. Adhesion molecule-coated cover slips were mounted into a
polycarbonate chamber with parallel plate geometry. The flow chamber
was placed onto the stage of an inverted microscope (Zeiss Canada, Don
Mills, Ontario, Canada) and monolayers were visualized at 100×
magnification using phase contrast imagery. The stage area was enclosed
in a warm air cabinet and maintained at 37°C. Blood was maintained
at 37°C using a water bath. A syringe pump (Harvard Apparatus,
Canada) was used to draw the blood through the flow
chamber at defined shear forces. Experiments were video recorded for
later analysis via a CCD camera (Hitachi Denshi, Ltd, Tokyo,
Japan) and a video cassette recorder (Panasonic, Secaucus, NJ) that were attached to the microscope.
Shear forces within the flow chamber are dependent on the dimensions of
the chamber and on the flow rate and viscosity of the perfused fluid.
The viscosity of whole blood is greater than that of isolated
leukocytes in buffer, resulting in a higher shear force at equivalent
flow rates for whole blood. The viscosity of whole blood can be derived
from the hematocrit,22 which allowed for calculation of
relative shear forces within the flow chamber according to the
following formula21: R = (6µQ)/(B2W) in which µ is the relative viscosity of the blood, Q is the flow rate through
the chamber, B is the chamber depth, and W is the chamber width.
Whole blood was perfused over adhesion molecule-coated cover slips for
5 minutes at different relative shear forces (2, 5, 10, 15, 20, 30, or
40 dynes/ cm2), followed by perfusion with Hanks' balanced
salt solution (HBSS) . Within 20 seconds of perfusion with buffer,
interacting leukocytes could be visualized on the cover slips.
Interacting cells were either rolling or adherent to the surface of the
cover slip. A leukocyte was defined as adherent if it remained
stationary for at least 10 seconds. To distinguish the types of
leukocytes that were interacting with protein-coated cover slips after
perfusion of whole blood, the cover slips were gently removed from the
chambers and differentially stained (Wright-Giemsa stain). This
procedure resulted in a minimal loss of interacting leukocytes as
determined by comparing the number of leukocytes per mm2 on
the cover slip before removal from the chamber with the number after
the staining procedure.
Neutrophil isolation and fluorescent labelling.
Human neutrophils were harvested from citrate anticoagulated venous
blood collected from healthy donors, as previously
described,23 with minor modification. All isolation steps
were performed at room temperature. Neutrophils were purified by
dextran sedimentation (Dextran 250 000; Spectrum Chemicals, New
Brunswick, NJ) followed by centrifugation through a density gradient
(6.07% Ficoll Type 400; Sigma) with 10% Hypaque Sodium
(Sterling-Winthrop, Markham, Ontario, Canada). Isolated neutrophils
were resuspended in HBSS at a density of 2 × 107
cells/mL. This yielded neutrophils that were 97% pure and 95% viable.
Neutrophils were labelled with rhodamine 6G (100mg/mL) (Sigma) for 15 minutes and washed twice using centrifugation before being added back
to whole blood (2 × 105 labelled neutrophils per 1 mL
of blood).
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RESULTS |
Leukocytes are recruited from whole blood when perfused over
immobilized VCAM-1.
Figure 1 shows the rolling (A) and adhesion
(B) data for leukocytes interacting with VCAM-1-coated cover slips.
Notable rolling occurred at 15 dynes/cm2, with maximal
rolling observed at 10 dynes/cm2 (A). At these shear
forces, most interacting leukocytes were rolling, but in experiments
where shear was 5 or 2 dynes/cm2, more of the cells were
adherent, with adhesion being highest at 5 dynes/cm2 (B).
At the lowest shear tested, fewer interactions occurred. Maximal total
interactions with VCAM-1 were observed at 5 dynes/cm2. An
important observation is that when the whole blood was pretreated with
the anti-4 antibody, HP1/2 (2 mg/mL), all leukocyte
interactions were eliminated at all shear forces tested (shown for 10 and 5 dynes/cm2 in Fig 2),
suggesting that these were specific 4-integrin/VCAM-1 interactions.
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| Fig 1.
Leukocyte rolling (A) and adhesion (B) observed on
soluble human VCAM-1-coated cover slips (5 µg/mL) after perfusion of
whole blood at different shear forces.
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| Fig 2.
Rolling (A) and adhesion (B) of leukocytes recruited from
whole blood onto VCAM-1 in the presence and absence of
anti-4-antibody HP1/2 (2 µg/mL). * P < .05 relative to no antibody condition.
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Figure 3 shows the percentage (bar graphs)
and absolute numbers (top table) of neutrophils, eosinophils,
lymphocytes, and monocytes recruited to VCAM-1 after whole blood
perfusion at different shear forces. The ratio of recruited leukocytes
varied depending on the shear force used in the experiment. Lymphocytes
were always the most prevalent leukocyte regardless of the shear force,
accounting for approximately 80% of leukocytes at the highest shear
and 60% at the lowest shear tested, with the greatest number being
recruited at 5 dynes/cm2. Monocytes were the second most
notable leukocyte on VCAM-1, contributing about 10% to 20% of
leukocytes recruited. Eosinophils were also able to attach to VCAM-1,
but only at lower shear forces. At the highest shear force tested, no
eosinophils were observed. Surprisingly, a small subset of neutrophils
always adhered to the substratum. This was an
4-integrin-dependent event because HP1/2 entirely
inhibited the interaction (Fig 2).
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| Fig 3.
Quantitative assessment of the types of leukocytes found
on VCAM-1-coated cover slips (5 µg/mL) after perfusion of whole
blood at different shear forces. Cover slips were differentially
stained and analyzed for the presence of neutrophils, eosinophils,
lymphocytes, and monocytes. Data is shown as the percentage of total
leukocytes found on the cover slips. Total leukocyte counts are the
actual number of leukocytes observed per mm2.
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To establish further selectivity for the different leukocyte
populations, the level of VCAM-1 densities was varied (10.0, 5.0, 2.5, and 1.0 µg/mL), and leukocyte recruitment from whole blood is
presented in Fig 4 and
Table 1. For these experiments, the shear
force was maintained at 10 dynes/cm2. Fig 4 (A) illustrates
that the number of rolling leukocytes was comparable at 10.0 and 5.0 µg/mL but the observable rolling decreased sharply at 2.5 µg/mL,
and was minimal at 1.0 mg/mL. The adhesion to VCAM-1-coated cover
slips followed a similar trend (B). Interestingly, the proportion of
mononuclear cell types on VCAM-1 were remarkably similar regardless of
the site density of VCAM-1 (Table 1). By contrast at 2.5 µg/mL of
VCAM-1, neutrophils no longer accumulated appreciably. Cover slips
coated at 1.0 µg/mL had very few leukocytes recruited from whole
blood, and it was not possible to reliably determine leukocyte ratios.
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| Fig 4.
Leukocyte rolling (A) and adhesion (B) observed on cover
slips coated with different concentrations of VCAM-1 after perfusion of
whole blood at 10 dynes/cm2. *P < .05 relative to
5.0 µg/mL
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Selective recruitment of leukocytes from whole blood onto E-selectin.
Leukocyte interactions with E-selectin occurred at higher relative
shear forces (30 dynes/cm2) and were recruited in much
larger numbers than that observed for VCAM-1
(Fig 5). Rolling was the predominant
interaction observed at higher shear forces but cells began to arrest
(without shape change) at relative shears less than 10 dynes/cm2 (panel B). No interactions were observed at 40 dynes/cm2 but the E-selectin was not removed by these high
shears because leukocyte interactions occurred at 10 dynes/cm2 after initial perfusion at 40 dynes/cm2. At the highest shears neutrophils were
essentially the only leukocyte type recruited onto E-selectin
(Fig 6), whereas at lower shear forces
lymphocytes were also recruited onto E-selectin, with the greatest
number of lymphocytes being recruited at 10 dynes/cm2. A
smaller number of monocytes and minimal numbers of eosinophils were
observed at all shears tested. All leukocyte interactions were blocked
with an anti-E-selectin antibody (ES-1, 5 µg/mL of whole blood),
whereas an isotype-matched control antibody (S-12) had no effect (not
shown).
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| Fig 5.
Leukocyte rolling (A) and adhesion (B) observed on
soluble human E-selectin-coated cover slips (5 µg/mL) after
perfusion of whole blood at different shear forces.
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| Fig 6.
Quantitative assessment of the types of leukocytes found
on E-selectin-coated cover slips (5 µg/mL) after perfusion of whole
blood at different shear forces. Total leukocyte counts are the actual
number of leukocytes observed per mm2.
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Selective recruitment of leukocytes from whole blood onto P-selectin.
P-selectin was able to recruit leukocytes from whole blood at even
higher shear forces than E-selectin (Fig
7A). Again most cells rolled on P-selectin until shear was reduced to
very low levels when arrest was apparent (Fig 7B). As observed for
E-selectin, neutrophils were the primary leukocyte recruited onto
P-selectin (Fig 8). By contrast, P-selectin
also supported eosinophil recruitment as shear forces were decreased.
Relative to neutrophils, a small number of lymphocytes and monocytes
were recruited at all shear forces tested, being greatest at 40 dynes/cm2 for lymphocytes and 10 dynes/cm2 for
monocytes. The P-selectin antibody (G1 at 2 µg/mL) completely blocked
leukocyte interactions. An isotype-matched control antibody that binds
to but does not block P-selectin (S-12) had no effect on leukocyte
interactions on any of the substratum tested (not shown).
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| Fig 7.
Leukocyte rolling (A) and adhesion (B) observed on
soluble human P-selectin-coated cover slips (5 µg/mL) after
perfusion of whole blood at different shear forces.
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| Fig 8.
Quantitative assessment of the types of leukocytes found
on P-selectin-coated cover slips (5 µg/mL) after perfusion of whole
blood at different shear forces. Total leukocyte counts are the actual
number of leukocytes observed per mm2.
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Selective recruitment is not due to gravity or change to buffer.
To determine if rolling cells remained on the cover slips when the
chamber was disassembled during assay takedown, isolated, purified
neutrophils were fluorescently labeled with rhodamine 6G and added back
to whole blood before perfusion over VCAM-1, E-selectin, or P-selectin.
Using fluorescent microscopy, labeled neutrophils were observed to
tether, roll, and adhere at 10 dynes/cm2 in whole blood.
The number of rolling and adherent neutrophils did not change during
the transfer to buffer conditions. After the cover slips were removed
and air dried, they were once again observed under fluorescence. The
number of labeled cells per mm2 did not change as a result
of manipulation of the cover slip indicating that rolling cells were
not lost during chamber disassembly.
It is conceivable that differential rates of leukocyte sedimentation
may influence the ratio of leukocytes recruited on cover slips, with
denser populations of leukocytes showing preferential recruitment. To
determine if gravity had an effect in our assay system, experiments
were performed in which the flow chamber was inverted, such that blood
cells would settle away from the cover slip. This resulted in a
decrease (approximately 30%) in the total number of leukocytes
recruited onto each of the adhesion molecules, but did not affect the
relative ratios of the leukocyte types. Thus differential sedimentation
was not a factor in this assay system.
The relative binding efficiencies of leukocytes at varying shear
forces.
To compare the relative recruitment efficiencies of the various
leukocytes onto cover slips, the ratio of leukocytes recruited onto the
cover slips was compared with that found in the whole blood being
perfused. The recruitment factor (R) was defined as R = (% of
leukocytes on cover slip)/(% of leukocytes in blood) and provides a
value for relative binding efficiency for a population of leukocytes.
An R-factor of 1.0 indicates no preferential recruitment when compared
with its presence in whole blood, whereas an R-factor greater than 1.0 suggests preference for that substrate.
Figure 9 shows that at all shears
neutrophils were less than preferentially recruited (R < 1.0) onto
VCAM-1. Monocytes, lymphocytes, and eosinophils (except at the highest
shear) were preferentially recruited onto VCAM-1. P-selectin provided
an entirely different preferential profile
(Fig 10); lymphocytes bound P-selectin
with minimal efficacy at all shear forces and monocytes were also not
very effective at binding P-selectin at the higher shears. Eosinophils
were not effective at binding P-selectin at the highest shear but
showed tremendous efficiency (R > 7.0) when the shears were lowered. Neutrophils bound to P-selectin just above an R-value of 1.0 under all
shear conditions. Finally, E-selectin showed yet another preferential profile (Fig 11); at higher shear forces
E-selectin preferentially recruited neutrophils, whereas all other cell
types were less effective at binding the selectin. As the shear was
reduced all leukocytes approached R = 1.0, suggesting a loss of
preference. It should be noted that neutrophils can only increase to a
maximum of 2.0 if 50% of leukocytes in blood are neutrophils,
therefore an R-value of 1.90 on E-selectin suggests an almost exclusive recruitment of neutrophils at high shears. Although reducing selectin densities resulted in a decrease in the number of rolling and adherent
leukocytes (Table 2), it did not alter the
relative ratios of the types of leukocytes recruited
(Table 3).
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| Fig 9.
Recruitment factors (R-factors) for the various
leukocytes found on VCAM-1-coated cover slips after perfusion of whole
blood. The R-factor was defined as percentage of leukocytes present on
the cover slip over the percentage found in the whole blood. An
R-factor of 1 means that the percentage of a particular leukocyte found
on the cover slip was equal to that found in the whole blood.
*P < .05 relative to an R-factor value of 1.
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| Fig 10.
Recruitment factors (R-factors) for the various
leukocytes found on P-selectin-coated cover slips after perfusion of
whole blood. *P < .05 relative to an R-factor value of 1.
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| Fig 11.
Recruitment factors (R-factors) for the various
leukocytes found on E-selectin-coated cover slips after perfusion of
whole blood. *P < .05 relative to an R-factor value of 1.
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Table 2.
Rolling and Adherent Leukocytes Recruited Onto Cover
Slips Coated With E-selectin and P-selectin at Varying
Concentrations
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DISCUSSION |
The capture of leukocytes to the endothelial cell surface is the
initial critical event in leukocyte recruitment. Studies have shown
that P-selectin, E-selectin, and VCAM-1 can capture leukocytes under
flow conditions. We extend this work to provide evidence that the
initial capture of leukocytes is in part a selective process,
particularly at the higher shear forces tested. Neutrophils were less
than preferentially recruited (R < 1.0) on VCAM-1 but favored
recruitment on the two selectins. P-selectin clearly preferred neutrophils and eosinophils but had much less affinity for lymphocytes and monocytes. E-selectin showed little affinity for any of the mononuclear cells but preferred neutrophils at higher shear forces. There, however, appeared to be some residual overlap for all of the
adhesion molecules suggesting that no adhesion molecule was entirely
exclusive for any leukocyte population at any shear.
There is some suggestion that there is selectivity for some adhesion
molecules by subpopulations of leukocytes. For example, eosinophils can
use all of the selectins to form attachments under shear conditions,
however, P-selectin is much better at mediating eosinophil recruitment
than E-selectin in vitro,7 our own data clearly support
this view. P-selectin but not E-selectin was extremely effective at
capturing eosinophils from whole blood. Moreover, at a relative shear
as high as 20 dynes/cm2 eosinophils were captured with
about twice the efficiency of neutrophils. This observation is
consistent with a previous report that eosinophils have at least twice
the P-selectin ligand (PSGL-1), that neutrophils express.7
Interestingly, the number of P-selectin ligands are not the only factor
at the highest shears (40 dynes/cm2). P-selectin
selectivity for eosinophils disappeared and neutrophils continued to be
recruited with tremendous avidity. Physical characteristics of
eosinophils may make this leukocyte less capable of being captured by
P-selectin at the highest shears. Eosinophils were also captured very
effectively onto VCAM-1. Alternatively, PSGL-1 may differ between
neutrophils and eosinophils such that eosinophils bind P-selectin with
greater efficacy at lower but not higher shear. Indeed, eosinophil
PSGL-1 has a different peptide backbone and has less and different
SLEX-containing molecules.24,25
Whether coexpression of P-selectin and VCAM-1 would further enhance
eosinophil recruitment in an additive or even a synergistic manner
remains to be determined. This increased selectivity for P-selectin and
VCAM-1 may contribute to the fact that even though eosinophils
represent a very minor fraction of circulating leukocytes, they are a
major component of the cellular infiltrate found in allergic diseases
such as asthma and late-phase cutaneous reactions in which both
P-selectin and VCAM-1 have been shown to play a role.26-28
This selective recruitment would be far more important in eosinophils
than neutrophils, which are 50 to 100 times more abundant in blood, and
neutrophils would therefore be recruited in sufficient quantities even
without any selectivity (R=1).
It is intriguing that neither selectin recruited monocytes or
lymphocytes as effectively at densities and shear forces that were
conducive to recruitment of other cell types. VCAM-1 did preferentially
recruit monocytes but not at the same shear as observed for the
selectins. A potential explanation may be related to a role for the
third selectin (L-selectin) and an as yet unidentified L-selectin
ligand. Indeed Lucinskas et al6 reported that L-selectin was absolutely critical for monocyte recruitment, and that the other
selectins played only a minor role. In those studies, P-selectin accounted for only 30% and E-selectin 0% of monocyte recruitment on
TNF-stimulated endothelium in spite of the fact that these were
isolated concentrated monocyte populations. Our data would agree that
these selectins are very inefficient at recruiting monocytes (R < 1).
Once the L-selectin ligand in the periphery is identified it will be
interesting to determine whether monocytes are preferentially recruited
by L-selectin.
A surprising observation in this study was that unstimulated
neutrophils could be recruited onto VCAM-1 via
4-integrin, a mechanism commonly thought to be
restricted to mononuclear leukocytes. Although previous work has
described 4-integrin-dependent recruitment of isolated
neutrophils,14 this required maximal stimulation with
dihydrocytochalasin B in combination with FMLP, and only occurred at
shears below 1.5 dynes/cm2. In contrast, VCAM-1-dependent
neutrophil recruitment from whole blood occurred without the need for
exogenous stimulation and was greatest at 15.0 dynes/cm2
(14% ± 2.6% of total leukocytes). This was not a nonspecific effect inasmuch as the 4-integrin antibody blocked these
interactions. Although human neutrophils are generally thought not to
express 4-integrin, neutrophil precursors in the bone
marrow express 4-integrin, with that expression
diminishing during terminal differentiation.29 It is
conceivable that a small percentage of circulating neutrophils that are
either not fully mature, still express 4-integrin, or
that some neutrophils re-express 4-integrin in the
circulation. Indeed, Issekutz et al30 has shown that 4-integrin can partially mediate neutrophil recruitment
into inflamed joints and dermal inflammatory sites in the rat. It is, however, important to note that in the current study, neutrophil capture was reduced relative to other leukocytes at lower VCAM-1 densities, suggesting disproportionately high levels of VCAM-1 were
required to recruit neutrophils. Nevertheless, the data suggests that
in disease states when VCAM-1 is over-expressed, neutrophils could
conceivably be recruited independent of selectins and perhaps even
independent of  2-integrins.
Discussion of model.
Blood is a non-Newtonian fluid, making it difficult to assess actual
values of the viscosity of whole blood. Therefore, based on the work of
Fan et al,22 who provided viscosity data for whole blood at
varying hematocrits, we provide relative shear stress values in our
flow chamber. Of course, as one moves from the mainstream of blood to
the vessel or chamber wall, viscosity drops markedly so that our shear
forces almost certainly overestimate the true values at the
leukocyte-substratum interface.21 Nevertheless, even if our
viscosity values were considered to be those of water (0.01 centipoise)
the leukocytes were tethering to P-selectin at 8 dynes/cm2,
to E-selectin at 6 dynes/cm2, and to VCAM-1 at 3 dynes/cm2. These values are as much as fourfold higher than
that previously reported for leukocyte tethering to any of the
aforementioned substratum. This is consistent with the view that the
smaller red blood cells tend to push the larger leukocytes from the
axial flow to the vessel wall and thereby enhance leukocyte-substratum interactions. Indeed, Melder et al31 showed that addition
of even 5% red blood cells enhanced leukocyte interactions under flow
conditions.
Additional issues raised are the potential differential rates of
leukocyte sedimentation, potential loss of rolling cells but not
adhering cells during disassembly of the chamber, and the use of
heparin, which may affect both primary and secondary interactions.
Inverting the flow chamber recruited 30% fewer leukocytes but the
proportions did not change. The addition of fluorescently labelled
neutrophils to this system has in the first instance allowed us to
observe these cells selectively in whole blood and to establish that
the number of rolling and adherent cells visualized under flow
conditions was equivalent to the total number of fluorescently labelled
cells after disassembly of the chamber. This suggests that neither
rolling nor adherent cells are lost during disassembly. Moreover,
addition of heparin at the same concentration used in this study to
neutrophils isolated from citrate-anticoagulated blood did not affect
neutrophil-selectin interactions (unpublished observations). Therefore,
it is unlikely that the concentration of heparin used was affecting
leukocyte interactions in whole blood. Indeed, preliminary work from
our laboratory suggests that addition of increasing concentrations of
heparin does not affect the total number of primary or secondary
interactions in whole blood (Mitchell, Reinhardt, and Kubes,
unpublished observation).
This novel approach of studying leukocyte-endothelium and
leukocyte-immobilized protein interactions has many advantages. First,
the shear forces clearly better reflect shear forces observed in vivo.
Second, this system incorporates red blood cells that, as already
discussed, clearly impact on leukocytes tethering to substratum.
Although it is well appreciated that initial flow chamber work of
single concentrated populations of cells was critical to the
understanding of the function of adhesion molecules, our approach may
now extend those studies to establish which adhesion molecules and
combinations of adhesion molecules will recruit selective populations
of leukocytes. Moreover, the perfusion of whole blood over endothelium
stimulated with various cytokines (for example IL-4 and TNF will
provide further insight into which cytokines recruit which type of
leukocytes. This technology can also be used for patient blood to
establish whether the pathology of a particular disease may be an
inherent humoral problem either of enhanced adhesivity of leukocytes or
enhanced activity of plasma. Indeed, preliminary work from our
laboratory suggests a very dramatic increase in leukocyte-substratum
interactions in septic patients (Ibbitson and Kubes, unpublished
observations). Finally, this technique can be used to
circumvent trying to isolate sufficient amounts of leukocytes from rats
and mice; 1 mL can be perfused over a cover slip and
leukocyte-substratum interactions can be observed. One limitation of
this technique is the inability to identify the type of rolling versus
adherent cells. Nevertheless, we show with this system that
tethering/rolling of leukocytes from whole blood onto various adhesion
molecules is a selective process.
| |
ACKNOWLEDGMENT |
We thank Danielle Davids for performing the E-selectin and P-selectin
dose-response experiments.
| |
FOOTNOTES |
Submitted March 16, 1998;
accepted August 6, 1998.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address correspondence to Paul Kubes, PhD, Immunology
Research Group, Department of Medical Physiology, Faculty of Medicine,
University of Calgary, Calgary, Alberta, T2N 4N1, Canada.
| |
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Top
Abstract
Introduction