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Blood, Vol. 95 No. 9 (May 1), 2000:
pp. 2954-2959
PHAGOCYTES
Importance of L-selectin-dependent
leukocyte-leukocyte interactions in human whole blood
Debra J. Mitchell,
Pauline Li,
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 investigate whether leukocytes
could be recruited by rolling leukocytes in a human whole blood model
system. In all experiments, either neutrophils, whole blood, or diluted
blood was perfused over immobilized E-selectin. With isolated
neutrophils (2 × 105/mL), the free-flowing neutrophils
were captured by attached neutrophils to form secondary interactions
that resulted in lines of rolling leukocytes. These secondary tethers
accounted for 50% to 60% of all interactions and were eliminated by
an L-selectin antibody, which also eliminated the lines of rolling
leukocytes. Perfusion of whole blood or diluted blood revealed no lines
of rolling leukocytes. The addition of red blood cells to isolated
neutrophils either in a 1000:1 or a 10:1 ratio also inhibited lines of
rolling leukocytes. Leukocytes were fluorescently labeled with
rhodamine-6G so that leukocyte-leukocyte interactions could be studied
in whole blood. A small number of secondary tethers (less than 20%)
occurred and could be reduced by more than 80% with an L-selectin
antibody. However, the overall impact on leukocyte recruitment was
negligible. Similar experiments were performed using murine whole blood
or isolated murine leukocytes. In the absence of red blood cells, murine leukocytes also formed lines of rolling leukocytes on
E-selectin, and secondary tethers accounted for 50% of total
interactions. However, when murine blood (diluted 1:5 with buffer) was
perfused over E-selectin, secondary tethers accounted for only 13% of
total interactions. These interactions were completely absent when
blood was used from L-selectin-deficient mice. These data demonstrate for the first time that the importance of L-selectin-dependent leukocyte-leukocyte interactions is greatly reduced in whole blood and
does not enhance overall recruitment of leukocytes in this physiologic milieu.
(Blood. 2000;95:2954-2959)
© 2000 by The American Society of Hematology.
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Introduction |
The recruitment of leukocytes from the mainstream of
blood is a critical event in response to injury or foreign pathogen
invasion. This process of leukocyte recruitment is a multistep cascade
that involves leukocyte tethering followed by rolling, firm adhesion, and, finally, emigration from the vasculature. The selectins (P, E, and
L) have been implicated as key mediators of the initial tethering and
rolling events. P- and E-selectin are expressed by the endothelium and
are thought to directly tether leukocytes.1-3 This
interaction is known as primary tethering (Figure
1A). By contrast, L-selectin and 1 of its
ligands, P-selectin glycoprotein ligand-1 (PSGL-1), are found
exclusively on leukocytes and can mediate leukocyte-leukocyte
interactions.4-7 This event has been postulated to allow
leukocytes rolling on endothelial monolayers, purified P-selectin, or
purified E-selectin to capture or tether free-flowing leukocytes to the
substratum. This event is termed secondary tethering (Figure 1B) and
results in the formation of lines of leukocytes accumulating in the
direction of flow.7,8 The importance of this phenomenon is
that secondary tethers have been reported to account for 60% to 70%
of recruitment of isolated neutrophil preparations in
vitro.7,8
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| Fig 1.
An overview of primary and secondary tethers.
The left side shows a primary tether of a leukocyte as it moves
from the mainstream of blood and binds to E-selectin expressed on the
endothelium (A), whereas the right side shows a leukocyte tethering to
a rolling leukocyte through L-selectin and PSGL-1 (B), which captures
the free-flowing leukocyte and causes it to interact with E-selectin on
the endothelium (secondary tether).
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In vivo, the importance of secondary tethers is more questionable. For
example, Kunkel et al9 proposed that leukocyte-leukocyte interactions account for only 1.2% of all recruited leukocytes in
mouse cremaster stimulated with tumor necrosis factor (TNF)- . In
that study, a secondary tether was defined as leukocytes that experience a major velocity change within 1 cell diameter of an already
adherent leukocyte. In contrast to the line formation observed in
vitro, Kunkel et al9 found that the leukocytes adhered in
clusters. This cluster formation was also observed in
L-selectin-deficient animals, suggesting that L-selectin may not be
involved in leukocyte recruitment in this model.9 These data raised serious questions about the importance of
leukocyte-leukocyte interactions enhancing leukocyte recruitment in
vivo. However, there are numerous critical differences that potentially
may explain the disparity between the aforementioned in vitro and in
vivo studies. Because there are 1000 red blood cells surrounding each leukocyte, the likelihood of 1 leukocyte interacting with another leukocyte may be less likely in vivo than with isolated cells. A second
critical difference is the use of human versus mouse models; there may
be crucial species differences for L-selectin function in these 2 species. Indeed, Zollner et al10 demonstrated that human,
not mouse, L-selectin can bind E-selectin. Whether similar differences
exist for L-selectin/PSGL-1 interactions remains unknown.
Using a standard in vitro laminar flow chamber system, we
systematically examined whether L-selectin-dependent
leukocyte-leukocyte interactions occurred in human whole or diluted
blood, whether this was an important mechanism of leukocyte recruitment
in blood, whether the role of red blood cells had anything to do with
respect to leukocyte-leukocyte interactions, and whether mouse
leukocytes behaved similarly to human leukocytes.
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Materials and methods |
Reagents and antibodies
E-selectin was bought from R&D Systems (Minneapolis, MN). Bovine
serum albumin and rhodamine-6G were obtained from Sigma Chemical (St.
Louis, MO). Heparin (Hepalean) from porcine intestinal mucosa was from
Organon Teknika (Toronto, ON, Canada). Hanks' balanced salt solution
(HBSS) containing Ca++ and Mg2+ was obtained
from Gibco BRL (Grand Island, NY). Blocking L-selectin antibody
(Dreg-56) was a kind gift of Dr M. Jutila (Montana State University,
Bozeman, MT).
Preparation of coverslips
Glass coverslips (Fisher Scientific, Ottawa, ON, Canada) were coated
with 5 µg/mL soluble human E-selectin (R&D Systems) and incubated
overnight at 4°C. To inhibit nonspecific interactions with glass,
the coverslips were then incubated with 1% bovine serum albumin for 2 hours at 37°C. All leukocyte interactions could be inhibited with
an E-selectin antibody in this system.11
Human blood cells for perfusion
Human neutrophils for perfusion over the protein-coated coverslips
were isolated from acetate-citrate-dextrose anticoagulated blood by
dextran sedimentation and density centrifugation on
Ficoll-Hypaque.12 The neutrophils were resuspended in HBSS
at a concentration of 2 × 105/mL. Whole blood for
perfusion over protein-coated coverslips was collected from healthy
donors and heparinized (30 U/mL) to prevent clotting. In whole blood,
the total number of neutrophils ranged from 1.5 to
6.0 × 106/mL. To ensure that the larger neutrophil
concentration present in whole blood was not a factor, we diluted whole
blood 5-fold and 10-fold with HBSS. The dilution of whole blood reduced
viscosity without altering the red blood cell-to-leukocyte ratio.
Experimental protocol using human neutrophils
To study leukocyte interactions with selectins under shear
conditions, a flow chamber assay was established as previously described.13 E-selectin coverslips were mounted into a
polycarbonate chamber with parallel-plate geometry. The flow chamber
was placed onto the stage of an inverted microscope (Zeiss,
Canada), and coverslips were visualized at
100 × magnification using phase-contrast imagery. This yielded
fields of view that measures 0.45 mm2. The stage area was
enclosed in a warm air cabinet and maintained at 37°C. Blood or
neutrophil suspensions were warmed to 37°C using a water bath. A
syringe pump (Harvard Apparatus, Canada) was used to
draw the cells through the flow chamber at an identical velocity for
every experiment (2 mL/min). The relative viscosity of whole and
diluted blood can be derived from the hematocrit,14 which allowed for calculations of relative shear forces within the flow chamber according to the formula 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. This has been used as the standard approach to calculate shear
forces in these flow chambers by us11 and others.1,13
Isolated human neutrophils (2 × 105/mL), whole
blood or blood diluted one fifth or one tenth with HBSS was perfused
over the E-selectin-coated coverslip. The perfused blood was chased by HBSS to allow for visualization of leukocyte interactions as
described.11 Five fields of view were recorded for each
experiment and were played back for off-line analysis. The values for
the 5 fields were averaged and treated as 1 value (n = 1). Each
experiment was repeated a minimum of 4 times. In some experiments, the
blood or neutrophil suspension was incubated at 37°C for 5 minutes
before perfusion over the coverslip with the L-selectin antibody
DREG-56 (3 µg/mL). Because heparin is known to bind to
L-selectin15 and could possibly inhibit
leukocyte-leukocyte interactions in the whole blood system, heparin
(30, 3, or 0.3 U/mL) was added back to isolated neutrophils to
determine whether this could have an impact on secondary tethers in the
isolated neutrophil system. Finally, it is possible that plasma-derived
proteins such as soluble L-selectin could inhibit L-selectin/PSGL-1
interactions. Therefore, in some experiments, neutrophils were
resuspended in plasma and perfused over E-selectin.
Mouse blood for perfusion
Mice were anesthetized by intraperitoneal injection of a mixture of
10 mg/kg xylazine (MTC Pharmaceuticals, Cambridge, Ontario, Canada) and
200 mg/kg ketamine hydrochloride (Rogar/STB, London, Ontario, Canada).
Blood was drawn from L-selectin-deficient mice (kind gift from Dr T. F. Tedder, Duke University Medical Center, Durham, NC) or from
wild-type mice (C57Bl/6) by cardiac puncture into heparinized (30 U/mL)
syringes. Because only a very small volume was obtained (less than 1 mL), we diluted the blood 5-fold with HBSS.
Experimental protocol using murine blood and isolated leukocytes
Diluted mouse blood (one fifth) labeled with rhodamine-6G (as
described for human blood) was drawn over human E-selectin-coated coverslips for 2 minutes. To test for the effect of L-selectin, mice
deficient in L-selectin were used. In a final series of experiments, red blood cells were removed from mouse leukocytes by lysis with distilled water resuspended in HBSS, and the white cells were perfused
over E-selectin.
Experiments were recorded on a video cassette recorder (Panasonic,
Secaucus, NJ) by a charge-coupled device (CCD) camera (Hatachi, Denshi,
Japan) for off-line analysis. In some experiments, the leukocytes in human whole blood or diluted mouse blood were labeled with rhodamine-6G (0.1 mg/mL) perfused over E-selectin and visualized at 200 × magnification for 2 minutes using a low-light-
sensitive CCD camera (Hamamatsu, Japan) and on an
inverted microscope (Zeiss, Dan Mills, ON) with an
AttoArc light source for epifluorescence. Total number
of interacting cells was measured for each field of view and then
averaged for each coverslip. Primary tethers were defined as flowing
leukocytes that tethered directly to the selectin substrate, whereas
secondary tethers were defined as leukocytes that tethered first to an
attached leukocyte before interacting with the substrate. Line
formation was defined as 4 or more leukocytes aligning on the substrate
in the direction of flow and not more than 2 leukocyte diameters apart.
Statistics
All data are presented as mean ± SEM. Student t test
was used to compare between groups. Statistical significance was set at
P < .05.
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Results |
Line formation with isolated neutrophils is L-selectin dependent
Figures 2A and 2B show the development
of lines of neutrophils, a hallmark feature of secondary interactions,
over the first 5 minutes on immobilized E-selectin. Neutrophils were
seen to interact briefly with an already attached neutrophil (primary tether) and to form a secondary tether within 2 leukocyte diameters of
the initial neutrophil. This process occurred repeatedly such that a
line of rolling leukocytes formed. At 3 minutes, 3 neutrophils in a row
can be clearly seen, and at 5 minutes, the number has more than doubled
(arrow). It is now generally accepted that these secondary interactions
are mediated by L-selectin. Indeed, Figure 2C illustrates 2 important
points the addition of the L-selectin antibody completely abolished
the line of rolling neutrophils, and this impacted on the total number
of neutrophils recruited. The cumulative data are summarized in Figure
3. Approximately 30 neutrophils were seen
per field of view, and L-selectin antibody reduced the total number of
cells by approximately 50% to 60% (Figure 3A). On average, 2 very
notable lines of rolling neutrophils were observed per field of view
from isolated neutrophil experiments, and an L-selectin antibody
essentially eliminated this pattern of neutrophil recruitment (Figure
3B). These data illustrate that the majority of cells accumulated as a
result of L-selectin and that these interactions are responsible for
the neutrophil line formation. Not shown is the fact that the
L-selectin antibody abolished secondary tethers in the isolated
neutrophil system.
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| Fig 2.
Interactions of isolated neutrophils with immobilized
E-selectin.
Line formation is seen at 3 minutes (A). This line is increased in
length by 5 minutes (B). The L-selectin antibody (C) eliminates this
line formation. The arrow indicates line formation.
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| Fig 3.
Role of L-selectin in isolated neutrophil recruitment.
Total interactions (A) and line formation (B) with isolated
neutrophils (n = 4). The L-selectin antibody significantly reduced
interactions and line formation. *P < .05 compared to
untreated.
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Lack of lines of rolling leukocytes with whole or diluted
blood
In contrast to isolated neutrophils, no lines of rolling leukocytes
were noted in whole blood or in blood diluted one fifth or one tenth
with buffer. Figure 4A illustrates the
pattern of leukocyte recruitment after perfusion of untreated one-fifth
diluted blood. Figure 4B demonstrates that in this system, the addition of L-selectin antibody did not affect the amount or pattern of leukocyte accumulation. The cumulative data are summarized in Figure
5. After perfusion of whole blood or
diluted blood (one fifth and one tenth), approximately 80 to 100 rolling leukocytes could be seen interacting with E-selectin per field
of view. The addition of L-selectin antibody did not significantly
reduce leukocyte recruitment in either whole blood or diluted blood.
Because the same perfusion rate was used with whole blood and the 2 diluted blood samples, the relative shear force was reduced from
approximately 10 dynes/cm2 to less than 3 dynes/cm2, suggesting that the importance of L-selectin did
not change over this range of shear forces.
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| Fig 4.
Interactions of one fifth diluted blood with immobilized
E-selectin.
No lines were observed with untreated blood (A). Inhibition of
L-selectin had no effect on the pattern of leukocyte interaction (B).
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| Fig 5.
Total interactions with blood on immobilized E-selectin
(n = 4 for each group).
The number of interactions was maintained with whole blood and
blood diluted one fifth and one tenth, regardless of the presence
or absence of L-selectin antibody.
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To determine whether the absence of lines of rolling leukocytes was
predicated on the presence of red blood cells, we added back to
isolated neutrophils various amounts of red blood cells from the same
donor to achieve final concentrations of 1 neutrophil/1000 red blood
cells (predicted physiologic concentration) or 1 neutrophil/10 red
blood cells. Figure 6 reveals that the
lines of rolling cells noted with isolated neutrophils were not
apparent at the 1:1000 neutrophil-to-red blood cell ratio.
Interestingly, even a ratio of 1:10 neutrophil-to-red blood cell was
sufficient to reduce significantly the lines of rolling leukocytes.
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| Fig 6.
Effect of red blood cell addition to line formation with
neutrophils on immobilized E-selectin (n = 4).
Lines of neutrophils were noted with isolated neutrophils but were
significantly reduced by the addition of red blood cells in the ratios
of 1000 or 10 red blood cells for every neutrophil.
*P < .05, compared to neutrophils alone.
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Rhodamine 6G treatment of whole blood permitted direct visualization of
leukocyte-leukocyte interactions. Fewer than 20 secondary interactions
(less than 25% of total interactions; primary plus secondary) could be
seen during the perfusion period (Figure
7). The addition of L-selectin antibody
reduced the secondary interactions by approximately 75% to 80%. As
already stated (Figure 5), this reduction in the leukocyte-assisted
capture of flowing leukocytes with L-selectin antibody was of minor
consequence because the total number of accumulated cells was not
reduced by this treatment (Figure 5). Secondary tethers occurred with
similar frequency in diluted blood (one tenth) as in whole blood, and
the majority of these interactions were again inhibitable with
L-selectin antibody (Figure 7).
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| Fig 7.
Secondary interactions with whole blood and one-tenth
blood (n = 4-17).
There were similar numbers of secondary tethers between the whole and
one-tenth diluted blood. L-selectin antibody significantly reduced the
number of secondary tethers in whole blood. *P < .05,
compared to untreated.
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Heparin and plasma do not affect line formation with neutrophils
In a final series of human leukocyte experiments, we ensured that
heparin did not affect line formation because the whole blood had to be
heparinized and this anticoagulant has been purported to inhibit
L-selectin function.15 We added heparin in varying concentrations directly to isolated human neutrophils and noted that
line formation still occurred, excluding heparin as the variable responsible for the lack of line formation in whole blood (Table 1).
To ensure that endogenous proteins in plasma (eg, soluble L-selectin or
soluble PSGL-1) were not altering the neutrophil-assisted capture of
free-flowing neutrophils, isolated neutrophils were resuspended in
either HBSS or plasma. The results revealed that the same number of
secondary interactions were noted per field of view, leading to similar
numbers of lines of rolling leukocytes (Table
2).
Isolated murine leukocytes form secondary tethers
Lines of rolling leukocytes were clearly visible on E-selectin using
isolated murine leukocytes (data not shown). Table
3 summarizes that approximately 50% of
total tethers were secondary, similar to what we reported with isolated
human neutrophils. By contrast, primary tethers were the dominant
interaction when mouse blood was used. Only 13% of the total
interactions were secondary (Table 3). An absence of L-selectin
(L-selectin-deficient mice) resulted in no secondary tethers (Table
3). No lines were observed when diluted mouse blood from wild-type or
L-selectin-deficient mice was perfused over E-selectin (data not
shown).
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Discussion |
There is a large body of evidence demonstrating that the initial or
primary tether of a leukocyte to endothelium and subsequent rolling is
mediated by the selectin family of adhesion molecules, including
P-selectin1,2,16 and E-selectin3 on endothelium and L-selectin17,18 on leukocytes. Laminar flow chambers
have been instrumental in this regard; perfusion of isolated leukocytes over endothelium-expressing selectins or over immobilized selectins has
clearly demonstrated the importance of selectins in this initial attachment.1,3 A consistent observation in these types of studies (regardless of substratum) is the formation of lines of rolling
leukocytes. Close examination of this phenomenon revealed that
free-flowing leukocytes frequently collided with a rolling leukocyte,
were captured by the rolling cell, and were secondarily tethered to the
endothelium or the immobilized selectin.7,8 This resulted
in 2 rolling leukocytes (Figure 1B) that would in turn recruit
additional free-flowing leukocytes to roll by the same process until a
line of rolling leukocytes had formed. It was demonstrated that
L-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) were the 2 molecules responsible for this phenomenon.7,8 Secondary
tethers forming lines of rolling leukocytes were potentially very
important because they accounted for 60% to 70% of total neutrophil,8 eosinophil,19
monocyte,20 or SKW3 T-cell8 recruitment in the
laminar flow chambers.
In this study, we report that perfusion of human whole blood or diluted
blood through the flow chamber revealed few secondary tethers and a
lack of lines of rolling leukocytes. The reintroduction of red blood
cells (but not of plasma or heparin) to isolated neutrophils was
sufficient to disrupt and prevent secondary tethers from occurring and
thereby prevented the characteristic lines of rolling leukocytes. A
number of key differences may explain the striking discrepancy in
behavior between isolated neutrophils versus whole blood. The viscosity
of whole blood is clearly much higher than the viscosity of isolated
neutrophils. This elevated viscosity would increase the shear stress
imposed on the leukocytes and could potentially disrupt or prevent
secondary tethers from successfully forming. Indeed, increasing the
viscosity of the perfusate with the addition of red blood cells to
isolated neutrophils eliminated secondary tethers. However, when whole
blood was diluted with buffer one fifth or one tenth, thereby reducing
the viscosity and the shear stress by more than 75% (Figure 4) without
altering the leukocyte-to-red blood cell ratio, no increase in
secondary tethers or lines of rolling leukocytes was observed. These
data suggest that across a reasonably broad range of viscosity and shear forces, secondary tethers do not contribute to overall leukocyte recruitment in blood. Although we cannot exclude the possibility that
the viscosity associated with isolated neutrophils in buffer is
required to allow for secondary tethers, this does not diminish the
observation that in the physiologic setting secondary tethers are less
likely to occur.
The presence of red blood cells may be responsible for the less
significant role of secondary tethers for leukocyte recruitment from
whole blood. There are approximately 1000 red blood cells for each
leukocyte in whole blood, and it is possible that with such large
numbers of erythrocytes surrounding each leukocyte, red blood cells
could physically interfere with leukocyte-leukocyte interactions.
Indeed, when red blood cells were added back to isolated neutrophils in
the physiologic ratio of 1000 erythrocytes to 1 leukocyte, line
formation was abolished. The addition of 10 red blood cells for every
leukocyte also interfered with line formation, albeit not to the same
extent. As mentioned previously, the reintroduction of red blood cells
would also increase the viscosity, which could have a direct impact on
the ability of leukocytes to interact with other leukocytes. However,
simply diluting the whole blood with buffer, but maintaining the red blood cell-to-leukocyte ratio at the present ratio in whole blood, did
not increase the importance of secondary tethers supporting the view
that the very large number of red blood cells for each leukocyte may
decrease the likelihood of leukocyte-leukocyte interactions in whole
blood. Another factor that may contribute to the reduction in secondary
tethers in the physiologic setting may be soluble L-selectin and
P-selectin that could bind PSGL-1 and prevent leukocyte-leukocyte interactions. This seems a less likely possibility because isolated neutrophils reconstituted in plasma had at least as many secondary tethers and lines of rolling neutrophils as neutrophils perfused in buffer.
In this study, we have amalgamated the beneficial aspects of an in
vitro laminar flow chamber, which permits excellent visualization of
leukocyte-leukocyte interactions using human leukocytes, with the in
vivo attributes of leukocyte-leukocyte interactions in whole
blood. Nevertheless, this still is not the in vivo situation wherein
the postcapillary venules are narrower and have more variable geometry.
Nevertheless, our data are in line with the only study to our knowledge
to address the importance of secondary interactions in
vivo.9 Kunkel et al9 examined secondary tethers
in an autoperfused murine cremaster preparation stimulated with
TNF-, and they defined a secondary tether as a leukocyte that
experienced a reduction in velocity within 1 cell diameter of an
already adherent leukocyte. Although this might actually
overestimate secondary tethers, the authors still concluded that
leukocyte-leukocyte interactions could only account for 1.2% of all
recruited leukocytes in this inflammatory situation. One could argue
that with high concentrations of TNF- administration, L-selectin was
shed, thereby eliminating L-selectin-dependent secondary tethers.
However, in our system in which inflammatory mediators were not
introduced, our data agree with the work of Kunkel et al9
in concluding that secondary tethers play a minor role in leukocyte
recruitment in whole blood. Finally, the fact that L-selectin antibody
or L-selectin deficiency is inhibitory under only some but not
all in vivo conditions21,22 is
consistent with the induction of a vascular L-selectin ligand under
those specific conditions. If L-selectin antibody inhibited
leukocyte-leukocyte interactions, one would predict that it would be
an effective inhibitor under all conditions (as L-selectin/PSGL-1 are
constitutively expressed).
Our own study suggested that human leukocyte-leukocyte secondary
tethers are more frequent (approximately 20%) than the 1.2% murine
leukocyte-leukocyte secondary tethers reported by Kunkel et
al.9 Although there may be different affinities between PSGL-1 and L-selectin in human versus mouse systems, we were able to
observe that secondary tethers were similar (50%) when either isolated
murine or human leukocytes were perfused through the laminar flow
chamber. However, when murine blood was perfused through the laminar
flow chamber, secondary tethers were decreased to approximately 13%
(in line with the 20% seen in human blood) and did not occur when
blood from L-selectin- deficient mice was used. Therefore, it appears
that the secondary interactions are similar in frequency within the
human and mouse systems. However, this study does not completely
exclude a role for L-selectin/PSGL-1-dependent leukocyte-leukocyte
interactions potentially playing a role in leukocyte aggregation in
places such as capillaries, which could have an impact on downstream
rolling events. Indeed, it remains unclear why in a number of animal
models L-selectin antibody reduces P-selectin-dependent rolling by
approximately 60%, despite the fact that L-selectin is not thought to
be a ligand for P-selectin.23-25
In conclusion, our results suggest that the secondary tethers observed
in isolated leukocyte populations, which account for a significant
amount of leukocyte recruitment in vitro, play a less dominant role in
human whole blood. Our data also suggest that red blood cells may
interfere with or impede the number of leukocyte-leukocyte
interactions that occur. This may be the result of a simple physical
barrier function of the red blood cell decreasing leukocyte-leukocyte
interactions. Alternatively, other physical properties, such as the
increased hydrodynamic dispersal forces imparted on a leukocyte by red
blood cells as it briefly tethers to a rolling leukocyte, may prevent
subsequent tethering to the endothelium. It is now well established
that red blood cells facilitate selectin-dependent leukocyte rolling on
endothelium.26,27 This event is thought to be caused by the
erythrocytes increasing the lateral displacement of leukocytes toward
the endothelial surface. The decrease in leukocyte secondary tethers in
the presence of red blood cells further underscores the importance of
erythrocytes in leukocyte-endothelium interactions.
| |
Footnotes |
Submitted February 22, 1999; accepted January 5, 2000.
Supported by a grant from the Medical Research Council. P.K. is a
Medical Research Council Scientist and an Alberta Heritage Foundation
for Medical Research Senior Scholar. D.M. is a fellow with the Alberta
Heritage Foundation for Medical Research.
Reprints: Paul Kubes, Immunology Research Group, Department of
Medical Physiology, Faculty of Medicine, University of Calgary,
Calgary, Alberta, T2N 4N1, Canada.
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.
| |
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Abstract
Introduction