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Department of Biomedical Engineering, University of Virginia Health Sciences Center, Charlottesville, VA 22908
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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50% less efficient in the absence of E-selectin. These data suggest
a model of leukocyte recruitment in which ß2 integrins
play a critical role in stabilizing leukocyte rolling during a
protracted cellular activation period before arrest and firm
adhesion. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ability of surface-bound chemoattractants to mediate arrest of rolling leukocytes has been demonstrated thus far only in vitro. Rainger et al. (5) demonstrated that neutrophils rolling on cultured HUVEC treated with IL-8 or platelet activating factor stopped within less than 1 s and less than 15 µm after initiation of rolling. In a reconstituted system, Campbell et al. (6) showed rapid arrest (<1 s) of lymphocytes rolling on a substrate containing peripheral node addressin (a ligand for L-selectin) and ICAM-1 (a ß2 integrin ligand) when appropriate chemokines were co-immobilized. Similarly, monocyte chemotactic protein-1 and IL-8 were shown to mediate rapid arrest of monocytes rolling on endothelial cells in a flow chamber system (7). Rapid arrest of rolling leukocytes can be observed in vivo when IL-8 (8) or one of its murine homologues, macrophage-inflammatory protein-2 (MIP-2)4 (9, 10), are injected adjacent to a venule using a micropipette. However, this mode of rapid activation may be typical of high local concentrations of chemoattractant, and rapid arrest of rolling leukocytes may not reflect the physiological process of leukocyte activation and arrest occurring during inflammation.
In previous work from our laboratory (11), we discovered
that the number of adherent leukocytes during inflammation correlates
with their venular transit time. This suggested that the amount of time
rolling leukocytes remain in contact with the venular endothelium and
are exposed to activating signals may determine arrest. To achieve
wild-type levels of leukocyte adhesion, rolling leukocytes require an
average rolling time of 30 s to pass a 100-µm segment of venule;
altering the rolling time using Abs against E-selectin or CD18
integrins reduced the number of adherent leukocytes. These data,
gathered as population averaged data, suggest that prolonged rolling
contact with the endothelium may be necessary to promote activation and
trigger integrin-mediated arrest and firm adhesion.
Using the TNF-
-treated mouse cremaster muscle as a
well-characterized model of acute cytokine-dependent inflammation
(12, 13, 14), we have begun to examine the transition from
rolling to firm adhesion under physiological conditions to understand
this apparent requirement for long endothelial contact times. In this
model, both P- and E-selectin are expressed on the vascular endothelium
(14), and all three selectins (L-, P-, and E-selectin)
contribute to leukocyte rolling (13, 15). About 97% of
all rolling and adherent leukocytes in this inflammatory model are
neutrophils (13). To examine the rolling-to-adhesion
transition, we have developed a new method involving tracking of
individual rolling leukocytes to test the hypothesis that rolling
leukocytes may become activated during the rolling process, leading to
progressively increasing engagement of ß2
integrins, and finally arrest and firm adhesion. In this paper we
present evidence that under the conditions of in situ TNF-
stimulation, leukocyte adhesion occurs not as rapid arrest in response
to chemoattractants, but a gradual deceleration process requiring
increased ß2 integrin adhesiveness.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All mice used were between 8 and 16 wk old and healthy under barrier vivarium conditions, although spontaneous inflammatory skin lesions have been reported in older CD18-/- mice (16) when kept in conventional facilities. CD18-/- and E-/- mice were back-crossed into a C57BL/6 background and were gifts of Dr. A. L. Beaudet (Baylor College of Medicine, Houston, TX) and D. C. Bullard (University of Alabama, Birmingham). Control mice were age- and strain-matched C57BL/6 wild-type mice purchased from Hilltop Lab Animals (Scottdale, PA). All animal experiments were conducted under a protocol approved by the University of Virginia institutional animal care and use committee.
Reagents
Recombinant murine TNF- was purchased from Genzyme
(Cambridge, MA). The mAb GAME-46 against the common mouse
ß2 integrin chain (30 µg per mouse i.v.)
reported to block LFA-1 binding to ICAM-1, -2, and -3 and Mac-1 binding
to ICAM-1 (17) was purchased from PharMingen (San Diego,
CA). The mAb LAM1-101 (30 µg per mouse i.v.), which binds to murine
L-selectin but does not inhibit rolling or lead to cell activation
(18), was a kind gift of Dr. T. F. Tedder (Duke
University, Durham, NC).
Intravital microscopy
Mice were pretreated 2.5 h before surgery with an
intrascrotal injection of 0.5 µg murine recombinant TNF- (Genzyme)
in 0.30 ml isotonic saline, then injected with 30 mg/kg sodium
pentobarbital (Nembutal; Abbott Laboratories, Abbott Park, IL), 0.1
mg/kg atropine (Elkins-Sinn, Cherry Hill, NJ), and 100 mg/kg ketamine
hydrochloride (Ketalar; Parke-Davis, Detroit, MI) i.p. for anesthesia,
and prepared for intravital miscrosopy (13). The cremaster
muscle was prepared as described and superfused with thermocontrolled
(35°C) bicarbonate-buffered saline (12). Microscopic
observations were made using an intravital microscope (Axioskop; Carl
Zeiss, Thornwood, NY) with a saline immersion objective (either SW
20/0.55 or SW 40/0.75). Individual leukocytes were chosen randomly and
without knowing their eventual outcomes as they exited 5-µm
capillaries into postcapillary venules. Rolling leukocytes were tracked
down a venular tree using a motorized stage (Märzhäuser,
Wetzlar, Germany) while recording through a charge-coupled device (CCD)
camera system (model VE-1000CD; Dage-MTI, Michigan City, IN) onto
videotape for offline analysis (S-VHS recorder; Panasonic, Osaka,
Japan). Distance-time tracings of tracked leukocytes were obtained at
15 µm intervals and used to calculate instantaneous rolling
velocity using a discrete center difference formula. The centerline
erythrocyte velocity and venular diameter were measured after each
point in the tree where two or more venules converged using a dual
photodiode and digital on-line cross-correlation program. Mean blood
flow velocities and wall shear rates were determined as described
(13). In the venules studied, wall shear rate increased
from small postcapillary venules (<10 µm diameter) toward larger
draining venules (>60 µm).
Data analysis
For each tracked leukocyte, the dependence of leukocyte rolling velocity on time and wall shear rate was analyzed by a multiple linear regression with no transformation. Distance-time and velocity-time curves were fit to both linear (two-parameter) and exponential (two-parameter) models to determine the best fit. Average decelerations, rolling times, distances, and velocities between experimental groups were compared using an ANOVA followed by a Student-Newman-Keuls multiple comparison procedure. All statistical analyses were performed using NCSS Statistical Software (Kaysville, UT).
Intracellular Ca2+ concentration ([Ca2+]i) measurements
Neutrophils were isolated from heparin-anticoagulated human venous blood over a Ficoll-Hypaque gradient and labeled with Fluo-3 (Molecular Probes, Eugene, OR) at a final concentration of 8 µM in PBS (with no calcium or magnesium) supplemented with 0.25% BSA and 0.1% glucose (Sigma, St. Louis, MO) for 30 min at room temperature. For flow cytometric measurement of calcium flux, labeled cells were resuspended at 1 x 106 cells/ml in calcium and magnesium-free PBS (with 0.25% BSA and 0.1% glucose). After measuring baseline fluorescence, chemoattractants were added to the tube of cells at the concentrations indicated and the change in cell fluorescence immediately recorded. Measurements were made on a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ) using CellQuest software, version 3.1 (Becton Dickinson).
For in vivo calcium flux measurements, Fluo-3 labeled human neutrophils (1 x 107 cells/ml) were injected into small catheters (pulled PE10 tubing) placed in the iliac arteries of C57BL/6 wild-type mice. Stroboscopic (30 flashes/s) epifluorescence microscopy showed very dimly fluorescent cells entering the smallest postcapillary venules from capillaries. Stroboscopic illumination renders pixel intensity independent of rolling velocity, because the images of the cells are frozen in time due to the very short duration of each flash (<<1 ms). Venules with fluorescent leukocytes were recorded on videotape using a SIT camera (SIT 66, Dage-MTI) without automtic gain control. Background tissue autofluorescence was negligible (and relatively constant throughout the tissue) at the low gains used here. To avoid major focal plane changes, venules were chosen in which leukocytes rolling along their top wall remained in focus while being tracked. Fluorescence intensity of individual leukocytes was measured repeatedly as the sum of all pixel intensities contained in a box measuring 120 x 120 pixels using NIH Image software on a Macintosh computer. In addition, distance-time data for each fluorescent cell were recorded.
Human LFA-1 can bind to murine ICAM-1 (19), and human neutrophils bind to murine E-selectin (20). Human neutrophils respond to murine MIP-2 and show chemotaxis (21). They also respond to two other ligands of murine CXCR2, murine KC (22), and murine granulocyte chemotactic protein-2 (GCP-2) (23), which could potentially be involved in activation of rolling leukocytes. Therefore, human neutrophils are likely to show many of the physiological responses relevant to rolling and attachment in the mouse system.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. A typical venular tree with diameter,
length, velocity, and shear rate measurements is shown in Fig. 1
. Venular trees were chosen randomly in
each cremaster, with the single condition that they had adequate flow
(flow velocities > 500 µm/s) in all segments from the smallest
postcapillary venules (5–12 µm) to large draining venules (>150
µm). Leukocytes to be tracked were chosen without bias to final
outcome (because their final outcome was unknown) by randomly picking a
leukocyte exiting from a capillary into a postcapillary venule. We
found that leukocytes made initial contact with the endothelium when
they exited from small capillaries and then rolled along the walls of
postcapillary and larger venules until they adhered, detached from the
endothelium, rolled out of the cremaster vasculature, or were lost due
to obstruction of microscopic view. We tracked a total of 127 rolling
leukocytes (67 in wild-type mice under various conditions, 28 in
CD18-/- mice, and 32 in
E-/- mice) of which 34 were lost due to
obstruction of the microscopic view. No tracked leukocyte rolled out of
the cremaster vasculature. We based our analysis on all 93 leukocytes
that had clear outcomes (adhered or detached).
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-treated venules eventually became adherent on the inflamed
endothelium (the efficiency could be as low as 74% in the unlikely
case that all 7 leukocytes lost during tracking eventually detached).
Arrest was dependent on TNF--induced inflammation, because none of
the 10 leukocytes tracked in untreated control mice became adherent. We
analyzed the behavior of rolling leukocytes by generating cumulative
distance vs time plots for each rolling leukocyte. A leukocyte steadily
engaged in rolling would be expected to have a linear distance vs time
curve (the slope is the average rolling velocity) and then a sudden
decrease to zero velocity when activation and arrest occurred. However,
almost all rolling leukocytes in TNF--treated wild-type mice had
nonlinear distance vs time curves (where the slope of the curve
decreases systematically along the curve), suggesting a decrease in
average rolling velocity was occurring before arrest (Fig. 2). To assess whether rolling velocity
was changing before arrest, we calculated the rolling velocity for
every point on the distance vs time curve for all tracked leukocytes
and conducted a multiple linear regression of velocity vs time and wall
shear rate. This analysis was designed to detect a variation of rolling
velocity with time and, as a control for hemodynamics, with wall shear
rate. For instance, a negative correlation between rolling velocity and
time reflects deceleration, a positive correlation reflects
acceleration, and no correlation means that rolling velocity is not
changing with time. A negative correlation between rolling velocity and
wall shear rate demonstrates that leukocytes are decelerating even as
wall shear rate increases, a positive correlation suggest that
leukocyte are accelerating as wall shear rate increases, and no
correlation means that rolling velocity is not affected by wall
shear rate.
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-treated wild-type mice showed a significant negative
correlation between rolling velocity and rolling time with an average
deceleration of 0.28 ± 0.13 µm/s2 (Table II). This means that an average rolling
leukocyte slowed down by about 0.3 µm/s each second it was rolling.
The starting velocity of these leukocytes was 9.0 ± 1.1 µm/s,
and the average velocity over the full distance tracked was 3.9 ±
2.5 µm/s. The rolling velocities in wild-type mice did not correlate
with wall shear rate. Leukocytes in wild-type mice rolled for 270
± 58 µm (30 cell diameters) before arrest and firm adhesion
(Table III). In contrast, the two
leukocytes in wild-type mice which detached from the endothelium
correlated positively with time and shear rate. Although surprisingly
few wild-type leukocytes detached during our observation period (too
few for relevant analysis), the few that did detach from the
endothelium behaved similarly to leukocytes from
CD18-/- mice and wild-type mice treated with an
anti-CD18 mAb (see below) in terms of rolling velocity correlation
with time and shear rate.
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). Interestingly, even though individual
leukocytes rolled for varying distances and times (Fig. 2
),
normalization allowed the superposition of the various curves,
highlighting the similarity of their behaviors. Leukocytes becoming
adherent exhibited a conspicuous convex shape of the distance over time
plot (Fig. 3A), reflecting the systematic decrease of
rolling velocity (Fig. 3B). In contrast, two leukocytes that
detached from the endothelium showed no such behavior (Fig. 3, C and D). Further statistical analysis of the
full data set showed that the distance-time plot for most leukocytes
eventually becoming adherent was best fit by an exponential model, and
the data for detaching leukocytes was best fit by a linear model (data
not shown). Deceleration before adhesion does not seem to be strictly
necessary, because 10% of leukocytes in wild-type mice arrested
abruptly. The fact that most of the distance-time curves (and
concomitantly, the velocity-time curves) were fit best by exponential
curves suggests a rolling behavior characterized by continual
deceleration of rolling leukocytes before arrest and firm adhesion.
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4 µm/s) as endogenous mouse leukocytes, suggesting the same or
similar adhesion molecules mediating the interactions. Using flow
cytometry, we found that human neutrophils labeled with Fluo-3 respond
to human IL-8 and leukotriene B4 (LTB4) with an
increase in [Ca2+]i as
well as exhibiting homologous desensitization upon restimulation (data
not shown). In addition, human neutrophils respond to even low doses
(10 nM) of murine MIP-2 (Fig. 4), which
is known to be present in TNF--stimulated inflammatory sites
(27) and plays a major role in neutrophil recruitment in
response to TNF- (28). Based on these findings and
previously published reports (19, 20, 21, 22, 23), the human
neutrophils appear to be a good model for the behavior of mouse
neutrophils in this system.
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a shows a typical leukocyte
that was very weakly fluorescent upon entering a postcapillary venule,
became brighter as it rolled down the venule, and showed an additional
burst of brightness when it finally became firmly adherent. Rolling
leukocytes in vivo showed increases in maximal cell intensity of
4.1 ± 1.2-fold. Human neutrophils labeled with calcein
acetoxymethyl ester (AM), a non calcium-sensitive dye, did not exhibit
changes in intensity while rolling along the endothelium (data not
shown). Fig. 5b shows an example of increasing
[Ca2+]i and decreasing
rolling velocity for a single neutrophil. Although not all neutrophils
showed such a clear and systematic increase of
[Ca2+]i, the pooled data
indicate a significant correlation between cell intensity and rolling
velocity (Fig. 5c). All neutrophils analyzed showed a
negative correlation between their fluorescence intensity and their
rolling velocity. The average slope was -0.3 ± 0.1
(µm/s)-1, indicating that an average
neutrophil would approximately double its fluorescence intensity from
baseline for a decrease of rolling velocity by 3 µm/s. Plotting
normalized distance as a function of time (Fig. 5d) reveals
that the Fluo-3-loaded neutrophils behave very similarly to the
unlabeled, endogenous neutrophils (Fig. 3), showing a systematic
decrease of rolling velocity before neutrophil arrest.
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) with 70% of rolling leukocytes not decelerating and
in fact increasing their rolling velocity with wall shear rate (Table II). The average rolling velocity was much higher in
CD18-/- mice (32.0 ± 6.8 µm/s) than in
wild-type mice (3.9 ± 2.5 µm/s) (Table III). This faster
rolling led to a dramatically reduced transit time of only 12 ±
2 s in CD18-/- mice, compared with an
average of 87 ± 22 s in wild-type mice (Table III). The
total distance over which cells were tracked in
CD18-/- mice (before detachment) was not
different from wild-type mice (Table III). These findings provide a
mechanistic explanation for data reported in vitro (29, 30) and in vivo (11, 31), showing that blockade of
CD18 integrins can increase the rolling velocity of leukocytes. We
confirmed this finding by blocking CD18 integrin function in wild-type
mice with mAb GAME-46. Five of five tracked leukocytes came to arrest
before GAME-46 injection, but only 3 of 20 arrested after GAME-46
injection (15% efficiency). The velocity of rolling leukocytes also
increased almost 4-fold after injection of GAME-46, consistent with the
velocity increase seen in CD18-/- mice. Rolling
leukocytes after GAME-46 treatment exhibited linear distance-time
curves similar to those seen in CD18-/- mice,
again demonstrating the role of CD18 integrins in rolling leukocyte
deceleration and arrest. As a negative control, we injected a
nonblocking L-selectin mAb (LAM1-101) (18) into wild-type
mice. This Ab did not influence adhesion (5 of 5 tracked leukocytes
with clear outcomes became adherent).
Finally, we investigated whether slow rolling mediated by engagement of
E-selectin (10, 13, 32) was required for arrest of rolling
neutrophils on inflamed endothelium. In E-/-
mice, 14 of 26 rolling leukocytes (54% efficiency) eventually became
adherent, whereas 12 cells detached. The distance traveled by rolling
leukocytes in E-/- mice was similar to that in
wild-type mice, but the average rolling velocity was significantly
elevated to 14 ± 2 µm/s (Table III), consistent with previously
published results (13). The endothelial contact time of
rolling leukocytes was reduced to 40 ± 14 s, compared with
87 ± 22 s in wild-type mice (Table III). Interestingly, most
leukocytes rolling in E-/- mice that eventually
adhered did not exhibit a clear decrease in rolling velocity before
arrest (Fig. 2), but a rather abrupt arrest after rolling a similar
distance as wild-type leukocytes (Table II).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The data presented in this paper provide direct evidence for and a
mechanistic explanation of previous data demonstrating a correlation
between rolling leukocyte transit time and the amount of firm adhesion
(11). When leukocytes are rolling slowly on
TNF--activated endothelium, CD18 integrins can participate in
stabilizing leukocyte rolling, and thus prevent detachment of rolling
leukocytes before they become activated enough to arrest. In fact, it
is exactly because the activation process appears to be protracted that
a relatively long endothelial contact time is necessary for efficient
arrest and normal levels of firm adhesion.
We also present the first data demonstrating changes in intercellular
calcium in rolling leukocytes. The gradual rise of
[Ca2+]i in rolling
leukocytes may depend on chemoattractant stimulation, adhesion molecule
cross-linking (for example, L-selectin), outside-in signaling through
integrin binding, or any combination of these activation signals.
Activation of rolling leukocytes appears to occur during the 86 s
that the leukocyte is rolling slowly along the endothelium, as opposed
to during capillary transit. Any additional stimulation, for example by
exogenous chemoattractant, would accelerate the cellular activation
process and lead to rapid arrest and firm adhesion (10).
Although more detailed studies of signaling events during leukocyte
rolling will be necessary, our observation of a gradual increase of the
[Ca2+]i in rolling cells
followed by a rapid rise upon arrest suggests that activation of
rolling leukocytes is only partial. More complete activation under
physiological conditions may require, or result from, firm adhesion, as
suggested by the pronounced increase of
[Ca2+]i upon
attachment.
Our observation of reduced efficiency of attachment and abrupt leukocyte arrest without a decrease in rolling velocity in E-/- mice is consistent with CD18 integrin involvement in leukocyte rolling and arrest. CD18 integrins have been shown to be unable to mediate leukocyte rolling independent of selectins (29), presumably because of the inability of CD18 integrins to bind rapidly to their endothelial ligands. However, it has been shown that the I domain of LFA-1, a ß2 integrin, can interact transiently with ICAM-1 and produce rolling interactions (33). This type of interaction may underlie the present observations. Leukocytes rolling on E-selectin appear to be moving slowly enough such that some CD18 integrins can bind to their ligands. In fact, the lack of cellular deceleration observed in E-/- mice suggests that the CD18 integrins are less efficient at decreasing rolling velocity in the absence of E-selectin, leading to a 3-fold elevation in rolling velocity in E-/- mice. When rolling leukocytes in E-/- become sufficiently activated (after rolling approximately the same distance as in wild-type mice), some of these leukocytes can arrest abruptly. This arresting mechanism is less efficient than the gradual deceleration process produced by CD18 integrin stabilization of the rolling process. These data are consistent with the moderate leukocyte recruitment defects seen in E-/- mice (10, 13, 32, 34).
Our data at first seems surprisingly at odds with previously published
results showing rapid leukocyte arrest in response to chemoattractant
stimulation (5, 6). One difference between these in vitro
systems and the current in vivo observations may be the level of
chemoattractant available to rolling leukocytes. In vivo, micropipette
applications of IL-8 (8) or MIP-2 (9) can
also cause rapid arrest of rolling leukocytes. However, this mode of
leukocyte arrest seems to be rare in cytokine-induced inflammation in
wild-type mice. TNF- stimulation alone may not result in
sufficiently high levels of chemoattractant expression to produce rapid
leukocyte arrest. In a previous study, 10 ng of TNF- injected into
an air pocket of a mouse caused a 3-fold increases in MIP-2 or KC mRNA
expression and produced 5–10 ng of secreted chemokine
(27). In our model, chemokine concentration may vary along
the venular tree, but it is currently not possible to measure the level
of chemoattractant present on the surface of inflamed venules in vivo.
Continuous superfusion of the tissue may also wash away some
chemoattractant. However, we commonly find that leukocyte adhesion
increases during the experiment, even with continual superfusion. If
there were high levels of chemoattractant washout, adhesion would be
expected to decrease, or at least remain constant as chemoattractant
levels were reduced.
Our data demonstrate that activation of rolling leukocytes by chemoattractants (including chemokines) can be gradual, and not necessarily an all-or-none response as proposed using various in vitro models (5, 6, 7). The gradual activation response may be due to low local chemoattractant concentrations on the vascular endothelium in combination with adhesion molecule-mediated activation. One interesting implication of gradual activation is that a leukocyte, such as a neutrophil, with distinct receptors for multiple chemoattractants, could potentially integrate activation signals from several different chemoattractants (e.g., platelet-activating factor (PAF), MIP-2, KC) while rolling along the endothelium, thus reinforcing cellular activation through several different receptor signaling pathways.
In summary, the surprisingly long endothelial contact time needed for gradual activation of leukocytes rolling in inflamed venules in vivo requires a revision of the concept that neutrophil activation is always rapid and follows rolling (2, 3). Rather, for neutrophils, rolling and activation appear to be intimately intertwined to produce neutrophil recruitment into inflamed tissues. More detailed cell biological analysis of the interplay between chemoattractants, rolling receptors, and integrins in reconstituted systems will be needed to fully understand this unusual pattern of neutrophil activation observed during physiological inflammation.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Pathology, Stanford University, 154-B VAMC, 3801 Miranda Avenue, Palo Alto, CA 94304. E-mail address:
3 Address correspondence and reprint requests to Dr. Klaus Ley, Department of Biomedical Engineering, University of Virginia, Box 377 Health Sciences Center, Charlottesville, VA 22908. E-mail address:
4 Abbreviations used in this paper: MIP-2, macrophage-inflammatory protein-2; [Ca2+]i, intracellular Ca2+ concentration.
Received for publication October 28, 1999. Accepted for publication January 3, 2000.
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References |
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. J. Immunol. 159:3595.[Abstract]
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M. Sperandio, M. L. Smith, S. B. Forlow, T. S. Olson, L. Xia, R. P. McEver, and K. Ley P-selectin Glycoprotein Ligand-1 Mediates L-Selectin-dependent Leukocyte Rolling in Venules J. Exp. Med., May 19, 2003; 197(10): 1355 - 1363. [Abstract] [Full Text] [PDF]
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A. E. R. Hicks, S. L. Nolan, V. C. Ridger, P. G. Hellewell, and K. E. Norman Recombinant P-selectin glycoprotein ligand-1 directly inhibits leukocyte rolling by all 3 selectins in vivo: complete inhibition of rolling is not required for anti-inflammatory effect Blood, April 15, 2003; 101(8): 3249 - 3256. [Abstract] [Full Text] [PDF]
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 M. AMON, M. D. MENGER, and B. VOLLMAR Heme oxygenase and nitric oxide synthase mediate cooling-associated protection against TNF-{alpha}-induced microcirculatory dysfunction and apoptotic cell death FASEB J, February 1, 2003; 17(2): 175 - 185. [Abstract] [Full Text] [PDF]
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A. J. Prorock, A. Hafezi-Moghadam, V. E. Laubach, J. K. Liao, and K. Ley Vascular protection by estrogen in ischemia-reperfusion injury requires endothelial nitric oxide synthase Am J Physiol Heart Circ Physiol, January 1, 2003; 284(1): H133 - H140. [Abstract] [Full Text] [PDF]
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T. Kadono, G. M. Venturi, D. A. Steeber, and T. F. Tedder Leukocyte Rolling Velocities and Migration Are Optimized by Cooperative L-Selectin and Intercellular Adhesion Molecule-1 Functions J. Immunol., October 15, 2002; 169(8): 4542 - 4550. [Abstract] [Full Text] [PDF]
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S. B. FORLOW, P. L. FOLEY, and K. LEY Severely reduced neutrophil adhesion and impaired host defense against fecal and commensal bacteria in CD18-/-P-selectin-/- double null mice FASEB J, October 1, 2002; 16(12): 1488 - 1496. [Abstract] [Full Text] [PDF]
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 T. S. Olson and K. Ley Chemokines and chemokine receptors in leukocyte trafficking Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2002; 283(1): R7 - R28. [Abstract] [Full Text] [PDF]
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 A. F. H. Lum, C. E. Green, G. R. Lee, D. E. Staunton, and S. I. Simon Dynamic Regulation of LFA-1 Activation and Neutrophil Arrest on Intercellular Adhesion Molecule 1 (ICAM-1) in Shear Flow J. Biol. Chem., May 31, 2002; 277(23): 20660 - 20670. [Abstract] [Full Text] [PDF]
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J. L. Dunne, C. M. Ballantyne, A. L. Beaudet, and K. Ley Control of leukocyte rolling velocity in TNF-alpha -induced inflammation by LFA-1 and Mac-1 Blood, January 1, 2002; 99(1): 336 - 341. [Abstract] [Full Text] [PDF]
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J. A. DiVietro, M. J. Smith, B. R. E. Smith, L. Petruzzelli, R. S. Larson, and M. B. Lawrence Immobilized IL-8 Triggers Progressive Activation of Neutrophils Rolling In Vitro on P-Selectin and Intercellular Adhesion Molecule-1 J. Immunol., October 1, 2001; 167(7): 4017 - 4025. [Abstract] [Full Text] [PDF]
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 M. J. Cotter, K. E. Norman, P. G. Hellewell, and V. C. Ridger A Novel Method for Isolation of Neutrophils from Murine Blood Using Negative Immunomagnetic Separation Am. J. Pathol., August 1, 2001; 159(2): 473 - 481. [Abstract] [Full Text]
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R. B. Henderson, L. H.K. Lim, P. A. Tessier, F. N.E. Gavins, M. Mathies, M. Perretti, and N. Hogg The Use of Lymphocyte Function-Associated Antigen (Lfa)-1-Deficient Mice to Determine the Role of Lfa-1, Mac-1, and {alpha}4 Integrin in the Inflammatory Response of Neutrophils J. Exp. Med., July 16, 2001; 194(2): 219 - 226. [Abstract] [Full Text] [PDF]
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K. Singbartl, J. Thatte, M. L. Smith, K. Wethmar, K. Day, and K. Ley A CD2-Green Fluorescence Protein-Transgenic Mouse Reveals Very Late Antigen-4-Dependent CD8+ Lymphocyte Rolling in Inflamed Venules J. Immunol., June 15, 2001; 166(12): 7520 - 7526. [Abstract] [Full Text] [PDF]
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E. E. ERIKSSON, X. XIE, J. WERR, P. THOREN, and L. LINDBOM Direct viewing of atherosclerosis in vivo: plaque invasion by leukocytes is initiated by the endothelial selectins FASEB J, May 1, 2001; 15(7): 1149 - 1157. [Abstract] [Full Text] [PDF]
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V. C. Ridger, B. E. Wagner, W. A. H. Wallace, and P. G. Hellewell Differential Effects of CD18, CD29, and CD49 Integrin Subunit Inhibition on Neutrophil Migration in Pulmonary Inflammation J. Immunol., March 1, 2001; 166(5): 3484 - 3490. [Abstract] [Full Text] [PDF]
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M. M. Mariscalco, W. Vergara, J. Mei, E. O'B. Smith, and C. W. Smith Mechanisms of decreased leukocyte localization in the developing host Am J Physiol Heart Circ Physiol, February 1, 2002; 282(2): H636 - H644. [Abstract] [Full Text] [PDF]
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),
CD18-/- (•), and E-/- (
) mice. Outcome
indicated by arrows: 
adhere, 
detach. Tracings are from two
representative leukocytes from each genotype.
