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Immunology Research Group, University of Calgary, Calgary, Alberta, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Indeed, a number of laboratories have reported that engagement of L-selectin, via Ab-mediated cross-linking or ligation, results in activation of signaling pathways and subsequent increases in cytokine mRNA expression, intracellular calcium, oxidant production, integrin activation, and firm adhesion (11, 12, 13, 14, 15). Simon et al. showed that cross-linking of L-selectin increased the ability of neutrophils to bind latex beads via increased expression of a ß2 integrin activation-dependent epitope (16). The same treatment also induced adhesion and transmigration of neutrophils across endothelial cells. Another in vitro study revealed that ligation of L-selectin potentiated the amount of adhesion and emigration induced by neutrophil-activating agonists such as platelet-activating factor (PAF)4 and IL-8 (17). The intracellular signaling molecules responsible for these include mitogen-activated protein (MAP) kinases, tyrosine kinases, and p21ras oncoprotein (Ras) (18, 19). Recently it has been observed that the p38 MAP kinase is phosphorylated within minutes of cross-linking L-selectin, and inhibition of p38 MAP kinase has been shown to inhibit L-selectin-dependent neutrophil shape change, adhesion, and degranulation (20, 21). Clearly, the absence of L-selectin and lack of activation of these pathways may explain the reduction in leukocyte recruitment observed in L-selectin-/- mice in vivo. However, to date an assessment of the role of L-selectin in the events that occur subsequent to leukocyte rolling in inflamed microvessels, and in the extravascular space, has not been performed.
The aim of this study was to systematically assess the role of L-selectin in the events downstream of rolling in vivo in the murine system. We used intravital microscopy to visualize leukocyte rolling, adhesion, emigration, and migration through the extravascular tissue of the cremaster muscle of wild-type and L-selectin-/- mice, induced by chemotactic inflammatory mediators. Based on preliminary data, we designed a novel in vivo assay system that establishes a unidirectional chemotactic gradient within the cremaster muscle. The data reveal a significant impairment in the ability of L-selectin-/- leukocytes to both emigrate and respond to directional cues within the interstitium. The lack of L-selectin did not affect rolling or adhesion in response to chemotactic mediators.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MEL-14, a mAb against murine L-selectin, was purchased from
PharMingen (San Diego, CA). KC, the murine homolog of
IL-8/growth-related oncogene-
, murine TNF-, and recombinant human
E-selectin were obtained from R&D Systems (Minneapolis, MN).
Animals
Male C57BL/6 wild-type mice were obtained from Charles River Breeding Laboratories (Wilmington, MA). Mice deficient in L-selectin were originally provided by Dr. Tom Tedder (Department of Immunology, Duke University Medical Center, Durham, NC). These mice were generated on a mixed background of 129/Sv x C57BL/6 (7) and have been backcrossed onto a C57BL/6 background for seven generations (9). All mice weighed between 20 and 35 g and were used between 6 and 10 wk of age.
Flow chamber assay
To study murine leukocyte behavior under shear conditions, a whole-blood flow chamber assay was used as previously described (22, 23). Blood was removed from anesthetized wild-type and L-selectin-/- mice via cardiac puncture, using acid-citrate dextrose as an anticoagulant. The blood was diluted 1:10 in HBSS, and the leukocytes were stained by the addition of rhodamine 6G 5 min before the flow experiment. Leukocyte interactions were examined on coverslips bearing immobilized human E-selectin, prepared as previously described (23). Recombinant human E-selectin has previously been shown to support interactions with murine leukocytes under flow conditions in vitro (23). Nonspecific interactions between the leukocytes and E-selectin substrate were blocked by incubation with 1% BSA (Sigma, St. Louis, MO) for 2 h at 37°C. E-selectin-coated coverslips were briefly dipped in buffer and then mounted into a polycarbonate chamber with parallel plate geometry as previously described (24). The flow chamber was placed onto the stage of an inverted microscope (Carl Zeiss, Don Mills, Ontario, Canada), and leukocyte interactions were visualized at x100 magnification using fluorescence microscopy. The stage area was enclosed in a warm air cabinet and maintained at 37°C.
The prepared mouse blood was maintained at 37°C using a water bath, and a syringe pump (Harvard Apparatus, South Anatik, MA) was used to draw blood through the flow chamber at defined wall shear stresses. All of the experiments described were performed at 4.0 dynes/cm2. To determine the number of leukocytes recruited, five randomly selected fields were examined during each experiment, and cells were classified as rolling or adherent. At the end of each experiment, the coverslips were gently removed from the flow chamber, dried, and stained with Wright-Giemsa stain to allow identification of the types of leukocytes recruited. Differential analysis of recruited leukocytes was performed on five separate fields per coverslip. Leukocyte rolling and adhesion and the types of leukocytes recruited were compared between wild-type and L-selectin-/- mice.
Intravital microscopy
The mouse cremaster preparation was used to study the behavior of leukocytes in the microcirculation (23). Mice were anesthetized by i.p. injection of a mixture of xylazine hydrochloride (10 mg/kg; MTC Pharmaceuticals, Cambridge, Ontario, Canada) and ketamine hydrochloride (200 mg/kg; Rogar/STB, London, Ontario, Canada). The jugular vein was cannulated and used to administer additional anesthetic and Abs. The cremaster muscle was dissected free of tissues and exteriorized onto an optically clear viewing pedestal. The muscle was cut longitudinally with a cautery and held flat against the pedestal by attaching silk sutures to the corners of the tissue. The muscle was then superfused with bicarbonate-buffered saline.
An intravital microscope (Axioskop; Carl Zeiss) with a x25 objective
lens (Weltzlar L25/0.35; E. Leitz, Munich, Germany) and a x10 eyepiece
were used to examine the cremasteric microcirculation. A video camera
(Panasonic 5100 HS; Osaka, Japan) was used to project the images onto a
monitor, and the images were recorded for playback analysis using a
videocassette recorder. Single unbranched cremasteric venules (25–40
µm in diameter) were selected and, to minimize variability, the same
section of cremasteric venule was observed throughout the experiment.
The number of rolling and adherent leukocytes was determined off-line
during video playback analysis. Rolling leukocytes were defined as
those cells moving at a velocity less than that of erythrocytes within
a given vessel. Leukocyte rolling velocity was determined by measuring
the time required for a leukocyte to roll along a 100-µm length of
venule. Rolling velocity was determined for 20 leukocytes at each time
interval. Leukocytes were considered adherent to the venular
endothelium if they remained stationary for 30 s or longer.
Leukocyte emigration was defined as the number of extravascular
leukocytes per microscopic field of view (x25 objective lens) adjacent
to the selected postcapillary venule. Venular diameter
(Dv) was measured on-line using a
video caliper (Microcirculation Research Institute, Texas A&M
University, College Station, TX). Centerline RBC velocity
(VRBC) was also measured on-line using
an optical Doppler velocimeter (Microcirculation Research Institute),
and mean RBC velocity (Vmean) was
determined as VRBC/1.6. Venular
wall shear rate (
) was calculated based on the Newtonian definition:
= 8
(Vmean/Dv)
(25).
Analysis of random extravascular migration (chemokinesis)
After an initial baseline recording, the cremasteric preparation was superfused with either PAF (Sigma) (100 nM with 0.5% BSA as a carrier) or KC (5.2 nM) dissolved in superfusion buffer. Experiments were performed in two stages. During the first hour, leukocyte rolling, adhesion, and emigration in single unbranched cremasteric venules were examined. This period served to allow leukocytes to enter the extravascular tissue. During the second hour, an avascular region of at least 175 µm width (one monitor screen), adjacent to a postcapillary venule, was examined. Migration of emigrated leukocytes within this area was recorded continuously using a time-lapse videocassette recorder (Panasonic AG-6730P). During playback analysis, the migration path of individual leukocytes was traced onto a monitor screen, then the path length was quantified using the Optimas image analysis system. In addition, the number of emigrated leukocytes present at 25-µm intervals lateral to the postcapillary venule was determined at the beginning and end of this period. Rolling, adhesion, and emigration responses of leukocytes induced by this nondirectional inflammatory cue were compared between wild-type and L-selectin-/- mice.
Analysis of directional extravascular migration (chemotaxis)
Agarose gel (2% in 50:50 HBSS/RPMI 1640) containing KC was prepared by boiling a concentrated agarose solution (8% in 10 ml distilled water) in a microwave, then adding a mixture of 10 ml of 2x HBSS and 20 ml RPMI 1640. Before cooling, a 100-µl aliquot of this solution was removed, and KC was added to this aliquot to achieve a final concentration of 5.2 µM. To enable visualization of the gel on the cremaster muscle, a small amount of India ink was added to each preparation. A 1-mm3 piece of the mixture was punched out using the tip of a Pasteur pipette. This piece of gel was carefully placed on the surface of the cremaster, in a preselected avascular area, 350 µm (two monitor screens wide) from a postcapillary venule. The gel was held in place using a coverslip, and the tissue was superfused beneath the coverslip at a minimum rate (<0.2 ml/min) so as not to disrupt the chemotactic gradient established adjacent to the agarose pellet. Pilot experiments determined that KC at a concentration of 5.2 µM in the agarose solution was effective at inducing leukocyte migration in the cremaster preparation. Experiments ran for 2 h. During the first hour, leukocyte migration was examined in the field adjacent to the postcapillary venule. During the second hour, as leukocyte migration progressed, the field adjacent to the gel pellet was examined.
Leukocyte migration responses induced by the directional chemotactic stimulus provided by the KC were compared between wild-type and L-selectin-/- mice. In addition, to confirm the role of L-selectin in this response, an additional group of wild-type mice were treated with an anti-L-selectin mAb (MEL-14, 100 µg i.v.) before the placement of the KC-loaded gel on the cremaster muscle, and leukocyte migration responses were compared with those observed in wild-type mice.
Circulating leukocyte counts
At the end of each experiment, whole blood was drawn via cardiac puncture. Total leukocyte counts were performed, using a Bright-line hemocytometer (Hausser Scientific, Horsham, PA).
Statistical analysis
All data are displayed as mean ± SEM. Comparisons between groups were performed using Student’s t tests. Paired analysis was used when appropriate. A value of p < 0.05 was deemed significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In initial experiments, we examined the role of L-selectin in
leukocyte recruitment onto human E-selectin using an in vitro flow
chamber assay. Leukocytes from wild-type and
L-selectin-/- mice rolled with equal efficiency
on this substrate (Fig. 1
A),
suggesting that the absence of L-selectin from leukocytes did not alter
their ability to undergo rolling interactions on E-selectin. In
addition, both the number and types of leukocytes recruited onto
E-selectin were comparable in wild-type and
L-selectin-/- blood (Fig. 1B),
indicating that the absence of L-selectin did not have a differential
effect on the ability of any one particular leukocyte population to be
recruited to this substrate.
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We next used intravital microscopy to determine whether the
absence of L-selectin affected in vivo leukocyte rolling, adhesion, and
emigration induced by the chemotactic mediator, PAF. To rule out any
effects of differences in circulating leukocyte numbers, leukocyte
counts were performed on all mice used in this study. These analyses
revealed that circulating leukocyte counts did not differ significantly
between wild-type (7.3 ± 1.1 x
106/ml) and L-selectin-/-
(8.8 ± 1.2 x 106/ml) mice. In
previous studies, we have seen that the values in
L-selectin-/- mice can vary from normal to as
much as double the values in wild-type mice (5). Fig. 2 shows leukocyte rolling flux, adhesion,
and emigration induced in the cremaster muscle by 60 min of PAF
superfusion. Leukocyte rolling flux decreased progressively over this
period, to a similar extent in wild-type and
L-selectin-/- mice, although at the 30-min time
point, leukocyte rolling was significantly lower in
L-selectin-/- mice. However, this minor
reduction in rolling in L-selectin-/- mice did
not impact PAF-induced adhesion. PAF induced marked increases in
leukocyte adhesion and emigration in wild-type mice, adhesion reaching
30 cells/100 µm, and emigration 15 cells/field by 60 min. In
L-selectin-/- mice, leukocyte adhesion
increased to comparable levels over the same time course, showing that
L-selectin was not important in determining the number of cells
accumulating on the endothelial surface. However, the number of
leukocytes that emigrated in L-selectin-/- mice
in response to 60-min PAF superfusion was markedly attenuated relative
to wild-type mice. These data indicated that L-selectin has a role in
mediating leukocyte exit from the vasculature.
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We next used time-lapse video microscopy to examine leukocyte
migration in the extravascular tissue during a second hour of PAF
superfusion. During superfusion with chemotactic mediators such as PAF
or KC, emigrated leukocytes commonly travel large distances (>100
µm), frequently changing directions, before ultimately stopping (Fig. 3A). During PAF superfusion,
the average velocity of locomotion observed (14 µm/min) (Table I) was similar to that previously
reported for PAF-induced neutrophil migration in the rat mesentery
(26). In wild-type mice, the average path length of
leukocyte locomotion during PAF superfusion was 120 µm (Fig. 4A), and migration of
individual wild-type leukocytes could be traced for an average of 13
min (Table I), after which the leukocyte became static or left the
field of view. In L-selectin-/- mice, the
average path length was reduced by almost 50%
(p < 0.05). This difference was not due to an
alteration in the migration velocity in
L-selectin-/- mice, as this parameter did not
differ from that observed in wild-type mice, but was attributable to a
comparable reduction in the period the leukocyte was actively migrating
(Table I). This finding indicated that L-selectin played a role in
determining the ability of an emigrated leukocyte to move in response
to a given inflammatory stimulus (PAF).
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(27) (Fig. 4
B). KC was even more effective at inducing leukocyte
migration than PAF. In wild-type mice, the average path length of
leukocyte locomotion was 180 µm (Fig. 4B). In
L-selectin-/- mice, this distance was reduced
by 50%. The difference in extravascular locomotion was not due to an
alteration in leukocyte migration velocity, as this was similar in both
types of mice (Table I). However, as seen in the PAF experiments, the
period of migration of individual leukocytes was again reduced by
50% in L-selectin-/- mice (Table I).
Furthermore, this difference did not appear to be due to upstream
effects as the level of leukocyte adhesion induced by KC superfusion
did not differ significantly between the two types of mice (data not
shown). These results show that the presence of a functional L-selectin
molecule plays an important role in facilitating extravascular
leukocyte migration induced by a nondirectional inflammatory stimulus.
However, these findings do not indicate at what point in the sequence
of leukocyte-endothelial cell interactions this L-selectin-dependent
effect is mediated. Responses to directional (chemotactic) cues are impaired in L-selectin-/- mice
Superfusion of the cremaster preparation with PAF and KC
indicated reduced chemokinesis in the absence of L-selectin, but
whether this impacted relevant biology, i.e., migration toward an
inflammatory site, was unclear. Therefore, we induced directional
migration of extravascular leukocytes using controlled release of KC
from agarose gel. Adhesion and emigration was noted immediately in the
postcapillary venule adjacent to the stimulus, and emigrated leukocytes
proceeded to migrate almost exclusively toward the source of KC (Fig. 3B). Upon nearing the chemotactic source (350 µm away from
the postcapillary venule), leukocytes either started to migrate in a
more random fashion, or became static.
Fig. 5A shows the number of
leukocytes present at increasing distances from the postcapillary
venule after 60 min of exposure to a directional cue of KC. In
wild-type mice, this treatment induced an even distribution of
emigrated leukocytes, with 20 leukocytes being present in each
25-µm segment from 25 to 150 µm lateral to the venule, suggesting
continuous migration toward the KC gel. In
L-selectin-/- mice, the largest number of
leukocytes was immediately adjacent to the vessel, and the numbers of
leukocytes rapidly dropped in each subsequent 25-µm segment.
Furthermore, the total number of leukocytes that emigrated in response
to this stimulus was reduced by >50% in
L-selectin-/- mice (Fig. 5B). As
seen previously, this difference was not due to a variation in
migration velocity as this was unchanged from wild-type mice (Table I).
To confirm that the impaired directional chemotactic response was due
to the lack of L-selectin, we treated wild-type mice with the
L-selectin mAb MEL-14, and examined the response. In these mice, the
directional migration response was reduced relative to untreated
wild-type mice, both in the extravascular distribution of emigrated
leukocytes (Fig. 6A) and in
the total number of emigrated cells (Fig. 6B). This effect
could not be explained by a reduction in circulating leukocytes, as
leukocyte counts were not affected by this dose of MEL-14 (MEL-14,
5.3 ± 0.8 x 106/ml; wild-type,
7.3 ± 1.1 x 106/ml) as observed
previously (5). Rolling and adhesion were also not
affected by MEL-14. These findings suggest that engagement of
L-selectin is critical in enabling leukocytes to respond effectively to
directional inflammatory stimuli.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There is a growing body of evidence that the function of L-selectin may be more complex than simply mediating leukocyte rolling. Following ligation or cross-linking, L-selectin can transduce activating signals into the cell that induce proinflammatory intracellular changes such as shape change and integrin-mediated intercellular adhesion (11, 12, 13, 14, 15, 17). Furthermore, previous work has revealed that immunoneutralization of L-selectin on human neutrophils and lymphocytes reduces adhesion and transmigration across activated endothelial monolayers (28), and mimicking L-selectin engagement by cross-linking L-selectin under flow conditions in vitro doubles the number of neutrophils that transmigrate across monolayers of endotoxin-stimulated HUVEC (16). However, in both of these studies, the changes in numbers of transmigrated leukocytes were thought to be reflective of changes in adhesion. More recently, Tsang et al. (17) normalized transmigration to leukocyte adhesion and reported that L-selectin ligation potentiated neutrophil transmigration across IL-1-stimulated HUVEC, but only at suboptimal levels of IL-1 stimulation (17). Our results extend these findings by providing direct in vivo evidence supporting a role for L-selectin in leukocyte emigration. By using intravital microscopy, we were able to clearly show that the absence of L-selectin did not reduce adhesion, but dramatically reduced leukocyte emigration. The work also suggests that L-selectin has an important role in promoting efficient migration of leukocytes after they have left the vasculature and entered the extravascular tissue. In an earlier publication, we noted in histologic samples of Ag-induced leukocyte recruitment that L-selectin-/- leukocytes were not found distributed throughout inflamed tissues, as was the case in inflamed wild-type tissue, but remained closely associated with postcapillary venules (5). The results herein provide an explanation for this observation by demonstrating an impairment in interstitial migration in L-selectin-/- mice. This may also provide an alternative explanation for the reduced leukocyte recruitment observed in L-selectin-/- mice during thioglycollate peritonitis and other inflammatory states (7, 8).
In addition, these experiments would suggest that in vivo, in response to acute chemotactic stimuli, absence of L-selectin does not affect rolling. More importantly, our whole blood in vitro flow assay revealed that the same percentage of neutrophils and mononuclear cells was recruited from wild-type and L-selectin-/- blood, arguing against a selective inhibition of rolling or adhesion of any one cell type. These data are consistent with the requirement for induction of an L-selectin ligand on endothelium rather than constitutive expression of such a molecule. Indeed, L-selectin inhibition does affect rolling following 4 h of Ag exposure and following cytokine stimulation of endothelium in various chronic inflammatory conditions (5, 29), but not following PAF or KC exposure. Our observations in this study suggest that, in addition to selective effects on rolling, the absence of L-selectin dramatically reduces emigration across endothelium in vivo, suggesting that a major function of L-selectin in the peripheral microvasculature may be to modulate events downstream of leukocyte rolling and adhesion, i.e., emigration and extravascular locomotion.
What cannot be determined from these observations is at what point in
the sequence of rolling, adhesion, and emigration L-selectin mediates
its effect. Results of many studies indicate that L-selectin expression
on emigrated leukocytes is dramatically reduced. In a model of
thioglycollate peritonitis, emigrated neutrophils have been shown to
express one-tenth the level of L-selectin expression of circulating
leukocytes (30). Furthermore, neutrophil transmigration
across TNF--treated endothelium reduces L-selectin expression by
>50% (31). In fact, adhesion of neutrophils to
IL-1-activated endothelial cells in vitro for only 10 min has been
shown to reduce L-selectin expression by 80% (32).
Although we did not examine L-selectin levels on emigrated leukocytes
in our experiments, it is probable that L-selectin expression was
greatly reduced on these cells. Presumably, this would preclude
L-selectin-dependent signaling by leukocytes that had already
emigrated, indicating that the signal by which L-selectin mediates this
effect must occur before or during the process of passing through the
endothelial layer. Moreover, this suggests that the signal transduced
by L-selectin has a prolonged effect on leukocytes, as it continues to
modulate their behavior well after they have left the vasculature while
they migrate to their target site. An alternative hypothesis may be
that despite L-selectin being expressed at greatly reduced levels on
emigrated leukocytes, residual L-selectin may continue to transduce
signals into the leukocyte that aid in the process of locomotion.
Further work is required to determine the relevance of L-selectin
shedding in these physiological responses.
CD18-dependent adhesion induced by L-selectin cross-linking has been shown to occur over a period of several minutes (17). In contrast, the process of activation by which rolling cells undergo the transition to adhesion happens very rapidly (within seconds). Campbell et al. showed that cells rolling on substrates consisting of immobilized adhesion molecules and chemokines underwent adhesion very rapidly after the initiation of rolling (33). Although this is not consistent with the in vitro observation (17) that L-selectin may contribute to adhesion over several minutes, these in vitro studies provide important information in that they demonstrate 1) that L-selectin can function as an activating molecule, and 2) that this leads to CD18-dependent events. Therefore, we would propose that L-selectin-mediated activation is unlikely to affect cells sufficiently quickly to impact adhesion within the microvasculature. However, given the time course required for emigration and migration, it is clearly more likely that L-selectin-mediated cellular activation may affect these processes. This hypothesis is supported by the observations from this study.
Whereas the molecular basis of the intravascular events of rolling and
adhesion have been extensively investigated, the process whereby
leukocytes move through the extravascular tissue to reach sites of
infection and/or injury is less well understood. Two classes of
molecules appear to be critically involved, chemoattractants/chemokines
and adhesion molecules. Bienvenu et al. examined leukocyte migration in
vivo in the rat mesentery and showed that stimulation with the
chemotactic mediators fMLP, leukotriene B4, and
PAF increased the rate of leukocyte migration significantly above
levels of unstimulated extravascular leukocytes (34).
Comparable rates of migration were observed in response to ischemia and
reperfusion, showing that the leukocyte responses observed with
superfusion of chemotactic mediators mimicked those observed during a
physiologically relevant inflammatory stimulus. More recently, Foxman
et al. found that migrating leukocytes are able to navigate through
complex sequential fields of chemotactic gradients to reach a
"target" agonist (2). Furthermore, it has recently
been shown that during the process of extravasation, leukocytes
up-regulate expression of several integrins and, subsequently, use
adhesion molecules such as the
1ß1 and
2ß1 integrins to
migrate through the interstitium (26, 35, 36, 37). These
studies indicate that leukocytes undergo a phenotypic change during the
process of inflammation-induced transmigration. A very appealing but as
yet untested possibility is that L-selectin transduces signals that
induce up-regulation of novel integrins and impact leukocyte
behavior.
It would be expected that for L-selectin to have a physiological function, it needs to interact with a ligand. Indeed, both Ab blockade and absence of L-selectin reduced migratory responses of murine leukocytes, providing support for the hypothesis that engagement of L-selectin is important in facilitating these responses. It has been shown that, under certain inflammatory conditions, an inducible endothelial L-selectin ligand capable of mediating leukocyte rolling can be expressed in the peripheral microvasculature (5, 28, 29, 38, 39, 40). However, it is doubtful that an inducible L-selectin ligand was functional in these experiments as expression of the inducible ligand for L-selectin conventionally requires cytokine stimulation over several hours, whereas these experiments only examined the first 2 h of the inflammatory response. This raises the question of what ligand, if any, was interacting with L-selectin to mediate the effects observed in this study. Although it is tempting to propose adhesion ligands as stimulators of L-selectin, it is possible that any molecule that can temporarily bind L-selectin may serve this function. For example P-selectin is thought not to function as an adhesion ligand for L-selectin, although it can present carbohydrates to L-selectin that may be sufficient to activate the L-selectin signaling pathway. An additional intriguing observation was the recent identification of the proteoglycan versican as an extravascular L-selectin ligand. This raises the possibility that L-selectin may continue to transduce signals once leukocytes have left the vasculature, perhaps providing directional cues for migrating cells (41).
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Footnotes |
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2 Current address: Baker Medical Research Institute, P.O. Box 6492, Saint Kilda Road Central, Melbourne, Victoria, Australia, 8008.
3 Address correspondence and reprint requests to Dr. Paul Kubes, Health Sciences Center, University of Calgary, 3330 Hospital Drive Northwest, Calgary, Alberta T2N 4N1, Canada.
4 Abbreviations used in this paper: PAF, platelet-activating factor; MAP, mitogen-activated protein.
Received for publication April 18, 2000. Accepted for publication September 20, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and interleukin-8 mRNA in human neutrophils: evidence for a role of L-selectin as signaling molecule. J. Biol. Chem. 269:4021.4-integrin supports leukocyte rolling and adhesion in chronically inflamed postcapillary venules in vivo. J. Exp. Med. 183:1995.4 integrin. Circ. Res. 81:196.2ß1 (VLA-2) is a principal receptor used by neutrophils for locomotion in extravascular tissue. Blood 95:1804.1ß1 and 2ß1 in models of hypersensitivity and arthritis. J. Clin. Invest. 105:721.[Medline]
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 K. Uchimura, K. Kadomatsu, F. M. El-Fasakhany, M. S. Singer, M. Izawa, R. Kannagi, N. Takeda, S. D. Rosen, and T. Muramatsu N-Acetylglucosamine 6-O-Sulfotransferase-1 Regulates Expression of L-Selectin Ligands and Lymphocyte Homing J. Biol. Chem., August 13, 2004; 279(33): 35001 - 35008. [Abstract] [Full Text] [PDF]
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W. Baker, B. A. St. P. Schneider, A. Kulkarni, G. Sloan, R. Schaub, J. Sypek, and J. G. Cannon P-selectin inhibition suppresses muscle regeneration following injury J. Leukoc. Biol., August 1, 2004; 76(2): 352 - 358. [Abstract] [Full Text] [PDF]
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S. Szanto, I. Gal, A. Gonda, T. T. Glant, and K. Mikecz Expression of L-Selectin, but Not CD44, Is Required for Early Neutrophil Extravasation in Antigen-Induced Arthritis J. Immunol., June 1, 2004; 172(11): 6723 - 6734. [Abstract] [Full Text] [PDF]
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D. Ogawa, K. Shikata, K. Honke, S. Sato, M. Matsuda, R. Nagase, A. Tone, S. Okada, H. Usui, J. Wada, et al. Cerebroside Sulfotransferase Deficiency Ameliorates L-selectin-dependent Monocyte Infiltration in the Kidney after Ureteral Obstruction J. Biol. Chem., January 16, 2004; 279(3): 2085 - 2090. [Abstract] [Full Text] [PDF]
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E. Galkina, K. Tanousis, G. Preece, M. Tolaini, D. Kioussis, O. Florey, D. O. Haskard, T. F. Tedder, and A. Ager L-Selectin Shedding Does Not Regulate Constitutive T Cell Trafficking but Controls the Migration Pathways of Antigen-activated T Lymphocytes J. Exp. Med., November 3, 2003; 198(9): 1323 - 1335. [Abstract] [Full Text] [PDF]
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Y. Ebisuno, T. Tanaka, N. Kanemitsu, H. Kanda, K. Yamaguchi, T. Kaisho, S. Akira, and M. Miyasaka Cutting Edge: The B Cell Chemokine CXC Chemokine Ligand 13/B Lymphocyte Chemoattractant Is Expressed in the High Endothelial Venules of Lymph Nodes and Peyer's Patches and Affects B Cell Trafficking Across High Endothelial Venules J. Immunol., August 15, 2003; 171(4): 1642 - 1646. [Abstract] [Full Text] [PDF]
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Z. Ding, T. B. Issekutz, G. P. Downey, and T. K. Waddell L-selectin stimulation enhances functional expression of surface CXCR4 in lymphocytes: implications for cellular activation during adhesion and migration Blood, June 1, 2003; 101(11): 4245 - 4252. [Abstract] [Full Text] [PDF]
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S. S. Tay, A. McCormack, C. Lawson, and M. L. Rose IFN-{gamma} Reverses the Stop Signal Allowing Migration of Antigen-Specific T Cells into Inflammatory Sites J. Immunol., March 15, 2003; 170(6): 3315 - 3322. [Abstract] [Full Text] [PDF]
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 C. E. Green, D. N. Pearson, N. B. Christensen, and S. I. Simon Topographic requirements and dynamics of signaling via L-selectin on neutrophils Am J Physiol Cell Physiol, March 1, 2003; 284(3): C705 - C717. [Abstract] [Full Text] [PDF]
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 L. Borsig, R. Wong, R. O. Hynes, N. M. Varki, and A. Varki Synergistic effects of L- and P-selectin in facilitating tumor metastasis can involve non-mucin ligands and implicate leukocytes as enhancers of metastasis PNAS, February 19, 2002; 99(4): 2193 - 2198. [Abstract] [Full Text] [PDF]
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D. C. Cara, J. Kaur, M. Forster, D.-M. McCafferty, and P. Kubes Role of p38 Mitogen-Activated Protein Kinase in Chemokine-Induced Emigration and Chemotaxis In Vivo J. Immunol., December 1, 2001; 167(11): 6552 - 6558. [Abstract] [Full Text] [PDF]
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E. E. Eriksson, X. Xie, J. Werr, P. Thoren, and L. Lindbom Importance of Primary Capture and L-Selectin-Dependent Secondary Capture in Leukocyte Accumulation in Inflammation and Atherosclerosis in Vivo J. Exp. Med., July 16, 2001; 194(2): 205 - 218. [Abstract] [Full Text] [PDF]
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H. J. Strausbaugh and S. D. Rosen A Potential Role for Annexin 1 as a Physiologic Mediator of Glucocorticoid-Induced L-Selectin Shedding from Myeloid Cells J. Immunol., May 15, 2001; 166(10): 6294 - 6300. [Abstract] [Full Text] [PDF]
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) and L-selectin-/- (•) mice during
60-min PAF (100 nM) superfusion. Leukocyte emigration is expressed as
the increase in the number of emigrated leukocytes per field from that
at the start of the experiment. Data represent mean ± SEM of four
mice per group. *, p < 0.05 relative to
wild-type mice.
