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4 Integrin1
*
Immunology Research Group, University of Calgary, Calgary, Alberta, Canada; and
Department of Molecular and Cellular Physiology, Louisiana State University Medical Center, Shreveport, LA 71130
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4 integrin. The objective of
this study was to determine the molecular mechanisms that underlie
IL-4-induced leukocyte recruitment in vivo. Mice received an
intrascrotal injection of IL-4 (100 ng). Twenty-four hours later,
leukocyte rolling, adhesion, and emigration in cremasteric
postcapillary venules were examined via intravital microscopy, and
expression of VCAM-1 and P- and E-selectin was quantitated using a
radiolabeled mAb technique. IL-4 increased VCAM-1 expression, but
P-selectin and E-selectin remained at constitutive levels. IL-4 induced
significant increases in leukocyte adhesion and emigration, with 50%
of the emigrated cells being eosinophils and the remainder being
mononuclear leukocytes. Leukocyte rolling in IL-4-treated mice was
>95% inhibitable using an anti-P-selectin Ab. However,
IL-4-induced leukocyte recruitment was unaltered in mice treated
chronically with P-selectin Ab or mice deficient in either P-selectin
or P- and E-selectin, suggesting that the residual rolling supported
all of the IL-4-induced recruitment. In IL-4-treated mice following
P-selectin blockade, tethering and rolling were not dependent on
L-selectin, but were abolished by 4 integrin blockade.
These findings show that the 4 integrin can initiate
leukocyte-endothelial cell interactions in the absence of selectins
under shear conditions in vivo, and that the absence of selectins does
not affect recruitment of eosinophils and mononuclear cells to
IL-4-treated tissue. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Leukocyte recruitment is a multistep cascade that involves initial
selectin-dependent leukocyte tethering (attachment) to endothelium,
which in vivo can occur at shear stresses between 2 and 15
dynes/cm2, followed by selectin-dependent
leukocyte rolling and finally integrin-dependent leukocyte adhesion.
From this simplistic model, one might predict that the integrin
pathway, 4 integrin/VCAM-1, would mediate firm
adhesion. However, this is far from incontestable, as in vitro the
4 integrin/VCAM-1 pathway has been shown to
mediate leukocyte tethering, rolling, and adhesion, albeit at the lower
end (0.7–2 dynes/cm2) of physiologic shear
(10, 11). Other in vitro studies have proposed initial
selectin-dependent leukocyte tethering and rolling, followed by
subsequent 4 integrin/VCAM-1-dependent
leukocyte adhesion (12, 13, 14). This group of in vitro
studies is more consistent with in vivo data from two inflammatory
models showing that selectins were absolutely required for the initial
attachment or tethering of leukocytes to endothelium, whereas the
4 integrin/VCAM-1 pathway was shown to play a
role in the subsequent rolling and adhesion (15, 16).
To date no one has examined whether a mediator such as IL-4, which
presumably induces expression of high levels of VCAM-1 and only limited
amounts, if any, of the selectins, induces leukocyte recruitment via
the 4 integrin/VCAM-1 pathway exclusively or
whether selectins are also involved. The adhesive mechanisms underlying
IL-4-induced leukocyte recruitment have generally been derived from in
vitro experiments. Initial studies using human umbilical vein
endothelium have consistently reported that IL-4 induced expression of
VCAM-1 independent of E-selectin, P-selectin, or other adhesion
molecules, and this was sufficient to tether eosinophils and
mononuclear leukocytes via the 4
integrin/VCAM-1 pathway, albeit at the lower range of physiologic shear
(17, 18, 19, 20). Others have proposed that selectin pathways
could be up-regulated on IL-4-stimulated HUVEC, which were then
required for initial tethering before VCAM-1-dependent leukocyte
adhesion (12, 21). Only one in vivo study to date has
demonstrated a role for the 4 integrin/VCAM-1
pathway in IL-4-induced leukocyte recruitment (9). Sanz et
al. used elicited, indium-labeled eosinophils to demonstrate that these
cells infiltrated IL-4-stimulated skin and 60% of this recruitment was
inhibited with an 4 integrin Ab. A number of
key questions have been raised by this study. First, the importance (if
any) of selectins in IL-4-induced leukocyte recruitment needs
investigation, and second, the mechanism by which
4 integrin/VCAM-1 pathway contributes to
IL-4-induced tethering/rolling, adhesion, or all mechanisms of
leukocyte recruitment also requires attention.
In this study, we made use of intravital microscopy that permits direct
visualization of the leukocyte recruitment cascade, to systematically
elucidate the mechanisms of IL-4-induced leukocyte recruitment in the
microcirculation. The results reveal that in wild-type mice, baseline
levels of P-selectin supported 
95–98% of all rolling cells
following IL-4 treatment. However, chronic immunoneutralization of
P-selectin with Ab or P-selectin gene deletion (or even
P-selectin/E-selectin gene deletion) had no impact on the ultimate
leukocyte accumulation in tissue. The 4
integrin was entirely responsible for the few tethering, rolling, and
adhering cells in E/P-selectin-deficient mice that underlie the
eosinophil and mononuclear cell recruitment in IL-4-treated tissue.
These findings also demonstrate that in peripheral microvessels IL-4
treatment allows the 4 integrin to mediate the
entire leukocyte-endothelial cell cascade under physiological shear
conditions in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male C57BL/6 wild-type mice and P-selectin-deficient mice on the C57BL/6 background were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice deficient in both P-selectin and E-selectin (P-sel/E-sel-/-) were generously provided by Dr. Dan Bullard (University of Alabama, Birmingham). These mice were generated on a mixed background of 129/Sv x C57BL/6 (22), and have been backcrossed onto a C57BL/6 background for six generations. All mice were used between 6–10 wk of age.
Abs and cytokines
The Abs used in this study were: RB40.34, an anti-murine
P-selectin mAb (PharMingen, San Diego, CA); R1-2, an anti-murine
4 integrin mAb (PharMingen); 9C10, a mAb that
recognizes the murine 4 integrin, but does not
block function (PharMingen); MK-1.9.1, a rat IgG1 against murine VCAM-1
(Bayer Laboratories, West Haven, CT); 9A9, an anti-murine
E-selectin Ab (generously provided by Dr. Barry Wolitzky, Hoffman
LaRoche Pharmaceuticals, Nutley, NJ); and P-23, a murine IgG1 against
human P-selectin (Pharmacia & Upjohn, Kalamazoo, MI). Murine IL-4 was
purchased from R&D Systems (Minneapolis, MN).
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 Canada, Don Mills,
Ontario, Canada) with a x25 objective lens (Wetzlar L25/0.35; E.
Leitz, Munich, Germany) and a x10 eyepiece was 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), and was
determined by averaging the data derived from four to five fields
adjacent to postcapillary venules. Leukocyte tethering was quantitated
as the number of new leukocyte-endothelium interactions initiated over
a 1-min period within a 100 µm length of 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)
(24).
Transmission electron microscopy
Cremaster specimens for transmission electron microscopy (TEM) were rapidly cut into 2-mm3 blocks and fixed by immersion in freshly prepared 4% glutaraldehyde in Millonig’s buffer (all materials for electron microscopy were supplied by Electron Microscopy Sciences, Cedar Lane Laboratories, Hornby, Ontario, Canada) (pH 7.25) overnight. Following washing in buffer and 1 h of postfixation in 1% osmium tetroxide in distilled water, the tissue was prestained in 2% uranyl acetate, then dehydrated through graded ethanol, embedded in Quetol resin, and polymerized at 60°C for 48 h. Areas of tissue containing extravascular leukocytes were identified by light microscopy in toluidine blue-stained 1-µm sections cut from randomly selected blocks. Thin sections (60–90 nm) of these areas were prepared, stained with 2% uranyl acetate and 0.35% mM lead citrate, and examined in a Hitachi H7000 electron microscope. Approximately 20 extravascular leukocytes were identified and classified per animal.
Quantitation of expression of VCAM-1, P-selectin, and E-selectin
Expression of the adhesion molecules VCAM-1, P-selectin, and E-selectin was quantified using a modified dual-radiolabeled Ab technique (25, 26). The Abs MK1.9.1 (against VCAM-1), RB40.34 (against P-selectin), 9A9 (against E-selectin), and P-23 (a murine IgG1 against human P-selectin) were labeled with either 125I (MK1.9.1, RB40.34, 9A9) or 131I (P-23) using the iodogen method, as previously described (25, 26). P-23 was used to detect nonspecific binding in the murine system. To measure VCAM-1, mice were injected with 10 µg 125I-labeled anti-VCAM-1 (MK1.9.1), 20 µg unlabeled anti-VCAM-1 (MK1.9.1), and a variable dose of 131I-labeled nonbinding Ab (P-23) calculated to achieve a total injected 131I activity of 400,000–600,000 cpm (total volume 200 µl). This Ab combination was chosen after pilot experiments, conducted over a range of doses of unlabeled MK1.9.1, showed that this protocol ensured receptor saturation under stimulated conditions.
To study P-selectin or E-selectin, animals were injected i.v. with a mixture of either 10 µg 125I-anti-labeled P-selectin (RB40.34) or 10 µg 125I-anti-labeled E-selectin (9A9), respectively, and a variable dose of 131I-labeled P-23, as above. The Abs were allowed to circulate for 5 min, then the animals were heparinized. A blood sample was obtained from a carotid artery catheter, then the mice were exsanguinated by blood withdrawal through the carotid artery catheter and simultaneous i.v. infusion with bicarbonate-buffered saline. The cremaster muscles were harvested and weighed, along with lungs, heart, and areas of skeletal muscle for control purposes. Both 131I and 125I activity were measured in plasma and tissue samples. VCAM-1 and P-selectin expression were calculated per gram of tissue, by subtracting the accumulated activity of the nonbinding Ab (131I-labeled P-23) from the accumulated activity of the binding Ab (125I-labeled MK1.9.1, 125I-labeled RB40.34, or 125I-labeled 9A9). Data for VCAM-1, P-selectin, and E-selectin expression were represented as the percentage of the injected dose of Ab per gram of tissue. We have previously demonstrated that this approach provides reliable quantitative values of adhesion molecule expression, that radiolabeled binding Ab can be displaced specifically with sufficient amounts of unlabeled Ab, and that in the case of P-selectin, values not different from zero are obtained in P-selectin-deficient mice (25, 26).
Experimental protocol
In initial experiments, a range of doses of IL-4 (1, 10, 100 ng) in 200 µl sterile saline was injected under the scrotal skin adjacent to the cremaster muscle. Twenty-four hours later, leukocyte accumulation in the cremaster muscle was assessed using intravital microscopy. Significant leukocyte accumulation was observed at 100 ng of IL-4, so all additional experiments were performed using this dose. For these and all subsequent IL-4 experiments, including those using gene-deficient mice, control animals were injected with the same volume of normal saline and examined 24 h later. To further characterize the effects of 100 ng IL-4, the expression of 1) VCAM-1, 2) P-selectin, and 3) E-selectin in the cremaster muscle was quantitated in three separate groups of mice 24 h after IL-4 treatment.
The aim of the next series of experiments was to examine the effects of IL-4 treatment on leukocyte trafficking. At 24-h IL-4 treatment, mice were prepared for intravital microscopy, and leukocyte rolling, adhesion, and emigration were examined in the cremasteric microvasculature. In addition, cremaster muscles from some of these mice were fixed for electron microscopy to identify the types of leukocytes recruited.
In subsequent experiments using intravital microscopy, IL-4-treated
mice were treated with Abs against the 4
integrin (R1-2, blocking Ab, 75 µg/mouse, i.v., or 9C10, nonblocking
Ab, 75 µg/mouse, i.v.) or P-selectin (RB40.34, 20 µg/mouse, i.v.),
or both, and the effects on leukocyte trafficking were recorded. To
characterize the role of P-selectin over the entire 24-h IL-4 treatment
period, additional mice were treated with two doses of 50 µg RB40.34
i.p., 0 and 12 h after IL-4 injection. Pilot experiments
determined that this treatment protocol blocked leukocyte rolling in
the unstimulated cremaster muscle (known to be P-selectin dependent)
over a 24-h period. Examination of postcapillary venules in the
exteriorized but otherwise untreated cremaster 12 h after RB40.34
administration revealed no rolling leukocytes. This Ab treatment
protocol did not affect circulating leukocyte counts. To further
characterize the role of P-selectin, a group of P-selectin-deficient
mice was also treated with IL-4 and examined 24 h later.
As inhibition or absence of P-selectin could not completely inhibit
IL-4-induced leukocyte rolling (only 95% effective) and subsequent
leukocyte recruitment, the aim of the final series of experiments was
to determine the ability of IL-4 treatment to induce leukocyte
recruitment in the complete absence of the endothelial selectins.
P-sel/E-sel-/- mice were treated with 100 ng
IL-4 and examined using intravital microscopy 24 h later. These
mice were treated with an Ab against the 4
integrin (R1-2, 75 µg/mouse, i.v.) during the microscopic
observation.
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. Normally distributed data were analyzed using Student’s t test, or when nonparametric analysis was appropriate, the Mann-Whitney test was used. 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 intitial experiments, we used intravital microscopy to examine
leukocyte trafficking in the IL-4-treated cremaster muscle. To
determine a dose of IL-4 that was effective at inducing leukocyte
recruitment, wild-type mice were treated with 1, 10, and 100 ng of
IL-4. At 100 ng, but not 1 or 10 ng, IL-4 induced a significant
increase in leukocyte adhesion (
15 cells/100 µm) (Fig. 1
). Furthermore, IL-4 at 100 ng induced a
profound accumulation of leukocytes in the extravascular tissue (50
cells/microscopic field) (Fig. 1). The numbers of both adherent and
extravascular leukocytes observed following IL-4 treatment were
comparable with that observed in mouse cremasteric venules in an
allergen model of inflammation 24 h after challenge (P. Kubes,
unpublished observations), indicating that IL-4 on its own was
extremely effective at inducing leukocyte recruitment. All additional
experiments were performed at this dose. IL-4 caused a reduction in
leukocyte rolling velocity of greater than 50% (65 µm/s in saline
treated vs 30 µm/s in IL-4 treated), but leukocyte rolling flux
was not affected (Fig. 2).
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). These data showed that 50% of the
cells recruited were eosinophils (Fig. 3), with the remaining cells consisting
of monocytes/macrophages and lymphocytes. Fig. 3 is an electron
micrograph demonstrating the level of resolution used to assess the
profile of leukocyte recruitment in IL-4-treated mice. Neutrophils were
not seen in IL-4-treated tissues. In addition, within postcapillary
venules, 60–70% of the leukocytes adherent on the vascular wall were
eosinophils.
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Analysis of the effect of IL-4 on expression of VCAM-1 in the
cremaster muscle revealed that after 24 h of IL-4 treatment,
VCAM-1 expression was significantly increased (Fig. 4). In contrast, P-selectin expression
did not change from constitutive levels. E-selectin expression was
negligible in both untreated and IL-4-treated mice. These data indicate
that in this model, leukocyte recruitment was occurring within a
microvasculature expressing high levels of VCAM-1, only constitutive
levels of P-selectin, and minimal levels of E-selectin. Previous work
from our laboratory has demonstrated that the constitutive levels of
P-selectin are indeed significantly higher than the degree of
P-selectin Ab binding in P-selectin-deficient mice. It is also worth
noting that the nonbinding Ab (P-23), infused with the binding Ab in
each experiment, accounts for alterations in blood flow or
microvascular permeability that may occur with IL-4 administration.
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4 integrin following
IL-4 treatment
We next determined the adhesion molecules responsible for the
leukocyte recruitment induced by IL-4. As IL-4 induced a significant
increase in VCAM-1, we first examined the importance of the ligand of
VCAM-1, the 4 integrin. After 24 h of
IL-4 treatment, acute Ab blockade of the 4
integrin did not affect the number of rolling leukocytes (Fig. 5A). As results from in vitro
experiments suggest that the 4 integrin is
able to mediate leukocyte tethering and rolling more effectively at the
low end of physiological shear rates, we examined the effectiveness of
4 integrin blockade relative to shear rate
within postcapillary venules. However, despite the shear rates in the
venules examined ranging from 160–600 s-1,
blockade of the 4 integrin was not observed to
reduce leukocyte rolling flux significantly, regardless of shear rate
(Fig. 5B). In contrast, inhibition of the
4 integrin did cause a significant (43%)
increase in leukocyte rolling velocity (Fig. 5C).
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). In
every animal, some rolling cells were noted, an event not seen when
P-selectin Ab is administered under baseline conditions in which
rolling is completely inhibited (27, 28). Fig. 6
(lower panel, note change in scale) illustrates that
the residual leukocyte rolling was inhibitable with a function-blocking
Ab against the 4 integrin (R1-2), but not with
an Ab that binds the 4 integrin but does not
affect function (9C10). Neither the
anti-4-integrin Abs nor the
anti-P-selectin Ab affected circulating leukocyte counts (data not
shown).
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The almost complete absence of leukocyte rolling in mice treated
acutely with anti-P-selectin Ab suggested that P-selectin was
critical for the overall IL-4-induced leukocyte recruitment. Therefore,
to determine the importance of P-selectin over the 24-h course of IL-4,
we next treated wild-type mice with IL-4 and inhibited P-selectin
throughout the 24-h period via chronic Ab treatment. In these mice,
leukocyte rolling was only 2–3 cells/min 24 h after IL-4
administration (Fig. 7A),
reproducing the findings from acute P-selectin blockade (Fig. 6
).
Treatment with additional P-selectin Ab did not affect leukocyte
rolling, indicating that P-selectin remained completely
immunoneutralized at the end of the 24-h Ab protocol (data not shown).
Despite the almost complete blockade of leukocyte rolling, a
significant amount of leukocyte adhesion was observed in P-selectin
Ab-treated mice (Fig. 7B). Most importantly, the number of
leukocytes recruited to the extravascular tissue was similar to
IL-4-treated animals not given anti-P-selectin Ab; more than 50
cells/field had emigrated out of the vasculature (Fig. 7C).
The chronic P-selectin Ab experiments were entirely confirmed in
P-selectin-deficient mice (Fig. 7). Although rolling was dramatically
reduced in these mice and adhesion was also 50% reduced, the ultimate
emigration was not affected. In accordance with previous experiments,
saline-injected mice in which P-selectin was inhibited or absent showed
negligible (<1 cell every 4 min) leukocyte rolling and minimal levels
of adhesion and emigration.
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), as previously reported
(16). Nevertheless, the number of rolling cells in
IL-4-treated P-sel/E-sel-/- mice was 2–3
cells/min, i.e., not different from wild-type mice treated with IL-4
and P-selectin Ab. Most importantly, similar to results with chronic
anti-P-selectin treatment or in P-selectin-deficient mice,
leukocyte emigration in IL-4-treated
P-sel/E-sel-/- mice did not differ from
IL-4-treated wild-type mice (Fig. 8).
Electron-microscopic analysis of the emigrated leukocytes in
IL-4-treated P-sel/E-sel-/- mice revealed that
similar percentages of leukocyte types were present in the
extravascular tissue, when compared with wild-type mice (Table I).
Saline-treated P-sel/E-sel-/- mice had
negligible (<1 cell every 5 min) leukocyte rolling, and minimal
adhesion and emigration.
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4 integrin is sufficient for IL-4-induced
leukocyte rolling
In IL-4-treated mice, leukocyte rolling velocity was not different
when P-selectin Ab was added or when the experiments were performed in
P-sel/E-sel-/- mice (Fig. 9A). However, in both
wild-type mice treated chronically with P-selectin Ab, and
P-sel/E-sel-/- mice, this low level of
leukocyte rolling was completely dependent on the
4-integrin, as an Ab against this molecule
blocked rolling (Fig. 9B). In contrast, following P-selectin
blockade, treatment of wild-type mice with the selectin-binding
polysaccharide, fucoidan, did not inhibit the residual leukocyte
rolling (data not shown). This finding indicated that the third
selectin, L-selectin, was not important in mediating this rolling. This
approach has been used previously to exclude a role for L-selectin in
leukocyte recruitment in P-sel/E-sel-/- mice
(29). Together these data show that the
4 integrin was responsible for sustaining
leukocyte rolling, independent of P-, E-, and L-selectin.
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4 integrin was also
responsible for initiating leukocyte rolling in these mice. Leukocyte
tethering was observed in 16 of 28 venules examined, at an average rate
of 1 tether/min/100 µm. Tethering was observed in venules exhibiting
shear rates ranging from 156–611 s-1, and was
blocked by an Ab against the 4 integrin. These
findings suggest for the first time that the 4
integrin can initiate tethering in vivo independent of endothelial
selectins.
Analysis of hemodynamic parameters in postcapillary venules of
saline-treated mice and mice treated with 100 ng IL-4 showed that IL-4
treatment did not significantly alter the diameters of the venules
examined or the venular shear rate relative to saline-treated controls
(Table II). In addition, local IL-4 administration did not affect
systemic circulating leukocyte counts (Table II).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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68 of 70
rolling cells/min in cremasteric microvessels. However, these 68
rolling cells/min do not appear to be crucial to the recruitment
process, as chronic inhibition of P-selectin, or P-selectin gene
deletion did not impact upon the ultimate number of emigrated
leukocytes. It is clear that a P-selectin-independent mechanism of
leukocyte recruitment was responsible for the 2 of 70 rolling cells/min
that ultimately accounted for all of the leukocyte recruitment. Our
data also reveal that the few cells rolling were not dependent on
E-selectin, as the rolling persisted in the
P-sel/E-sel-/- mice, in accordance with our
observation that E-selectin expression was negligible following IL-4
treatment. Additionally, we ruled out L-selectin, as the addition of an
L-selectin inhibitor (fucoidan) to mice following P-selectin blockade
also did not impact on the residual 2 rolling cells induced by IL-4.
However, an 4 integrin Ab inhibited all of the
P-selectin-independent rolling. This demonstrates that following IL-4
treatment, the 4 integrin is able to induce
leukocyte rolling independently of the three selectins.
Although at first glance one could argue that leukocyte rolling was not
affected by IL-4, this is not the case. The 24-h exposure of the
microcirculation to IL-4 did not change the flux of rolling leukocytes,
but did reduce leukocyte rolling velocity by almost 50%. This is not a
trivial amount inasmuch as reducing the rolling velocity by half
doubles the transit time (time for a cell to traverse a given length of
vessel) and therefore doubles the number of cells within the
microvasculature interacting with venules at any given time. When the
4 integrin Ab was given to wild-type mice
treated with IL-4, a significant reduction in leukocyte rolling was not
observed, but this is not surprising and in fact consistent with only a
few cells rolling via the 4 integrin per
minute. What is noteworthy is that addition of the
4 integrin Ab increased leukocyte rolling
velocity by 50% back to near control values. Of course, this reduces
transit time of rolling cells and reduces the number of cells
interacting with venules within the microvasculature. Finally, it is
worthwhile noting that the number of leukocytes recruited to the
cremaster 24 h after Ag challenge is approximately the same as
that induced by IL-4 treatment, but in the Ag challenge model leukocyte
rolling velocity is not reduced (unpublished observations). Unlike in
the IL-4 experiments, blocking the endothelial selectins (for 8 h) in
the Ag model is sufficient to entirely inhibit leukocyte emigration
(16). Therefore, the different endothelial phenotype
induced by IL-4 treatment allows leukocytes to be recruited
independently of selectin function.
An unexpected finding based on previous work is that IL-4 treatment
allows for leukocytes to use the 4 integrin to
not only roll, but also to tether to endothelium in vivo. Tethering
(initial capture or attachment) and rolling are distinct adhesive
mechanisms inasmuch as rolling can occur in vitro at shear stress,
where tethering does not. For example, Alon et al. demonstrated that
VCAM-1 could support rolling via 4 integrin up
to very high shear stresses in vitro (7
dynes/cm2), but tethering would not occur at 2
dynes/cm2, which is a shear that supports
selectin-dependent tethering (11). These data suggest a
requirement for selectins as tethering molecules for
4 integrin-dependent rolling to occur.
Moreover, there is a lack of evidence that the
4 integrin can mediate tethering of leukocytes
in vivo. In a model of Ag-induced inflammation of the cremaster
microcirculation, intravital microscopy revealed that 50% of the
leukocyte rolling was 4 integrin dependent
(16). However, the rolling was entirely inhibited by an Ab
against P-selectin, demonstrating an absolute requirement for this
selectin. In the P-selectin-deficient mouse, the
4 integrin supported 100% of the rolling, but
again all rolling could be inhibited by an E-selectin Ab. From that
work, it was concluded that 4
integrin-mediated leukocyte/endothelial cell interactions required
selectins for tethering and/or rolling. In the present study, IL-4 was
able to induce 4 integrin-dependent leukocyte
tethering and rolling in the absence of selectin function over a broad
range of physiologic shear stresses in vivo. An obvious difference
between the IL-4 and Ag model is that in the latter, P-selectin
expression is greatly increased in response to Ag (27),
but VCAM-1 expression remains near control levels (our unpublished
results). This different profile of adhesion molecule expression
induced by each of these treatments provides a potential explanation
for the absence of a role for selectins in IL-4-induced inflammation,
but a prominent role in Ag-induced inflammation.
It is tempting to conclude that the increased VCAM-1 expression was
supporting the 4 integrin-dependent leukocyte
recruitment. However, there is some recent evidence that IL-4-induced
up-regulation of VCAM-1 does not necessarily correlate with a
functional role for this molecule in leukocyte recruitment. In a model
of IL-4-induced eosinophil accumulation, Larbi et al. (30)
compared the functional roles of the 4
integrin and VCAM-1 in the skin and pleural cavity. They observed that
while recruitment was dependent on the 4
integrin in both tissues, VCAM-1 blockade reduced recruitment in the
skin, but not in the pleural cavity. These observations suggest that
despite elevated expression of VCAM-1, in some tissues the
4 integrin is able to use a ligand other than
VCAM-1 to mediate leukocyte recruitment. The role of VCAM-1 in the
IL-4-stimulated murine cremaster remains to be fully characterized;
however, our preliminary results are equivocal. Whereas VCAM-1
antisense oligonucleotides blocked 50% of the IL-4-induced leukocyte
recruitment, an anti-VCAM-1 mAb (M/K-2), which binds to a single
domain of VCAM-1, had no effect on the 4
integrin-dependent rolling (M. Hickey and P. Kubes, unpublished
observations). The latter observation may be due to the ability of the
4 integrin to bind to multiple domains on
VCAM-1 (19).
Our data also suggest that P-selectin-mediated leukocyte rolling
following IL-4 treatment is of limited importance to subsequent
leukocyte recruitment. Although the great majority of leukocytes rolled
in IL-4-treated postcapillary venules via P-selectin, leukocyte
recruitment was unaffected in mice treated with P-selectin Ab,
P-selectin-deficient mice, or P-sel/E-sel-/-
mice. This raises the possibility that the leukocytes that use
P-selectin for rolling are not destined to undergo firm adhesion and
emigration in an IL-4-treated microvasculature and preventing rolling
of these cells does not impact upon the ultimate emigration. The data
also suggest that the few remaining leukocytes interacting with the
endothelium by tethering and rolling via 4
integrin are the population that are recruited to this tissue. Although
it is impossible to determine the type of leukocyte rolling (using
intravital microscopy), we previously performed in vitro experiments
using whole blood that revealed that 90% of cells tethered by
P-selectin were neutrophils, whereas VCAM-1 selectively tethered
mononuclear leukocytes and eosinophils (31). It is
therefore conceivable that with the presence of both P-selectin and
VCAM-1, all leukocytes are tethered to the venular wall and downstream
activation events select for rolling eosinophils and mononuclear
leukocytes, but not rolling neutrophils. As 1–3% of all circulating
leukocytes are eosinophils, but 50% of the emigrated leukocytes
were eosinophils, the results lend credence to a very highly selective
process of recruitment of eosinophils, but not neutrophils at the
endothelial cell surface. Specific chemokines induced by IL-4 such as
eotaxin (32, 33) are likely to underlie the very selective
recruitment process induced by IL-4.
A final issue that needs to be raised is the less than uniform data regarding IL-4-induced VCAM-1 expression in vitro. Initial work using human umbilical vein endothelium has consistently reported that IL-4 induced expression of VCAM-1 independently of P-selectin, E-selectin, or other adhesion molecules (17, 18, 19). Use of microvascular endothelium from dermal (34) or intestinal origin revealed no up-regulation of VCAM-1 following IL-4 treatment (35), but VCAM-1 expression was observed on nasal polyp-derived microvascular endothelium in response to IL-4 (36). Cultured microvascular endothelium requires passaging to increase numbers of cells, but passaging of cells may result in the loss of P-selectin (37) and perhaps inappropriate alteration of other adhesion molecules including VCAM-1. Other investigators have argued that primary cultures of HUVEC may not always reflect expression of adhesion molecules on microvascular endothelium. In the present study using the dual radiolabeling technique, wherein the nonbinding Ab accounts for such factors as 1) nonspecific binding, 2) microvascular permeability changes, and 3) alterations in blood flow, we reveal that VCAM-1 does indeed increase in microvessels in vivo in response to IL-4 (wherein no culture artifact was introduced) and that P-selectin and E-selectin remain at constitutive levels. The constitutive levels of P-selectin are small but functional as these P-selectin values are significantly higher than those generated in P-selectin-deficient animals, which reflect complete absence of P-selectin (25). In addition, the P-selectin is present regardless of whether surgery is performed on the cremaster muscle (27), suggesting constitutive expression of P-selectin and constitutive rolling in this microvascular bed.
In summary, to date in vitro work has revealed an important role for
4 integrin-dependent leukocyte rolling and
adhesion and at lower shear forces for the initial tethering process.
However, in vitro experiments may differ from the in vivo setting in
several critical aspects. First, it is unknown whether the site
densities of VCAM-1 protein used, either in purified form or expressed
by tranfected cells, correspond to the amount of protein expressed by
microvascular endothelial cells under inflammatory conditions in vivo.
Second, whether the shear stresses at which 4
integrin-dependent leukocyte tethering has been shown to occur in vitro
overlap with the lower end of the physiological range or differ from
those present in vivo in postcapillary venules also remains unclear.
Our data have for the first time demonstrated that the cytokine IL-4
does indeed alter the endothelial phenotype in microvessels in vivo, to
a sufficient extent to induce 4
integrin-dependent leukocyte recruitment across the physiologic range
of shear forces in vivo to allow for significant leukocyte recruitment
independent of selectin function.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Paul Kubes, Health Sciences Centre, University of Calgary, 3330 Hospital Drive N.W., Calgary, AB, T2N 4N1, Canada. E-mail address:
Received for publication February 11, 1999. Accepted for publication July 7, 1999.
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, n = 7) or mice treated with IL-4 at a
range of doses (24 h, 
). *, p < 0.05 relative
to saline-treated mice.
).
