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*
Immunology Research Group, University of Calgary, Alberta, Canada;
Department of Physiology and Biophysics, Louisiana State Medical Center, Shreveport, LA 71130; and
Preclinical Research and Development, Genetics Institute, Cambridge, MA 02140
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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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In vitro, there is ample evidence that the same endothelium stimulated
with different stimuli will induce different patterns of endothelial
selectin expression. A very good example to date is TNF-
, which
causes profound expression of E-selectin but not P-selectin over the
first 24 h in human umbilical vein endothelium (7), whereas IL-4
induces expression of VCAM-1 and perhaps P-selectin but not E-selectin
(8, 9) over this same time frame in the same cells. Heterogeneity in
the induction of the adhesion molecules can also be seen in different
endothelial cells. Activation with protein kinase C induced significant
E-selectin expression in human umbilical vein endothelium but not in
dermal microvascular endothelium (10). Adhesion molecule expression
also differs distinctly between stimulated human umbilical vein
endothelium and intestinal microvascular endothelium (11). There is
less evidence in vivo that distinct patterns of selectin expression
exist in different vascular beds. Eppihimer et al. reported increases
in both P-selectin and E-selectin expression in all organs in response
to LPS (12). Ley and colleagues have demonstrated that TNF- induces
leukocyte rolling dependent upon both P-selectin and E-selectin in the
cremaster muscle (13, 14). Clearly, administration of exogenous LPS and
TNF- appear to induce expression of both endothelial selectins in
numerous tissues, and, at least in the cremaster microvasculature, they
both contribute to leukocyte rolling.
Although these studies are seminal in demonstrating the importance of
both selectins, exogenous application of TNF- and LPS almost
certainly do not reflect all responses in all tissues. In this study,
we examined the endogenous immune response to foreign Ag in both the
cremaster muscle and the skin microvasculature. To achieve this aim, we
used a combination of two approaches to quantitate responses to Ag: 1)
we quantitatively measured selectin expression using a radiolabeled-mAb
technique, and 2) we examined leukocyte recruitment using intravital
microscopy in skin and skeletal muscle of sensitized mice. We report
diametrically opposing responses in the expression of the two
endothelial selectins in the two microvascular beds in response to Ag
and demonstrate that these differences underlie striking differences in
the basic mechanisms of leukocyte recruitment within a particular
vascular bed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All animals used in this study were male C57BL/6 mice weighing between 20–35 g and were used between 6 and 10 wk of age.
Sensitization and challenge protocol
To develop a model of acute, allergic inflammation, a type I hypersensitivity reaction was elicited by systemically (i.p. injection) sensitizing animals with 10 µg chicken OVA (Sigma, St. Louis, MO) and 10 mg Grade V aluminum hydroxide (AlOH; Sigma) in a total volume of 0.2 ml saline. Two weeks later, animals were locally challenged, either intradermally or intrascrotally, with the sensitizing Ag. Intrascrotal injection localizes the Ag solution beneath the scrotal skin directly adjacent to the cremaster muscle and results in the Ag solution directly bathing the cremaster tissue. In the skin, challenge with 10 µg OVA had no effect on leukocyte rolling flux; therefore, another series of animals were challenged with 100 µg OVA. Sham sensitization and sham challenge involved systemic and local injection of 0.2 ml saline, respectively. The animals were prepared for either fluorescent or light intravital microscopy to visualize the dermal (skin flap) or skeletal (cremaster) muscle microcirculations, respectively. Leukocyte kinetics were quantified in untreated mice and at 4 h post-Ag challenge in the skin flap and cremaster muscle.
Intravital microscopy
Animals were anesthetized by i.p. injection of a mixture of 10 mg/kg xylazine (MTC Pharmaceuticals, Cambridge, Ontario, Canada) and 200 mg/kg ketamine hydrochloride (Rogar/STB, London, Ontario, Canada). The left jugular vein was cannulated to administer anesthetic, fluorescent dyes, and various drugs. Animals were then prepared as follows to view either the dermal (skin flap) or skeletal muscle microcirculation.
Skin flap preparation. A midline abdominal incision was made beginning at the level of the diaphragm and extending to the pelvic region. The skin was carefully separated from the underlying tissue, remaining attached laterally. Blood supply to the skin flap remained intact. The skin flap was then extended over a viewing pedestal, secured along the edges using 4-0 suture exposing the dermal microvasculature. The exposed skin was continuously superfused with bicarbonate-buffered saline to avoid tissue dehydration. Due to the thickness of the skin flap, leukocyte-endothelial cell interactions were not visible by transillumination. Therefore, for this protocol, animals were injected with the fluorescent dye, rhodamine 6G (0.3 mg/kg i.v.; Sigma), immediately before microscopic visualization. Rhodamine 6G at the dose used labels leukocytes and platelets and has been shown to allow detection of the same number of rolling leukocytes as transmitted light and have no effect on leukocyte kinetics (15, 16). Therefore, it allows for quantification of leukocyte rolling flux, leukocyte rolling velocity, and leukocyte adhesion via epifluorescence microscopy. Rhodamine 6G-associated fluorescence was visualized by epi-illumination at 510–560 nm using a 590-nm emission filter (16, 17). Although window chambers have been used to study the dermal microcirculation, this is the first documentation of this acute method of examination of the skin microcirculation.
Cremaster muscle preparation. An incision was made in the scrotal skin to expose the left cremaster muscle, which was then carefully removed from the associated fascia. A lengthwise incision was made on the ventral surface of the cremaster muscle. The testicle and epididymis were separated from the underlying muscle and reintroduced into the abdominal cavity. The muscle was then spread out over an optically clear viewing pedestal and secured along the edges with 3-0 suture. The exposed tissue was superfused with warm bicarbonate-buffered saline (pH 7.4).
The dermal and cremasteric microcirculations were observed through an intravital microscope (Nikon-Optiphot-2, Japan) with a 40x water immersion lens (skin flap; 40/0.55 WI; Nikon, Tokyo, Japan) or a 25x objective lens (skeletal muscle; Leitz Wetzlar L25/0.35) and a 10x eyepiece. The image of the dermal microcirculatory bed was recorded using a silicon-intensified fluorescent camera (model C-2400-08; Hamamatsu Photonics, Hamamatsu City, Japan), and the cremasteric microcirculation was recorded using a video camera (Panasonic-Digital 5100, Panasonic, Secaucus, NJ). Images of the dermal and skeletal muscle microcirculation were recorded before and after administration of various Abs in untreated mice and 4 h after Ag challenge. All experimental parameters were quantified at these time points.
Single unbranched venules (20–40 µm in diameter) were selected for
each study. Venular diameter (Dv) was measured using a
video caliper (Microcirculation Research Institute, Texas A&M
University, College Station, TX). Rolling leukocytes were defined as
those leukocytes that rolled at a velocity slower than that of RBC.
Leukocyte rolling velocity was measured for the first 20 leukocytes
entering the field of view at the time of recording and was determined
as the time required for a leukocyte to traverse a given length of
venule. Leukocyte adhesion was quantified as the number of leukocytes
that adhered to the vessel wall for 30 s or more within a given
segment of the vessel. RBC velocity (Vrbc) was measured
online using an optical Doppler velocimeter (Microcirculation Research
Institute) and was only measured for the skeletal muscle preparation,
as determination of RBC velocity using fluorescence in the skin flap
was not possible. Venular blood flow in the skeletal muscle was
calculated from the product of cross-sectional area and mean RBC
velocity (Vmean = Vrbc/1.6), assuming
cylindrical geometry. Venular wall shear rate (
) was calculated
based on the Newtonian definition, =
8(Vmean/Dv), and venular wall shear stress was
x blood viscosity, where blood viscosity was assumed to be 0.025
poise (18).
Experimental protocol
In the first series of experiments leukocyte kinetics were established in the dermal microcirculation of untreated mice. In a separate series of mice, an Ag-induced late-phase response was elicited in the skin, and leukocyte rolling, leukocyte rolling velocity, and leukocyte adhesion in the challenged area were examined upon exteriorization 4 h later. A role for selectins in leukocyte recruitment in the skin flap was studied in both untreated skin and following Ag-challenge. After an initial recording, animals were treated i.v. with an anti-P-selectin Ab (RB40.34; PharMingen, San Diego, CA; 20 µg/animal), an anti-E-selectin Ab (9A9; 100 µg/animal, generously provided by Dr. Barry Wolitzky, Hoffmann-La Roche Pharmaceuticals, Nutley, NJ), or both Abs simultaneously. Leukocyte kinetics were assessed before and after administration of these Abs. An identical protocol was followed for the mouse cremasteric microvasculature. Analysis of blood samples from animals treated with the Abs showed that Ab administration did not affect circulating leukocyte counts.
Passive cutaneous anaphylaxis (PCA)3 reaction
Serum was obtained from all OVA-sensitized animals at the end of the experiment by intracardiac puncture. Serial dilutions (1/8–1/64) of the serum samples were prepared, and 200 µL of each sample was injected intradermally into the shaved backs of control, untreated mice and Sprague-Dawley rats. Serum from sensitized mice elicited the same response in Sprague-Dawley rats as it did in C57BL/6 mice. Therefore, rats were used for all subsequent PCA reactions primarily because PCA was easier to detect. After 72 h, animals were challenged with an intracardiac injection of a solution containing 2.5 mg Evan’s Blue dye and 5 mg chicken OVA in a total volume of 1.5 ml (saline). The final reaction was read 60 min later as the highest dilution that produced a distinct blue region (Evan’s Blue dye extravasation) at the center of the injection site (19). Sensitized animals had serum anti-OVA Ab titers of at least 1/64, whereas sham-sensitized animals had no anti-OVA Abs.
P- and E-selectin expression in the skin and skeletal muscle
In an additional series of experiments, expression of P-selectin and E-selectin was quantified in the skin and skeletal muscle of untreated mice and at 4 h post-Ag challenge. This was accomplished using a modified dual-radiolabeled Ab technique (12). Briefly, animals were injected i.v. with a mixture of either 10 µg 125I-anti-P-selectin (RB40.34) or 10 µg 125I-anti-E-selectin (10E6, generously provided by Dr. Barry Wolitzky, Hoffmann-La Roche Pharmaceuticals) Abs, 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). 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. Whole organs (cremaster muscle and dorsal skin flap) were harvested and weighed. Both 131I and 125I activity were measured in plasma and tissue samples. Both P- and E-selectin expression were calculated per gram of tissue by subtracting the accumulated activity of the nonbinding Ab (131I P-23) from the accumulated activity of the binding Ab (125I RB40.34 or 125I 10E6). Data for P- or E-selectin expression was represented as the percent 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 values not different from zero are obtained for P-selectin in P-selectin-deficient mice and for E-selectin in E-selectin-deficient mice (12).
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) in untreated and sensitized mice.
Statistical analysis
Data are presented as mean ± SEM. A student’s t test with bonferroni correction was used for multiple comparisons. Statistical significance was set at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All Ag-sensitized animals in this study had anti-OVA Ab titers
of at least 1/64, as assessed by PCA reaction. Fig. 1
illustrates the kinetics of leukocyte
recruitment in the dermal microvasculature of untreated and
Ag-sensitized mice at 4 h post-Ag challenge. In untreated animals,
there were 
30 rolling cells per min in dermal postcapillary venules
(Fig. 1A). Intradermal OVA challenge (10 µg) in sensitized
animals caused no change in leukocyte rolling flux. To determine
whether a higher concentration of Ag would induce an increase in
rolling flux, another series of animals was challenged with 100 µg of
OVA. This concentration also had no effect on the leukocyte rolling
flux (Fig. 1A), suggesting either that Ag challenge in the
skin does not increase rolling flux or that insufficient stimulus was
being used. The latter seems unlikely as Ag challenge at both doses
induced dramatic changes in leukocyte rolling velocity (Fig. 1B). In untreated animals, leukocytes rolled at a velocity
of 35 µm/s. Intradermal Ag challenge with 10 µg OVA dramatically
reduced (60–70%) leukocyte rolling velocity
(p < 0.05). A 10-fold higher dose of Ag did
not further reduce leukocyte rolling velocity. Leukocyte adhesion in
dermal postcapillary venules was significantly increased 4 h
post-Ag challenge (10 µg) relative to untreated animals (Fig. 1C). As 10 µg induced a higher number of adherent cells
than the 100 µg concentration, 10 µg was used for all additional
experiments including those in muscle. Accumulation of platelets within
the challenged dermal microvasculature was undetectable by intravital
microscopy. Comparison of circulating leukocyte counts in control mice
(6.3 ± 2.8 x 106/ml) vs sensitized mice 4
h after challenge (9.5 ± 3.4 x 106/ml) suggests
that Ag challenge may have induced release of leukocytes from the
noncirculating pool.
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Fig. 2 demonstrates that there is
some P-selectin expression in skin under basal conditions, and that
this did not increase following Ag stimulation. In direct contrast,
E-selectin expression 4 h after Ag challenge increased 10-fold
(Fig. 2). Blockade of E-selectin alone had no effect on the leukocyte
rolling flux at 4 h post-Ag challenge in skin (Fig. 3A). The effect of P-selectin
blockade was also unremarkable and reduced leukocyte rolling flux by
only 30% in dermal postcapillary venules (Fig. 3A).
However, tandem inhibition of both E- and P-selectin function reduced
leukocyte rolling flux in the skin by 80% at 4 h post-Ag
challenge. Interestingly, a small amount of rolling persisted in the
skin even in the presence of E-selectin and P-selectin Abs, suggesting
an additional rolling pathway in the Ag-challenged dermal
microvasculature.
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B); only a 1.5- to 2.0-fold increase in rolling velocity
was noted (NS). Blockade of both endothelial selectins caused a
striking sevenfold increase in leukocyte rolling velocity, suggesting
that E-selectin and P-selectin were working in tandem to induce the
dramatic reduction in rolling velocity in Ag-challenged dermal
microvessels. Ag-induced leukocyte rolling profile in cremaster microvasculature differs from skin
The kinetics of leukocyte recruitment in the cremaster
microvasculature of untreated and Ag-sensitized mice at 4 h
post-Ag challenge are shown in Fig. 4. In
untreated animals, there were 50 cells/min rolling in muscle
postcapillary venules. Local challenge with chicken egg OVA (10 µg)
in sensitized mice caused a large increase (200 cells/min) in leukocyte
rolling flux (Fig. 4A). This is in direct contrast to the
small flux of rolling cells (<40 cells/min) in Ag-challenged dermal
postcapillary venules. However, unlike the response in dermis,
leukocyte rolling velocity was not significantly altered in the
cremasteric microvasculature (Fig. 4B). Leukocyte adhesion
was increased significantly in this microvasculature 4 h post-Ag
challenge (Fig. 4C). Similar to the response in dermal
vessels, no platelet accumulation was discernible in the cremasteric
microvasculature following challenge.
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Similar to the skin microvasculature, there were constitutive
levels of P-selectin expression but very low amounts of E-selectin
expression in muscle (Fig. 5). In
striking contrast to skin, the muscle microvasculature responded
to Ag challenge by significantly increasing P-selectin expression
without an increase in E-selectin expression (Fig. 5). Consistent with
this observation were the functional data that blockade of P-selectin
function eliminated leukocyte rolling (Fig. 6). Because an anti-P-selectin Ab
alone completely inhibited leukocyte rolling in the cremaster, the
combination of E-selectin and P-selectin was not performed. E-selectin
blockade did not affect leukocyte rolling flux (Fig. 6) and leukocyte
rolling velocity remained unchanged (data not shown). Blockade of
P-selectin and/or E-selectin had no effect on the existing leukocyte
adhesion in either tissue (data not shown) consistent with the view
that the selectins mediate rolling but not adhesion.
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Leukocyte kinetics in sham-sensitized and sham-challenged animals
were identical to untreated animals in both tissues (data not shown).
Inhibition of P-selectin function in untreated mice eliminated
leukocyte rolling flux in both skin and muscle (Fig. 7). This is consistent with previous work
showing that constitutive values for P-selectin expression in various
tissues of wild-type mice were greater than P-selectin values obtained
from P-selectin-deficient mice in the absence of stimulus (12). These
data suggest that this low level of constitutive P-selectin expression
is capable of mediating leukocyte rolling. In contrast, E-selectin
blockade imparted no effect on any of the leukocyte parameters (data
not shown). Again, this supports earlier studies in which E-selectin
expression values in unstimulated wild-type mice were shown to be
approximately the same as those in E-selectin-deficient mice,
suggesting no constitutive expression of this selectin (12).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Putting together the quantitative expression data and functional rolling data reveals very important information. First, in untreated mice there is minimal constitutive expression of E-selectin in skin and cremaster, and E-selectin does not appear to support any basal leukocyte rolling in either tissue. This is consistent with the work of Eppihimer et al. (12) that E-selectin expression in heart, intestine, and lungs was not different between wild-type and E-selectin-deficient animals in the absence of stimulus. On the other hand, this study reveals that there is constitutive expression of P-selectin in both skin and cremaster muscle and this accounts for all of the rolling in untreated animals. We have previously seen P-selectin expression in unperturbed lung, mesentery, and intestine, but not heart or brain, suggesting that some tissues, including in this study skin and cremaster muscle, can constitutively express sufficient P-selectin to support functional rolling. The presence of constitutive P-selectin-dependent rolling has been documented in the dorsal skin chamber and dermal venules of mice, further supporting this view (15).
Following Ag challenge, the ratio of P-selectin to E-selectin in skin
approaches 1:1, whereas the ratio in muscle was greater than 40:1.
Functionally, in muscle we observed a dramatic increase in the number
of rolling cells without a change in rolling velocity, whereas in the
skin there was no increase in the number of rolling cells but a very
significant reduction in rolling velocity. At first glance, it is
tempting to conclude that in these systems increases in P-selectin
expression elevate the flux of rolling cells, whereas an increase in
E-selectin expression is associated with a reduction in rolling
velocity. Indeed, there is some strong evidence to support the latter:
Kunkel and Ley (14) demonstrated that slow rolling induced by TNF-
was evident in L-selectin-deficient and P-selectin-deficient, but not
E-selectin-deficient, mice. These data support the view that E-selectin
is an absolute requirement for the slow rolling observed with TNF-.
Although our data are consistent with a need for E-selectin for slow
rolling, they do not support an exclusive role for E-selectin as a
mediator of slow rolling. In the skin, blockade of E-selectin or
P-selectin alone was not sufficient to significantly affect the
extremely slow rolling velocity, whereas combined administration of the
Abs increased rolling velocity almost sevenfold.
It is intriguing that a very significant increase in P-selectin up-regulation could occur independent of a change in rolling velocity and that this increase was sufficient to recruit more rolling cells. Clearly, this is an example of dissociation between an increase in adhesion molecule expression and an decrease in rolling velocity and belies in vitro work that demonstrates that increasing the concentration of P-selectin protein incorporated into lipid bilayers decreases rolling velocity (2). It is possible that nonrandom selectin expression on microvascular endothelium in vivo, such as clustering or dimerization, may not be entirely replicated by adhesion molecule distribution on glass coverslips or in transfected cell systems. These data also differ from the rat mesentery, where within minutes of exogenous application of leukotriene C4 (LTC4) both increased leukocyte rolling and a dramatic reduction in rolling velocity were apparent, and both were dependent upon P-selectin (20). However, in that model, nonselectin molecules also contributed to the slow rolling with LTC4. Both inhibitors of platelet-activating factor (PAF) and the ß2 integrin increased rolling velocity, suggesting that P-selectin alone was not sufficient to decrease rolling velocity. Therefore, the data presented in this study highlight the complexity of factors that underlie leukocyte rolling velocity in vivo and also suggest that increased expression of a single selectin does not necessarily reduce the speed of a rolling leukocyte. However, the presence of two selectins at least in the skin was sufficient to reduce rolling velocity in a synergistic manner.
We believe that rolling velocity is an extremely important parameter inasmuch as slow rolling leukocytes respond to chemotactic stimuli with at least 10-fold greater sensitivity than fast rolling cells (20). To illustrate this point, we have previously shown in the rat mesentery that the velocity of leukocytes induced to roll by LTC4 is 50% lower than those induced by histamine. Subsequently, the histamine-induced fast rolling cells were able to undergo firm adhesion in response to 10 nM PAF but not to 1 nM PAF. In contrast, LTC4-induced slow rolling cells were able to respond to PAF at the lower concentration and adhere (20). In this study, a similar concept is presented: in the cremaster microvasculature the milieu is such that many fast rolling cells induce a small proportion of cells to adhere, whereas in the skin a small number of slow rolling cells is sufficient to also increase cell adhesion.
Patel et al. (21) have shown that the length of the P-selectin molecule is critical in its ability to induce leukocyte rolling. Shortening of P-selectin by deletion of several of the consensus repeat regions caused P-selectin to become unable to tether free-flowing leukocytes and initiate rolling. This raises some interesting questions about whether a cell would roll exclusively upon the longer P-selectin and not interact with the much shorter E-selectin molecule if both molecules are present. Indeed, Luscinskas et al. (22, 23) demonstrated in vitro that when both P-selectin and E-selectin were present, monocytes and CD4+ T cells preferred P-selectin, whereas if only E-selectin was available then leukocytes would roll on this molecule. Our results suggest in a system wherein the expression of both P-selectin and E-selectin exists, as is the case in skin, cells use both selectins. Therefore, the longer P-selectin does not preclude leukocyte interactions with E-selectin. These data are consistent with the work of other investigators who have demonstrated an overlapping role for both P-selectin and E-selectin in allergy-induced leukocyte recruitment in the skin (24, 25, 26). However, our own work extends those observations to suggest that not all tissues use both endothelial selectins to recruit leukocytes. Second, the use of intravital microscopy of skin and muscle revealed that the E-selectin and P-selectin expression ensemble may dictate a pattern of leukocyte recruitment not seen with P-selectin alone.
Although the elucidation of the mechanism underlying differences in
endothelial selectin expression between tissues was not within the
scope of this study, at least two explanations are possible. First, the
endothelium from cremaster muscle may not have the machinery to express
E-selectin, whereas the skin microvasculature can express both
P-selectin and E-selectin. This is very unlikely in light of work by
Ley and colleagues that demonstrates that TNF- can induce
E-selectin expression in the cremaster muscle (14, 27). It is also
unlikely that 4 h was not sufficient for E-selectin synthesis
inasmuch as TNF- could induce much E-selectin expression in the
cremaster vasculature within this time frame. Although one could argue
that the stimulus was too weak to induce E-selectin in the cremaster,
the same stimulus caused strong expression of E-selectin in the skin
and was sufficient to activate cremasteric endothelium to express
P-selectin. Finally, the same amount of Ag was able to induce
E-selectin expression in the cremaster muscle of P-selectin-deficient
mice, suggesting that sufficient amounts of Ag were used but in the
presence of P-selectin the system did not express E-selectin (5). The
second more likely explanation is that the immune response is
differentially regulated by the local environment in each tissue, and
that this dictates whether or not E-selectin is expressed. Although
there is no direct evidence to support this view, Galli and coworkers
have shown that leukocyte recruitment during cutaneous IgE-dependent
late-phase responses is mast cell-dependent (28). Furthermore, in human
skin grafted onto SCID mice, mast cell degranulation can induce
E-selectin-dependent leukocyte recruitment (29). These observations are
noteworthy as our preliminary data show no impairment of leukocyte
recruitment in mast cell-deficient mice in the cremaster muscle, where
all of the leukocyte recruitment is dependent upon P-selectin (P.K.,
unpublished observations). Whether these extravascular immunocytes
dictate the adhesion molecule profile in different microvascular beds
during the immune response to Ag is an important future consideration.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Paul Kubes, Immunology Research Group, University of Calgary, Health Sciences Centre, 3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1. E-mail address:
3 Abbreviations used in this paper: PCA, passive cutaneous anaphylaxis; LTC4, leukotriene C4; PAF, platelet-activating factor.
Received for publication June 5, 1998. Accepted for publication October 7, 1998.
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-activated vascular endothelium under flow. J. Exp. Med. 181:1179.This article has been cited by other articles:
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S. Cuzzocrea, G. Nocentini, R. Di Paola, E. Mazzon, S. Ronchetti, T. Genovese, C. Muia, A. P. Caputi, and C. Riccardi Glucocorticoid-induced TNF receptor family gene (GITR) knockout mice exhibit a resistance to splanchnic artery occlusion (SAO) shock J. Leukoc. Biol., November 1, 2004; 76(5): 933 - 940. [Abstract] [Full Text] [PDF]
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 T. Stokol, P. O'Donnell, L. Xiao, S. Knight, G. Stavrakis, M. Botto, U. H. von Andrian, and T. N. Mayadas C1q Governs Deposition of Circulating Immune Complexes and Leukocyte Fc{gamma} Receptors Mediate Subsequent Neutrophil Recruitment J. Exp. Med., October 4, 2004; 200(7): 835 - 846. [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. SEELIGER, C. K. DERIAN, N. VERGNOLLE, N. W. BUNNETT, R. NAWROTH, M. SCHMELZ, P.-Y. VON DER WEID, J. BUDDENKOTTE, C. SUNDERKOTTER, D. METZE, et al. Proinflammatory role of proteinase-activated receptor-2 in humans and mice during cutaneous inflammation in vivo FASEB J, October 1, 2003; 17(13): 1871 - 1885. [Abstract] [Full Text] [PDF]
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M. U. Norman, N. C. Van De Velde, J. R. Timoshanko, A. Issekutz, and M. J. Hickey Overlapping Roles of Endothelial Selectins and Vascular Cell Adhesion Molecule-1 in Immune Complex-Induced Leukocyte Recruitment in the Cremasteric Microvasculature Am. J. Pathol., October 1, 2003; 163(4): 1491 - 1503. [Abstract] [Full Text] [PDF]
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S. Cuzzocrea, A. Rossi, I. Serraino, E. Mazzon, R. Di Paola, L. Dugo, T. Genovese, B. Calabro, A. P. Caputi, and L. Sautebin 5-Lipoxygenase knockout mice exhibit a resistance to pleurisy and lung injury caused by carrageenan J. Leukoc. Biol., June 1, 2003; 73(6): 739 - 746. [Abstract] [Full Text] [PDF]
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K. Y. Larbi, J. P. Dangerfield, F. J. Culley, D. Marshall, D. O. Haskard, P. J. Jose, T. J. Williams, and S. Nourshargh P-selectin mediates IL-13-induced eosinophil transmigration but not eotaxin generation in vivo: a comparative study with IL-4-elicited responses J. Leukoc. Biol., January 1, 2003; 73(1): 65 - 73. [Abstract] [Full Text] [PDF]
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W. G. James, D. C. Bullard, and M. J. Hickey Critical Role of the {alpha}4 Integrin/VCAM-1 Pathway in Cerebral Leukocyte Trafficking in Lupus-Prone MRL/faslpr Mice J. Immunol., January 1, 2003; 170(1): 520 - 527. [Abstract] [Full Text] [PDF]
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G. Andonegui, S. M. Goyert, and P. Kubes Lipopolysaccharide-Induced Leukocyte-Endothelial Cell Interactions: A Role for CD14 Versus Toll-Like Receptor 4 Within Microvessels J. Immunol., August 15, 2002; 169(4): 2111 - 2119. [Abstract] [Full Text] [PDF]
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N. W. Lukacs, A. John, A. Berlin, D. C. Bullard, R. Knibbs, and L. M. Stoolman E- and P-Selectins Are Essential for the Development of Cockroach Allergen-Induced Airway Responses J. Immunol., August 15, 2002; 169(4): 2120 - 2125. [Abstract] [Full Text] [PDF]
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D.-M. McCafferty, A. W. B. Craig, Y. A. Senis, and P. A. Greer Absence of Fer Protein-Tyrosine Kinase Exacerbates Leukocyte Recruitment in Response to Endotoxin J. Immunol., May 15, 2002; 168(10): 4930 - 4935. [Abstract] [Full Text] [PDF]
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M. J. Hickey, D. C. Bullard, A. Issekutz, and W. G. James Leukocyte-Endothelial Cell Interactions Are Enhanced in Dermal Postcapillary Venules of MRL/faslpr (Lupus-Prone) Mice: Roles of P- and E-Selectin J. Immunol., May 1, 2002; 168(9): 4728 - 4736. [Abstract] [Full Text] [PDF]
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 C. Tremblay, M. Paradis, and M. Dore Expression of E- and P-selectin in Tumor Necrosis Factor-induced Dermatitis in Dogs Vet. Pathol., May 1, 2001; 38(3): 261 - 268. [Abstract] [Full Text] [PDF]
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 S. Praprotnik, M. Blank, Y. Levy, S. Tavor, M.-C. Boffa, B. Weksler, A. Eldor, and Y. Shoenfeld Anti-endothelial cell antibodies from patients with thrombotic thrombocytopenic purpura specifically activate small vessel endothelial cells Int. Immunol., February 1, 2001; 13(2): 203 - 210. [Abstract] [Full Text] [PDF]
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S. A. Briaud, Z.-M. Ding, L. H. Michael, M. L. Entman, S. Daniel, and C. M. Ballantyne Leukocyte trafficking and myocardial reperfusion injury in ICAM-1/P-selectin-knockout mice Am J Physiol Heart Circ Physiol, January 1, 2001; 280(1): H60 - H67. [Abstract] [Full Text] [PDF]
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 J. Carvalho-Tavares, M. J. Hickey, J. Hutchison, J. Michaud, I. T. Sutcliffe, and P. Kubes A Role for Platelets and Endothelial Selectins in Tumor Necrosis Factor-{alpha}-Induced Leukocyte Recruitment in the Brain Microvasculature Circ. Res., December 8, 2000; 87(12): 1141 - 1148. [Abstract] [Full Text] [PDF]
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M. Ho, M. J. Hickey, A. G. Murray, G. Andonegui, and P. Kubes Visualization of Plasmodium falciparum-Endothelium Interactions in Human Microvasculature: Mimicry of Leukocyte Recruitment J. Exp. Med., October 16, 2000; 192(8): 1205 - 1212. [Abstract] [Full Text] [PDF]
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O. A. Harari, J. F. McHale, D. Marshall, S. Ahmed, D. Brown, P. W. Askenase, and D. O. Haskard Endothelial Cell E- and P-Selectin Up-Regulation in Murine Contact Sensitivity Is Prolonged by Distinct Mechanisms Occurring in Sequence J. Immunol., December 15, 1999; 163(12): 6860 - 6866. [Abstract] [Full Text] [PDF]
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M. J. Hickey, D. N. Granger, and P. Kubes Molecular Mechanisms Underlying IL-4-Induced Leukocyte Recruitment In Vivo: A Critical Role for the {alpha}4 Integrin J. Immunol., September 15, 1999; 163(6): 3441 - 3448. [Abstract] [Full Text] [PDF]
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