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* Center for Inflammatory Diseases, Monash University, Clayton, Victoria, Australia;
Department of Genomics and Pathobiology, University of Alabama, Birmingham, AL 35294; and
Departments of Pediatrics, Microbiology-Immunology, and Pathology, Dalhousie University, Halifax, Nova Scotia, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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10%) leukocyte rolling persisted following
P-selectin blockade. This residual rolling was predominantly
inhibitable with an anti-E-selectin mAb; however, treatment with
anti-E-selectin mAb alone had a minimal effect.
P-selectin-deficient MRL/faslpr mice
also displayed leukocyte rolling that was significantly lower than in
wild-type MRL/faslpr mice. However, in
these mice, leukocyte adhesion remained at the elevated levels observed
in wild-type MRL/faslpr mice. This
adhesion was eliminated by chronic treatment with anti-E-selectin
mAb. These findings indicate that leukocyte-endothelial cell
interactions are enhanced in the dermal microvasculature of
MRL/faslpr mice above the age of 12
wk. Furthermore, the data suggest that the endothelial selectins share
overlapping roles in mediating this enhanced leukocyte
recruitment. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The process of leukocyte recruitment to sites of inflammation requires
that leukocytes undergo a well-defined sequence of interactions with
endothelial cells lining the microvasculature (5).
Initially leukocytes, moving rapidly in the blood, tether and roll
along the endothelial surface. Once rolling along the endothelium,
cells are able to detect and respond to activating stimuli such as
chemokines or other chemoattractant molecules, and subsequently arrest
on the endothelial surface, via the process of firm adhesion. Adherent
cells are then able to emigrate out of the vasculature. Evidence from
many in vitro and in vivo studies indicates that the tethering and
rolling steps are predominantly mediated by the selectin
family of adhesion molecules, and in some cases, the leukocyte
4 integrin (6, 7, 8, 9). In particular,
the endothelial selectins, P- and E-selectin, appear to play
prominent roles in initiating leukocyte attachment to the vascular wall
during inflammatory responses in the peripheral
microvasculature (8, 10, 11). The adhesion step is
mediated by interaction of leukocyte integrins
(
2 and 1) with their
respective endothelial ligands, including ICAM-1 and VCAM-1
(5). Although the above paradigm is well-established for
acute responses, recent work has suggested that molecular pathways of
leukocyte recruitment can be profoundly altered during chronic
inflammatory states (12, 13). Some degree of progress has
been made in identifying the critical adhesion molecules in the chronic
inflammatory disease affecting MRL/faslpr
mice. Expression of ICAM-1 and VCAM-1 has been shown to increase
progressively in the heart, brain, and kidneys of these mice during
disease development (14, 15, 16). Furthermore,
ICAM-1-deficient MRL/faslpr mice exhibit
prolonged survival and reduced tissue inflammation, indicating a
functional role for this molecule in disease development (17, 18). However, as ICAM-1-deficient
MRL/faslpr mice are not entirely protected
from disease, it is clear that additional adhesion molecule pathways
are functioning in these animals.
To fully understand the unique roles played by adhesion molecules in
the multistep process of leukocyte recruitment in these animals, it is
necessary to directly examine the affected microvasculature. Therefore,
the aim of these studies was to characterize the adhesion molecule
pathways responsible for the vasculitis in the skin of
MRL/faslpr mice, via the use of intravital
microscopy. We chose to focus on the dermal microvasculature, as the
skin is one of the organs affected most commonly in human SLE.
Furthermore, previous studies have shown that
MRL/faslpr mice develop inflammatory
lesions of the skin which have features in common with those in human
lupus erythematosus (19). In these experiments, it was
observed that infiltration of mononuclear leukocytes into the dermis
commences at 2–3 mo. Therefore, we examined mice at 8 wk of age,
when inflammation was likely to be minimal, and 12 and 16 wk, when
further increases in leukocyte trafficking were anticipated. In
contrast to earlier studies which had focused on adhesion molecules
thought to be responsible for mediating leukocyte adhesion (e.g.,
ICAM-1 and VCAM-1), the focus of these studies was on the potential
role of P- and E-selectin in initiating rolling interactions between
leukocytes and endothelial cells. The involvement of these molecules
was examined using function-blocking Abs, and
MRL/faslpr mice possessing a gene-targeted
mutation in the P-selectin gene. These experiments revealed that
leukocyte-endothelial cell interactions in dermal postcapillary venules
of lupus-prone mice progressively increase as the mice are affected by
active disease, and that P- and E-selectin share overlapping roles in
mediating these interactions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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"Lupus-prone" MRL-MpJ/faslpr (MRL/faslpr) mice and MRL-MpJ+/+ (MRL+/+) mice were supplied by The Jackson Laboratory (Bar Harbor, ME). MRL/faslpr mice have recently been renamed MRL/Tnfrsf6lpr; however, we will use the more familiar nomenclature in describing these experiments. MRL+/+ mice are susceptible to autoimmune disease, especially later in life. However, addition of the lpr mutation to this background (MRL/faslpr mice) causes a marked acceleration in development of autoimmune disease and vasculitis. Therefore, both MRL+/+ and MRL/faslpr mice were examined to determine whether the MRL background alone contributed to any alterations in dermal leukocyte trafficking. A similar approach has been used in previous studies (16). BALB/c mice (purchased from the University of Adelaide, Adelaide, Australia) were used as controls to indicate the level of basal leukocyte trafficking in nondiseased wild-type mice. Mice were housed under quarantine conditions, and used at 8, 12, and 16 wk of age.
Generation of P-selectin-/- MRL/faslpr mice
P-selectin-/--MRL/faslpr mice were generated by backcrossing a gene-targeted P-selectin mutation onto the MRL/faslpr strain background for eight generations (20). Mice were then intercrossed to produce double homozygotes (P-selectin-/-/faslpr/lpr) (21).
Intravital microscopy
Animals were anesthetized by i.p. injection of a mixture of 10 mg/kg xylazine (Bayer Pharmaceuticals, Pymble, Australia) and 200 mg/kg ketamine hydrochloride (Parke-Davis, Caringbah, Australia). The left jugular vein was cannulated to administer anesthetic, fluorescent dyes, and Abs. The animal was placed on a thermocontrolled heating pad, regulating the core temperature to 37°C. The microcirculation of the ventral abdominal skin was then prepared for microscopy as previously described (22). Briefly, a midline abdominal incision was made extending from the level of the diaphragm to the pelvic region. The skin was carefully separated from the underlying tissue, remaining attached laterally to ensure the blood supply remained intact. The area of skin was then extended over a viewing pedestal and secured along the edges using 2–0 suture. The loose connective tissue on the dermal undersurface was carefully removed by dissection under an operating microscope. The exposed dermal microvasculature was immersed in normal saline and covered with a coverslip held in place with vacuum grease. To visualize leukocytes, animals were injected with 50 µl of 0.05% (i.v.) rhodamine 6G (Sigma-Aldrich, St. Louis, MO) immediately before microscopy. 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 has no effect on leukocyte kinetics (23, 24). 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 (24, 25).
In an additional series of experiments, the dermal microvasculature of the ear was examined using a technique which causes minimal disruption to the tissue (26). Briefly, following anesthesia and insertion of a catheter in the tail vein, the hair on the right ear was removed using depilatory cream. The ear was then gently positioned on a heated pad, immersed in saline, and covered with a coverslip held in place with vacuum grease. To aid in visualization of the vasculature, 10 µl of 5% FITC-70-kDa dextran (Sigma-Aldrich) was administered i.v. as a plasma marker. This fluorochrome was visualized by epi-illumination at 450–490 nm, with a 520-nm emission filter. Detection of rhodamine-6G-labeled leukocytes was performed as for the ventral skin preparation.
For preparations of both types, the microvasculature was visualized using an intravital microscope (Axioplan 2 Imaging; Carl Zeiss, Carnegie, Australia) with a x40 water immersion objective lens (Achroplan x40/0.80 NA, Carl Zeiss) and a x10 eyepiece. A SIT video camera (Dage-MTI VE-1000; SciTech Pty. Ltd., Preston South, Australia) was used to project the images onto a monitor (Sony PVM-20N5E; Carl Zeiss), and the images were recorded for playback analysis using a videocassette recorder (Panasonic NV-HS950; Panasonic, Secaucus, NJ). One to four dermal venules (25–40 µm in diameter) were selected in each experiment, and to minimize variability, the same section of venule was observed throughout the experiment. Venular diameter and the number of rolling and adherent leukocytes were determined offline 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 whether they remained stationary for 30 s or longer.
RBC velocity was determined via analysis of the velocity of 1-µm
diameter fluorescent polystyrene microspheres
(FluoSpheres-yellow/green, Molecular Probes, OR) injected i.v. in small
boluses (27). Beads were visualized via epifluorescence as
for FITC-dextran. Video sequences showing microspheres
moving through postcapillary venules were digitized using an image
analysis computer with an IC-PCI video capture card (Imaging
Technology, Bedford, MA) controlled by the Sequence Snap video
acquisition software (Adept Electronic Solutions, Perth, Australia).
Following calibration appropriate to the magnification
under examination, RBC velocity was measured using Scion Image
(Scion, Frederick, MD) in combination with a macro developed by
Dr. K. Norman (University of Sheffield, Sheffield, U.K.)
(28). The mean velocity (VMEAN)of
10 randomly selected microspheres was determined, and venular wall
shear rate (
) was calculated based on the Newtonian definition:
= 8
(VMEAN/Dv)
(29).
Circulating leukocyte counts
At the end of each experiment, whole blood was drawn via cardiac puncture. Total leukocyte counts were performed, using a Neubauer hemocytometer (U-Lab, Eltham, Australia).
Antibodies
The Abs used in vivo in this study were RB40.34, an mAb against
murine P-selectin (BD Biosciences, San Diego, CA; 20 µg/mouse);
R1–2, an mAb against the murine 4 integrin
(BD Biosciences; 75 µg/mouse); and RME-1, an mAb against rat and
mouse E-selectin (Issekutz Laboratory, Halifax, Nova Scotia,
Canada). The doses of RB40.34 and R1–2 used have been shown previously
to be effective in specifically blocking their respective target
molecules in vivo (11, 30). RME-1 inhibits binding of
HL-60 myeloid cells to recombinant murine E-selectin, and has been
shown to block E-selectin function in vivo in a murine model of
endotoxin-induced leukocyte rolling (31, 32). For acute
blockade of E-selectin, 100 µg of RME-1 was administered i.v., as
previously described (32). For studies assessing effects
of chronic E-selectin blockade, 200 µg RME-1 was administered i.v.
via the tail vein, 18 h before microscopic observation. This dose
has been observed to provide effective E-selectin blockade for at least
22 h (A. Issekutz, unpublished observations). Abs used for flow
cytometry and immunohistochemistry were: PE-conjugated anti-murine
P-selectin glycoprotein ligand-1 (PSGL-1) (2PH1), FITC-conjugated
anti-murine CD3 (17A2), and anti-murine E-selectin (10E9.6)
(all from BD Biosciences); Cy5-conjugated RB6-8C5 (Gr-1) purified from
hybridoma supernatant; and affinity-purified rabbit polyclonal Ab
raised against human P-selectin, generated as previously described
(generously provided by Dr. M. Berndt, Baker Medical Research
Institute, Melbourne, Australia) (33).
Histopathology
Areas of ventral skin were fixed in formalin, and 3-µm sections were prepared and stained with H&E according to standard techniques. Profiles of postcapillary venules located immediately subjacent to the dermis, corresponding to those viewed in vivo, were identified and the presence of leukocytes in close apposition to the endothelium determined. All venular profiles with at least one leukocyte closely apposed to the endothelium were defined as containing leukocytes interacting with the endothelium. These leukocytes were then classified as either granulocytic or mononuclear according to their morphology.
Flow cytometry and immunohistochemistry
Expression of PSGL-1 on circulating leukocytes was examined via flow cytometry. Heparinized blood samples were collected via cardiac puncture and 100-µl blood samples were treated with PE-conjugated 2PH1 at 1:100 for 20 min. To determine PSGL-1 expression by lymphocytic and granulocytic populations, samples were coincubated with FITC-conjugated anti-murine CD3 and Cy5-conjugated anti-Gr-1 (RB6-8C5, granulocyte marker). Leukocyte fixation and erythrocyte lysis were performed using a Q-Prep Workstation (Beckman Coulter, Miami, FL) and samples were analyzed using a MoFlo flow cytometer (Cytomation, Fort Collins, CO). In addition, mononuclear and granulocytic populations were differentiated on the basis of forward and side scatter.
For immunohistochemical analysis of endothelial selectin expression, skin samples were fixed in periodate-lysine-paraformaldehyde for 4 h at 4°C and cryoprotected via washing in 7% sucrose/PBS for 48 h. Samples were then embedded in OCT compound, frozen over liquid nitrogen, and 7-µm cryostat sections prepared. To determine P-selectin expression, sections were stained using a three-layer peroxidase-anti-peroxidase technique, as previously described (34). Briefly, rabbit anti-P-selectin Ab (10 µg/ml, overnight at 4°C) was used as primary Ab. The secondary Ab was peroxidase-conjugated swine anti-rabbit IgG (1:50, 60 min), followed by rabbit peroxidase anti-peroxidase (1:100, 60 min). To determine E-selectin expression, rat anti-mouse E-selectin (10E9.6) (1:25, overnight at 4°C) was used as primary Ab, rabbit anti-rat IgG (1:100) as secondary Ab, and HRP-conjugated swine anti-rabbit IgG as tertiary Ab. All secondary and tertiary Abs were supplied by DAKO (Carpinteria, CA). All sections were developed by incubation in diaminobenzidine, and counterstained with hematoxylin. Selectin expression was analyzed by quantitating the number of vascular profiles displaying positive staining per x10 field.
Statistics
For parameters such as leukocyte rolling flux, rolling velocity, and adhesion, comparison between the three mouse strains were performed using one-way analysis of variance, or Student’s t tests using the Bonferroni correction for multiple comparisons. Velocity analyses before and after administration of mAbs were performed using paired t tests. 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, leukocyte trafficking was characterized in
dermal postcapillary venules of BALB/c, MRL+/+,
and MRL/faslpr mice at 8, 12, and 16 wk of
age using the acute ventral skin preparation. Leukocyte rolling flux is
shown in Fig. 1
. In BALB/c and
MRL+/+ (nondiseased) mice, leukocyte rolling flux
remained at a constant level of 10–15 cells/min at each of the ages
examined, indicating that there were no age-dependent alterations in
this parameter over this 8-wk period. In 8-wk-old
MRL/faslpr mice, the level of leukocyte
rolling was similar to that seen in the two control mouse strains.
However, at both 12 and 16 wk, the number of rolling leukocytes was
significantly enhanced in the lupus-prone mice. This was not due to an
elevation in the circulating leukocyte count, as this parameter did not
differ between the three strains of mice at these time points. At 16 wk
of age, leukocyte counts in BALB/c, MRL+/+, and
MRL/faslpr mice were 8.6 ± 1.0,
7.9 ± 0.7, and 9.1 ± 0.8 x
106/ml, respectively. In addition, venular shear
rate was significantly reduced in 16-wk
MRL/faslpr mice relative to BALB/c mice,
but not relative to MRL+/+ mice (Table I). Given that the rolling observed in
16-wk MRL+/+ mice was <50% than in age-matched
MRL/faslpr mice despite comparable shear
rates, this indicates that reduced shear rate alone was insufficient to
explain the elevation in rolling in the lupus-prone mice.
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However, in MRL/faslpr mice, leukocyte
adhesion was significantly enhanced in 12- and 16-wk-old mice,
indicating that the alteration in leukocyte rolling observed in these
mice was associated with an increase in the number of cells ultimately
progressing to adhesion (Fig. 3).
Although the increased number of adherent cells in 12- and 16-wk-old
MRL/faslpr mice (4/100-µm venule) is
quite low relative to other tissues, this is comparable to the amount
of adhesion we have seen in skin with optimal levels of Ag challenge
(22). Leukocyte-endothelial cell interactions in arteries
were also assessed in these mice. However, leukocytes were rarely
observed to interact with the endothelium in arterial vessels, apart
from an occasional cell in severely diseased
MRL/faslpr mice at 16 wk of age (data not
shown).
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).
Histopathological analysis of skin in 16-wk-old
MRL+/+ and
MRL/faslpr mice revealed that in
MRL+/+ mice, 14% of postcapillary venular
profiles displayed leukocytes in close apposition to the endothelial
surface, whereas in MRL/faslpr mice, 46%
of venules displayed leukocytes adjacent to the endothelium. However,
in both strains of mice, 60–75% of these cells were mononuclear,
suggesting that while the number of interacting cells was increased in
MRL/faslpr mice, this increase was not due
to a relative increase in the proportion of one particular subgroup of
leukocytes interacting with the endothelium.
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To determine whether the elevation in leukocyte rolling and
adhesion in MRL/faslpr mice was related to
trauma induced by surgical preparation of the ventral skin, leukocyte
rolling was also analyzed in the microvasculature of the ear in the
absence of surgical manipulation (26). Similar to findings
using the ventral skin preparation, leukocyte rolling in dermal
postcapillary venules in the noninflamed ears of 16-wk-old
MRL/faslpr mice was greater than double
that in age-matched MRL+/+ mice (Fig. 4).
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To determine a role for the endothelial selectins in leukocyte
rolling in these animals, mice were treated with function-blocking Abs
to P- and E-selectin. Fig. 5 illustrates
the effect of P-selectin inhibition on dermal leukocyte rolling in the
three mouse strains. At both 8 and 12 wk, leukocyte rolling in all
three strains was almost entirely eliminated by P-selectin blockade. In
contrast, at 16 wk, residual rolling of the order of 3–5 cells/min
persisted following P-selectin blockade. This finding was the same in
all three strains examined. Finally, in BALB/c,
MRL+/+ (data not shown), and
MRL/faslpr mice at 16 wk (Fig. 6), the residual P-selectin-independent
rolling was eliminated by subsequent E-selectin blockade.
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). However, in accord with previous
studies (35), E-selectin blockade displayed some effects
on rolling velocity (Table III). At 8 wk, E-selectin blockade increased
rolling velocity significantly in both BALB/c and
MRL+/+ mice. The same treatment was without
effect on rolling velocity in MRL/faslpr
mice. At 12 wk of age, small but significant increases were observed in
BALB/c and MRL/faslpr mice following
E-selectin blockade, but not in MRL+/+ mice. At
16 wk, E-selectin blockade was without effect on any of the strains
examined.
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4
integrin in mediating the increased rolling in venules in dermal
venules of MRL/faslpr mice was
examined. Treatment of 12- and 16-wk-old
MRL/faslpr mice with a mAb against the
4 integrin had no effect on either leukocyte
rolling flux or rolling velocity (Table III
). Role of E-selectin in mediating leukocyte rolling and adhesion in P-selectin-/--MRL/faslpr mice
Finally, to further examine the relative roles of P- and
E-selectin in mediating leukocyte rolling and subsequent adhesion in
MRL/faslpr mice, we examined the dermal
microvasculature of
P-selectin-/--MRL/faslpr
mice (Fig. 7). In agreement with the
results of P-selectin blockade in wild-type
MRL/faslpr mice, leukocyte rolling was
dramatically reduced, although not entirely absent in
P-selectin-/--MRL/faslpr
mice. At all time points examined, some residual rolling was observed.
However, despite the marked reduction in leukocyte rolling in the
P-selectin-deficient mice, leukocyte adhesion remained at the elevated
levels observed in wild-type MRL/faslpr
mice, indicating that leukocyte adhesion could occur in these animals
via a P-selectin-independent pathway (Fig. 7B).
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C). We next wanted to
test whether E-selectin-dependent rolling resulted in leukocyte
adhesion in this model. The relatively short time course of the acute
experiments with RME-1 was insufficient to observe the impact of
E-selectin blockade on leukocyte adhesion, as adherent cells which may
have required E-selectin for rolling no longer use E-selectin to remain
firmly attached to the endothelium, and consequently would not be
affected by E-selectin blockade. Therefore, we chronically treated both
wild-type MRL/faslpr mice and
P-selectin-/--MRL/faslpr
mice with RME-1 to prevent E-selectin-dependent rolling for 18 h, thus
preventing cells from progressing to adhesion via this mechanism (Fig. 8). In wild-type
MRL/faslpr mice, extended E-selectin
blockade had no effect on rolling flux, rolling velocity (data not
shown), and leukocyte adhesion (Fig. 8A). In sharp contrast,
in
P-selectin-/--MRL/faslpr
mice treated chronically with RME-1, leukocyte rolling and adhesion
were eliminated (Fig. 8B).
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One potential mechanism to explain the increase in
selectin-dependent leukocyte rolling was an alteration in endothelial
expression of P- and E-selectin. This possibility was assessed by
immunohistochemical analysis of selectin expression in
MRL+/+ and
MRL/faslpr mice. Fig. 9 shows the level of P-selectin
expression observed in the skin of the two strains of mice at various
ages. At all ages examined, the number of vascular profiles which
stained positively for P-selectin was consistently higher in
MRL/faslpr mice than in
MRL+/+ mice. In contrast, E-selectin expression
was detected only sporadically in both strains of mice, with the number
of labeled vessels not differing between the two strains. However,
after systemic treatment with LPS, E-selectin expression was observed
in dermal vessels in both strains of mice (data not shown), indicating
that these tissues had the capacity to express this molecule given
appropriate stimulation.
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Alterations in leukocyte expression of PSGL-1, the major ligand of
P-selectin, could conceivably also be responsible for alterations in
selectin-dependent interactions in the microvasculature. Therefore, we
compared PSGL-1 expression by circulating leukocytes in
MRL+/+ and
MRL/faslpr mice. Combined analysis of both
the granulocyte and mononuclear populations in the two strains
indicated that in MRL+/+ mice at 16 wk, 76
± 2% of circulating leukocytes were positive for PSGL-1, whereas in
similarly aged MRL/faslpr mice, 93 ±
1% of the cells expressed PSGL-1 (p < 0.001).
In both strains of mice, essentially all granulocytes
(Gr-1+) and T cells (CD3+)
expressed high levels of PSGL-1. However, analysis of the CD3-negative
cells within the mononuclear population, which include B cells and NK
cells, showed a significant increase in the proportion expressing
PSGL-1 (37 ± 3% in MRL+/+ vs 62 ±
1% in MRL/faslpr; p <
0.001) (Fig. 10). Similar but less
pronounced changes were observed in 8- and 12-wk
MRL/faslpr mice (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study enabled us to determine the role of the endothelial selectins in mediating leukocyte recruitment to inflamed venules undergoing a chronic inflammatory stimulus. Several observations suggested that P-selectin was highly important in mediating leukocyte rolling in these vessels. Firstly, P-selectin blockade was highly effective at preventing rolling in dermal vessels in MRL/faslpr mice. Even when rolling was significantly elevated above basal levels at 12 and 16 wk, P-selectin blockade inhibited rolling by <90%. Furthermore, the number of rolling leukocytes was reduced to a comparable degree in P-selectin-/--MRL/faslpr mice. Thirdly, our data indicated that P-selectin expression was increased in the dermal microvasculature of MRL/faslpr mice. Using an immunohistochemical approach, we found that although P-selectin was rarely expressed at detectable levels in MRL+/+ mice, in MRL/faslpr mice vascular expression of P-selectin was consistently higher. This is in contrast to previous experiments which indicated that expression of P- and E-selectin, determined using an in vivo radiolabeled immunoassay, did not differ between BALB/c, MRL+/+, and MRL/faslpr mice (36). The reasons for this discrepancy are unclear, but could include differences in the sensitivity of the detection technique, or differences in housing conditions of the mice. Nevertheless, together the current data support the hypothesis that P-selectin has a key role in mediating leukocyte rolling in dermal postcapillary venules in these chronically inflamed mice.
Analysis of PSGL-1 expression by circulating leukocytes provided an additional potential mechanism to explain the increase in selectin-dependent interactions observed. In the present study, the proportion of circulating cells expressing PSGL-1 was significantly higher in MRL/faslpr mice compared with MRL+/+ mice. This alteration was due to an increase in the proportion of CD3-negative mononuclear cells in the lupus-prone strain expressing the molecule. The increased PSGL-1 expression in the lupus-prone strain correlated well with the increase in selectin-dependent interactions observed in vivo in the dermal microvasculature of these mice. PSGL-1 is the major leukocyte ligand for P-selectin, and is also capable of interacting with E-selectin (37, 38, 39). The critical role of PSGL-1 in mediating P-selectin-dependent leukocyte rolling is demonstrated by the observation that PSGL-1-deficient mice show markedly reduced leukocyte rolling in the acutely inflamed microvasculature and delayed neutrophil recruitment in the thioglycollate model of peritonitis, comparable to that seen in P-selectin-deficient mice (39). Therefore, it is conceivable that alterations in the distribution or expression of this molecule may lead to comparable changes in leukocyte rolling in peripheral vascular beds. Further work is required to determine the functional role of PSGL-1 in the altered leukocyte trafficking in this model.
However, despite the suggestions of a key role for P-selectin in mediating leukocyte recruitment in this response, in P-selectin-/--MRL/faslpr mice the number of leukocytes which progressed onto adhesion was not different from that observed in wild-type MRL/faslpr mice. This indicated that P-selectin was not required for leukocyte adhesion to reach the elevated levels observed in these animals. Therefore, to determine the role of the other endothelial selectin (E-selectin) in this response, we analyzed adhesion after chronic anti-E-selectin treatment. Chronic blockade of E-selectin completely eliminated both rolling and adhesion in P-selectin-/--MRL/faslpr mice, clearly illustrating that in the absence of P-selectin, E-selectin-dependent rolling was required to enable leukocytes to become adherent. Interestingly, when wild-type MRL/faslpr mice underwent the same anti-E-selectin treatment, leukocyte adhesion was not reduced. Presumably, in wild-type MRL/faslpr mice in which E-selectin function was inhibited, rolling mediated by P-selectin was sufficient to enable leukocyte adhesion to reach elevated levels. This indicates that in the dermal microvasculature in this model of chronic inflammation, the functions of P- and E-selectin are interchangeable. These findings provide a plausible explanation for the observation that P-selectin-/--MRL/faslpr mice are not protected from the pathology and premature death which affects wild-type MRL/faslpr mice (D. Bullard, manuscript in preparation). In these animals, the absence of P-selectin does not affect the ability of pathogenic leukocytes to be recruited to vasculitic sites. Our data indicate that in the absence of P-selectin, at least in the dermal microvasculature, E-selectin provides an alternative molecular pathway which is capable of mediating leukocyte recruitment. However, this function is not apparent in wild-type MRL/faslpr mice, under conditions of normal P-selectin expression.
It was clear from the examination of the P-selectin-/--MRL/faslpr mice, that a dramatic reduction in the number of rolling leukocytes did not affect the number of cells able to undergo adhesion in dermal postcapillary venules. This indicates that in the prolonged inflammatory response occurring in these animals, the number of leukocytes which undergo adhesion is not affected by marked reductions in leukocyte rolling flux. This finding is supported by studies of leukocyte recruitment induced by IL-4 over a 24-h time course, in which reduction in leukocyte rolling to as low as 2 cells/min (>95% reduction) did not reduce the number of leukocytes recruited to the cremaster muscle (30). Together with the present findings, these data indicate that in contrast to acute inflammatory responses, during more prolonged or chronic inflammatory responses, leukocyte adhesion and subsequent entry of leukocytes to the inflamed area can occur in an efficient manner even when leukocyte rolling has been almost entirely abolished. This has important implications if leukocyte rolling is to be the target of pharmacological therapy for chronic inflammatory disease.
In peripheral vascular beds under basal conditions, and indeed during most inflammatory responses, leukocytes rarely interact with the endothelium on the arterial side, but predominantly undergo rolling and adhesion on the venular side of the microcirculation (40, 41). However, given that arterial vessels are one of the primary targets of the vasculitis in these mice, it is conceivable that under these inflammatory conditions, leukocytes will undergo rolling and adhesive interactions on the arterial endothelial surface. In the present study, direct observation of arterial vessels revealed that leukocytes rarely interacted with the endothelial surface of dermal arterioles in these animals. On the rare occasions that these interactions were detected, it was only in the most severely diseased MRL/faslpr mice. This might be considered a surprising observation; however, similar findings were observed in a study of adjuvant-induced vasculitis in rats in which leukocytes did not undergo interactions in arterioles, despite a 10-fold increase in leukocyte rolling and adhesion in adjacent postcapillary venules (9). There are several possible explanations for our observations in the MRL/faslpr model. Arterial interactions contributing to vasculitis may occur rarely and over extended periods, making detection of these events beyond the scope of conventional intravital microscopy experiments. Alternatively, there is evidence to suggest that leukocytes infiltrating the wall of arteries affected by vasculitis do not enter by interacting with the endothelial lining of the affected vessel, but instead emigrate from alternative vascular sites such as the adventitial microvasculature or adjacent postcapillary venules (4). Histopathological studies of MRL/faslpr mice show that that the initial mononuclear vasculitis predominantly affects the perivascular/adventitial zones of arteries, i.e., the area of the vessel most distant from the endothelial surface (4). Finally, the lack of lumenal interactions in arteries in these vasculitic mice could be unique to the tissue under examination. The skin of MRL/faslpr mice has been shown to have a low, or at least delayed, incidence of vasculitis compared with more severely affected organs such as the lungs, kidneys, and salivary glands (3). It is possible that the dermal arteries examined in the present study were only minimally affected by vasculitis, and consequently, the rate of leukocyte entry into the vascular wall was exceedingly low. These issues may be resolved in the future by direct examination of arteries in more severely affected tissues of MRL/faslpr mice.
In conclusion, we have shown that one of the consequences of the systemic inflammatory disease which affects MRL/faslpr mice is an enhancement in leukocyte-endothelial cell interactions in the dermal microvasculature. This increase in leukocyte trafficking is potentially an important contributor to the development of the systemic inflammation which affects these mice. Currently it remains unknown whether comparable alterations in leukocyte trafficking occur in other organs. However, it is clear that in the MRL/faslpr model of systemic autoimmune disease, and indeed in clinical SLE, a wide range of organs can be affected by imflammatory vascular disease. The aim of future studies will be to determine the existence and mechanisms of aberrant leukocyte trafficking in other critical organs in these mice.
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2 Address correspondence and reprint requests to Dr. Michael J. Hickey, Centre for Inflammatory Diseases, Department of Medicine, Monash Medical Centre, Monash University, Block E, 246 Clayton Road, Clayton, Victoria, 3168, Australia. E-mail address: michael.hickey{at}med.monash.edu.au
3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; PSGL-1, P-selectin glycoprotein ligand-1.
Received for publication August 9, 2001. Accepted for publication February 27, 2002.
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-activated vascular endothelium under flow in vitro. J. Immunol. 156:326.
4-integrin supports leukocyte rolling and adhesion in chronically inflamed postcapillary venules in vivo. J. Exp. Med. 183:1995.4-integrin, P-selectin, and E-selectin in an allergic model of inflammation. J. Exp. Med. 185:1077.4-integrin in acute, subacute and chronic leukocyte recruitment in vivo. J. Immunol. 159:4514.[Abstract]
and IL-1 sequentially induce ICAM-1 and VCAM-1 expression in MRL/lpr lupus-prone mice. J. Immunol. 163:3993.4-integrin. J. Immunol. 163:3441.(1,3)-fucosyltransferase-IV and FucT-VII during leukocyte rolling in dermal microvessels. Immunity 12:665.[Medline]
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)
and MRL/faslpr mice (
) at 8, 12,
and 16 wk of age. Each data point represents an individual animal. Data
represent the number of positively labeled vascular profiles observed
per 10x field.