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4 Integrin/VCAM-1 Pathway in Cerebral Leukocyte Trafficking in Lupus-Prone MRL/faslpr Mice1
* Centre for Inflammatory Diseases, Monash University, Victoria, Australia; and
Department of Genomics and Pathobiology, University of Alabama, Birmingham, AL 35294
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4
integrin or VCAM-1 eliminated P-selectin-independent leukocyte rolling.
4 Integrin blockade also significantly inhibited
leukocyte adhesion. These studies demonstrate that the systemic
inflammatory response that affects
MRL/faslpr mice results in leukocyte
rolling and adhesion in the cerebral microcirculation, and that the
4 integrin/VCAM-1 pathway plays a central role in
mediating these interactions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many insights into the immunological basis of SLE have been made by examination of various mouse strains affected by systemic autoimmune disease. One of the most widely studied of these, the MRL/faslpr mouse, displays many features in common with SLE patients, including increased autoantibody production, immune complex deposition, systemic vasculitis, and glomerulonephritis (4). The cerebral vasculature is also affected in these mice, with mononuclear cell infiltrates present surrounding cerebral microvessels and evidence of extravascular accumulation of IgG and albumin, suggestive of a disruption of the blood-brain barrier (5). Furthermore, MRL/faslpr mice develop evidence of neurological dysfunction illustrated by defects in balance, learning ability, and other cognitive impairments, temporally correlated with the onset of leukocyte recruitment (6, 7, 8). These observations raise the possibility that leukocyte recruitment to the CNS is a key factor in the neurologic complications in MRL/faslpr mice. However, the mechanisms responsible for recruitment of these leukocytes to the cerebral microvasculature have not been investigated.
There is now extensive evidence that leukocyte recruitment to sites of
inflammation involves a sequence of interactions between circulating
leukocytes and endothelial cells (9). Initially,
leukocytes must tether and roll along the endothelial surface, before
undergoing adhesion and emigrating out of the vasculature. The
tethering and rolling steps are mediated by members of the selectin
family of adhesion molecules, and the 4
integrin expressed on specific leukocyte populations
(10, 11, 12, 13, 14). Subsequent leukocyte adhesion is mediated by
interaction of leukocyte integrins (
2 and
1) with their respective endothelial ligands,
including ICAM-1 and VCAM-1 (9). Although this paradigm
has been supported by repeated observations in tissues, such as the
mesentery and skeletal muscle, there is a growing body of evidence that
the cerebral microvasculature responds to inflammatory stimulation in a
highly unique manner.
Direct observation of the cerebral (pial) microcirculation has shown
that constitutive leukocyte rolling is almost entirely absent in
cerebral microvessels, in marked contrast to most other organs
(15, 16). In addition, local injection of proinflammatory
agents such as fMLP and TNF-, which readily induce leukocyte
recruitment to peripheral microvascular beds, fails to induce
significant leukocyte recruitment in the brain (17).
Systemic activation with TNF- can induce leukocyte rolling and
adhesion within the cerebral microvasculature, although the molecular
mechanisms used in this process are distinct from those observed in the
periphery (15). Finally, recent experiments have indicated
that leukocyte rolling is not required for recruitment of
encephalitogenic T cell blasts to the CNS microcirculation
(18). Given these observations, it is conceivable that the
mechanisms of leukocyte recruitment to the CNS during a systemic
autoimmune disease such as lupus may be highly divergent from those at
work in peripheral organs exposed to the identical stimulus. Therefore,
the aim of these experiments was to examine the cerebral
microvasculature of lupus-prone MRL/faslpr
mice to determine the mechanisms of the leukocyte recruitment that
occurs in the CNS of these mice. These experiments revealed that
leukocyte-endothelial cell interactions are increased in cerebral
microvessels of MRL/faslpr mice, and that
these interactions are critically dependent on the
4 integrin/VCAM-1 pathway.
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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.) P-selectin-/--MRL/faslpr mice were generated by backcrossing a gene-targeted P-selectin mutation onto the MRL/faslpr strain background, as previously described (19). As controls for the lupus-prone MRL/faslpr mice, we used MRL+/+ mice. This mouse strain has displayed a susceptibility to autoimmune disease later in life. However, at the ages examined in this study, we have previously observed no signs of inflammation in the dermal vasculature (19). Mice were used at 8, 12, and 16 wk of age and weighed between 30 and 50 g.
Intravital microscopy
Animals were anesthetized by i.p. injection of a cocktail of 10 mg/kg xylazine (Bayer Pharmaceuticals, Pymble, New South Wales, Australia) and 200 mg/kg ketamine hydrochloride (Caringbah, New South Wales, Australia). A catheter was inserted in the tail vein to administer anesthetic, fluorescent dyes, and Abs. The animal was placed on a thermo-controlled heating pad, regulating the core temperature to 37°C.
The cerebral microcirculation was then prepared for microscopy, as previously described (15). Briefly, a craniotomy was performed on the right parietal bone using a high speed drill (Fine Science Tools, North Vancouver, British Columbia, Canada). A superfusion chamber was held in place over the craniotomy by securing the incised scalp around the lower lip of the chamber with dental cement and Loctite 401 rapid adhesive (Loctite Australia, Caringbah, New South Wales, Australia). Before removal of the dura and exposure of the pial microvasculature, artificial cerebrospinal fluid (CSF) (ionic composition in mmol/L: NaCl, 132; KCl, 2.95; CaCl2, 1.71; MgSO4, 1.4; NaHCO3, 24.6; glucose, 3.71; urea, 6.7; pH 7.4) was continuously pumped through the superfusion chamber at 37°C to maintain the exposed brain. A pH and gas tension similar to that of normal CSF was replicated in the artificial equivalent by constant bubbling with 12% O2, 5% CO2, and 83% N2. Animals were examined for a maximum of 1 h.
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 (20, 21). It therefore 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 (21, 22). To aid in visualization of the vasculature, 10 µl of 5% FITC/250-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.
The brain microvasculature was visualized using an intravital microscope (Axioplan 2 Imaging; Carl Zeiss, Carnegie, Victoria, 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; Sci Tech Pty., 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; Klapp Electronics, Prahran, Victoria, Australia). One to four pial postcapillary venules (30–50 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 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. In some animals, however, less than 20 leukocytes were observed rolling in a vessel during the period of recording. In these animals, the velocity of each of the leukocytes observed to be rolling was measured. Leukocytes were considered adherent to the venular endothelium if they remained stationary for 30 s or longer. To account for variability of venular diameter, leukocyte adhesion was expressed as cells/mm2 of venular surface area, as shown previously (15).
RBC velocity was determined via analysis of the velocity of
1-µm-diameter fluorescent polystyrene microspheres (FluoSpheres,
yellow/green; Molecular Probes, Eugene, OR) injected i.v. in 10 µl
boluses (23). 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 MVC-IC-PCI video capture card (Coreco Imaging, St.
Laurent, Quebec, Canada) controlled by the Sequence Snap video
acquisition software (Adept Electronic Solutions, Perth, Western
Australia, Australia). Following calibration appropriate to the
magnification under examination, RBC velocity was measured using Scion
Image (Scion, Frederick, MD), as previously described
(19). 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) (24).
Antibodies
The Abs used in vivo in this study were RB40.34, a mAb against
murine P-selectin (BD Biosciences, San Diego, CA; 20 µg/mouse); R1-2,
a mAb against the murine 4 integrin (BD
Biosciences; 75 µg/mouse); RME-1, a mAb against rat and mouse
E-selectin (100 µg/mouse; generously provided by A. Issekutz,
Dalhousie University, Halifax, Nova Scotia, Canada); and MK/2.7,
a mAb against murine VCAM-1 (100 µg/mouse; R&D Systems, Minneapolis,
MN). The doses of all function-blocking Abs used have been shown
previously to be effective in specifically blocking their respective
target molecules in vivo (12, 14, 19, 25).
Histopathology
Whole brains were fixed in formalin, and 7-µm sections were prepared and stained with H&E according to standard techniques. Profiles of pial postcapillary venules, corresponding to those viewed in vivo, were identified, and the presence of leukocytes in close apposition to the endothelium was 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 nuclear morphology.
Flow cytometric analysis of 4 integrin expression
Samples (100 µl) of whole blood from MRL+/+ and MRL/faslpr mice underwent erythrocyte lysis and paraformaldehyde fixation using a Q-Prep Workstation (Beckman Coulter, Miami, FL). Samples were then incubated with R1-2 (0.5 µg/106 cells) for 25 min. Cells were washed, then incubated with FITC-conjugated sheep anti-rat IgG (Silenus, Melbourne, Victoria, Australia) (1/100, 25 min), following preincubation with 5% mouse serum to prevent nonspecific binding. Cells were subsequently washed and analyzed using a MoFlo flow cytometer (Cytomation, Fort Collins, CO).
Cerebral microvascular permeability
Microvascular permeability of the cerebral microvasculature was
assessed utilizing a modification of a technique used previously in
rats (26). Mice were anesthetized and maintained at 37°C
with a heating blanket, as for the intravital microscopy experiments.
The left femoral artery and the tail vein were catheterized for
arterial blood sampling and delivery of molecular tracers and
additional anesthetic, respectively. The pial microvasculature was
accessed via a cranial window, as for microscopy experiments.
Artificial CSF was continuously superfused across the exposed
vasculature at 0.8 ml/min and collected afterward via an outflow port
in the superfusion chamber. At the completion of the surgery, the
tissue underwent a 30-min equilibration period to allow any bleeding
from the dural vasculature to cease, before administration of the
intravascular tracer. Permeability of the exposed vasculature was
assessed by measuring the clearance of 70-kDa FITC dextran
(Sigma-Aldrich) from the pial vessels into the superfused artificial
CSF (26). At the start of the experimental period, FITC
dextran was given as a bolus dose (1.25 mg/10 g of 5% solution, in 200
µl heparinized saline, i.v.). Subsequently, the artificial CSF was
collected for the last minute of each 5-min period throughout the
30-min experimental period. Arterial blood samples (50 µl) were taken
at 15 and 30 min, and 10 µl plasma samples were collected. The
concentration of FITC-derived fluorescence in the CSF solution and
plasma samples was measured on a 96-well plate fluorimeter
(
ex485 and em538)
(PolarStar Optima; BMG Labtechnologies, Mount Eliza, Victoria,
Australia), and concentrations were determined by reference to a
standard curve. FITC dextran clearance was determined by multiplying
the ratio of CSF solution concentration to plasma concentration by the
CSF flow rate.
Statistics
For parameters such as leukocyte rolling flux, rolling velocity, and adhesion, within strain comparisons of mice at different ages were performed with one-way ANOVA. Comparisons between the two mouse strains were performed using Student’s t tests. Comparisons of rolling and adhesion 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, we examined the pial microcirculation of
MRL+/+ and
MRL/faslpr mice at 8, 12, and 16 wk.
Previously, we had observed that these time points were before the
onset of severe disease in MRL/faslpr
mice, and animals remained sufficiently healthy to undergo the
anesthesia and microscopy procedures. In pial postcapillary venules of
MRL+/+ mice at all ages examined, leukocyte
rolling was rarely observed (Fig. 1
).
This is in concert with previous observations of the pial circulation
in uninflamed, wild-type mice (15). In
MRL/faslpr mice at 8 and 12 wk, leukocyte
rolling was similarly infrequent (1–2 cells/min). However, in
16-wk-old mice, leukocyte rolling was significantly increased to an
average of 20 cells/min (Fig. 1). Leukocyte adhesion followed a similar
pattern. Minimal adhesion was observed in MRL+/+
mice at any age (Fig. 2). In
MRL/faslpr mice at 8 and 12 wk, leukocyte
adhesion was at comparable levels to that observed in
MRL+/+ mice. However, at 16 wk, leukocyte
adhesion was significantly increased above levels in comparably aged
MRL+/+ mice, and above levels in
MRL/faslpr mice at 8 and 12 wk (Fig. 2).
These differences in leukocyte-endothelial cell interactions in
16-wk-old mice were not due to divergences in venular diameters or
hydrodynamic shear rates, as these did not differ significantly between
the two strains (Table I). Furthermore,
we have previously reported that the number of circulating white blood
cells did not significantly differ between the two strains of mice at
the ages under examination (19).
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. Although the levels of adherent cells
in 16-wk MRL/faslpr mice are low compared
with some other types of inflammatory response, they are comparable to
the increased levels of adhesion observed previously in the dermal
microcirculation of 16-wk MRL/faslpr mice
(19). Furthermore, the levels of adhesion observed in this
model of chronic, systemic inflammation reach 
50% of that achieved
in this microvasculature by systemic treatment with high doses of the
proinflammatory cytokine, TNF- (15). Histological
analysis of pial vessels in brains from each strain also showed that in
16-wk MRL/faslpr mice, the number of
venules containing leukocytes apposed to the endothelial surface was
significantly increased relative to MRL+/+ mice
(Table II). Morphological analysis of
these leukocytes indicated they were almost exclusively mononuclear.
Finally, as previous studies of the cerebral microvasculature have
observed that platelets readily undergo interactions with this vascular
bed, and indeed are capable of promoting interactions between
leukocytes and the endothelium, we assessed platelet recruitment in
16-wk MRL/faslpr mice (15).
However, very few platelets were observed to undergo interactions in
the cerebral microvasculature of these animals, apart from occasional
transient tethering interactions in some mice.
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We next attempted to determine the roles of P- and E-selectin in
mediating the increased leukocyte rolling observed in 16-wk
MRL/faslpr mice. Two approaches were used
to examine the role of P-selectin. First, the effect of
function-blocking mAbs against P-selectin in wild-type
MRL/faslpr mice was determined (Fig. 4A). In mice with the most
markedly elevated leukocyte rolling (20–30 cells/min), P-selectin
blockade reduced leukocyte rolling to 4 cells/min. However, in 50%
of mice examined, leukocyte rolling was in the range 4–10 cells/min.
In these animals, anti-P-selectin had no appreciable effect. To
further delineate the role of P-selectin, we examined
P-selectin-deficient MRL/faslpr mice at 16
wk. The level of leukocyte rolling in these mice was consistently
observed to be 4 cells/min (Fig. 4B), comparable to that
seen in wild-type MRL/faslpr mice after
treatment with anti-P-selectin mAb. Analysis of leukocyte rolling
velocities in wild-type MRL/faslpr mice
showed that before P-selectin blockade, leukocytes were rolling at a
wide range of velocities, from <10 µm/s to >100 µm/s (mean =
40.5 µm/s) (Fig. 5). However, following
P-selectin blockade, the remaining leukocytes rolled more slowly, with
no rolling observed above a velocity of 60 µm/s (mean = 28.0
µm/s). Similarly, in
P-selectin-/--MRL/faslpr
mice, leukocyte rolling was not observed at velocities greater than 50
µm/s (mean = 6.6 µm/s) (Fig. 5). These observations suggested
that the main function of P-selectin in the cerebral microvasculature
of MRL/faslpr mice was to support the
rolling of leukocytes at velocities above 50–60 µm/s. However,
alternative molecules appear to be more important in supporting rolling
at lower velocities.
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).
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). Given that E-selectin has previously
been shown to be important in reducing leukocyte rolling velocity in
inflamed microvessels (27), velocity was also assessed in
these animals. Mean rolling velocity did not alter following E-selectin
blockade (27.2 ± 4.2 µm/s pre-mAb vs 25.9 ± 3.1 µm/s
post-mAb; mean ± SEM, n = 5). Together these
observations indicated that E-selectin played no role in these
interactions.
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4 integrin in leukocyte-endothelial
cell interactions in cerebral microvasculature of lupus-prone mice
As the 4 integrin has been shown to
mediate leukocyte rolling in other models of chronic inflammatory
disease (13), we next examined the effect of
4 integrin blockade.
4 Integrin blockade in 16-wk wild-type
MRL/faslpr mice significantly reduced
leukocyte rolling, and also reduced leukocyte adhesion to basal levels
within a 20-min period of mAb treatment (Fig. 8, A and B).
Residual leukocyte rolling was observed in two of six animals. This
rolling displayed the elevated rolling velocity characteristic of
P-selectin-dependent rolling observed earlier, and was eliminated by
P-selectin blockade (data not shown). The effect of
4 integrin blockade was more striking in 16-wk
P-selectin-/--MRL/faslpr
mice. In these animals, treatment with R1-2 eliminated all leukocyte
rolling (Fig. 8C). In addition, R1-2 significantly reduced
adhesion, as seen in wild-type
MRL/faslpr mice (Fig. 8D).
These observations indicate that the 4
integrin is essential for P-selectin-independent rolling in this model,
and may also directly mediate leukocyte adhesion.
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4 integrin
expression by circulating leukocytes contributed to the observed
increase in 4 integrin-dependent interactions,
we measured 4 integrin expression in
leukocytes isolated from 16-wk MRL/faslpr
and MRL+/+ mice. These experiments revealed no
quantitative differences in 4 integrin
expression by circulating leukocytes from the two strains of mice (data
not shown). VCAM-1 mediates leukocyte-endothelial cell interactions in the cerebral microvasculature of lupus-prone mice
As it has been previously reported that the potential
4 integrin ligand VCAM-1 is expressed at
elevated levels in the brains of
MRL/faslpr mice at 14 wk of age
(28), we examined the role of VCAM-1 in cerebral leukocyte
trafficking in these mice. Treatment of 16-wk
MRL/faslpr mice with the anti-VCAM-1
mAb MK/2.7 reduced leukocyte rolling in most of the mice examined (Fig. 9A). Moreover, all
VCAM-1-independent residual rolling had a high rolling velocity (80
µm/s) characteristic of the P-selectin-dependent rolling previously
observed in MRL/faslpr mice after
treatment with an 4 integrin mAb. This
residual rolling was abolished by P-selectin blockade (Fig. 9A). Examination of leukocyte adhesion revealed a similar
pattern in that adhesion was almost completely eliminated in four of
six mice examined (Fig. 9B). Finally, VCAM-1 blockade also
profoundly reduced rolling in
P-selectin-/--MRL/faslpr
mice (data not shown), similar to the effect of
4 integrin blockade in these animals. These
data suggest that, as observed for the 4
integrin, VCAM-1 plays a critical role in mediating cerebral leukocyte
trafficking in lupus-prone mice.
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To determine whether the leukocyte recruitment observed in
MRL/faslpr mice was associated with a
disruption in the blood-brain barrier, we assessed cerebral
microvascular permeability in 16-wk
MRL/faslpr mice, and compared it with that
in similarly aged MRL+/+ mice. No difference in
leakage of 70-kDa FITC dextran was observed between the two strains of
mice (Fig. 10). Similarly, cerebral
microvascular permeability in
P-selectin-/--MRL/faslpr
mice was found to be indistinguishable from that in
MRL+/+ and
MRL/faslpr mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4 integrin/VCAM-1
pathway was critical in mediating both rolling and adhesion in these
animals. In contrast, experiments in both wild-type
MRL/faslpr mice and
P-selectin-/--MRL/faslpr
mice showed that P-selectin mediated some rolling in cerebral
microvessels, but this rolling was mainly of a high velocity that was
not critical in allowing leukocytes to ultimately adhere. These
observations indicate that in this spontaneous model of systemic
inflammatory disease, the 4 integrin/VCAM-1
pathway plays a key role in allowing recruitment of mononuclear
leukocytes to the cerebral microvasculature.
We have previously documented a progressive increase in
leukocyte-endothelial cell interactions in the dermal microcirculation
of MRL/faslpr mice, involving both
granulocytic and mononuclear cells (19). The enhanced
rolling interactions in dermal microvessels were mediated predominantly
via P-selectin, with E-selectin playing an additional minor role. These
findings were associated with alterations in P-selectin glycoprotein
ligand-1 expression by circulating cells, as well as changes in
P-selectin expression in the dermal microvasculature. In contrast, in
the present study, examination of the cerebral microcirculation in the
same population of mice revealed that enhanced leukocyte recruitment in
the cerebral microvasculature was mediated by the
4 integrin/VCAM-1 pathway, independently of P-
and E-selectin. This divergence is not unexpected, in that several
studies have shown that the molecular mechanisms of leukocyte
recruitment vary between individual tissues, even in response to an
identical inflammatory stimulus (29, 30, 31). Moreover, it has
previously been demonstrated that expression of VCAM-1 is significantly
increased in the brains of MRLfaslpr mice
at 14 wk (28). The timing of this increase in VCAM-1
expression correlates well with the observed increase in
VCAM-1-dependent interactions observed in the present study. Together,
these findings suggest that the increase in VCAM-1 expression in the
cerebral microcirculation is of key importance in mediating the
observed increase in cerebral leukocyte trafficking in
MRL/faslpr mice. It is noteworthy that
despite previous evidence of alterations in P-selectin glycoprotein
ligand-1 expression by circulating leukocytes in these mice, the
minimal role of the endothelial selectins indicates that alterations in
selectin ligand expression were without effect on leukocyte recruitment
to the cerebral vasculature. Clearly, in this model of systemic
inflammatory disease, changes in selectin ligand expression by
circulating cells alone are insufficient to mediate an increase in
leukocyte recruitment to this vascular bed. Additional alterations in
adhesion molecule expression in the local microvasculature must also be
required.
Although there are few other studies that have used intravital
microscopy to examine the microvasculature in animals affected with a
chronic systemic inflammatory disease, some novel observations of
aberrant leukocyte trafficking have been made in rats with chronic
adjuvant-induced vasculitis. Johnston et al. (13, 32)
examined leukocyte trafficking in mesenteric postcapillary venules in
rats following immunization with CFA, and noted that
leukocyte-endothelial cell interactions increased markedly in the days
following immunization. The molecular mechanisms responsible for these
increased interactions were quite distinct from those at work under
acute inflammatory conditions. As in the present study, it was found
that the 4 integrin was of key importance in
mediating the increases in both rolling and adhesion. Given that the
4 integrin is most highly expressed on
mononuclear leukocytes and eosinophils, but not neutrophils, this
suggested that these interacting cells were predominantly mononuclear
leukocytes. This contention was supported by the observation that
rolling and adhesion were not diminished by neutrophil depletion
strategies (13). Similarly, in the present study, a role
for the 4 integrin/VCAM-1 pathway was observed
concurrent with an exclusively mononuclear infiltrate. This is
consistent with a more prominent role for mononuclear leukocytes in
chronic inflammatory responses.
Previous analysis of the cerebral microvasculature has shown that it
responds to inflammatory stimulation in a highly unique manner.
Constitutive leukocyte rolling is rarely detectable in the pial
circulation, and intracerebral injection of acute inflammatory agents
does not induce leukocyte recruitment to the CNS parenchyma
(15, 16, 17). Nevertheless, the cerebral microvasculature is
not entirely refractory to inflammatory stimuli. Systemic activation
with TNF- induces leukocyte rolling and adhesion in the pial
circulation, via a highly unusual mechanism involving nonoverlapping
roles for P-selectin, E-selectin, and platelets (15).
Also, recruitment of encephalitogenic T cell blasts to the uninflamed
microcirculation of the spinal cord has been observed to bypass the
normally critical process of rolling, with these leukocytes capable of
undergoing immediate capture and arrest on the endothelial surface in
both capillaries and postcapillary venules (18).
Interestingly, as seen in CNS microvessels in
MRL/faslpr mice, in the latter study, the
4 integrin/VCAM-1 pathway was solely
responsible for recruitment of encephalitogenic T cell blasts
(18). The observation of the 4
integrin/VCAM-1 pathway mediating leukocyte recruitment independently
of selectin function in these two studies further emphasizes the
distinctive nature of recruitment to the cerebral vascular bed. This
pathway has only been observed to operate rarely, in unique situations
such as rolling of hemopoietic progenitor cells in bone marrow
microvessels (25). However, our data show a further
distinction from the observations of Vajkoczy et al.
(18), in that we did not observe immediate capture
of leukocytes in the cerebral microvasculature of
MRL/faslpr mice. All recruited leukocytes
were observed to undergo rolling in postcapillary venules. These
findings further emphasize that even under the unique conditions of the
cerebral microvasculature, different inflammatory states invoke
alternative recruitment mechanisms.
The minimal role of P-selectin in this model may be considered somewhat
surprising in light of previous examinations of cerebral inflammation.
P-selectin blockade has been observed to reduce brain injury induced by
permanent middle cerebral artery occlusion (33).
Furthermore, direct observation of the cerebral microcirculation has
revealed a critical role for P-selectin in short-term models, such as
exposure to nicotine, systemic TNF-, and LPS (15, 34, 35). Also, in a recent study of the experimental autoimmune
encephalomyelitis model of CNS inflammation, leukocyte trafficking
during active disease was shown to be highly dependent on
P-selectin-mediated initiation of rolling (36). It is
interesting to note that this study also revealed a role for the
4 integrin in mediating leukocyte rolling and
adhesion, but in contrast to the present study, these
4 integrin-mediated interactions were
critically dependent on initiation of rolling via P-selectin. There are
several potential explanations for this difference. One possibility is
that distinct leukocyte populations are being recruited in each of
these studies. Indeed, neutrophils were the principal type of leukocyte
recruited following systemic treatment with TNF-, in contrast to the
mononuclear leukocytes observed in the present study (15).
Alternatively, the level of P-selectin expression in the brains of
MRL/faslpr mice may not be elevated to the
same extent as has been demonstrated in several of these models,
including the TNF-, LPS, and experimental autoimmune
encephalomyelitis studies (15, 35, 36). It is noteworthy
that measurement of P-selectin expression in various organs of
MRL/faslpr mice has shown no increase from
basal levels, despite the presence of ongoing inflammation in these
animals (37). This suggests that the chronic inflammatory
conditions present in these mice are not conducive to increased
expression of P-selectin.
It has previously been reported that MRL/faslpr mice display evidence of enhanced macromolecular leakage across the blood-brain barrier (5). This was based on immunohistochemical analysis that revealed that IgG and IgM accumulated in extravascular areas in the brains of MRL/faslpr mice, with the amount of extravascular Ig increasing progressively from 8 to 26 wk. However, this observation was associated with a concomitant 5-fold increase in serum Ig. It is possible that the presence of these high levels of Ab in the serum may have falsely exaggerated the level of permeability assessed using this approach. Moreover, significant numbers of lymphocytes were also detectable in the brain parenchyma, where they may have acted as cellular sources of Ab behind the blood-brain barrier. In the present experiments, we assessed cerebral microvascular permeability using an in vivo technique that provides an instantaneous measurement of macromolecular leakage that is unaffected by the level of serum Ig or Ab production within the brain. Using this approach, we observed no difference between MRL+/+ and MRL/faslpr mice at 16 wk of age, despite the existence of significant levels of leukocyte adhesion in the latter group. This suggests that the level of inflammatory insult to the brain in MRL/faslpr mice at 16 wk is insufficient to compromise the function of the blood-brain barrier. However, this does not exclude the possibility that increases in permeability do occur in older mice, as the inflammatory response progresses.
Treatment of MRL/faslpr mice with
immunosuppressive agents such as cyclophosphamide reduces both
leukocyte recruitment into the brain and behavioral deficits
(38). This suggests that attenuation of leukocyte
recruitment to the brain may also be beneficial in SLE patients. The
results of the present study illustrate that the
4 integrin/VCAM-1 pathway is of key importance
in mediating leukocyte recruitment to the CNS in
MRL/faslpr mice. This raises the
possibility that this specific molecular pathway may also be of
relevance in mediating CNS leukocyte recruitment in SLE patients.
Clearly, further examination of the recruitment mechanisms at work in
the CNS of lupus patients may determine whether these molecules present
a relevant therapeutic target in cerebral lupus.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael J. Hickey, Centre for Inflammatory Diseases, Department of Medicine, Monash University, Block E, Monash Medical Centre, 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; CSF, cerebrospinal fluid.
Received for publication August 9, 2002. Accepted for publication October 23, 2002.
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4 integrin to recruit leukocytes to the central nervous system in experimental autoimmune encephalomyelitis. J. Immunol. 169:1000.This article has been cited by other articles:
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, Denotes p < 0.05 vs
8- and 12-wk MRL/faslpr mice. *,
Denotes p < 0.05 vs 16-wk MRL+/+
mice.
