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*
Department of Pathology, University of Verona, Verona, Italy;
Department of Neurological Sciences, Istituto di Ricovero e Cura a Carattere Scientifico, Ospedale Maggiore Policlinico, and "Dino Ferrari" Center, Milan, Italy;
Department of Pediatrics, Pathology, and Microbiology-Immunology, Dalhousie University, Halifax, Nova Scotia, Canada;
Institute of Cell Biology, Center for Molecular Biology of Inflammation, University of Muenster, and Max-Planck-Institut, Muenster, Germany; and
¶ Department of Pathology, Stanford University, Stanford, CA 94305
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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or LPS, proteolipid protein
139–151 autoreactive T lymphocytes rolled and arrested; notably, only
a few peripheral lymph node cells rolled and firmly adhered. Abs
anti-P-selectin glycoprotein ligand-1 and anti-E- and
P-selectin blocked tethering and rolling of autoreactive lymphocytes,
suggesting that P-selectin glycoprotein ligand-1/endothelial selectins
are critical in the recruitment of lymphocytes in inflamed brain
venules. E- and P-selectin were expressed on cerebral vessels upon in
vivo activation and had a patchy distribution during the preclinical
phase of active and passive experimental autoimmune encephalomyelitis.
LFA-1/ICAM-1 and 4 integrins/VCAM-1 supported rolling,
but were not relevant to rolling velocity. Firm arrest was mainly
mediated by LFA-1 and ICAM-1. Pretreatment of autoreactive lymphocytes
with pertussis toxin blocked integrin-dependent arrest, implicating a
requirement for Gi protein-dependent signaling in vessels
from nonlymphoid districts. In conclusion, our data unveils the
molecular mechanisms controlling the recruitment of autoreactive
lymphocytes in inflamed cerebral vessels and suggest new insights into
the pathogenesis of autoimmune inflammatory diseases of the
CNS. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The process leading to lymphocyte extravasation is a finely
regulated sequence of steps controlled by both adhesion molecules and
activating factors. It involves: 1) initial contact (tethering or
capture) and rolling along the vessel wall mediated by selectins and
integrins, and their ligands; 2) chemoattractant-induced heterotrimeric
Gi protein-dependent intracellular biochemical
changes leading to integrin activation; 3) integrin-dependent firm
arrest; and 4) diapedesis (5, 6, 7). Although this sequence
of events likely represents a stereotyped leukocyte response to
proadhesive and chemotactic stimuli, selective recruitment of various
lymphocyte subtypes under physiological and pathological situations
depends on the action of different classes of adhesion molecules and
activating factors whose combination generates a tissue-specific
"area code" (8). Previous immunohistologic studies
have shown that resting lymphocytes do not breach the BBB. In contrast,
activated T lymphocytes, can cross the BBB regardless of their Ag
specificity, but only T cells that recognize the CNS Ag persist and can
recruit other inflammatory cells (9). Therefore, it is
believed that the state of activation of lymphocytes is more important
in inflammatory cell homing to the brain than Ag specificity
(10). In vitro adhesion assays have shown that binding of
lymphocytes to inflamed brain vessels is mainly mediated by
LFA-1/ICAM-1, very late activation antigen (VLA)-4/VCAM-1
(11, 12). Moreover, blocking Abs against adhesion
molecules involved in the interactions between leukocytes and
endothelium are able to inhibit the development of EAE,
suggesting that interfering with not yet well elucidated steps of
leukocyte extravasation can be therapeutic for autoimmune inflammatory
diseases of the brain (12, 13, 14, 15). It was previously shown
that anti-P-selectin Abs are able to block leukocyte rolling on
nicotine-activated endothelium in pial vessels (16).
Recent intravital microscopy work has shown that both E- and P-selectin
are required for efficient tethering and rolling of neutrophils in
brain vessels in mice treated with TNF- (17). Moreover,
a recent immunohistologic study suggested that P-selectin may be
involved in the early recruitment of encephalitogenic lymphocytes into
the brain (18).
All intravital microscopy studies used until now to determine the interactions between blood-circulating leukocytes and brain endothelium were conducted by performing a cranial window into the skull using a drill. In these experiments, animals need to be artificially ventilated, while bleeding and tissue overheating from cautery may occur. So far, no study has been performed to determine the behavior of different leukocyte subpopulations and of cells in different states of activation in cerebral vessels. The present study was designed to assess the adhesion cascade of lymphocytes in brain microcirculation. We set up a novel intravital microscopy model allowing visualization of cerebral vessels through the skull and analysis of the interactions between different lymphocyte subpopulations and the endothelium in the brain microvasculature of mice. Our findings show that brain endothelium in EAE-susceptible mice requires activation to support adhesive events and that activated, but not resting, lymphocytes interact efficiently. We also provide evidence that P-selectin glycoprotein ligand-1 (PSGL-1) and endothelial selectins represent key molecules in the recruitment of lymphocytes into the brain. E- and P-selectin are expressed during preclinical EAE suggesting a role for these molecules in the recruitment of leukocytes in the pathology of brain inflammation. Moreover, we show that LFA-1 and ICAM are critical in lymphocyte adhesion and that integrin-dependent arrest requires heterotrimeric Gi protein-dependent signaling. We conclude that the brain endothelium expresses a combination of adhesion ligands and activating factor(s) for Gi-linked receptors that together mediate lymphocyte recruitment, and that the combination of molecules involved in this CNS venule-adhesion cascade strongly favors the arrest of activated vs naive T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anti-L-selectin Mel-14, anti-4
PS/2, anti-LFA-1 -chain TIB213, anti-VCAM-1 MK 2.7, and
anti-ICAM-1 YN 1.1.7.4 mAbs were obtained from American Type
Culture Collection (Manassas, VA). Anti-E- and P-selectin
(anti-E-selectin and anti-P-selectin) and anti-PSGL-1 (4RA10) Abs
were obtained as previously described (19, 20, 21). Green
5-chloromethylfluorescein diacetate or orange
5-(and-6)-(((chloromethyl)benzoyl)amino)tetramethylrhodamine (Molecular
Probes, Eugene, OR) were prepared as a stock solution in DMSO
and kept at -20°C until the moment of use. Pertussis toxin (PTX) was
purchased from Calbiochem (Inalco, Italy) and the mutant PTX having two
amino acid substitution (PT9K/129G) was a generous gift of Dr. R.
Rappuoli (Chiron Biocine, Siena, Italy).
Preparation of lymphocytes and fluorescent labeling
Resting lymphocytes were obtained from peripheral lymph nodes (PLN) from young adult SJL mice. Cell number and viability were assessed by trypan blue exclusion.
Production, characterization, and maintenance of proteolipid protein (PLP) 139–151-specific T cell lines was as previously described (22). In brief, SJL/J mice were immunized with 250 µg of peptide PLP139–151. Ten days later, draining lymph nodes were removed and stimulated with 30 µg/ml peptide for 4 days. T cell lines were obtained by stimulation of these cultures every 14 days with irradiated syngenic spleen cells at a ratio of 1:10 (T cell vs irradiated spleen cells). For the intravital microscopy experiments, lymphocytes were Ag-stimulated for 4 days and then kept for 1–5 days in Ag-free medium. Lymphocytes were starved of Ag after antigenic stimulation to reduce background adhesiveness and to be able to reveal inside-out signaling generated by local proadhesive agonists.
For intravital microscopy experiments, lymphocytes were suspended at a concentration of 0.5 x 106/ml in DMEM without sodium bicarbonate (Sigma-Aldrich, St. Louis, MO), supplemented with 20 mM of HEPES, 5% FCS (pH 7.1) and were labeled with either green 5-chloromethylfluorescein diacetate (Molecular Probes) or orange 5-(and-6)-(((chloromethyl)benzoyl)amino)tetramethylrhodamine (Molecular Probes) for 20 min at 37°C.
Animal preparation for intravital microscopy
SJL young females were purchased from Harlan-Nossan (Udine,
Italy) and were housed and used according to current European community
rules for the usage of laboratory animals. Mice received no treatment
or were injected i.p. with 12 µg of LPS (Escherichia coli,
026:B6; Sigma-Aldrich) or i.v. with 1 µg of TNF-, 5–6 h or 3–4
h, respectively, before starting the intravital experiment. Animals
were anesthetized by i.p. injection (10 ml/kg) of physiologic saline
containing with ketamine (5 mg/ml) and xylazine (1 mg/ml). The
recipient was maintained at 37°C by a stage-mounted strip heater
Linkam CO102 (Olympus, Melville, NY). A heparinized PE-10
polyethylene catheter was inserted into the right common carotid artery
toward the brain. To exclude the noncerebral vessels from the analysis,
the right external carotid artery and the pterygopalatine artery, a
branch from the internal carotid, were ligated. The scalp was
reflected, the skull was bathed with sterile saline, and a 24 mm
x 24 mm coverslip was applied and fixed with silicon grease. A round
chamber with an 11-mm internal diameter was attached onto the coverslip
and filled with water.
Intravital videomicroscopy
The preparation was placed on an Olympus BX50WI microscope and a
water immersion objective with long focal distance (Olympus Achroplan,
focal distance 3.3 mm, NA 0.5 
) was used. Blood vessels were
visualized by using fluorescent dextrans: 3 mg of FITC-dextran (148
kDa; Sigma-Aldrich) and/or 6 mg of tetramethylrhodamine
isothiocyanate-dextran (155kDa; Sigma-Aldrich) were diluted in
0.3 ml of sterile physiologic saline and centrifuged for 5 min at
14,000 x g (each mouse received 0.05 ml of
supernatant). Fluorescent-labeled cells (2.5 x
106)/condition were slowly injected into the
carotid artery by a digital pump at a flow rate of 0.13–1 µl/s. The
images were visualized by using a silicon-intensified target
videocamera (VE-1000 SIT; Dage-MTI, Michigan, IL) and a Sony SSM-125CE
monitor (Tokyo, Japan). Recordings were digitalized and stored
on videotapes using a digital VCR (Panasonic NV-DV10000; Secaucus,
NJ) and Casablanca digital system (MS MacroSystem Computer,
Witten, Germany). The recordings were made during the injection of the
cells and for a few minutes after the injection ended.
Image analysis
Video analysis was performed by playback of digital videotapes
in real time, or at reduced speed, and frame by frame. Vessel diameter
(D), hemodynamic parameters, and the velocities of rolling
were determined by using a PC-based system including a LG-3 frame
grabber (Psion Corporation, Frederick, MD), NIH Image software 1.61,
and a Casablanca digital system (MS MacroSystem Computer). The
velocities of 
20 consecutive freely flowing cells/venule were
calculated and from the velocity of the fastest cell in each venule
(Vfast), we calculated the mean blood
flow velocities (Vm):
Vm =
Vfast/(2 -
2), where is the ratio of the lymphocyte
diameter to the vessel diameter (23). The wall shear rate
(
) was calculated from = 8 x
Vm/D
(s-1) and the shear stress (
) acting on
rolling cells was approximated by x 0.025
(dyne/cm2), assuming a blood viscosity of 0.025
poise. Lymphocytes were considered as rolling if they traveled at
velocities below Vcrit
(Vcrit =
Vm x x (2 - ))
(23). Lymphocytes that remained stationary on the venular
wall for 30 s were considered adherent. At least 140 consecutive
cells/venule were examined. Rolling and firm arrest fractions were
determined as the percentage of cells that rolled or firmly arrested
within a given venule in the total number of cells that enter that
venule during the same period (24).
In vivo staining of endothelial adhesion molecules
Anti-E- and P-selectin, anti-VCAM-1, and anti-ICAM-1 mAbs and an isotype-matched control Ab (anti-human Ras) were labeled using an Alexa Fluor 488 labeling kit (Molecular Probes). For some experiments, control mAb was also labeled using an Alexa Fluor 566 kit. Fifty micrograms of fluorescent mAb were injected i.v. Twenty minutes later, the animal was anesthetized and perfused through the left ventricle and through a catheter inserted into the right carotid with cold PBS. The skull and meninges were removed and cerebral vessels were visualized using the intravital microscopy setting.
Induction of active and passive EAE
Six- to 8-wk-old SJL/J females were purchased from Harlan-Nossan. PLP139-151-specific T cell lines were produced as previously described (22). Ag-stimulated T cells (25 x 106) were injected i.v. into mice. In the case of active EAE induction, mice were immunized s.c. with 300 µg of PLP139–151 as previously described (25). Mice were checked daily and scored for EAE (22, 25).
Statistics
Statistical analysis of the results was performed by using SPSS 10.0 software (SPSS, Chicago, IL). A two-tailed Student’s t test was used for statistical comparison of two samples. Multiple comparisons were performed using the Kruskall-Wallis test with the Bonferroni correction of P. Velocity histograms were compared using the Mann-Whitney U test and the Kolmogorov-Smirnov test. Linear regressions were analyzed using the Spearman rank correlation test. Differences were regarded significant with a value of p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The parietal skull was sufficiently transparent to allow for the
observation of superficial cerebral vessels. To exclude the bone
vessels, bone marrow vessels, and meningeal vessels from our analysis,
we ligated both the right external carotid and the right
pterygopalatine artery (Fig. 1
A). The pterygopalatine
artery is an extracranial branch of the internal carotid and it
vascularizes mostly extracranial structures, with the notable exception
of the middle meningeal artery, a branch we needed to exclude because
the analysis was performed in parietal vessels (26).
Arterioles were identified as vessels with divergent bifurcations,
whereas confluent branches defined venular segments (23).
Many of the branches of superficial cerebral venules have a
characteristic "interrupted" shape with convex origins which are
due to their emergence on the brain surface from the more profound
layers (Fig. 1B). This peculiarity of cerebral veins makes
them easily recognizable and clearly distinguishes them from bone and
meningeal vessels. We compared the hemodynamic characteristics of right
and left main cerebral veins and no significant differences were
observed during the time course of our experiments (not shown). The
analysis of the injected fluorescent cells could be performed in
cerebral vessels of the ipsilateral hemisphere of the brain after
carotid ligation, but also in the contralateral hemisphere where cells
arrive through the circle of Willis.
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Two populations of lymphocytes were used to study the interactions
with nonactivated brain endothelium: resting cells freshly isolated
from PLN cells and autoreactive T cells specific for PLP139–151 (Table I; data not shown). The behavior of
autoreactive T cells was studied in 30 venules with diameters ranging
from 18 to 104 µm in 9 animals. Similar experiments were performed
with PLN cells (data not shown). Fluorescently labeled lymphocytes were
slowly injected by using a digital pump. In each venule, at least 140
consecutive cells were examined. Neither of the two lymphocyte
populations rolled or firmly adhered to normal brain endothelium (Table I; data not shown). Some autoreactive lymphocytes were mechanically
entrapped in capillaries, but this caused no significant modification
of the hemodynamic parameters. We concluded that nonactivated brain
endothelium does not support lymphocyte interactions.
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Behavior of lymphocytes in activated brain venules
It was previously shown that LPS or TNF treatment is able to
up-regulate adhesion molecules on the endothelium in vivo
(28, 29, 30). We next treated the mice with LPS or TNF 5–6 or
3–4 h, respectively, before starting the experiments. Fluorescently
labeled lymphocytes were able to roll and arrest (Fig. 1
C)
in inflamed brain venules. The rolling fraction of PLP139–151-specific
T cells was significantly higher than for PLN cells (mean ± SD of
8.09 ± 3.78% vs 1.24 ± 1.20%; p < 0.01),
showing that the activation of lymphocytes increases the frequency of
interactions with the brain endothelium (Table I; Fig. 2). The percentages of autoreactive
lymphocytes that rolled in mice treated with LPS or TNF were similar
(Table I). Rolling fractions inversely correlated with venule diameter
but were independent of mean blood flow velocity and wall shear stress
(Fig. 3).
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;
Fig. 4). In mice pretreated with LPS, the
median rolling velocity for autoreactive lymphocytes was 5.1 µm/s
(Table I). In contrast, the median rolling velocity of PLN cells was
137 µm/s and significant reduction in the fraction of rolling cells
<50 µm/s was observed when compared with activated lymphocytes
(p < 0.001).
Vroll from different experiments were
pooled in velocity class and cumulative
Vroll histograms were plotted to
better analyze the rolling differences between PLN cells and
Ag-specific T cells. As shown in Fig. 4B, 47% of
autoreactive T cells have a Vroll of
<5 µm/s. Considering the pool of cells that have a
Vroll <10 µm/s, the difference
between PLN cells and autoreactive lymphocytes is striking: the
percentage of autoreactive cells is 70%, while PLN cells account only
for 9% (p < 0.001), suggesting different
molecular mechanisms mediating rolling interactions in the two
populations. No significant differences in
Vroll of encephalitogenic lymphocytes
was observed between mice treated with LPS and mice treated with TNF
(the median, 6.4 µm/s, was obtained by analyzing 69 cells/6 venules/3
mice).
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PSGL-1/endothelial selectins are critical for tethering and rolling of autoreactive lymphocytes
We next endeavored to identify the molecular mechanisms of
autoreactive lymphocyte rolling. Lymphocytes were Ag-stimulated for 4
days and then kept for 1–5 days in Ag-free medium before intravital
microscopy experiments. Flow cytometry experiments showed a constantly
high expression of VLA-4 during the starvation period, whereas LFA-1
and PSGL-1 expression were constantly high between Ag stimulations
(data not shown). As we expected, treatment of cells with a mAb to
L-selectin had no effect on Ag-activated lymphocyte rolling; in
contrast, 96% of tethering and rolling was blocked by an
anti-PSGL-1 Ab (Fig. 5A).
Blocking effects similar to those observed with anti-PSGL-1 Abs
were obtained with mAbs to P-selectin and anti-P- and E-selectin
used together (inhibition of 96 and 90%, respectively) (Table II and Fig. 5B). The
anti-E-selectin mAb, when used separately, was also able to
substantially block lymphocyte rolling (77% inhibition). These data
show that PSGL-1 expression on lymphocytes is critical for capture and
rolling and that both E- and P-selectin are required on the endothelium
for efficient primary adhesion.
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In vivo experiments have shown that integrins and their
endothelial ligands may also participate in primary adhesion in vivo
(31, 32, 33). To determine whether VCAM-1 and
4 integrins mediate rolling on the activated
brain endothelium, we used mAbs against these molecules. Both Abs
inhibited rolling fractions by 
50% in SJL mice suggesting that
4
1/VCAM-1 are
involved in rolling (Fig. 5, A and B).
Furthermore, to a similar extent, anti-LFA-1 and anti-ICAM-1
Abs were also able to inhibit rolling by 48 and 52%, respectively
(Fig. 5, A and B).
It was previously shown in other experimental models that LFA-1 and
4 integrins are able to strengthen rolling
interactions and to reduce rolling velocity (33, 34, 35). We
asked whether there might be a similarly significant contribution by
4 and
L1 integrins to the
strength of rolling of PLP139–151 encephalitogenic lymphocytes in
brain microcirculation. To address this question, we compared
microvascular hemodynamics and Vroll
(the velocities of at least 10 cells/venule were measured) before and
after Ab treatment. No significant effect using the anti-CD11a mAb,
anti-4 integrins, anti-ICAM-1, and
anti-VCAM-1 could be ascertained (Table II).
Firm arrest is mediated by LFA-1-ICAM-1 and 4
integrin-VCAM-1
To investigate firm arrest, autoreactive lymphocytes were
pretreated with an anti-CD11a Ab (TIB213) or with PS/2 mAb
anti-4 integrins. In other experiments,
mice received an anti-ICAM-1 or anti-VCAM-1 mAb. Firm arrest
was also examined after the treatment of lymphocytes with the
anti-PSGL-1 mAb or after the mice were challenged with anti-E-
or P-selectin mAbs. Importantly, sticking was highly blocked by
treatment with anti-PSGL-1/endothelial selectins, even when LFA-1
function was not blocked (Fig. 6). Firm
arrest was blocked by anti-LFA-1 and anti-ICAM-1 mAbs by 74 and
70%, respectively (p < 0.001) (Fig. 6, A and B). VCAM-1 and 4
integrins were also involved in firm arrest and anti-VCAM-1 and
4 integrin Abs blocked 40% of sticking
(Fig. 6, A and B). These findings suggest that in
a subacute model of inflammation, LFA-1/ICAM-1 are central molecules
involved in firm arrest in inflamed cerebral vessels.
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To determine whether E- and P-selectin are expressed on cerebral
vessels after in vivo activation of the endothelium, we injected Alexa
488-labeled anti-E or P-selectin mAbs. Mice received 12 µg of LPS
i.p. 5–6 h before the injection of Alexa-labeled mAbs. Twenty minutes
after mAb administration, either anti-E- or P-selectin Ab
accumulation was detectable with videomicroscopy at the luminal surface
of cerebral venules, but not arteries (Fig. 7 and Table III). However, no positivity for
anti-selectin mAbs was detected in normal mice (not shown). Alexa
488-labeled anti-ICAM-1 and anti-VCAM-1 Abs accumulated in both
arterioles and venules, supporting the data we obtained with blocking
Abs. An isotype-matched control Ab revealed no positivity in inflamed
cerebral venules (Fig. 7).
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To understand if our intravital microscopy data might be relevant
to the pathogenesis of EAE, we next studied the expression of E- and
P-selectin during preclinical and clinical phases of both transfer and
active EAE. In addition to previous immunohistochemical studies, our
videomicroscopy experimental setting allowed us to better explore the
expression of selectins on vessels that can be visualized for the
majority of their length. Fluorescently labeled Abs were injected in
mice and localization to venules was assessed as described above. As
shown in Table III, we found venular accumulation of the
anti-E-selectin mAb in actively induced EAE on day 5
postimmunization and also during clinical disease (day 9). In transfer
EAE, positive vessels for the anti-E-selectin mAb were apparent 2
days postinjection of encephalitogenic cells and E-selectin staining
was also detected when disease was clinically evident (day 5; Table III). Previous work suggested a role for P-selectin in the early
recruitment of encephalitogenic cells (2 h postinjection of T cells;
Ref. 18). However, we found no anti-P-selectin Ab
accumulation in cerebral vessels after 2 h postinjection of
encephalitogenic cells (Table III). In active EAE, positivity for
anti-P-selectin Abs was detected on day 5 postimmunization, while
during transfer EAE, positivity was revealed after 2 days postinjection
of encephalitogenic lymphocytes. However, positivity for
anti-P-selectin during preclinical EAE was present on fewer vessels
than for the E-selectin mAb. No anti-P-selectin binding was
detected during clinical EAE in either transfer or active models (Table III). Positivity for the anti-P-selectin mAb was not encountered
even when the clinical score was 3 or 4 (data not shown). Alexa 488- or
Alexa 566-labeled control Ab revealed no positivity during preclinical
or clinical EAE (Fig. 8; data not shown).
To obtain accurate results, in some experiments during the preclinical
phase of EAE, we injected both Alexa 488-labeled anti-E-selectin
mAb and Alexa 566 control mAb (data not shown). Interestingly, our
experimental setting allowed us to observe that anti-selectin mAb
binding had a patchy distribution and often positivity was encountered
at sites where vessels branch, suggesting the existence of preferential
recruitment areas.
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It is widely accepted that integrins require activation to promote
rapid arrest under flow. It was previously shown that both LFA-1 and
VLA-4 can mediate firm arrest after Gi-linked
signaling to trigger activation of these integrins (24, 36, 37). We hypothesized that lymphocyte sticking in inflamed
cerebral vessels may also involve PTX-sensitive signaling events. PTX
treatment of autoreactive lymphocytes had no effect on rolling, but
abolished 80% of the sticking fraction (Fig. 9). In contrast, a mutant holotoxin
(PTX9K/129G) had no effect on arrest in brain vessels (24, 36, 38).
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i proteins were blocked by PTX. There
was no significant difference between
Vroll of control lymphocytes and cells
treated with PTX or mutant toxin (Table II; data not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our experiments, we adopted two sets of lymphocytes: PLN cells and brain-specific lymphocytes. We found that an inflamed brain endothelium, but not a nonactivated endothelium is able to mediate adhesion of lymphocytes; thus, we concluded that in our experimental model, the endothelium requires in vivo activation to support adhesive events. In inflamed cerebral vessels, we observed a low frequency of adhesive interactions using PLN cells (the majority naive), whereas Ag-specific lymphocytes efficiently interacted with the endothelium. Thus, efficient lymphocyte recruitment to brain microcirculation requires endothelium, as well as lymphocyte, preactivation. Rolling fractions of autoreactive lymphocytes were higher in vessels with small and medium calibers, but did not correlate with hemodynamic parameters such as mean blood flow velocity and wall shear stress, providing evidence that endothelial, but not hemodynamic, differences are responsible for lymphocyte rolling in mouse brain venules (23).
P- and E-selectin on endothelial cells are primary adhesion molecules for capture and the initiation of slow inflammatory rolling (39, 40). We showed that both P- and E-selectin are expressed on the brain endothelium after in vivo activation with LPS or TNF. In contrast to other intravital microscopy studies in other organs, where either E- or P-selectin are alone able to support leukocyte rolling, participation of both E- and P-selectin is required for efficient tethering and rolling in brain venules. Our data are in accordance with recent results showing that granulocytes also need both endothelial selectins to roll in cerebral vessels (17). We found that anti-PSGL-1 Abs, block 96% of capture and rolling in inflamed brain vessels, suggesting that PSGL-1 represents a key molecule in the tethering and rolling of lymphocytes in inflamed brain vessels. Moreover, recent results obtained by Carrithers et al. (18), suggested that P-selectin, a PSGL-1 ligand, may be involved in the early recruitment of encephalitogenic lymphocytes into the brain. Our results are also consistent with recent data showing that PSGL-1 is able to efficiently mediate tethering and rolling in vivo on both P- and E-selectins (41). Abs to PSGL-1 inhibit interactions of leukocytes to areas of inflammation in animal models (42, 43). Moreover, recombinant soluble forms of PSGL-1 inhibit selectin-mediated inflammatory responses in models of inflammation and thrombosis in vivo (44, 45). Taken together, these results suggest that inhibition of PSGL-1 interactions with endothelial selectins might prevent the accumulation of activated lymphocytes into the brain, having a protective role in autoimmune inflammatory diseases of the CNS. This is also supported by previous studies showing that sulfated polysaccharides are able to inhibit EAE presumably by interfering with the passage of lymphocytes across the brain endothelium (46, 47).
In our experimental model rolling interactions are also partially blocked by Abs to LFA-1 and VLA-4, suggesting that selectins and integrins must cooperate to ensure an efficient rolling. We found that Abs to these integrins or to ICAM-1 and VCAM-1 do not increase rolling velocity, suggesting that residual PSGL-1/endothelial selectin interactions are "per se" sufficient to mediate slow rolling. As shown for anti-integrin Abs, rolling velocity is not increased by PTX pretreatment, thus Gi-protein-linked signaling is not relevant for the strength of rolling interactions. However, it was previously shown under in vitro flow conditions that VLA-4 integrin-mediated rolling requires Gi-protein-linked signaling (37). The lack of involvement of integrins and Gi-protein-linked signaling in rolling velocity in our model could suggest that Ag-activated lymphocytes may have a significant proportion of integrins already organized in clusters, which is critical to integrin-dependent rolling (48). Notably, preliminary confocal microscopy data have revealed that the autoreactive lymphocytes that we used for our study already have LFA-1 and VLA-4 organized into big clusters (M. Majeed and G. Constantin, unpublished observation). Although integrins mediate part of the rolling, their engagement does not compensate for the inhibition induced by anti-PSGL-1 Abs, which block 96% of capture and rolling of autoreactive lymphocytes. Thus, we hypothesize that PSGL-1, but not integrins, is the critical molecule that mediates activated lymphocyte tethering on the inflamed brain endothelium.
We documented positivity for both P- and E-selectin before the onset of EAE. Only E-selectin was expressed during clinical EAE. This was also previously found in vessels from acute plaques in MS patients (49). In contrast to our study, previous work by Engelhardt et al. (50) has shown that E- and P-selectin could not be up-regulated on the brain endothelium after LPS or TNF treatment of mice or during EAE. This discrepancy may be due to the fact that we used different mAbs and a more sensitive detection method of fluorescent videomicroscopy. It was previously shown that P-selectin is constitutively absent from cultured brain endothelial cells, but is up-regulated after 4 h of TNF or IL-1 stimulation (51). These data support our findings that a cytokine-activated, but not a normal, brain endothelium is able to express P-selectin. Our results are also in agreement with previous intravital microscopy studies in brain microcirculation showing a critical role of P- and E-selectin in granulocyte recruitment after activation of the endothelium (16, 17). It was previously shown that E- and P-selectin expression on the endothelium preferentially recruits Th1 cells (52). Thus, it is possible that during the preclinical phase of EAE, Th1, but not Th2, cells may be recruited. Further studies are required to clarify this issue. We also provide evidence that during the preclinical phase of EAE, potential "recruitment areas" that express endothelial selectins are up-regulated. These areas were often seen where vessels branch. Thus, as also shown by the exclusive expression of selectins on venules after in vivo LPS treatment, lower shear stress may favor the up-regulation of selectins on the brain endothelium. The presence of these recruitment areas may be of crucial importance to lymphocyte entry into the CNS and might explain the disposition in plaques of inflammation in MS patients.
LFA-1 and ICAM-1 were critical for lymphocyte sticking, whereas
4 integrins and VCAM-1 have a lesser role,
suggesting that during early inflammation in brain microvessels, LFA-1
and ICAM-1 are mainly responsible for firm arrest (Fig. 10).
Moreover, we found that integrin-dependent firm
arrest in brain microcirculation is
blocked by PTX. Thus, as previously shown in studies on naive
lymphocytes homing to Peyer’s patches and lymph nodes (24, 36), Ag-specific lymphocytes also require in situ activation by
an adhesion-triggering agonist which exerts its effect via a
Gi-coupled surface receptor. This is the first
demonstration showing the in vivo requirement for lymphocyte adhesion
of a Gi-protein-linked signaling pathway in brain
vessels. Notably, chemokines, which play a central role in lymphocyte
adhesion triggering in secondary lymphoid organs, have been recently
shown to trigger rapid LFA-1-dependent lymphocyte adhesion by inducing
a high-affinity state and heterodimer lateral mobility leading to
massive clustering (53). Interestingly, autoreactive
lymphocytes already have a pool of LFA-1 and VLA-4 organized in
clusters (M. Majeed and G. Constantin, unpublished observation). As
stated above, this could be sufficient to mediate rolling, but not firm
adhesion, as the inhibitory effect of PTX suggests, leading us to
speculate that, as also suggested in Peyer’s patches, integrin
affinity triggering by locally presented chemokines may be required for
rapid autoreactive lymphocyte arrest on cerebral vessels.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gabriela Constantin, Department of Pathology, Section of General Pathology, University of Verona, Strada le Grazie 8, Verona 37134, Italy. E-mail address: gabriela.constantin{at}univr.it
3 Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental autoimmune encephalomyelitis; BBB, blood-brain barrier; VLA, very late activation Ag; PSGL, P-selectin glycoprotein ligand; PTX, pertussis toxin; PLN, peripheral lymph nodes; PLP, proteolipid protein.
Received for publication September 6, 2001. Accepted for publication December 5, 2001.
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4 integrin clustering by immobilized chemokines stimulates leukocyte tethering and rolling on endothelial vascular cell adhesion molecule 1 under flow conditions. J. Exp. Med. 192:495.2 integrin affinity and mobility changes: differential regulation and roles in lymphocyte arrest under flow. Immunity 16:759.This article has been cited by other articles:
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