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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 27, 2001 as doi:10.1096/fj.00-0881fje. |
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Department of Molecular Cell Biology and Immunology, VUMC, Amsterdam, The Netherlands;
* Solvay Research Laboratories, Solvay Pharmaceuticals BV, Weesp, The Netherlands; and
# Endothelial and Epithelial Cell Biology, Institute of Ophthalmology, University College London, London, UK
2Correspondence: Department of Molecular Cell Biology, VUMC, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. E-mail: HE.de_Vries.Cell{at}med.vu.nl
SPECIFIC AIM
Reactive oxygen species (ROS) may play a role in the development of multiple sclerosis (MS) lesions by influencing monocyte migration across the blood–brain barrier (BBB), a critical and early event in MS lesion formation. We studied the role of ROS on the migration of monocytes across a monolayer of cerebral endothelial cells (CEC).
PRINCIPAL FINDINGS
1. ROS inhibitors and scavengers influence monocyte migration
Monocyte migration was monitored in the absence and presence of
specific inhibitors of ROS producing enzymes and scavengers of ROS.
Diphenyleneiodonium (DPI) inhibits the formation of ROS by the NADPH
oxidase complex and allopurinol blocks the activity of xanthine oxidase
(XO). The various ROS can be scavenged with superoxide dismutase (SOD),
which converts superoxide (O2-)
into hydrogen peroxide
(H2O2) or catalase, a
scavenger of hydrogen peroxide
(H2O2). Only allopurinol
and SOD significantly decreased the migration of monocytes across the
CEC monolayer by 40–50%; DPI and catalase showed no effect. This
suggests that O2- production by
XO enhances the migration of monocytes.
2. ROS treatment enhances migration and adhesion of monocytes
During CNS inflammation, different forms of ROS can be produced by
the various cell types of the CNS. To further identify which ROS form
specifically influence monocyte migration, CEC monolayers were treated
with ROS before the migration experiment. Treatment of the CEC for
1 h with O2- significantly
increased monocyte migration (171.7% and 131.6% for nonstimulated and
cytokine-stimulated CEC, respectively) whereas
H2O2 and
OH- revealed no effect (Fig. 1A
).
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Before migration of monocytes across a monolayer of CEC, monocytes
first have to adhere to the monolayer. We tested whether ROS treatment
also had an effect on the adhesion of monocytes to a CEC monolayer. In
Fig. 1B
, it is shown that
O2- also significantly
increased the adhesion of monocytes to a CEC monolayer (148.3% and
138.2% for nonstimulated and cytokine-stimulated CEC, respectively),
whereas H2O2 and
OH- treatment again had no effect. The enhanced
adhesion (as shown in Fig. 1B
) was not due to increased
expression of adhesion molecules (ICAM-1 and VCAM-1), however.
3. O2- treatment leads to tight junction
rearrangement and cytoskeletal changes of CEC
Since no changes in adhesion molecule expression could be
detected, we studied the effect of ROS treatment on the cytoskeletal
arrangement of the CEC. As shown in Fig. 1A
, B
, treatment of
a CEC monolayer with O2-
increased both adhesion and migration of monocytes across the CEC
monolayer. A prominent feature of the BBB is the presence of the tight
junctions between the CEC. A tight junction is a complex of several
proteins, including zona occludens-1 (ZO-1), which is linked to the
cytoskeleton through other proteins (actin binding). We tested whether
O2- treatment disturbed the
arrangement of the tight junctions of CEC. CEC monolayers were treated
with O2- for 1 h and
stained for ZO-1. ZO-1 staining of untreated monolayers shows a fine
smooth network of the borders between adjacent cells, representing a
normal distribution of tight junctions. However, monolayers treated
with O2- shows ruffled borders,
indicating a disruption of the tight junction.
H2O2 and
OH- treatment showed only minor alterations of
the ZO-1 expression.
A clear re-arrangement of F-actin into the formation of stress fibers was observed after incubating the CEC for 1 h with O2–, whereas untreated cells showed no significant changes. H2O2 and OH- treatment of the CEC for 1 h did not induce significant changes in F-actin.
4. PI-3 kinase and PLC inhibitors partly abolish the enhanced
migration of monocytes across the BBB after
O2- treatment
Cytoskeletal changes are often mediated by intracellular signaling
events and it is known that ROS may induce second messengers. We tested
whether the observed effects of
O2- treatment on CEC was
regulated via PI-3 kinase and phospholipase C (PLC). CEC were treated
with O2- for 1 h in the
presence of PI-3 kinase inhibitors (Wortmannin or LY29004) or PLC
inhibitor (ET18OCH3). The treatment of CEC with
O2- again increased the
migration of monocytes across CEC (up to 246.7%). Both the PI-3 kinase
inhibitors Wortmannin (164.2%) and LY29004 (163.3%) could partly
abolish the effect of O2- on
the migration of monocytes across a CEC monolayer (Fig. 2
). The PLC inhibitor could completely abolish the
effect of O2- on the migration
of monocytes across a CEC monolayer (102.9%). The combination of a
PI-3 kinase inhibitor (LY29004) and a PLC inhibitor
(ET18OCH3) could even reduce the migration of
monocytes across O2-treated CEC until under the
basal migration of monocytes across untreated CEC (80.8%). The
inhibitors alone or in combination had no significant effect on the
migration, suggesting that O2-
alone activates signaling events in the CEC.
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5. O2- induces PLC activation in CEC
To confirm whether the signal transduction pathway via PLC is
indeed activated by O2-
treatment of CEC, several second messengers were measured during the
O2- treatment of CEC.
IP3 enhancement is associated with the activation
of PLC. An accumulation of IP3 was found during
O2- treatment of CEC cells
(twofold compared to control). The IP3 found
during O2- treatment was equal
to the IP3 found during treatment with DOI, which
served as a positive control (284.7% and 287.2%, respectively).
Because IP3 production is known to lead to
mobilization of intracellular calcium, we investigated calcium
signaling in CEC during O2-
treatment. We showed that intracellular calcium transient occurred
(1–1/2- to 2-fold) within a few seconds after the addition of
O2-. The peak length was 
1
min, after which the transient reduced to basal levels. The baseline
was stable for at least 10 min after the transient. ATP, which served
as a positive control, showed a fivefold
transient.
DISCUSSION
In this study we showed for the first time that only O2- enhanced both monocyte adhesion to and monocyte migration across a CEC monolayer, which served as an in vitro model for the BBB. In contrast, H2O2 and hydroxyl radical (OH-) did not affect either monocyte adhesion or monocyte migration. We also observed that allopurinol (inhibitor of xanthine oxidase) and SOD (scavenger of O2-) could decrease the migration of monocytes across a CEC monolayer, whereas DPI (inhibitor of NADPH oxidase complex) and catalase (scavenger of H2O2) showed no effect. Furthermore, we showed that O2- activated PLC in CEC, as measured by IP3 accumulation and Ca2+ mobilization, which ultimately lead to rearrangements of the tight junctions and the cytoskeleton of the CEC. Therefore, it can be concluded that O2- is the active ROS responsible for the effect on both adhesion and permeabilization of a CEC monolayer. Moreover, we have provide evidence to suggest that XO is the main source of O2- in this system.
From our data, it has become evident that XO-produced O2- triggers cytoskeletal rearrangements in CEC, which facilitate the migration of monocytes across the BBB. It is likely that small extracellularly amounts of ROS are able to activate signal transduction pathways that subsequently can trigger changes in the CEC. In fact, we were unable to detect ROS production during adhesion of monocytes to CEC. Indeed, it has recently been reported that ROS can play an essential role in signal transduction. Here, we have identified (one of) the signal transduction pathway involved in the enhanced migration of monocytes across the BBB after O2- treatment, since inhibitors of PI-3 kinase and PLC were able to block the O2-enhanced monocyte migration across the BBB in vitro. This was confirmed by observations showing that O2- treatment of CEC significantly induced IP3 accumulation and Ca2+ mobilization, which are markers for PLC activation. Recently, it was shown that IP3 accumulation and Ca2+ mobilization in CEC initiated by ligation of ICAM-1 lead to the cytoskeletal changes. Accordingly, we suggest that in our experiments the Ca2+ mobilization, most likely by activation of PLC, resulted in cytoskeletal changes of the CEC and rearrangement of the intercellular tight junctions. Indeed, in epithelial cells it was shown that intracellular Ca2+ affected cellular distribution of the tight junction protein ZO-1.
We established that O2- produced in inflammatory lesions during MS is the only ROS that triggers signal transduction pathways in CEC, such as the activation of PLC and an intracellular calcium transient. Our data support the hypothesis that upon the firm adhesion of monocyte to and migration across CEC, XO alone becomes activated to secrete O2-, leading to cytoskeletal changes in the CEC that facilitate migration of monocytes across the BBB. After migration and subsequent differentiation to infiltrated monocytes/macrophages, however, other ROS may also contribute to lesion development. In a previous study we showed that myelin is able to activate the NADPH oxidase complex of macrophages to produce various forms of ROS, of which only H2O2 and OH- stimulate macrophages to phagocytose myelin. It is obvious that during the different stages of lesion development in MS (migration of monocytes across the BBB and demyelination by myelin phagocytosis by macrophages), different ROS producing enzymes and their products are involved. Results from these studies may lead to potential new pharmacological strategies to intervene in the interaction of monocyte and CEC, thereby diminishing monocyte recruitment into the brain during early lesion formation and limiting the ensuing demyelination process.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0881fje ; to cite this article, use FASEB J. (June 27, 2001) 10.1096/fj.00-0881fje 
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and
Il-1 (black bars) for 48 h before the migration/adhesion
experiments. A) Migration: Results are given as a
percentage of the control migration (n
4). Migration across
(non)stimulated CEC served as controls and were regarded as 100%.
Nonstimulated CEC corresponds 100% to 11.5% ± 0.3% of migrated
cells of total cells, as stimulated CEC corresponds 100% to 18.3% ±
2.3% migrated cells of total cells. **P < 0.001.
B) Adhesion: Results are given as a percentage of the
control adhesion (n=12). Adhesion to (non)stimulated CEC
served as controls and were regarded as 100%. For nonstimulated CEC,
100% corresponds to 16.9% ± 1.3% of adhered cells of total cells;
stimulated CEC corresponds 100% to 28.8% ± 2.1% adhered cells of
total cells. **P < 0.001.
