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* Department of Cell Biology, Institute of Ophthalmology, and
Neuroinflammation Group, Department of Neurochemistry, Institute of Neurology, University College London, London, United Kingdom;
Drug Discovery Program, Departments of Oncology and Biochemistry and Molecular Biology, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, FL 33612; and
Department of Chemistry, Yale University, New Haven, CT 06520
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Leukocytes must be able to leave the circulation and traffic through
the solid tissues of the body. A critical component of this activity is
their ability to migrate through the vascular endothelial cell
(EC)4 wall. Lymphocyte
transendothelial migration in the CNS has been shown to be dependent on
both lymphocyte activation (4) and an ability to
effectively elicit signaling responses in ECs (5, 6, 7). CNS
endothelia, unlike endothelia from most other tissues, are connected
together by impermeable tight junctions forming both the blood-brain
and inner blood-retinal barriers, respectively. Despite these cellular
barriers, a low level of leukocyte traffic into the CNS occurs
(8), which can be dramatically up-regulated during the
development of immune-mediated diseases. It has previously been
reported that the interaction between ICAM-1 (CD54) expressed on brain
ECs and the T cell integrin
L
2 (LFA-1,
CD11a/CD18) is pivotal in mediating transendothelial migration of
lymphocytes through CNS EC monolayers (9, 10, 11). Recent
studies have also demonstrated that ICAM-1 on brain EC not only serves
as a leukocyte adhesion molecule, but upon engagement results in EC
intracellular signaling responses, leading to endothelial facilitation
of transendothelial lymphocyte migration (5, 6, 7).
The efficient transduction of CD54-mediated signaling responses in brain ECs, and consequently, transendothelial migration of T lymphocytes has been shown to be critically dependent on functional EC Rho proteins (5, 6). Rho proteins undergo posttranslational modification which entails prenylation of the C terminus, which is necessary for their correct subcellular localization (12, 13) and function (14). Both RhoA and RhoC are prenylated with a geranylgeranyl isoprenoid group, whereas RhoB is prenylated by either geranylgeranyl or farnesyl isoprenoids (13).
The aim of this study was to determine whether pharmacological targeting of CNS ECs, through the inactivation of Rho proteins with protein prenyltransferase inhibitors (15), are effective in reducing lymphocyte migration through monolayers of CNS ECs in vitro, and the recruitment of lymphocytes to the CNS in vivo. We report in this study, for the first time, that treatment of brain ECs in vitro with inhibitors of protein prenyltransferases inhibits the migration of T lymphocytes through CNS EC monolayers. Moreover, treatment of Biozzi ABH mice with inhibitors of protein prenyltransferases following induction of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, results in substantially reduced leukocyte recruitment to the CNS and is accompanied by a significant attenuation of clinical disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-Deoxy-D-[2,6-3H]glucose, [3H]thymidine, HRP-coupled rabbit anti-mouse and goat anti-rabbit IgG, and ECL reagents were obtained from Amersham International (Bucks, U.K.). Polyclonal anti-Rho Ab (which recognizes RhoA, B, and C by immunoblot analysis) was obtained from Autogenbioclear (Wilts, U.K.). Anti-ICAM-1 (1A29) mAb and antimacrophage mAb were obtained from Serotec (Oxford, U.K.), and anti-CD3 KT3 mAb was from K. Tomonari, Matsuoka (Fukui, Japan). Unless otherwise stated, all chemicals used were obtained from the Sigma Aldrich (Dorset, U.K.).
Adhesion of peripheral lymph node cells to endothelia and transendothelial migration of Ag-specific T lymphocytes
The extensively characterized immortalized Lewis rat brain EC line GP8/3.9 (5, 6, 7), which retains phenotypic characteristics of primary cultures, were maintained as previously described (16). Rat aortic ECs were isolated from aortic explants and cultured as reported previously (17). The encephalitogenic myelin basic protein (MBP) T cell line (a gift from Dr. E. Beraud, Universite de la Mediterranee, Marseille, France) was established from guinea pig MBP-primed Lewis rat lymph nodes and maintained as previously described (18). These cells have been characterized as MHC-class II-restricted CD4+ T cells (19, 20). Lymphocyte adhesion and transendothelial migration assays were conducted as described in detail elsewhere (4, 6).
Preparation of plasma membranes and Western blotting
Ice-cold lysis buffer containing 10 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 1 mM DTT, and 1 mM PMSF was added to cells and incubated on ice for 10 min. Cells were subsequently homogenized and centrifuged at 5000 x g for 10 min to remove nuclei. Supernatants were then centrifuged at 100,000 x g in a Beckman Ultracentrifuge (Beckman Coulter, Fullerton, CA) for 30 min to obtain crude membranes. Membrane pellets were washed with buffer containing 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl2, 1 mM DTT, and 1 mM PMSF, and recentrifuged at 100,000 x g for 30 min. Membrane pellets were then resuspended in sample buffer and proteins resolved on 12.5% SDS-PAGE gels. Proteins were electroblotted on nitrocellulose membranes and immunblotted with either anti-Rho polyclonal Ab (Santa Cruz Biotechnology, Wilts, U.K.) or anti-ICAM-1 mAb (Serotech). Proteins within membrane fractions were visualized following incubation with a 1/15,000 dilution of goat anti-rabbit or goat anti-mouse-HRP (Pierce, Chester, U.K.) and ECL development (Amersham International). Protein concentration was determined using bicinchoninic acid reagent (Pierce).
Induction and treatment of EAE in Biozzi ABH mice
Six- to 8-wk-old Biozzi ABH mice were purchased from Harlan Olac (Bicester, U.K.), and maintained on RM-1(E) diet and water ad libitum. EAE was induced as previously described (21) by s.c. inoculation in the flank with 1 mg of syngeneic spinal cord homogenate in CFA (day 0) and a further inoculation 7 days later (day 7). Animals were monitored daily and clinical signs ranked as follows: normal = 0, limp tail = 1, impaired righting reflex = 2, partial hind limb paralysis = 3, and complete hind limb paralysis = 4 (21, 22). EAE-induced animals were split into three groups; untreated, vehicle-treated, and those treated with protein prenylation inhibitors. Treated animals were injected daily with 0.1 ml i.p. of vehicle (1/1 PBS/DMSO) or inhibitors (25 mg/kg of both FTI-276 and GGTI-297 dissolved in vehicle) from day 9 to 24 postinduction. FTI-276 and GGTI-297 represent the free acid forms of FTI-277 and GGTI-298, respectively, and were used in animal studies. Differences in clinical score between groups were assessed using Mann Whitney U nonparametric statistics.
Following remission of disease, animals that had not spontaneously relapsed were reinoculated s.c. in the flank with 1 mg of spinal cord homogenate in CFA (day 68 after the initial challenge) and the time course to induce relapse assessed. Two groups, consisting of seven control animals (previously exhibiting grade 4 disease) and six protein prenyltransferase-treated animals (previously showing no sign of disease) were monitored daily for disease relapse and were assigned clinical scores, as described above. To evaluate leukocyte influx into the CNS, control EAE animals and protein prenyltransferase inhibitor-treated EAE animals were sacrificed on day 19 postinoculation. The brain and spinal cords were removed and either snap-frozen in liquid nitrogen for immunocytochemistry, or were fixed in formal saline, embedded in paraffin wax, sectioned, and stained with H&E. Cryostat sections were stained using an indirect immunoperoxidase technique for CD3 and antimacrophage marker as described previously (22).
Oxazolone and Con A-stimulated leukocyte proliferation
Leukocyte proliferation in Biozzi ABH mice was initiated
following application of 25 µl of 2.5% oxazolone in acetone:olive
oil (4:1) to the ear. Animals were sacrificed 3 days later and
peripheral lymph node leukocytes isolated from the auricular lymph
nodes. Isolated cells were washed in RPMI 1640 and 200 µl of cells
(2.5 x 106/ml) in RPMI 1640 plus 10% FCS
placed in microtiter plates. Leukocytes from oxazolone-treated animals
were cultured with 1 µCi [3H]thymidine for
8 h or assessed for increase in cell numbers using the aqueous kit
(Promega, Madison, WI). Alternatively, in vitro leukocyte proliferation
assays were performed following isolation of splenic lymphocytes
stimulated with 5 µg/ml Con A before culture with 1 µCi
[3H]thymidine for 8 h. Cells were
harvested using a cell harvester and
[3H]thymidine incorporation determined by
-scintillation spectrometry.
Animal studies
All animal studies were conducted in accordance with our Institutional Review Committee and U.K. Home Office (London, U.K.) guidelines.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To establish the length of treatment with protein
prenyltransferase inhibitors necessary to prevent the majority of Rho
protein being prenylated, and hence inactivated, whole-cell membranes
were prepared from control and treated brain ECs by high-speed
centrifugation. Isoprenylation of Rho proteins are essential for their
efficient association with cell membranes (12). Western
blot analysis of control brain EC membranes showed that Rho proteins
were associated with cell membranes. Following treatment with 10 µM
FTI-277 (23) and 10 µM GGTI-298 (24) for
48 h, there was a significant reduction in the amount of Rho
protein associated with the cell membrane fraction derived from CNS ECs
(Fig. 1
a). This effect was not
observed when the ECs were treated for 24 h, which suggests that
48-h pretreatment with protein prenyltransferase inhibitors is required
to fully prevent the prenylation of cellular Rho proteins and their
subsequent localization at cell membranes. Conversely, inactivation of
endothelial Rho proteins through ADP-ribosylation with C3-transferase
is independent of protein prenylation, and thus, did not affect the
subcellular distribution of Rho proteins (Fig. 1a).
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b). Treatment of cells with
FTI-277/GGTI-298 for 48 h did not effect either the expression or
membrane localization of ICAM-1 (Fig. 1c). Treatment of ECs with inhibitors of protein prenyltransferase inhibits T lymphocyte migration through brain, but not aortic EC monolayers
Rat EC monolayers derived from brain (GP8/3.9 cells) and aorta
were able to support the transendothelial migration of Ag-specific T
lymphocytes over a 4-h period with 43.0 ± 4.6% and 31.4 ±
5.4% of the lymphocytes migrating through the EC monolayers,
respectively. GP8/3.9 cells, which are derived from Lewis rat primary
brain ECs have previously been shown to model primary cultures with
respect to facilitation of transendothelial lymphocyte migration
(16). In addition, it has also previously been
demonstrated that both GP8/3.9 cells and aortic ECs express comparable
levels of Ig superfamily molecules (16). Treatment of
GP8/3.9 cell monolayers with 10 µM FTI-277 for 24 h before and
during the 4-h T lymphocyte coculture did not result in a significant
alteration in T cell migration through the EC monolayer. However, under
identical conditions, treatment with 10 µM GGTI-298 resulted in a
significant inhibition of transendothelial lymphocyte migration to
73.5 ± 6.0% of control migration (p <
0.005 vs controls, n = 30; Fig. 2a). When 10 µM FTI-277 was
used in combination with 10 µM GGTI-298, this resulted in a further
inhibition of lymphocyte migration to 60.4 ± 5.1% of control
migration (p < 0.005 vs controls,
n = 30; Fig. 2a). This level of inhibition
did not approach that achieved with C3 transferase, which inhibited T
cell migration to 18.4 ± 4.1% of control values
(p < 0.005 vs controls, n
= 12).
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b). A combination of both FTI-277 and GGTI-298 resulted in
a further reduction of T cell migration to 39.3 ± 6.4% of
controls (p < 0.005 vs controls,
n = 30, and p < 0.02 vs 24-h-treated
animals; Fig. 2b). This temporal observation is consistent
with the demonstration that inhibition of Rho protein prenylation
required 48-h pretreatment to prevent its association with membrane
fractions. The degree of inhibition of T cell migration with combined
FTI-277/GGTI-298 treatment approached that obtained following
C3-transferase treatment of ECs, which results in an inhibition of
transendothelial lymphocyte migration to 18.4 ± 4.1% of control
value (p < 0.005 vs controls,
n = 12). None of the observed inhibitory effects on migration were due to the prenyltransferase inhibitors affecting the T cells during the 4-h coculture as the presence of the inhibitor during a 4-h coculture alone had no effect on migration (data not shown). Furthermore, treatment of the MBP T cell line for a total of 52 h (48-h pretreatment plus 4-h coculture with EC) with a combination of 10 µM FTI-277 and 10 µM GGTI-298 brought about only a small reduction in T cell migration to 87.4 ± 2.8% of control migration (vehicle-treated T cells = 100.7 ± 1.9%), and did not approach the level of inhibition achieved when the EC monolayer was treated with the inhibitors for the same duration.
Treatment of brain endothelia with either FTI-277 or GGTI-298 for 24 or
48 h, plus the 90 min adhesion assay, did not have any significant
effect on the ability of T lymphocytes to adhere to brain ECs. However,
a combination of both 10 µM FTI-277 and 10 µM GGTI-298 under
identical conditions resulted in small but significant reduction in T
lymphocyte adhesion to brain ECs following both 24 and 48 h
treatments (Fig. 2, a and b).
The finding that treatment of the GP8/3.9 brain EC line with protein prenyltransferase inhibitors is effective in causing a significant reduction in the transendothelial migration of T lymphocytes, but not their adhesion, demonstrates that this effect is predominantly due to the inhibition of EC support of lymphocyte migration.
Contrary to the findings with brain ECs, the treatment of aortic ECs
with a combination of 10 µM FTI-277 and 10 µM GGTI-298 for 48
h and during the T cell coculture did not have any effect on either T
lymphocyte adhesion or migration (Fig. 2c). The inability of
protein prenyltransferase inhibitors to significantly attenuate T
lymphocyte migration through aortic EC cultures suggests that Rho
proteins are not functionally important for facilitating lymphocyte
migration in aortic EC.
Treatment of Biozzi ABH mice with a combination of protein prenyltransferase inhibitors attenuate the clinical signs of EAE
Biozzi ABH mice induced with EAE began to show clinical signs of
disease 13 days after initial inoculation with syngeneic spinal cord
homogenate, with the peak of disease occurring at day 17 (Fig. 3a). Of a total of 15 positive
EAE control animals, 13 developed disease, 2 of which progressed to
grade 3 disease (partial hind limb paralysis) and 11 progressed to
grade 4 (complete hind limb paralysis) (Table I). The remaining two animals showed no
observable signs of disease. The mean clinical score for the whole
group was 3.2 ± 0.4 (n = 15), and of the animals
that developed disease, 3.7 ± 0.1 (n = 13) with a
mean day of disease onset of 15.1 ± 1.2 days.
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a;
Table I). Neither of these disease indices were significantly different
from untreated EAE control animals.
Animals that had been treated from day 9 to 24 with a combination of 25
mg/kg FTI-276 and 25 mg/kg GGTI-297 showed grade 4 disease in only 2 of
the 13 animals with one animal developing grade 3 disease, one animal
showing grade 2 disease (impaired righting reflex), and a further 2
animals with grade 1 disease (limp tail). Seven of the 13 animals,
which were treated with combined prenyltransferase inhibitors, showed
no signs of clinical disease. The mean group score of clinical disease
was 1.3 ± 0.5 (n = 13), which was significantly
different from both untreated control EAE animals
(p < 0.01), and vehicle-treated control EAE
animals (p < 0.05). Of the animals which
developed disease in this group, the mean disease index was 2.4 ±
0.6 (n = 6), which was also significantly different
from untreated control EAE animals (p < 0.05)
(Fig. 3a; Table I). The onset of disease in treated animals
was unchanged at 15.3 ± 0.8 days.
Prior treatment of Biozzi ABH mice with a combination of protein prenyltransferase inhibitors during the acute phase does not prevent subsequent rapid induction of disease following Ag challenge
Biozzi ABH mice, which were induced for EAE but showed no signs of
disease following treatment with the combined protein prenyltransferase
inhibitors during days 9–24, or control animals in which active
disease had remitted after day 30, were reinoculated with spinal cord
homogenate in CFA at day 68. Day 68 represents 44 days since the last
treatment with prenyltransferase inhibitors. Animals were selected that
had not relapsed spontaneously. Both EAE control animals
(n = 7) and protein prenyltransferase
inhibitor-pretreated animals (n = 6) developed severe
disease relapse equating to a mean disease score of 3.6 ± 0.1 and
3.8 ± 0.1, respectively. Disease induction was rapid in both
groups with mean day of onset being 8.0 ± 1.5 and 8.9 ± 2.2
days post reinoculation, respectively (Fig. 3b). Neither
mean group clinical disease score nor mean day of onset was
significantly different between the two groups (Student’s t
test).
Treatment of Biozzi ABH mice with combined protein prenyltransferase inhibitors at disease onset is ineffective
Animals which were first treated with protein prenyltransferase inhibitors at the first clinical manifestation of disease (animals showing at least 0.5 disease index) did not influence the subsequent progression of the disease. Untreated animals showed disease in five of five animals with a mean group score of 3.4 ± 0.2. These values were not significantly different from those of animals treated with protein prenyltransferase inhibitors at disease onset, where six of six animals showed symptoms of EAE with a mean group score of 3.4 ± 0.2. In animals displaying grade 1 disease, significant numbers of leukocytes have already infiltrated the CNS (data not shown).
Treatment of Biozzi ABH mice with a combination of protein prenyltransferase inhibitors restricts leukocyte migration into the CNS
Biozzi ABH mice were sacrificed for histology and
immunohistochemical analysis on day 19 postinoculation, during which
clinical disease is normally severe (25). Sections of
spinal cord, cerebrum, and cerebellum were examined from control EAE
animals (n = 5) and combined protein prenyltransferase
inhibitor-treated EAE animals (n = 6). In EAE control
animals (all grade 4 disease) characteristic lesions were observed
around vessels that consisted of substantial perivascular cuffing of
leukocytes and infiltration into the parenchyma of the spinal cord.
Examination of the cerebrum and cerebellum also revealed leukocyte
cuffing of vessels, although to a much lesser extent. Similar
observations were seen with the vehicle-treated animals. In contrast,
animals which were treated daily between day 9 and 19 with a
combination of 25 mg/kg FTI-276 and 25 mg/kg GGTI-297 (grade 0 disease)
showed no evidence of perivascular cuffing of leukocytes or
infiltration into the parenchyma in any of the tissues examined (Fig. 4, a–h). In contrast to
control groups, immunohistochemical analysis of spinal cord sections
demonstrated the absence of both T lymphocytes and macrophages in
protein prenyltransferase-treated animals (Fig. 4, i–l).
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Isolation of splenic lymphocytes from normal mice, which were
subsequently cultured for 1–3 days including an 8 h pulse with 1
µCi [3H]thymidine, demonstrated that
concentrations of FTI-277/GGTI-298 up to 10 µM were unable to prevent
proliferation of lymphocytes in response to stimulation with 5 µg/ml
Con A (Fig. 5a). However,
culture of splenic lymphocytes in the presence of 100 µM
FTI-277/GGTI-298 showed a significant inhibition of Con A-stimulated
lymphocyte proliferation at all time points tested. To demonstrate that
in vivo concentrations of FTI-276/GGTI-297 are unable to dramatically
inhibit leukocyte proliferation, Biozzi ABH mice were treated with 25
mg/kg FTI-276/GGTI-297 for 2 days before stimulation of lymphocyte
proliferation with 25 µl 2.5% oxazolone applied to the ear. Animals
were maintained on 25 mg/kg FTI-276/GGTI-297 for a further 3 days.
Auricular lymph nodes were isolated and lymph node cells harvested and
cultured with 1 µCi [3H]thymidine for 8
h or within the aqueous cell proliferation assay (Promega) to assess
proliferative responses. Similar numbers of cells were isolated from
nodes in both FTI-276/GGTI-297 and vehicle-treated animals. Populations
of lymph node cells also showed similar oxazalone-stimulated
proliferative responses by both methods in vehicle- and
FTI-276/GGTI-297-treated animals, with treated animals showing a
significant inhibition of leukocyte proliferation (Fig. 5b).
However, significant levels of lymphocyte proliferation were still
observed in the presence of prenyltransferase inhibitors.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rho proteins are substrates for protein geranylgeranyltransferase type I, which catalyzes the addition of a geranylgeranyl group to the C terminus of RhoA and RhoC (26). It has been proposed that RhoB exists in two distinct forms resulting in cellular populations which are either geranylgeranylated or farnesylated (13). The farnesylation and geranylgeranylation of RhoB appear to be catalyzed by farnasyltransferase (27) and geranylgeranyltransferase type I, respectively. Consequently, it has been observed that inhibition of RhoB farnesylation results in a concomitant increase in the levels of geranylgeranylated RhoB (27). Such protein prenylation of Rho proteins, within a C-terminal CAAX box motif, is essential for its effective targeting to cellular membranes (12) and interaction with specific effector molecules (14).
This study has investigated the effect of agents that prevent Rho protein prenylation on leukocyte migration through brain EC monolayers, and recruitment to the CNS in an animal model of multiple sclerosis. The CAAX box peptidomimetic protein prenyltransferase inhibitors FTI-277 and GGTI-298, which are effective in blocking both protein farnesylation and protein geranylgeranylation of Rho proteins, respectively (15), effectively inactivate Rho function. Treatment of brain EC monolayers with these inhibitors results in a significant reduction in transendothelial T lymphocyte migration, which is further increased when used in combination. Rho proteins undergo cycles of prenylation and deprenylation with specific half-lives of the prenylated form. It has previously been determined that the half-life of RhoA prenylation is in the order of 31 h (28). This correlates with the ability of protein prenyltransferase inhibitors to affect both membrane association of Rho and transendothelial lymphocyte migration following treatment for 48 h. RhoB is an immediate early gene (29), and ICAM-1 cross-linking, which mimics leukocyte adhesion to ECs (5, 6), results in the rapid induction of RhoB mRNA (our unpublished observations). In keeping with its role as an immediate early gene, the half-life of this protein is between 2 and 4 h (30). Therefore, this necessitates the continued presence of the protein prenyltransferase inhibitors during the 4-h coculture of ECs and lymphocytes to prevent the prenylation of newly translated RhoB.
Unlike CNS ECs, pretreatment of aortic ECs with a combination of protein prenyltransferase inhibitors was ineffective in attenuating transendothelial lymphocyte migration. This is not due to differential expression of ICAM-1 and suggests that the effects of these agents may be specific to CNS ECs which demonstrate the heterogeneity of endothelia derived from different vascular sites (16). These studies also indicate that lymphocyte migration into the brain paranchyma may be more actively regulated by the vascular endothelium than into other tissues, and therefore, may partly explain the lower level of leukocyte infiltration in the CNS under normal conditions.
To test the efficacy of these prenyltransferase inhibitors in vivo, EAE was induced in the Biozzi ABH mouse. In this chronic relapsing and remitting model of multiple sclerosis (22), the progression of the disease is a direct consequence of a large-scale infiltration of leukocytes into the brain and spinal cord (25). Leukocyte recruitment to the CNS in this model occurs at a precise time postinoculation, and has been reported in detail elsewhere (21, 31). Treatment of Biozzi ABH mice with prenyltransferase inhibitors was able to attenuate dramatically and significantly the infiltration of leukocytes into the CNS of Biozzi ABH mice and to substantially alleviate clinical signs of disease. This treatment regimen reduced both the number of animals showing clinical signs of EAE and the severity of the disease without delaying onset. However, these agents were ineffective in attenuating subsequent disease if first administered after the onset of disease. This is likely to be due to the fact that significant numbers of leukocytes have already entered the CNS and will continue to do so for at least 24–48 h before protein prenyltransferase inhibtors are able to limit further extravasation. These observations are consistent with the in vitro effects of these agents in which 48 h is required to effectively inhibit transendothelial lymphocyte migration. The rapid reinduction of disease in both untreated and animals previously pretreated with prenyltransferase inhibitor demonstrates that initial sensitization to spinal cord homogenate Ag was not affected following treatment with prenyltransferase inhibitors before the acute phase of the disease. As it has previously been noted that induction of disease in naive animals is slower (32), this suggests that these agents act in this model by inhibiting leukocyte migration to the CNS and not by affecting T lymphocyte priming. The fact that animals treated with prenyltransferase inhibitors are still able to develop a delayed-type hypersensitivity response to contact-sensitizing agents, suggests that the major effect of prenyltransferase inhibitors is on the process of leukocyte trafficking and not leukocyte proliferation. It is unlikely that the observed reduction in lymphocyte proliferation could account for the total absence of leukocytes within the CNS. However, it is also likely that the partial inhibition of leukocyte proliferation by prenyltransferase inhibitors may contribute to the effectiveness of these agents in controlling neuroinflamation. Because protein prenyltransferase inhibitors are able to inhibit leukocyte entry into the CNS, it is likely that these agents would not also be effective in attenuating the spontaneous relapse phase of EAE, as this is also the result of leukocyte migration into the CNS. However, because there is a requirement for prenyltransferase inhibitors to be present for 48 h before leukocyte migration into the CNS, and the difficulty in predicting the specific time of relapse in individual animals, these experiments may be technically difficult. Similarly, the requirement for 48 h of preincubation accounts for the ineffectiveness of prenyltransferase inhibitors in attenuating disease when given at onset of clinical signs.
The precise mechanism by which Rho proteins may mediate transvascular migration of leukocytes is currently unknown. However, Rho proteins have been implicated in both the organization of tight junction function (33) and phagocytosis (34) and therefore, may be important for either a transcellular or paracellular migration of lymphocytes through the blood-CNS barriers.
Abs directed against
41 integrin
(35, 36) have previously been shown to inhibit the
clinical signs of EAE and have also demonstrated a marked improvement
in the incidence of new lesions in multiple sclerosis patients with
secondary progressive disease (37). The effectiveness of
these agents in both mouse and human studies suggest that observations
in mouse EAE studies may be accurately extrapolated to human
neuroinflammatory disease. Recently, studies aimed at affecting
intracellular signaling pathways have used general inhibitors of
tyrosine kinases, which were also found to alleviate the clinical signs
of EAE (38, 39), although whether these agents are
affecting leukocytes or vascular ECs was not clear. The observation
that protein prenyltransferase inhibitors are effective in reverting
ras-mediated tumors in mice (40, 41) through
inhibition of Rho prenylation, coupled with the fact that these agents
are believed to possess low toxicity, suggest that multiple sclerosis
in humans may potentially be managed using inhibitors of Rho
prenylation. Alternative therapies targeted at blood-CNS barrier
EC-signaling pathways central to the facilitation of transvascular
migration may also provide future opportunities for controlling
neuroinflammatory disease.
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Acknowledgments |
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Footnotes |
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2 J.G. and P.A. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Peter Adamson or Prof. John Greenwood, Department of Cell Biology, Institute of Ophthalmology, Bath Street, London EC1V 9EL, U.K. E-mail address: padamson{at}hgmp.mrc.ac.uk
4 Abbreviations used in this paper: EC, endothelial cell; EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein.
Received for publication May 14, 2001. Accepted for publication February 1, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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41 integrin. Nature 356:63.[Medline]
4-integrin reverses the MRI-detectable signs of experimental allergic encephalomyelitis. J. Neuroimunol. 58:1.
4 integrin antibody on brain lesion activity in MS. Neurology 53:466.This article has been cited by other articles:
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  A. Reijerkerk, G. Kooij, S. M. A. van der Pol, T. Leyen, B. van het Hof, P.-O. Couraud, D. Vivien, C. D. Dijkstra, and H. E. de Vries Tissue-Type Plasminogen Activator Is a Regulator of Monocyte Diapedesis through the Brain Endothelial Barrier J. Immunol., September 1, 2008; 181(5): 3567 - 3574. [Abstract] [Full Text] [PDF]
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 G. Schreibelt, G. Kooij, A. Reijerkerk, R. van Doorn, S. I. Gringhuis, S. van der Pol, B. B. Weksler, I. A. Romero, P.-O. Couraud, J. Piontek, et al. Reactive oxygen species alter brain endothelial tight junction dynamics via RhoA, PI3 kinase, and PKB signaling FASEB J, November 1, 2007; 21(13): 3666 - 3676. [Abstract] [Full Text] [PDF]
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G. Schreibelt, R. J. P. Musters, A. Reijerkerk, L. R. de Groot, S. M. A. van der Pol, E. M. L. Hendrikx, E. D. Dopp, C. D. Dijkstra, B. Drukarch, and H. E. de Vries Lipoic Acid Affects Cellular Migration into the Central Nervous System and Stabilizes Blood-Brain Barrier Integrity J. Immunol., August 15, 2006; 177(4): 2630 - 2637. [Abstract] [Full Text] [PDF]
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 E. M. Frohman, M. Filippi, O. Stuve, S. G. Waxman, J. Corboy, J. T. Phillips, C. Lucchinetti, J. Wilken, N. Karandikar, B. Hemmer, et al. Characterizing the Mechanisms of Progression in Multiple Sclerosis: Evidence and New Hypotheses for Future Directions Arch Neurol, September 1, 2005; 62(9): 1345 - 1356. [Abstract] [Full Text] [PDF]
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M. E. Gegg, R. Harry, D. Hankey, H. Zambarakji, G. Pryce, D. Baker, P. Adamson, V. Calder, and J. Greenwood Suppression of Autoimmune Retinal Disease by Lovastatin Does Not Require Th2 Cytokine Induction J. Immunol., February 15, 2005; 174(4): 2327 - 2335. [Abstract] [Full Text] [PDF]
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J. Greenwood, C. L. Amos, C. E. Walters, P.-O. Couraud, R. Lyck, B. Engelhardt, and P. Adamson Intracellular Domain of Brain Endothelial Intercellular Adhesion Molecule-1 Is Essential for T Lymphocyte-Mediated Signaling and Migration J. Immunol., August 15, 2003; 171(4): 2099 - 2108. [Abstract] [Full Text] [PDF]
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P. ADAMSON, B. WILBOURN, S. ETIENNE-MANNEVILLE, V. CALDER, E. BERAUD, G. MILLIGAN, P.-O. COURAUD, and J. GREENWOOD Lymphocyte trafficking through the blood-brain barrier is dependent on endothelial cell heterotrimeric G-protein signaling FASEB J, August 1, 2002; 16(10): 1185 - 1194. [Abstract] [Full Text] [PDF]
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) and adhesion of mitogen activated rat peripheral
lymph node lymphocytes (
) to brain and aortic ECs was evaluated as
described in detail in the text. a, Brain ECs were
pretreated for 24 h with the protein prenyltransferase inhibitors
FTI-277 (FTI), GGTI-298 (GGTI), FTI/GGTI combination, or with C3
transferase before coculture with lymphocytes. Protein
prenyltransferase inhibitors were maintained during incubation with
lymphocytes. b, Brain ECs pretreated for 48 h with
FTI, GGTI, and FTI/GGTI combination as described above. c,
Cultures of rat aortic ECs were pretreated for 48 h with FTI,
GGTI, and FTI/GGTI combination, and the inhibitors were maintained in
the media during T cell coculture. In all cases, observations are a
minimum of three independent experiments using 10 wells per assay for
the FTI-277/GGTI-298 inhibitors and 4 wells per assay for
C3-transferase treatment. Data are expressed as mean ± SEM
percentage of control migration. Significant differences between groups
were determined by Student’s t test. *,
p < 0.005 vs controls.
