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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 5, 2003 as doi:10.1096/fj.02-1014fje. |
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,3
* Division of Cell Biology, Institute of Ophthalmology, University College London, London, EC1V 9EL, UK;
Department of Neuroinflammation, Institute of Neurology, University College London, London, WC1N 1PJ, UK; and
Laboratoire d’Immunologie, Faculté de Medécine, Universite de la Méditerranée, Marseille, France
2Correspondence: Division of Cell Biology, Institute of Ophthalmology, University College London, Bath Street, London, EC1V 9EL, UK. E-mail: j.greenwood{at}ucl.ac.uk
SPECIFIC AIMS
Lymphocyte migration across the blood–brain barrier (BBB) is mediated by a CD54-induced signaling pathway in brain endothelial cells that incorporates the small GTPase Rho. As brain EC require functional Rho to support lymphocyte migration, we investigated whether a reduction in the supply of isoprenoids through inhibition of HMG-CoA reductase with lovastatin leads to reduced Rho prenylation, functional inactivation, and subsequent inhibition of both trans-endothelial lymphocyte migration and neuroinflammation.
PRINCIPAL FINDINGS
1. Lovastatin treatment of brain endothelial cells results in the inhibition of trans-endothelial lymphocyte migration
Brain endothelial cell (EC) monolayers were pretreated for 24 h with the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor lovastatin to deplete isoprenoid synthesis, subsequent post-translational prenylation, and hence functional inactivation of EC Rho. This resulted in a significant inhibition of antigen-specific T lymphocyte migration during a 4 h coculture (Fig. 1
) similar to that observed after treatment with C3 transferase, a toxin that specifically ribosylates and inactivates Rho. In this paradigm, the inhibition of migration was due to the effect of lovastatin on EC and not T cell metabolism, as we were unable to provoke inhibition by exposing the T cells to lovastatin during the 4 h coculture (Fig. 1)
. In excess of 4 h exposure to lovastatin was necessary to elicit an effect; this is consistent with the reported half-lives of Rho GTPases. We also determined whether the inhibitory effect of lovastatin on lymphocyte migration was independent of its action on HMGCo reductase. Treatment of EC with the downstream product of HMG-CoA reductase mevalonic acid (mevalonolactone) was able to reverse lovastatin-induced inhibition of lymphocyte migration, demonstrating that its effect on migration was due to the inhibition of HMG-CoA reductase (Fig. 1)
.
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2. Lovastatin-induced inhibition of lymphocyte migration is through its indirect inhibition of post-translational prenylation of brain EC Rho
To determine whether lovastatin-induced inhibition of lymphocyte migration operates through depletion of EC isoprenoids, and thus prenylation of EC Rho, a brain EC line was generated expressing myristoylated Rho. This modification enables Rho to associate with cellular membranes within the correct compartment and function in the absence of prenylation. T cell migration through EC expressing myristoylated Rho was enhanced by 50% over control vector-transfected EC (Fig. 1)
, which is consistent with endothelial Rho being necessary for lymphocyte migration through the BBB. Crucially, however, when EC expressing myristoylated Rho, which is functionally active in the absence of prenylation, was pretreated with lovastatin there was no inhibition in lymphocyte transmigration (Fig 1)
. Conversely, C3 treatment of the myristoylated Rho EC was, as predicted, still able to effectively inhibit lymphocyte migration as this inhibits Rho through ADP ribosylation. When we prepared membrane preparations from EC lines and probed for membrane associated Rho, we found that endogenous EC Rho did not associate with EC membranes after exposure to lovastatin whereas myristolated Rho was still able to associate with the membrane fraction under these conditions (Fig. 1)
. This demonstrates that a key mechanism of lovastatin-mediated inhibition of lymphocyte trans-endothelial migration is due to the prevention of Rho prenylation within brain EC.
3. Lovastatin treatment attenuates disease in a relapsing-remitting mouse model of multiple sclerosis
Our observation that lovastatin is a potent antimigratory compound in vitro suggested that it may possess therapeutic potential in controlling central nervous system (CNS) inflammation. Using a relapsing-remitting mouse model of multiple sclerosis (Biozzi ABH mouse), we found that lovastatin administration caused a significant dose-dependent reduction in the severity of clinical experimental autoimmune encephalomyelitis (EAE) (Table 1
) whereas vehicle-treated animals displayed characteristic paralysis. As predicted from the proposed mechanism (namely, an effect on lymphocyte migration) when lovastatin treatment was discontinued, EAE developed rapidly, suggesting that lovastatin had not restricted initial T cell priming events. Consistent with our in vitro data, the therapeutic effects of lovastatin were also partially reversed in mice after coadministration of mevalonolactone (Table 1)
. Histological examination of the CNS of vehicle-treated control animals at the peak of clinical disease (day 18) revealed characteristic inflammatory lesions that were absent in lovastatin-treated animals.
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To investigate whether lovastatin could attenuate disease relapse, the drug was administered during remission from the acute (grade 4) paralytic phase. This resulted in complete abrogation of disease relapse compared with untreated mice (Table 1)
. However, as with treatment of the acute phase, upon cessation of statin treatment on day 46 postinoculation, most animals rapidly (within 5 days) developed paralytic relapse.
4. Migration of donor lymphocytes into the CNS of lovastatin-treated recipient mice is severely inhibited
To test further the premise that a critical therapeutic effect of lovastatin is independent of its potential immunosuppressive action on T cells, mice were pretreated with vehicle or lovastatin before intravascular delivery of fluorescently labeled antigen-activated lymphocytes from an untreated donor animal. After 14 h, labeled lymphocytes were observed in the cerebral parenchyma of those animals treated with vehicle alone but were rarely seen in animals treated with lovastatin. The average number of labeled lymphocytes per 100 µm coronal section was significantly greater for vehicle-treated animals (14.2±3.0 mean ± SE) than for the lovastatin-treated animals (0.75±0.3, P<0.001). Leucocytes observed in statin-treated animals were primarily restricted to the vascular lumen of small blood vessels.
CONCLUSIONS AND SIGNIFICANCE
A critical stage in the development of neuroinflammatory diseases such as multiple sclerosis is the infiltration of leucocytes from the vasculature to the neural parenchyma, a process that depends on both lymphocyte activation and an ability of leucocytes to effectively elicit signaling responses in EC through surface receptors such as ICAM-1 (CD54). A central constituent of the ICAM-1-mediated signaling pathway in brain EC is the small GTPase Rho, which requires post-translational prenylation to be functionally active (Fig. 2
). Depleting the supply of isoprenyl groups derived from isoprenoid pyrophosphate substrates (that are part of the cholesterol synthesis pathway) can therefore effectively inhibit Rho function. Here we have shown that a reduction in the supply of isoprenoids through inhibition of HMG-CoA reductase with lovastatin leads to reduced Rho prenylation, loss of correct subcellular localization, and subsequent inhibition of trans-endothelial lymphocyte migration. Crucially, ectopic expression of myristoylated Rho in brain EC renders the cells insensitive to lovastatin, as myristoylation of Rho and its correct subcellular localization is independent of the supply of isoprenoid precursors.
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Treatment of EAE-induced Biozzi ABH mice with lovastatin dramatically attenuated the infiltration of leucocytes into the CNS and substantially alleviated clinical signs in the acute and (more important) the relapse phase of disease. This treatment regimen reduced both the number and severity of animals showing clinical signs of EAE and in most cases delayed disease onset. Although our in vitro data would suggest that a major action of lovastatin is via inhibition of leucocyte migration across the BBB, we recognize that due to the pleiotropic action of statins, other inhibitory pathways may also contribute to the amelioration of disease in vivo. However, in support of an inhibitory effect of lovastatin on the migration of T cells across the BBB is the observation that migration of T cells from untreated donor animals into the brain of lovastatin-treated recipient animals is significantly less than into untreated recipients.
We conclude that leucocyte migration across the BBB is a pivotal stage in the development of inflammatory lesions; as statins inhibit this process, their use in the treatment of multiple sclerosis may prove to be of significant therapeutic potential.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1014fje; to cite this article, use FASEB J. (March 5, 2003) 10.1096/fj.02-1014fje 
3 These authors contributed equally to the work.
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