Lovastatin inhibits brain endothelial cell Rho-mediated lymphocyte migration and attenuates experimental autoimmune encephalomyelitis

Abstract

Neuroinflammatory diseases, such as multiple sclerosis (MS), result from aberrant leukocyte traffic into the central nervous system (CNS). To breach the specialized blood-brain barrier, activated leukocytes interact with CNS endothelial cells (EC) and activate a CD54-mediated signaling pathway controlling the Rho GTPase. To function correctly Rho requires posttranslational prenylation, and this can be inhibited by depleting the supply of isoprenoids through inhibition of the cholesterol synthesis pathway with 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase) inhibitors (statins). Here we show that treatment of brain EC in vitro with lovastatin inhibits Rho-mediated transendothelial T cell migration. This effect can be reversed by supplementation with mevalonolactone, the downstream product of HMG-CoA reductase, or by ectopic expression of myristoylated Rho, which remains active in the absence of prenylation. In a relapsing-remitting mouse model of MS, lovastatin treatment inhibited leukocyte migration into the CNS and significantly attenuated the development of both acute and relapsing clinical disease. These studies demonstrate that the indirect pharmacological inhibition of Rho proteins in brain EC by statins can inhibit a key stage in the pathogenesis of neuroinflammation, namely leukocyte migration across the blood-brain barrier. These studies demonstrate a novel effect of statins in modulating the immune response in neuroinflammtory diseases and may provide additional rationale for their use in the treatment of MS.

Figures

Figure 1

(a) Lovastatin treatment during the 4h EC/lymphocyte co-culture does not affect lymphocyte adhesion (shaded bars) or transendothelial migration (solid bars). (b) Pre-treatment of brain EC for 24 h with lovastatin or C3 transferase inhibits transendothelial lymphocyte migration without affecting adhesion. *p<0.0001 cf. control. (c) Mevalonolactone reverses lovastatin-induced inhibition of lymphocyte migration. *p<0.02, **p<0.001 cf. control. †p<0.0001 cf. lovastatin treated cells. (d) Over expression of myristolated Rho B confers resistance to the inhibitory effect of lovastatin but not C3 transferase. *p<0.02, **p<0.0001 cf. control migration through pBabepuro cell line. †p<0.0001 cf. EC expressing myristoylated RhoB. (e) Myristolated Rho, but not endogenous Rho, associates with EC membranes following treatment with lovastatin.

Figure 2

(a) Lovastatin treatment (10mg/kg; days 8-22) inhibits the development of disease until cessation of treatment. (b) Spinal cord and cerebrum from control and lovastatin treated (10mg/kg) ABH mice. EAE in untreated mice showed marked perivascular leucocyte cuffing (arrows) that was absent in treated mice (arrow heads). Scale bar = 20μm. (c) Lovastatin treatment (10mg/kg) of acute phase EAE after onset does not attenuate disease progression whereas (h) treatment during remission (20mg/kg) prevents the onset of disease relapse.

Figure 3

(a) Proliferation of auricular lymph node cells evaluated by [3H]thymidine incorporation (left abscissa, solid bars) or by a colorimetric cell proliferation assay (right abscissa, shaded bars) from vehicle and lovastatin treated ABH mice. *p<0.05 cf. vehicle-treated animals. (b) Representative confocal fluorescence projected images of cerebral vasculature (red) and labelled lymphocytes (green). Top panel (bar = 100 μm) shows extravasated lymphocyte and middle panel (bar = 20 μm), a lymphocyte in the cerebral parenchyma adjacent to a microvessel from vehicle treated animals. Insert represents projection of middle panel Z stack. Lower panel (bar = 20 μm) shows a lymphocyte confined to the intravascular compartment from a lovastatin pre-treated mouse (20 mg/kg/day for 4days). Insert shows higher power X-Y section detail of lower panel (bar = 10 μm). (c) Delayed type hypersensitivity function in vehicle or lovastatin treated (20mg/kg) mice expressed as the percentage increase in ear thickness over baseline measurements. **p<0.001, *p<0.01 cf. non-oxazolone primed animals.