RECOMENDACIONES FINALES

To determ ine w hether lovastatin has an effect on leukocyte recruitm ent to peripheral tissues Biozzi ABH mice were treated with 25pl 2.5% oxazolone applied to the ear and treated with 10 mg/kg/day lovastatin for 2 days prior to an application o f 25pl 2.5% oxazolone to the ear. Animals were m aintained on 10 mg/kg/day lovastatin for a further 3

C la ire E W alters C h a p te r 6 - In h ib itio n o f R ho G T P a s e s w ith io v astatin : e ffe c t on n e u ro in fla m m a tio n days and ear thickness measured. Animals subsequently re-stim ulated w ith oxazolone showed normal skin cell trafficking responses (figure 6.7).

Figure 6.7: HMGCoA reductase inhibitors do not alter cell trafficking into peripheral sites.

Biozzi ABH m ice were treated with 10 m g/kg/day lovastatin for 2 days prior to an application o f 25 pi 2.5 % oxazolone to the ear. Animals were m aintained on 10 m g/kg/day lovastatin for a further 3 days and ear thickness m easured as discussed in section 6.3.9). Significant differences between groups w ere determ ined by S tudent’s t test. *p< 0.01, **/?<0.001 significantly different from responses in non-oxazolone prim ed anim als, NS: not significantly different from each other.

6.4 Discussion

To test the efficacy o f the HM GCoA reductase inhibitor lovastatin in vivo, EAE was induced in Biozzi ABH mice. In this well characterised chronic relapsing and remitting model o f multiple sclerosis (Baker et.al, 1990) the progression o f the disease is a direct consequence o f a large-scale infiltration o f leukocytes into the brain and spinal cord (Brennan et.al, 1999). Leukocyte recruitm ent to the CNS in this model occurs at a precise tim e post inoculation and has been reported in detail elsewhere (Butter et.al,

1991; Allen et.al, 1993). The treatm ent o f Biozzi ABH mice with the HMGCoA reductase inhibitor lovastatin was able to dramatically attenuate the infiltration o f leukocytes into the CNS o f Biozzi ABH mice and to alleviate clinical signs in a dose-

___________________________________________________________________________ effect on neuroinflammation

dependent manner. Furthermore, lovastatin treatment was effective in attenuating both the acute and relapse phase of the disease. This treatment regime reduced both the number o f animals showing clinical signs of EAE, the severity of the disease, and in most cases also delayed onset of the disease. It is interesting to note that following withdrawal from lovastatin therapy animals developed disease suggesting that leukocytes were fully activated and may be awaiting access to the CNS.

The action o f lovastatin appears to be most prominent in restricting trafficking to the CNS as opposed to being anti-inflammatory since it did not prevent infiltration of leukocytes into skin. These observations also suggest that the effect of lovastatin was not via a general mechanism such as down regulation of adhesion molecule expression since this would be expected to alter trafficking into peripheral sites. These in vivo observations are supported by our in vitro data that illustrates that brain endothelial cells can support leukocyte adhesion in the presence or absence of lovastatin to the same degree (Chapter 5). However, these findings are contrary to previous studies by Stanislaus et.al (2001). Although, Stanislaus et.al (2001) has also shown that lovastatin treatment decreases the mononuclear cell infiltration into the CNS of Lewis rats with EAE along with the amelioration of disease, they suggest a different mechanism. Their study suggests that in EAE lovastatin acts by blocking the induction of pro-inflammatory cytokines (TNF-a, IL-lp, IFN-y and iNOS) and attenuating the expression of adhesion molecules such as LFA-1. In our study we do not observe that lovastatin is able to alter either basal or IFN- y stimulated ICAM-1 expression and since the adhesion of leukocytes to endothelial cells was not affected by lovastatin treatment our studies suggest a different mechanism to that proposed by Stanislaus et al.

It is also possible that the efficacy of lovastatin in EAE is due to an inhibition of T- lymphocyte proliferation. However, although such effects of statins have been widely reported, in our experience lovastatin only slightly reduced leukocyte proliferation both in vitro and in vivo. These observations point to an alternative action other than T- lymphocyte immunomodulation for statins in limiting infiltration o f leukocytes to the CNS. This study suggests that lovastatin restricts the transendothelial migration of leukocytes across the BBB and into the CNS by inhibiting endothelial cell Rho signalling proteins. However, direct evidence for lovastatin inhibiting Rho signalling in this model

Claire E Walters Chapter 6 - Inhibition o f Rho GTPases with lovastatin: ___________________________________________________________________________ effect on neuroinflammation

has not been shown and at present this is only implicit since another HMGCoA reductase inhibitor (cerivastatin) has been shown to reduce Rho A activation (Yoshida et.al, 2001).

Acknowledgements

The work described in this chapter was carried out in collaboration with Dr David Baker and Mr Gareth Pry ce (from the Institute of Neurology, UCL, London). Mr Gareth Pryce carried out the EAE animal experiments, whereas Dr David Baker analysed the data. I am extremely grateful for their invaluable help with this part of my thesis.