Experimental autoimmune encephalomyelitis (EAE), the best available animal model of multiple sclerosis, is a CD4+ T-cell mediated disease of the central nervous system (CNS) that can be induced by inoculation with CNS antigens, such as myelin basic protein (MBP) and adjuvants (MBP-EAE). The disease is characterized by the infiltration of the nervous system with lymphocytes and macrophages, with subsequent demyelination of nerve fibres.
The first theme of this thesis is based on the interesting observation that EAE is less severe or delayed in onset when induced in pregnant animals. Pregnancy also appears to have a protective effect on multiple sclerosis, with evidence of fewer relapses during gestation. The exact nature of such beneficial effects remains unclear, but is thought to be related to a selective immunosuppression during pregnancy. The present study has demonstrated that rats inoculated during
early pregnancy displayed less severe MBP-EAE than non-pregnant rats and that pregnancy sera down regulated the proliferation of both MBP-specific lymph node cells and MBP-specific T cell lines. In an attempt to gain further insight in the immunoregulatory mechanisms responsible for the apparent protection afforded by pregnancy on EAE, cytokine mRNA expression by inflammatory cells contained in the spinal cords of pregnant and non-pregnant rats with EAE was studied using real-time RT-PCR techniques. We found that pregnancy was associated with significant increases in expression of IL-4, IL-10, and TNF-α mRNA.
We also designed experiments to explore the role of the pregnancy hormone early pregnancy factor (EPF) in EAE. Since EPF has been found to have both immunosuppressive and growth factor properties, we hypothesised that EPF may be upregulated during the latter phase of EAE where it may play a role in recovery from disease. By using the standard
bioassay for EPF, the Rosette Inhibition Test, and by semi-quantitative RT-PCR techniques, the present study has now shown that inflammatory cells from the spinal cord of rats with EAE can produce and secrete EPF, with production of EPF being greatest during early recovery. Clinical studies were also performed to assess the suitability of EPF as a candidate therapy for EAE and possibly MS. Similar to the pregnancy study, EPF was found to be beneficial in reducing severity of disease and delaying onset of acute MBP-EAE. EPF also suppressed the proliferation of MBP-specific lymph node cells and T cell lines in a similar fashion to pregnancy sera. Administration of EPF also resulted in a significant increase in the expression of IL-4 and IL-10 and a significant decrease in IFN-γ mRNA expression in spinal cord inflammatory cells of rats with MBP-EAE.
The second theme of the thesis centered on the development of a new model of chronic relapsing EAE
(CR-EAE). In Lewis rats, MBP-EAE is usually an acute monophasic illness from which rats spontaneously recover. However, as people with MS tend to experience recurrent relapses that later evolve into a secondary progressive course of disease, a better animal model of MS would be one in which the animals show signs of relapse. The present study reported a new relapsing model of EAE whereby CR-EAE was induced in rats with MBP-EAE by the administration of cyclosporin A (CsA) every other day. It was found that treatment with CsA 8mg/kg reliably delayed the onset of disease, increased the frequency of second episodes and resulted in 50% of rats having a third episode or following a chronic persistent course. In an attempt to investigate how low-dose CsA treatment induces relapses in CR-EAE, we studied the effect of a single dose of CsA on the cells and the cytokines produced in the inflammatory infiltrate of rats with MBP-EAE using flow cytometry and RT-PCR methods. There was an increase
in apoptosis of the total inflammatory cell population in the spinal cord in MBP-EAE 16 hours after treatment with CsA along with an increase in the percentages of CD5+ cells and TCRαβ* cells that were apoptotic. CsA therapy suppressed expression of IL-2 and IFN-γ mRNA in inflammatory cells from the spinal cords of rats with MBP-EAE, but induced a significant increase in expression of TGF-β mRNA.
Taken together, the experiments in the present study have shown that pregnancy, EPF and CsA can have profound effects on the regulation of EAE. Pregnancy and EPF-treatment down regulated the clinical signs of EAE and this suppression was in part mediated through the down regulation of MBP-specific lymphocytes and through alterations in cytokine expression in the spinal cord inflammatory infiltrate of rats with EAE. CsA therapy induced a chronic relapsing form of MBP-EAE and caused changes in the cellular
composition, frequency of apoptosis and cytokine mRNA expression of the spinal cord inflammatory infiltrate of rats with MBP-EAE. These experiments have given an initial insight into how the immunological effects of single dose CsA may contribute to the observed clinical effects of low-dose CsA therapy on EAE and have also provided a reliable relapsing animal model of MS to allow further studies into the mechanisms of relapse.