A flow cytometric approach in the characterization of actively induced EAE in Lewis rat

Actively induced Experimental Autoimmune Encephalomyelitis (EAE) is a well reproducible model for portraying the acute phase of multiple sclerosis as well as good model to develop new promising treatments. Both lymphnode and spinal cord (s.c.) have been widely described by means of molecular biology and immunohistochemistry. The aim of this study was to characterize both spleen and s.c. lymphocyte populations and to highlight possible useful markers to be monitored during new therapeutic treatments. Thus at the onset (day 10 after EAE induction) and at the peak of the disease (day 14 after EAE induction) we investigated CD4, CD8 and encephalitogenic TCRVb8.2+ T cells prevalence as well as the expression of adhesion molecules CD49d (VLA-4) and CD11a (LFA-1). EAE was actively induced by subcutaneously inoculation of 50μg guinea pig Myelin Basic Protein (gifted by P. Riccio) in complete Freund’s adjuvant with inactivated Mycobacterium tuberculosis. The spleen and s.c. were dissected from both healthy (controls) and EAE-induced animals, cells were collected, stained with different combinations of conjugated antibodies and acquired using a flow cytometer. In the spleen of EAE animal we reported a significant decrease in the mean absolute cell number on day 14, together with the presence of a CD45+ population other than lymphocytes as soon as day 10. Furthermore we observed an increase in CD4 and decrease in CD8 T cells compared to the controls on day 14, while no changes were reported on day 10. Moreover no alteration in the TCRVb8.2+ lymphocytes percentage as well as in CD49d (VLA-4) and CD11a (LFA-1) expression on CD4 and CD8 T cells were never observed. In the spinal cord no infiltrating lymphocytes were detected until day 14. Therefore the results on s.c. were compared to those obtained in the spleen on day 14. Thus CD4 lymphocytes percentage was higher in the s.c., while CD8 lymphocytes prevalence decreased. We also observed an increase in CD4 TCRVb8.2+ T cells percentage together with a significant increase in the expression of CD49d (VLA-4) which was more accentuated for CD4 than for CD8 T cells. Our results confirm that EAE in Lewis rats is a CD4 T cell mediated disease in which the expression of TCR Vb8.2 and adhesion molecules is probably regulated mainly in the target organ. We show that multiparametric flow cytometry can be an informative method to describe immune system cells during the EAE ongoing. Supported by a MIUR 2006 grant (prot. 2006064219-002)