Characterization of spleen and spinal cord infiltrating lymphocytes during actively induced EAE in Lewis rats.

Experimental Autoimmune Encephalomyelitis (EAE) is an inflammatory T cell mediated disease of the central nervous system (CNS) that serves as model to understand the pathogenesis of multiple sclerosis (MS). An acute form of EAE can be actively induced in Lewis rat by the inoculation of Myelin Basic Protein resuspended in complete Freud’s adjuvant. This model is highly reproducible and it has been commonly used to study the immunopathogenesis as well as to develop different promising treatments for MS. The aim of the present study is to characterize both splenic and CNS infiltrating lymphocytes during the course of the disease mainly by means of flow cytometry. Thus we investigated if there were differences in the prevalence of encephalitogenic T cells (TCR Vbeta8.2+) and in the expression of the adhesion molecule VLA-4 both in the main organ of memory immune system (spleen) and in the target organ of EAE (spinal cord) in both healthy and EAE animals on day 10 and 14. EAE was actively induced in Lewis rats by subcutaneously inoculation of 50 μg guinea pig MBP (gifted by P. Riccio) in 100 μg complete Freund’s adjuvant with 3 mg/ml of inactivated Mycobacterium tuberculosis in both hind limb footpads. Rats were evaluated every 24 hours for neurological signs. The spleen and spinal cord were dissected from both healthy and EAE-induced animals at the onset of clinical signs (day 10) and at the peak of the disease (day 14). Cells from spleen and spinal cord were collected, stained with different combination of conjugated monoclonal antibodies (anti CD3, CD4, CD8, CD45, CD49d, TCR αβ, TCR Vβ8.2) and then acquired using a FACSCanto flow cytometer. We observed a significant decrease in splenic mean absolute cell number in EAE animals on day 14 in comparison with the controls and an inverse correlation between the decrease in mean cell number value and the increase in the clinical score evaluation. Moreover in the spleen we reported the presence of an unexpected leukocyte population as soon as day 10 after treatment while in spinal cord we did not observed infiltrating lymphocytes or leukocytes until day 14; no leukocytes were never present in the spinal cord of healthy animals as expected. Moreover during the course of the disease, in the spleen we did not report an increase in the percentage of the encephalitogenic T cells, both CD4+ and CD8+, bearing TCR Vβ8.2 in the EAE animals compared with the controls. Furthermore we observed an increase in the percentage of CD4+ TCR Vβ8.2+ T cells in the spinal cord of EAE animals at day 14 compared with the spleen values of the same animals. No significant changes in the expression of CD49d (VLA-4) was observed in the spleen on day 10 or at day 14 on both CD4+ and CD8+ T lymphocytes. Furthermore a significant increase in the expression of this protein was evident in infiltrating T cells. This increase was more accentuated for CD4+ than for CD8+ T cells. Flow cytometry is a powerful and very informative method to describe the variation in immune system elements during the ongoing of EAE. It clearly confirms that EAE in Lewis rats is a CD4 T cell mediated disease in which the expression of TCR Vβ8.2 and adhesion molecules is probably regulated mainly in the target organ (CNS). Supported by a MIUR 2006 grant (prot. 2006064219)