Mechanism of CpdA action in the treatment of EAE.

(A) EAE was induced in C57Bl/6 mice followed by treatment with 1.5 mg/kg or 5 mg/kg CpdA dissolved in water for 3 consecutive days after the mice had developed an average clinical score of 3. On the following morning the mice were sacrificed and the leukocytes isolated from the spinal cord. Cellularity was determined by microscopic counting; staining for AnnexinV binding and surface expression of LFA-1 on CD3⁺CD4⁺ Th cells was performed by flow cytometry. n = 3−7. (B) The spleen was isolated from the same animals as in panel A and the leukocytes analyzed for AnnexinV binding as well as LFA-1 and CD44 surface expression on CD3⁺CD4⁺ Th cells by flow cytometry. n = 3−8. (C, D) Splenocytes from the same animals as in panels A and B were cultured in the presence or absence of ConA or MOG_35–55 peptide. Proliferation was measured by ³[H]-thymidine incorporation assay and expressed as a proliferation index relative to the values obtained in the absence of any stimulus (C); IL-17 and IFNγ levels in the supernatant were determined by ELISA (D); n = 7−10. *: p<0.05, ***: p<0.001, n.s.: p>0.05.