Perforin and granzyme mediated toxicity on cultured oligodendrocytes

Oligodendrocytes (OLs) are major targets of immune‐mediated demyelination in multiple sclerosis. Perforin, a pore‐forming protein released from cytolytic granules of cytotoxic T‐cells and NK cells, is implicated as an effector in inflammatory conditions leading to demyelination. Cytolytic granules contain other proteins which may also promote demyelination, including the serine dependent protease granzyme B. We used a repeated measures assay to test effects of enriched perforin, granzymes and total granule extracts on survival of individual OLs over 72 h. Granules were isolated from rat RNK‐16 cells. Granzymes and perforin were isolated by Cu + 2‐immobilized metal affinity chromatography. Lytic activity/mL at 4 h was calculated for all preparations. We assayed survival of both mature and immature OLs to determine developmental sensitivity. Cells were exposed to perforin, granzymes, perforin + granzymes, granule extract or control buffers for 1–3 h. Survival of mature OLs with large processes and membrane expansions was reduced by granule extracts but unaffected by perforin and granzymes. Immature OLs with a bipolar morphology or with multiple, thin processes were vulnerable to both perforin and granzymes alone and in combination, and to granule extracts. The majority of cell death in all cases occurred by 24 h. Results suggest that cytolytic granules contain factors that are toxic to OLs at all developmental stages and that immature OLs are differentially sensitive to perforin and/or granzyme mediated toxicity. Cytolytic granules may thus have a dual effect, to promote demyelination and to limit remyelination by targeting newly generated OLs. Acknowledgements: Supported by Natl. MS Soc. (PEK) & NIH R01 CA38942 (DH).