Perforin granule release from cytotoxic lymphocytes ex vivo is inhibited by ciclosporin but not by methotrexate

The 70-kD plasma membrane pore-forming protein perforin is a key component of lymphocyte cytotoxicity mediated by lytic granules. It represents a majo r player in the regulation of various immune reactions like immunoglobulin synthesis, T-cell activation and homeostasis, and in the elimination of vir us-infected and tumor cells. Dysregulation of the perforin-granule system, i.e. an increase of perforin-containing lymphocytes, was recently demonstra ted in exacerbated psoriasis and generalized drug reactions. In contrast, i n patients with exacerbated atopic dermatitis or unsymptomatic rhinitis all ergica, a severe perforin depletion in cytotoxic T cells was demonstrated. In addition, these cells displayed a remarkable transport defect of lytic g ranules, i.e. a perforin hyperreleasability. Thus, the process of perforin- granule release may represent an attractive target for therapeutic immune m odulation in various dermatological diseases. Ficoll isolated peripheral bl ood mononuclear cells (PBMCs) of healthy volunteers were preincubated with different concentrations of ciclosporin or methotrexate (MTX) for 1 h. A ne wly developed flow cytometry based perforin release assay was used to quant ify the velocity of ionomycin/phorbol 12-myristate 13-acetate stimulated pe rforin-granule release in the presence or absence of pharmacological agents . The immunosuppressant MTX did not influence perforin-granule release. Cic losporin, in contrast, was found to inhibit perforin-granule release signif icantly and dose dependently: whereas release from CD8(+) lymphocytes was a lmost maximal for the untreated control after 60 min (41 % of CD8(+) perfor in(+) cells at time zero), ciclosporin at 20, 4 and 2 mug/ml elevated the a forementioned parameter up to 73, 65 and 53%, respectively. Our data demons trate that (i) perforin-granule release can be targeted efficiently by phar macological agents which can be monitored directly in a newly developed per forin-granule release assay, and (H) suppression of perforin-granule based cytotoxicity by ciclosporin might contribute to the beneficial therapeutic effects of this drug as an immunomodulating and immunosuppressant target.