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 major player in the regulation of various immune reactions like immunoglobulin synthesis, T-cell activation and homeostasis, and in the elimination of virus-infected and tumor cells. Dysregulation of the perforin-granule system, i.e. an increase of perforin-containing lymphocytes, was recently demonstrated in exacerbated psoriasis and generalized drug reactions. In contrast, in patients with exacerbated atopic dermatitis or unsymptomatic rhinitis allergica, a severe perforin depletion in cytotoxic T cells was demonstrated. In addition, these cells displayed a remarkable transport defect of lytic granules, i.e. a perforin hyperreleasability. Thus, the process of perforin-granule release may represent an attractive target for therapeutic immune modulation in various dermatological diseases. Ficoll isolated peripheral blood mononuclear cells (PBMCs) of healthy volunteers were preincubated with different concentrations of ciclosporin or methotrexate (MTX) for 1 h. A newly developed flow cytometry based perforin release assay was used to quantify the velocity of ionomycin/phorbol 12-myristate 13-acetate stimulated perforin-granule release in the presence or absence of pharmacological agents. The immunosuppressant MTX did not influence perforin-granule release. Ciclosporin, in contrast, was found to inhibit perforin-granule release significantly and dose dependently: whereas release from CD8(+) lymphocytes was almost maximal for the untreated control after 60 min (41% of CD8(+) perforin(+) cells at time zero), ciclosporin at 20, 4 and 2 microg/ml elevated the aforementioned parameter up to 73, 65 and 53%, respectively. Our data demonstrate that (i) perforin-granule release can be targeted efficiently by pharmacological agents which can be monitored directly in a newly developed perforin-granule release assay, and (ii) suppression of perforin-granule based cytotoxicity by ciclosporin might contribute to the beneficial therapeutic effects of this drug as an immunomodulating and immunosuppressant target.