Abstract
Pyrimethamine (2,4-diamino-5-p-chlorophenyl-6-ethyl-pyrimidine), a folic acid antagonist, may exert, in addition to antiprotozoan effects, immunomodulating activities, including induction of peripheral blood lymphocyte apoptosis. However, the molecular mechanisms underlying this proapoptotic activity remain to be elucidated. Here we show that pyrimethamine, used at a pharmacologically relevant concentration, induced per se apoptosis of activated lymphocytes via the activation of the caspase-8- and caspase-10-dependent cascade and subsequent mitochondrial depolarization. Importantly, this seems to occur independently from CD95/Fas engagement. The proapoptotic activity of pyrimethamine was further confirmed in a patient with autoimmune lymphoproliferative syndrome, an immune disorder associated with a defect of Fas-induced apoptosis. In this patient, pyrimethamine treatment resulted in a “normalization” of lymphocyte apoptosis with a significant amelioration of laboratory parameters. Altogether, these results suggest a mechanism for pyrimethamine-mediated apoptosis that seems to bypass CD95/Fas engagement but fully overlaps CD95/Fas-induced subcellular pathway. On these bases, a reappraisal of the use of pyrimethamine in immune lymphoproliferative disorders characterized by defects in CD95/Fas-mediated apoptosis should be taken into account.
Footnotes
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This work was supported by a Grant from Ricerca Corrente ISS-2003 (Studio dei Meccanismi Cellulari e Molecolari di Aumentata Suscettibilità alle Infezioni, Malattie Autoimmuni, e Neoplasie nelle Immunodeficienze Primarie) (to M.P.).
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M.P. and A.M.G. equally contributed to this work.
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W.M. and A.G. are considered senior investigators.
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doi:10.1124/jpet.105.086736.
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ABBREVIATIONS: ΔΨ, mitochondrial membrane potential; ALPS, autoimmune lymphoproliferative syndrome; TCR, T cell receptor; FasL, Fas ligand; PBL, peripheral blood lymphocyte(s); HD, healthy donor(s); PBS, phosphate-buffered saline; PHA, phytohemagglutinin; IL, interleukin; DMSO, dimethyl sulfoxide; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; HLA-DR, human leukocyte antigen-DR; PE, phycoerytrin; PerCP, peridinin chlorophyll protein; APC, allophycocyanin; PMA, phorbol 12-myristate 13-acetate; IFN-γ, interferon-γ; PI, propidium iodide; JC-1, 5–5′,6–6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide; ELISA, enzyme-linked immunosorbent assay; Cyt c, cytochrome c; SEM, scanning electron microscopy; FasL, Fas ligand; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; JC-1, 5–5′,6–6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide.
- Received March 23, 2005.
- Accepted September 6, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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