Procainamide, a drug causing lupus, induces prostaglandin H synthase-2 andformation of T cell-sensitizing drug metabolites in mouse macrophages

Procainamide (PA) may cause drug-induced lupus, and its reactive metabolite s, hydroxylamine-PA (HAPA) and nitroso-PA, are held responsible for this. H ere, we show that N-oxidation of PA to these metabolites can take place in macrophages and lead to formation of neoantigens that sensitize T cells. Mu rine peritoneal macrophages (PM phi), exposed to PA in vitro, generated neo antigens related to HAPA as indicated by(1) their capacity to elicit a spec ific recall response of HAPA-primed T cells in the adoptive transfer poplit eal lymph node (PLN) assay and (2) the appearance of metabolite-bound prote in in PA-pulsed PM phi, as determined by Western blot. Analysis of five pha se I enzymes that might be responsible for HAPA formation by PM phi pointed to prostaglandin H synthase-2 (PGHS-2) as a likely candidate. Experimental evidence that PA can be oxidized to HAPA by PGHS was obtained by exposing PA to PGHS in vitro. The resulting metabolites were identified by mass spec tral analysis and covalent protein binding in ELISA. In vitro, PA exposure of PM phi of slow acetylator A/J and fast acetylator C57BL/6 mice failed to show significant strain differences in enzyme mRNA expression, enzyme acti vities, or formation of HAPA-related neoantigens. By contrast, after long-t erm PA treatment in vivo only in slow acetylators the PM phi harbored HAPA- related neoantigens and T cells were sensitized to them. PM phi of fast ace tylator C57BL/6 mice only contained HAPA-related neoantigens, and their T c ells were only sensitized to them if, in addition to long-term PA treatment , their donors had received injections of phorbol myristate acetate (PMA), a known enhancer of oxidative enzymes in phagocytes. In conclusion, PA trea tment leads to N-oxidation of PA by enzymes, in particular PGHS-2, present in antigen-presenting cells (APC) and, hence, to generation of neoantigens which sensitize T cells. The enhanced neoantigen formation and T cell sensi tization seen in slow acetylators might be explained by their higher concen tration of PA substrate that is available for extrahepatic N-oxidation in A FC.