Procainamide, a Drug Causing Lupus, Induces Prostaglandin H Synthase-2 and Formation of T Cell-Sensitizing Drug Metabolites in Mouse Macrophages

Abstract

Procainamide (PA) may cause drug-induced lupus, and its reactive metabolites, hydroxylamine-PA (HAPA) and nitroso-PA, are held responsible for this. Here, 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. Murine peritoneal macrophages (PMvarphi), exposed to PA in vitro, generated neoantigens related to HAPA as indicated by (1) their capacity to elicit a specific recall response of HAPA-primed T cells in the adoptive transfer popliteal lymph node (PLN) assay and (2) the appearance of metabolite-bound protein in PA-pulsed PMvarphi, as determined by Western blot. Analysis of five phase I enzymes that might be responsible for HAPA formation by PMvarphi 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 spectral analysis and covalent protein binding in ELISA. In vitro, PA exposure of PMvarphi of slow acetylator A/J and fast acetylator C57BL/6 mice failed to show significant strain differences in enzyme mRNA expression, enzyme activities, or formation of HAPA-related neoantigens. By contrast, after long-term PA treatment in vivo only in slow acetylators the PMvarphi harbored HAPA-related neoantigens and T cells were sensitized to them. PMvarphi of fast acetylator C57BL/6 mice only contained HAPA-related neoantigens, and their T cells 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 treatment 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 sensitization seen in slow acetylators might be explained by their higher concentration of PA substrate that is available for extrahepatic N-oxidation in APC.

Enzyme-Mediated Protein Haptenation of Dapsone and Sulfamethoxazole in Human Keratinocytes: II. Expression and Role of Flavin-Containing Monooxygenases and Peroxidases

“…In the first of these two companion articles, we demonstrate that cytochromes P450 do not play a major role in the protein haptenation observed in NHEKs exposed to SMX or DDS. In addition, although other investigators have reported that cyclooxygenase-2 is able to oxidize arylamine compounds (Goebel et al, 1999), our results indicate that neither cyclooxygenase-1 nor -2 is involved in the protein haptenation observed in these cells (Vyas et al, 2006). Hence, we probed the role of flavin-containing monooxygenases (FMOs) and peroxidases (PRXs) in this phenomenon.…”

“…Oxidation of SMX and DDS to their arylhydroxylamine and then subsequently to arylnitroso metabolites is believed to be a critical step in their ability to cause CDRs (Naisbitt et al, 1999;Svensson et al, 2001). Various hepatic enzymes, such as the cytochrome P450-dependent monooxygenases, cyclooxygenases, and PRXs, have been shown to be active in SMX and DDS metabolism (Cribb et al, 1990;Mitra et al, 1995;Goebel et al, 1999;Winter et al, 2000). Although survival of reactive metabolites during transit from liver to skin has not been demonstrated, evidence suggests the presence of the hydroxylamine metabolites of these drugs in plasma and urine after therapeutic doses (Coleman et al, 1992;Mitra et al, 1995;Winter et al, 2000).…”

Formation and Uptake of Arylhydroxylamine-Haptenated Proteins in Human Dendritic Cells

“…Bioactivation of these drugs to their reactive metabolites followed by binding to intracellular proteins has been suspected to play a key role in eliciting the immune response. In addition to liver microsomes, formation of these drug-protein covalent adducts have already been demonstrated in various nonhepatic cellular models, including keratinocytes , fibroblasts (Bhaiya et al, 2006), monocytes (Goebel et al, 1999), and a T cell line (Manchanda et al, 2002).…”

Enzyme-Mediated Protein Haptenation of Dapsone and Sulfamethoxazole in Human Keratinocytes: I. Expression and Role of Cytochromes P450

“…Several arylamine drugs, including procainamide, are oxidized to arylhydroxylamine metabolites by COX-2 in vitro (Liu and Levy, 1998;Goebel et al, 1999). Which of these enzymes, if any, mediates the bioactivation of SMX and DDS in NHEKs is unknown.…”

“…Hydroxylation of SMX and DDS by various CYP450s has been reported (Cribb et al, 1995;Mitra et al, 1995;Gill et al, 1999;Winter et al, 2000). Several arylamine drugs, including procainamide, are oxidized to arylhydroxylamine metabolites by COX-2 (Liu and Levy, 1998;Goebel et al, 1999).…”