N‐acetylprocainamide is a less potent inducer of t cell autoreactivity than procainamide

Abstract

We have reported that an inhibitor of DNA methylation, 5-azacytidine, makes cloned, antigenspecific CD4+ T cells autoreactive, and that procainamide and hydralazine mimic this effect. Those results suggested that procainamide and hydralazine may induce autoimmunity by inhibiting DNA methylation and causing T cell autoreactivity. We report now that Nacetylprocainamide, a procainamide derivative that does not induce lupus, is also a DNA methylation inhibitor, but it is 100 times less potent than procainamide in inducing T cell autoreactivity.DNA methylation inhibitors induce expression of genes normally suppressed by mechanisms associated with cytosine methylation (1) and, in certain systems, can change cellular phenotype and alter differentiation (2,3). We have previously reported that 5-azacytidine (5-azaC), a DNA methylation inhibitor, made 4 cloned antigen-reactive T cells respond to autologous class I1 major histocompatibility complex (MHC) determinants without specific antigen, presumably by inducing expression of suppressed genes (4). This response was termed autoreactivity.In those studies, we demonstrated that the 5- azaC-treated T cells responded to autologous but not allogeneic macrophages, and that monoclonal antibodies to class I1 but not class I MHC determinants would inhibit activation (4). Those results suggested the possibility that similar inhibition of T cell DNA methylation in vivo could lead to an autoimmune disease.To determine if inhibitors of DNA methylation could produce an autoimmune disease, we tested whether 2 drugs known to induce a lupus-like syndrome inhibit DNA methylation and induce T cell autoreactivity. We found that hydralazine (Hyd) and procainamide (Pca) mimicked the effects of 5-azaC on T cells by inducing T cell autoreactivity in these cloned lines and decreasing deoxymethylcytidine (d"C) content in T cell DNA, thus supporting the hypothesis that DNA methylation inhibitors may induce autoimmune disease (5).To further test this hypothesis, we compared Pca and Hyd with structural analogs, using the assays previously developed. N-acetylprocainamide (Napa) is a procainamide derivative that rarely induces lupus ( 6 4 , and hydralazine is a phthalazine (Phth) derivative containing a hydrazine side chain (l-hydrazinophthalazine) (9,lO). Previously, investigators speculated that the hydrazine side chain of hydralazine may be responsible for the lupus-like syndrome (9); therefore, we compared Pca with Napa and Hyd with Phth, for the ability to induce T cell autoreactivity and inhibit DNA methylation. MATERIALS AND METHODST cell cultures. CD4 + , cloned, tetanus toxoid (TTh reactive, interleukin-2 (IL-2tdependent T cell lines were generated and maintained as previously described (4,ll).

Treating activated CD4+ T cells with either of two distinct DNA methyltransferase inhibitors, 5-azacytidine or procainamide, is sufficient to cause a lupus-like disease in syngeneic mice.

“…This represents a decrease of 0.26% or -160,000 methylated bases (36). Similar to our previous report (37), the decrease caused by tiM Pca was smaller and not statistically significant. Controls in which EL-4 cells were treated with 0.25 tM 5-azaC demonstrated a 0.30% decrease in overall dmC content.…”

Mechanisms of drug‐induced lupus. IV. Comparison of procainamide and hydralazine with analogs in vitro and in vivo

“…The 5% decrease in d"C content induced by Pca was less than the 14% previously reported using Jurkat cells (1,2). The reason for this is unclear, but may reflect a species-specific difference in the sensitivity of T cells to DNA methylation inhibitors, since Pca also caused an identical 5% decrease in murine EL-4 cells (3).…”

“…D10 cells were then treated for 6 days with 10 p M HU, 50 Pca or Napa, or 10 pA4 Hyd or Phth, and examined for LFA-1 expression by flow cytometry. These concentrations were chosen because they are close t o the thera- peutic concentrations of Pca (10-30 pA4) and Hyd (0.5-5.0 p h f ) (1,2), and because autoreactivity was detected at these concentrations. The results are shown in Figures 2A-F. Untreated (control) cells expressed relatively low levels of LFA-1, as did HU-treated cells.…”

Drug-induced anti-histone autoantibodies display two patterns of reactivity with substructures of chromatin.

“…In this way drugs might be thought to bind to histone-containing self material, altering it to produce nonnative or even foreign epitopes, breaking B cell tolerance to autoantigens. T cell help could derive from a drug-specific response such as the experimental lymphoproliferation induced in mice by injection of penicillamine, streptozotocin, and gold salts (43), or from nonspecific, direct lymphocyte activation observed in vitro by drugs such as procainamide (44) or its metabolite, procainamide-hydroxyl-amine (45). However, autoantibody binding does not require the presence of drug since the nucleohistones used as antigens were derived from calf thymus, and drug carry-over from patients' sera would be insignificant after dilution or a few days after withdrawal of therapy.…”