Brief Report: Drugs Implicated in Systemic Autoimmunity Modulate Neutrophil Extracellular Trap Formation.

OBJECTIVE: Aberrant neutrophil extracellular trap (NET) formation has been implicated as a mechanism to induce autoreactivity in individuals at risk of autoimmune diseases. The objective of this study was to assess whether medications implicated in cases of drug-induced autoimmunity (hydralazine and procainamide) and medications less commonly associated with drug-induced autoimmunity (minocycline and clozapine) induce NET formation and/or prevent NET degradation. METHODS: Human neutrophils were incubated with the drugs of interest and resultant NET formation was quantified by fluorescent microscopy. The ability of these drugs to interfere with NET degradation by serum nuclei was assessed. Pathways of drug-induced NET formation were studied with pharmacologic inhibitors of reactive oxygen species (ROS), peptidylarginine deiminases (PADs), and muscarinic receptors, and by assessment of intracellular calcium levels by flow cytometry. To determine if NET protein cargo varies by drug stimulus and/or neutrophil source, proteomic analysis of NET lysates induced by specific medications was compared using neutrophils from healthy donors and from patients with autoimmune diseases. RESULTS: Hydralazine and procainamide significantly induced NET formation while minocycline and clozapine did not. None of the medications significantly impaired NET degradation. NETosis induced by these drugs required NADPH oxidase and PAD4 activation. Procainamide triggered NETs via muscarinic receptor engagement on neutrophils, while hydralazine modulated calcium release from intracellular stores. Differences in protein cargo, particularly histone content, were observed in NETs induced by hydralazine and procainamide. CONCLUSION: Medications commonly implicated in drug-induced autoimmunity trigger NET formation displaying distinct protein cargo, via common and specific pathways. NETosis may play a role in the pathogenesis of drug-induced autoimmunity.

A comparison of the covalent binding of clozapine, procainamide, and vesnarinone to human neutrophils in vitro and rat tissues in vitro and in vivo.

Covalent binding of drug reactive metabolites to neutrophils or their precursors is thought to play a role in the development of drug-induced agranulocytosis. In this study, we used immunochemical techniques to compare the covalent binding of clozapine, vesnarinone, and procainamide (three drugs associated with agranulocytosis) to phorbol-12,13-myristate acetate (PMA)-activated human neutrophils in vitro and rat tissues in vivo. In PMA-activated human neutrophils in vitro, clozapine and procainamide modified neutrophil proteins with molecular masses ranging from 30 to 200 kDa, while vesnarinone predominately formed adducts with molecular masses greater than 70 kDa. All three drugs formed adducts at 126, 98, and 58 kDa, and they all covalently bound to human myeloperoxidase when incubated with this enzyme and H2O2 in vitro. Covalent binding to PMA-activated neutrophils was inhibited by nucleophiles, such as glutathione and N-acetylcysteine, but not by N-acetyllysine. In the presence of the PMA, all three drugs covalently bound to activated rat bone marrow cells in vitro, while in its absence only clozapine did. Covalently modified liver proteins were observed in rats treated for 6 weeks with clozapine (25 or 50 mg/kg/day), vesnarinone (300 mg/kg/day), or procainamide (50 mg/kg/day). Clozapine extensively modified proteins in all subcellular fractions; procainamide formed a 99 kDa adduct in a membrane-containing fraction and 57, 47, and 36 kDa adducts in a cytosolic fraction, while vesnarinone formed liver-protein adducts with molecular masses of 82, 62, 49, and 40 kDa in membrane, cytosolic, and S9 fractions. In addition, clozapine and procainamide, but not vesnarinone, formed a 49 kDa drug-protein adduct in the bone marrow of treated rats. Furthermore, procainamide covalently bound to a 58 kDa protein in neutrophils of a patient treated with the drug. We suspect that covalent modification of common targets in the neutrophils by these three drugs plays a role in the development of drug-induced agranulocytosis.

Antibodies to non-bilayer phospholipid arrangements induce a murine autoimmune disease resembling human lupus.

Antibodies recognizing non-bilayer phospholipid arrangements (NPA) in membrane models and in cell membranes in vivo, triggered an autoimmune-like disease in mice. This exhibited features similar to human lupus and was induced by injecting mice either with the H308 monoclonal antibody specific to NPA, with sera from mice which already had developed the autoimmune disease, or with liposomes treated with the NPA inductors chlorpromazine or procainamide; or with these NPA inductors alone. All these procedures revealed the involvement of antibodies to non-bilayer phospholipids in inducing this autoimmune-like disease. Unraveling the mechanisms of these antibodies might contribute to a better understanding of the molecular and immunological basis of autoimmune diseases like lupus and, hopefully, towards the development of better therapeutic strategies.

Persistence of autoreactive T cell drive is required to elicit anti-chromatin antibodies in a murine model of drug-induced lupus.

Long-term treatment with procainamide and numerous other medications is occasionally associated with the development of drug-induced lupus. We recently established a murine model for this syndrome by disrupting central T cell tolerance. Two intrathymic injections of procainamide-hydroxylamine (PAHA), a reactive metabolite of procainamide, into (C57BL/6 x DBA/2)F1 mice resulted in the appearance of chromatin-reactive T cells and anti-chromatin autoantibodies. The current study explores in this model the role of autoreactive T cells in autoantibody production and examines why autoantibodies after a single intrathymic drug injection were much more limited in isotype and specificity. Injection of as few as 5000 chromatin-reactive T cells into naive, syngeneic mice induced a rapid IgM anti-denatured DNA response, while injection of at least 100-fold greater number of activated T cells was required for induction of IgG anti-chromatin Abs, suggesting that small numbers of autoreactive T cells can be homeostatically controlled. Mice subjected to a single intrathymic PAHA injection after receiving splenic B cells from an intrathymic PAHA-injected syngeneic donor also developed anti-chromatin Abs, but adoptive transfer of similarly primed T cells or of B cells without intrathymic PAHA injection of the recipient failed to produce an anti-chromatin response. However, anti-chromatin Abs developed after a single intrathymic PAHA injection in Fas-deficient C57BL/6-lpr/lpr mice, suggesting that activation-induced cell death limited autoimmunity in normal mice. Taken together, these results imply that chromatin-reactive T cells produced by intrathymic PAHA created a B cell population primed to somatically mutate and Ig class switch when subjected to a heavy load or second wave of autoreactive T cells.

Genetic and immunologic studies of patients on procainamide.

Forty (40) patients with cardiac arrhythmias receiving procainamide (PA) therapy and 24 patients who were receiving other drugs for their cardiac disorders were investigated for class II HLA phenotypes and their DRB1*04 and DQB1*03 subtypes. Other genetic marker evaluations in the PA patients included: 1) class III MHC C4A and C4B null alleles of complement; and, 2) acetylation phenotype. Twenty (20) of the PA patients were also tested for the ability of their stimulated cells to secrete Interleukin-1 (IL-1 beta) and tumor necrosis factor (TNF alpha). We also examined the spontaneous production of these cytokines by peripheral blood leukocytes (PBL) from patients who were receiving chronic PA treatment. The results revealed no association of acetylation phenotypes with the class II HLA phenotypes nor class III MHC C4 allotypes in these patients. The results did show a significant increase in class III C4 complement allotypes in the PA patients when compared to the controls. The results also showed a significant increase in autoantibodies and DQw3 phenotypes in the PA patient group when compared to control populations. Results of spontaneous IL-1 and TNF production suggested there may be an association of select class II HLA phenotypes in some patients and this may be relevant to host responsiveness to PA treatment.

Study of procainamide hapten-specific antibodies in rabbits and humans.

Procainamide (PA) is the drug most commonly associated with the induction of autoantibodies and drug-related lupus (DRL). While the majority of these patients express autoantibodies, antibodies to the parent drug and metabolites, PA-hydroxylamine (PAHA) or nitroso-PA (NOPA), have not been reported in humans. Hapten-carrier conjugates were prepared using human hemoglobin (HgB) or autologous rabbit erythrocytes with PAHA or NOPA. PA was conjugated to rabbit serum albumin (RSA) or egg albumin (OVA) via diazotization and condensation methods. Rabbits were immunized with hapten conjugates in Freund's adjuvant. These hapten-carrier compounds (5-10 micrograms/ml) were used as test antigens for antibodies in sera from the rabbits and 40 patients on chronic PA treatment. 10 SLE patients, 33 elderly and 20 young normal controls by ELISA. Type I and II collagens were also used as test antigens for human sera. Sera from rabbits immunized with the PA compounds had elevated IgG antibody values to PA, PAHA and NOPA, but no autoantibodies. Absorption of the rabbit sera with the PA compounds reduced the antibody levels; ssDNA and histones failed to inhibit the total binding values. Mean binding to PA-OVA was 0.95 +/- 0.41 for PA patients and 1.37 +/- 0.26 standard error of means (S.E.M.) in the SLE patients compared to 0.37 +/- 0.14 S.E.M. in the normal sera (P < or = 0.05); similar binding values to PAHA-HgB and NOPA-HgB were also observed. Sixty-eight percent of the PA patients had antibodies to type II collagen. Elevated binding values to PA compounds were inhibited by absorption of human sera with ssDNA or total histones; absorption with PA or PAHA had no significant effect. These findings suggest that sera from PA patients containing high titers of autoantibodies cross-react in vitro with unrelated antigens.

T lymphocytes ignore procainamide, but respond to its reactive metabolites in peritoneal cells: demonstration by the adoptive transfer popliteal lymph node assay.

The drug procainamide (PA) is notorious for causing drug-induced systemic lupus erythematosus (SLE) in humans. Indirect evidence suggests that metabolism of PA to a reactive intermediate metabolite is involved in the pathogenesis of drug-induced SLE in that N-hydroxylation of the arylamine group of PA favors this condition, whereas N-acetylation prevents it. If this is correct, one would expect hydroxylamine-PA (HAPA) to be immunogenic, whereas N-acetyl-PA (N-ac-PA) should be nonimmunogenic. This hypothesis was confirmed by means of the popliteal lymph node assay (PLNA) in mice: injection of PA and N-ac-PA failed to induce a reaction in the direct PLNA, whereas HAPA induced a vigorous reaction. Using the adoptive transfer PLNA, splenic T cells of mice that had received three injections of HAPA were shown to be specifically sensitized to this metabolite, but not to PA or N-ac-PA. In this system, an anamnestic T cell response could also be elicited when homogenized peritoneal cells of mice that had been treated with PA for 4 months were used as the challenging antigen, indicating that the peritoneal cells of PA-treated animals contained or had been exposed to the reactive intermediate metabolite HAPA. Whereas in slow acetylator mice this 4-month PA treatment sufficed to generate HAPA in peritoneal cells, fast acetylators required additional stimulation of their oxidative metabolism in order to produce enough HAPA detectable by sensitized T cells. These findings clearly support the concept that reactive intermediate metabolites, such as HAPA, are generated by the oxidative metabolism of phagocytic cells and are immunogenic for T cells.

Genetic, immunologic and biotransformation studies of patients on procainamide.

This report represents follow-up observations of a unique long-term study of patients on procainamide (PA) for various cardiac arrhythmias. Serologic and clinical evaluations associated with drug-related autoimmunity were assessed and patients were characterized for factors postulated to influence susceptibility to autoimmunity, including acetylator phenotype, oxidative metabolism of PA, HLA class profile, and production of interleukin-1 (IL-1) and tumor necrosis factor (TNF). Fifty-two percent had IgM and 70% IgG antibodies to total histones; 67% had IgG antibodies to histone H2A/H2B. Patients were equally divided between fast and slow acetylators. N-oxidative metabolism of PA was indicated by the presence of urinary nitroprocainamide, which correlated with elevated titers of antihistone antibodies. There was a significant incidence of the DQw7 split of DQw3 in PA patients when compared to controls, and the frequency of antibodies to total histones and H2A/H2B was significantly increased in the DQw7 patients. C4A*QO and C4B*QO alleles were more frequent in the PA patients than in controls. IL-1 and TNF production was not different in patients compared to controls. These data suggest that certain genetic factors may serve as markers for PA-related autoimmunity.

Drug-specific immune responses induced by procainamide, hydralazine and isoniazid in guinea-pigs.

The drug-induced graft vs host reaction (GVHR) hypothesis requires, as its first step, specific T-cell immune responses to the drug-modified self. Procainamide, isoniazid and hydralazine are known to provoke various allergic reactions including GVHR-like adverse effects in man. We now report that drug-specific immune responses can easily be induced by these drugs in guinea-pigs. Twenty-five milligrams of each of these drugs and penicillin G, which is known to make covalent bonds with proteins and to also induce drug-specific immune responses, were mixed with complete Freund's adjuvant (CFA) and subcutaneously (s.c.) injected twice at an interval of 2 weeks into female Hartley guinea-pigs. The antibodies to these drugs were assessed by means of an enzyme-linked immunosorbent assay (ELISA). Two weeks after the last injection, all animals treated with isoniazid, hydralazine and penicillin G produced high titers of antibodies to these drugs. Antibodies to procainamide were also detected, although their antibody titers were low. The specificity of the antibodies produced were tested by the inhibition of ELISA and concentration-dependent inhibition was observed. Delayed type hypersensitivity (DTH) reactions were also observed in the animals treated with procainamide, isoniazid and hydralazine 2 weeks after the last injection. These results suggest that the allergic reactions observed in clinical use are related to the inducing potential of drug-specific immune responses in an animal system. Therefore, immunization of guinea-pigs with test drugs and CFA may give useful information for predicting the occurrence of allergic reactions in man.

Immunomodulatory effects of procainamide metabolites: their implications in drug-related lupus.

Evidence suggests that N-oxidized metabolites of procainamide may be responsible for the development of lupus-like symptoms associated with procainamide therapy. The human hepatic microsomal metabolism of procainamide has been previously reported to result in formation of the N-hydroxylamine derivative of procainamide (procainamide hydroxylamine [PAHA]). The objective of this study was to examine the effects of PAHA on human lymphocytes and adherent cells (monocytes and macrophages). When incubated with lymphocytes in whole blood, PAHA enhanced the response to mitogen and immunoglobulin secretion at lower concentrations (less than or equal to 4 mumol/L) but suppressed these functions at higher concentrations. The cytotoxic effects were nonselective for T lymphocytes and B lymphocytes and appeared to involve an interaction between PAHA and hemoglobin. When erythrocytes were removed or when hemoglobin was converted to carboxyhemoglobin, the suppressive effects of PAHA on lymphocytes were reduced. PAHA stimulated interleukin-1 production by adherent cells at 25 mumol/L but had no effect at lower concentrations. Superoxide anion release was unaffected by PAHA in "resting" adherent cells. Pretreatment with PAHA (2 mumol/L) diminished superoxide release in response to stimulation by phorbol myristate acetate (PMA) or latex bead phagocytosis but augmented superoxide release when coincubated with PMA or latex. These observations indicate that PAHA produces complex, concentration-dependent interactions with human immunoregulatory cells, and they suggest that the effects of PAHA on lymphocyte function may result from the further oxidation of PAHA by hemoglobin, perhaps to the nitroso form.

Development of Fluoroimmunoassays for the determination of individual or combined levels of procainamide and N-acetylprocainamide in serum.

A fluoroimmunoassay has been developed for the simultaneous determination of serum levels of procainamide and its active metabolite N-acetylprocainamide. It employs procainamide linked through its aromatic amino group to fluorescein isothiocyanate as tracer and an antiserum raised against procainamide conjugated to human thyroglobulin through the same position. Separation is rapidly and simply achieved by covalently linking the antiserum to magnetisable microparticles and use of a magnet. Specific magnetisable particle fluorimmunoassays were also developed for procainamide and for N-acetylprocainamide by the use of suitable immunogens and fluorescein-labelled tracers. That for procainamide uses an antiserum raised to a procainamide-enzyme conjugate and fluorescein-labelled p-aminobenzoic acid while the fluoroimmunoassay for N-acetylprocainamide employs an antiserum against a N-acetylprocainamide-enzyme conjugate and fluorescein-labelled p-acetamidobenzoic acid.

Experience with immunological tests in drug-induced hepatitis.

Nine patients are reported who developed hepatic injury following the administration of therapeutic agents. The drugs incriminated were prajmalium bitartrate (4 patients), quinidine (3 patients), procainamide and nifedipine (one patient each). In all patients the clinical features indicated an allergic process, which in three cases was substantiated by positive response to reexposure. The detection of immunologic processes in our patients by various in vitro methods supports the assumption that immune mechanisms may be involved in the production of drug-induced hepatic disease and confirm allergy.

Production and characterization of specific antibody for radioimmunoassay of procainamide.

The production and characterization of a specific antibody for use in the radioimmunoassay of procainamide are described. Cross-reactivity was measured by a nonequilibrium competitive procedure. Procainamide analog concentrations resulting in 50% inhibition were: procainamide, 1.59 nmoles/ml; N-acetylprocainamide, 3.55 nmoles/ml; a propyl analog of procainamide, 398 nmoles/ml; procaine, 316 nmoles/ml; lidocaine, greater than 8000 nmoles/ml; and practolol, greater than 16,000 nmoles/ml. Variations in the ability to inhibit binding of labeled procainamide were related to structural similarities and differences. The affinity constant of the antibody for procainamide was 2.9 x 10(8) liters/mole as measured from a Scatchard plot. The assay allows the direct measurement of procainamide in a 0.1-ml aliquot of diluted serum. The advantages of this method over currently available techniques are its sensitivity, specificity, and simplicity. Furthermore, prior extraction of serum samples is not required. As little as 1 ng of drug/ml of serum can be detected by this method. The accuracy and precision were determined by adding known amounts of procainamide to human serum and then assaying five replicates of each concentration. The within-day and between-day coefficients of variation were 2 and 5%, respectively. The proposed method was used to determine the serum concentration after an intravenous dose of procainamide. A comparison of the radioimmunoassay results with values obtained by a GLC procedure showed excellent agreement.

Lymphocyte alteration by procainamide: relation to drug-induced lupus erythematosus syndrome.

Sera from 11 (65%) of 17 patients with newly diagnosed procainamide-induced lupus contained cold-reactive lymphocytotoxic antibodies to normal human lymphocytes in titres of 1/2 to 1/128. In contrast, only 3 of 15 patients on long-term procainamide therapy without lupus and 3 of 65 normal men had serum lymphocytotoxic antibodies, none at a titre higher than 1/2. Antibody levels in the lupus patients declined quickly after procainamide was stopped, in parallel with their clinical improvement. Procainamide (3.75 x 10(-3) mol/l) suppressed by more than 80% in-vitro phytohaemagglutinin-induced 3H-thymidine incorporation by normal human blood lymphocytes. At 3.75 x 10(-4) mol/l, procainamide enhanced the mitogenic response to 160 +/- 20% of normal. Thus procainamide may interact with the lymphocyte membrane, possibly producing a lupus syndrome directly, by altering lymphocyte function, or indirectly, by generating autoantibodies reactive with normal membrane structures.