Characterization of Peroxidases Expressed in Human Antigen

Article pubs.acs.org/crt

Characterization of Peroxidases Expressed in Human Antigen Presenting Cells and Analysis of the Covalent Binding of Nitroso Sulfamethoxazole to Myeloperoxidase Monday O. Ogese,† Rosalind E. Jenkins,† James L. Maggs,† Xiaoli Meng,† Paul Whitaker,‡ Daniel Peckham,‡ Lee Faulkner,† B. Kevin Park,† and Dean J. Naisbitt*,† †

MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, University of Liverpool, Ashton Street, Liverpool L69 3GE, United Kingdom ‡ Regional Adult Cystic Fibrosis Unit, St James’s University Hospital, Leeds LS9 7TF, United Kingdom S Supporting Information *

ABSTRACT: Drug hypersensitivity remains a major concern, as it causes high morbidity and mortality. Understanding the mechanistic basis of drug hypersensitivity is complicated by the multiple risk factors implicated. This study utilized sulfamethoxazole (SMX) as a model drug to (1) relate SMX metabolism in antigen presenting cells (APCs) to the activation of T-cells and (2) characterize covalent adducts of SMX and myeloperoxidase, which might represent antigenic determinants for T-cells. The SMX metabolite nitroso-SMX (SMX-NO) was found to bind irreversibly to APCs. Time- and concentration-dependent drug− protein adducts were also detected when APCs were cultured with SMX. Metabolic activation of SMX was significantly reduced by the oxygenase/ peroxidase inhibitor methimazole. Similarly, SMX-NO-specific T-cells were activated by APCs pulsed with SMX, and the response was inhibited by pretreatment with methimazole or glutaraldehyde, which blocks antigen processing. Western blotting, real-time polymerase chain reaction (RT-PCR), and mass spectrometry analyses suggested the presence of low concentrations of myeloperoxidase in APCs. RT-PCR revealed mRNA expression for flavin-containing monooxygenases (FMO1−5), thyroid peroxidase, and lactoperoxidase, but the corresponding proteins were not detected. Mass spectrometric characterization of SMX-NO-modified myeloperoxidase revealed the formation of N-hydroxysulfinamide adducts on Cys309 and Cys398. These data show that SMX’s metabolism in APCs generates antigenic determinants for T-cells. Peptides derived from SMX-NO-modified myeloperoxidase may represent one form of functional antigen.

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INTRODUCTION Sulfamethoxazole (SMX) hypersensitivity is a classical immunemediated reaction involving activation of antigen-specific Tlymphocytes.1−4 Antigenic determinants, i.e., drug−peptide conjugates, are generated through processing, following binding of the chemically reactive derivative and putative metabolite nitroso SMX (SMX-NO) to protein. Haptenation of serum protein in SMX hypersensitive patients and drug naiv̈ e volunteers has been detected using an anti-drug antibody.5,6 The haptenation appeared to be highly selective, but neither the protein nor the adduct structure was identified.6 More recently, the model proteins glutathione S-transferase π (GSTP) and human serum albumin (HSA) were incubated with SMX-NO and subjected to mass spectrometric analysis to characterize the adduction chemistry. SMX-NO was found to modify both proteins at cysteine residues; sulfinamide, N-hydroxysulfinamide, and N-hydroxysulfonamide adducts were detected (Scheme 1),7 but only the N-hydroxysulfinamide adduct, derived putatively from cysteine sulfenic acids, was found on both proteins. © 2014 American Chemical Society

Although the liver is the major organ involved in drug metabolism, the skin is the main target affected in most hypersensitivity reactions.8 Certain reactive metabolites and precursor intermediates are translocated from the liver to distal organs.9 Nevertheless, localized generation of reactive species from the parent drug could be the trigger for the pronounced involvement of the skin in adverse reactions.10 Hepatic oxidative metabolism of SMX to its hydroxylamine in humans is catalyzed predominantly by CYP2C9.11,12 The hydroxylamine undergoes spontaneous oxidation to form SMX-NO. Reactive metabolites of SMX capable of protein modification are also formed by oxidative pathways in various human skin cells and in antigen presenting cells (APCs).10,13−17 However, from transcriptomic analyses and the effects of metabolic inhibitors, it seems unlikely P450 isoforms contribute appreciably to the bioactivation of SMX in those cells.16,17 Isolated prostaglandin H synthase and myeloperoxidase Received: November 14, 2014 Published: December 22, 2014 144

DOI: 10.1021/tx500458k Chem. Res. Toxicol. 2015, 28, 144−154

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Chemical Research in Toxicology

Scheme 1. N-4-Oxidatation of SMX to Hydroxylamine and Nitroso Derivatives, and the Proposed Adduction Reactions of SMXNO with Cysteine, Cysteine Sulfenic Acid, and Cysteine Sulfinic Acid Residues of Proteins

catalyze the formation of SMX hydroxylamine.18 Expression of myeloperoxidase in human kupffer cells has been claimed,19 but later analyses of acutely injured human liver found myeloperoxidase only in recruited neutrophils.20 Certainly the enzyme is not known to contribute to the hepatic oxidation of SMX. Additional studies on the enzymology of SMX’s oxidative bioactivation in epidermal keratinocytes suggested substantial roles for flavin-containing monoxygenase (FMO) 3 and unidentified peroxidases; immunoblot analysis and reverse transcription polymerase chain reactions failed to detect lactoperoxidase, thyroid peroxidase, or myeloperoxidase.17 Although the metabolism of SMX has been characterized, it is still not known whether SMX-NO, generated within the liver, can evade hepatic detoxification, circulate in the periphery, and bind to proteins in skin (the main site of SMX-mediated tissue injury) or immune cells. The nitroso’s immediate precursor, SMX hydroxylamine,21 is a plasma metabolite,22 but its transfer to skin has also not been reported. Circulating SMX does penetrate the skin,23 and the drug’s susceptibility to enzymatic

oxidation, combined with the expression of oxidative and peroxidative enzymes by extra-hepatic cells, suggests that SMX metabolites might be formed locally in skin and/or immune cells. Given the intrinsic reactivity of SMX-NO, we hypothesized that modification of cysteine residues would occur in molecular proximity to the site of metabolite formation. Hence, it is possible that myeloperoxidase could catalyze the bioactivation of SMX and also serve as a source of antigenic determinants for an immune response. This proposition is supported by the finding that 7-hydroxyfluperlapine, a pre-reactive metabolite of fluperlapine, is oxidized by human myeloperoxidase in vitro and covalently modifies the protein.24 Therefore, this study aimed to determine the expression profile of oxygenases and peroxidases in human immune cells, investigate SMX’s bioactivation in those cells and its functional consequences, and characterize adduction of myeloperoxidase by SMX-NO in vitro. A panel of immunochemical assays (ELISA, Western blotting) was used to detect drug−protein adducts formed in immune cells (EBV145


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inhibitor of FMOs, peroxidases, and cytochrome P450 isoforms,27−29 before incubation with SMX. Enzyme-Linked Immunosorbent Assay (ELISA) of Drug− Protein Adducts. Cell lysates from incubations of EBV-transformed B-cells, dendritic cells, and HL60 cells (100 μL, 250 μg/mL) were plated in duplicate onto high-capacity 96-well ELISA plates and incubated for 16 h at 4 °C. In other experiments, the haptenation of recombinant myeloperoxidase (50 μL, 100 μg/mL; approximately 0.6 μM) that had been incubated with either SMX (2 mM) and hydrogen peroxide (10 μM) or SMX-NO (50 μM) for 1 h was also assessed by ELISA. Those incubations were performed in 0.1 M phosphate buffer, pH 7.4, at 37 °C, under an atmosphere of 5% CO2. Wells were washed with phosphate-buffered saline (PBS) containing 0.001% (v/v) Tween-20 and blocked for 1 h with 2.5% (w/v) skimmed milk in PBS-Tween. They were then washed with PBS-Tween and incubated in 100 μL of anti-SMX antiserum (1:2000) overnight at 4 °C. After overnight incubation, the wells were washed and incubated in 100 μL of alkaline phosphatase-conjugated anti-rabbit IgG (1:1000) for 2 h. Final washes were followed by a 30 min incubation in alkaline phosphatase substrate (100 μL/well). Absorbance values in incubation samples and solvent controls were measured at 405 nm. Western Blot Detection of Drug−Myeloperoxidase Adducts. Solutions of unmodified and SMX-modified myeloperoxidase (100 μg/mL; 10 μL/lane) were prepared in RIPA buffer. They were treated with reducing Laemmli buffer and then separated by electrophoresis on a 12% SDS-PAGE gel (300 V, 60 mA, 1 h). Separated proteins were then transferred from the gel onto nitrocellulose membranes (300 V, 250 mA, and 1 h). Membranes were then blocked with 2.5% (w/v) skimmed milk prepared in Tris-saline-Tween buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.6, 0.05% (v/v) Tween-20). Immunodetection of SMX−protein adducts was performed by incubating the blot with anti-SMX rabbit antiserum (1:2000) in phosphate buffer overnight at 4 °C. Unbound antibody was removed by washing with PBS-Tween, and the membrane was incubated with peroxidase-conjugated anti-rabbit IgG antibody (1:10,000 in Trissaline-Tween buffer) for 2 h at room temperature. The membrane was finally developed using an enhanced chemiluminescent substrate. Reversed-phase column fractions containing haptenated tryptic peptides of myeloperoxidase were spotted directly onto nitrocellulose membrane, which were then processed in the same way as the Western blots. Western Blot Detection of Myeloperoxidase in Immune Cells. EBV-transformed B-cells, dendritic cells, and HL60s were lysed with RIPA buffer, and protein concentration was determined using the Bradford assay. Western blotting for myeloperoxidase, FMO3, and thyroid peroxidase was performed using the procedure described above. Estimation of Cellular Peroxidase Activity. The total peroxidase activity in HL60 cells, dendritic cells, and EBV-transformed B-cells was assayed using RIPA buffer lysates. Cell lysates were first clarified by centrifugation. Clarified cell lysate samples (250 μg/mL, 50 μL) and standard myeloperoxidase (0−200 ng/mL; 50 μL) were plated into a 96-well microtiter plate in duplicate. An equal volume of 2× Amplex UltraRed reagent working solution (Life Technologies, Paisley, United Kingdom) was added to all the samples and myeloperoxidase standards, and the wells were developed according to the manufacturer’s instructions. Peroxidase activity in lysates was expressed as myeloperoxidase equivalents. Analysis of Oxidative Enzymes by Real-Time Polymerase Chain Reaction (RT-PCR). Total RNA was extracted from EBVtransformed B-cells, HL60 cells, and dendritic cells (5 × 106) with the RNeasy Mini kit. On-column DNase digestion (Qiagen, Manchester, United Kingdom) was used to remove genomic DNA. RNA (1 μg) was used for cDNA synthesis with M-MLV reverse transcriptase and oligodT primers. qRT-PCR was performed using Power SYBR Green Mastermix (Applied Biosystems, Foster City, CA). Reactions were analyzed using an ABI 7000 real-time PCR machine with the following cycle conditions: 10 min at 95 °C, followed by 45 cycles of 15 s at 95 °C and 60 s at 60 °C. Results were then normalized to the β-actin expression.

transformed B cells, neutrophil-derived HL60 cells, and dendritic cells). Protein and mRNA of drug-metabolizing enzymes were compared in the different cell types. Finally, LCMS/MS analysis was used to characterize the modification of myeloperoxidase by SMX-NO.

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EXPERIMENTAL PROCEDURES

Cells, Chemicals, and Reagents. HL60 promyelocytic leukemia cells were obtained from Sigma-Aldrich (Dorset, United Kingdom). All chemicals and reagents were purchased from Sigma-Aldrich unless otherwise stated. Anti-myeloperoxidase antibody, anti-β actin antibody, anti-thyroid peroxidase antibody, and recombinant human myeloperoxidase were obtained from Abcam (Cambridge, United Kingdom). Anti-FMO2 and anti-FMO3 antibodies were obtained from BD Bioscience (Oxford, United Kingdom). Anti-SMX antiserum was prepared by Panigen Inc. (Blanchardville, WI). M-MLV reverse transcriptase was supplied by Promega (Southampton, United Kingdom). Laemmli buffer was obtained from Bio-Rad (Hemel Hempstead, United Kingdom). Chemiluminescent substrate for immunoblotting was purchased from Thermo Scientific (Cramlington, Northumberland, United Kingdom). SMX-NO was purchased from Dalton Chemical Laboratories Inc. (Toronto, Canada). Generation of Dendritic Cells. CD14+ monocytes were isolated from peripheral blood mononuclear cells (PBMCs) using magnetic MicroBeads and columns according to the manufacturer’s instructions (Miltenyi Biotech, Bisley, United Kingdom) and then cultured in dendritic cell culture medium consisting of RPMI-1640, penicillin (100 μg/mL), streptomycin (100 U/mL), transferrin (25 μg/mL), 10% (v/ v) human AB serum, HEPES buffer (25 mM), and L-glutamine (2 mM), supplemented with GM-CSF (800 U/mL) and IL-4 (800 U/ mL) for 7−8 days to encourage differentiation to dendritic cells. Generation of EBV-Transformed B-Cells. PBMCs were transformed into B-cell lines using supernatant from the virus-producing cell line B9.58. PBMCs (5 × 106) were resuspended in supernatant from B9.58 cells (5 mL) that had been passed through a 0.45-μm syringe filter. Cyclosporin A (1 μg/mL) was added to inhibit the proliferation of T-lymphocytes, and the PBMCs were incubated overnight at 37 °C under an atmosphere of 95% O2/5% CO2. Cells were then washed with Hank’s balanced salt solution (HBSS), resuspended in medium [RPMI 1640 supplemented with 10% (v/v) fetal bovine serum, HEPES (25 mM), penicillin (1000 U/mL), streptomycin (0.1 mg/mL), and L-glutamine (2 mM)] supplemented with cyclosporin A (1 μg/mL), and cultured in a 24-well plate. Fresh culture medium was added twice a week to maintain the cells. Cyclosporin was omitted from the culture medium after 2 weeks to enhance the proliferation of the B-cells. Cells were transferred to a tissue culture flask when confluent and maintained with fresh culture medium twice a week. Cell Incubations To Assess Formation of Drug−Protein Adducts. Epstein−Barr virus (EBV)-transformed B-cells, dendritic cells, and HL60 cells (2 × 106 cells/mL) were incubated with either SMX (0.5−3 mM; 0.1% (v/v) DMSO) or SMX-NO (5−50 μM; 0.1% (v/v) DMSO) in culture medium in a 24-well plate at 37 °C under an atmosphere of 5% CO2 for 16 h. The control incubations contained solvent alone. HL60 cells were used as a positive control for the expression of myeloperoxidase.25 The cells were washed three times with HBSS by centrifugation at 1500 rpm for 5 min to remove noncovalently bound drug. Final cell pellets were re-suspended in 200 μL of RIPA buffer [50 mM Tris, pH 7.5, 150 mM NaCl, 2.5 mM EDTA, 10% (w/v) glycerol, 1% (w/v) Triton X-100, 1 mM Na3VO4, 10 μg/ mL aprotinin, 10 μg/mL leupeptin, 1 mM phenylmethanesulfonyl fluoride, 0.1% (w/v) SDS, and 0.5% (w/v) Na deoxycholate] and placed on ice for 30 min to lyse. The cells were given three bursts of sonication on ice. The lysates were centrifuged at 14000g for 10 min at 4 °C. Supernatants were collected and their protein concentrations determined using the Bradford assay.26 Protein concentrations were standardized to 250 μg/mL. In certain experiments, EBV-transformed B-cells were pre-incubated for 30 min with methimazole (1 mM), an 146


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Figure 1. Bioactivation of SMX in antigen presenting cells. (A) Time-dependent protein adduction by SMX. EBV-transformed B cells (2 × 106/mL) were incubated with SMX (2 mM) for 1, 4, and 16 h. ELISA was performed with anti-SMX antibody on cell lysate to determine the levels of drug− protein adducts. (B,C) EBV-transformed B-cells (2 × 106/mL) were incubated with either SMX-NO (5 to 50 μM) or SMX (0.5−3 mM) for 16 h. ELISA was performed on lysate to determine levels of drug−protein adducts. (D) EBV-transformed B-cells (EBVs), dendritic cells (DC), or HL60 cells (2 × 106/mL) were incubated with SMX (2 mM) for 16 h. ELISA was performed on lysates to determine levels of protein adduct. (E) EBVtransformed B-cells (2 × 106/mL) were pre-incubated with methimazole (1 mM) for 30 min, followed by a 16 h incubation with SMX (2 mM). ELISA was performed to determine the extent of protein haptenation. Mass Spectrometric Analysis of Myeloperoxidase in Immune Cells. Proteins were extracted from pooled samples of EBV-transformed B-cells and processed for untargeted LC-MS/MS protein identification as described previously.31 Samples were delivered into a Triple TOF 5600 mass spectrometer (AB Sciex) by automated in-line reversed-phase liquid chromatography (LC) using an Eksigent NanoUltra cHiPLC system mounted with a microfluidic trap and an analytical column (15 cm × 75 μm) packed with ChromXP C18-CL 3 μm. A NanoSpray III source was fitted with a 10 μm inner diameter PicoTip emitter (New Objective). A gradient of 2− 50% (v/v) ACN, 0.1% (v/v) FA over 90 min was applied to the column at a flow rate of 300 nL/min. Spectra were acquired automatically in positive ion mode using information-dependent acquisition powered by Analyst TF 1.5.1. software. Proteins were identified using ProteinPilot software v4.0 using the ParagonTM algorithm and the most recent version of the SwissProt database. After the failure to detect myeloperoxidase in EBV-transformed B-cells, a targeted mass spectrometric approach was developed. Briefly, in-gel trypsin digests of selected protein bands resolved by SDS-PAGE were subjected to multiple reaction monitoring (MRM) MS. A 5500 QTRAP hybrid triple-quadrupole/linear ion trap instrument was used with a Nanospray II source (AB Sciex). Samples were delivered into the QTRAP by automated in-line liquid chromatography (Ultimate 3000 LC system with Nanoflow splitter, 5 mm C18 nano-pre-column and 75 μm × 15 cm C18 PepMap column (Dionex, Sunnyvale, CA) via a 10 μm inner diameter PicoTip. The ion spray potential was set to 2200−3500 V, the nebulizer gas to 19, and the interface heater to 150 °C. A gradient from 2% acetonitrile/0.1% formic acid (v/v) to 50% acetonitrile/0.1% formic acid (v/v) in 60 min was applied at a flow rate of 300 nL/min. MRM transitions were acquired in positive ion mode and at unit resolution in

both the Q1 and Q3 quadrupoles, and dwell times were 50 ms. MRM transitions were designed for two myeloperoxidase peptides, 490 IANVFTNAFR499 (2+ m/z 576.6 with fragment ions of m/z 608, 755, 854, and 968) and 560QNQIAVDEIR569 (2+ m/z 593.3 with fragment ions of m/z 288, 631, 702, and 815). Detection of a transition triggered the acquisition of a product ion spectrum of the peptide. Mass Spectrometric Analysis of Nitroso-sulfamethoxazoleModified Myeloperoxidase. Myeloperoxidase was incubated with SMX-NO (molar ratio, 1:5) in 0.1 M phosphate buffer, pH 7.4, at 37 °C for 1 h under an atmosphere of 5% CO2. The protein was subjected to SDS-PAGE to remove unbound drug. The excised gel band was reduced, alkylated, and digested with trypsin. Extracted peptides from five myeloperoxidase bands were resuspended in 500 μL of 0.1% trifluoroacetic acid (TFA), injected onto a Prodigy C18 5-μm column (150 × 4.6 mm; Phenomenex, Macclesfield, Cheshire, United Kingdom), and eluted using a gradient from 95% solvent A (5% acetonitrile/0.1% TFA)/5% solvent B (95% acetonitrile/0.1% TFA v/ v), to 50% v/v solvent B in 40 min at a flow rate of 1 mL/min. Fractions of 1 mL were collected and dried in a SpeedVac vacuum evaporator (Eppendorf, Cambridge, United Kingdom). Aliquots of reconstituted fractions were spotted onto nitrocellulose membrane for immunodetection of SMX-peptide adducts, and drug-positive fractions were analyzed by LC-MS/MS on a Triple TOF 5600, as described above. Generation of Drug-Specific T-Cell Clones. PBMCs were isolated from three patients who had developed maculopapular skin eruptions following SMX exposure, for the generation of drug-specific T-cell clones and autologous EBV-transformed B-cells. Patient demographics and details of the adverse reactions have been described previously.32 Approval for the study was obtained from the local 147


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Chemical Research in Toxicology research ethics committee, and informed written consent was obtained from the patients. For the generation of clones, PBMCs (1 × 106/well; 0.5 mL) were cultured with SMX-NO (25 and 50 μM). On days 5 and 9, culture medium was supplemented with IL-2 (200 IU/mL) to expand the number of antigen-specific T-cells prior to cloning on day 14. Autologous EBV-transformed B-cell lines were used as APCs in assays with the clones. Antigen specificity was assessed by culturing irradiated EBVtransformed B-cells (1 × 104 cells/well) and either SMX (2 mM) or SMX-NO (50 μM) and clones (5 × 104 cells/well; 200 μL) for 48 h. Proliferation was measured by the addition of [3H]-thymidine followed by scintillation counting. Clones with a stimulation index of greater than 2 were expanded by repetitive stimulation with irradiated allogeneic PBMCs (5 × 104 cells/well; 200 μL) and phytohemeagglutinin (5 μg/mL) in IL-2-containing medium. T-cell activation was also investigated using an interferon-γ ELISpot assay. Spots were visualized and counted using an ELISpot reader (Autoimmun Diagnostika GmbH, Strassberg, Germany). Antigen Presenting Cell Pulsing and Inhibition Assays. EBVtransformed B-cells were pulsed with SMX for 16 h, washed extensively with HBSS to remove free drug, and co-incubated with SMX-NO-specific T-cell clones in a 96-well U-bottom microplate for 48 h under an atmosphere of 5% CO2 at 37 °C. [3H]-thymidine was added for the final 16 h of incubation, and T-lymphocyte proliferation was evaluated using scintillation counting. A mixture of APCs and Tcells incubated with SMX-NO (50 μM) for 48 h served as a positive control because the nitroso haptenates cellular proteins spontaneously. The role of intracellular antigen processing in the activation of the Tcells was investigated by fixing APCs with glutaraldehyde. Briefly, autologous EBV-transformed B-cells (2 × 106 cells/mL) were suspended in HBSS (1 mL), glutaraldehyde (25% aqueous solution, v/v, 1 μL; final concentration, 2.6 mM) was added, and the cells were mixed gently for 30 s. Glycine (1 mL of 1 M aqueous solution) was then added to quench excess glutaraldehyde, and the cells were mixed for a further 45 s. The cells were washed three times to remove residual glutaraldehyde, suspended in culture medium, and used in Tcell assays as outlined above. Finally, EBV-transformed B-cells were pre-incubated with methimazole (1 mM) for 30 min, washed, and cultured with SMX (2 mM) for 16 h. Cells were washed extensively to remove free SMX and then co-cultured with SMX-NO-specific T-cell clones. Statistical Analysis. Data are presented as means and their standard deviations. Statistical comparisons of experimental groups were made using the paired t tests method (SigmaPlot 12 software, Systat Software Inc., San Jose, CA). A value of P < 0.05 was considered to be significant.

response and to explore mechanisms of antigen presentation. APCs pulsed with SMX (2 mM) for 16 h induced T-cell proliferation and interferon-γ secretion (Figure 2A,B). Fixation

Figure 2. Bioactivation of SMX in immune cells and T-cell activation. (A,B) As positive and negative controls, five SMX-NO-specific T-cell clones from one patient (5 × 104 cells, 50 μL) were co-incubated with EBV-transformed B-cells (1 × 104 cells, 50 μL) in the presence and absence of SMX-NO (50 μM, 100 μL) for 48 h. APCs were pulsed with SMX (2 mM) for 16 h, washed repeatedly to remove unbound drug, and added to the T-cell assay as a source of drug-derived antigen. APCs were also fixed with glutaraldehyde (final concentration, 0.025% v/v) or pre-treated with methimazole (1 mM) prior to SMX exposure. (A) T-cell proliferation was measured by [3H]-thymidine incorporation. Data represent the mean of duplicate wells and their standard deviations. (B) Interferon-γ release was measured using an ELISpot assay (images shown are from one clone, representative of the five clones used in panel A).

of APCs with glutaraldehyde or treatment with the general oxygenase/peroxidase inhibitor methimazole before a 16 h incubation with SMX (2 mM) blocked T-cell activation, while glutaraldehyde and methimazole themselves failed to activate T-cells (Figure 2A). Expression of Peroxidases and FMO in Immune Cells. Expression of myeloperoxidase14 by human dendritic cells and of FMO17,33 by keratinocytes has been reported previously. Herein, Western blotting demonstrated the expression of myeloperoxidase in EBV-transformed B-cells and dendritic cells (Figure 3A). As expected, HL60 cells expressed much greater quantities of myeloperoxidase.25 FMO3 and thyroid peroxidase were not detected in any of the cell types by Western blotting (results not shown). Peroxidase activity in the cells was investigated using the fluorogenic Amplex UltraRed assay of H2O2 as a general peroxidase assay. The rank order of the activity was HL60 ≫ dendritic cells > B-cells (Figure 3B). RT-PCR was then used to verify the presence of mRNAs for peroxidases and FMO isoforms (Figure 3C). Messenger RNA for FMO4 and FMO5 was detected in all three cell types, whereas FMO2 and FMO3 transcripts were only detected in the B-cells. Expression of lactoperoxidase mRNA in dendritic cells was highly selective and quite abundant. In agreement with

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RESULTS Characterization of the Bioactivation of Sulfamethoxazole in Immune Cells and Its Functional Consequences. Protein haptenation by SMX in EBV-transformed B-cells was both time- and concentration-dependent (Figure 1A,B). Nevertheless, haptenation progressed slowly; the greatest binding of SMX was observed at 16 h (p = 0.004). Significantly higher levels of binding were detected with SMX-NO (Figure 1C). Bioactivation of SMX, as determined by covalent binding, was observed in the three types of immune cells (Figure 1D), but HL60 cells showed approximately 3-fold and 2-fold greater haptenation compared with EBV-transformed B-cells and dendritic cells, respectively (Figure 1D). A significant decrease in SMX-protein adduct formation was observed when EBVtransformed B-cells were incubated with methimazole for 30 min before incubation with SMX for 16 h (Figure 1E). We have previously described the generation and phenotypic characterization of SMX-NO-responsive CD4+ T-cell clones from hypersensitive patients.32 In this study, five clones were used to confirm that SMX-pulsed APCs activate a T-cell 148


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oxidase adducts in vitro was dependent on drug concentration and the presence of the cofactor hydrogen peroxide (Figure 5A,B). The modification of myeloperoxidase by SMX-NO was time-dependent over 60 min, with the highest level of modification occurring between 30 and 60 min (Figure 5C). A time point of 1 h was used for subsequent myeloperoxidase haptenation studies. Haptenation was detected weakly at the 1:1 molar ratio of protein to drug and more readily with higher molar ratios (1:5 and 1:10) (Figure 5D). Since no SMX-NO-modified peptides were detected when the entire protein digest was analyzed by LC-MS/MS, the mixture of peptides was subjected to off-line reversed-phase chromatography in order to enhance the sensitivity of MS detection (Supporting Information, Supplementary Figure 1A). Each fraction was spotted onto a sheet of nitrocellulose and probed with anti-SMX antibody to identify those that contained drug-modified peptides (Supplementary Figure 1B). The positive fractions were then analyzed by LC-MS/MS, and the data were searched using ProteinPilot software with SMX-NO included as a user-defined modification of cysteine. Two cysteine-containing peptides were observed with a mass addition corresponding to the N-hydroxysulfinamide adduct of SMX (Scheme 1), namely 303IKNQADCIPFFR314 and 396 IPCFLAGDTR405. However, no modification of Cys316 was observed, despite the fact that this is considered to be the only free cysteine residue in myeloperoxidase (Figure 5E). Scheme 1 shows the putative mechanism of N-hydroxysulfinamide formation wherein preformed cysteine sulfenic acids are subsequently modified by SMX-NO. Neither the sulfinamide nor the N-hydroxysulfonamide adduct of a cysteine that is formed when SMX-NO reacts with GSTP in vitro7 was detected on myeloperoxidase.

Figure 3. Expression of peroxidases and FMOs in antigen presenting cells. (A) For Western blotting of myeloperoxidase expressed in the immune cells and HL60 cells, cell lysates were electrophoresed on 12% SDS-PAGE gel and blot probed for myeloperoxidase using an antimyeloperoxidase antibody (1:1000). Expression of β-actin was used as a control. (B) Total peroxidase activity assay showing the myeloperoxidase equivalent concentrations in HL60 cells, dendritic cells (DC), and EBV-transformed B-cells (EBVs). Clarified cell lysates and standard myeloperoxidase solutions were incubated with Amplex UltraRed substrate. (C) RT-PCR determination of mRNA of various FMOs and peroxidases expressed in HL60 cells, dendritic cells, and EBV-transformed B-cells. RNA extracts were subjected to RT-PCR using primers for FMO1−5, lactoperoxidase, thyroid peroxidase, myeloperoxidase, and β-actin. Relative mRNA levels were normalized to the corresponding β-actin mRNA expression.

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DISCUSSION Certain chemically inert drugs undergo oxidative biotransformation to generate protein-reactive intermediates capable of “provoking” the immune system. SMX is the drug used most frequently to study the role of metabolism in immunological drug reactions, as its oxidative biotransformations are welldefined and the putative protein-reactive metabolite SMX-NO can be synthesized.34−36 SMX-NO is highly immunogenic in experimental animals,37 activates the vast majority of T-cells in ̈ T-cells from patients with hypersensitivity,32 and primes naive healthy human donors when dendritic cells present the drugderived antigen.38,39 Recently, T-cells from SMX hypersensitive patients with cystic fibrosis were found to be selectively activated by SMX-NO.40 Although adduction of cysteine residues on the model proteins HSA and human GSTP by SMX-NO has been characterized,7 SMX-NO’s ability to modify cysteine-containing proteins which might be involved in the immune response has not been researched. Since the stability of hepatic chemically reactive metabolites in extracellular transit or in the systemic circulation is questionable,41 extra-hepatic metabolism leading to localized generation of reactive intermediates might be more relevant to the activation of the adaptive immune system. In this respect, myeloperoxidase and other peroxidases might be important targets for SMX-NO in hypersensitive patients. Lavergne et al.42 reported a significantly higher level of anti-myeloperoxidase antibodies in sulfonamide-hypersensitive compared with sulfonamide-tolerant dogs. These data indicate that SMX-modified myeloperoxidase may be involved in the drugspecific humoral response and provide an immunological

the SMX haptenation study and Western blot analysis, mRNA for myeloperoxidase was differentially expressed, HL60 cells ≫ dendritic cells > EBV-transformed B-cells. Mass Spectrometric Detection of Myeloperoxidase in Immune Cells. An untargeted proteomic LC-MS/MS approach to detect myeloperoxidase failed to reveal the presence of the protein in either dendritic cells or EBVtransformed B-cells. Consequently, a targeted LC-MS/MS approach was attempted. A tryptic digest of recombinant myeloperoxidase was used to design protein-specific MRM transitions (Figure 4). Lysates from APCs and HL60 cells were then subjected to SDS-PAGE, and protein bands of the correct molecular weight were excised, in-gel digested, and analyzed using the same MRM method. Peptides 490IANVFTNAFR499 and 560QNQIAVDEIR569 were detected in the digest of protein from HL60 cells at retention times of 32.95 and 25.18 min, respectively, and full-scan MS/MS spectra were acquired to confirm the peptide’s identity (Figure 4). Analysis of the corresponding B-cell protein digest revealed low-intensity peaks at the correct retention times for the two peptides, although acquisition of the MS/MS spectrum was only triggered for 490 IANVFTNAFR499. Although the product ion spectrum was weak, sufficient fragment ion correspondence was observed between MS/MS spectra acquired for HL60 cells and B-cells to conclude that myeloperoxidase was present in low abundance in the latter. Characterization of SMX-NO-Modified Recombinant Myeloperoxidase. Formation of SMX-derived myeloper149


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Figure 4. LC-MS/MS characterization of myeloperoxidase in antigen presenting cells. LC-MRM MS/MS of tryptic peptides (IANVFTNAFR and QNQIAVDEIR) of the myeloperoxidase isolated from HL60 cells and EBV-transformed B-cells. Positive MRM survey scans triggered the generation of product ion spectra of the peptides.

context for the present investigations of myeloperoxidase modification by SMX. Although the skin is highly vascularized, allowing dispersed penetration of circulating drugs, its oxidative drug metabolism capability is limited. Nevertheless, mRNAs of FMO isoforms are expressed in human skin and epidermal keratinocytes, in

amounts similar to or greater than those of mRNA of P450.17,29,33 Furthermore, myeloperoxidase has been found in human skin43 and dendritic cells,14 and unidentified peroxidases (but not lactoperoxidase, thyroid peroxidase, or myeloperoxidase) have been found in epidermal keratinocytes.17 An early analysis of the enzymology of SMX’s bioactivation in 150


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Figure 5. Characterization of SMX-NO-modified recombinant myeloperoxidase. (A) Oxidation of SMX to protein-reactive intermediates by myeloperoxidase ± hydrogen peroxide. SMX (2 mM) was incubated with myeloperoxidase (100 μg/mL) with or without hydrogen peroxide (10 μM) for 1 h. Myeloperoxidase was also incubated with SMX-NO (50 μM; positive control). ELISA was then performed with rabbit anti-SMX antiserum to determine the relative abundances of SMX-derived and SMX-NO-derived adducts. (B) Myeloperoxidase (1.5 mg/mL) was incubated with or without SMX-NO (50 μM) or with various concentrations of SMX (0.05−2 mM) in the presence of hydrogen peroxide (10 μM) for 1 h at 37 °C. Samples were then processed and separated using a 12% SDS-PAGE gel before Western blotting with rabbit anti-SMX antiserum. (C) Myeloperoxidase (1.5 mg/mL) was incubated with SMX-NO (50 μM) in a 1:5 molar ratio for 0−60 min. Samples were processed and separated on a 12% SDS-PAGE gel before Western blotting with an anti-SMX rabbit antibody. (D) Myeloperoxidase was incubated with SMX-NO (1:1, 1:5, and 1:10 molar ratio) for 1 h at 37 °C. Samples were then processed and separated using a 12% SDS-PAGE gel before Western blotting with an antiSMX rabbit antibody. Coomassie Blue staining of myeloperoxidase was used as a loading control. (E) Representative MS3 spectra of haptenated myeloperoxidase-derived tryptic peptides showing characteristic fragment ions from N-hydroxysulfinamide adducts. The modifications by SMX-NO occurred on Cys309 and Cys398. 151


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Analyses of myeloperoxidase haptenated in vitro revealed that the protein had apparently been modified by SMX-NO only at oxidized cysteine residues. Hypothetically, a cysteine adduct of the general type NSO2H (mass increment, 283 amu) might be formed by either of two distinct mechanisms, namely via oxidation of the semi-mercaptal and/or sulfinamide product of the familiar coupling reaction between arylnitroso and cysteine thiolate,7 or by nucleophilic addition at a cysteine sulfenic acid. The latter mechanism is analogous to the well-documented reaction of a nitroso with a sulfenic acid that yields an Nhydroxysulfonamide.49,50 However, the neutral, buffered, aqueous solution in which the modification of MPO occurred apparently did not contain an agent that might have effected the required post-adduction oxidation. m-Chloroperoxybenzoic acid has been required to stabilize hydrolytically labile sulfinamide adducts of human surum albumins Cys34 and heterocyclic nitroso compounds by oxidation to the sulfonamide derivative.51 When reacting with an arylnitroso, a sulfenic acid will prefer to act as a soft nucleophile by virtue of a nucleophilic character of its sulfur atom,52 attacking the nitrogen to form an N-hydroxysulfinamide. Sulfinic acids are known to be soft nucleophiles because they are fully deprotonated at physiological pH. On reacting with nitroso compounds, sulfur attack is favored and forms the more thermodynamically stable sulfonamide.49,50 The wide-spread existence of protein sulfenic acids has been documented,52 and there is some evidence that a cysteine of MPO can be oxidized to a sulfenic acid.53 Nevertheless, neither the sulfenic acid derivative of cysteine 316 or 319 was the target. Instead, cysteines 309 and 398 were modified, despite normally being linked to cysteines 285 and 387, respectively, by intra-chain disulfide bonds. It is not clear how those residues were accessible to covalent modification; it is possible that the recombinant protein did not exhibit the same conformation as the native protein, but their similar enzymatic properties belie this suggestion. Alternatively, mild oxidative damage during expression and isolation of the myeloperoxidase could have influenced the redox status of individual cysteine residues. The exclusive formation of N-hydroxysulfinamide adducts of SMXNO on both human serum albumin7 and myeloperoxidase in vitro contrasts with the additional formation of sulfinamide and N-hydroxysulfonamide adducts on the exceptionally electrophilic Cys47 of GSTP (Scheme 1).7 This difference between proteins might, ultimately, reflect the stability of their cysteine sulfenic and sulfinic acids. Collectively, these data show that myeloperoxidase is potentially an enzyme that bioactivates SMX in human APCs, and because of the proximity of its cysteine residues to the site of metabolite formation, it might represent an important target for drug antigen formation. In ongoing experiments, we are conducting immuno-precipitation/mass spectrometry experiments on drug-treated antigen presenting cells in an attempt to detect myeloperoxidase adducts in viable cells.

APCs demonstrated that protein haptenation was blocked with methimazole. In order to extend our understanding of extrahepatic oxidative metabolism of SMX, peroxidase activity and expression in APCs were investigated. Most of the experiments focused on EBV-transformed human B-cells, as these are often used in ex vivo T-cell assays for antigen presentation. Initially, SMX’s bioactivation in APCs was assessed indirectly using immunochemical assays to quantify the formation of SMXderived protein adducts. Progressive haptenation by SMX was detected over 16 h. Adduct formation was drug concentrationdependent and blocked by methimazole. The levels of adduct formation were higher, and adducts were detected at much earlier time points when cells were exposed to SMX-NO directly. The activation of SMX-NO-specific T-cell clones by APCs pulsed with SMX for 16 h suggests that enzymatic biotransformation of SMX to SMX-NO is involved in the activation of T-cells. Glutaraldehyde fixation of APCs and preincubation of APCs with methimazole significantly decreased T-cell proliferative responses and IFN-γ secretion. Each type of immune cell used in this study displayed detectable peroxidase activity; hence, RT-PCR was used to investigate the relative expression of mRNAs for myeloperoxidase, lactoperoxidase, and thyroid peroxidase. mRNA of lactoperoxidase was not detected in the B-cells. Thyroid peroxidase can probably be excluded as a candidate enzyme in the B-cell metabolism of SMX, because unlike myeloperoxidase it was not detected by Western blotting. The metabolism of SMX by recombinant FMO1 and FMO3 has been reported.17 FMO1 seemingly N-oxygenates only tertiary amines efficiently, whereas human FMO3 N-oxygenates primary, secondary, and tertiary amines.44 The mRNA of both isoforms can be found in human skin,45 but immunoblotting detected only FMO3 in epidermal keratinocytes.17 FMO3 transcripts were detected in the B-cells, and methimazole is inter alia an inhibitor of FMO3.46 However, an appreciable involvement of FMOs in SMX’s metabolism in APCs can be excluded by the negative Western blotting analysis. While methimazole is additionally an inhibitor of P450, the main isoform that catalyzes the N-oxidation of SMX, namely CYP2C9,14 is not expressed by EBV-transformed B-cells.31 This interpretation effectively limits the observed action of methimazole to inhibition of myeloperoxidase and other, presently unidentified enzymes, as has been suggested in studies exploring metabolism of amodiaquine and clozapine.47 In order to consolidate the identification of myeloperoxidase in cell lysates, mass spectrometric analysis was performed. Recombinant human myeloperoxidase was used as a source of signature tryptic peptides for MRM, and also as a model of the native protein for characterization of the adducts formed by SMX-NO. Recombinant myeloperoxidase and the mature native enzyme are certain to differ in particular respects, such as glycosylation, but they have similar enzymatic properties.48 The half-molecule of the recombinant human enzyme consists of 697 amino acid residues, including 15 cysteine residues, 13 of which exist as intra- or inter-chain disulfides. The cysteine residue at position 316 can exist as a sufenic acid which reacts readily with SMX-NO. Hence, this position was initially identified as a probable target for SMX−myeloperoxidase adduct formation. A targeted MRM-MS approach provided evidence for the presence of myeloperoxidase in EBV-transformed B-cells which, together with Western blot and RT-PCR data, suggested that the protein is expressed basally at low levels in these cells.

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S Supporting Information *

Characterization of SMX-NO-modified MPO. This material is available free of charge via the Internet at http://pubs.acs.org.

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Corresponding Author

*Telephone: 0044 151 7945346. Fax: 0044 151 7945540. Email: [email protected]. 152


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This work was supported by a grant from the CF Trust (PJ533) as part of the Centre for Drug Safety Science supported by the Medical Research Council (G0700654). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the patients who donated blood for this project. ABBREVIATIONS ADRs, adverse drug reactions; SMX, sulfamethoxazole; SMXNO, nitrososulfamethoxazole; FMO, flavin-containing monooxygenase; GSTP, glutathione S-transferase π; HSA, human serum albumin; EBV, Epstein−Barr virus; HBSS, Hank’s balanced salt solution; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; RT-PCR, realtime polymerase chain reaction; TFA, trifluoroacetic acid; MRM, multiple reaction monitoring

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Characterization of Peroxidases Expressed in Human Antigen

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