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

Division of Pharmaceutics, College of Pharmacy, The University of Iowa, Iowa City, Iowa (S.R., P.M.V., C.K.S.); Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, Purdue University, West Lafayette, Indiana (C.K.S.)

(Received November 3, 2006; Accepted January 9, 2007)

	Abstract

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Bioactivation of sulfonamides and the subsequent formation of haptenated proteins is believed to be a critical step in the development of hypersensitivity reactions to these drugs. Numerous lines of evidence suggest that the presence of such adducts in dendritic cells (DCs) migrating to draining lymph nodes is essential for the development of cutaneous reactions to xenobiotics. Our objective was to determine the ability of human DCs to form drug-protein covalent adducts when exposed to sulfamethoxazole (SMX), dapsone (DDS), or their arylhydroxylamine metabolites [sulfamethoxazole hydroxylamine (S-NOH) and dapsone hydroxylamine (D-NOH)] and to take up preformed adduct. Naive and immature CD34+ KG-1 cells were incubated with SMX, DDS, or metabolites. Formation of haptenated proteins was probed using confocal microscopy and ELISA. Cells were also incubated with preformed adduct (drug-bovine serum albumin conjugate), and uptake was determined using confocal microscopy. Both naive and immature KG-1 cells were able to bioactivate DDS, forming drug-protein adducts, whereas cells showed very little protein haptenation when exposed to SMX. Exposure to S-NOH or D-NOH resulted in protein haptenation in both cell types. Both immature and naive KG-1 cells were able to take up preformed haptenated proteins. Thus, DCs may acquire haptenated proteins associated with drugs via intracellular bioactivation, uptake of reactive metabolites, or uptake of adduct formed and released by adjacent cells (e.g., keratinocytes).

Drug-protein adducts formed may be taken up by the antigen presenting cells (APCs) in the skin as neoantigens, followed by processing and presentation to T cells. In the skin, Langerhans cells (LC) are the primary APC, in addition to other skin dendritic cells (DCs). LC/DCs collect antigens in the skin, process them, and transport them to the lymphoid organ in a synchronized fashion (Stoitzner et al., 2006). During this journey to draining lymph nodes, DCs undergo a maturation process that equips them to present processed antigen to T cells in the draining lymph node. In humans, DCs represent a heterogeneous population of APC that seem to arise from different distinct hematopoietic progenitor cells, such as CD14⁺ monocytes or CD34⁺ bone marrow cells. From the cellular perspective, these progenitor cells undergo two main stages of differentiation. The first stage involves differentiation of the progenitor cells to the immature or unactivated DC that show a high capacity for antigen uptake. In the second stage, immature DCs undergo a maturation process wherein they become more potent mediators of T cell activation.

The ability of LC/DCs to bioactivate xenobiotics has not been carefully examined. Limited studies have demonstrated the presence of mRNA for different drug-metabolizing enzymes in LC or monocyte-derived DCs, including different cytochrome P450s (Saeki et al., 2002) and myeloperoxidase (Strobl et al., 1993). These data indicate that DCs may also play a direct role in drug metabolism leading to drug-protein adduct formation, which may be subsequently processed and presented to T cells. Based on these observations, we hypothesized that DCs can also bioactivate chemically inert drugs to reactive metabolites with subsequent protein haptenation.

Human donor-derived DCs have a short life-span and exhibit diverse phenotypic as well as functional variability (Bracho et al., 2003; O'Neill and Wilson, 2004). To circumvent these problems, we have used immortalized myelogenous CD34⁺ KG-1 cells to study the bioactivation of drugs in a LC/DC-like cellular model (Koeffler and Golde, 1978; Hulette et al., 2001). Our results demonstrate that KG-1 cells are able to bioactivate drugs, leading to protein haptenation. Moreover, the differentiation status of these cells (immature versus mature) does not seem to substantially affect the process of protein adduction.

	Materials and Methods

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Materials. Arylhydroxylamine metabolites of DDS and SMX were synthesized as described previously (Vyas et al., 2005) and determined by high-performance liquid chromatography to be >97% pure. Rabbit antisera were raised against SMX- and DDS-keyhole limpet hemocyanin conjugates and specificity assessed as described previously (Reilly et al., 2000). Rat-tail collagen (type I) was obtained from Sigma (St. Louis, MO). KG-1 cells were obtained from Dr. David Kusner (University of Iowa, Iowa City, IA). Microtiter ELISA plates (96 wells) were obtained from Rainin Instruments (Woburn, MA). Goat-anti-rabbit IgG conjugated with Alexa Fluor-488, goat anti-mouse IgG conjugated with Alexa Fluor-568, and goat anti-rabbit antibody conjugated with alkaline phosphatase were purchased from Invitrogen (Carlsbad, CA). Anti-HLA DR antibody was purchased from eBioscience (San Diego, CA). Bradford assay reagent was purchased from Pierce Chemical Company (Rockford, IL). Immunomount was obtained from Vector Laboratories (Burlingame, CA). All other chemicals were purchased from Sigma (St. Louis, MO) or Fisher Scientific Co. (Pittsburgh, PA).

Cell Culture. KG-1 cells, from a human dendritic cell line, were cultured as described previously (Herrmann et al., 2005), with minor modifications. Cells (2 x 10⁵/ml) were grown as suspension culture, in L-glutamine-supplemented minimum essential media containing 20% fetal bovine serum, 100 units/ml penicillin and 100 µg/ml streptomycin. Media was changed every second day. Cells were maintained at concentration 2 x 10⁵/ml to 1 x 10⁶/ml. Cells were tested for HLA-DR staining, a marker for KG-1 cells, using flow cytometric analysis.

Flow Cytometry. Cultured cells were washed, centrifuged at 550g for 10 min, suspended at 1 x 10⁵ cells in 200 µl of cold Dulbecco^'s phosphate-buffered saline (PBS), and incubated with fluorescein isothiocyanate-conjugated HLA-DR antibody or its isotypic control for 30 min at room temperature. Cells were washed thrice and resuspended in 800 µl of Dulbecco^'s PBS. Cells were passed through a nylon mesh to eliminate cell-aggregates, and staining was analyzed with a fluorescence-activated cell sorting scanner (BD Biosciences, San Jose, CA). A minimum of 10⁴ cells was analyzed three times for each sample.

Cell Differentiation. KG-1 cells were harvested by centrifugation and resuspended in complete culture media at a concentration of 2 x 10⁵ cells/ml in a 25 cm² culture flask.

Immature DC-Like Cells. To induce immature DC-like cell differentiation, recombinant human interleukin-4 (rh-IL-4; 100 ng/ml) and rh-granulocyte macrophage–colony-stimulating factor (GM-CSF; 100 ng/ml) were added to the cultures. Cells were cultured for 5 days at 37°C and 5% CO₂. Media was changed every third day with fresh cytokine cocktail-supplemented media.

Mature DC-Like Cells. To induce mature DC-like cell differentiation, rh-IL-4 (100 ng/ml), rh-GM-CSF (100 ng/ml), and tumor necrosis factor- (20 ng/ml) were added to the cultures. Cells were cultured for 5 days at 37°C and 5% CO₂. Media was changed every third day with fresh cytokine cocktail-supplemented media.

ELISA Analysis of Drug/Metabolite-Protein Adducts. Formation of haptenated proteins after SMX or DDS exposure was determined by cultivating KG-1 cells (5 x 10⁵ cells/ml) for 3 h in 50-ml centrifuge tubes containing 5 ml of complete growth medium. Cells were then incubated with SMX or DDS (250 µM). After 3 h, tubes were centrifuged at 550g for 10 min to pellet the cells. The supernatant containing the medium was drained off, and the cell pellets were lysed in 1 ml of deionized water, using repeated cycles of freezing and thawing (three times) and ultrasonication to ensure complete lysis. The cell suspension was then thoroughly vortexed and centrifuged at 550g for 10 min and the pellet containing the cell debris was discarded. The supernatant containing cellular soluble proteins was collected for protein assay and subsequent ELISA.

ELISA analysis for detection of drug-protein adduct formation was performed as described previously (Reilly et al., 2000) with minor modifications. After protein content measurement using the Bradford reagent kit, all samples were diluted to contain 100 µg/ml protein. An aliquot of 100 µl was adsorbed onto 96-well polystyrene microtiter plates for 16 h at 4°C. Wells were washed three times with Tris-casein buffer (0.5% casein, 0.9% NaCl, 0.01% thimerosal, and 10 mM Tris-HCl, pH 7.6) and then blocked with Tris-casein buffer for 1 h. After an additional wash, wells were incubated for 16 h at 4°C with 100 µl of anti-SMX or anti-DDS rabbit serum (1:500 diluted with Tris-casein buffer) previously characterized in our laboratory (Reilly et al., 2000). Wells were subsequently washed four times with Tris-casein buffer and incubated with alkaline phosphatase-conjugated goat anti-rabbit antibody (1:1000 diluted in Tris-casein buffer) for 2 h at room temperature. After washing four times with Tris-casein buffer, antibody binding was detected with colorimetric alkaline phosphate substrate reagent. After 1 h of incubation at room temperature, optical density was measured at 405 nm using a V_max kinetic microplate reader (Molecular Devices, Sunnyvale, CA).

To determine the effect of flavin monooxygenase (FMO) and peroxidase substrate/inhibitor on drug-protein covalent adduct formation after DDS exposure, KG-1 cells (2 x 10⁶ cells) were incubated with DDS (250 µM) in the presence or absence of the selective enzyme substrate/inhibitor for 3 h (as specified under Results). The FMO competitive substrate methimazole (MMZ; 5 mM) was coincubated with DDS, and the peroxidase inhibitor 4-aminobenzoic acid hydrazide (ABH; 5 mM) was added 1 h before DDS exposure. After incubation, tubes were centrifuged at 550g for 10 min to pellet the cells. Covalent adducts were determined as described previously (Vyas et al., 2005).

Determination of the Cytotoxicity of Enzyme Inhibitors in KG-1 Cells. To determine whether the concentrations of MMZ or ABH used in our studies were cytotoxic to KG-1, cells were exposed to the agents and cytotoxicity determined using a cell impermeable DNA binding dye (YOYO-1) as described previously (Vyas et al., 2005).

Immunocytochemistry. To detect protein haptenation in KG-1 cells of varying differentiation status, cells were cultured in the presence or absence of cytokines (GM-CSF and IL-4) for 5 days and then plated onto collagen-coated coverslips. Cells were allowed to attach to the collagen-coated (0.1 mg/ml) coverslips and then placed in Petri dishes containing 2 ml of complete growth medium. After 24 h, cultures were subjected to different drug treatments for varying times (specified under Results) followed by washing (three times) with PBS (0.05 M sodium phosphate and 0.15 M NaCl, pH 7.4) and fixation for 20 min with 4% paraformaldehyde in PBS. After fixation, cultures were washed three times with PBS followed by blocking for 60 min with Tris-casein buffer containing 0.3% Triton-X-100 and overnight incubation with the anti-DDS or anti-SMX antisera (1:500 diluted in blocking buffer) at 4°C. Coverslips were then washed with PBS, incubated for 3 h at 37°C with the fluorochrome-conjugated secondary antibody (Alexa Fluor-488–labeled goat-anti-rabbit IgG, 1:500 diluted in blocking buffer), and mounted on glass slides using Immunomount containing anti-fade reagent.

To differentiate between intracellular and cell surface drug-protein adducts, cells were treated with the native drug (DDS or SMX, 800 µM, 24 h) or its arylhydroxylamine metabolite (D-NOH or S-NOH, 25 µM, 3 h), followed by routine immunocytochemical procedure except that the permeabilization step was deleted.

Fluorescence images were acquired with a Zeiss Laser Scanning Microscope (LSM 510, Axiovert stand, 63x oil lens; Carl Zeiss Inc., Thornwood, NY) using excitation at 488 nm. Emission was set to a long-pass filter at 505 nm.

View larger version (18K): [in this window] [in a new window]   FIG. 1. Detection of protein haptenation in naive KG-1 cells after incubation with parent drugs or their arylhydroxylamine metabolites. Naive KG-1 cells were incubated either with DDS (250 µM for 3 h)/D-NOH (25 µM for 3 h) (A) or SMX (250 µM for 3 h)/S-NOH (25 µM for 3 h) (B). At the end of the incubation, cells were fixed, permeabilized, immunostained, and imaged on a confocal microscope for DDS-specific covalent adduct formation. Top micrographs were obtained via immunofluorescent confocal microscopy; bottom micrographs are the corresponding phase contrast for the same view field.

	Results

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Detection of Protein Haptenation in Naive KG-1 cells Exposed to DDS or SMX. To detect drug-induced protein haptenation in naive KG-1 cells, cells were exposed to DDS (250 µM, 3 h) or its reactive metabolite D-NOH (25 µM, 3 h), followed by washing, fixation, and immunocytochemical detection using confocal microscopy. Formation of haptenated proteins were evident in both naive and immature KG-1 cells exposed to either DDS or D-NOH (Fig. 1A, top). Simultaneous differential integration coefficient images were acquired from the same field to show the cellular morphology of the particular cells (Fig. 1A, bottom). In contrast to DDS, SMX (250 µM, 3 h) showed no detectable protein haptenation in naive KG-1 cells (Fig. 1B), whereas haptenated proteins were readily detected in cells exposed to S-NOH.

To assess the localization of intracellular versus cell surface drug-protein adduction, cells were treated with the native drug (DDS or SMX, 800 µM, 24 h) or its arylhydroxylamine metabolite (D-NOH or S-NOH, 25 µM, 3 h) followed by routine immunocytochemical procedure without prior permeabilization of cells (meaning antisera could only access surface adducts). Under these conditions, negligible fluorescence was observed (data not shown), suggesting the absence of drug-protein adducts on the surface of these cells.

To confirm these observations, additional experiments were conducted employing ELISA to determine the formation of haptenated proteins in naive KG-1 cells exposed to DDS or SMX. We observed significant protein haptenation when KG-1 cells were exposed to either DDS or D-NOH (Fig. 2A). As observed when protein haptenation was assessed via confocal microscopy, we found a relatively low level of haptenation in cells exposed to SMX, but significant haptenation in those exposed to S-NOH (Fig. 2B). Moreover, DDS-treated naive KG-1 cells showed much higher haptenation compared with the SMX-treated counterparts.

View larger version (16K): [in this window] [in a new window]   FIG. 2. Assessment of protein haptenation via ELISA in naive KG-1 cells exposed to drugs or their metabolites. Cells (5 x 105 cells/ml) were incubated in the presence or absence of 250 µM DDS/SMX or 25 µM DNOH/SNOH and DDS/SMX-protein adducts determined using ELISA. Results shown are the optical density (OD) mean (SD) from three separate incubations for each condition. DDS/SMX-BSA was added to cell free wells as a positive control, but it was not included in the statistical analysis. A, Results for incubations using DDS or D-NOH, B, results for incubations using SMX or S-NOH. *, p < 0.05 compared with control incubations; **, p < 0.05 compared with control and DDS incubations.

View larger version (21K): [in this window] [in a new window]   FIG. 3. Protein haptenation in immature versus mature KG-1 cells exposed to DDS or D-NOH. KG-1 cells were exposed to different cytokine combinations to achieve specific cellular differentiation. After differentiation, both immature (A) and mature (B) cells were incubated with DDS (250 µM) or D-NOH 25 µM for 3 h while corresponding control groups were exposed to vehicle (dimethyl sulfoxide) only. At the end of the incubation cells were fixed, permeabilized, immunostained, and imaged on a confocal microscope for DDS-specific protein haptenation. Top row, micrographs were obtained via confocal microscopy; bottom row, corresponding micrographs were obtained via phase contrast.

Role of Peroxidase and FMO on Protein Haptenation in Naive KG-1 Cells. Because peroxidases and FMO have been shown to bioactivate DDS and SMX in human epidermal keratinocytes (Vyas et al., 2006b), leading to protein haptenation, we sought to determine the role of these enzymes in the DDS-specific protein haptenation in naive KG-1 cells. For this purpose, naive KG-1 cells were exposed to DDS in the presence or absence of a peroxidase inhibitor (ABH) or a competitive substrate for FMO (MMZ). MMZ was coincubated with DDS, and ABH was added 1 h before the drug was added. Formation of haptenated proteins were quantitatively compared using ELISA. Both MMZ and ABH were able to markedly reduce (by >50%) the protein haptenation observed in KG-1 cells exposed to DDS (Fig. 4A). Because neither agent increased cell death above that seen with DDS alone (Fig. 4B), this reduction in bioactivation could not be attributed to cytotoxicity.

View larger version (12K): [in this window] [in a new window]   FIG. 4. Effect of MMZ and ABH on protein haptenation in naive KG-1 cells exposed to DDS. A, cells (5 x 105 cells/ml) were incubated with 250 µM DDS in the presence or absence of MMZ or ABH and DDS-protein adducts determined using ELISA. MMZ was coincubated with DDS, whereas the incubation with ABH started 1 h before the addition of DDS. Results shown are the optical density (OD) mean (SD) from three separate incubations for each condition. *, p < 0.05 compared with cells exposed to DDS alone. B, cytotoxicity of DDS, in presence and absence of MMZ or ABH was examined in KG-1 cells using a cell impermeable nucleic acid binding dye (YOYO-1). Results shown are the mean (SD) percentage cytotoxicity of three replicate incubations for each condition.

Uptake of Preformed Haptenated Proteins by KG-1 Cells. To compare the efficacy of naive and immature cells to uptake preformed drug-protein conjugates, cells were exposed to drug-BSA conjugates for 10 and 60 min followed by washing, fixation, and immunostaining with the corresponding drug-specific antisera. Our results demonstrated that both naive and immature KG-1 cells were able to uptake the drug-BSA conjugates (Fig. 5 and 6). KG-1 cells exposed to DDS-BSA seem to show higher immunostaining than those exposed to SMX-BSA. This could be a consequence of the higher sensitivity of the anti-DDS-antisera compared with the anti-SMX antisera or a true difference in drug-protein conjugate uptake. Moreover, exposure of DDS-BSA for 60 min resulted in higher immunostaining in both naive and immature KG-1 cells compared with the cells exposed for 10 min. Neither naive nor immature KG-1 cells exposed to SMX-BSA showed such time-dependent uptake of the drug-BSA conjugate.

View larger version (19K): [in this window] [in a new window]   FIG. 5. DDS-BSA conjugate uptake in naive (A) and immature (B) KG-1 cells. Both naive and immature KG-1 cells were incubated with DDS-BSA (100 µg/ml) for 10 and 60 min. Controls were incubated with vehicle (PBS) only. At the end of the incubation cells were fixed, permeabilized, immunostained, and imaged on a confocal microscope for DDS-specific covalent adduct formation. Top row, images obtained via confocal microscopy; bottom row, corresponding micrographs of the same view field visualized via phase contrast.

View larger version (22K): [in this window] [in a new window]   FIG. 6. SMX-BSA conjugate uptake in naive (A) and immature (B) KG-1 cells. Both naive and immature KG-1 cells were incubated with SMX-BSA (100 µg/ml) for 10 and 60 min. Controls received the vehicle (PBS) only. At the end of the incubation, cells were fixed, permeabilized, immunostained, and imaged on a confocal microscope for SMX-specific covalent adduct formation. Top row, images obtained via confocal microscopy; bottom row, corresponding micrographs of the same view field visualized via phase contrast.

	Discussion

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DCs represent a unique group of cells with high phenotypic as well as functional diversity. DCs are believed to originate from CD34⁺ hematopoietic stem cells and differentiate to circulating precursor cells that give rise to multiple cell types with distinct tissue localization and function (Reid et al., 1990). The primary function of DCs is to engulf particles and soluble compounds from their tissue environment, process these proteins into smaller peptides, and present them to T cells upon activation. Various stimuli, including local tissue damage, inflammation, stress, or altered metabolic products, can activate DCs. Activation of DCs is associated with various phenotypic as well as functional alterations, including reduction in phagocytic ability, induction of costimulatotry molecules, induction of cytokines and chemokines, and higher mobility (Ardavin et al., 2001). After such changes, DCs become highly efficient in priming both helper and cytotoxic T cells and are referred to as mature DCs.

LCs are the major APCs in skin (Jakob et al., 2001). LCs/DCs are known as the sentinels of skin. In addition to their role in antigen uptake, processing and presentation, these cells may play an important, as-yet unexplored metabolic role. Limited data have shown metabolic activity in DCs and LCs (Sieben et al., 1999). The presence of either mRNA and/or protein of some metabolic enzymes (e.g., cytochrome 450s or prostaglandin E2 synthase) have been found in monocyte-derived DCs or LCs (Norgauer et al., 2003), but no detailed study has been carried out to elucidate the role of these cells in drug metabolism.

CD34⁺ KG-1 cells have been used as a cellular model for DCs in a variety of studies (St Louis et al., 1999; Ackerman and Cresswell, 2003; Berges et al., 2005). Our data show that KG-1 cells are able to metabolize DDS, leading to protein haptenation (Figs. 1 and 2), whereas protein adduction in response to SMX exposure was very low. Exposure to the reactive metabolites of these drugs also resulted in adduction to cellular proteins, as evidenced from both our confocal images and ELISA data.

Because KG-1 cells undergo differentiation upon exposure to different inflammatory stimuli that mimic immature or mature DC-like characteristics (Santiago-Schwarz et al., 1992; Romani et al., 1994), we determined whether the extent of protein adduction varies with the differentiation status of the KG-1 cells. Our results have clearly demonstrated that KG-1 cells of the three different differentiation states exhibit appreciable adduct formation when exposed to DDS or D-NOH. These data suggest that there is not a substantial alteration in metabolic activity of KG-1 cells with progression of differentiation.

To elicit an immune response, processing of the antigen followed by presentation on the surface in conjunction with costimulatory molecules is an essential step. Because previous studies have shown that on transition from naive to immature stages KG-1 cells express costimulatory molecules on their surface, we anticipated that during this stage drug-protein adducts may also be processed and presented on the surface in the context of major histocompatibility complex. However, we found little evidence that these adducts are expressed on the surface of KG-1 cells at all three differentiation stages (data not shown).

Because our previous studies have shown the involvement of FMO and peroxidases in the bioactivation of DDS in keratinocytes (Vyas et al., 2006a,b), we sought to determine whether these enzymes play a role in formation of DDS-protein adducts in KG-1 cells. Because there was no difference in DDS-protein adduct formation among KG-1 cells of various differentiated states, we used naive cells to test this hypothesis. Our data demonstrate that DDS-protein haptenation was significantly reduced in naive KG-1 cells in the presence of the peroxidase inhibitor ABH, as well as the competitive FMO substrate MMZ. These results suggest that, similar to our observations in keratinocytes, peroxidases and FMO play an important role in the bioactivation of DDS in KG-1 cells.

On transition from naive to the immature stage, the endocytotic capacity of KG-1 cells is known to increase severalfold (Berges et al., 2005). We therefore determined the localization of haptenated proteins formed intracellularly versus the localization when cells were exposed to prehaptenated proteins. Our results demonstrate that both naive and KG-1 cells were able to take up preformed drug-BSA adducts. When cells were exposed to DDS-BSA, haptenated proteins were found in the cytosol. In the case of SMX-BSA, adducts were traced intracellularly as punctated vesicle like structures.

In summary, our data demonstrate that model human DCs are able to acquire drug-associated haptenated proteins through three potential mechanisms: 1) intracellular bioactivation of parent drug to reactive metabolites, 2) uptake of extracellular reactive metabolite, or 3) uptake of preformed haptenated proteins. Because it has been previously demonstrated that LCs can cross-present keratinocyte-derived antigens in a physiological environment (Stoitzner et al., 2006), our prior demonstration of the capacity of such cells to give rise to haptenate proteins suggests that keratinocyte-LC exchange of antigens may play an important role in the mediation of CDRs.

	Acknowledgments

 
We thank Dr. David Kusner for supplying the KG-1 cells and Tara Herrman for her assistance in establishing this cell line in our lab. We also thank the staff of the Central Microscopy Research Facility at The University of Iowa, which is supported by the Office of the Vice President for Research, for their technical assistance.

	Footnotes

 
This work was supported in part by National Institutes of Health Grants AI41395 and GM63821 (to C.K.S.).

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.106.013680.

ABBREVIATIONS: CDR, cutaneous drug reaction; APC, antigen presenting cell; LC, Langerhans cell; DC, dendritic cell; DDS, dapsone; SMX, sulfamethoxazole; D-NOH, dapsone hydroxylamine; S-NOH, sulfamethoxazole hydroxylamine; ELISA, enzyme-linked immunosorbent assay; HLA, human leukocyte antigen; FMO, flavin monooxygenase; MMZ, methimazole; ABH, 4-aminobenzoic acid hydrazide; rh, recombinant human; IL, interleukin; GM-CSF, granulocyte macrophage–colony-stimulating factor; PBS, phosphate-buffered saline; BSA, bovine serum albumin.

¹ Current affiliation: Department of Pathobiology / NC22, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195

² Current affiliation: Riley Hospital for Children, Wells Center for Pediatric Research, 1044 West Walnut, R4 Room W344, Indianapolis, IN 46202

Address correspondence to: Dr. Craig K. Svensson, Office of the Dean, College of Pharmacy, Nursing and Health Sciences, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 49707. E-mail: svensson{at}purdue.edu

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