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IL‑8 Release from Human Neutrophils Cultured with Pro-Haptenic Chemical Sensitizers Eva Kiorpelidou, Brian Foster, John Farrell, Monday O. Ogese, Lee Faulkner, Chris E. Goldring, B. Kevin Park, and Dean J. Naisbitt* MRC Centre for Drug Safety Science, Department of Pharmacology, University of Liverpool, Sherrington Building, Ashton Street, Liverpool L69 3GE, England ABSTRACT: Cytokine release from dendritic cells in vitro is a useful marker to discriminate between sensitizing and irritant haptenic chemicals. Unfortunately, pro-haptens, which gain reactivity following metabolic/auto activation, yield negative results. To overcome this, we exposed human neutrophils and THP-1 cells to haptens/pro-haptens and measured IL-8 release. Haptenic compounds stimulated IL-8 release in neutrophils and THP-1 cells. In contrast, the prohaptens eugenol, isoeugenol, and 2-aminophenol stimulated high levels of IL-8 release from neutrophils alone. Neutrophil cytokine release was reduced when glutathione was added. Cyp1A1/1B1/3A4 were not detectable in THP-1 cells or neutrophils; however, neutrophils expressed high levels of myeloperoxidase.
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whether measurement of neutrophil IL-8 release might represent a useful single cell readout for the prediction of sensitizing pro-haptens. Neutrophils were selected because they are recruited in the skin during allergic sensitization. THP-1 cells, cultured in RPMI-1640 supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin, were used before passage 16. Neutrophils were isolated from heparinized blood by density separation using established methods.6 All results show mean data using neutrophils isolated from a minimum of three subjects. Assays with both cell types were conducted using the medium listed above. In initial experiments, enzyme expression in THP-1 and neutrophils was measured by immunoblotting using antibodies displaying reactivity against the cytochrome P450 enzymes Cyp1A1, 1B1, and 3A4, and myeloperoxidase. Cyp expression was not detectable in THP-1 cells or neutrophils (limits of detection using recombinant Cyps: 1A1, 1 fmol; 1B1 10 fmol; 3A4, 4fmol). As described previously,7 myeloperoxidase was detected in neutrophils (from 0.75 μg/mL neutrophil protein) but not THP-1 cells (up to 40 μg/mL THP-1 cell protein). Thus, oxidation of pro-haptenic chemical sensitizers by neutrophil peroxidases might result in the formation of protein-reactive intermediates that stimulate IL-8 release. In the next series of experiments, THP-1 cells and neutrophils (1 × 106/ml; 24 well plates) were cultured separately with the haptenic chemical sensitizer cinnamaldehyde (1−1000 μM) for 24 h, and IL-8 release and cell viability were measured using ELISA and MTS reagent, respectively. These assays were conducted to (1) demonstrate that chemical treatment stimulates THP-1 and neutrophil cytokine secretion and (2) compare the levels of cytokine secreted from the two
egislation prohibiting animal testing for evaluating the sensitizing potential of cosmetics has resulted in the search for alternative in vitro approaches. Gerberick et al.1 developed a peptide reactivity assay that measures the binding of haptenic chemical sensitizers to nucleophilic amino acids and demonstrated that a significant correlation exists between sensitizer potency and reactivity against cysteine and lysine. More recently, the same team demonstrated that it was possible to incorporate a peroxidase activating system to explore the reactivity of pro-haptens, compounds that gain protein reactivity following metabolic/autoactivation.2 Dendritic cells are known to play a pivotal role in the development of immune responses that drive contact sensitization. Following epicutaneous allergen exposure, numerous signaling pathways are activated that drive dendritic cells to undergo phenotypic and functional changes that result ultimately in the transport of antigenic determinants to draining lymph nodes and the ̈ T-lymphocytes.3 This knowledge has led to priming of naive the development of in vitro assays that involve exposing primary dendritic cells or dendritic cell-like cell lines to chemical sensitizers and analysis of cellular maturation (changes in cell surface phenotype, gene transcription, or cytokine release) as a predictor of sensitization. Initial results show the activation of dendritic cells with sensitizing chemicals but not with irritant controls, which indicates that dendritic cell based assays might represent an important strategy to characterize sensitizer potential in vitro.4 One important limitation of dendritic cell based assays is that pro-haptens generate negative or inconsistent results. Hennen et al.5 recently reported a promising coculture system that involves the exposure of chemicals to HaCaT keratinocytes and the monocytic cell line THP-1. HaCaT cells apparently metabolize pro-haptens such as eugenol to reactive intermediates that subsequently stimulate increased CD86 expression on THP-1 cells. The aim of this study was to compare metabolic activity in THP-1 cells and primary human neutrophils and explore © 2012 American Chemical Society
Received: August 8, 2012 Published: September 4, 2012 2054
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Chemical Research in Toxicology
Rapid Report
Once the assay conditions had been optimized with haptenic chemicals, IL-8 release from THP-1 cells and neutrophils was measured after 24 h incubation with the pro-haptens eugenol, isoeugenol, and 2-aminophenol. The fragrance components eugenol and isoeugenol are classified according to the mouse local lymph node assay as weak and moderate sensitizers, respectively.8 Both compounds are metabolized to quinine methide and/or o-quinone intermediates.9 Thompson et al.10 demonstrated that peroxidase-catalyzed eugenol metabolism generates metabolites that rapidly deplete neutrophil glutathione and covalently modify cellular protein. 2-Aminophenol is a strong sensitizer that has been shown to react much more avidly with cysteine-containing peptides in the presence of a peroxidase metabolizing system.2 Pro-hapten treatment of THP-1 cells was associated with the release of only low levels of IL-8. In contrast, IL-8 release from pro-hapten-treated neutrophils was significantly higher (Figure 2a-c). The relatively high concentrations needed to stimulate IL-8 release likely relate to the low metabolic turnover in the neutrophil assay. IL8 release was not detected in THP-1 cells or neutrophils with the nonsensitizer control phenol (Figure 2d). Figure 3a compares neutrophil IL-8 release with 2-aminophenol in the presence and absence of glutathione (1 mM). The release of IL-8 was significantly reduced with glutathione, which is consistent with the hypothesis that a reactive intermediate derived from 2-aminophenol is involved in activating neutrophils. Similar results were obtained with Nacetyl cysteine (results not shown). Although most pro-hatpenic chemical sensitizers gain reactivity through peroxidase-catalyzed reactions, it is important to note that there are a few exceptions. For example, cinnamic alcohol is metabolized by alcohol dehydrogenase and CYPs to the hapten cinnamaldehyde.11 These enzymes are unlikely to be expressed in sufficient quantities in neutrophils to catalyze the biotransformations needed to activate IL-8 release. Indeed, cinnamic alcohol did not stimulate IL-8 release from neutrophils (Figure 3b). Despite this, the future development of a neutrophil cytokine release assay may be useful for predicting the sensitizing potential of most chemicals that gain reactivity through metabolic activation.
cell types. Significant quantities of IL-8 were measured in the culture supernatant when cinnamaldehyde (150−500 μM; loss of membrane integrity was observed at a concentration of 500 μM and above) was incubated with THP-1 cells and neutrophils. The level of IL-8 released from neutrophil cultures was significantly lower (P < 0.05; Student's t test) when the 2 cell types were compared (Figure 1a). Higher levels of THP-1
Figure 1. IL-8 release from THP-1 cells and neutrophils cultured with (a) cinnamaldehyde and (b) DNCB.
IL-8 secretion was also detected when the model haptenic chemical sensitizer 2,4-dinitrochloro-benzene (DNCB; 7.5 μM) was cultured with the 2 cell types. DNCB was used as a positive control in the IL-8 release assays; thus, Figure 1b shows measurements from 24 independent experiments. Phorbol-12myristate-13-acetate, an activator of neutrophil peroxidases, could not be included in the experimental design as phorbol12-myristate-13-acetate treatment stimulated IL-8 release (results not shown). To show that peroxidases are metabolically active in nonstimulated neutrophils, cells (1 × 106) from 3 subjects were cultured with the drug sulfamethoxazole (1 mM; 24 h) in the presence and absence of the peroxidase inhibitor methimazole, and protein adduct formation was measured by ELISA with an antidrug antibody. Neutrophils were found to metabolize sulfamethoxazole to a proteinreactive intermediate, and the level of adduct formation was significantly reduced in the presence of methimazole (OD values: no drug, 0.27 ± 0.07; sulfamethoxazole, 1.73 ± 0.29; sulfamethoxazole+methimazole, 0.73 ± 0.07; P < 0.05).
Figure 2. IL-8 release from THP-1 cells and neutrophils cultured with (a) eugenol, (b) isoeugenol, (c) 2-aminophenol, and (d) phenol. IL-8 levels were measure in a culture supernatant by ELISA. Data derived from 3 independent experiments (*P < 0.05, Student's t test; coefficient of variation less than 20%). 2055
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(5) Hennen, J., Aeby, P., Goebel, C., Schettgen, T., Oberli, A., Kalmes, M., and Blomeke, B. (2011) Cross talk between keratinocytes and dendritic cells: impact on the prediction of sensitization. Toxicol. Sci. 123, 501−510. (6) Hawley, S. R., Bray, P. G., O’Neill, P. M., Naisbitt, D. J., Park, B. K., and Ward, S. A. (1996) Manipulation of the N-alkyl substituent in amodiaquine to overcome the verapamil-sensitive chloroquine resistance component. Antimicrob. Agents Chemother. 40, 2345−2349. (7) Uetrecht, J. P. (1994) Metabolism of drugs by leukocytes. Drug Metab. Drug Interact. 11, 259−282. (8) Gerberick, G. F., Ryan, C. A., Kern, P. S., Schlatter, H., Dearman, R. J., Kimber, I., Patlewicz, G. Y., and Basketter, D. A. (2005) Compilation of historical local lymph node data for evaluation of skin sensitization alternative methods. Dermatitis 16, 157−202. (9) Bertrand, F., Basketter, D. A., Roberts, D. W., and Lepoittevin, J. P. (1997) Skin sensitization to eugenol and isoeugenol in mice: possible metabolic pathways involving ortho-quinone and quinone methide intermediates. Chem. Res. Toxicol. 10, 335−343. (10) Thompson, D., Constantin-Teodosiu, D., Norbeck, K., Svensson, B., and Moldeus, P. (1989) Metabolic activation of eugenol by myeloperoxidase and polymorphonuclear leukocytes. Chem. Res. Toxicol. 2, 186−192. (11) Elahi, E. N., Wright, Z., Hinselwood, D., Hotchkiss, S. A., Basketter, D. A., and Pease, C. K. (2004) Protein binding and metabolism influence the relative skin sensitization potential of cinnamic compounds. Chem. Res. Toxicol. 17, 301−310.
Figure 3. (a) IL-8 release from neutrophils cultured with 2aminophenol ± glutathione. Data derived from 3 experiments. Student's t test was used to compare the data. (b) IL-8 release from neutrophils and THP-1 cells cultured with cinnamic alcohol.
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AUTHOR INFORMATION
Corresponding Author
*Tel: 0044 151 7945346. Fax: 0044 151 7945540. E-mail:
[email protected]. Funding
This work received funding from the EU sixth framework program for the development of novel strategies for in vitro assessment of allergens (Sens-it-iv − LSHB-CT-2006-018681) and the Centre for Drug Safety Science supported by the Medical Research Council. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the blood donors who participated in the project. ABBREVIATIONS DNCB, 2,4-dinitrochlorobenzene REFERENCES
(1) Gerberick, G. F., Vassallo, J. D., Bailey, R. E., Chaney, J. G., Morrall, S. W., and Lepoittevin, J. P. (2004) Development of a peptide reactivity assay for screening contact allergens. Toxicol. Sci. 81, 332− 343. (2) Gerberick, G. F., Troutman, J. A., Foertsch, L. M., Vassallo, J. D., Quijano, M., Dobson, R. L., Goebel, C., and Lepoittevin, J. P. (2009) Investigation of peptide reactivity of pro-hapten skin sensitizers using a peroxidase-peroxide oxidation system. Toxicol. Sci. 112, 164−174. (3) Martin, S. F., Esser, P. R., Weber, F. C., Jakob, T., Freudenberg, M. A., Schmidt, M., and Goebeler, M. (2011) Mechanisms of chemical-induced innate immunity in allergic contact dermatitis. Allergy 66, 1152−1163. (4) Kimber, I., Dearman, R. J., and Basketter, D. A. (2012) Dendritic cells and the assessment in vitro of skin sensitizing potential. Cutan. Ocul. Toxicol., in press. 2056
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