Trimethoprim Stimulates T-Cells through Metabolism-Dependent and

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Trimethoprim Stimulates T-Cells through Metabolism-Dependent and -Independent Pathways Sabah El-Ghaiesh,†,‡ Joseph P. Sanderson,† John Farrell,† Sidonie N. Lavergne,† Wing-Kin Syn,§,|| Munir Pirmohamed,† B. Kevin Park,† and Dean J. Naisbitt*,† †

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MRC Centre for Drug Safety Science, Department of Pharmacology, University of Liverpool, Sherrington Building, Ashton Street, Liverpool, L69 3GE, England ‡ Department of Pharmacology, University of Tanta, Tanta, Egypt § Centre for Liver Research, Institute of Biomedical Research, University of Birmingham, B15 2TT, England Department of Physiology, University of the Basque Country, Bilbao, Spain

bS Supporting Information ABSTRACT: Pathways of drug-specific T-cell stimulation have not been fully defined. The aim of this study was to use T-cell clones from a patient hypersensitive to the drug trimethoprim to characterize the involvement of drug metabolism and processing in antigen presentation and cross-reactivity patterns. The MHC-restricted CD4þ and CD8þ T-cell response was dependent on the presence of antigen-presenting cells, and both processing-dependent and -independent pathways of antigen presentation were detected. Stimulation of certain clones was blocked through inhibition of drug-metabolizing enzyme activity. Trimethoprim clones were additionally stimulated with diaveridine and pyrimethamine but not other closely related structures.

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human urine (50
60%); however, several stable metabolites have been detected.14 TMP also forms a protein reactive iminoquinone methide species through oxidation of the pyrimidine ring via a peroxidase-catalyzed reaction.15 To investigate the role of metabolic drug activation in the presentation of trimethoprim to T-cells, we analyzed first the necessity for APC; second, MHC restriction; third, the ability of enzyme inhibitors to block the T-cell response; and finally, the involvement of processing in the presentation of drug-derived antigens. T-cell clones were generated by serial dilution from a 20 year old female who developed fever, desquamating rash, pruritus, and severe liver damage [Bilirubin, 283 (reference 2-14); ALT (IU/mL), 599 (reference 44-147); INR 2.4 (reference 0.9-1.2)] 2 weeks after initial exposure to TMP. Blood results on admission were as follows: white cell count 24.9  106/mL (reference range, 4
11) with eosinophilia of 13% (reference range, 0
7%). Blood samples for cloning were obtained 1 year after clinical symptoms receded. EBV-transformed B-cell lines, used as APC, were generated by incubating blood lymphocytes with supernatant from the B9-58 cell line. To characterize of the involvement of drug metabolism and antigen processing in the stimulation of T-cells, the standard proliferation assay, which involves culture of T-cell clones (5  104; total volume 200 μL) with irradiated APC (1  104) and titrated TMP for 48 h prior to the addition of [3H]thymidine, was modified in various ways. First, APC were omitted; second, inhibitors of drug-metabolizing

ecause the majority of drugs associated with a high incidence of hypersensitivity are metabolized into reactive intermediates in patients, it has been inferred that such metabolites are involved in the disease pathogenesis through the generation of drug
protein conjugates, which stimulate specific T-cells following processing and the liberation of antigenic peptides. T-cell cloning methodology using lymphocytes isolated from hypersensitive patients has made it possible to define pathways of drugspecific T-cell activation at individual T-cell receptors.1 Using sulfamethoxazole as a model drug antigen and the synthetic metabolite nitroso sulfamethoxazole, we and others have shown that T-cells can indeed be stimulated to proliferate and secrete cytokines by peptides derived from drug metabolite-modified protein.2
10 However, sulfamethoxazole the parent drug also binds directly to MHC and specific T-cell receptors through as yet undefined noncovalent interactions, with sufficient binding energy to stimulate a T-cell response.1,6
9 Furthermore, we have shown that antigen-presenting cells (APC) express peroxidase activity, and sulfamethoxazole metabolites generated in APC act as functional drug-derived antigens for T-cells.10
13 Thus, for sulfamethoxazole, the weight of evidence suggests that metabolism and formation of irreversibly APC bound protein adducts in APC are important for the initial stimulation of T-cells, while the parent drug stimulates memory T-cells where the threshold for activation is lower. The aim of this study was to use T-cell clones from a patient hypersensitive to the drug trimethoprim (TMP) to further characterize the involvement of drug metabolism and processing in antigen presentation. TMP is secreted largely unchanged in r 2011 American Chemical Society

Received: March 18, 2011 Published: April 14, 2011 791

dx.doi.org/10.1021/tx2001256 | Chem. Res. Toxicol. 2011, 24, 791–793

Chemical Research in Toxicology

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Figure 2. Role of antigen processing in the stimulation of TMP-specific T-cell clones. (A) TMP stimulates T-cell clones via both processingdependent and -independent pathways. T-cell clones were incubated with the drug and irradiated (gray bars) or fixed (black bars) APC. (B) The kinetics of T-cell receptor internalization following stimulation with TMP. T-cell receptor expression was monitored by flow cytometry following staining with an anti-CD3þ antibody. Each graph shows the response of a representative T-cell clone at 1 hour (gray) or 16 hours (black). The coefficient of variation was consistently less than 20%. The Mann
Whitney test was used to compare T-cell proliferation (*P < 0.05).

Figure 1. Role of drug metabolism in the stimulation of TMP-specific T-cell clones. (A) Stimulation of clones with TMP in the presence or absence of APC. (B) Stimulation of clones with TMP in the presence or absence of inhibitors of drug-metabolizing enzyme activity. (C) Stimulation of clones with TMP in the presence of methimazole added to culture before (1 h), at the same time, and after (16 h) TMP exposure. (D) Concentration-dependent inhibition of the metabolism-dependent (gray) or independent (black) TMP-specific T-cell response with methimazole. The coefficient of variation was consistently less than 20%. The Mann
Whitney test was used to compare T-cell proliferation (*P < 0.05).

The response of T-cells to TMP was dependent on the presence of APC (Figure 1A). CD4þ and CD8þ responses were inhibited by anti-MHC class II and class I blocking antibodies, respectively (results not shown). The majority of clones was stimulated with TMP in the presence and absence of inhibitors of drug-metabolizing enzyme activity, indicating, as described for several other drugs (e.g., sulfamethoxazole and carbamazepine), that the parent drug can stimulate specific responses directly (Figure 1B).6,7,16 However, inhibitors blocked the response of approximately 20% of the clones. The inhibitory effect was time- (i.e., only detected if the enzyme inhibitor was added prior to or at the time of drug exposure; Figure 1C) and concentration-dependent (inhibitor concentrations below 100 μM did not block the proliferative response; Figure 1D). Thus, a drug metabolite, which presumably forms antigenic determinants through covalent modification of protein, stimulates T-cells. With clones responding exclusively to TMP-derived metabolites, fixation of APC blocked the T-cell response, and internalized T-cell receptors were only detected after 16 h, the time required for processing (Figure 2).6 In contrast, clones responding to the parent compound were stimulated with TMP and fixed or irradiated APC. Furthermore, T-cell receptors were internalized rapidly, which is not feasible if antigen processing is a prerequisite for presentation. These data are similar to the

enzymes, specifically methimazole (an inhibitor of peroxidases and flavin-monooxygenases) and 1-aminobenzotriazole (a nonselective suicide inhibitor of a wide range of metabolic enzymes including myeloperoxidase4), were added to the cell culture medium at a concentration that blocks the enzyme activity (both 1 mM);4 and third, APC were fixed with glutaraldehyde—fixation inactivates the protease enzyme activity and blocks antigen processing.6 T-cell receptor internalization is a sensitive marker of T-cell activation. Thus, flow cytometric analysis of the kinetics (1
16 h) of down-regulated T-cell receptor expression was used to further define the involvement of processing in the presentation of TMP. Written informed consent was obtained, and the study was approved by the Liverpool local research ethics committee. Forty-five CD4þ and CD8þ clones expressing different Vβ receptors were identified as TMP-specific based on their proliferative response following TMP stimulation (0, 982 ( 350 cpm; 25 μg/mL trimethoprim, 5718 ( 3727 cpm). Proliferative responses were detected with TMP between 1 and 300 μg/mL (steady-state TMP plasma concentrations, 1
2 μg/mL). All subsequent experiments were performed with optimum stimulatory concentrations of the drug. 792

dx.doi.org/10.1021/tx2001256 |Chem. Res. Toxicol. 2011, 24, 791–793

Chemical Research in Toxicology recently described processing-dependent and -independent presentation of iodinated contrast media to specific T-cells from hypersensitive patients.17 The degree of T-cell cross-reactive recognition of antigen is a critical parameter for understanding adaptive immunity. It has recently been estimated that the frequency of cross-reactivity to unrelated peptide antigens is approximately 1 in 30000.18 The reactivity of T-cells responding to drugs has been studied to a much lesser extent. Published data suggest that T-cells are highly specific and only respond to structurally similar drugs of the same chemical class.17,19,20 To explore this subject in greater detail, a panel of clones responding to TMP directly were cultured with titrated concentrations of six structurally related compounds [diaveridine, pyrimethamine, 2,4-diamino-pyrimidine, 3,4,5-trimethoxy-benzoic acid, 3,4,5-trimethoxy-cinnamic acid, and 2,4,6-triamino-pyrimidine (all 34
172 μM)]. TMP is a weak base21 and therefore able to participate in relatively strong ionic interactions. Van der Waals forces and hydrogen bonds will also be involved in the interaction of TMP with MHC and the T-cell receptor. Almost 70% of the TMP-specific clones were additionally stimulated with diaveridine and pyrimethamine (see Supporting Information). These compounds are relatively divergent in a chemical sense but contain the moieties involved in the formation of a relatively strong reversible interaction with protein. In contrast, compounds containing a single six-membered ring did not stimulate T-cells. Thus, both ring structures of TMP contribute toward the binding interaction with MHC and/or the T-cell receptor. These data are consistent with the work of Baldo and colleagues who characterized IgE binding determinants in both ring structures using serum from patients with TMP-mediated immediate hypersensitivity22 and highlight that the nature of the immune response induced is generally a property of the genetics and/or phenotype of the individual and not the drug molecule. To conclude, our results describe the response of T-cells from a hypersensitive patient against TMP and TMP metabolites generated by APC. In future experiments to define the chemical entity that stimulated the primary response, it might be useful to establish an animal model as recently described with nevirapine.22 In this study, the authors clearly demonstrate that the parent drug must be metabolized to induce a reaction, but T-cells from affected animals were stimulated with both parent drug and metabolites.

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’ ACKNOWLEDGMENT We thank the CDSS nurses who helped to collect samples, as well as the patients who participated in the project. ’ REFERENCES (1) Schnyder, B., Mauri-Hellweg, D., Zanni, M., Bettens, F., and Pichler, W. J. (1997) J. Clin. Invest. 100, 136–141. (2) Castrejon, J. L., Berry, N., El-Ghaiesh, S., Gerber, B., Pichler, W. J., Park, B. K., and Naisbitt, D. J. (2010) J. Allergy Clin. Immunol. 125, 411–418. (3) Callan, H. E., Jenkins, R. E., Maggs, J. L., Lavergne, S. N., Clarke, S. E., Naisbitt, D. J., and Park, B. K. (2009) Chem. Res. Toxicol. 22, 937–948. (4) Naisbitt, D. J., Gordon, S. F., Pirmohamed, M., Burkhart, C., Cribb, A. E., Pichler, W. J., and Park, B. K. (2001) Br. J. Pharmacol. 133, 295–305. (5) Schnyder, B., Burkhart, C., Schnyder-Frutig, K., von Greyerz, S., Naisbitt, D. J., Pirmohamed, M., Park, B. K., and Pichler, W. J. (2000) J. Immunol. 164, 6647–6654. (6) Burkhart, C., von Greyerz, S., Depta, J. P., Naisbitt, D. J., Britschgi, M., Park, K. B., and Pichler, W. J. (2001) Br. J. Pharmacol. 132, 623–630. (7) Castrejon, J. L., Lavergne, S. N., El-Sheikh, A., Farrell, J., Maggs, J. L., Sabbani, S., O'Neill, P. M., Park, B. K., and Naisbitt, D. J. (2010) Chem. Res. Toxicol. 23, 184–192. (8) Nassif, A., Bensussan, A., Boumsell, L., Deniaud, A., Moslehi, H., Wolkenstein, P., Bagot, M., and Roujeau, J. C. (2004) J. Allergy Clin. Immunol. 114, 1209–1215. (9) Lavergne, S. N., Whitaker, P., Peckham, D., Conway, S., Park, B. K., and Naisbitt, D. J. (2010) Chem. Res. Toxicol. 23, 1009–1011. (10) Elsheikh, A., Castrejon, L., Lavergne, S. N., Whitaker, P., Monshi, M., Callan, H., El-Ghaiesh, S., Farrell, J., Pichler, W. J., Peckham, D., Park, B. K., and Naisbitt, D. J. (2011) J. Allergy Clin. Immunol. Epub ahead of print. (11) Elsheikh, A., Lavergne, S. N., Castrejon, J. L., Farrell, J., Wang, H., Sathish, J., Pichler, W. J., Park, B. K., and Naisbitt, D. J. (2010) J. Immunol. 185, 6448–6460. (12) Lavergne, S. N., Wang, H., Callan, H. E., Park, B. K., and Naisbitt, D. J. (2009) J. Pharmacol. Exp. Ther. 331, 372–381. (13) van't Klooster, G. A., Kolker, V, Woutersen-van Nijnanten, F. M., Noordhoek, J., and van Miert, A. S. (1992) J. Chromatogr. 579, 354–360. (14) Lai, W. G., Zahid, N., and Uetrecht, J. P. (1999) J. Pharmacol. Exp. Ther. 291, 292–299. (15) Wu, Y., Sanderson, J. P., Farrell, J., Drummond, N. S., Hanson, A., Bowkett, E., Berry, N., Stachulski, A. V., Clarke, S. E., Pichler, W. J., Pirmohamed, M., Park, B. K., and Naisbitt, D. J. (2006) J. Allergy Clin. Immunol. 118, 233–241. (16) Keller, M., Lerch, M., Britschgi, M., Tache, V., Gerber, B. O., Luthi, M., Lochmatter, P., Kanny, G., Bircher, A. J., Christiansen, C., and Pichler, W. J. (2010) Clin. Exp. Allergy 40, 257–268. (17) Ishizuka, J., Grebe, K., Shenderov, E., Peters, B., Chen, Q., Peng, Y., Wang, L., Dong, T., Pasquetto, V., Oseroff, C., Sidney, J., Hickman, H., Cerundolo, V., Sette, A., Bennink, J. R., McMichael, A., and Yewdell, J. W. (2009) J. Immunol. 183, 4337–4345. (18) Lerch, M., Keller, M., Britschgi, M., Kanny, G., Tache, V., Schmid, D. A., Beeler, A., Gerber, B. O., Luethi, M., Bircher, A. J., Christiansen, C., and Pichler, W. J. (2007) J. Allergy Clin. Immunol. 119, 1529–1536. (19) von Greyerz, S., Zanni, M. P., Frutig, K., Schnyder, B., Burkhart, C., and Pichler, W. J. (1999) J. Immunol. 162, 595–602. (20) Nowak, A., Klimowicz, A., and Kadykow, M. (1985) Eur. J. Clin. Pharmacol. 29, 231–234. (21) Pham, N. H., Baldo, B. A., Manfredi, M., and Zerboni, R. (1996) Clin. Exp. Allergy 26, 1155–1160. (22) Chen, X., Tharmanathan, T., Mannargudi, B., Gou, H., and Uetrecht, J. P. (2009) J. Pharmacol. Exp. Ther. 331, 836–841.

’ ASSOCIATED CONTENT

bS

Supporting Information. Figure showing the stimulation of TMP-responsive T-cell clones with structurally-related compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Tel: 0044 151 7945346. Fax: 0044 151 7945540. E-mail: [email protected]. Funding Sources

S.E.G. is a Ph.D. student funded by the Egyptian government. M.P. is a NIHR Senior Investigator and is funded by the NHS Chair of Pharmacogenetics from the UK Dept of Health. This work was funded by a grant from the Wellcome Trust (078598/ Z/05/Z) as part of the Centre for Drug Safety Science supported by the Medical Research Council (G0700654). 793

dx.doi.org/10.1021/tx2001256 |Chem. Res. Toxicol. 2011, 24, 791–793