Chem. Res. Toxicol. 2010, 23, 1009–1011
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Drug Metabolite-Specific Lymphocyte Responses in Sulfamethoxazole Allergic Patients with Cystic Fibrosis Sidonie N. Lavergne,†,§ Paul Whitaker,‡ Daniel Peckham,‡ Steve Conway,‡ 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, and Cystic Fibrosis Unit, St. James Hospital, Leeds, U.K. ReceiVed April 27, 2010
Sulfamethoxazole (SMX) is an important antibiotic in the management of patients with cystic fibrosis, but allergic reactions may develop thus restricting therapy. The aim of this study was to utilize drug (metabolite) antigens to diagnose SMX-mediated allergic reactions in patients with cystic fibrosis. Lymphocytes from 2/12 allergic patients were stimulated to proliferate strongly with the SMX metabolite nitroso SMX (SMX-NO). In contrast, responses to SMX were weak. The introduction of an antigendriven T-cell enrichment step prior to the analysis of proliferation increased the sensitivity of the assay. SMX-NO responses were detected with lymphocytes from all patients with cutaneous signs. Potentiated sulfonamides, such as trimethoprim-sulfamethoxazole (SMX1), are inexpensive antimicrobials with a wide spectrum of action and good tissue penetration. They are commonly used in immuno-compromised patients (e.g., HIV infected and post-transplant patients) and patients with cystic fibrosis. Their usefulness as therapeutic agents can be limited by the occurrence of delayed-type allergic reactions (1). Symptoms usually involve the skin, varying in severity from a simple rash to Stevens-Johnson syndrome or toxic epidermal necrolysis. Reactions can also target other organs including the liver, blood, lungs, or the meninges (1, 2). Drug-specific T-cells are believed to play an important role in the pathogenesis of cutaneous reactions (3-9). However, despite decades of research drug allergy continues to represent a challenge to diagnose. The lymphocyte transformation test is an in Vitro research tool used to help confirm drug allergy diagnosis. Lymphocytes from allergic patients and drug-exposed controls are cultured with suspected drug antigens for 6 days prior to the analysis of T-cell stimulation. The assay has a quantitative end-point. Typically, lymphocyte proliferation is assessed by incorporation of [3H]thymidine; however, recent studies have modified the end-point to analyze secreted cytokines or markers of immunemediated killing (10-14). The lymphocyte transformation test has a relatively high sensitivity (65% or above; percentage of allergic patients with a positive lymphocyte transformation test) and specificity (85% or above; percentage of nonallergic patients with negative lymphocyte transformation test) when patient cohorts have been carefully selected based on clinical manifestations, patch test data, and/or drug class (10). The sensitivity falls dramatically when patient selection is based on selfreporting of drug allergy or general practitioner records. Diagnosis of drug allergy is complicated by the fact that the nature of the antigen stimulating immune cells is not fully defined. According to the hapten theory, drugs must bind covalently to amino acid residues on protein to generate a T-cell antigen (15). This theory holds for β-lactam antibiotics; penicil* Corresponding author. Tel: 0044 151 7945346. Fax: 0044 151 7945540. E-mail:
[email protected]. † University of Liverpool. ‡ St. James Hospital. § Current address: University of IllinoissUrbana. 1 SMX, sulfamethoxazole; SMX-NO, nitroso sulfamethoxazole; SI, stimulation index.
lin-serum adducts and designer MHC binding peptides modified with penicillin have been shown to stimulate T-cells from allergic patients (16, 17). However, most drugs are not protein reactive but acquire protein reactivity through drug metabolism. Independent research groups have shown that synthetic proteinreactive metabolites stimulate T-cells from allergic patients (3, 5, 7-9). Pichler and colleagues have also demonstrated that lymphocytes from allergic patients can be stimulated with the parent drug presumably through a direct binding interaction with MHC molecules and/or specific T-cells (5, 18-22). It is clear that T-cell responses are directed against multiple drug antigens. Therefore, it is difficult to interpret a negative lymphocyte transformation test result as it may be due to incorrect clinical diagnosis or the use of an irrelevant antigen in the in Vitro assay. In the current study, we have explored the use of the lymphocyte transformation test and a novel indirect proliferation assay that incorporates an antigen-driven T-cell enrichment step prior to the analysis of proliferation to diagnose drug allergy in a cohort of sulfamethoxazole (SMX) allergic patients with cystic fibrosis. Patients attended the Regional Adult Cystic Fibrosis Unit in Leeds. The case records of 349 patients with cystic fibrosis were reviewed. Twelve patients were identified with a previous delayed-type allergic reaction (this represents a prevalence of 3.4%). Reactions followed oral treatment in 11 cases and intravenous treatment in one patient. Clinical features of the allergic reactions are described in Table 1. Nine patients developed drug-induced skin reactions (maculopapular exanthema [n ) 6], urticaria [n ) 1], fixed drug eruption [n ) 1], and vasculitic rash [n ) 1]). The other patients developed thrombocytopenia (n ) 2), another clinical sign commonly associated with sulfonamide reactions (23, 24), and an unusual periorbital edema (n ) 1). All patients received trimethoprimSMX for pulmonary exacerbations. Patient 2 was receiving intravenous trimethoprim-SMX in hospital when the reaction occurred. The remaining patients were taking oral medication. The median time from first exposure and the development of the allergic reaction was 3 years (range 0.5-8 years). The number of treatment courses received prior to the reaction was 3 (range 2-6). In this cohort of patients, prick tests were all negative, and only one patient (out of 10 tested) showed a positive reaction to intradermal testing. These negative results may relate to the use of an irrelevant antigen in the skin test
10.1021/tx100151v 2010 American Chemical Society Published on Web 05/20/2010
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Chem. Res. Toxicol., Vol. 23, No. 6, 2010
LaVergne et al.
Table 1. Clinical Characteristics of Patients and Lymphocyte Transformation Test (LTT) Dataa patient
age (years)/sex
route
reaction
time to reaction (days)
1 2 3 4 5 6 7 8 9
18/M 24/F 25/M 45/F 22/F 27/F 17/M 23/M 24/F
oral IV oral oral oral oral oral oral oral
maculopapular rash maculopapular rash maculopapular rash maculopapular rash maculopapular rash fixed drug reaction maculopapular rash vasculitic rash urticarial rash
10 11 12
21/F 41/M 22/F
oral oral oral
periorbital edema thrombocytopenia thrombocytopenia
time since reaction (years)
LTT SMX SMX-NO
cutaneous eruptions 10 6 7 10 4 3 5 16 2
17 5 7 8 2 3 5 2 4
+b NT + -
++ NT +++
nonclassical symptoms 3 14 7
3 7 20
+
-
a F, female; M, male; IV, intravenous; nt, not tested. b Data expressed as stimulation index (SI) (proliferation in drug-treated wells/proliferation in control wells): -, SI less than 2.5; +, SI 2.5-10; ++, SI 10-20; +++, SI 20-30.
Figure 1. Representative responses from 2 allergic patients and tolerant controls. Lymphocytes from the allergic patients were stimulated to proliferative in the presence of SMX-NO but not the parent drug. The coefficient of variation was consistently less than 20%. The Mann-Whitney test was used to compare lymphocyte proliferation in the presence and absence of antigen (*P < 0.05).
(i.e., the parent drug) and add to a growing body of data showing that skin testing offers only limited benefit in the diagnosis of delayed-type SMX reactions (25) (26). The tolerant patient cohort (n ) 6) was composed of patients with cystic fibrosis exposed to repeated courses of trimethoprim-SMX without any adverse event. There was no statistical difference in age, gender, lung function, or sputum analysis, between tolerant and allergic patients. The naı¨ve cohort (n ) 4) was healthy volunteers that had not been exposed to trimethoprim-SMX. Written informed consent was obtained from all patients, and the study was approved by The Leeds East Ethics Committee.
To measure lymphocyte responses, blood lymphocytes were isolated using Ficoll gradient centrifugation. Isolated lymphocytes were suspended in culture medium (RPMI-1640 supplemented with penicillin-streptomycin [100 IU/mL and 100 µg/mL, respectively], HEPES [25 mM], L-glutamine [2 mM], 10% pooled human AB serum, and 12.5 mg of holo-transferrin) at 1.5 × 106 cells/mL and dispensed into a 96-well U-bottom plate (100 µL/well). Following the addition of SMX (125 µM-1 mM), SMX metabolites (SMXhydroxylamine and SMX-NO; 10-80 µM), trimethoprim (10-250 µM), or tetanus toxoid (0.5 µg/mL; positive control), lymphocytes were cultured in a CO2 (5%) ventilated incubator at 37 °C for 6 days. On the fifth day of culture, [3H]thymidine (0.5 µCi/well) was added to each well to measure antigen-specific lymphocyte proliferation. The cells were harvested onto filter membranes (PerkinElmer, Cambridge, U.K.), and incorporated radioactivity was quantified using a β-counter (MicroBeta Trilux, PerkinElmer, Cambridge, U.K.). Results are expressed as stimulation index (SI, cpm in antigen treated cultures/cpm in solvent controls). To increase the sensitivity of the lymphocyte transformation test, lymphocytes (4.5 × 106; 2 mL, 24 well culture plates) were also cultured with either SMX (500 µM) or SMX-NO (40 µM) at 37 °C for 11 days. On day 4, the medium was supplemented with human IL-2 (10 IU/mL). On completion of the enrichment step, cells were washed prior to dispensing into 96-well plates (0.1 × 106/well). Frozen autologous lymphocytes (0.025 × 106/well) were irradiated (30 Gy) and added to the culture plates as a source of antigen presenting cell, in the presence or absence of SMX (250 µM-1 mM), SMXNO (20-80 µM), TMP (25 µM), and PHA (2 µg/mL; positive control). Cells were cultured for 2 days prior to the addition of [3H]thymidine to measure lymphocyte proliferation. (Figure 1 summarizes the methodological approach used.) Results were analyzed using the Mann-Whitney test or a Wilcoxon test for paired data sets. The Fisher exact test was used to compare
Table 2. Stimulation of Drug Antigen-Enriched Lymphocytes from Allergic Patients with Sulfamethoxazole and Nitroso Sulfamethoxazole SMX prestimulation
SMX (mM)
SMX-NO
SMX-NO (µM)
SMX (mM)
SMX-NO (µM)
poststimulation condition
Cpm in control wells
0.25
0.5
1
20
40
80
Cpm in control wells
0.25
0.5
1
20
40
80
2 4 5 6 8 9
96 ( 33.9 714.5 ( 169 1240.0 ( 320.2 5523.3 ( 343.2 217.0 ( 50.9 36.0 ( 17.0
NTb
