Chem. Res. Toxicol. 2005, 18, 1799-1813
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Articles Evidence of an Immune-Mediated Mechanism for an Idiosyncratic Nevirapine-Induced Reaction in the Female Brown Norway Rat Jacintha M. Shenton,†,§ Marija Popovic,† Jie Chen,† Mary Jane Masson,†,| and Jack P. Uetrecht*,†,‡ Faculties of Pharmacy and Medicine, University of Toronto, Toronto, Ontario, Canada Received April 28, 2005
Previously, we reported a new animal model of an idiosyncratic drug reaction in which nevirapine causes a skin rash in some rats that has characteristics similar to the reaction that occurs in humans. Strong evidence that the reaction is immune-mediated was found; specifically, low-dose pretreatment induced tolerance, while with rechallenge, the time to onset decreased and the severity increased. Furthermore, splenocytes from rechallenged rats transferred rash susceptibility to naı¨ve recipients. We now report the results of studies to explore the immune aspects of this reaction. T cells were found to play an important role, as demonstrated by their ability to adoptively transfer susceptibility to the skin reaction. Of these T cells, CD4+ cells are the likely effectors because they were capable of transferring susceptibility and the reaction was delayed in rats partially depleted of CD4+ T cells. In contrast, it appears that CD8+ T cells are not essential, as CD8+ T cells were unable to transfer sensitivity to a naı¨ve animal and rats depleted of CD8+ T cells still developed skin rash. Unlike the penicillamine model, where we have demonstrated that the tolerance induced by low-dose treatment is immune-mediated, tolerance induced by low-dose nevirapine appears to be largely due to induction of metabolism as it can be overcome by inhibition of cytochrome P450. Pretreatment with the immunosuppressants, cyclosporine and tacrolimus, prevented the rash and even led to resolution of the rash during nevirapine treatment. These studies reinforce the hypothesis that the reaction in this model is similar to that which occurs in humans. In particular, the finding that CD4+ T cells may play a central role in this model fits with the observation that the incidence of idiosyncratic reactions to nevirapine in humans appears to be lower in patients with low CD4+ counts.
Introduction Nevirapine (Viramune) is a nonnucleoside reverse transcriptase inhibitor used in combination therapy to treat human immunodeficiency virus (HIV)1 infections. Nevirapine is known to cause a significant incidence of liver toxicity and skin rashes (1). Among the rashes caused by nevirapine are the severe and potentially lifethreatening rashes, Stevens-Johnson syndrome and toxic epidermal necrolysis (1-3). * To whom correspondence should be addressed at Faculty of Pharmacy, University of Toronto, 19 Russell Street, Toronto, Ontario, M5S 2S2. Telephone: 416-978-0185. E-mail:
[email protected]. † Faculty of Pharmacy, University of Toronto. ‡ Faculty of Medicine, University of Toronto. § Presently at Department of Immunotoxicology, Bristol Myers Squibb Company, 6000 Thompson Road, East Syracuse, NY 13057. | Presently at National Institutes of Health, Molecular and Cellular Toxicology Section, Bethesda, MD. 1 Abbreviations: HIV, human immunodeficiency virus; BN, Brown Norway; RPMI, Roswell Park Memorial Institute; DME, Dulbecco’s Modified Eagles; poly(I:C), polyinosinic-polycytidylic acid; FBS, fetal bovine serum; ABT, aminobenzotriazole; SPE, solid-phase extraction; TCR, T cell receptor; OPD, o-phenylenediamine dihydrochloride; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate; PE, phycoerythrin; HRP, horseradish peroxidase; PBS, phosphate-buffered saline.
Animal models have the potential to greatly facilitate our understanding of the mechanisms of such idiosyncratic drug reactions (4). To this end, we previously reported the discovery of a new animal model of an idiosyncratic drug reaction: nevirapine-induced skin rash in the rat (5). It was shown that 100% of female Brown Norway (BN) rats administered nevirapine (150 (mg/kg)/ day) developed red ears on days 7-10 and skin lesions on days 10-21 of treatment (5). Furthermore, the characteristics of the reaction suggested an immunemediated mechanism. First, histology of skin sections from nevirapine-treated female BN rats revealed the existence of an inflammatory cell infiltrate, which was determined to be composed primarily of T cells and macrophages. Second, a 2-week low dose (40 (mg/kg)/day) lead-in treatment prior to starting the full dose prevented the rash, conceivably due to immune tolerance. Furthermore, rats with skin rash that were removed from nevirapine treatment and then reexposed several weeks later, clearly developed symptoms faster on rechallenge (red ears in 0.99 for each curve. The HPLC was carried out using the column described above. A gradient elution was used in which mobile phase A consisted of water with 1% acetic acid and 2 mM
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Chem. Res. Toxicol., Vol. 18, No. 12, 2005
Shenton et al.
Table 1. Numbers and Types of T Cells Used for Adoptive Transfer splenocyte number of number of subset recipients cells injected
antibody clone
1 1 1 3
30 × 106 73 × 106 88 × 106 100 × 106
yes yes no yes
CD4 T cells CD4 T cells CD4 T cells CD4 T cells CD4 T cells CD4 T cells
1 2 1 2 1 1
13 × 106 20 × 106 50 × 106 85 × 106 100 × 106 200 × 106
no no yes no; 1 out of 2 red ears by 36 h slightly; but clearly by 48 h yes
CD8 T cells CD8 T cells CD8 T cells
2 1 1
5 × 106 6 × 106 8 × 106
no no no
Table 2. Percent Gated Cells before (None) and after Passage of Splenocytes through Various T Cell Enrichment Columns enrichment T CD8β+ CD4+ CD8R+ column cells T cells T cells cells B cells macrophages 39 88 97 75
7 15 0 40
27 60 95 1
suppliera
volb of Ab (µL)
specificity
CedarLane CedarLane CedarLane CedarLane CedarLane Serotec CedarLane CedarLane
35 35 5 35 20 35 20 10
Rβ T cells Rβ and γδ T cells CD4+ T cells, macrophages B cells CD8+ T cell, NK cells CD8+ T cells macrophages NK cells
red ears in
