Study of the Sequence of Events Involved in Nevirapine-Induced Skin

Chem. Res. Toxicol. 2006, 19, 1205-1214

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Study of the Sequence of Events Involved in Nevirapine-Induced Skin Rash in Brown Norway Rats Marija Popovic,†,⊥ Jeff L. Caswell,§ Baskar Mannargudi,† Jacintha M. Shenton,| and Jack P. Uetrecht†,‡ Faculty of Pharmacy, UniVersity of Toronto, Toronto, Ontario M5S 3M2, Canada, Faculty of Medicine, UniVersity of Toronto, Toronto, Ontario M5S 3M2, Canada, Department of Pathobiology, Ontario Veterinary College, UniVersity of Guelph, Guelph, Ontario N1G 2W1, Canada, and Department of Immunotoxicology, Pharmaceutical Research Institute, Bristol-Myers Squibb Company, East Syracuse, New York ReceiVed May 26, 2006

Nevirapine, used for the treatment of HIV infection, is associated with development of skin rash and liver toxicity. The mechanism of these idiosyncratic reactions is unknown. We have previously reported the discovery of a new animal model of nevirapine-induced skin rash in rats. When treated with nevirapine, Brown Norway rats developed red ears on about day 7 and skin rash on about day 21. On rechallenge, ears turn red within 24 h, and skin lesions develop by day 9. In the current study, we analyzed the time course of the sequence of events involved in the development of skin rash. Rats were treated with nevirapine for 7, 14, or 21 days or rechallenged with it for 0, 1, or 9 days. This treatment led to an increase in the total number of auricular lymph node T, B, and macrophage cells. There was also an increase in the activation/infiltration marker ICAM-1 and activation/antigen presentation marker MHC II in these cells compared with those from control rats. Immunohistochemistry analysis showed macrophage infiltration and ICAM-1 expression in the ears of treated rats as early as day 7 of treatment. Macrophage infiltration preceded T cell infiltration, which was not apparent until the onset of rash. Both MHC I and MHC II expression increased in the skin of nevirapine-treated rats that developed rash. A major inducer of MHC is IFNγ. Although rechallenge with nevirapine led to a large increase in serum levels of IFNγ, this was not observed during the treatment of naı¨ve rats with nevirapine. These observations provide further clues to the mechanism of nevirapine-induced skin rash. Introduction Nevirapine (NVP1, Figure 1) belongs to a group of nonnucleoside reverse transcriptase inhibitor (NNRTI) drugs used for the treatment of HIV-1 infection (1). Soon after being marketed, NVP use was found to cause skin rash and liver toxicity (1). In early studies, the incidence of skin rash was 16% and that of clinically evident hepatotoxicity was 1% (1); however, the incidence of rash is probably somewhat less at present. The skin rash varied from mild to severe, with a combined incidence of Stevens-Johnson syndrome and toxic epidermal necrolysis of 0.3%. Clinically, most patients on nevirapine present with erythematous rashes, often accompanied by fever, and in some cases with internal organ involvement (1). A two week period of low dose NVP treatment followed by the full dose treatment reduces the risk of rash, while treatment with steroids increases the incidence of rash (2). Also, the risk of severe rashes is higher in women than in men (3). * Corresponding author. Tel: 416-978-8939. Fax: 416-978-8511. Email: [email protected]. † Faculty of Pharmacy, University of Toronto. ‡ Faculty of Medicine, University of Toronto. § University of Guelph. | Bristol-Myers Squibb Company. ⊥ Current address: Department of Investigative Toxicology, Novartis Pharma AG, Basel, Switzerland. 1 Abbreviations: NVP, nevirapine; 12-OH NVP, 12-hydroxynevirapine; NNRTI, nonnucleoside reverse transcriptase inhibitor; FCS, fetal calf serum; BN, Brown Norway; ALN, auricular lymph node; PBS, phosphate-buffered saline, 2-ME, 2-mercaptoethanol; MHC, major histocompatibility complex; FACS, fluorescence-activated cell sorting or fluorescence-assisted cell sorting.

Figure 1. Structure of nevirapine

There have recently been reports of severe hepatotoxicity and rash in individuals given NVP as part of postexposure prophylaxis (4); therefore, the use of NVP for postexposure prophylaxis is no longer recommended. Although reactions to nevirapine are termed allergic, few studies have been done to determine whether the reactions are truly immune-mediated. We have previously reported the discovery of a novel animal model of NVP-induced skin rash in rats, which is strain- and sex-dependent (5). When female Brown Norway (BN) rats are fed a diet resulting in a dose of ∼150 mg/kg/day of NVP, they develop red ears in about 7 days and skin rash in 14-21 days with an incidence of 100%. The incidence in female Sprague Dawley rats was 20%, whereas none of the male rats of either strain developed the rash. When the treatment is discontinued, the syndrome resolves. If animals are rechallenged with NVP, red ears develop within 24 h and skin lesions accompanied by malaise by day 9 of the treatment. This rapid onset of skin rash on rechallenge suggested an immune mechanism (5). Involvement of the immune system was confirmed when we were able to transfer this sensitivity with splenocytes or just splenic CD4+ T cells (6). Similarly, partial depletion of CD4+ T cells

10.1021/tx0601152 CCC: $33.50 © 2006 American Chemical Society Published on Web 09/01/2006

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decreased the incidence and severity of the rash, whereas the depletion of CD8+ T cells increased the severity of the rash (6). The characteristics of the rash induced by nevirapine in humans are very similar to those in this model, which suggests that the mechanisms are similar. Our ultimate goal is to understand the mechanism of nevirapine-induced rash in humans, but it is very difficult to study the mechanisms of idiosyncratic drug reactions in humans. In this study, we analyzed the sequence of events leading up to the rash in the rat model. The results of our studies provide clues to the mechanism of nevirapine-induced skin rash in rats, which will be used to elucidate the mechanisms of this rash in humans.

Table 1. Antibodies Used in Flow Cytometry Studies

Materials and Methods Materials. NVP was kindly provided by Boehringer Ingelheim Chemicals, Inc. (Petersburg, VA). Fetal calf serum (FCS), 1640 RPMI-HEPES modified, acetone, anhydrous ethyl alcohol, 2-mercaptoethanol (2-ME), and sodium azide were obtained from Sigma Aldrich (Oakville, ON). PBS (10 mM, pH 7.4) media used for flow cytometry studies were prepared by the University of Toronto Media Services. MEM nonessential amino acids and antibiotics (penicillin G and streptomycin) were purchased from Invitrogen Canada, Inc. (Burlington, ON). Detection and isotype control antibodies used for flow cytometry analysis were purchased from Cedarlane (Hornby, ON): PE mouse anti-rat Rβ TCR IgG1, PE mouse antirat CD45RA IgG2a, PE mouse anti-rat CD11b/c IgG2a, FITC mouse anti-rat CD4 IgG1, FITC mouse anti-rat RT1.B (major histocompatibility complex MHC II) IgG1, biotin mouse anti-rat CD54 (ICAM-1) IgG1, APC streptavidin, PE mouse IgG1 isotype control, PE mouse IgG2a isotype control, and FITC mouse IgG1 isotype control. Purified mouse anti-rat CD32 (FcγII Receptor) IgG1, biotin mouse anti-rat CD80 IgG1, biotin mouse anti-rat CD86 IgG1, and biotin mouse IgG1 isotype control were purchased from BD Biosciences (Mississauga, ON). FITC mouse anti-rat CD8β IgG1 was obtained from Serotec (Raleigh, NC). Tissue-Tek Optimal Cutting Temperature (OCT) compound embedding medium for frozen tissue specimens was purchased from Sakura Finetek USA, Inc. (Torrance, CA). Hydrogen peroxide and xylenes for immunohistochemical staining were bought from EM Science (Gibbstown, NJ). Most of the antibodies used for the immunohistochemistry analysis were purchased from Cedarlane, unless otherwise specified: purified mouse anti-rat RβTCR IgG1, purified mouse anti-rat CD2 IgG2a, purified mouse anti-rat CD4 IgG1, purified mouse anti-rat CD8a IgG1, purified mouse anti-rat CD54 (ICAM1) IgG1, purified mouse anti-rat RT1.B IgG1, and purified mouse anti-rat RT1.Ac IgG2a. Mouse anti-rat CD68 (ED1) purified IgG1 antibody was obtained from Serotec. Rat IFNγ ELISA kits and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate reagent kits were obtained from BD Biosciences. Animal Care. Female BN rats (150-175 g) were obtained from Charles River (Montreal, QC) and housed in pairs in standard cages with free access to water and Agribrands powdered lab chow diet (Leis Pet Distributing Inc., Wellesley, ON). After a one-week acclimation period during which food intake was monitored, the rats were either continued on the powdered lab chow diet (control) or switched to a diet containing NVP such that the approximate daily dose was 150 mg/kg. Rats were monitored for the development of red ears, skin rash, food intake, and body weight. After the appearance of red ears or prominent skin lesions, the rats were anesthetized by an ip injection (1-2 mL/kg) of a ketamine (100 mg/mL) and xylazine (20 mg/mL) mixture (5:3 ratio by volume) and killed by exsanguination. Time Course Study. Female BN rats (n ) 48) were either fed a powdered lab chow diet (n ) 24, four rats per time point) or a diet delivering 150 mg/kg/day of NVP (n ) 24, four rats per time point), until they developed red ears or skin lesions. Shenton et al. previously reported that after 7 days of NVP treatment rats develop red ears and by day 21, skin lesions (5). Upon rechallenge with

Primary Antibodya

Catalog No./Supplier

PE RβTCR IgG1 PE CD45RA (B) IgG2a PE CD11b/c IgG2a FITC CD4 IgG1 FITC CD8β IgG1 FITC RT1.B IgG1 BIOTIN ICAM IgG1 BIOTIN CD80 IgG1 BIOTIN CD86 IgG1 APC-STV Purified CD32 (FcyII Receptor)

CL057/Cedarlane CL033/Cedarlane CL042/Cedarlane CL003/Cedarlane MCA938/Serotec CL010/Cedarlane CL054/Cedarlane 555013/BD Biosciences 555017/BD Biosciences CLCSA 1005/Cedarlane 550270/BD Pharmingen

Isotypic Controlb

Catalog No./Supplier

PE IgG1 PE IgG2a FITC IgG1 BIOTIN IgG1

CLCMG104/Cedarlane CLCMG2A04/Cedarlane CLCMG101/Cedarlane 550615/BD Pharmingen

a All primary antibodies are mouse anti-rat. b All isotypic control antibodies are made in mouse.

Table 2. Antibodies Used in Immunohistochemistry

a

primary antibodya

catalog no./supplier

CD68 (ED1) IgG1 ICAM-1 IgG1 RT1.Ac IgG2a RT1.B IgG1 CD4 IgG1 CD8a IgG1 RβTCR IgG1 CD2 IgG2a

MCA34lR/Serotec CL054AP/Cedarlane CL027AP/Cedarlane CL010AP/Cedarlane CL003AP/Cedarlane CL004AP/Cedarlane CL057AP/Cedarlane CL034AP/Cedarlane

All primary antibodies are purified mouse anti-rat.

Figure 2. Total number of auricular lymph node cells in control (CON) and NVP-treated rats over time. Control animal data for days 7, 14, or 21 (CON 7, CON 14, and CON 21) and rechallenge days 0, 1, or 9 (CON R0, CON R1, and CON R9) are displayed with clear bars. NVPtreated animal data for days 7, 14, or 21 (NVP 7, NVP 14, and NVP 21), and rechallenge days 0, 1, or 9 (NVP R0, NVP R1, and NVP R9) are displayed with black bars. Cells were counted manually using a hemocytometer, and data are expressed as the group mean of four rats ( SEM. Statistical significance between treatment groups was determined using the Mann Whitney test; *p e 0.05 compared to respective control.

NVP, rats previously treated with nevirapine develop red ears within 1 day and skin lesions within 9 days. Control rats were fed commercially supplied powdered chow diet on both primary and rechallenge exposures. On the basis of this data, animals were sacrificed after 7, 14, and 21 days of primary NVP exposure and 0, 1, and 9 days of rechallenge, and sera, ALNs, and ear sections were collected. Preparation of ALNs for Flow Cytometry Analyses. ALNs were excised, put into Petri dishes containing culture medium (50 mL of FCS, 5 mL of MEM nonessential amino acids, 5 mL of antibiotics, 5 mL of diluted 2-ME (35 µL of 2-ME in 100 mL of distilled water) and 435 mL of 1640 RPMI-HEPES modified). ALN

Immune-Mediated Mechanism for NeVirapine Skin Rash

Chem. Res. Toxicol., Vol. 19, No. 9, 2006 1207

Figure 3. Total numbers of T cells (CD4 or CD8), B cells, and macrophages in ALNs of control (C) and NVP (N)-treated rats. Control animal data for days 7, 14, or 21 (C7, C14, and C21) and rechallenge days 0, 1, or 9 (CR0, CR1, and CR9) are displayed with clear bars. NVP-treated animal data for days 7, 14, or 21 (N7, N14, and N21), and rechallenge days 0, 1, or 9 (NR0, NR1, and NR9) are displayed with black bars. The levels are expressed as the group mean of four rats ( SEM. Statistical significance between treatment groups was determined using the Mann Whitney test; *p e 0.05 compared to respective control.

cells were teased out of the nodal capsule and filtered through a 70 µm mesh (VWR). Cells were spun down at 200g, 4 °C for 6 min, resuspended in FACS buffer (100 µL/10 mL FCS, 5 mL of sodium azide, 485 mL of PBS), and counted. One million cells were plated in each well of the 96-well V bottom polystyrene microplates (Hospital Logistics, Inc., Oakville ON) and stained at 4 °C for 15 min, first with the anti-FcγII receptor antibody to prevent nonspecific staining, followed by the primary antibodies for various cell surface markers (outlined in Table 1). In the case of biotin-labeled primary antibodies, the cells were incubated for 15 min with streptavidin-APC and then washed once with the FACS buffer (100 µL). Data were acquired by FACSCalibur (BD Biosciences) and analyzed using CellQuest software (BD Biosciences). Preparation of Ear Sections for Immunohistochemistry. This procedure was conducted as described by Shenton et al. (5). Skin Patch Test. Female BN rats were fed rat chow containing NVP (∼150 mg/kg/day dose, n ) 15) until they developed skin lesions. After a period of 28 days during which skin lesions resolved, a single dose of NVP, 12-hydroxynevirapine (12-OH NVP), or vehicle control was administered onto the inner side of the rat’s right ear. 12-OH NVP is a metabolite of NVP and potentially the precursor to a reactive quinone methide, which could be responsible for the rash. Then, 100 µL of a vehicle (acetone/ olive oil, 1:1/v:v), 2.5 mg/mL, 0.5 mg/mL of NVP, or 12-OH NVP were applied to the right ear of a rat (n ) 3). A day after applying NVP or 12-OH NVP, any change in the rats’ ear color was noted. Three days later, the rats were sacrificed and both right and left ears were longitudinally dissected and prepared for the immunohistochemistry analyses as described above. ELISA. Blood was obtained from these rats by cardiac puncture, stored overnight at 4 °C in Vacutainer red top 10 mL tubes (VWR; Mississauga, Ontario), and centrifuged the following morning at 150g at 4 °C for 10 min. Rat serum IFNγ analysis was performed following the instructions on the BD Biosciences kit. The kit sensitivity was determined to be 26.5 pg/mL. Statistics. Statistical significance between treatment groups was determined using the Mann Whitney test; values of p e 0.05 were considered statistically significant.

Results Time Course of ALN Cellular Composition. The total ALN cell count from both control and NVP-treated female BN rats is displayed in Figure 2. At all time points, except for day 14 of the primary treatment, the total numbers of ALN cells in the NVP-treated rats were greater than those in the control rats. The numbers of CD4+ T, CD8+ T, B, and macrophage cells were higher in the NVP-treated rats than those in the controls (Figure 3). In addition, the ALNs of the NVP-treated rats contained more activated macrophages and B cells, which expressed MHC II cell surface activation markers (Figures 4 and 5). Furthermore, there was a higher percent of activated cells, B cells in particular, in the NVP-treated group than that in the control group (Figure 5). Immunohistochemical Analyses of Ear Sections. Our analysis confirmed the previously reported observation by Shenton et al. that CD4+ and CD8+ T cell infiltrates are observed in the ears of rats that developed skin lesions on day 21 of primary and day 9 of rechallenge exposure to NVP (5). In addition, we observed CD4+ and CD8+ T cell dermal infiltration at day 14 of the primary exposure; at this time point, the rats still had red ears and had started developing skin lesions (Figure 6). We did not observe infiltration of CD4+/CD8+ cells in the ears of rats before they developed skin lesions, that is, day 7 of the primary treatment, days 0 and 1 of rechallenge, or at any time point in the control rats. In contrast, staining for macrophages showed that these cells are present in the ear dermis as early as day 7 of primary and day 1 of rechallenge, time points at which animals were present with red ears, but not skin lesions (Figure 7). At the time points when rats develop skin lesions (day 14-21 of the primary exposure and day 9 of rechallenge), dermal macrophage infiltrates are even more prominent (Figure 7). In rats with skin lesions, macrophages were mostly observed throughout the dermis, around the hair


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Figure 4. Evidence of activated immune cells in auricular lymph nodes, total number of cells expressing (A) ICAM-1 (CD54 marker), (B) MHC II (RT1.B marker), (C) total number of B cells expressing MHC II, or (D) macrophages (CD11b/c) expressing MHC II cell surface markers in the ALNs of control and NVP-treated rats. Control animal data for days 7, 14, or 21 (C7, C14, and C21) or rechallenge days 0, 1, or 9 (CR0, CR1, and CR9) are displayed with clear bars. NVP-treated animal data for days 7, 14, or 21 (N7, N14, and N21) or rechallenge days 0, 1, or 9 (NR0, NR1, and NR9) are displayed with black bars. The levels are expressed as the group mean of four rats ( SEM. Statistical significance between treatment groups was determined using the Mann Whitney test; *p e 0.05 compared to respective control.

Figure 5. Evidence of activated immune cells in auricular lymph nodes. Percent of cells expressing (A) MHC II (RT1.B), (B), ICAM-1 (CD54), (C) double-stained macrophage and MHC II (macrophage/MHC II), or (D) double-stained B and MHC II (B/MHC II) cells in the ALNs of control and NVP-treated rats. Control rat data for days 7, 14, or 21 (C7, C14, and C21) or rechallenge days 0, 1, or 9 (CR0, CR1, and CR9) are displayed with clear bars. NVP-treated rat data for days 7, 14, or 21 (N7, N14, and N21) or rechallenge days 0, 1, or 9 (NR0, NR1, and NR9) are displayed with black bars. Data are expressed as the group mean of four rats ( SEM. Statistical significance between treatment groups was determined using the Mann Whitney test; *p e 0.05 compared to respective control.

follicles, and surrounding sebaceous and apocrine glands. Some macrophages were also detected in the skin muscle layer. We observed similar findings with ICAM-1 cell surface marker staining (Figure 8). ICAM-1 is present on activated skin cells that mediate lymphocyte infiltration into the skin (7). Like macrophages, increased ICAM-1 expression was detected as

early as day 7 of the primary exposure and day 1 of rechallenge. Elevated ICAM-1 expression was mostly observed around the skin blood vessels, hair follicles in the dermis, and in the dermalepidermal junction (Figure 8). By the time rats developed skin lesions, staining was prominent throughout the entire dermis of the ear (Figure 8).



Figure 6. Immunohistochemistry analysis of ear sections for the infiltration of (A) CD4+ and (B) CD8a+ cells in control (C) and nevirapine (N)-treated rats on day 14 of the primary exposure. The magnification is 200×.

Figure 7. Immunohistochemistry analysis of ear sections for the presence of macrophages (ED1) in control (C) and NVP (N)-treated rats on days 7, 14, and 21 and rechallenge (R) days 0, 1, and 9 of the time course study. The magnification is 200×.

When ear sections were stained for the MHC I and MHC II, the increase in expression of both markers was observed mostly around the blood vessels and hair follicles in the dermis of rats that showed skin lesions (Figures 9 and 10). As early as day 7 of treatment, leukocytes infiltrating the dermis had increased MHC I expression. By day 14, prominent upregulation of MHC I expression was detected on cells of the epidermis, dermalepidermal junction, and around the hair follicles in the dermis. On rechallenge, extensive epidermal and dermal MHC I expression, particularly around hair follicles, was detected. A modest increase in MHC II expression was also observed on leukocytes infiltrating the dermis as early as day 7. By day 14, dermal leukocytes, follicular infundibulum, and some fibroblasts had increased expression of MHC II. Also, parts of the epidermis had both scattered single cells and patchy areas staining positive

for MHC II. On rechallenge, epidermal cells, follicular infundibulum, and the dermis stained positive for this marker. Skin Patch Test. Previously sensitized BN rats were used for skin patch testing. After challenging sensitized rats by applying a higher dose (0.25 mg) of NVP or 12-OH NVP to the ear, both ears turned red within 24 h with either treatment. For rats challenged with the lower dose (0.05 mg) of either chemical, the ears did not become red. Dermal T cell and macrophage infiltrates were observed in both rats exposed to the high and low dose of NVP and 12-OH NVP; nevertheless, the infiltration was more prominent in the ears of rats challenged with the higher dose than those with the lower dose (Figures 11 and 12). ICAM-1 cell activation marker expression was increased on cells surrounding dermal blood vessels in both high and low dose NVP- and 12-OH NVP-challenged rats (Figure


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Figure 8. Immunohistochemistry analysis of ear sections for the expression of the ICAM-1 (CD54) cell surface marker in control (C) and NVP (N)-treated rats on days 7, 14, and 21 and rechallenge (R) days 0, 1, and 9 of the time course study. The magnification is 200×.

Figure 9. Immunohistochemistry analysis of ear sections for the expression of the MHC I (RT1.Ac) cell surface marker in control (C) and NVP (N)-treated rats on days 7, 14, and 21 and rechallenge (R) days 0, 1, and 9 of the time course study. The magnification is 200×.

13). Increased expression of both MHC I and MHC II cell surface proteins was detected in the dermis of both NVP- and 12-OH NVP-treated rats (Figures 14 and 15). When left (contralateral) ear sections were analyzed for ICAM-1 or MHC I and MHC II, the same staining pattern as that seen in the right ears (patch test ears) was observed, however, to a lesser extent (data not shown). Control rats, which were initially sensitized with nevirapine but rechallenged with the vehicle (acetone/olive oil) solution, did not have their ears turn red 24 h postexposure. No immune cell infiltration was observed in the patch test or in the contra-lateral ear of the control rats. Only a slight increase in MHC I staining (Figure 14, V) was observed in the control

rat patch test ear; however, this staining was less prominent than that in the NVP and 12-OH NVP-tested rats. IFNγ ELISA. Analyses of rat sera showed increased IFNγ levels in rats rechallenged with NVP but not in rats exposed to the drug during primary treatment (Figure 16).

Discussion The mechanisms by which drugs cause idiosyncratic adverse reactions are unknown. Many of the characteristics of these reactions suggest that they are immune-mediated, but in most cases, this has never been demonstrated. One major impediment



Figure 10. Immunohistochemistry analysis of ear sections for the expression of the MHC II (RT1.B) cell surface marker in control (C) and NVP (N)-treated rats on days 7, 14, and 21 and rechallenge (R) days 0, 1, and 9 of the time course study. The magnification is 200×.

Figure 11. Skin Patch test immunohistochemistry analysis of right ear sections for the expression of CD2 (T, natural killer, dendritic, and macrophage cell) surface marker in vehicle (V), 0.25 mg of NVP (N)-, 0.25 mg of 12-OH NVP (12-OH N)-, 0.05 mg of NVP (N)-, and 0.05 mg of 12-OH NVP (12-OH N)-treated rats on day 3 post-testing. The magnification is 200×.

to the study of this type of adverse reaction is the paucity of animal models. Previous work by Shenton et al. (5) demonstrated that the NVP-induced skin rash in the female BN rat is immune-mediated. In the present study, we investigated the sequence of events involved in the development of skin rash in female BN rats treated with NVP in order to better understand the mechanistic steps leading to the onset of this immunemediated reaction. Elucidation of the chain of immunologic events, which precede the onset of skin rash and occur both in the secondary lymphoid tissues and the skin, will further help

us understand the mechanism leading to the onset of rash in the NVP-treated rats. Analyses of rat ALNs showed that NVP-treated rats have higher total lymphocyte and macrophage cell counts than control rats (Figures 2 and 3). Approximately one-third of the total ALN cells were activated in the NVP-treated rats at every point during the study, either expressing ICAM-1 or MHC II cell surface markers. Specifically, increased numbers of macrophages and B cells expressed MHC II surface marker, indicating that these cells were activated at all time points tested, and these cells


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Figure 12. Skin patch test immunohistochemistry analysis of right ear sections for the expression of the macrophage (ED1) cell surface marker in vehicle (V), 0.25 mg of NVP (N)-, 0.25 mg of 12-OH NVP (12-OH N)-, 0.05 mg of NVP (N)-, and 0.05 mg of 12-OH NVP (12-OH N)-treated rats on day 3 post-testing. The magnification is 200×.

Figure 13. Skin patch test immunohistochemistry analysis of right ear sections for the expression of the ICAM-1 cell surface marker in vehicle (V), 0.25 mg of NVP (N)-, 0.25 mg of 12-OH NVP (12-OH N)-, 0.05 mg of NVP (N)-, and 0.05 mg of 12-OH NVP (12-OH N)-treated rats on day 3 post-testing. The magnification is 200×.

may have played a role in antigen presentation in the course of skin rash development (8). Infiltration of macrophages into the dermis and upregulation of ICAM-1 on the surface of skin cells were observed as early as day 7 of primary and day 1 of rechallenge exposures, the time points at which rats presented with red ears. These events preceded lymphocyte infiltration into the ear, which occurred when rats developed skin rash on day 21 of the primary exposure and day 9 of rechallenge. This suggests an important role for macrophages in the early stages of this immune response.

Macrophages may respond as representatives of the innate immune system, necessary in the first line of immune response against foreign antigens. Once in the skin, they may also act as antigen-presenting cells, taking up foreign skin antigens (NVP, its metabolites, or covalently modified skin tissue proteins), processing them and presenting on the surface of their MHC molecules for naı¨ve infiltrating T cells to recognize and respond to. In order for CD8+ T and CD4+ T cells to mount an immune response against a foreign antigen, they need to directly interact



Figure 14. Skin patch test immunohistochemistry analysis of right ear sections for the expression of the MHC I cell surface marker in vehicle (V), 0.25 mg of NVP (N)-, 0.25 mg of 12-OH NVP (12-OH N)-, 0.05 mg of NVP (N)-, and 0.05 mg of 12-OH NVP (12-OH N)-treated rats on day 3 post-testing. The magnification is 200×.

Figure 15. Skin patch test immunohistochemistry analysis of right ear sections for the expression of the MHC II cell surface marker in vehicle (V), 0.25 mg of NVP (N)-, 0.25 mg of 12-OH NVP (12-OH N)-, 0.05 mg of NVP (N)-, and 0.05 mg of 12-OH NVP (12-OH N)-treated rats on day 3 post-testing. The magnification is 200×.

with the MHC I and MHC II surface proteins, respectively, on antigen-presenting cells (9). Because CD4+ T and CD8+ T cell infiltrates were only detected in the ears of rats that developed skin lesions (Figure 6), we analyzed the expression of MHC I and MHC II proteins in ears at the same time points (Figures 9 and 10). Both MHC I and MHC II receptor expression significantly increased in the dermis of rats that developed skin lesions. Previous work has shown that IFNγ levels are a major determinant of both MHC I and MHC II expression (10, 11). Analyses of rat sera showed increased IFNγ levels in rats rechallenged with nevirapine, but not during the development of rash in rats treated with NVP for the first time. This also correlates with the observed clinical picture. Rats develop red ears and skin lesions during both primary and rechallenge exposures to NVP; however, the rash and other clinical

characteristics of the reaction differ between primary exposure and rechallenge. During primary exposure, rats develop a relatively severe rash, but with no signs of discomfort, behavioral changes, weight loss, or malaise. On rechallenge, rats do not present with as many skin lesions as those on the primary exposure, but they lose weight, experience hair loss, and lack curiosity about their surroundings; by day 9 of rechallenge, they are euthanized for humane reasons. The sequence of events observed in these studies is that which might be expected of an immune-mediated reaction, that is, first an upregulation of adhesion molecules and an infiltration of macrophages followed by an infiltration of T cells. Previous experiments suggest that it is CD4+ T cells that mediated the rash (6). The next step is to determine how the drug or reactive metabolite initiates this sequence of events. It is likely that initial


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was surprising is that there was a relatively sharp dose-response curve, and once a sufficient amount of drug was applied to the ear to cause it to turn red, both ears turned red, which suggests a more systemic reaction. In future studies, we should be able to determine whether it is the parent drug or quinone methide that initiates the immune response. Acknowledgment. This work was supported by a grant from Canadian Institutes of Health Research (CIHR). M. Popovic is a recipient of a doctoral fellowship awarded by Rx&D HRF CIHR, and Dr. J. P. Uetrecht is a recipient of Canada Research Chair in Adverse Drug Reactions. Figure 16. ELISA analysis showing the time course of IFNγ secretion in the serum of female BN rats treated with 150 mg/kg of NVP in the diet for 5, 7, 14, or 21 days on primary exposure and 0, 1, or 9 days on rechallenge. The levels are expressed as the group mean of four rats ( SEM. Statistical significance between treatment groups was determined using the Mann Whitney test; values of p e 0.05 were considered statistically significant.

steps involve the resident cells of the dermis such as fibroblasts, which may express cytokines or activation markers that lead to the activation of the immune system that is documented in the present study, and this will be the focus of future studies. Such studies are only feasible in an animal model and are essential for mechanistic understanding. A major controversy is the degree to which reactive metabolites play a role in the mechanism of most idiosyncratic drug reactions. There is a large amount of circumstantial evidence to suggest that most idiosyncratic drug reactions are due to reactive metabolites. We have postulated that nevirapine-induced skin rash is due to the oxidation of NVP to 12-OH NVP in the liver followed by the sulfation of this benzylic alcohol in the skin and the loss of sulfate to form a quinone methide. In contrast, Pichler has found T cells in patients with a recent history of an idiosyncratic reaction to a drug that are activated by the offending drug in the absence of metabolism (12). This suggests that the parent drug can initiate an idiosyncratic drug reaction. An alternate hypothesis is that it is a reactive metabolite that is responsible for initiating an immune response, and once the immune response has been initiated, T cells are generated that also respond to the parent drug. It would be very difficult to test these competing hypotheses in humans. Our findings in this model are consistent with the findings of Pichler, that is, we found that application of either nevirapine or 12-hydroxynevirapine to the ears of previously sensitized animals led to a local infiltration of T cells. Although oxidation of NVP could occur in the skin, it is unlikely that the amount formed would be comparable to the amount present when the 12-hydroxy metabolite is directly applied to the ear; yet, the response to both was similar. This suggests that there were T cells in sensitized animals that recognized the parent drug. What

References (1) Pollard, R. B., Robinson, P., and Dransfield, K. (1998) Safety profile of nevirapine, a nonnucleoside reverse transcriptase inhibitor for the treatment of human immunodeficiency virus infection. Clin. Ther. 20, 1071-1092. (2) Montaner, J. S., Cahn, P., Zala, C., Casssetti, L. I., Losso, M., Hall, D. B., Wruck, J., McDonough, M., Gigliotti, M., Robinson, P. A., and 1100.1286 Study Team. (2003) Randomized, controlled study of the effects of a short course of prednisone on the incidence of rash associated with nevirapine in patients infected with HIV-1. J. Acquired Immune Defic. Syndr. 33, 41-46. (3) Bersoff-Matcha, S. J., Miller, W. C., Aberg, J. A., Van Der Horst, C., Hamrick, H. J., Jr., Powderly, W. G., and Mundy, L. M. (2001) Sex differences in nevirapine rash. Clin. Infect. Dis. 32, 124-129. (4) Johnson, S., and Baraboutis, J. G. (2000) Adverse effects associated with use of nevirapine in HIV postexposure prophylaxis for 2 health care workers. JAMA 284, 2722-2723. (5) Shenton, J. M., Teranishi, M., Abu-Asab, M. S., Yager, J. A., and Uetrecht, J. P. (2003) Characterization of a potential animal model of an idiosyncratic drug reaction: nevirapine-induced skin rash in the rat. Chem. Res. Toxicol. 16, 1078-1089. (6) Shenton, J. M., Popovic, M., Chen, J., Masson, M. J., and Uetrecht, J. P. (2005) Evidence of an immune-mediated mechanism for an idiosyncratic nevirapine-induced reaction in the female Brown Norway rat. Chem. Res. Toxicol. 18, 1799-1813. (7) Ackermann, L., and Harvima, I. T. (1998) Mast cells of psoriatic and atopic dermatitis skin are positive for TNF-alpha and their degranulation is associated with expression of ICAM-1 in the epidermis. Arch. Dermatol. Res. 290, 353-359. (8) Harding, C. V., and Unanue, E. R. (1990) Quantitation of antigenpresenting cell MHC class II/peptide complexes necessary for T-cell stimulation. Nature 346, 574-576. (9) Janeway, C. A., Jr., Bottomly, K., Horowitz, J., Kaye, J., Jones, B., and Tite, J. (1985) Modes of cell:cell communication in the immune system. J. Immunol. 135, 739s-742s. (10) Steiniger, B., Van der Meide, P. H., Falk, P., and Klempnauer, J. (1988) Induction of class II MHC antigen expression in rat organs after systemic application of recombinant gamma interferon. AdV. Exp. Med. Biol. 237, 795-799. (11) Hao, L. M., Wang, Y., Gill, R. G., La Rosa, F. G., Talmage, D. W., and Lafferty, K. J. (1989) Role of gamma-interferon in islet allograft rejection: class I MHC antigen induction. Transplant Proc. 21, 26972699. (12) Zanni, M. P., Mauri-Hellweg, D., Brander, C., Wendland, T., Schnyder, B., Frei, E., Von Greyerz, S., Bircher, A., and Pichler, W. J. (1997) Characterization of lidocaine-specific T cells. J. Immunol. 158, 1139-1148.

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Study of the Sequence of Events Involved in Nevirapine-Induced Skin

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