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AIDS:
doi: 10.1097/QAD.0b013e3280119579
Basic Science

Immune responses to abacavir in antigen-presenting cells from hypersensitive patients

Martin, Annalise Ma; Almeida, Coral-Anna; Cameron, Paulb; Purcell, Anthony Wc; Nolan, Davida; James, Iana; McCluskey, Jamesd; Phillips, Elizabethe; Landay, Alanf; Mallal, Simona,b

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Author Information

From the aCentre for Clinical Immunology and Biomedical Statistics, Royal Perth Hospital and Murdoch University, Western Australia

bDepartment of Clinical Immunology and Biochemical Genetics, Royal Perth Hospital, Western Australia

cDepartment of Biochemistry and Molecular Biology, Australia

dDepartment of Microbiology and Immunology, University of Melbourne, Victoria, Australia

eBritish Columbia Centre of Excellence in HIV/AIDS, Vancouver, University of British Columbia, Canada

fDepartment of Immunology/Microbiology Rush University Medical Center, Chicago, Illinois, USA.

Received 8 November, 2005

Revised 7 September, 2006

Accepted 5 October, 2006

Correspondence to Simon Mallal, Centre for Clinical Immunology and Biomedical Statistics, 2nd floor, North Block, Royal Perth Hospital, Perth, Western Australia, 6000, Australia. E-mail: S.Mallal@murdoch.edu.au

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Abstract

Objectives: A potentially life-threatening hypersensitive reaction accompanies the use of HIV nucleoside analogue abacavir (ABC) in 4–8% of Caucasian individuals. HLA-B*5701 and Hsp70 493T alleles have been shown to predict susceptibility to this hypersensitivity.

Design and methods: This study was undertaken to provide a mechanistic understanding of the highly significant genetic association of HLA Class I and Hsp70 alleles with ABC hypersensitivity.

Results: In this study an ABC-induced localization of intracellular HSP70 to endosomal vesicles of antigen-presenting cells was demonstrated. This ABC-stimulated redistribution of endogenous HSP70 was substantially higher in the genetically homogenous HLA-B*5701+, Hsp70 493T+ ABC-hypersensitive individuals and ABC-naive individuals in comparison with the heterogenous tolerant patients (P = 0.023). Increased expression of HSP70 was also detected in the hypersensitive group as measured by flow cytometry (P = 0.032). Blocking of HSP70 and HSP70 cell surface receptors CD14 and TLR2 abrogated ABC-stimulated HSP70 redistribution in sensitized individuals to basal levels (P < 0.004). In addition, the use of TcRαβ and HLA-B57/58 antibodies also ablated the expression of HSP70. Cells expressing the activation markers CD40 were increased after ABC stimulation in the hypersensitive patients (P = 0.006). ABC-stimulated interferon-gamma levels were higher in hypersensitive patients in comparison with ABC-tolerant individuals with a mean of 123.54 versus 0 pg/ml (P = 0.001).

Conclusion: The present data indicates that ABC stimulates an innate immune response and activates antigen-presenting cells via the endogenous HSP70-mediated Toll-like receptor pathway in genetically susceptible individuals potentially initiating the immuno-pathological hypersensitive response.

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Introduction

Abacavir (ABC) is a commonly prescribed nucleoside analogue reverse transcriptase inhibitor (NRTI) drug used for the treatment of HIV-1 infection. Although this drug has proved highly effective and generally has a favourable toxicity profile, potentially life-threatening hypersensitivity reactions have emerged as a major treatment-limiting toxicity in approximately 4–8% of ABC recipients [1,2]. This clinical syndrome bears the hallmarks of an idiosyncratic drug hypersensitivity reaction in that symptoms and signs of a multi-organ inflammatory syndrome appear rapidly (9–11 days), evolve and intensify with sustained therapy, and improve within 24–48 h of terminating ABC treatment [3]. Symptoms also reappear rapidly and with greater severity following ABC rechallenge [4].

Genetic association studies have also provided strong support for the possibility that this hypersensitivity syndrome reflects a specific HLA Class I-restricted immune response to ABC, in that susceptibility is strongly linked to carriage of HLA-B*5701 and related genetic markers within the major histocompatibility complex (MHC) gene cluster [5–9]. Carriage of this genetic (HLA-B*5701) risk factor is associated with a positive predictive value of between 70 and 90% for the development of drug hypersensitivity [5,6,9] in Caucasian populations rather than individuals of African descent where this allele is uncommon [10]. Recombinant haplotype mapping has also implicated central MHC alleles such as a heat shock protein variant (Hsp70 Hom M493T) and a tumour necrosis factor-alpha (TNF-alpha) promoter polymorphism (TNF–238A), in addition to HLA-B*5701, in conferring susceptibility to ABC hypersensitivity [5,6,8,9]. In this context, the presence of multiple susceptibility loci existing in strong linkage disequilibrium within a common MHC haplotype organization [referred to as the 57.1 ancestral haplotype (AH)] may provide a basis for the remarkably high penetrance associated with genetic predisposition to ABC hypersensitivity, compared with other idiosyncratic drug reactions.

We have previously hypothesized that in susceptible individuals, ABC or its metabolites may be involved in the haptenation of endogenous peptides leading to presentation of ‘altered self’ and induction of pathogenic T-cell responses [9]. This is consistent with a direct role for HLA-B*5701 in ABC hypersensitivity reaction (HSR) and supports the CD8+ T-cell infiltration in skin biopsies obtained after epicutaneous patch testing [11] and CD8+ T-cell-dependent production of TNF following exposure of mononuclear cells from HIV-infected, HLA-B*5701-positive patients to ABC in vitro [9]. Associations between the risk of developing ABC HSR and elevated CD8+ T cells also support this possibility [12]. Presentation of drug-haptenated endogenous peptides has conventionally been thought to occur in antigen presenting cells initiating drug-specific immune responses. The role of HSP70 chaperoning and assisting in presentation of HLA Class I ligands and as an initiator of the innate immune response is well documented.

These data provide a rational basis for genetic screening strategies to minimize the risk of ABC hypersensitivity. However, at this stage little is known of the functional, immunological correlates of these genetic associations. In the present study a series of experiments designed to elucidate the functional role of genetic background in determining the immunological response to ABC were undertaken. The roles of endogenous HSP70 and its cell surface receptors as critical factors in the induction of ABC-specific immune responses have also been examined.

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Materials and methods

Subjects

HIV-positive individuals who had a hypersensitive reaction to ABC fulfilled the criteria of clinical symptoms involving multiple systems, a positive epicutaneous skin patch reaction and elevated TNF response to ABC in peripheral blood mononuclear cell (PBMC) cultures [9]. Cryopreserved PBMCs from ABC HSR (patients with genotypes HLA-B*5701, TNF-238A, Hsp70 Hom 493T, n = 9), ABC-tolerant patients [with genotypes HLA-B*5701, TNF-238A, Hsp70 Hom 493T, n = 1, HLA-B*5701, TNF-238G, HspHom 493M, n = 1, HLA-B*5701, TNF-238G, HspHom 493M, HLA-DRB1*0701, –DQ*0303, n = 4] and individuals carrying the entire or partial 57.1 ancestral haplotype unexposed to ABC (n = 8) were obtained from consenting individuals from the West Australian HIV Cohort cultures [9].

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Cell culture

PBMCs from individuals were cultured in RPMI with 10% fetal calf serum (FCS). Cells were stimulated with ABC or mock [phosphate-buffered saline (PBS)] treatments. Monocyte-derived dendritic cells (MDDCs) were positively selected from PBMCs with Miltenyi GAM beads and MACs columns after labelling with CD14 monoclonal antibodies. Cells were cultured at 5 × 105 cells/ml with RPMI + 10% FCS, granulocyte-macrophage colony stimulating factor (GM-CSF) (40 ng/ml) and interleukin-4 (20 ng/ml) for 5 days. More than 90% of the MDDCs exhibited a CD14, CD1a+ immunophenotype.

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Biotinylated abacavir

Biotin was covalently conjugated to the free amino group of abacavir (Moravek Fine Chemicals, Moravek Biochemicals Inc., Brea, California, USA) by GenScript Corp, Piscataway, New Jersey, USA. The biotinylated derivative (> 95% high-performance liquid chromatography pure, with structural confirmation by nuclear magnetic resonance) was used at 4 μg/ml re-suspended in PBS (pH 7.4) in all stimulation assays. PBMCs of one HSR patient and tolerant control were cultured with 0, 1, 4 and 10 μg/ml ABC-biotin and interferon (IFN)-γ levels were monitored. Mock (PBS) treatment controls was set up using an equivalent volume of PBS (pH 7.4). ABC at 4 μg/ml concentration gave the maximum IFN-γ levels at 48 h.

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Immunofluorescence and confocal microscopy

Intracellular localization was determined by co-labelling PBMCs with primary antibodies to HLA-B57 [B17 alloantiserum (Nick Acquarola, Department of Clinical Immunology and Biochemical Genetics, Western Australia, Australia.) or B17 monoclonal antibodies (One Lambda Inc., Canoga Park California, USA)] and HSP70Hom [13] (kind gift from Anne Fourie, Johnson and Johnson, San Diego, California, USA) or HSP70 [Abcam1428 (Abcam Ltd, Cambridge, UK)] [14]. Detection was performed using secondary antibodies labelled with appropriate fluorophores [Alexa fluor dyes AF-350, 546, 488, or streptavidin-AF546 conjugates and Hoechst dye to label nuclei (Molecular Probes, Eugene, Oregon, USA)] and subsequent visualization by confocal microscopy. PBMCs (0.5 × 106 cells/ml) from all individuals with sufficient cells available were cultured with ABC (4 μg/ml) in RPMI and 10% FCS. The cells were centrifuged at 1500 rpm for 5 min and re-suspended in PBS (pH 7.4), 1% bovine serum albumin (BSA), 0.1% Triton X 100 and anti-HSP70 primary antibody (5 μg/ml) and B17 alloantisera or B17 monoclonal antibodies (reacts with HLA-B57/B58 molecules) at 4°C for 4 h. The cells were washed for 2–3 h in PBS with 1% BSA at 4°C, and incubated overnight at 4°C with an appropriate secondary antibody solution and 0.4 μmol/l Hoechst dye. Cells were washed with 500 μl of PBS with 1% BSA for 2–4 h and re-suspended in 10 μl of 90% glycerol, 1% formaldehyde solution. Eight microlitres of the cell suspension was mounted on glass slides and sealed. Immuno-phenotyping of cells utilized mouse anti-CD4-PE, -CD8-PE, -CD14-FITC, CD56-PE, CD19-PE (Beckman Coulter, Brea, California, USA). Identification of early and late endocytic compartments utilized primary antibodies directed to Rab4 protein [15] (StressGen Biotechnologies, Victoria, Canada; KAP-GP005) and Rap1 protein [16] (StressGen Biotechnologies; KAP-GP120), while tapasin antibodies (StressGen Biotechnologies; CSA-630) were used to identify endoplasmic reticulum [17]. A fluorescent microscope equipped with a Bio-Rad MRC 1000/1024 UV confocal laser scanning system (BioRad Laboratories Inc., Hercules, California, USA), controlled by Lasersharp image acquisition (Lasersharp Image acquisition system; BioRad Laboratories), was used to capture digital images of stained cells. All images were acquired using similar experimental conditions and identical instrument settings that avoided saturation of the brightest pixels. Each image was collected using 2.5 mm Iris, 1290 Gain, 5 black level microscope settings. Specimens were imaged using a Nikon 40× NA 1.3 oil immersion objective and an optical zoom of 1.0–2.5. Images were generally collected from four scans at slow scan speed using Kalman averaging. Confocal Assistant software (Confocal Assistant Version 4.02; Todd Clarke Brelje, Centre for Microscopy, Characterisation and Analysis, Western Australia, Australia) was used to compile the images and where possible a series of optical sections was collected at increments of 0.5 μm along a z-axis. Red and green, red and blue and green and blue fluorophores colocalized as yellow, magenta and cyan, respectively, while white was obtained with equimolar red, blue and green fluorophores. HSP70 redistribution after ABC stimulation was quantitatively estimated using the NIH Image J v 1.32j program {Wayne Rasband, National Institutes of Health, Bethesda, Maryland, USA; http://rsb.info.gov/ij/}. The area of each cell was selected and the mean (sum of the grey values of all pixels divided by the number of pixels) was measured. The intensity was estimated as the difference between the maximum and minimum grey values. The program output was then analysed and expressed as arbitrary fluorescence units = mean pixels/unit area * intensity.

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Cytokine/protein assays

PBMCs (0.5 × 106 cells/ml) were cultured as described previously and exposed to 4 μg/ml abacavir [9]. IFN-γ levels were measured from culture supernatants by an enzyme-linked immunosorbent (ELISA) assay kit (Pharmacia, Peapack, New Jersey, USA) using the manufactures instructions. Phorbol 12-myristate 13-acetate (PMA) stimulation of one of the patients was used as a positive control. Extracellular HSP70 levels were detected in culture supernatants by ELISA (StressGen Biotechnologies).

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Blocking of CD14, TLR4, TLR2 HSP70, HLA–B17, TcRαβ

Blocking experiments with antibodies to human CD14 (clone My4-RD1; Coulter), TLR4 (Abcam AB9610, HTA125 monoclonal antibodies), TLR2 (clone TL2.1, Abcam AB9100) [18] and HSP70 (Abcam AB1482) were performed using antibodies at a concentration of 5–10 μg/ml. Briefly, PBMCs (0.5 × 106 cell/ml) were exposed to the antibodies for 30 min at 37°C and then cultured in the presence of ABC (4 μg/ml) for 48 h and their effects on HSP70 redistribution, expression of CD40, CD40L, CD83, and IFN-γ were studied. Redistribution into subcellular compartments was also studied following culturing of PBMCs from a HSR patient with PBS (mock treatment control), ABC, and ABC together with puromycin (Sigma, St Louis, Missouri, USA), Brefeldin A (Sigma), and wortmannin (Calbiochem, San Diego, California, USA) at a final concentration of 1 μg/ml, 10 μg/ml and 100 nmol/l [19]. Blocking with TcRαβ (Becton Dickinson, Franklin Lakes, New Jersey, USA) and B17 (One Lambda) was also performed as described above and intracellular HSP expression was estimated by flow cytometry using the Beckton Dickinson Flow cytometer. Cells were also cultured with IgG isotype antibody (10 μg/ml) similarly. HSP70 immunofluorescence was measured following stimulation of PBMCs from an ABC-hypersensitive patient with PBS (pH 7.4), mitogens including lipopolysaccharide (LPS), phytohaemagglutinin (PHA), PMA and abacavir to exclude the possibility of endotoxin or mitogen induced redistribution of HSP70.

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Statistical analysis

Comparison of the means was performed using the Mann–Whitney statistical test or paired Student t-tests.

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Results

Abacavir-induced redistribution of HSP70

The effect of ABC stimulation on expression of HSP70 in cultured PBMCs of individuals with ABC HSR was examined by confocal microscopy. A significant increase in fluorescence for HSP70 was seen at 3 h after exposure to ABC in all ABC HSR patients examined (n = 9) (with a mean HSP70 fluorescence intensity of 21.19 arbitrary units compared with 1.50 units in tolerant controls (n = 6, P = 0.023), as presented in Fig. 1a. This redistribution was also evident at 48 h after ABC stimulation (data not shown). Increased HSP70 was also evident in ABC-stimulated PBMCs from ABC-naive individuals carrying HLA-B*5701 and Hsp70 Hom 493T (Fig. 1a) suggesting redistribution is an immediate early innate response and may not require prior ABC sensitization. Representative patterns of HSP70 distribution from an ABC HSR and a tolerant individual is presented in Fig. 1b where HSP70 is shown within discrete vesicles or aggregates of 0.5–1 μm in diameter distributed within the cell periphery and perinuclear region. Increased expression of MHC class I molecules was also observed (Fig. 1b). Levels of HSP70 immunofluorescence were also measured in PBMCs from a hypersensitive patient cultured in the presence of ABC, demonstrating a mean immunofluorescence of 34.7 arbitrary units (SD = 18.8), compared with negative controls [PBS (pH 7.4) (mean 2.9, SD = 4.6), LPS (mean 3.9, SD = 5.4), PHA (mean 2.8, SD = 2.5), and PMA (mean 2.95, SD = 4.6)] (Fig. 1c). Hence, the heat shock protein response in this context appears to be stimulated specifically by the presence of ABC.

Fig. 1
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Immunophenotyping of cells with HSP70 redistribution

Blood mononuclear cells from ABC HSR patients were examined for their ability to redistribute HSP70 in response to ABC. Cells were co-stained for HSP70 and antibodies to CD14, CD4, CD8, CD19 and CD56 after ABC stimulation to phenotype the cells showing HSP70 redistribution. Redistribution was seen primarily in CD14+ (Fig. 1d) and CD19+ (data not shown) antigen presenting cells of monocytic and B cells lineage. HSP70 redistribution was also observed in cells expressing TNF following abacavir stimulation and not in mock (PBS)-treated PBMCs (Fig. 1e). Dendritic cells derived from peripheral blood CD14+ cells also showed the redistribution phenotype (Fig. 1f). In contrast HSP70 redistribution was not detected in CD4+ or CD8+ T cells or CD56+ NK cells (data not shown).

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Co-localization of HSP70 in endoplasmic reticulum and endosomes

Subcellular distribution of HSP70 was examined by staining ABC-cultured PBMCs for tapasin, a protein present largely in the endoplasmic reticulum [17]; Rab4 that colocalizes with early endosomes [15]; and Rap1 that is located within phagosomal or late endosomal vesicles [16]. Colocalization of HSP70 and HLA-B57 was detected in the tapasin-positive region in the endoplasmic reticulum probably representing newly synthesized molecules (Fig. 2a). Staining of early endosomes with Rab4 GTPase showed a peripheral distribution of stained vesicles (Fig. 2b). These vesicles contained HSP70 molecules. A few of the vesicles contained HLA-B57 and some of the Rab4 positive vesicles stained for HSP70 and HLA-B57. In contrast, almost equivalent proportions of HSP70 and HLA-B57 were seen in late endosomal vesicles marked by Rap1 (Fig. 2c). Incorporation of a protein synthesis inhibitor, puromycin decreased ABC-stimulated HSP70 immunofluorescence in some cells while accumulation of HSP70 in large aggregates occurred following ABC stimulation with Brefeldin A, an inhibitor of de-novo protein translocation from the endoplasmic reticulum (Fig. 2d). Immunofluorescence within clustered vesicles following addition of wortmannin is consistent with its proposed role in altering intracellular endosomal trafficking (Fig. 2d). Diffuse intracellular staining of biotinylated abacavir was detected in ABC-cultured PBMCs suggestive of drug diffusion. Biotinylated ABC also colocalized with HLA-B57 as well as Rab4 and Rap1 within early and late endosomes/phagosomal cellular compartments as well as within tapasin-positive compartments (Fig. 2e–h).

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Fig. 2
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Blocking of cell surface receptors and extracellular HSP70 abrogate endogenous HSP70 redistribution and CD40 expression

Blocking of extracellular cell receptors and HSP70 was performed to determine whether the abacavir stimulated endogenous HSP70 redistribution functions via an intracellular pathway or is released into external milieu and transduces a signal via extracellular receptors to stimulate activation of immune cells.

In these experiments, HSP70 redistribution in ABC HSR patients (averaging 12.3 arbitrary units) was effectively abrogated by the presence of either CD14 antibodies (1.47 units), HSP70 antibodies (1.74 units) or TLR4 antibodies, (1.26 units) (all P-values < 0.004) (Fig. 3a). Utilizing FACS analysis, the difference in the number of CD14+ monocytes expressing the cell surface CD40 costimulatory molecule was increased after ABC stimulation in HSR versus tolerant patients (P = 0.006). This effect was also blocked with antibodies to HSP70 (P = 0.004) and TLR2 (P = 0.01), but not by TLR4 blockade (P = 0.8) (Fig. 3b). In addition, CD40 expression in CD14+, CD83+ cells of ABC HSR patients was significantly higher than the tolerant group after ABC exposure (P = 0.0008) (Fig. 3c), while the number of cells co-expressing CD83 marker remained constant (data not shown). This response was also abrogated after blocking with antibodies to HSP70 (P = 0.003) and TLR2 (P = 0.01) but not TLR4 (Fig. 3c). Figure 3d depicts smoothed histograms of MFI of CD40 expression of CD83+, CD14+ cells from an ABC-stimulated cultures of an HSR patient and a tolerant control. Extracellular HSP70 levels were measured from culture supernatants by ELISA (StressGen Biotechnologies) to detect the excreted protein. However, similar levels of free HSP70 were detected in both abacavir-stimulated culture supernatants from abacavir-hypersensitive and tolerant patients. It should be noted that HSP70 can be released in membrane-bound exosomes [20].

Fig. 3
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Changes in intracellular HSP70 expression after 48 h of continuous culture with ABC or mock (PBS) stimulation were also detected by flow cytometry. Similar results were obtained with increased levels of HSP70 detected in ABC-stimulated PBMCs demonstrated in HLA-B*5701+ ABC-hypersensitive cases (n = 8) but not in tolerant patients (n = 5) (mean 35.9 versus −24.6 mean fluorescence intensity, P = 0.02) (Fig. 3e). Blocking with B17 and TcRαβ monoclonal antibodies decreased this MFI of HSP70 in some of the hypersensitive patients (Fig. 3f). Similarly depletion of CD8+ T cells and CD14+ cells ablated HSP70 immunofluorescence (Fig. 3g).

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Interferon-γ expression after exposure to abacavir correlates with HSP70 redistribution

The expression of IFN-γ was assessed following the exposure of cultured PBMCs from ABC-hypersensitive, ABC-tolerant and unexposed individuals to ABC and biotinylated ABC, utilizing ELISA⋅ The levels of ABC-stimulated IFN-γ were higher in patients with ABC HSR compared with ABC-tolerant individuals, with a mean of 123.54 versus 0 pg/ml (P = 0.001). Similar results were obtained using biotinylated ABC (89.34 versus 0 pg/ml; P = 0.002; Mann–Whitney test (Fig. 4). Cells expressing intracellular IFN-γ were CD8+ T cells, but not CD4+ T cells or B cells (Almeida et al. manuscript in preparation). These results suggest that both the biotinylated derivative and unlabelled ABC stimulate an immune response. Detectable levels of IFN-γ after 48 h of ABC stimulation were not observed in genetically susceptible ABC-naive individuals carrying various regions of the 57.1 AH (Fig. 4).

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Discussion

This study builds on our previous finding that alleles on the 57.1 ancestral haplotype including HLA-B*5701 and Hsp70 Hom 493T haplotypic variant are associated with susceptibility to ABC hypersensitivity. Here we observe that rapid HSP70 redistribution occurs after ABC exposure in cultured PBMCs of genetically homogenous ABC hypersensitive patients carrying HLA-B*5701, Hsp70 Hom 493T alleles, and not in genetically heterogenous tolerant controls. In this context, the Hsp70 Hom variant that is highly associated with HLA-B*5701 within a common MHC haplotype could be involved in driving an innate response in antigen presenting cells. Hence, this Hsp70 variant may facilitate HLA-B*5701-restricted presentation of ABC-specific immunogens to CD8+ T cells, thereby contributing to the systemic cell-mediated immune response that underlies the clinical drug hypersensitivity syndrome.

Immunophenotyping of PBMCs revealed that CD14+ and CD19+ cells demonstrate increased HSP70 expression in response to ABC stimulation. Furthermore, HSP70 appears to localize to subcellular compartments that are positive for early and late endosomal and phagosomal marker proteins, consistent with previously reported endosomal localization of HLA class I and HSP70 in cross priming [21]. The colocalization of HLA-B57 and HSP70 and ABC suggests a potential role for HSP70 in chaperoning ABC and or its haptenated derivative in antigen processing, thereby facilitating HLA-restricted presentation of ABC antigens. It should also be noted that intracellular TNF-alpha was elevated in monocytes from ABC-stimulated PBMCs from ABC HSR compared with the tolerant patients [9], consistent with TNF induction from activated monocytes via toll-like receptors [22].

These data suggest that either native ABC or its reactive intermediate trigger immune cell activation via a mechanism that involves increased HSP70 mobilization into subcellular compartments. In this context HLA class I molecules have been identified in endosomal compartments [21,23–25] and have been thought to participate in alternate pathways of antigen presentation [21,26–28]. Colocalization of HSP70 and HLA class I molecules within endosomal compartments [21,24,29–31] of dendritic cells [32,33] and with proteins involved in antigen presentation [34–36] has also been previously documented. Heat shock proteins can function both in cross-presentation and as adjuvants for immune response by providing ‘danger signals’ and a source of antigens that serve to stimulate the innate and adaptive immune system [37–46] during infections, apoptosis, heat stress and perhaps drug-induced stress [47]. In this instance, the rapid induction of HSP70 in response to ABC stimulation suggests that the increased HSP70 immunofluorescence may provide a ‘danger signal’ that induces an inflammatory rather than a tolerogenic immune response to ABC exposure.

Redistribution of HSP70 may occur from existing cytoplasmic stores or endogenous HSP70 excreted into the external milieu, where it may subsequently trigger activation and maturation of antigen presenting cells via cell surface receptors [48]. In this study, the involvement of extracellular HSP70 was suggested indirectly as the redistribution was blocked by antibodies to HSP. Interestingly, while blocking with antibodies to HSP70, CD14 and TLR2 reduced HSP70 redistribution and CD40 expression in CD83+, CD14+ monocytes, it was found that blocking TLR4 did not reduce HSP70 redistribution or CD40 expression. These data are consistent with previous reports indicating HSP70-mediated signal transduction specifically via CD14, TLR2 receptors [49,50]. The role of additional receptors operating in alternate pathways of signal transduction is currently being investigated.

Whereas the immunofluorescence of HSP70 detected at 3 h after ABC stimulation may represent an immediate innate response in PBMCs from 57.1 AH, ABC-naive individuals, the response detected at 48 h in the ABC-exposed individuals may reflect a secondary immune response requiring T-cell help consistent with data observed in Fig. 3f and g. Blocking of HLA-B57 receptor with B17 monoclonal antibodies decreased the levels of HSP70 in ABC-stimulated PBMCs from a hypersensitive patients (n = 4). In contrast, a synergistic inhibitory effect was observed following blocking with monoclonal antibodies to HLA-B57 and the T-cell receptor. These data suggest an involvement of HLA-B57 restricted presentation of ABC-specific ligand to a cognate T-cell receptor in the immune response.

In conclusion, these data support a pivotal role for ABC-stimulated early redistribution of HSP70 in subcellular compartments of antigen-presenting cells via TLR2 and CD14 receptors. This is accompanied by an increase in immune cell activation and maturation as measured by CD40 expression, thereby preparing the APCs for HLA-B*5701-restricted presentation of ABC-specific ligands. Together, these data suggest a positive feedback loop initiated by HSP70 redistribution and presumed extracellular release, involving the activation and maturation of APCs via increased expression of co-stimulatory molecules CD40 and TNF through well characterised pathways of Toll-like receptor-mediated signal transduction. The involvement of TLRS may have therapeutic implications. Whatever the precise pathways involved, it is clear from our data that ABC is able to induce early changes in antigen-presenting cells. This observation may provide further explanation for the highly predictable development of ABC HSRs in genetically susceptible individuals who carry the 57.1 ancestral haplotype.

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Acknowledgments

The authors wish to thank the participants and clinical staff of the Western Australian HIV Cohort Study. The efforts of Dr Paul Rigby, Department of Confocal Microscopy, UWA and Lotteries Commission are greatly appreciated.

Sponsorship: A.M., D.N., S.M. are supported by the Australian National Health and Medical Research Council Project grant ID 237408.

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Keywords:

abacavir hypersensitivity; monocytes; cell activation; HLA-B*5701; interferon gamma

© 2007 Lippincott Williams & Wilkins, Inc.

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