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Toxicology Letters 185 (2009) 153–159

Contents lists available at ScienceDirect

Toxicology Letters

journa l homepage: www.e lsev ier .com/ locate / tox le t

nvestigation of the immunogenicity of p-phenylenediamine and Bandrowski’sase in the mouse

ohn Farrell, Claire Jenkinson, Sidonie N. Lavergne, James L. Maggs, B. Kevin Park, Dean J. Naisbitt ∗

RC Centre for Drug Safety Science, Department of Pharmacology and Therapeutics, The Sherrington Building, Ashton Street,he University of Liverpool, Liverpool L69 3GE, UK

r t i c l e i n f o

rticle history:eceived 18 September 2008eceived in revised form 10 December 2008ccepted 12 December 2008vailable online 24 December 2008

eywords:mmunogenicity-Phenylenediamine-lymphocytes

a b s t r a c t

p-Phenylenediamine (PPD) exposure is associated with T-cell mediated contact dermatitis. T-cells fromallergic patients proliferate following exposure to PPD and the oxido-conjugation product Bandrowski’sbase (BB). Both compounds are classified as sensitizers in the local lymph node assay; however, becauseof their instability the nature of the antigenic determinant remains ill-defined. The aim of this studywas to explore the immunogenic potential of PPD and BB in mice. Spleen cell proliferation and cytokinesecretion was measured ex vivo following antigen recall with soluble PPD or BB and either irradiated orglutaraldehyde fixed, antigen pulsed dendritic cells from syngeneic mice. Glutathione was added to cer-tain incubations. LC–MS analysis and solvent extraction were used to monitor the fate of [14C]BB in cultureand the extent of BB binding, respectively. Spleen cells from BB exposed, but not PPD- or vehicle-exposed,

ontact dermatitis mice proliferated when stimulated with BB. Proliferating cells secreted high levels of IFN-�, GM-CSF andIL-2. Stimulation with PPD instigated low levels of proliferation. Irradiated, but not fixed, dendritic cellspulsed with BB stimulated proliferation signifying a classical hapten mechanism involving irreversibleBB binding to protein and processing. BB bound preferentially to serum protein when incubated togetherwith cells and serum. Degradation of BB in the presence of glutathione was associated with a strongerstimulation of specific T-cells at higher BB concentrations. These data demonstrate that BB is a potent

.

immunogen in the mouse

. Introduction

Exposure to p-phenylenediamine (PPD), a central componentf most permanent hair dye formulations, is associated with theevelopment of T-cell-mediated allergic contact dermatitis. The

ncidence of such reactions is on the increase, especially in younghildren (McFadden et al., 2007).

PPD is extremely unstable and susceptible to oxidation in aque-us solution; yielding p-benzoquinonediimine by two electronxidation. 2,5-Dimethyl-p-benzoquinonediimine, a less reactiveerivative, has recently been shown to react with nucleophilic sidehains of amino acids via a complex series of conjugative and oxido-eductive mechanisms (Eilstein et al., 2006, 2007, 2008), suggestinghat protein conjugates of electrophilic PPD oxidation products are

ntigenic determinants for immune cells. Despite this, the onlyompound sufficiently stable to be synthesized is a rearrange-ent product of the trimer, namely Bandrowski’s base (BB; Fig. 1A;

oulter et al., 2007).

Abbreviations: PPD, p-phenylenediamine; BB, Bandrowski’s base.∗ Corresponding author. Tel.: +44 151 794 5346; fax: +44 151 794 5540.

E-mail address: dnes@liv.ac.uk (D.J. Naisbitt).

378-4274/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.toxlet.2008.12.008

© 2008 Elsevier Ireland Ltd. All rights reserved.

In dendritic cell studies assessing the ability of PPD to promoteco-stimulatory signalling, activation has been shown to be depen-dent on PPD oxidation and the generation of unstable intermediates(Aeby et al., 2008). BB provides co-stimulatory signals to dendriticcells directly.

Topical exposure of mice to both PPD and BB leads to immunecell activation, as measured by an increased cellular infiltration ofdraining lymph nodes (Aeby et al., 2008; Warbrick et al., 1999;White et al., 2006). However, since auto-oxidation of PPD is likely tooccur on skin, the sensitizing potential of different PPD derivativesremains unresolved.

In allergic patients, contact dermatitis is normally diagnosedby patch testing using 1% PPD (Ho et al., 2005), which has a highpositive predictive value. Patch testing positivity with PPD is time-dependent (Basketter et al., 2006) indicating that oxidation on skinmight be important for sensitization. Recently, only 16% of PPDpatch test positive patients were shown to be responsive to BB, andin these cases the response was weak (White et al., 2006). These

data suggest that a compound other than BB may be the ultimateantigenic determinant in most human subjects.

Lymphocytes isolated from PPD allergic patients proliferate vig-orously following in vitro exposure to both PPD and BB (Coulter etal., 2008; Sieben et al., 2002). Somewhat surprisingly, lymphocytes,

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ut not cord bloods, from tolerant individuals are also stimulated toroliferate, but only in the presence of BB (Coulter et al., 2008). Theresence of BB-specific T-cells in both allergic patients and tolerant

ndividuals suggests that the acquired immune response to BB isot translated invariably into an allergic reaction.

Human exposure to PPD is an almost unavoidable occurrence.onsequently, it is difficult to define the nature of the antigeniceterminant that stimulates naïve T-cells using human samples.hus, the aim of the present study was to explore the immunogenicotential of PPD and BB, the functionality of antigen-specific T-cellsnd mechanism of antigen presentation to T-cells using a mouseodel. BALB/c strain mice were immunized with either PPD or BB

ia a single sub-cutaneous injection, and antigen-specific T-cell pro-iferation was analyzed after 7 days. This immunization protocolvoids potential PPD air (auto) oxidation processes on the skin oruring cutaneous absorption, which is thought to be important forbtaining concentrations of PPD derivatives needed to induce skinensitization (Aeby et al., 2008).

. Materials and methods

.1. Chemicals, radiochemicals and reagents

Dimethyl sulfoxide (DMSO), l-glutamine, glutaraldehyde, HEPES, penicillin,treptomycin, RPMI 1640 medium, [3H]thymidine, foetal bovine serum, and PPDere obtained from Sigma–Aldrich (Poole, Dorset, UK). BB was obtained from ICNiomedicals Inc. (Aurora, OH). Lymphoprep was obtained from Nycomed (Birming-

am, UK). [14C]BB was purchased from Selcia Ltd. (Essex, UK).

.2. Sensitization protocol for determination of immune activation in the mouse

PPD and BB (5–25 mg/kg, 25% (v/v) DMSO in PBS; 50 �l) were administered,n the presence or absence of complete Freunds adjuvant, via a single subcutaneous

ig. 1. Immunogenicity of PPD and BB in the mouse. (A) Structures of PPD and BB. (B) Prultured with PPD or BB. Proliferation was determined by incorporation of [3H]-thymidineour mice, incubations carried out in triplicate.

ers 185 (2009) 153–159

injection to young adult (8–12 weeks old) female BALB/c stain mice (Charles River UKLtd., Kent, UK) for analysis of immunogenicity. The adjuvant served to deliver the co-stimulatory signals that may not be provided by the chemical per se. All experimentswere carried out under the provisions of the United Kingdom Animals (ScientificProcedures) Act 1986. Concurrent controls received the appropriate vehicle with orwithout adjuvant. Seven days after initial chemical exposure, mice were sacrificedand the spleen removed.

2.3. Generation of bone marrow derived dendritic cells

Dendritic cells were generated from femur and tibia bone marrow cells. Mar-row was flushed from bones with culture medium and cells (3 × 106) cultured inPetri dishes containing medium enriched with murine GM-CSF (20 ng/ml). Culturemedium was refreshed on days 3 and 6. On day 8 cells were harvested and used as asource of antigen presenting cell in the proliferation assay. Dendritic cells routinelyexpressed high levels of CD11c and LPS inducible MHC class II, CD40 and CD86 (datanot shown).

2.4. Determination of the in vitro proliferative response of splenocytes top-phenylenediamine and Bandrowski’s base

A suspension of single spleen cells was prepared under sterile conditionsby gentle disaggregation through sterile mesh. The lymphocyte fraction was iso-lated by centrifugation through Lymphoprep. Cells with viability greater than 95%were suspended in RPMI-1640 supplemented with HEPES (25 mM), streptomycin(400 �g/ml), penicillin (400 �g/ml) and 10% heat inactivated foetal bovine serumand incubated (1.5 × 105) in 96-well U-bottom cell culture plates with either PPD(0.01–10 �g/ml) or BB (0.01–10 �g/ml) at 37 ◦C under 5% CO2. In certain experi-ments, the compounds were incubated with cells in media containing glutathione(1 mM), which has recently been shown to prevent the conversion of PPD to BB

(Coulter et al., 2007). After 3 days, proliferation was measured by the additionof [3H]thymidine (0.5 �Ci) for the final 16 h of culture. Cells were harvested, andincorporated radioactivity was measured as counts per minute on a beta counter(PerkinElmer Life Sciences, Cambridge, UK). Proliferative responses were calculatedas stimulation indices (SI; cpm in treated cultures/cpm in cultures containing vehiclealone).

oliferative response of spleen cells from vehicle control, PPD and BB-treated miceover an additional 16 h. Results are presented as mean proliferative response from

J. Farrell et al. / Toxicology Letters 185 (2009) 153–159 155

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ig. 2. Cytokine secretion from antigen-stimulated spleen cells isolated from BB-exalue from 3 BB-sensitized mice.

.5. Measurement of lymphocyte cytokine/chemokine secretion

Supernatants (100 �l) were collected from antigen-stimulated and unstim-lated spleen cells, prior to the addition of [3H]thymidine, for the analysisf cytokine/chemokine secretion using a LINCOplex multiplex assay kit (LINCOesearch Inc., Hampshire, UK). Protein (IL-2, IL-4, IL-5, IL-6, IL-10, IFN�, GM-CSF)ontent was measured using a Liquichip 100 workstation (Qiagen Ltd., West Sussex,K) with LiquiChip IS 2.3 software.

.6. Determination of the mechanism of antigen presentation to T-cells

To determine whether the BB-specific proliferative response was due to irre-ersible binding of BB to protein, spleen cells were pulsed with BB for 2 h. Pulsedells were washed repeatedly to remove unbound BB and cultured for the remainder

f the incubation period in drug-free medium. In addition, dendritic cells from naïveyngeneic mice were pulsed with BB (0.5–2 �g/ml) and used as a source of antigenresenting cell. Pulsed cells were washed, irradiated (4500 rads; to prevent any pos-ible non-specific proliferation) and added (0.5 × 105/well) to the proliferation assayontaining splenocytes (1.5 × 105/well) from sensitized mice. To explore the impor-ance of processing in BB presentation, glutaraldehyde-fixed dendritic cells were

mice. Each data point is the mean of duplicate cultures. Bar represents the mean

used as antigen presenting cells. Fixation blocks processing, but does not alter theexpression of surface MHC molecules or presentation of pre-processed antigens (Wuet al., 2006; Zanni et al., 1998). To measure proliferation, [3H]thymidine (0.5 �Ci) wasadded to the cultures for 16 h.

2.7. Irreversible binding of Bandrowski’s base to protein

To determine whether BB binds irreversibly to protein, [14C]BB (5 �g/ml, 1 �Ci)was incubated with spleen cells in complete medium for 2–96 h, and total irre-versible binding was measured by exhaustive solvent extraction using a methodadapted from (Kitteringham et al., 1988). Cells and serum were separated by cen-trifugation. Protein was then extracted using acetonitrile (2 ml × one wash and1 ml × three washes) and dissolved in sodium hydroxide (1 M; 2 ml) for quantifi-cation of bound radioactivity by liquid scintillation counting. Protein concentration

was measured according to the method of Bradford (1976).

Irreversible BB binding to human serum albumin was also assessed by incu-bating [14C]BB (400 (M [1 �Ci]) with protein (1 mg) for 16 h. On completion of theincubation period, unbound BB was removed by exhaustive solvent extraction andprotein was precipitated by the addition of acetonitrile (100 �l). An aliquot con-taining 20 �g of total protein was taken and added to an equal volume of reducing

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ample buffer. Samples were then boiled at 95 ◦C for 10 min and protein separatedy electrophoresis on SDS-polyacrylamide gels using the discontinuous buffer sys-em described by Laemmli. The gel was cut into 0.5 cm sections and digested witholuene at 50 ◦C for 1 h before the addition of 30 �l glacial acetic acid. Incorporated14C]BB in each band was then determined by scintillation counting.

.8. Determination of the chemical fate of Bandrowski’s base in the presence andbsence of glutathione

The chemical fate of BB in culture has not been explored. Thus, [14C]BB (1 mM;�Ci) was incubated with cell culture medium in the presence and absence of glu-

athione (250 mM) at 37 ◦C. At various time-points, 100-�l aliquots were taken andnalyzed by parallel radiometric HPLC/LC–MS. The eluent was delivered by JascoU980 pumps (Great Dunmow, Essex, UK). Analytes in the eluate were monitoredith a Jasco UV-975 spectrophotometer (� = 254 nm). Radiolabeled analytes were

uantified using a Radiomatic Flo-One �A-250 flow detector (PerkinElmer, Pang-ourne, Berkshire, UK). The eluate was mixed with Ultima-Flo AP scintillant at aate of 1 ml/min. The split flow of eluate to the mass spectrometer was ∼50 �l/min.ass spectra were acquired between m/z 50 and 1050 Micromass with a Quattro

I instrument (Waters Corp., Manchester, UK) at 1 scan/5 s. The source temperatureas 80 ◦C, the capillary voltage was 3.9 × 103, and the standard cone voltage was0 V. Data were processed via MassLynx 3.5 software (Micromass Ltd., Manchester,K). Aliquots of the solution (10 �l; 100 × 103 dpm) were eluted without treatment

rom a Prodigy 5 �m ODS(2) column (150-mm × 4.6-mm i.d.; Phenomenex, Mac-lesfield, Cheshire, UK) at room temperature with a gradient of acetonitrile (5% formin; 5–85% over 20 min) in 10 mM ammonium acetate, pH 3.5. The flow rate was.9 ml/min.

.9. Statistical analysis

The Mann–Whitney test was used for comparison of control and test values.

. Results

.1. Bandrowski’s base, but not p-phenylenediamine, stimulates arimary T-cell response in the mouse

The principal aim of these studies was to explore the immuno-enicity of PPD and BB in the mouse by assessment of ex vivo

ig. 3. Processing-dependent presentation of BB-modified protein to T-cells from sensitizerotein following exhaustive solvent extraction. (B) Analysis of [14C] BB binding to humanf bands and solvent extraction. (C) Proliferative response of spleen cells from BB-treatedf spleen cells from BB-treated mice treated with BB-pulsed (c, 0.1 �g/ml BB; d, 1 �g/mann–Whitney test for non-parametric data compares proliferation of spleen cells from

ers 185 (2009) 153–159

proliferation following antigen recall. In this respect, mice wereimmunized with PPD, BB or vehicle alone via a single sub-cutaneous injection. Seven days later, mice were killed, spleencells isolated and incubated with titrated concentrations of PPDand BB. Splenocytes from BB-treated mice (1–25 mg/kg) prolifer-ated in a concentration-dependent fashion when cultured with BB(0.01–1 �g/ml; Fig. 1B). In vitro stimulation with PPD produced avery weak response, which is likely due to the in situ generation ofBB, which has been shown previously (Coulter et al., 2007). Freund’sadjuvant did not alter the antigen-specificity of responding spleno-cytes and thus in subsequent experiments to explore the mecha-nism of antigen presentation, mice were immunized with BB alone.

There was no discernible PPD- or BB-mediated stimulation ofspleen cells from mice administered either PPD or vehicle alone(Fig. 1B).

3.2. Bandrowski’s base-specific spleen cells secrete IFN-� , IL-2and GM-CSF following antigen stimulation

Spleen cells from BB-exposed mice secreted high levels of IFN-�,IL-2 and GM-CSF and moderate levels of IL-6 and IL-10 following BBstimulation in vitro (Fig. 2). Levels of IL-4 and IL-5 did not exceed30 pg/ml. PPD stimulation of BB-specific spleen cells was associatedwith secretion of a similar panel of cytokines, albeit at lower levelsthan that seen with BB.

3.3. Bandrowski’s base stimulates T-cells via a classical haptenmechanism involving protein binding and antigen processing

To quantify the possible irreversible binding of BB to protein,[14C]BB was incubated with cells in complete medium for 96 h.[14C]BB was found to bind in a time-dependent fashion to cellu-lar and serum protein (Fig. 3A); resulting in approximately 25%

d mice. (A) Comparison of the irreversible binding of BB to cellular and extracellularserum albumin by scintillation counting following gel electrophoresis, extraction

mice stimulated with either soluble or pulsed BB. (D and E) Proliferative responsel BB) and irradiated or glutaraldehyde-fixed dendritic cells. Statistical analysis by

treated and untreated mice.

J. Farrell et al. / Toxicology Letters 185 (2009) 153–159 157

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ig. 4. Co-culture of spleen cells, from BB treated mice, with glutathione prevents imhowing the 30 min incubations of [14C]BB (1 mM; Rt 14.5 min) in culture medium,pleen cells from BB-treated mice cultured with BB in the presence or absence of gluroups. For full dose-response, see Fig. 1B.

rreversibly bound material at the conclusion of the incubationeriod. Binding of [14C]BB to serum protein was approximatelyvefold higher than to cellular protein.

Gel electrophoresis followed by band excision and scintillationounting for incorporated radioactivity revealed [14C]BB associatedxclusively with human serum albumin, which eluted in lane 7Fig. 3B). Levels of radioactivity associated with other bands didot exceed background counts.

The role of irreversible binding and processing in BB-specific T-ell proliferation was determined by: firstly, pulsing splenocytesrom BB-treated mice with BB for 2 h; and secondly, co-incubatingB-pulsed irradiated or glutaraldehyde-fixed naïve dendritic cellsith BB-specific spleen cells in the absence of soluble antigen. Puls-

ng spleen cells from BB-treated mice with BB for 2 h was associatedith levels of proliferation similar to those seen with soluble BB

Fig. 3C). Similarly, significant spleen cell proliferation was observedsing BB (0.1 and 1 �g/ml)-pulsed dendritic cells as a source of anti-en (Fig. 3D and E). Fixation of dendritic cells with glutaraldehyderior to the addition of BB inhibited T-cell proliferation.

.4. Glutathione inhibits the immunosuppressive effect associatedith high Bandrowski’s base concentrations

The chemical interaction of glutathione with BB and its effectn BB-specific T-cell proliferation was studied. Parallel radiometricPLC/LC–MS analyses revealed that [14C]BB (Rt 14.5 min; [M+1] at/z 319) was degraded completely when it was incubated with a

50-fold molar excess of glutathione (250 mM) for 30 min (Fig. 4A).he only radiolabeled product produced was highly polar, and couldot be associated confidently with a putative parent ion. A 10-

old molar excess of glutathione effected complete degradation ofB, and apparently without formation of intermediate products,etween the first and third hour of incubation.

Addition of glutathione (1 mM; approximately 60-fold molarxcess) to cell culture medium did not inhibit the proliferative

osuppression associated with high BB concentrations. (A) Radiometric HPLC tracespresence (2) and absence (1) of glutathione (250 mM). (B) Proliferative response ofne. Dose-response curve reduced to emphasize the distinction between treatment

response of BB-specific spleen cells, but actually increased the rangeof stimulatory BB concentrations (Fig. 4B).

4. Discussion

PPD and BB are categorized as strong and potent sensitizers inanimal tests involving topical exposure, followed by assessment ofdraining lymph node cell proliferation (Warbrick et al., 1999; Whiteet al., 2006). Moreover, PPD and BB-specific T-cells have been iso-lated from peripheral blood of allergic human patients (Coulteret al., 2008; Krasteva et al., 1993; Sieben et al., 2002). Thus, theinduction of an antigen-specific T-cell response is thought to beessential for the development of contact dermatitis. Despite this,controversy exists as to the nature of the antigenic determinantthat interacts with T-cells in both humans and experimental ani-mals. To address this issue and to explore mechanisms of antigenpresentation PPD and BB were administered to BALB/c strain mice.Subcutaneous injection was deemed appropriate because PPD is acommon component of permanent hair dye formations and cloth-ing dyes (i.e., skin is the primary route of human exposure). Micewere not exposed topically to prevent oxidation of PPD prior toabsorption.

Exposure of mice to BB resulted in marked T-cell stimulationas assessed by measurement of spleen cell proliferation followingin vitro antigen stimulation (Fig. 1B). Spleen cells from BB-treatedmice were also stimulated to proliferate weakly following in vitroPPD exposure, which relates to the well characterized instabilityof PPD and generation of BB in situ (Aeby et al., 2008; Coulter etal., 2007; Picardo et al., 1990). Importantly, spleen cells from naïvemice were not specifically stimulated, indicating that BB exposure

is not associated with mitogenic T-cell stimulation.

Low molecular weight chemicals interact with and stimulateT-cells via three independent pathways, all involving an associa-tion with MHC molecules expressed on antigen presenting cells(Naisbitt et al., 2007; Pichler, 2003). The first pathway, often

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eferred to as “hapten recognition”, involves irreversible (oftenovalent) binding of the chemical to protein, prior to processingnd the liberation of peptides for presentation to T-cells by MHColecules. Hapten-specific T-cells have been characterized in the

irculation of human patients with a diagnosis of allergic reactionso drugs and/or chemicals such as dinitrochlorobenzene (Pickard etl., 2007), phenytoin (Cooper et al., 2005) and nickel (Moulon et al.,995). The second pathway involves the direct irreversible bindingf chemicals to MHC or a peptide embedded within the peptideinding groove of MHC (Burkhart et al., 2002; Schnyder et al.,000). Finally, non-reactive chemicals have been shown to associateeversibly to MHC molecules and specific T-cell receptors with suf-cient binding energy to stimulate a T-cell response. Using the drugllergen sulfamethoxazole as a model test compound, three inde-endent research groups have shown that individual T-cell clonestimulated directly with reversibly and irreversibly bound antigeno-exist in allergic patients (Farrell et al., 2003; Nassif et al., 2004;chnyder et al., 2000). In terms of BB, certain T-cell clones fromllergic human patients are stimulated with BB via a processing-ependent “hapten-like” mechanism (Sieben et al., 2002). Our data,sing dendritic cells for antigen presentation, show that T-cellsrom BB-exposed mice are stimulated via a pathway involving bothrreversible binding of BB to protein and antigen processing. Time-ependent irreversible binding of BB to cellular and serum proteinas detected under conditions identical to those used in the prolif-

ration assay. Antigen presenting cells pulsed with BB and thereforexpressing only irreversibly bound BB and/or peptides derived fromB-modified protein stimulated spleen cell proliferation. Finally,endritic cell fixation with glutaraldehyde, which prevents pro-essing of protein adducts, but not presentation of pre-processedntigens (Zanni et al., 1998), inhibited the BB-specific proliferativeesponse. In on-going studies we are investigating the nature of theinding interaction between BB and protein and the amino acidesidues preferentially modified.

Glutathione, which has recently been used in cell culture assayso limit irreversible protein binding of nucleophilic drug metabo-ites (Burkhart et al., 2001; Naisbitt et al., 1999, 2003; Schnydert al., 2000), was used to supplement incubations containing BB.he response of spleen cells to higher concentrations of BB wasnhanced in the presence of glutathione (molar excess, 318–64-old). Use of parallel radiochromatographic mass spectrometricnalysis to explore the fate of [14C]BB in cell culture mediumevealed that glutathione readily and radically degrades BB underild conditions. Although the product could not be characterized

rom the available mass spectrometric data, the degradation of BBrovided a rational explanation for our observed biological results.either dihydrogenated BB nor PPD was found in the reaction mix-

ure. Notably, and notwithstanding BB has a diimine structure, theroduct was not a simple adduct of glutathione and either BB, theimeric analog of BB or PPD.

Cytokines are small signalling proteins that control the strengthnd nature of the induced immune response through their inter-ction with cognate receptors expressed on immune cells. T-cellsrom allergic human patients secrete a panel of cytokines includinghe Th2 cytokines IL-4, -5 and -13, following PPD or BB stim-lation (Coulter et al., 2008; Sieben et al., 2002). Comparisonf cytokine secretion profiles from allergic patients and tolerantndividuals recently revealed that stimulated lymphocytes from tol-rant individuals secrete significantly lower levels of Th2 cytokinesunpublished data). BB stimulation of sensitized mouse spleen cellsas associated with secretion of high levels of IFN-�, IL-2 and GM-

SF, moderate levels of IL-6 and IL-10, but little or no IL-4 and IL-5,hich is consistent with data obtained from tolerant individuals.

In contrast to BB, a single sub-cutaneous PPD injection didot induce an immune response in the mouse. Spleen cells wereot stimulated to proliferate following in vitro stimulation with

ers 185 (2009) 153–159

either PPD or BB. These data clearly imply that immuno-stimulatoryconcentrations of BB were not generated following sub-cutaneousexposure to PPD. Furthermore, irreversible binding of primary PPDoxidation products to protein did not stimulate a T-cell responsein this mouse model. These data clearly indicate that mice andallergic human patient lymphocytes react differently following PPDexposure.

Several previous studies have used human dendritic cells as asource of antigen presenting cell to measure PPD and BB-specificprimary T-cell sensitization in vitro (Krasteva et al., 1996; Rougier etal., 1998, 2000). Stimulation with BB induced reproducible immuneresponses in most human volunteers, but PPD treatment was eithernot associated with a significant response or induced a weakresponse in a limited cohort. These data are in agreement withour experiments in the mouse and emphasize that BB is a potentT-cell immunogen. PPD-specific, Th2 secreting T-cells are foundonly in human patients with clinically diagnosed allergic dermati-tis (Coulter et al., 2008; Sieben et al., 2002). The reasons for thisare unclear, but may relate to (1) specific MHC molecules beinginvolved in the presentation of PPD to T-cells a similar phenomenais associated with susceptibility to certain forms of drug allergy(Chessman et al., 2008; Chung et al., 2004; Mallal et al., 2002); (2)stimulation of specific T-cell receptors; and/or (3) differential cel-lular distribution of PPD in allergic patients. Each of these avenuesof research warrant further investigation.

Conflicts of interest statement

The authors declare no conflicts of interest

Acknowledgement

This work was funded by The British Skin Foundation as partof the Centre for Drug Safety Science supported by the MedicalResearch Council [grant number G0700654].

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