Murine bone marrow-derived dendritic cells as a potential in vitro model for predictive identification of chemical sensitizers

Available online at www.sciencedirect.com

Toxicology Letters 175 (2007) 89–101

Murine bone marrow-derived dendritic cells as a potential in vitro model for predictive identification of chemical sensitizers Elsa P´epin ∗ , Mich`ele Goutet, Masarin Ban Department of Pollutants and Health, National Institute for Research and Safety, 54501 Vandoeuvre-les-Nancy, France Received 5 June 2007; received in revised form 26 September 2007; accepted 26 September 2007 Available online 2 October 2007

Abstract The identification of potential sensitizing chemicals is a key step in the safety assessment process. To this end, predictive tests that require no or few animals and that are reliable, inexpensive and easy to perform are needed. The aim of this study was to evaluate the performance of murine bone marrow-derived dendritic cells (BMDCs) in an in vitro skin sensitization model. BMDCs were exposed to six well-known allergens (dinitrochlorobenzene, DNCB; dinitrofluorobenzene, DNFB; Bandrowski’s base, BB; paraphenylenediamine, PPD; nickel sulfate, NiSO4 ; cinnamaldehyde, Cinn). Surface expression of MHC class II, CD40, CD54, and CD86 was measured by flow cytometry after 48 h exposure to these chemicals. All the allergens tested induced a significant increase in marker expression, with an augmentation in the percentage of mature cells ranging from 2.3- to 10.5-fold change over control. The level of up-regulation was dependent on the concentration and the strength of the allergens. In contrast, the irritants (sodium dodecyl sulfate, SDS and 4-aminobenzoic acid, pABA) and the negative control (zinc sulfate, ZnSO4 ) tested induced either no modification or a down-regulation of membrane marker expression. Taken together, our data suggest that murine BMDCs may represent a new and valuable in vitro model to predict the sensitizing properties of chemicals. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Contact allergy; Dendritic cells; In vitro sensitization test

1. Introduction Allergic contact dermatitis (ACD) is a common occupational and environmental health problem, and many chemicals are known to cause skin sensitization. The mechanisms required for the acquisition of skin sensitization, and for the subsequent elicitation of allergic reactions in the skin, are complex and dependent upon highly orchestrated molecular and cellular interactions. ∗ Corresponding author at: D´ epartement Polluants et Sant´e, Institut National de Recherche et S´ecurit´e (INRS), Avenue de Bourgogne BP27, 54501 Vandoeuvre-les-Nancy Cedex, France. Fax: +33 383 50 20 96. E-mail address: [email protected] (E. P´epin).

In common with other types of allergic diseases, contact allergy develops in two temporally discrete phases. During the ‘induction’ phase, topical exposure of an inherently susceptible individual to a skin sensitizer results in a primary cutaneous immune response. Briefly, the first step is the uptake of allergen by professional antigen presenting cells such as dendritic cells (DCs). DCs reside throughout the body in a relatively immature state that allows the efficient capture of antigens. The antigen is internalized and processed into peptides, which bind major histocompatibility complex molecules (MHC). Following the encounter with the antigen, the DCs become activated and migrate to the draining lymph nodes. During transit, the DCs undergo maturation, which is characterized, among other changes, by

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an increase in the cell surface expression of class II MHC (MHC II) and co-stimulatory molecules, and a modulation in other cell surface marker expression and cytokine production (Schuurhuis et al., 2006). Mature DCs are able to present the processed antigen to na¨ıve T lymphocytes, which become activated and expand. The ‘elicitation’ phase is triggered by the subsequent challenge with the same allergen. The DCs reactivate memory specific T cells, inducing a succession of events including the recruitment of other leukocytes and the secretion of cytokines and chemokines. This process leads to a dermal inflammation clinically recognized as ACD. Prior to being introduced onto the market, new products likely to involve skin contact must be tested to ensure they will not induce ACD. To help achieve this objective, thorough skin sensitization risk assessment is needed. Traditionally, sensitization hazard tests have been conducted in guinea pigs (Buehler, 1965; Magnusson and Kligman, 1969). More recently, the murine local lymph node assay (LLNA) has been developed and validated as a test method for assessing skin sensitization potential (ICCVAM, 1999; Gerberick et al., 2000). However, as animal tests are very expensive, time-consuming and have ethical implications, a great deal of effort has been put into establishing alternative systems to predict the sensitizing potential of new substances. Computational prediction tools may be useful to some extent in the initial screening of the potential sensitization hazard of chemicals. However, these tools are based mainly on theoretical predictions of the reaction of chemicals with proteins and therefore do not estimate potency, making them unsuitable as risk assessment tools in safety support. As a result of the recent advances in the mechanistic understanding of ACD, the development of in vitro assays has been underway for the past few years. Most of these alternative methods use cells involved in the sensitization phase of contact allergy, and include the in vitro culture of DC-like cells, which is one of the most promising. The aim is to reconstitute the chemical-induced maturation process of DCs in vitro, and to monitor early activation markers such as modification of cell surface molecule expression (i.e. MHC II, CD54 and CD86), production of cytokines (i.e. IL-1␤), and activation of autologous T cells (for review, see Kimber et al., 2004; Ryan et al., 2005). One approach consists in the direct use of Langerhans cells purified from human skin explants and exposed to chemicals in vitro. Several modifications have been observed following cell treatment with contact allergens, including a decrease in MHC II molecule and

E-Cadherin expression on cell surface (Verrier et al., 1999), an up-regulation of CD54, CD86 and MHC II expression (Tuschl and Kovac, 2001), and differences in the MHC II endocytosis patterns (Rizova et al., 1999). However, this model suffers from a shortage of available human skin, difficulty in isolating DCs, and weak cell viability. For these reasons, it has been proposed to derive DC-like cells from peripheral blood mononuclear cells (PBMC). In this second approach, which has been the most frequently used, the effects of sensitizers on surface marker modulation, mRNA expression and cytokine production have been investigated. An up-regulation of MHC II, CD86, CD54 or CD40 has been frequently observed (Aiba et al., 1997; Coutant et al., 1999; Manome et al., 1999; Tuschl et al., 2000; Hulette et al., 2002; Staquet et al., 2004; Jugde et al., 2005), as well as an increase in IL-1␤ mRNA expression (Pichowski et al., 2000; Aeby et al., 2004). One major limitation of this model is the common finding of considerable donor-to-donor variability in responsiveness. More recently, a third approach has been proposed using DCs derived from CD34+ cord blood hematopoietic progenitors. Several authors have reported an increased surface expression of MHC II, CD86, CD83 or CCR7 by cells exposed to chemicals (De Smedt et al., 2001, 2005; Boisleve et al., 2004), as well as a down-regulation of CD1a or E-Cadherin (Boisleve et al., 2004) or an upregulation of CCL2, CCL3, CCL4 chemokine mRNA expression (Verheyen et al., 2005). However, because cord blood samples are difficult to obtain and CD34+ cells are rare in adult blood, this type of approach does not lend itself easily to the development of a routine testing procedure. One alternative to circumvent interdonor variability and cell acquisition difficulty consists in using cell lines. Unfortunately, DC lines are very difficult to produce. Several groups have therefore postulated that myeloid human cell lines, which belong to the same broad hematopoietic lineage as DCs, may have the capacity to respond to chemical sensitizers. Experiments carried out with leukemia-derived THP-1 and MUTZ-3 cell lines in their undifferentiated state showed an up-regulation of CD54 or CD86 in the presence of sensitizers (Ashikaga et al., 2002; Yoshida et al., 2003; Azam et al., 2006). These monocytic cell lines may provide useful tools to develop in vitro skin sensitization test methods, although further investigation is warranted. The aim of the present report was to explore the potential usefulness of DCs generated from mouse bone marrow for an alternative in vitro sensitization test. Mouse bone marrow-derived DCs (BMDCs) have been used by many investigators to characterize the molecular mechanisms involved in antigen internalization and

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processing. For example, Olasz et al. showed that haptenpulsed BMDCs are able to stimulate specific secondary proliferation of primed T lymphocytes, and are able to induce skin sensitization in na¨ıve mice (Olasz et al., 2002). We selected this source of DCs because it fulfills the need for a material that is easy to obtain, available in a sufficient quantity and homogeneous. In this study, immature DCs generated from mouse bone marrow were exposed to several well-known chemical allergens and non-allergens. The activation/maturation of the cells was assessed by analyzing changes in the expression of phenotypic surface markers.

All the chemicals were used at the highest concentrations leading to at least 80% viability, as assessed by trypan blue exclusion and propidium iodide staining. The contact allergens were used at 3–5 ␮M DNCB, 3–5 ␮M DNFB, 50 ␮M PPD, 5–10 ␮M BB, 200–300 ␮M NiSO4 and 25–50 ␮M Cinn. The irritants and negative control were used at 100–200 ␮M SDS, 10 mM pABA and 200 ␮M ZnSO4 . DNCB, DNFB, BB and Cinn were first dissolved in dimethyl sulfoxide (DMSO, Sigma) and added to the medium to give a final DMSO concentration of less than 0.03%. The control samples were treated with the vehicle for the same period of time. LPS was used as BMDC activating positive control at 10–50 ng/ml for 24 h.

2. Materials and methods

2.4. Flow cytometric analysis of surface antigen expression

2.1. Chemicals 1-Chloro-2,4-dinitrobenzene (DNCB), 1-fluoro2,4-dinitrobenzene (DNFB), nickel sulfate (NiSO4 ), p-phenylenediamine (PPD), sodium dodecyl sulfate (SDS), Cinnamaldehyde (Cinn), 4-aminobenzoic acid (pABA) and lipopolysaccharide (LPS) were purchased from Sigma (Saint Quentin Fallavier, France). Bandrowski’s base (BB) was purchased from MP Biomedicals (Illkirch, France). Zinc sulfate (ZnSO4 ) was purchased from Acros Organics (Halluin, France). 2.2. Generation of immature DCs from bone marrow The method for generating murine BMDCs was carried out according to previous publications (Inaba et al., 1992; Lutz et al., 1999) with slight modifications. Briefly, the femurs and tibiae of 8–10-week-old BALB/C mice were removed, both ends were cut and the bone marrow tissue was flushed with Iscove’s modified Dulbecco’s medium (IMDM) containing 10% fetal calf serum (FCS). The cells were then suspended in 90% FCS–10% DMSO and frozen in liquid nitrogen until use. After thawing, they were cultured in 150 mm Petri dishes in IMDM supplemented with 2 mM l-glutamine, 100 U/ml penicillin, 100 ␮g/ml streptomycin, 50 ␮M 2-mercaptoethanol (all from Life Technologies, Cergy-Pontoise, France), 10% heatinactivated FCS (BioWest, Nuaill´e, France), and 15% culture supernatant from mouse GM-CSF-producing J558 cells (a kind gift of D. Gray, University of Edinburgh). On day 4, fresh medium was added to the cultures. On day 7, the cells were briefly trypsinized (0.05%Trypsin–0.02% EDTA, Sigma), centrifuged and re-suspended in fresh medium in new dishes. On day 9, approximately 90% of the cells were CD11c+ , with an immature phenotype. 2.3. Treatment of cultured BMDCs BMDCs were collected on day 7, washed, counted and plated in 60 mm Petri dishes in complete medium. Chemicals were added for 48 h.

The analysis of treated BMDCs was performed by twocolour immunofluroscence staining with R-phycoerythrine (R-PE)-conjugated monoclonal antibody against CD11c (clone HL3) and fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies against IAd (clone AMS-32.1), CD40 (clone 3/23), CD54 (clone 3E2), CD86 (clone GL1) or isotype-matched control monoclonal antibodies. All the antibodies were purchased from Pharmingen BD Biosciences (Le Pont de Claix, France) and were used at saturating concentration. Cell harvesting took place on day 9, and surface staining was performed on fresh cells. The cells were distributed in a 96-well round bottom plate, washed with staining buffer (PBS–0.5% BSA–0.02% sodium azide) and incubated with FcBlock (Pharmingen) at 4 ◦ C for 15 min to reduce nonspecific staining. They were then stained with R-PE- and FITC-conjugated antibodies at 4 ◦ C for 20 min, washed with staining buffer and immediately analyzed on a FACStarPLUS flow cytometer using CELLQuest software (Becton Dickinson). Debris and dead cells were eliminated from the analysis using a gate based on forward and side scatters. Only CD11c+ cells were analyzed for FITC marker expression. At least 20,000 cells were analyzed for each sample. The analyses were carried out by measuring either the mean fluorescence intensity (MFI) or the percentage of highly positive (mature) cells, as explained in Fig. 1. Where indicated, the results are expressed as fold change over control cells. Data are presented as mean ± S.D. from at least three independent experiments, unless mentioned otherwise. 2.5. Statistical analysis A Student’s t-test was used to determine whether the alterations in surface marker expression after chemical exposure were significant. Values of p < 0.05 were considered significant. The statistical analysis was performed on three samples per group.

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Fig. 1. Example of flow cytometry analysis of a marker on control cells and chemical treated cells. Following Cellquest software data processing, the results are analyzed in two ways: (1) mean expression of a given marker, which ensues from the mean fluorescence intensity and is expressed as fold change over control cells (upper part of the diagram), (2) percentage of mature cells, which corresponds to the cells highly positive for a given marker (lower part of the diagram).

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Fig. 2. Expression of CD11c, CD19, CD3, MHC II, CD40, CD54 and CD86 on BMDCs following 9 days of culture (control cells). Example of representative histograms (dotted lines: isotype control and filled histograms: marker staining).

3. Results 3.1. Phenotypic characterization of BMDCs After 9 days of culture in the presence of GM-CSF, the BMDCs developed into a homogeneous population, with 90% of the cells expressing the DC marker CD11c. They also expressed low levels of the co-stimulatory molecules CD40 and CD86 and intermediate levels of CD54 and MHC II (Fig. 2), both of which are consistent with an immature phenotype. The cells were negative for the expression of B and T cell markers, i.e. CD19 and CD3. All the 9th day DC derivations led to a reproducible expression of all these molecules. To evaluate the capacity of our cultured BMDCs to respond in vitro to a stimulus known to induce maturation, LPS (50 ng/ml) was applied to the cells during the last 24 h of culture. Following the addition of LPS, the cells acquired the phenotype of mature DCs, as evidenced by the strong up-regulation of MHC II, CD40, CD54 and CD86 expression (Fig. 4). The MFIs of the four markers were increased by between 4.5- and 18.8fold (Fig. 5), with more than 80% of the CD11c+ cells displaying a highly positive staining (Fig. 6A). 3.2. Phenotypic characterization after chemical treatment Immature BMDCs were treated for 48 h with six chemical sensitizers (DNCB, DNFB, PPD, BB, NiSO4 and Cinn), two irritants (SDS and pABA) or a negative control substance (ZnSO4 ).

The highest concentrations of chemicals leading to less than 20% of cells positively stained with propidium iodide were selected (Fig. 3). Fig. 4 shows representative flow cytometry histograms and illustrates that all the allergens induced a cell maturation characterized by an up-regulation of MHC II, CD40, CD54 and CD86 expression. In contrast,

Fig. 3. Selection of chemical concentrations which allow a viability > 80%. The percentage of cells positively stained with propidium iodide was counted following 48 h of incubation with the chemicals. Concentrations inducing more than 20% of PI positive cells were not selected (in italics). The highest chemical concentrations leading to less than 20% of positive cells were selected for the subsequent experiments. Mean ± S.D. from at least three experiments.

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Fig. 4. Effect of LPS, sensitizers DNCB, DNFB, PPD, BB, NiSO4 and Cinn, negative control ZnSO4 , and irritants SDS and pABA on BMDC maturation. Expression of MHC II, CD40, CD54 and CD86 was analyzed by flow cytometry. Histograms result from one representative experiment (see Fig. 5 for conditions). Filled histograms: control cells and solid black lines: treated cells.

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Fig. 5. Effect of LPS, sensitizers DNCB, DNFB, PPD, BB, NiSO4 and Cinn, negative control ZnSO4 , and irritants SDS and pABA on the phenotypic response of BMDCs: analysis of the mean fluorescence intensity. MHC II, CD40, CD54 and CD86 expression was analyzed by flow cytometry, and MFI (treated over control cells) was calculated for each marker, as described in Fig. 1. Cell analysis was performed following 24 h of incubation with LPS (50 ng/ml) or 48 h incubation with DNCB (5 ␮M), DNFB (5 ␮M), PPD (50 ␮M), BB (10 ␮M), NiSO4 (300 ␮M), Cinn (50 ␮M), ZnSO4 (200 ␮M), SDS (200 ␮M), or pABA (10 mM). Mean ± S.D. from at least three independent experiments. * Treated cells > control cells (P < 0.05) and # treated cells < control cells (P < 0.05).

neither the irritants nor the negative control provoked such an increased expression. Two analytical methods were applied to quantify these observations: MFI and percentage of highly positive (mature) cells. 3.3. MFI In the first type of analysis, we measured the MFI of each marker following chemical exposure, and compared it to that observed with the control cells. As shown in Fig. 5, all the sensitizers induced an up-regulation of the maturation markers, but with a different range in MFI increase. Exposure to DNCB and DNFB induced a strong up-regulation of the expression of the four surface markers. MFI for the co-stimulatory molecule CD86 increased by 7.0- and 9.1-fold, respectively compared to control cells. PPD led to a slight increase in marker expression, causing a maximum 1.8fold increase in MFI relative to CD86, whereas its oxidation product BB induced a more pronounced upregulation (2.6- and 4.6-fold increase for CD40 and CD86 MFI, respectively). NiSO4 induced a significant increase in maturation marker expression, in contrast

to ZnSO4 which tended to give a ratio lower than one. Exposure to Cinn induced a slight but significant increase in expression of the four markers, which was more pronounced for CD86 (2.4-fold increase). Cells treated with the irritant SDS always displayed marker expression levels lower than those observed on control cells, whereas the second irritant, pABA, resulted in no significant modification of MHC II, CD40 and CD86 expression. Interestingly, it can be seen that the highest increase in expression was observed with all the sensitizers for the co-stimulatory molecule CD86, whereas the lowest upregulation was related to the adhesion molecule CD54. Thus, all six sensitizers tested induced cell maturation characterized by an increase in the MFI of CD86, MHC II, CD40 and CD54. In contrast, neither the irritants nor the negative control substance induced a significant upregulation of maturation marker expression. 3.4. Percentage of mature cells As two peaks of fluorescence were observed after chemical exposure, which does not make the MFI anal-

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Fig. 6. Effect of LPS, sensitizers DNCB, DNFB, PPD, BB, NiSO4 and Cinn, negative control ZnSO4 , and irritants SDS and pABA on the phenotypic response of the BMDCs: analysis of the percentage of highly positive cells. MHC II, CD40, CD54 and CD86 expression was analyzed by flow cytometry. Cell analysis was performed following 24 h of incubation with LPS (50 ng/ml) or 48 h incubation with DNCB (5 ␮M), DNFB (5 ␮M), PPD (50 ␮M), BB (10 ␮M), NiSO4 (300 ␮M), Cinn (50 ␮M), ZnSO4 (200 ␮M), SDS (200 ␮M), or pABA (10 mM). Grey bars: treated cells and white bars: respective control cells. (A) The percentage of highly positive cells was calculated for each marker, as described in Fig. 1. Mean ± S.D. from at least three independent experiments. (B) The percentage of mature cells was obtained by calculating the mean ± standard deviation of the percentages of cells highly positive for the four markers analyzed in (A). * Treated cells > control cells (P < 0.05) and # treated cells < control cells (P < 0.05).

ysis entirely satisfactory, we also analyzed our flow cytometry data by measuring the percentage of cells that became highly positive (right peak) for the maturation markers, i.e. mature cells. Fig. 6A shows the percentage of cells highly positive for each of the markers MHC II, CD40, CD54 and CD86. The control cells displayed a mean percentage of spontaneous maturation of 6.1 ± 2.2. Treatment with DNCB and DNFB made 50–61% of the cells highly positive for MHC II, CD40, CD54 and CD86. PPD led to about 14% of highly positive cells, whereas its oxidation product, BB, resulted in almost 25%. Following NiSO4 exposure, approximately 20% of the cells had a ++ phenotype. Exposure to Cinn resulted in almost

8% of mature cells. In contrast, the negative control ZnSO4 and the irritant SDS induced percentages of highly positive cells lower than those observed with the control cells, whereas pABA induced no significant changes. We observed that the percentages of highly positive cells following exposure to a given chemical were equivalent for the four markers analyzed, suggesting that there was one single population of mature cells expressing high levels of MHC II, together with CD40, CD54 and CD86. This was confirmed by performing multiple staining (data not shown) and led to our determining the mean of the four marker percentages and proposing it as a second procedure of analysis (Fig. 6B).

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Fig. 7. Comparison of two analytical methods: CD86 MFI (grey bars) and mean % of mature cells (white bars), expressed as fold change over control. Cell analysis was performed following 48 h incubation with DNCB (5 ␮M), DNFB (5 ␮M), PPD (50 ␮M), BB (10 ␮M), NiSO4 (300 ␮M), Cinn (50 ␮M), ZnSO4 (200 ␮M), SDS (200 ␮M) or pABA (10 mM). Mean ± S.D. from at least three independent experiments.

3.5. Comparison of our two analytical methods We then compared our two analytical methods, i.e. best MFI marker (CD86) and mean percentage of mature cells, both expressed as fold change over control. As shown in Fig. 7, these two analytical means led to similar values, except for NiSO4 which yielded a significantly higher increase when analyzed by percentage of mature cells (3.6 ± 0.7 vs. 1.8 ± 0.2). DNFB induced the highest maturation effect, and displayed an equivalent augmentation with the two analytical means (9.1 ± 1.7 vs. 10.5 ± 2.8). The negative control ZnSO4 and the irritant SDS gave values lower than the vehicle with both methods. 3.6. Chemical concentration effect Cell response intensity was proportional to chemical concentration. Fig. 8 shows the results obtained with the analysis of the percentage of mature cells. For example, doubling the BB concentration led to a 2.3-fold increase in the percentage of mature cells, whereas the same increase in SDS concentration led to a 1.5-fold decrease in mature cell percentage. Analysis of the MFI led to the same relationship (data not shown). 4. Discussion The establishment of in vitro sensitization methods for the screening of new industrial chemicals is of major importance both in terms of reducing animal testing and ensuring product safety. Due to their critical role

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in the induction of allergic contact dermatitis, DCs have become a focal point in the effort to develop in vitro skin sensitization assays. Human DCs derived from PBMC or CD34+ cord blood cells have been widely used these past few years, but these models suffer from limitations such as interdonor variability and cell supply inconveniences. In the present study, we elected to test the capacity of mouse bone marrow-derived DCs to respond to sensitizers in vitro. The activation/maturation of immature BMDCs was assessed by analyzing the expression of the phenotypic markers MHC II, CD40, CD54 and CD86. We chose to perform our phenotyping experiments on day 9 of BMDC culture, as this was the optimal time to obtain a high percentage of CD11c+ dendritic cells displaying an immature phenotype. Before examining the effect of chemical allergens on BMDC maturation, we first checked the flexibility of maturation marker expression in our DC culture system. In this respect, we treated BMDC with LPS, which has been shown to induce DC maturation by triggering TLR4. In our system, LPS consistently up-regulated MHC II, CD40, CD54 and CD86 expression to 5–19-fold compared to control. With these data in hand, we assumed that if a chemical allergen were capable of up-regulating marker expression on cultured murine BMDC, we should be able to detect it. We next examined a number of well-characterized contact sensitizers, including DNCB, DNFB, PPD, BB, NiSO4 , and Cinn. Each of these chemicals is known to cause allergic contact dermatitis in humans and to be positive in animal sensitization models. We also tested pABA and SDS, which are exclusively irritant in human pathology. SDS has been widely used as a negative control for the validation of a number of animal sensitizing experiments, although it induces false positive results in LLNA (Basketter et al., 1998). Our study showed that the in vitro murine BMDC system was able to reproducibly discriminate all the allergens tested from non-allergens using cell surface marker expression. Indeed, the expression of MHC II, CD40, CD54 and CD86 was significantly increased on cells exposed to sensitizers, whereas cells exposed to irritants showed a diminished or unchanged expression compared to vehicle control. Moreover, murine BMDCs responded in a dose-dependent manner. The MFI analysis showed that the up-regulation of all four surface markers was qualitatively but not quantitatively equivalent among the allergens. Indeed, whatever the sensitizer, CD86 always appeared as the marker displaying the highest increase in expression, followed by CD40 and MHC II, whereas CD54 exhibited the lowest up-regulation. In contrast with the sensitizers, LPS induced a higher up-regulation of CD40 than CD86, sug-

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Fig. 8. Concentration effect of LPS, sensitizers DNCB, DNFB, BB, NiSO4 and Cinn or irritant SDS on BMDC phenotype. Mean percentage of mature cells was calculated as described in Fig. 6B. Mean ± S.D. from at least two independent experiments. * Treated cells > control cells (P < 0.05) and # treated cells < control cells (P < 0.05).

gesting a different activation pathway. Up-regulation of the co-stimulatory molecule CD86 has been the most predictive parameter described in the literature, with several cellular models and numerous sensitizers (Aiba et al., 1997; Boisleve et al., 2004; Jugde et al., 2005; Azam et al., 2006; Sakaguchi et al., 2006). However, some authors have mentioned that marker regulation is qualitatively dependent on the nature of the chemical. For example, Azam et al. observed on MUTZ-3 cells that DNCB strongly induced CD86 but not CD40,

whereas the moderate sensitizer hexylcinnamaldehyde (HCA) induced both CD86 and CD40 to a similar extent (Azam et al., 2006). Using a PBMC-derived DC model, Aiba et al. detected that NiCl2 exposure up-regulated CD54, MHC II and CD86, while DNCB up-regulated only CD86 (Aiba et al., 2000). Interestingly, Sakaguchi et al. observed different responses depending not only on the sensitizers but also on the cellular models (Sakaguchi et al., 2006). In their hands, the U937 cell line caused no significant CD54 induction. However, they showed a

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strong up-regulation of CD54 by metal allergens, but not by PPD, on the THP-1 cell line. All these data suggest that sensitizers act differently depending on the cellular system, and underline the importance of choosing an appropriate cellular model for the development of an in vitro skin sensitization predictive test. Thus, unlike other models which strongly require the analysis of several surface markers to identify different sensitizers (De Smedt et al., 2005; Sakaguchi et al., 2006), CD86 investigation may allow the detection of several classes of allergens in our murine BMDC model. Interesting results could also be provided by our second analytical procedure based on examining the percentage of cells highly positive for the maturation markers. In some cases, the measurement of the percentage of mature cells gave more discriminative results than the data obtained by analysing CD86 MFI. Moreover, as two fluorescence peaks were observed after chemical exposure, this analytical procedure may be more accurate. A prospective investigation including a broader panel of chemicals would allow the proposal of a positivity threshold for the test. It is now accepted that an ideal method for skin sensitization testing should not only identify the hazard but should also provide some indication of relative sensitization potency. In our model, the intensity of up-regulation seems to be correlated to the class of allergen. Indeed, if we calculate effective concentration values, which represent extrapolated concentrations at which the cells start to react to the chemicals, it appears that the murine BMDC response induced by extreme sensitizers like DNCB or BB is much higher than that induced by the weaker allergen NiSO4 (not shown). This preliminary assumption must of course be confirmed with a broader panel of allergens, but our model does seem to be more suitable than others, which were often shown to be more reactive to the moderate sensitizer NiSO4 than to the strong sensitizer DNCB (Coutant et al., 1999; Manome et al., 1999; Aiba et al., 2000; Tuschl et al., 2000; Boisleve et al., 2004; De Smedt et al., 2005). However, it should be borne in mind that some sensitizers must be given careful consideration. Indeed, although many skin sensitizers are inherently protein-reactive, some contact allergens must be converted to a protein-reactive species for the induction of sensitization. For example, PPD is ranked as a strong to extreme sensitizer by all human and animal predictive tests, but has been shown to induce little or no effect in vitro, in contrast to its oxidation product BB (Krasteva et al., 1996; Rougier et al., 2000; Pichowski et al., 2001; Hulette et al., 2005; Sakaguchi et al., 2006; Toebak et al., 2006). Our data show that PPD can induce some BMDC maturation, which suggests that PPD does have some intrinsic allergenic potential and/or that our

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cell system possesses some metabolic activity. Nevertheless, PPD is less efficient than BB at inducing BMDC maturation. The requirement of metabolic competence, which is an issue relevant to all in vitro test methods, might be overcome by supplying the cell culture with an exogenous metabolization system such as the S9 fraction. Compound solubility may also represent a limitation to the validity of in vitro models. For example, we could not test oxazolone, a well-known strong sensitizer, because of its low solubility in culture medium, which has already been reported (Azam et al., 2006). Another major issue is related to cell viability. The paradigm commonly used in cell-based in vitro assays is to deliver the test chemical at the maximum nontoxic concentration. However, some investigators noticed that up-regulation of surface marker expression was more robust when DCs were treated with concentrations of chemical allergen that induce slight to moderate cytotoxicity (Yoshida et al., 2003; Hulette et al., 2005; Sakaguchi et al., 2006). They speculated that some cytotoxicity could be necessary for optimal DC activation in order to obtain the equivalent of a danger signal, which is required for the initiation of an innate immune response (Matzinger, 2002; Ryan et al., 2005). On the other hand, Straube and colleagues observed an induction of CD86 and MHC II expression with irritants at cytotoxic concentrations, and therefore recommend quantifying marker expression and cytotoxicity in parallel (Straube et al., 2005). With a view to limiting unspecific artifacts that could be caused by a substantial toxicity, we chose chemical concentrations which did not yield less than 80% cell viability. It is worth noting that, even at concentrations where they induce detectable toxicity of BMDCs, the irritant and negative control substances never induced an up-regulation pattern equivalent to that obtained with the sensitizers (not shown). Thus, our data suggest that the responsiveness of murine BMDCs may require borderline concentrations of sensitizers in terms of cell viability, but that toxicity itself is not responsible for the observed effects. Ideally, potential human skin sensitizers should be tested on human cells, but the use of murine cells may turn out to be an additional method to identify markers suitable for the identification of sensitizing chemicals. Moreover, practical advantages might make murine BMDCs well-suited to the development of an in vitro sensitization test. Above all, murine BMDCs are easy to derive in sufficient numbers and as a homogeneous population. Interestingly, mouse bone marrow cells can be frozen immediately after removal without affecting their biological properties in the subsequent assays. In our hands, cell proliferation and differentia-

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