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Moderate skin sensitizers can induce phenotypic changes on in vitro generated dendritic cells
Toxicology in Vitro 18 (2004) 493–500 www.elsevier.com/locate/toxinvit
Moderate skin sensitizers can induce phenotypic changes on in vitro generated dendritic cells M.J. Staquet a, M. Sportouch a, C. Jacquet a, D. Schmitt a, J. Guesnet b, J. Peguet-Navarro a,* a
INSERM U 346, Pavillon R, H^opital E. Herriot, 69437, Lyon 03, France b YSL Beaute, Neuilly/Seine, France Received 1 September 2003; accepted 4 December 2003
Abstract In the present study, we analyzed the phenotypic alterations induced by several allergens on immature dendritic cells (DC), with the aim to develop a potential in vitro alternative for predicting the sensitizing potential of chemicals. DC were generated from human monocytes cultured in the presence of GM-CSF, IL-4 and TGF-b1 and treated for 2 or 4 days with different chemicals. Surface marker expression (HLA-DR, CD1a, CD40, CD54, CD83, CD86, CCR7 and E-cadherin) was analyzed by flow cytometry. Results showed that a 2-day treatment with the representative allergens DNCB and NiSO4 induced significant changes of most antigens while other chemicals such as balm of Peru (strong allergen), kathon (moderate allergen), cinnamic aldehyde (mild allergen) or the irritant SLS had no significant effect. In contrast, the 4-day treatment with allergens substantially improved the results. Indeed, despite a large variability according to the donors, the number of modified antigens was significantly higher with all the tested chemicals, except kathon, as compared to that observed with the irritant SLS. The present study indicates that, in this model, the screening of mild or moderate allergens requires both the consideration of many antigens and a prolonged time of incubation with the chemicals. 2004 Elsevier Ltd. All rights reserved. Keywords: Contact sensitization; Alternatives in vitro; Dendritic cells; Phenotype
1. Introduction Contact of the skin with chemicals may result in a large variety of harmful effects at the site of contact. The two most frequent manifestations are irritant contact dermatitis and allergic contact dermatitis. Irritant contact dermatitis is a form of toxic skin inflammation induced by primary contact with chemicals that is thought not to be mediated by lymphocytes. By contrast, allergic contact dermatitis represents a lymphocyte-mediated delayed type hypersensitivity reaction that requires previous sensitization by the same chemical. Langerhans cells (LC), the dendritic cells (DC) of the epidermis, play a pivotal role in the initiation of cuta-
Abbreviations: CHS, contact hypersensitivity; DC, dendritic cells; DNCB, 2,4-dinitrochlorobenzene; HSR, hypersensitivity reactions; LC, Langerhans cells; SLS, sodium lauryl sulfate * Corresponding author. Tel.: +33-4-72-11-02-87; fax: +33-4-72-1102-90. E-mail address: [email protected] (J. Peguet-Navarro). 0887-2333/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2003.12.005
neous immune responses including the induction of allergic sensitization to chemicals. Following primary contact with the skin, LC bind allergens and migrate to regional lymph nodes where they trigger specific T cell activation and proliferation, generating effector cells of contact hypersensitivity (CHS) (Kripke et al., 1990). During this course, LC undergo a maturation process that includes phenotypic and functional changes such as increased cell surface expression of major histocompatibility complex (MHC) antigens, co-stimulatory and adhesion molecules which correlates with their ability to activate na€ıve T cells (Steinman, 1991; Maurer and Stingl, 2001). Concomitantly, E-cadherin and CCR7, some markers involved in the migration process, are modified (Tang et al., 1993; Schwarzenberger and Udey, 1996; Dieu et al., 1998). Contact dermatitis is one of the most common skin diseases with a great socio-economic impact, and a major problem remains the evaluation of new compounds for their potential to cause allergic sensitization. The increase in commercialized chemicals and
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pharmaceuticals on one hand, and the demand to reduce animal experiments on the other hand, require the development of in vitro models for the prediction of sensitizing potential of new substances. Several biological assays have tried to reproduce in vitro the different sequences implicated in the initiation of CHS by human LC, such as alteration of phenotypic marker expression (Rambukkana et al., 1996, Verrier et al., 1999), production of cytokines (Reutter et al., 1997), migration (Kobayashi et al., 1994; Pistoor et al., 1996) or activation of autologous T cells (Moulon et al., 1993; Krasteva et al., 1996). More recently, similar studies have been conducted with DC or LC generated in vitro, derived either from CD34+ progenitors (Rougier et al., 1998, 2000; De Smedt et al., 2001) or from monocytes (Degwert et al., 1997; Reutter et al., 1997; Aiba et al., 1997, 2000; Aiba and Tagami, 1999; K€ uhn, 1998, Manome et al., 1999; Coutant et al., 1999; Guironnet et al., 2000, Arrighi et al., 2001; Tuschl and Kovac, 2001, Hulette et al., 2002). All these studies concluded that the changes observed, indicative of DC activation, may be predictive of the contact sensitizing potential of a chemical and serve as the basis for an in vitro test model for evaluating new molecules. Indeed, these data essentially showed that it is possible to distinguish severe allergens (NiSO4 , DNCB, DNFB which result in the development of CHS reaction in a large percent of individuals) from irritants. However, the challenge remains to distinguish moderate and weak allergens from irritants. In the present study, immature DC generated by culturing monocytes with GM-CSF, IL-4 and TGF-b1 (Geissmann et al., 1998) were used to analyze phenotypic changes that occur following exposure to severe (NiSO4 , DNCB, balm of Peru), moderate (kathon), weak (cinnamic aldehyde) chemical allergens, and the irritant sodium lauryl sulfate (SLS). Results confirmed the differential changes in surface marker expression by severe allergens and irritant. They also provided promising results in the screening between mild allergens and irritants.
2. Materials and methods 2.1. Culture medium and cytokines Culture medium was RPMI-1640 supplemented with (GIBCO BRL, Grand Island, NY, USA), 1% antibiotic solution (Sigma, St Louis, MO), 10% heatinactivated fetal calf serum (FCS Myoclone, GIBCO BRL), thereafter referred to as complete medium. Recombinant human TGF-b1 (specific activity 16 · 106 – 50 · 106 U/mg) was from R&D Systems (Minneapolis, MN), recombinant human GM-CSF (specific activity: 2 · 106 U/mg) and IL-4 (specific activity: 2 · 107 U/mg)
were a generous gift from Schering-Plough Research Institute (Kenilworth, NJ). 2.2. Chemicals NiSO4 , 2,4-dinitrochlorobenzene (DNCB) and SLS were from Sigma Chemicals (St Louis, Mo). The balm of Peru, kathon and cinnamic aldehyde were provided by YSL Beaute (Neuilly/Seine, France). All chemicals were used at the highest non-toxic concentration, as assessed by trypan blue staining of DC suspensions after 18 h treatment with the chemicals. DNCB, cinnamic aldehyde and balm of Peru were initially prepared in DMSO (Sigma) and subsequently diluted in complete medium to give a final concentration of DMSO of 1/10 000. NiSO4 , Kathon and SLS were prepared in complete medium. 2.3. Monocyte purification and culture Mononuclear cells were obtained from the peripheral blood of healthy donors by centrifugation on FicollHypaque (Pharmacia, St Quentin en Yvelines, France). Monocytes were depleted of T and B cells using haptenconjugated anti-CD3, CD7, CD19, CD45RA, CD56 mAbs and anti-hapten Ig coupled to magnetic microbeads according to the manufacturerÕs instructions (Monocyte Isolation Kit, Miltenyi Biotec, Bergisch Gladbach, Germany). The technique routinely resulted in more than 80% purity, as assessed by flow cytometry. Purified monocytes (106 cells/ml) were cultured for 5 days in 6-well tissue culture plates (Costar Corp., Cambridge, MA) in complete medium supplemented with rhGM-CSF (200 ng/ml), IL-4 (33 ng/ml) and TGFb1 (10 ng/ml). 2.4. DC treatment with chemicals At day 5, the cells were enumerated and re-plated (106 cells/ml) for 2 or 4 days in complete medium supplemented with the cytokines, in the presence of the different chemicals: DNCB (7.6 lg/ml), NiSO4 (50 lg/ml), balm of Peru (10 lg/ml), Kathon (10 lg/ml), cinnamic aldehyde (10 lg/ml) and SLS (10 lg/ml). Control cells received either DMSO or medium alone. Cells were then recovered for phenotypic analysis. 2.5. Flow cytometry
L -glutamine
Cells were incubated for 30 min at 4 C with affinity purified mouse mAbs at the appropriate concentration or with irrelevant isotype-matched mouse Igs at the same concentration. Cells were washed and, for indirect staining, further incubated for 30 min at 4 C with FITC-conjugated F ðab0 Þ2 fragments of goat anti-mouse Ab. The following monoclonal antibodies were used:
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anti-HLA-DR-FITC (B8.12.2, IgG1), anti-CD54-FITC (84H10, IgG1), anti-CD83-FITC (HB15A, IgG1), antiCD40 (mAb89, IgG1) all from Immunotech (Marseille, France); anti-CD1a-FITC (BB-1, IgG1), anti-CD86FITC (IT2.2, IgG1), anti-CCR7 (2H4, IgM) from Pharmingen (San Diego, CA); anti-CD14 FITC (TUK 4, IgG1) from Dako (Glostrup, Denmark); anti Ecadherin (HECD-1, IgG1) from Takara (Shiga, Japan); anti-Langerin (DCGM4, IgG1), a generous gift from Schering-Plough (Dardilly, France). Intracellular staining for HLA-DR was carried out by using the fix and perm cell permeabilization kit (Caltag Laboratories, San Francisco, CA), according to the manufacturerÕs instructions. Flow cytometry was performed with a FACScan and data were analyzed using the Cell Quest software (Becton Dickinson, Le Pont de Claix, France). 2.6. Statistical analysis Statistical analysis was carried out using the nonparametric WillcoxonÕs test for paired values.
3. Results Two-day treatment of monocyte-derived DC only reveals the sensitizing potential of the strong allergens NiSO4 and DNCB: As previously reported (Guironnet et al., 2002), monocytes cultured for 5 days in the presence of GMCSF, IL-4 and TGF-b1 develop into non-adherent DC which express CD1a, HLA-DR, CD54 and CD40 while the costimulatory molecules CD80 and CD86 are only faintly or not expressed at the cell surface. The cells lack the monocytic marker CD14 and express the Langerhanscell related antigen E-cadherin. In most experiments, however, no staining was obtained using the anti-Langerin mAb. The cells display an immature phenotype, as revealed by the low percentage of cells expressing either CD83 or CCR7 antigens (data not shown). We first analyzed the phenotypic alterations induced on DC after a 2-day culture in the cytokine-supplemented medium, in the presence of several allergens or medium alone. In each experiment and for a given antigen, the phenotypic alterations were quantified by making the ratio of the MFI in the presence of the allergen to the MFI in the absence of the allergen. Similarly, we considered the ratio of the percentage of positive cells in the presence of the allergen to the percentage of positive cells in the absence of the allergen. The differences in the MFI, as well as in the percentage of positive cells, were considered as significant when the ratio exceeded 1.2 or was below 0.8 for up-regulated or down-regulated antigens, respectively. Table 1 summarizes the results of six experiments carried out with different donors, by indicating for each experiment and
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each chemical which antigen(s) was significantly altered. Although tested in only one or two out of six experiments, both NiSO4 and DNCB induced modifications of most of the antigens tested, such as up-regulation of extra and intracellular HLA-DR, CD54, CD83 and CCR7 antigens and down-regulation of E-cadherin (Fig. 1). These modifications were highly significant. For example, in the experiments using NiSO4 , the mean ratio of the MFI for CD1a, HLA-DR and CD54 averaged 0.72 ± 0.23, 3.9 ± 1.4 and 2.2 ± 0.37, respectively. Interestingly, the phenotypic alterations induced by SLS were unusual and concerned the expression of more than one antigen in only one out of six experiments (Table 1). However, quite similar results were obtained with the balm of Peru, although considered as a strong allergen. Indeed, the chemical only modified the expression of one or two antigens, which varied largely according to the donors. With kathon and cinnamic aldehyde, individual variations in the identity of the modified antigen(s) were also noticed. The number of modified antigens exceeds one in only one and three out of six experiments using kathon and cinnamic aldehyde, respectively. Globally, the number of modified antigens observed with kathon, cinnamic aldehyde and balm of Peru was not significantly different from that obtained with SLS, as assessed by the non-parametric WillcoxonÕs test for paired values (not shown). Four-day treatment of monocyte-derived DC allows the discrimination of both strong and mild allergens from irritant: We then tried to improve the results by treating DC for four days with the chemicals, in the presence of the cytokines GM-CSF, IL-4 and TGF-b1. Fig. 2 illustrates a representative experiment and Table 2 gives the entire results. Data showed that, in all experiments, NiSO4 and DNCB induced very significant alteration of nearly all the antigens tested. Regarding NiSO4 , the mean ratio of the MFI for CD1a and E-cad were 0.4 ± 0.14 and 0.56 ± 0.1, respectively, while those for HLA-DR, CD54 and CCR7 averaged 3.32 ± 0.97, 3.24 ± 1.03 and 1.9 ± 0.7, respectively. Concerning DNCB, this ratio averaged 0.58 ± 0.38 for CD1a and 4.68 ± 3.21 for HLA-DR. For the other chemicals, the 4-day treatment improved the results, in that it considerably increased the number of the modified allergens (Table 2). Indeed, while the irritant SLS never modified more than one antigen at the cell surface, three antigens at least were modified with the mild allergen cinnamic aldehyde, in most experiments. At least two antigens were modified in two out of six and five out of six experiments using kathon and balm of Peru, respectively. Statistical analysis was carried out using the nonparametric WillcoxonÕs test for paired values. As compared to SLS, the number of modified antigens was significantly higher using all the tested chemicals, with an exception for kathon (Fig. 3).
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Table 1 Phenotypic alterations induced by a 2-day DC treatment with the different chemicals Exp 1
Exp 2
Exp 3
Exp 4
Exp 5
Exp 6
SLS
–
CD86
–
–
CD40 CCR7
–
Cin ald
–
CD86
CD1a E-cad
CD40 E-Cad
E-Cad
DR E-cad CCR7
Kathon
–
CD1a DR CD54 CD83a CD86
–
E-Cad
CD40
–
Balm of Peru
CD1a
E-Cad
–
E-Cad
CD54 CCR7
CD83 CCR7
DNCB
ND
ND
ND
ND
ND
DRa CD54 CD86 E-cad CCR7
NiSO4
ND
ND
ND
ND
CD1a DRa CD54 CD86a CD40 E-cad CCR7a Intra-DR
DR CD54 CD83a CD40 E-cad Intra-DR
For each chemical and experiment, the antigens which are significantly modified are indicated. The expression of a given antigen was considered as significantly altered when the ratio of the MFI (or, alternatively the % of positive cells) in the presence versus in the absence of chemical was below 0.8 for E-cadherin and CD1a or exceeded 1.2 for the other antigens. a For these antigens, the above-defined ratio exceeds 2.
Fig. 1. A 2-day treatment of monocyte-derived DC with the strong allergens DNCB and NiSO4 induced significant modifications of most antigens. Monocytes were cultured for 5 days in the presence of GM-CSF, IL-4 and TGF-b1 and then treated for 2 days with the chemicals in the cytokinesupplemented medium. Cells were stained with a panel of monoclonal antibodies and analyzed by flow cytometry. Isotype controls are shown as empty histograms.
4. Discussion Despite active research, there is still a lack of reliable in vitro assays for defining chemical-induced allergic reactions. In the present study, we analyzed the phenotypic alterations induced on immature DC by several
allergens with the aim to develop a potential in vitro alternative for predicting the sensitizing potential of chemicals. Within the skin, LC display an immature phenotype, characterized by the low expression of MHC class II and co-stimulatory molecules. After application of allergens
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Fig. 2. The 4-day treatment of monocyte-derived DC allows the discrimination of both strong and mild allergens from irritant. Monocytes were cultured for 5 days in the presence of GM-CSF, IL-4 and TGF-b1 and then treated for 4 days with the chemicals in the cytokine-supplemented medium. Cells were stained with a panel of monoclonal antibodies and analyzed by flow cytometry. Isotype controls are shown as empty histograms. Vertical lines corresponding to the peak chanel of fluorescence intensity of a given antigen have been drawn to allow comparison of antigen expression levels on control and treated DC. Results are representative of six experiments.
on the skin, LC migrate to regional lymph nodes where they acquire the capacity to prime specific autologous T cells and thus initiate CHS reactions. During this process, LC phenotype is altered: the cells down-regulate some LC typical markers such as CD1a and E-cadherin whereas they acquire or up-regulate adhesion and costimulatory molecules such as CD54, CD80, CD40 and CD86 (Maurer and Stingl, 2001). Accordingly, many studies have tried to reproduce in vitro the phenotypic alterations induced on immature DC by allergens, in order to develop a simple and valuable tool for the screening of chemicals. These investigations were recently conducted with DC generated in vitro from either human CD34+ progenitors or monocytes, the later being favored because they provide a more homogeneous population of immature DC. Aiba and co-workers first reported very convincing data showing that 48 h-treatment with DNCB and NiCl2 induced up-regulation of HLA-DR, CD86 and CD54 on DC generated from monocytes by in vitro culture in the presence of GM-CSF and IL-4 (Aiba et al., 1997, 2000; Aiba and Tagami, 1999; Manome et al., 1999). TNCB could induce similar responses but in less numerous donors. By contrast, the irritants benzalkonium chloride, ZnCl2 or SLS had no effect. Others have confirmed the results, provided the same representative severe allergens DNCB, NiSO4 or NiCl2 were used (Degwert et al., 1997; Coutant et al., 1999; Tuschl and Kovac, 2001). However, the results were far less convincing when considering other strong allergens and, especially, chemicals which display only moderate or mild sensitizing potential. Coutant et al. (1999) reported that the moderate allergen chlorpromazine induced sig-
nificant up-regulation of HLA-DR, CD54 and CD86 on monocyte-derived DC. In this study, however, the authors did a cell selection and discarded DC cultures with high spontaneous HLA-DR expression, which gave poor responses to the sensitizers. Using a similar model, Tuschl and Kovac (2001) found that a-hexylcinnamaldehyde and eugenol increase the expression of CD86, CD54 and HLA-DR but the magnitude of the response was far less important than that obtained with NiSO4 . Finally, the measurement of surface marker changes as an alternative method to detect sensitizers was recently challenged, since both the severe allergen dinitrofluorobenzene and methylchloroisothiazolinone/methylisothiazolinone, i.e. kathon, only slightly up-regulated HLA-DR on monocyte-derived DC (Hulette et al., 2002). In the present study, we used an alternative cytokine cocktail to generate DC from monocytes, namely GMCSF, IL-4 and TGF-b1, which polarizes DC differentiation towards the LC pathway (Borkowski et al., 1996; Geissmann et al., 1998). We found that, in every experiments, the cells respond to a 2 or 4-day incubation with NiSO4 and DNCB by up-regulating most molecules involved in antigen presentation such as HLA-DR, CD40, CD54 and CD86, while down-regulating the expression of molecules specific of DC residing in the epidermis such as CD1a and E-cadherin. Moreover, we found significant up-regulation of CCR7, necessary for DC to migrate to regional lymph nodes. The results extend those published by Aiba et al. (2000), who earlier used this cytokine cocktail to generate LC-like DC and test the effects of NiCl2 and DNCB. In contrast with our results, Aiba et al. found that DNCB, at the same
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Table 2 Phenotypic alterations induced by a 4-day DC treatment with the different chemicals SLS
Exp 1
Exp 2
Exp 3
Exp 4
Exp 5
Exp 6
CD83
CD86
CD54
CCR7
–
CCR7
DR CD54 CD86
CD54 CD86 CCR7 Intra-DR
a
Cin ald
CD1a CD54a E-Cadh Intra-DR
CD1a DR CD54 CD83a CD86a Intra-DR
DR CD83 Intra-DR
DR
Kathon
CCR7
CD54a CD86 E-cad
CD40 CCR7
DR
–
CD86
Balm of Peru
CD1a CD54 E-cad CCR7a Intra-DR
CD54 CD83a CD86a E-cad Intra-DR
CCR7 Intra-DR
CD86a CCR7 E-cad
Intra-DR
CD54 CD83a CCR7
DNCB
CD1a CD54 E-cad CCR7
DR CD54 CD83 CD86a Intra-DR
DRa CD83 CD86a E-cad Intra-DRa
CD1a DRa CD86a E-cad CCR7a Intra-DRa
CD1a DRa CD54 CD86a E-cad CCR7 Intra-DRa
DR CD54 CD83 CD86
NiSO4
CD1a DRa CD54a CD83a CD86a CD40 E-cad CCR7 Intra-DR
ND
CD1a DR CD54a CD83 CD86a CD40a E-cad CCR7a Intra-DRa
CD1a DRa CD54 CD86a CD40 E-cad CCR7a Intra-DRa
CD1a DRa CD54a CD86a CD40 E-cad CCR7 Intra-DRa
CD1a DRa CD54a CD83a CD86a E-cad CCR7 Intra-DRa
For each chemical and experiment, the antigens which are significantly modified are indicated. The modification of expression for a given antigen was considered as significant when the ratio of the MFI (or the percentage of positive cells) in the presence versus in the absence of chemical was below 0.8 for E-cadherin and CD1a or exceeded 1.2 for the other antigens. a For these antigens, the above-defined ratio exceeded 2.
p< 0,005
NiSO4 p< 0,005
DNCB p< 0,01
balm Peru ns
kathon
p< 0,01
cin. ald SLS 0
2
4
6
8
0
number of modified antigens
Fig. 3. Mean number of modified antigens after a 4-day DC treatment with the chemicals. Results were obtained from six experiments performed with different donors. Statistical analysis was carried out using the non-parametric WillcoxonÕs test for paired values. p values were obtained by comparing the data obtained from DC treated with chemicals to those treated with SLS. ns: mean value not statistically different from mean of SLS.
concentration as we used, only modified the expression of CD86 and E-cadherin on DC. The time of incubation with the allergens was 2 days, whereas most of our results are from a 4-day incubation. Moreover, Aiba et al. did not add cytokines during incubation, while we did, which may explain the discrepancies in the results. Indeed, the presence of TGF-b1, known to maintain the cells in an immature stage (Geissmann et al., 1999), is likely to emphasize the maturating effect of allergens on DC. With regard to the other chemicals, we found here a very high variability in the response of DC, according to the donors, a result that has been regularly reported elsewhere (Aiba et al., 1997, 2000; Aiba and Tagami, 1999; Manome et al., 1999; Coutant et al., 1999; Tuschl and Kovac, 2001, Hulette et al., 2002). Indeed, for a given chemical, the number, as well as the identity of the modified antigens, are highly variable among experiments. Accordingly, no significant results could be
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obtained when considering the MFI. This leads us to propose an alternative for the results analysis. We first considered that an antigen is significantly modified when the relative MFI, or the relative percentage of positive cells exceeds 1.2 or is less than 0.8, which is quite restrictive. This allows us to quantify, in each experiment, the number of modified antigens. We found that a 2-day incubation with the chemicals different from NiSO4 or DNCB did not induce significant alterations of this number. However, the interesting finding is that the 4-day treatment with allergens substantially improves the results. Indeed, as compared to the irritant SLS, the number of modified allergens was significantly increased with all the tested chemicals, with an exception for kathon. This later result might be related to the high instability of the compound in culture medium (Rastogi, 1990). That prolonged time of incubation improves the results might be related to an accumulation of TNFa. Indeed, allergens were shown, on monocyte-derived DC, to induce the synthesis of TNFa (Aiba et al., 2000, Coutant et al., 1999), a cytokine known to induce DC maturation. In conclusion, the present study confirms that the use of phenotypic alterations on immature DC as an in vitro alternative to discriminate allergens from irritants presents some limits. Indeed, a 2-day DC incubation with the chemicals only allows the screening of some strong allergens. However, although our experiments were based on only few chemicals, the results demonstrate that both the consideration of many antigens and a prolonged time of incubation in the presence of the cytokines substantially improve the results, especially for differentiating mild or moderate allergens from irritants.
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Moulon, C., Peguet-Navarro, J., Courtellemont, P., Redziniak, G., Schmitt, D., 1993. In vitro primary sensitization and restimulation of hapten-specific T cells by fresh and cultured human epidermal LangerhansÕ cells. Immunology 80, 373–379. Pistoor, F.H.M., Rambukkana, A., Kroezen, M., Lepoittevin, J.P., Bos, J.D., Kapsenberg, M.L., Das, P.K., 1996. Novel predictive assay for contact allergens using human skin explant cultures. American Journal of Pathology 149, 337–343. Rambukkana, A., Pistoor, F.H.M., Bos, J.D., Kapsenberg, M.L., Das, P.K., 1996. Effects of contact allergens on human Langerhans cells in skin organ culture: migration, modulation of cell surface molecules, and early expression of interleukin-1b protein. Laboratory Investigation 74, 422–436. Rastogi, S.C., 1990. Kathon CG and cosmetics products. Contact Dermatitis 22, 155–160. Reutter, K., J€ ager, D., Degwert, J., Hoppe, U., 1997. In vitro model for contact sensitization: II. Induction of IL-1b mRNA in human blood-derived dendritic cells by contact sensitizers. Toxicology In Vitro 11, 619–626. Rougier, N., Redziniak, G., Schmitt, D., Vincent, C., 1998. Evaluation of the capacity of dendritic cells derived from cord blood CD34+ precursors to present haptens to unsensitized autologous T cells in
vitro. The Journal of Investigative Dermatology 110, 348– 352. Rougier, N., Redziniak, G., Mougin, D., Schmitt, D., Vincent, C., 2000. In vitro evaluation of the sensitization potential of weak contact allergens using Langerhans cell-like dendritic cells and autologous T cells. Toxicology 145, 73–82. Schwarzenberger, K., Udey, M.C., 1996. Contact allergens and epidermal proinflammatory cytokines modulate Langerhans cell E-cadherin expression in situ. The Journal of Investigative Dermatology 106, 553–558. Steinman, R.M., 1991. The dendritic cell system and its role in immunogenicity. Annual Review of Immunology 9, 271–296. Tang, A., Amagai, M., Granger, L.G., Stanley, J.R., Udey, M.C., 1993. Adhesion of epidermal Langerhans cells to keratinocytes mediated by E-cadherin. Nature 361, 82–85. Tuschl, H., Kovac, R., 2001. Langerhans cells and immature dendritic cells as model systems for screening of skin sensitizers. Toxicology In Vitro 15, 327–331. Verrier, A.C., Schmitt, D., Staquet, M.J., 1999. Flagrance and contact allergens in vitro modulate the HLA-DR and E-cadherin expression on human epidermal Langerhans cells. International Archives of Allergy and Immunology 120, 56–62.
Moderate skin sensitizers can induce phenotypic changes on in vitro generated dendritic cells M.J. Staquet a, M. Sportouch a, C. Jacquet a, D. Schmitt a, J. Guesnet b, J. Peguet-Navarro a,* a
INSERM U 346, Pavillon R, H^opital E. Herriot, 69437, Lyon 03, France b YSL Beaute, Neuilly/Seine, France Received 1 September 2003; accepted 4 December 2003
Abstract In the present study, we analyzed the phenotypic alterations induced by several allergens on immature dendritic cells (DC), with the aim to develop a potential in vitro alternative for predicting the sensitizing potential of chemicals. DC were generated from human monocytes cultured in the presence of GM-CSF, IL-4 and TGF-b1 and treated for 2 or 4 days with different chemicals. Surface marker expression (HLA-DR, CD1a, CD40, CD54, CD83, CD86, CCR7 and E-cadherin) was analyzed by flow cytometry. Results showed that a 2-day treatment with the representative allergens DNCB and NiSO4 induced significant changes of most antigens while other chemicals such as balm of Peru (strong allergen), kathon (moderate allergen), cinnamic aldehyde (mild allergen) or the irritant SLS had no significant effect. In contrast, the 4-day treatment with allergens substantially improved the results. Indeed, despite a large variability according to the donors, the number of modified antigens was significantly higher with all the tested chemicals, except kathon, as compared to that observed with the irritant SLS. The present study indicates that, in this model, the screening of mild or moderate allergens requires both the consideration of many antigens and a prolonged time of incubation with the chemicals. 2004 Elsevier Ltd. All rights reserved. Keywords: Contact sensitization; Alternatives in vitro; Dendritic cells; Phenotype
1. Introduction Contact of the skin with chemicals may result in a large variety of harmful effects at the site of contact. The two most frequent manifestations are irritant contact dermatitis and allergic contact dermatitis. Irritant contact dermatitis is a form of toxic skin inflammation induced by primary contact with chemicals that is thought not to be mediated by lymphocytes. By contrast, allergic contact dermatitis represents a lymphocyte-mediated delayed type hypersensitivity reaction that requires previous sensitization by the same chemical. Langerhans cells (LC), the dendritic cells (DC) of the epidermis, play a pivotal role in the initiation of cuta-
Abbreviations: CHS, contact hypersensitivity; DC, dendritic cells; DNCB, 2,4-dinitrochlorobenzene; HSR, hypersensitivity reactions; LC, Langerhans cells; SLS, sodium lauryl sulfate * Corresponding author. Tel.: +33-4-72-11-02-87; fax: +33-4-72-1102-90. E-mail address: [email protected] (J. Peguet-Navarro). 0887-2333/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2003.12.005
neous immune responses including the induction of allergic sensitization to chemicals. Following primary contact with the skin, LC bind allergens and migrate to regional lymph nodes where they trigger specific T cell activation and proliferation, generating effector cells of contact hypersensitivity (CHS) (Kripke et al., 1990). During this course, LC undergo a maturation process that includes phenotypic and functional changes such as increased cell surface expression of major histocompatibility complex (MHC) antigens, co-stimulatory and adhesion molecules which correlates with their ability to activate na€ıve T cells (Steinman, 1991; Maurer and Stingl, 2001). Concomitantly, E-cadherin and CCR7, some markers involved in the migration process, are modified (Tang et al., 1993; Schwarzenberger and Udey, 1996; Dieu et al., 1998). Contact dermatitis is one of the most common skin diseases with a great socio-economic impact, and a major problem remains the evaluation of new compounds for their potential to cause allergic sensitization. The increase in commercialized chemicals and
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pharmaceuticals on one hand, and the demand to reduce animal experiments on the other hand, require the development of in vitro models for the prediction of sensitizing potential of new substances. Several biological assays have tried to reproduce in vitro the different sequences implicated in the initiation of CHS by human LC, such as alteration of phenotypic marker expression (Rambukkana et al., 1996, Verrier et al., 1999), production of cytokines (Reutter et al., 1997), migration (Kobayashi et al., 1994; Pistoor et al., 1996) or activation of autologous T cells (Moulon et al., 1993; Krasteva et al., 1996). More recently, similar studies have been conducted with DC or LC generated in vitro, derived either from CD34+ progenitors (Rougier et al., 1998, 2000; De Smedt et al., 2001) or from monocytes (Degwert et al., 1997; Reutter et al., 1997; Aiba et al., 1997, 2000; Aiba and Tagami, 1999; K€ uhn, 1998, Manome et al., 1999; Coutant et al., 1999; Guironnet et al., 2000, Arrighi et al., 2001; Tuschl and Kovac, 2001, Hulette et al., 2002). All these studies concluded that the changes observed, indicative of DC activation, may be predictive of the contact sensitizing potential of a chemical and serve as the basis for an in vitro test model for evaluating new molecules. Indeed, these data essentially showed that it is possible to distinguish severe allergens (NiSO4 , DNCB, DNFB which result in the development of CHS reaction in a large percent of individuals) from irritants. However, the challenge remains to distinguish moderate and weak allergens from irritants. In the present study, immature DC generated by culturing monocytes with GM-CSF, IL-4 and TGF-b1 (Geissmann et al., 1998) were used to analyze phenotypic changes that occur following exposure to severe (NiSO4 , DNCB, balm of Peru), moderate (kathon), weak (cinnamic aldehyde) chemical allergens, and the irritant sodium lauryl sulfate (SLS). Results confirmed the differential changes in surface marker expression by severe allergens and irritant. They also provided promising results in the screening between mild allergens and irritants.
2. Materials and methods 2.1. Culture medium and cytokines Culture medium was RPMI-1640 supplemented with (GIBCO BRL, Grand Island, NY, USA), 1% antibiotic solution (Sigma, St Louis, MO), 10% heatinactivated fetal calf serum (FCS Myoclone, GIBCO BRL), thereafter referred to as complete medium. Recombinant human TGF-b1 (specific activity 16 · 106 – 50 · 106 U/mg) was from R&D Systems (Minneapolis, MN), recombinant human GM-CSF (specific activity: 2 · 106 U/mg) and IL-4 (specific activity: 2 · 107 U/mg)
were a generous gift from Schering-Plough Research Institute (Kenilworth, NJ). 2.2. Chemicals NiSO4 , 2,4-dinitrochlorobenzene (DNCB) and SLS were from Sigma Chemicals (St Louis, Mo). The balm of Peru, kathon and cinnamic aldehyde were provided by YSL Beaute (Neuilly/Seine, France). All chemicals were used at the highest non-toxic concentration, as assessed by trypan blue staining of DC suspensions after 18 h treatment with the chemicals. DNCB, cinnamic aldehyde and balm of Peru were initially prepared in DMSO (Sigma) and subsequently diluted in complete medium to give a final concentration of DMSO of 1/10 000. NiSO4 , Kathon and SLS were prepared in complete medium. 2.3. Monocyte purification and culture Mononuclear cells were obtained from the peripheral blood of healthy donors by centrifugation on FicollHypaque (Pharmacia, St Quentin en Yvelines, France). Monocytes were depleted of T and B cells using haptenconjugated anti-CD3, CD7, CD19, CD45RA, CD56 mAbs and anti-hapten Ig coupled to magnetic microbeads according to the manufacturerÕs instructions (Monocyte Isolation Kit, Miltenyi Biotec, Bergisch Gladbach, Germany). The technique routinely resulted in more than 80% purity, as assessed by flow cytometry. Purified monocytes (106 cells/ml) were cultured for 5 days in 6-well tissue culture plates (Costar Corp., Cambridge, MA) in complete medium supplemented with rhGM-CSF (200 ng/ml), IL-4 (33 ng/ml) and TGFb1 (10 ng/ml). 2.4. DC treatment with chemicals At day 5, the cells were enumerated and re-plated (106 cells/ml) for 2 or 4 days in complete medium supplemented with the cytokines, in the presence of the different chemicals: DNCB (7.6 lg/ml), NiSO4 (50 lg/ml), balm of Peru (10 lg/ml), Kathon (10 lg/ml), cinnamic aldehyde (10 lg/ml) and SLS (10 lg/ml). Control cells received either DMSO or medium alone. Cells were then recovered for phenotypic analysis. 2.5. Flow cytometry
L -glutamine
Cells were incubated for 30 min at 4 C with affinity purified mouse mAbs at the appropriate concentration or with irrelevant isotype-matched mouse Igs at the same concentration. Cells were washed and, for indirect staining, further incubated for 30 min at 4 C with FITC-conjugated F ðab0 Þ2 fragments of goat anti-mouse Ab. The following monoclonal antibodies were used:
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anti-HLA-DR-FITC (B8.12.2, IgG1), anti-CD54-FITC (84H10, IgG1), anti-CD83-FITC (HB15A, IgG1), antiCD40 (mAb89, IgG1) all from Immunotech (Marseille, France); anti-CD1a-FITC (BB-1, IgG1), anti-CD86FITC (IT2.2, IgG1), anti-CCR7 (2H4, IgM) from Pharmingen (San Diego, CA); anti-CD14 FITC (TUK 4, IgG1) from Dako (Glostrup, Denmark); anti Ecadherin (HECD-1, IgG1) from Takara (Shiga, Japan); anti-Langerin (DCGM4, IgG1), a generous gift from Schering-Plough (Dardilly, France). Intracellular staining for HLA-DR was carried out by using the fix and perm cell permeabilization kit (Caltag Laboratories, San Francisco, CA), according to the manufacturerÕs instructions. Flow cytometry was performed with a FACScan and data were analyzed using the Cell Quest software (Becton Dickinson, Le Pont de Claix, France). 2.6. Statistical analysis Statistical analysis was carried out using the nonparametric WillcoxonÕs test for paired values.
3. Results Two-day treatment of monocyte-derived DC only reveals the sensitizing potential of the strong allergens NiSO4 and DNCB: As previously reported (Guironnet et al., 2002), monocytes cultured for 5 days in the presence of GMCSF, IL-4 and TGF-b1 develop into non-adherent DC which express CD1a, HLA-DR, CD54 and CD40 while the costimulatory molecules CD80 and CD86 are only faintly or not expressed at the cell surface. The cells lack the monocytic marker CD14 and express the Langerhanscell related antigen E-cadherin. In most experiments, however, no staining was obtained using the anti-Langerin mAb. The cells display an immature phenotype, as revealed by the low percentage of cells expressing either CD83 or CCR7 antigens (data not shown). We first analyzed the phenotypic alterations induced on DC after a 2-day culture in the cytokine-supplemented medium, in the presence of several allergens or medium alone. In each experiment and for a given antigen, the phenotypic alterations were quantified by making the ratio of the MFI in the presence of the allergen to the MFI in the absence of the allergen. Similarly, we considered the ratio of the percentage of positive cells in the presence of the allergen to the percentage of positive cells in the absence of the allergen. The differences in the MFI, as well as in the percentage of positive cells, were considered as significant when the ratio exceeded 1.2 or was below 0.8 for up-regulated or down-regulated antigens, respectively. Table 1 summarizes the results of six experiments carried out with different donors, by indicating for each experiment and
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each chemical which antigen(s) was significantly altered. Although tested in only one or two out of six experiments, both NiSO4 and DNCB induced modifications of most of the antigens tested, such as up-regulation of extra and intracellular HLA-DR, CD54, CD83 and CCR7 antigens and down-regulation of E-cadherin (Fig. 1). These modifications were highly significant. For example, in the experiments using NiSO4 , the mean ratio of the MFI for CD1a, HLA-DR and CD54 averaged 0.72 ± 0.23, 3.9 ± 1.4 and 2.2 ± 0.37, respectively. Interestingly, the phenotypic alterations induced by SLS were unusual and concerned the expression of more than one antigen in only one out of six experiments (Table 1). However, quite similar results were obtained with the balm of Peru, although considered as a strong allergen. Indeed, the chemical only modified the expression of one or two antigens, which varied largely according to the donors. With kathon and cinnamic aldehyde, individual variations in the identity of the modified antigen(s) were also noticed. The number of modified antigens exceeds one in only one and three out of six experiments using kathon and cinnamic aldehyde, respectively. Globally, the number of modified antigens observed with kathon, cinnamic aldehyde and balm of Peru was not significantly different from that obtained with SLS, as assessed by the non-parametric WillcoxonÕs test for paired values (not shown). Four-day treatment of monocyte-derived DC allows the discrimination of both strong and mild allergens from irritant: We then tried to improve the results by treating DC for four days with the chemicals, in the presence of the cytokines GM-CSF, IL-4 and TGF-b1. Fig. 2 illustrates a representative experiment and Table 2 gives the entire results. Data showed that, in all experiments, NiSO4 and DNCB induced very significant alteration of nearly all the antigens tested. Regarding NiSO4 , the mean ratio of the MFI for CD1a and E-cad were 0.4 ± 0.14 and 0.56 ± 0.1, respectively, while those for HLA-DR, CD54 and CCR7 averaged 3.32 ± 0.97, 3.24 ± 1.03 and 1.9 ± 0.7, respectively. Concerning DNCB, this ratio averaged 0.58 ± 0.38 for CD1a and 4.68 ± 3.21 for HLA-DR. For the other chemicals, the 4-day treatment improved the results, in that it considerably increased the number of the modified allergens (Table 2). Indeed, while the irritant SLS never modified more than one antigen at the cell surface, three antigens at least were modified with the mild allergen cinnamic aldehyde, in most experiments. At least two antigens were modified in two out of six and five out of six experiments using kathon and balm of Peru, respectively. Statistical analysis was carried out using the nonparametric WillcoxonÕs test for paired values. As compared to SLS, the number of modified antigens was significantly higher using all the tested chemicals, with an exception for kathon (Fig. 3).
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Table 1 Phenotypic alterations induced by a 2-day DC treatment with the different chemicals Exp 1
Exp 2
Exp 3
Exp 4
Exp 5
Exp 6
SLS
–
CD86
–
–
CD40 CCR7
–
Cin ald
–
CD86
CD1a E-cad
CD40 E-Cad
E-Cad
DR E-cad CCR7
Kathon
–
CD1a DR CD54 CD83a CD86
–
E-Cad
CD40
–
Balm of Peru
CD1a
E-Cad
–
E-Cad
CD54 CCR7
CD83 CCR7
DNCB
ND
ND
ND
ND
ND
DRa CD54 CD86 E-cad CCR7
NiSO4
ND
ND
ND
ND
CD1a DRa CD54 CD86a CD40 E-cad CCR7a Intra-DR
DR CD54 CD83a CD40 E-cad Intra-DR
For each chemical and experiment, the antigens which are significantly modified are indicated. The expression of a given antigen was considered as significantly altered when the ratio of the MFI (or, alternatively the % of positive cells) in the presence versus in the absence of chemical was below 0.8 for E-cadherin and CD1a or exceeded 1.2 for the other antigens. a For these antigens, the above-defined ratio exceeds 2.
Fig. 1. A 2-day treatment of monocyte-derived DC with the strong allergens DNCB and NiSO4 induced significant modifications of most antigens. Monocytes were cultured for 5 days in the presence of GM-CSF, IL-4 and TGF-b1 and then treated for 2 days with the chemicals in the cytokinesupplemented medium. Cells were stained with a panel of monoclonal antibodies and analyzed by flow cytometry. Isotype controls are shown as empty histograms.
4. Discussion Despite active research, there is still a lack of reliable in vitro assays for defining chemical-induced allergic reactions. In the present study, we analyzed the phenotypic alterations induced on immature DC by several
allergens with the aim to develop a potential in vitro alternative for predicting the sensitizing potential of chemicals. Within the skin, LC display an immature phenotype, characterized by the low expression of MHC class II and co-stimulatory molecules. After application of allergens
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497
Fig. 2. The 4-day treatment of monocyte-derived DC allows the discrimination of both strong and mild allergens from irritant. Monocytes were cultured for 5 days in the presence of GM-CSF, IL-4 and TGF-b1 and then treated for 4 days with the chemicals in the cytokine-supplemented medium. Cells were stained with a panel of monoclonal antibodies and analyzed by flow cytometry. Isotype controls are shown as empty histograms. Vertical lines corresponding to the peak chanel of fluorescence intensity of a given antigen have been drawn to allow comparison of antigen expression levels on control and treated DC. Results are representative of six experiments.
on the skin, LC migrate to regional lymph nodes where they acquire the capacity to prime specific autologous T cells and thus initiate CHS reactions. During this process, LC phenotype is altered: the cells down-regulate some LC typical markers such as CD1a and E-cadherin whereas they acquire or up-regulate adhesion and costimulatory molecules such as CD54, CD80, CD40 and CD86 (Maurer and Stingl, 2001). Accordingly, many studies have tried to reproduce in vitro the phenotypic alterations induced on immature DC by allergens, in order to develop a simple and valuable tool for the screening of chemicals. These investigations were recently conducted with DC generated in vitro from either human CD34+ progenitors or monocytes, the later being favored because they provide a more homogeneous population of immature DC. Aiba and co-workers first reported very convincing data showing that 48 h-treatment with DNCB and NiCl2 induced up-regulation of HLA-DR, CD86 and CD54 on DC generated from monocytes by in vitro culture in the presence of GM-CSF and IL-4 (Aiba et al., 1997, 2000; Aiba and Tagami, 1999; Manome et al., 1999). TNCB could induce similar responses but in less numerous donors. By contrast, the irritants benzalkonium chloride, ZnCl2 or SLS had no effect. Others have confirmed the results, provided the same representative severe allergens DNCB, NiSO4 or NiCl2 were used (Degwert et al., 1997; Coutant et al., 1999; Tuschl and Kovac, 2001). However, the results were far less convincing when considering other strong allergens and, especially, chemicals which display only moderate or mild sensitizing potential. Coutant et al. (1999) reported that the moderate allergen chlorpromazine induced sig-
nificant up-regulation of HLA-DR, CD54 and CD86 on monocyte-derived DC. In this study, however, the authors did a cell selection and discarded DC cultures with high spontaneous HLA-DR expression, which gave poor responses to the sensitizers. Using a similar model, Tuschl and Kovac (2001) found that a-hexylcinnamaldehyde and eugenol increase the expression of CD86, CD54 and HLA-DR but the magnitude of the response was far less important than that obtained with NiSO4 . Finally, the measurement of surface marker changes as an alternative method to detect sensitizers was recently challenged, since both the severe allergen dinitrofluorobenzene and methylchloroisothiazolinone/methylisothiazolinone, i.e. kathon, only slightly up-regulated HLA-DR on monocyte-derived DC (Hulette et al., 2002). In the present study, we used an alternative cytokine cocktail to generate DC from monocytes, namely GMCSF, IL-4 and TGF-b1, which polarizes DC differentiation towards the LC pathway (Borkowski et al., 1996; Geissmann et al., 1998). We found that, in every experiments, the cells respond to a 2 or 4-day incubation with NiSO4 and DNCB by up-regulating most molecules involved in antigen presentation such as HLA-DR, CD40, CD54 and CD86, while down-regulating the expression of molecules specific of DC residing in the epidermis such as CD1a and E-cadherin. Moreover, we found significant up-regulation of CCR7, necessary for DC to migrate to regional lymph nodes. The results extend those published by Aiba et al. (2000), who earlier used this cytokine cocktail to generate LC-like DC and test the effects of NiCl2 and DNCB. In contrast with our results, Aiba et al. found that DNCB, at the same
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Table 2 Phenotypic alterations induced by a 4-day DC treatment with the different chemicals SLS
Exp 1
Exp 2
Exp 3
Exp 4
Exp 5
Exp 6
CD83
CD86
CD54
CCR7
–
CCR7
DR CD54 CD86
CD54 CD86 CCR7 Intra-DR
a
Cin ald
CD1a CD54a E-Cadh Intra-DR
CD1a DR CD54 CD83a CD86a Intra-DR
DR CD83 Intra-DR
DR
Kathon
CCR7
CD54a CD86 E-cad
CD40 CCR7
DR
–
CD86
Balm of Peru
CD1a CD54 E-cad CCR7a Intra-DR
CD54 CD83a CD86a E-cad Intra-DR
CCR7 Intra-DR
CD86a CCR7 E-cad
Intra-DR
CD54 CD83a CCR7
DNCB
CD1a CD54 E-cad CCR7
DR CD54 CD83 CD86a Intra-DR
DRa CD83 CD86a E-cad Intra-DRa
CD1a DRa CD86a E-cad CCR7a Intra-DRa
CD1a DRa CD54 CD86a E-cad CCR7 Intra-DRa
DR CD54 CD83 CD86
NiSO4
CD1a DRa CD54a CD83a CD86a CD40 E-cad CCR7 Intra-DR
ND
CD1a DR CD54a CD83 CD86a CD40a E-cad CCR7a Intra-DRa
CD1a DRa CD54 CD86a CD40 E-cad CCR7a Intra-DRa
CD1a DRa CD54a CD86a CD40 E-cad CCR7 Intra-DRa
CD1a DRa CD54a CD83a CD86a E-cad CCR7 Intra-DRa
For each chemical and experiment, the antigens which are significantly modified are indicated. The modification of expression for a given antigen was considered as significant when the ratio of the MFI (or the percentage of positive cells) in the presence versus in the absence of chemical was below 0.8 for E-cadherin and CD1a or exceeded 1.2 for the other antigens. a For these antigens, the above-defined ratio exceeded 2.
p< 0,005
NiSO4 p< 0,005
DNCB p< 0,01
balm Peru ns
kathon
p< 0,01
cin. ald SLS 0
2
4
6
8
0
number of modified antigens
Fig. 3. Mean number of modified antigens after a 4-day DC treatment with the chemicals. Results were obtained from six experiments performed with different donors. Statistical analysis was carried out using the non-parametric WillcoxonÕs test for paired values. p values were obtained by comparing the data obtained from DC treated with chemicals to those treated with SLS. ns: mean value not statistically different from mean of SLS.
concentration as we used, only modified the expression of CD86 and E-cadherin on DC. The time of incubation with the allergens was 2 days, whereas most of our results are from a 4-day incubation. Moreover, Aiba et al. did not add cytokines during incubation, while we did, which may explain the discrepancies in the results. Indeed, the presence of TGF-b1, known to maintain the cells in an immature stage (Geissmann et al., 1999), is likely to emphasize the maturating effect of allergens on DC. With regard to the other chemicals, we found here a very high variability in the response of DC, according to the donors, a result that has been regularly reported elsewhere (Aiba et al., 1997, 2000; Aiba and Tagami, 1999; Manome et al., 1999; Coutant et al., 1999; Tuschl and Kovac, 2001, Hulette et al., 2002). Indeed, for a given chemical, the number, as well as the identity of the modified antigens, are highly variable among experiments. Accordingly, no significant results could be
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obtained when considering the MFI. This leads us to propose an alternative for the results analysis. We first considered that an antigen is significantly modified when the relative MFI, or the relative percentage of positive cells exceeds 1.2 or is less than 0.8, which is quite restrictive. This allows us to quantify, in each experiment, the number of modified antigens. We found that a 2-day incubation with the chemicals different from NiSO4 or DNCB did not induce significant alterations of this number. However, the interesting finding is that the 4-day treatment with allergens substantially improves the results. Indeed, as compared to the irritant SLS, the number of modified allergens was significantly increased with all the tested chemicals, with an exception for kathon. This later result might be related to the high instability of the compound in culture medium (Rastogi, 1990). That prolonged time of incubation improves the results might be related to an accumulation of TNFa. Indeed, allergens were shown, on monocyte-derived DC, to induce the synthesis of TNFa (Aiba et al., 2000, Coutant et al., 1999), a cytokine known to induce DC maturation. In conclusion, the present study confirms that the use of phenotypic alterations on immature DC as an in vitro alternative to discriminate allergens from irritants presents some limits. Indeed, a 2-day DC incubation with the chemicals only allows the screening of some strong allergens. However, although our experiments were based on only few chemicals, the results demonstrate that both the consideration of many antigens and a prolonged time of incubation in the presence of the cytokines substantially improve the results, especially for differentiating mild or moderate allergens from irritants.
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