MUTZ-3-derived dendritic cells as an in vitro alternative model to CD34+ progenitor-derived dendritic cells for testing of chemical sensitizers

Toxicology in Vitro 23 (2009) 1477–1481

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MUTZ-3-derived dendritic cells as an in vitro alternative model to CD34+ progenitor-derived dendritic cells for testing of chemical sensitizers Inge Nelissen *, Ingrid Selderslaghs, Rosette Van Den Heuvel, Hilda Witters, Geert R. Verheyen 1, Greet Schoeters Flemish Institute for Technological Research (VITO NV), Environmental Risk and Health, BE-2400 Mol, Belgium

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Article history: Received 4 November 2008 Accepted 25 August 2009 Available online 2 September 2009 Keywords: Dendritic cell MUTZ-3 Skin sensitization

a b s t r a c t The cytokine-dependent CD34+ human acute myeloid leukaemia cell line MUTZ-3 was used to generate immature dendritic-like cells (MUTZ-3 DC) and their validity as an alternative to primary CD34+ progenitor-derived DC (CD34–DC) for testing chemical-induced sensitization was assessed. Expression levels of the DC maturation markers HLA-DR, CD86, CD83 and CD11c were studied using flow cytometry after 24 and 48 h exposure to the model compound nickel sulphate (100 and 300 lM). No maturation of MUTZ-3 DC was observed, whereas significantly upregulated expression levels of CD83 and CD86 were noticed in CD34–DC after 24 h treatment with 300 lM nickel sulphate compared to control cells. Differential expression of the cytokine genes IL1b, IL6, IL8, CCL2, CCL3, CCL3L1, CCL4 was analyzed using real-time RT-PCR after 6, 10 and 24 h of nickel sulphate exposure. In response to 100 lM nickel sulphate MUTZ-3 DC revealed slightly upregulated mRNA levels after 24 h, whereas 300 lM induced transcription of CCL3, CCL3L1 and IL8 significantly after 6 or 10 h. These cytokine data correspond to the previously observed effects of 100 lM nickel sulphate in CD34–DC. Our findings underline the stimulatory capacity of nickel sulphate in MUTZ-3 DC with regard to cytokine mRNA induction, but not surface marker expression. Compared to CD34–DC, however, the studied endpoint markers seemed to be less inducible, making the MUTZ-3 DC model in its presented form less suitable for in vitro testing of sensitization. Further assessment of MUTZ-3 DC using other differentiation protocols and an extended set of chemicals will be required to reveal whether this cell line may be a valid alternative model system to primary CD34–DC. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Many chemicals are capable of inducing allergic contact dermatitis (ACD) in susceptible individuals. ACD represents an important occupational and consumer health problem. Therefore reliable methods that allow the prospective identification of chemical skin allergens are needed. Currently these methods are based on in vivo animal experiments, with their economical and ethical drawbacks. The availability of validated in vitro test systems, which are based on (a) relevant cell model(s) and specific endpoint markers that predict the sensitizing potency of chemicals, is therefore of high importance to the pharmaceutical, chemical, cosmetic, pesticide and food industry. A promising in vitro alternative to animal testing is the use of cultured human antigen-presenting cells (APC). Dendritic cells

* Corresponding author. Tel.: +32 14 33 51 07; fax: +32 14 58 26 57. E-mail address: [email protected] (I. Nelissen). 1 Present address: Johnson & Johnson Pharmaceutical Research and Development, Department of Mechanistic Toxicology, Turnhoutseweg 30, BE-2340 Beerse, Belgium. 0887-2333/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2009.08.022

(DC) represent professional APC that are accepted to be essential in allergic sensitization, due to their unique ability to induce antigen-specific primary T cell responses, and deliver co-stimulatory signals necessary for T cell activation. In vitro, myeloid DC generated from human cord blood CD34+ progenitor cells (CD34–DC) can reproduce the maturation process of skin Langerhans cells or interstitial DC (Caux et al., 1996), and are therefore highly relevant as a model system for in vitro testing of skin sensitizing compounds. Various primary DC models have been described so far, and most of them were found to be able to identify the sensitizing potential of chemicals to some extent by monitoring either allogenic T cell stimulation, expression of activation markers or cytokine production (Aiba et al., 1997; De Smedt et al., 2005; Tuschl et al., 2000). CD34–DC have furthermore proven to be able to discriminate skin sensitizing from non-sensitizing chemicals based on gene expression profiles (Schoeters et al., 2006, 2007; Verheyen et al., 2005) and a classification model was recently developed (Hooyberghs et al., 2008). Although the latter model looks promising, the development of a high-throughput test system based on human donor-derived CD34–DC is hampered by a number of

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drawbacks. Current preparation methods yield limited DC numbers, require long expansion periods, and suffer from the inherent difficulties of availability and biological variability associated with the study of primary human material. Human cell lines with dendritic-like properties coping with most of these limitations may provide valuable in vitro models for the development of standardized high-throughput assays for sensitization. DC are non-dividing terminally differentiated cells, and ex vivo leukaemic cells or cell lines show little similarity to DC. However, leukaemic cells retain a degree of lineage plasticity and many of them differentiate further in response to defined stimuli (Rasaiyaah et al., 2007). The potential to differentiate into dendriticlike cells has already been studied for several cell lines of lymphoid or myeloid origin, such as KG-1, THP-1, U937, Mono Mac 6, MUTZ3 and K562 (Azam et al., 2006; Berges et al., 2005; Chang et al., 2005; Hulette et al., 2002; Larsson et al., 2006; Masterson et al., 2002; Yoshida et al., 2003). The most ‘dendritic-like’ cells appear to be derived from less differentiated myelomonocytic, rather than monocytic leukaemias, such as the CD34+ human acute myeloid leukaemia cell line MUTZ-3 (Rasaiyaah et al., 2007). The latter cell line uniquely represents a ready and unlimited source of CD34+ DC progenitors, and has been described to possess the capacity to acquire a functional cytokine-induced immature DC phenotype, which displays the full range of functional antigen-processing and presentation pathways (Santegoets et al., 2008). Furthermore, immature MUTZ-3-derived DC (MUTZ-3 DC) were found to mature similar to primary monocyte-derived dendritic cells, both at the phenotypic and gene induction level (Kim et al., 2006; Larsson et al., 2006). Interestingly, naive MUTZ-3 cells have been observed to potently detect and classify contact sensitizers by means of CD86 activation (Azam et al., 2006). Thus, we investigated whether immature MUTZ-3 DC are useful as an alternative cell source to primary CD34–DC for the identification of skin allergens. To this end, the model sensitizing chemical nickel, which previously has been shown to potently induce CD34–DC maturation (De Smedt et al., 2001; Schoeters et al., 2006), was used to evaluate the response of MUTZ-3 DC both at the phenotypic and mRNA level. This response was compared to data obtained with CD34–DC.

2. Material and methods 2.1. Cell culture and chemical exposure The human acute myeloid leukaemia cell line MUTZ-3 (DSMZ; Braunschweig, Germany) was maintained at 37 °C and 5% CO2 in a-Minimal Essential Medium (MEM) supplemented with ribonucleosides and deoxyribonucleosides (Invitrogen, Merelbeke, Belgium), 20% (v/v) heat-inactivated fetal bovine serum (FBS) and 40 ng/ml granulocyte macrophage-colony stimulating factor (GM-CSF; Leukomax, Novartis, Basel, Switzerland). Culture medium was replaced every 2–3 days, and cells were counted and assessed for viability with the use of a Nucleocounter (ChemoMetec, Allerød, Denmark). Viability of the cultures was consistently more than 95%. MUTZ-3 cultures were subcultured before reaching the saturation cell density of 106 cells/ml. To obtain immature dendritic-like cells starting from naive MUTZ-3 cultures, cells (2  105 cells/ml) were differentiated for 7 days in a-MEM supplemented with 20% (v/v) FBS, 100 ng/ml GM-CSF (Novartis), 10 ng/ml interleukin (IL)-4 (Biosource, Nivelles, Belgium) and 2.5 ng/ml tumor necrosis factor (TNF)-a (Roche, Basel, Switzerland) (Masterson et al., 2002). CD34+ cell isolation and culture procedures were as described before (Schoeters et al., 2007). Briefly, human cord blood samples were collected from the umbilical blood vessels of placentas of nor-

mal, full-term infants. Informed consent was given by the mothers and the study was approved by the ethical commission of the University of Anwerp (Belgium). Mononuclear cells were separated from the cord blood by density gradient centrifugation and subsequently CD34+ progenitor cells were extracted by positive immunomagnetic selection. These cells were cultured for 12 days in Iscove’s modified Dulbecco’s medium (IMDM; Invitrogen) supplemented with 10% (v/v) FBS, 500 ng/ml GM-CSF (Novartis), 50 ng/ ml stem cell factor (SCF; Biosource), 2.5 ng/ml TNF-a (Roche), and 34 ng/ml IL-4 (Biosource) to induce proliferation and differentiation towards immature CD34–DC, according to the method described by Lardon et al. (1997). At the end of the differentiation period, immature DC cultures were exposed for 6, 10, 24 and/or 48 h to 100 and/or 300 lM nickel(II) sulphate hexahydrate (Merck, Overijse, Belgium) dissolved in bidistilled water, while being kept in conditioned (CD34–DC) or fresh differentiation medium (MUTZ-3 DC). Exposure to 300 lM nickel sulphate inhibited MUTZ-3 DC cell proliferation by 10% (IC10) compared to control cells at 24 h exposure as determined by the Alamar BlueTM fluorescence assay (Immunosource, HalleZoersel, Belgium). Besides this sub-toxic concentration, 100 lM did not result in inhibition of cell proliferation and was used as a non-toxic concentration. Control cultures consisted of CD34–DC in conditioned or MUTZ-3 DC in fresh differentiation medium supplemented with bidistilled water. MUTZ-3 DC and CD34–DC were seeded at a density of 106 cells/ml and exposed in 24-well culture plates (1 ml/well) for flow cytometric studies, whereas for RNA extraction and real-time RT-PCR analyses 6-well plates (4 ml/well) were used. For each exposure condition one well was used. 2.2. Flow cytometry To examine the expression of DC-associated cell surface markers, cells from each exposure condition were harvested, counted and aliquots of 105 cells were prepared in 50 ll cold phosphate buffered saline (PBS) supplemented with 10% FBS. These aliquots were each incubated with a mouse anti-human monoclonal antibody conjugated to either fluorescein isothiocyanate (FITC) or phycoerythrin (PE) at 4 °C for 30 min in the dark. The following antibodies (Becton Dickinson, Erembodegem, Belgium) were used: PE-conjugated HLA-DR, CD83, CD86, CD11c, and CD14, and FITCconjugated CD1a. Mouse isotype controls were PE-conjugated IgG1, IgG2a and IgG2b, and FITC-conjugated IgG1 (Becton Dickinson). Cells were then washed and resuspended at 2  105 cells/ml in PBS. Flow cytometry was performed on a FACStar Plus flow cytometer (Becton Dickinson). Data were collected on-line and analyzed using the Cell Quest software (Becton Dickinson). Gates were set to exclude dead cells and cell debris, which were determined using propidium iodide staining. Percentages of positive cells in M1 as compared to the respective isotype control (99% threshold method) and the geomean fluorescence intensity (MFI) were calculated. For assessment of phenotypical changes stimulation indices (SI) were calculated as follows: (% positive cells  MFI) of chemicaltreated cells, divided by (% positive cells  MFI) of time-related control cells. Results from three to four independent experiments are shown. 2.3. Total RNA extraction MUTZ-3 DC from each exposure condition were collected from 6-well plates, isolated by centrifugation (400 g, 10 min), and lyzed in guanidine isothiocyanate-containing lysis buffer (Qiagen, Hilden, Germany), supplemented with 0.025% (w/v) 8-hydroxychinolin and 50% (v/v) phenol. To enhance cell disruption, cells were kept in lysis buffer for 24 h at 80 °C. Total RNA was extracted with the use of a RNeasy Mini kit (Qiagen), according to a modified

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protocol. Phenol was extracted by addition of 10% (v/v) chloroform to the lyzed cell suspension, and the nucleic acids were bound to silica-based membranes in the presence of 35% ethanol. After treatment with DNase I and two washing steps, RNA was eluted in RNase-free water and stored at 80 °C. The RNA concentration was measured by UV spectrophotometry (NanoDropTM Technologies; Thermo Scientific, Wilmington, US) and its quality was assessed by agarose gel electrophoresis. 2.4. Real-time RT-PCR Transcription of the genes IL1b, IL6, IL8, CCL2, CCL3, CCL3L1 and CCL4 was analyzed in MUTZ-3 DC using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Synthesis of cDNA by reverse transcription, real-time RT-PCR reactions, and primer sequences of all genes studied were as previously described in Verheyen et al. (2005). Gene expression levels were normalized against the expression levels of the housekeeping gene b-actin. Gene expression levels were quantified relative to time-related, control cell cultures (Livak and Schmittgen, 2001) and expressed as fold-change. Results from two independent experiments, each consisting of triplicate measurements are shown. 2.5. Statistical analysis The Student’s t-test was used to assess statistically significant differences between chemical-treated and control cells of surface marker expression in flow cytometric measurements and of gene expression levels obtained in real-time RT-PCR analyses. P-values less than or equal to 0.05 were considered statistically significant. 3. Results and discussion 3.1. Morphologic and phenotypic characterization of the MUTZ-3 cell line after differentiation Morphologically, naive MUTZ-3 cells appeared as polygonally shaped cells in suspension. Differentiation of the cells using a well-defined mixture of cytokines which is known to stimulate

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generation of DC from monocytes and CD34+ stem cells (Caux et al., 1996; Lardon et al., 1997) resulted in elongated cells bearing dendritic processes and forming clusters of high cell density, similar to immature CD34–DC. Expression levels of the surface markers CD1a, HLA-DR, CD86, CD83, CD11c and CD14, specific for DC differentiation and activation, were analyzed in these MUTZ-3 DC by flow cytometry and compared to those of immature CD34–DC (Fig. 1). MUTZ-3 DC were observed to consistently show higher basal expression of the studied markers than CD34–DC, thus representing a more ‘matured’ phenotype. Statistically significant differences in expression levels between both cell models were observed for CD1a (p = 0.002), CD83 (p = 0.001) and CD11c (p = 0.0008). Similar or even higher expression levels of the studied markers have been described by others in immature MUTZ-3 DC when using the same (Chang et al., 2005; Santegoets et al., 2006) or a different cytokine mixture (Kim et al., 2006) to drive DC differentiation. 3.2. Nickel sulphate-induced surface marker expression in MUTZ-3 DC To compare MUTZ-3 DC and CD34–DC for their potential to display a maturation response upon recognition of a skin sensitizing compound, the cells were exposed to the model chemical nickel sulphate at 100 and 300 lM for 24 or 48 h. To assess a positive maturation response, increase of expression of HLA-DR, CD86, CD83 and CD11c was evaluated in exposed compared to control cells using flow cytometry. No stimulatory effect of 100 lM nickel sulphate on the studied markers was observed (data not shown). Using 300 lM nickel sulphate a tendency to maturation was observed in MUTZ-3 DC, but this response (log2 SI < 1) was less evolved compared to CD34–DC (log2 SI > 2), which showed significantly upregulated CD86 and CD83 expression levels after 24 h (p 6 0.05) (Fig. 2). The observed lack of surface marker stimulation in MUTZ-3 DC is also in contrast with findings in the naive MUTZ-3 cell line (Azam et al., 2006), which was previously assessed for its validity as in vitro model to screen potential sensitizers. In the latter study, CD86 (but not HLA-DR, CD40, CD54, CD80, B7-H1, B7-H2, and B7DC) was shown to be able to identify five sensitizing chemicals, but

Fig. 1. Comparison of surface marker expression in unstimulated, immature MUTZ-3 DC with CD34–DC. Gray histograms show flow cytometric analyses in MUTZ-3 DC after 7 days of differentiation from one representative experiment, whereas open histograms are from CD34–DC at day 12. Mean percentages of positive cells in M1 and the corresponding standard deviations (n = 3) are indicated (italic values below are from MUTZ-3 DC, regular values on top from CD34–DC).

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Fig. 2. Surface marker induction in MUTZ-3 DC and CD34–DC after exposure to nickel sulphate. Immature MUTZ-3 DC (A) and CD34–DC (B) were treated with 300 lM nickel sulphate or bidistilled water (control) for 24 and 48 h, and analyzed by flow cytometry. Mean log2 SI as compared to control cultures (mean log2 SI = 0) and the corresponding standard deviations (n = 3–4) are shown. * Indicates |log2 SI| > 1 and p 6 0.05 as evaluated by a Student’s t-test.

not two irritants. The observed differences may be explained by higher basal expression levels of the activation markers in unstimulated MUTZ-3 DC compared to CD34–DC (see Section 3.1) and naive MUTZ-3 cells (Azam et al., 2006). 3.3. Differential cytokine gene expression in MUTZ-3 DC induced by nickel sulphate Previous studies demonstrated that several cytokine genes (IL1b, IL6, IL8, CCL2, CCL3, CCL3L1 and CCL4) were found to be consistently upregulated in response to 100 lM nickel sulphate in CD34–DC derived from different donor samples at selected time points (Schoeters et al., 2006). Further real-time RT-PCR analyses confirmed these results, and identified CCL2, CCL3 and CCL4 as potential markers to discern skin sensitizers from irritants (Verheyen et al., 2005). In this study these seven cytokines were assessed for their responsiveness to nickel sulphate in MUTZ-3 DC to further assess the validity of the cell model as an alternative to primary CD34–DC. Differential analysis of cytokine transcription in MUTZ-3 DC in response to 100 lM nickel sulphate compared to control cells revealed time-dependent upregulated mRNA levels of the chemokines CCL2, CCL3 and CCL4 after 24 h of exposure (Fig. 3A). The threshold of twofold upregulation (log2 foldchange >1) was crossed only in the case of CCL2 and CCL3, but these effects were statistically not significant. In contrast, 300 lM nickel sulphate (Fig. 3B) induced transcription of all genes more than twofold above control levels already after 6 h of exposure, except for IL6. Significantly upregulated levels of IL8, CCL3 and CCL3L1 were observed after 6 or 10 h. This effect gradually decreased with time, reaching background levels again after 24 h. No stimulation

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of IL6 mRNA expression was observed. The observed difference in kinetics of some of the gene responses (e.g. CCL3) to the non-toxic (100 lM) as compared to the sub-toxic concentration (300 lM) of nickel sulphate may be explained by a dose effect and the previously made observation that some degree of cytotoxicity promotes a sensitization response (Hooyberghs et al., 2008; Hulette et al., 2005). The MUTZ-3 DC cytokine data obtained with 300 lM nickel sulphate are in line with earlier observations in CD34–DC (Verheyen et al., 2005). In the latter cell model the maximal stimulatory effect of nickel sulphate treatment was also observed after short incubation periods of 3 or 6 h, except for IL6 expression, which showed its highest increased levels only after 24 h. Similar induction levels (expressed as fold-changes) were obtained in both DC models, although a different ranking of the most inducible cytokines in response to nickel sulphate was observed. In MUTZ-3 DC the three most inducible genes were CCL3L1 (mean fold-change of 63.1 after 6 h), CCL3 (29.3 after 6 h) and CCL4 (13.7 after 6 h), versus CCL4 (45.9 after 3 h), IL-6 (29.5 after 24 h) and IL-8 (20.5 after 6 h) in CD34–DC. However, despite this analogy, the stimulatory effects described above in CD34–DC were already obtained using 100 lM nickel sulphate, thus providing evidence of a more sensitive response to nickel sulphate exposure of this primary DC model. 3.4. Conclusions To our knowledge, this is the first time the response of MUTZ-3 DC to a model sensitizing chemical is investigated and compared with that of a well-established primary DC model. Our findings

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Fig. 3. Cytokine mRNA induction in MUTZ-3 DC after exposure to nickel sulphate. Immature dendritic cells were treated with 100 lM (A) and 300 lM (B) nickel sulphate, or bidistilled water (control), and analyzed by real-time RT-PCR after 6, 10 and 24 h of exposure. Mean log2 fold-change in gene expression levels compared to control levels (mean log2 fold-change = 0) and the corresponding standard deviations (n = 2) are shown. * Indicates |log2 fold-change| > 1 and p 6 0.05 as evaluated by a Student’s t-test.

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demonstrate the stimulatory capacity of nickel sulphate in MUTZ-3 DC with regard to cytokine, but not surface marker expression. Compared to CD34–DC, overexpression of the studied activation markers and loss of responsiveness to nickel sulphate were observed in MUTZ-3 DC. This could be due to pretreatment with differentiating cytokines and maintenance of cytokine treatment during the exposure experiment, as has previously been described in cytokine-treated THP-1 and KG-1 myeloid cell line-based DC models (Hulette et al., 2002; Yoshida et al., 2003). Apparently, CD34–DC that are obtained through differentiation from primary stem cells do not suffer from such ‘pre-maturing’ effects, and therefore remain superior to the MUTZ-3 DC model presented here for in vitro testing of sensitization. It would be interesting to evaluate the effects of different differentiation protocols, as well as individual preconditioning cytokines on the assay sensitivity in MUTZ-3 DC to further assess the validity of this cell model to replace CD34–DC. Furthermore, identification of more sensitive endpoint markers, for instance based on transcriptome responses of MUTZ-3 DC, and additional experiments with an extended set of chemicals may contribute to the development of a MUTZ-3 DCbased test system. Acknowledgements The authors would like to thank Hilde Leppens and Daniëlla Ooms for expert technical assistance. This work was supported by a Grant of the John Hopkins Center for Alternatives to Animal Testing (CAAT, 2005–2006) and the EU FP6 project Sens-it-iv (LSHB-CT-2005-018861). References Aiba, S., Terunuma, A., Manome, H., Tagami, H., 1997. Dendritic cells differently respond to haptens and irritants by their production of cytokines and expression of co-stimulatory molecules. Eur. J. Immunol. 27, 3031–3038. Azam, P., Peiffer, J.-L., Chamousset, D., Tissier, M.-H., Bonnet, P.-A., Vian, L., Fabre, I., Ourlin, J.-C., 2006. The cytokine-dependent MUTZ-3 cell line as an in vitro model for the screening of contact sensitizers. Toxicol. Appl. Pharmacol. 212, 14–23. Berges, C., Naujokat, C., Tinapp, S., Wieczorek, H., Hoh, A., Sadeghi, M., Opelz, G., Daniel, V., 2005. A cell line model for the differentiation of human dendritic cells. Biochem. Biophys. Res. Commun. 333, 896–907. Caux, C., Vanbervliet, B., Massacrier, C., Dezutter-Dambuyant, C., de Saint-Vis, B., Jacquet, C., Yoneda, K., Imamura, S., Schmitt, D., Banchereau, J., 1996. CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to GM-CSF + TNF-alpha. J. Exp. Med. 184, 695–706. Chang, C.C., Satwani, P., Oberfield, N., Vlad, G., Simpson, L.L., Cairo, M.S., 2005. Increased induction of allogeneic-specific cord blood CD4+CD25+ regulatory T (Treg) cells: a comparative study of naïve and antigenic-specific cord blood Treg cells. Exp. Hematol. 33, 1508–1520. De Smedt, A.C., Van Den Heuvel, R.L., Berneman, Z.N., Schoeters, G.E., 2001. Modulation of phenotype, cytokine production and stimulatory function of CD34+-derived DC by NiCl(2) and SDS. Toxicol. In Vitro 15, 319–325.

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