Toxicology 206 (2005) 257–272

Detection of immunotoxicity using T-cell based cytokinereporter cell lines (“Cell Chip”)�

Tove Ringerikea, Erik Ullerasb, Rene Volkerc, Bert Verlaanc, Ase Eikeseta,Dominika Trzaskad, Violetta Adamczewskad, Maciej Olszewskid,

Aurelia Walczak-Drzewieckae, Joanna Arkuszf, Henk van Loverenc,Gunnar Nilssonb, Martinus Lovika, Jarosław Dastychd, Rob J. Vandebrielc,∗

a Division of Environmental Medicine, Norwegian Institute of Public Health, Oslo, Norwayb Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden

c Laboratory for Toxicology, Pathology and Genetics, National Institute of Public Health and the Environment,P.O. Box 1, 3720 BA, Bilthoven, The Netherlands

d International Institute of Molecular and Cell Biology, Warsaw, Polande Center for Medical Biology of the Polish Academy of Sciences, Ł´odz, Poland

f Nofer Institute of Occupational Medicine and WHO/Collaborating Centre, Ł´odz, Poland

Received 25 June 2004; accepted 30 August 2004Available online 18 October 2004

Abstract

effects ofc or in vitroa rtunately,t ions as thes homact hanges infl ell Chip”t cytokinee tm latinate,

0d

Safety assessment of chemicals and drugs is an important regulatory issue. The evaluation of potential adverseompounds on the immune system depends today on animal experiments. An increasing demand, however, exists flternatives. Cytokine measurement is a promising tool to evaluate chemical exposure effects on the immune system. Fo

his type of measurement can be performed in conjunction with in vitro exposure models. We have taken these considerattarting point to develop an in vitro method to efficiently screen compounds for potential immunotoxicity. The T-cell lympell line EL-4 was transfected with the regulatory sequences of interleukin (IL)-2, IL-4, IL-10, interferon (IFN)-� or actin fusedo the gene for enhanced green fluorescent protein (EGFP) in either a stabile or a destabilised form. Consequently, cuorescence intensity represent changes in cytokine expression with one cell line per cytokine. We used this prototype “Co test, by means of flow cytometry, the immunomodulatory potential of 13 substances and were able to detect changes inxpression in 12 cases (successful for cyclosporine, rapamycin, pentamidine, thalidomide, bis(tri-n-butyltin)oxide, house dusite allergen (Der p I), 1-chloro-2,4-dinitrobenzene, benzocaine, tolylene 2,4-diisocyanate, potassium tetrachlorop

� Supported by the EU, contract #QLK4-CT-2000-00787.∗ Corresponding author. Tel.: +31 30 2742610; fax: +31 30 2744437.E-mail address:r.vandebriel@rivm.nl (R.J. Vandebriel).

300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.tox.2004.08.014

258 T. Ringerike et al. / Toxicology 206 (2005) 257–272

sodium dodecyl sulphate and mercuric chloride; unsuccessful for penicillin G). In conclusion, this approach seems promisingfor in vitro screening for potential immunotoxicity, especially when additional cell lines besides T-cells are included.© 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Cell Chip; In vitro; Immunotoxicology; Immunosuppression; Sensitisation; Transfection; EL4; Lymphoma; Cytokine; IL-2; IL-4;IL-10; IFN-�; Actin

1. Introduction

Safety assessment of chemicals and drugs is an im-portant regulatory issue. The evaluation of potentialadverse effects of these compounds on the immune sys-tem depends today on animal experiments. An increas-ing demand, however, exists for in vitro alternatives,one aim being to reduce, refine and replace the use oflaboratory animals. This is also important because thesafety evaluation of cosmetics no longer allows the useof laboratory animals. A second reason is the need forhigh-throughput screening methods that will increasethe speed and reduce the cost per chemical entity forsafety evaluation.

if1ebRe

srfetreaooocasmat

Technology For Fluorescent “Cell Chip” Immunotox-icity testing” (QLK4-CT-2000-00787). A comprehen-sive rationale and a proof-of-principle report have beenlaid down in an accompanying paper (Ulleras et al.,2004).

From the panel of cell types included in the project(lymphocytes, mast cells, keratinocytes, fibroblasts) wehave chosen to first use the T-cell lymphoma EL4, sincethe exposure effects of many compounds often com-prise alterations in T-cell activity (Luster et al., 1992).

We have chosen to measure the expression of fourcytokines that together indicate effects on the size ofthe T-cell response and the balance between T-helper1 (Th1) and T-helper 2 (Th2) response (Abbas et al.,

n

o-celler-it

Assessment of cytokine expression and productions a promising tool to evaluate chemical exposure ef-ects on the immune system (Cohen et al., 1999; House,999; Vandebriel et al., 1998). Importantly, cytokinexpression is a parameter that can be readily com-ined with in vitro approaches (Corsini et al., 1998;

1996; O’Garra, 1998). (1) IL-2 plays a pivotal role ithe growth and function of T-cells (Smith, 1988). (2)IFN-� is produced by Th1 cells only. (3) IL-4 is prduced by Th2 cells only. (4) IL-10 suppresses T-function. Besides, IL-10 plays a key role in the diffentiation and function of regulatory T-cells that lim

yan et al., 2001; Vandebriel et al., 1999; Van Wauwet al., 1996).

Effects on gene expression can be efficiently mea-ured by using a reporter construct, consisting of theegulatory region(s) and promoter of a certain geneused to a promoterless reporter gene. Effects on genexpression are measured as effects on the amount ofranslated product, most often fluorescent proteins. Theesulting fluorescence can be measured by flow cytom-

r vi-cellsongeo-eachon--

uit-era-ew

immune responses (Moore et al., 2001).Here we show that using a set of transfected cell

lines derived from the murine T-cell lymphoma cellline EL4, we were able to identify immunotoxicity for12 out of 13 tested compounds.

2. Methods

2

2o

t omB 5GA tw tor( nedi ech,

try. When the cells are simultaneously stained fobility, fluorescence can be measured in viablenly. In addition, flow cytometry allows the acquisitif data for a large number of individual cells. A ranf cell lines derived from different origins (lymphytes, mast cells, keratinocytes, fibroblasts) thatre transfected with various cytokine reporter cell ctructs (making a “Cell Chip”) may thus identify imunotoxicity. In addition, this system should be sble for high-throughput screening. These consid

ions have been the rationale for the EU project “A N

.1. Construction of EGFP reporter vectors

.1.1. Interleukin-2 reporter vectorThe IL-2 upstream region (−2686/+25 relative t

he transcription start site) was PCR amplified frALB/c mouse genomic DNA using the primers′TA AGC AAC AGG TGA CAG GT 3′ and 5′ GTGGG AGT GAT TAG CAA GG 3′. The PCR producas directly cloned into the pGEM-T EASY vec

Promega, Madison, WI, USA) and then sub-clonto the pd2EGFP-1 and pEGFP-1 vectors (Clont

T. Ringerike et al. / Toxicology 206 (2005) 257–272 259

Palo Alto, CA, USA) using Xho I and Bam HI. Itsidentity was confirmed by sequencing.

2.1.2. Interleukin-4 reporter vectorThe IL-4 upstream region (−797/+5 relative to the

transcription start site) was obtained by PCR from the−797 pCAT plasmid (Henkel et al., 1992) and clonedinto the TA-cloning vector pTAdvance (Clontech). TheCMV promoter in pd1EGFP-N1 (Clontech) was re-placed by the IL-4 upstream region using Sma I andVsp I.

2.1.3. Interleukin-10 reporter vectorThe IL-10 upstream region (−1626/+92 relative to

the transcription start site) was PCR amplified from thepIL10pro(−1626)CAT plasmid (Gollnick et al., 2001)using the primers 5′ AAT TCT CGA GCA GTC AGGAGA GAG GGC AGT 3′ and 5′ AAT TGG ATC CGATGG AGC TCT CTT TTC TGC 3′. The PCR productwas directly cloned into the pGEM-T EASY vector andthen sub-cloned into the pd2EGFP-1 vector using Xho Iand Bam HI. Its identity was confirmed by sequencing.

2.1.4. Interferon-� reporter vectorThe IFN-� upstream region (−2001/+34 relative to

the transcription start site) was PCR amplified fromBALB/c mouse genomic DNA using the primers 5′AGA TTG CTG GCT TCT GTC AC 3′ and 5′ TCTTCT CTA GGT CAG CCG AT 3′. The PCR productwas directly cloned into the pGEM-T EASY vectora FP-1v edb

2cer

a e Ia en)a ) tor amer

2

pre-p ctedb asm a4 per-

ature (RT). Medium was RPMI 1640 withl-Glutamine(GibcoBRL, Invitrogen Corp., Paisley, UK), supple-mented with 100 U/mL penicillin, 100�g/mL strepto-mycin (PAA Laboratories, Linz, Austria) and 10% fe-tal bovine serum (FBS; Gibco BRL). Electroporationwas performed using a Gene Pulser (BioRad, Hercules,CA, USA) with the settings 320 V, 960�F. The cellswere incubated for 10 min at RT, transferred to 10 mLmedium in a Petri dish and allowed to recover for 48 h.Medium was then replaced with medium containing1 mg/mL G418 (PAA). G418 containing medium wasreplaced every second day. Selection was continueduntil all the mock transfected cells were dead (approx-imately 1 week).

2.3. Cloning of reporter cell lines

The G418 resistant cells were expanded. Limitingdilution cloning was performed by seeding the cells in96-well plates to a final concentration of 0.5 cell perwell in G418 containing medium. Resulting single cellderived clones were expanded and frozen. The presenceof the integrated reporter plasmid was verified by PCRfor the EGFP coding region on genomic DNA from theclones (data not shown).

2.4. Cell culture

T-cell lymphoma cells (EL-4, ATCC number TIB-39) (Hyman et al., 1972; Ralph, 1973) were stablet ionsf eg ndI sta-b

tu-t eyw 2w sedt d at1 rew

2

as-s ) ac-t ics,M mi-

nd then sub-cloned into the pd2EGFP-1 and pEGectors using Apa I and Pst I. Its identity was confirmy sequencing.

.1.5. Actin reporter vectorThe CAG promoter (containing the CMV enhan

nd chicken�-actin promoter) was excised using Asnd Eco47III from the pQE-TriSystem vector (Qiagnd ligated into the pd1EGFP-N1 vector (Clontecheplace the CMV promoter (excised using the sestriction enzymes).

.2. Stable transfections

Reporter plasmids and mock plasmid wereared using a plasmid midi kit (Qiagen) and transfey electroporation. Twenty micrograms plasmid wixed with 1× 107 EL4 cells in 0.4 mL medium inmm cuvette and incubated for 10 min at room tem

ransfected with the regulatory and promoter regrom IL-2, IL-4, IL-10, IFN-� and actin fused to thene for destabilised EGFP. In addition, for IL-2 a

FN-� clones containing these regions but fused toile EGFP were made.

Cells were cultured in medium. After reconstiion from storage in liquid nitrogen, however, there cultured in G418 containing medium foreeks to eliminate cells that no longer expres

he transfected construct. Cells were maintaine× 105–1× 106 cells/mL in a humidified atmospheith 5% CO2 at 37◦C.

.5. Selection of in vitro exposure concentrations

Cell death/cell lysis was established using anay that measures lactate dehydrogenase (LDHivity released from the cytosol (Roche Diagnostannheim, Germany). The concentration of a che

260 T. Ringerike et al. / Toxicology 206 (2005) 257–272

cal corresponding to 10% cytotoxicity was chosen tobe the most concentrated one to be tested.

2.6. Exposure to xenobiotics

Cells were exposed in 24-well plates at a densityof 0.5× 106 cells/mL. Two controls were included foreach cell line. One control consisted of cells in mediumonly, while the second control consisted of cells in thepresence of 1�M ionomycin (Sigma, St Louis, MO,USA) and 10 ng/mL PMA (Sigma). Cells were incu-bated with the different chemicals at the concentrationthat led to 10% cytotoxicity, as well as concentrations10× and 100× more diluted, in the absence or the pres-ence of ionomycin/PMA. Cells were incubated in a hu-midified atmosphere, 5% CO2 and 37◦C, and after 24 hthe samples were analysed by flow cytometry.

2.7. Flow cytometry

Samples were analysed in an EPICS® XL-MCLCoulter flow cytometer with Expo v.2 Analysis Soft-ware/Expo32 Analysis Software (Applied CytometrySystems, Sheffield, UK). After staining with propidiumiodide (0.75�g/mL, Sigma) two distinct cell popula-tions were apparent, representing dead and viable cells,and they were gated separately. The results presentedare from viable cells only. Regions were set in plots ofviable control cells, and the same regions were usedfor cells exposed to ionomycin/PMA only, as well asf o-m

2

Aw n flu-o di-t = 1a cal-c oleda Stat2

2

esw ne

way analysis of variance on Ranks. If the dif-ferences between groups were statistically signifi-cant (p< 0.05), we continued with tests for multiplecomparisons. In normally distributed data we usedBonferroni t-test and multiple comparisons versusthe control. When normality failed we used Dun-nett’s Method for multiple comparisons versus thecontrol.

2.10. Chemicals and reagents

Cyclosporine (minimum purity 95%, Sigma) wasdiluted in ethanol to 5 mg/mL. Rapamycin (mini-mum purity 95%, Sigma) was dissolved in DMSO to2 mg/mL. Pentamidine isethionate salt (Sigma) wasdissolved in DMSO to 25 mg/mL. (±)-Thalidomide(purity >98%, Sigma) was dissolved in DMSO to0.2 M. Prior to experiments, the solution was dilutedin medium, which led to a white precipitate. The so-lution was resuspended and used for further dilutions.Bis(tri-n-butyltin)oxide (TBTO; purity 96%, Aldrich)was diluted in ethanol to 10 mM. House dust mited-pteronyssinus (Alutard SQ Depot allergen extract;suspension for injection, 100,000 SQ-U/mL solution,ALK-Abell o, Hørsholm, Denmark) was kept at 4–8◦C.1-Chloro-2,4-dinitrobenzene (DNCB; purity minimum98%, Sigma) was diluted in ethanol to 2 mM. Ben-zocaine (ethyl-4-aminobenzoate, Sigma) was dilutedin ethanol to 0.5 M. Tolylene 2,4-diisocyanate (TDI;Sigma, purity 95%) was first dissolved in DMSOt lu-t e-fI ofh bot-t ble.T evens oro-p inm n)s di-l ate( Ot y9 llc tocks ited

or cells exposed to xenobiotics (with or without ionycin/PMA).

.8. Data presentation

Cells incubated with or without ionomycin/PMere analysed separately. In each case, the mediarescence intensity from the control (i.e. without ad

ion of xenobiotics) was defined as index numbernd the index numbers for the exposed cells wereulated. Data from replicate experiments were pond statistical analysis was performed using Sigma.03 software (SPSS Inc., Chicago, IL, USA).

.9. Statistical analysis

One way ANOVA was performed. In cashere normality failed, we used Kruskal–Wallis o

o 0.1 M on the day of the experiment. This soion was further diluted in medium to 1 mM, bore final dilution in medium (Pons et al., 1999).t is noteworthy that the TDI/DMSO solution isigher density than medium and sinks to the

om of the tube as well as becoming insoluhe white precipitate was resuspended to anuspension before dilution. Potassium tetrachllatinate(II) (purity 99.99%, Aldrich) was dilutededium to 30 mM. Penicillin G (benzylpenicilli

odium salt (activity >1477 U/mg, Sigma) wasuted in medium to 200 mM. Sodium dodecyl sulphSDS; purity >85%, Merck) was diluted in DMSo 100 mM. Mercury(II) chloride (minimum purit9.5%, Merck) was diluted in ethanol to 6 mM. Ahemicals were diluted in medium before use. Solutions were kept at−20◦C, except house dust m-pteronyssinus.

T. Ringerike et al. / Toxicology 206 (2005) 257–272 261

3. Results

To study the use of cells transfected with cytokinereporter constructs as a possible tool to screen chemi-cals for potential immunomodulatory or immunotoxicactivity, the mouse T-cell line EL4 was transfected withthe regulatory sequences of IL-2, IL-4, IL-10, IFN-�and actin fused to the gene for EGFP. Thereby, changesin fluorescence intensity represent changes in cytokineexpression (Ulleras et al., 2004). For each compoundthe toxic concentration was defined as the concen-tration that led to 10% cytotoxicity in LDH-releaseassays after 24 h (data not shown). The two condi-tions, without and with induction by ionomycin/PMA,were used to identify stimulatory and suppressive ac-tivities, respectively. Ionomycin/PMA induces a pro-ductive cell activation that includes expression ofIL-2, IL-4, IL-10 and IFN-� (Macian et al., 2002;Noble et al., 1993). The actin clone was used as a con-trol for specificity. In this clone, EGFP is expressedconstitutively, but it is also further inducible. To con-trol for non-specific changes in fluorescence uponadding the different compounds, we also used non-transfected cells. The substances were selected basedon knowledge of their in vivo effects, to cover a range

Table 1Summary of results

A cells tra dilution;L no effe

of immunotoxic effects and thus potential outcomes(Table 1).

3.1. Immunosuppressants

Cyclosporine (Cs) and rapamycin are well-knownimmunosuppressive drugs used in organ transplanta-tion. Pentamidine is used as an antiprotozoal drug, buthas also been shown to reduce expression of several cy-tokines. Thalidomide is used as an anti-inflammatorydrug. Bis(tri-n-butyltin)oxide (TBTO) has also beenshown to be immunosuppressive, both in vivo and invitro.

3.1.1. CyclosporineIn the absence of ionomycin/PMA, we found a sta-

tistically significant dose-dependent decrease in IL-4and IL-10 at 1 and 0.1�M. Surprisingly, we found asignificantly higher expression of actin at the highestconcentration (1�M) (Fig. 1A). In the presence of ion-omycin/PMA we found significantly lower induction inall clones, at all concentrations, except for IL-4 at thelowest concentration (Fig. 1B). The suppressive actionof Cs in stimulated cells was confirmed for IL-2 andIFN-�, by using clones transfected with the same reg-

bbreviations:D, cells transfected with destabilised EGFP; S,, 1:100 dilution;↑, stimulatory effect;↓, inhibitory effect; blank,

nsfected with stable EGFP; H, highest concentration; M, 1:10cts observed; grey field, not tested.

262 T. Ringerike et al. / Toxicology 206 (2005) 257–272

Fig. 1. Cyclosporine. Cells transfected with the regulatory elements from actin (black bars), IL-2 (grey bars), IL-4 (wide striped bars), IL-10(diamond bars) and IFN-� (narrow striped bars) fused to EGFP were exposed to 1�M Cs, which led to 10% cytotoxicity, and 1:10 or 1:100dilution of this for 24 h in the absence (A) or presence (B) of ionomycin/PMA (1�M/10 ng/mL). In non-induced cells (A) at the highestconcentration, we found statistically significant differences compared to the control for actin, IL-4 and IL-10. At the 1:10 dilution, only IL-4and IL-10 were statistically significantly different from their control. In stimulated cells (B) expression of all genes tested were statisticallysignificantly different from their control, except for IL-4 at the 1:100 dilution. The graphs show the mean values with S.E.M. (n= 3). Statisticallysignificant findings with*p< 0.05,** p< 0.01 and*** p< 0.001. The dashed line represents the control level.

ulatory elements, but fused to a stabile form of EGFP.In these clones in the presence of ionomycin/PMA,Cs exposure resulted in a dose-dependent reductionof IL-2 expression at 0.01 and 0.1�M (p< 0.01)and of IFN-� expression at 0.01�M (p< 0.01) and0.1�M (p< 0.001). In the absence of ionomycin/PMAthese cells failed to show exposure effects (data notshown).

3.1.2. RapamycinIn the absence of ionomycin/PMA, we found a sig-

nificant reduction of IL-4 at all tested concentrations ofrapamycin (10, 1 and 0.1�g/mL), while IL-10 showedsuppression only at the highest concentration. How-ever, we also found a significant increase of actin at thetwo lowest concentrations (Fig. 2A). In the presenceof ionomycin/PMA, at the highest concentration, ra-pamycin induced suppression of IL-2, IL-4 and IL-10.IFN-� was also suppressed at the highest concentration,but statistical analysis did not confirm the trend, due tolow power of the test (Fig. 2B). At the two lowest con-centrations (1 and 0.1�g/mL) IL-2 and actin showed asignificant increase. IL-10 and IFN-� showed a trendtowards an increase, while IL-4 was not affected.

3.1.3. PentamidineWe found an increase in actin at the two highest con-

centrations of pentamidine (30 and 3�g/mL), while IL-10 was clearly inhibited at these concentrations in theabsence of ionomycin/PMA (Fig. 3A). In the presenceof ionomycin/PMA, pentamidine exerted suppressiveactivity, since at the highest concentration all cytokineswere suppressed, while at the 1:10 dilution all cytokinesexcept IFN-� were suppressed (Fig. 3B). The expres-sion of actin was not altered in the presence of pen-tamidine at any of the concentrations tested in iono-mycin/PMA activated cells.

3.1.4. ThalidomideWe did not find thalidomide to be toxic in the LDH-

assay and used 1 mM as the highest concentration.Cells exposed to thalidomide in the absence of ion-omycin/PMA did not show any significant changescompared to their controls, except for actin at 1 mM(p< 0.05) and even there the reduction was very small(5–10% inhibition; data not shown). The same resultwas apparent in the presence of ionomycin/PMA, withonly actin inhibited (p< 0.01). However, experimentsusing IL-2 and IFN-� fused to a stabile form of EGFP

T. Ringerike et al. / Toxicology 206 (2005) 257–272 263

Fig. 2. Rapamycin. Cells transfected with the regulatory elements from actin (black bars), IL-2 (grey bars), IL-4 (wide striped bars), IL-10(diamond bars) and IFN-� (narrow striped bars) fused to EGFP were exposed to 10�g/mL rapamycin, which led to 10% cytotoxicity, and 1:10or 1:100 dilution of this for 24 h in the absence (A) or presence (B) of ionomycin/PMA (1�M/10 ng/mL). In non-induced cells (A) at the highestconcentration, we found a statistically significant decrease in fluorescence compared to the control for IL-4 and IL-10. At the 1:10 dilution, IL-4was still inhibited while actin was statistically significantly increased. In stimulated cells (B) at the highest concentration IL-2, IL-4 and IL-10were statistically significantly different from their controls. At the 1:10 and 1:100 dilutions, actin and IL-2 showed a statistically significantincrease compared to their controls. The graphs show the mean values with SEM (n= 3 for IL-10 and IFN-�, n= 4 for actin and IL-4,n= 5 forIL-2). Statistically significant findings with*p< 0.05,** p< 0.01 and*** p< 0.001. The dashed line represents the control level.

Fig. 3. Pentamidine. Cells transfected with the regulatory elements from actin (black bars), IL-2 (grey bars), IL-4 (wide striped bars), IL-10(diamond bars) and IFN-� (narrow striped bars) fused to EGFP were exposed to 30�g/mL pentamidine, which led to 10% cytotoxicity, and1:10 or 1:100 dilutions of this for 24 h in the absence (A) or presence (B) of ionomycin/PMA (1�M/10 ng/mL). In non-induced cells (A) at thehighest concentration and 1:10 dilution, we found statistically significant differences compared to the control for actin and IL-10. In stimulatedcells (B) at the highest concentration all tested cytokines were statistically significantly different from their controls. At the 1:10 dilution, IL-2,IL-4 and IL-10 were statistically significantly different from their controls. The graphs show the mean values with SEM (n= 3 exceptn= 4 forIL-2). Statistically significant findings with*p< 0.05,** p< 0.01 and*** p< 0.001. The dashed line represents the control level.

264 T. Ringerike et al. / Toxicology 206 (2005) 257–272

in the absence of ionomycin/PMA showed a signif-icant inhibition of 10% for IL-2 at 1 mM (p< 0.05).In the presence of ionomycin/PMA both IL-2 andIFN-� showed a dose-dependent inhibition at 1 mM(p< 0.001). Thalidomide exposure did not show de-tectable effects at the two lowest concentrations.

3.1.5. Bis(tri-n-butyltin)oxide (TBTO)Experiments using IL-2 and IFN-� fused to a stabile

form of EGFP, in the absence of ionomycin/PMA at anyTBTO concentration tested (50, 5 and 0.5 nM), failed toshow altered expression of both IL-2 and IFN-�. In thepresence of ionomycin/PMA, a statistically significantinhibition of IL-2 at the 1:10 dilution (5 nM) was found(p< 0.01).

3.2. Allergens and autoimmunity inducing agents

We also wanted to examine the possible use ofthe “Cell Chip” panel to detect (and distinguish)different kinds of allergens. Substances representingthree classes of allergens were included: (1) IgE-mediated respiratory allergy to protein allergens, ex-emplified by the mite allergen Der p I, (2) contactallergy to low molecular weight substances mediatedby hapten-protein conjugate specific T lymphocytes,

F ry elem ed bars),I GFP w tion, and1 ence ( eh rescen -10. In thep und (Ba ificant vel.

exemplified by 1-chloro-2,4-dinitrobenzene (strong al-lergen) and benzocaine (weak allergen), and (3) low-molecular weight chemical allergens, causing clinicalsymptoms similar to IgE-mediated allergy but withunknown mechanisms, exemplified by tolylene 2,4-diisocyanate where specific IgE is demonstrable onlyin a minority of cases and potassium tetrachloroplati-nate where specific IgE is regularly demonstrable. Peni-cillin G was included because it is known to inducedrug hypersensitivity in humans. Furthermore, an ir-ritant (sodium dodecyl sulphate) was included, be-cause the distinction between irritants and contact al-lergens is an important problem in contact allergy test-ing. Finally, mercuric chloride was included since itis known to induce Th1 and Th2 subsets leading toautoimmunity.

3.2.1. Der p-dust mite allergenWe used a Der p solution produced for skin prick

testing, and added it to cells at 3000, 300 and 30 Stan-dard Quality U/mL as previously described byRusznaket al. (1999). We found that Der p increased the fluo-rescence at the highest concentration in the absence ofionomycin/PMA for all clones except IL-10 (Fig. 4A).No significant changes were observed in the presenceof ionomycin/PMA (Fig. 4B).

ig. 4. Der p-mite allergen. Cells transfected with the regulatoL-10 (diamond bars) and IFN-� (narrow striped bars) fused to E:10 or 1:100 dilution of this for 24 h in the absence (A) or presighest concentration, a statistically significant increase in fluoresence of stimulation no alterations in fluorescence were foctin and IL-4,n= 5 for IFN-� andn= 7 for IL-2). Statistically sign

ents from actin (black bars), IL-2 (grey bars), IL-4 (wide stripere exposed to 3000 SQU/mL Der p, as the highest concentraB) of ionomycin/PMA (1�M/10 ng/mL). In non-induced cells (A) at thce compared to the control was found for all clones except IL

). The graphs show the mean values with SEM (n= 3 for IL-10, n= 4 forfindings with*p< 0.05. The dashed line represents the control le

T. Ringerike et al. / Toxicology 206 (2005) 257–272 265

Fig. 5. DNCB. Cells transfected with the regulatory elements from actin (black bars), IL-2 (grey bars), IL-4 (wide striped bars), IL-10 (diamondbars) and IFN-� (narrow striped bars) fused to EGFP were exposed to10�M DNCB, which led to 10% cytotoxicity, and 1:10 or 1:100 dilutionsof this for 24 h in the absence (A) or presence (B) of ionomycin/PMA (1�M/10 ng/mL). In non-induced cells (A) at the highest concentration, wefound a statistically significant increase in fluorescence compared to the control for actin, IL-2, IL-4 and IFN-�. In the presence of stimulation,no statistically significant effects were found (B). The graphs show the mean values with SEM (n= 3). Statistically significant findings with*p< 0.05,** p< 0.01 and*** p< 0.001. The dashed line represents the control level.

3.2.2. 1-Chloro-2,4-dinitrobenzene (DNCB)In the absence of ionomycin/PMA, at the highest

concentration (10�M) of DNCB, an increased expres-sion was seen for all clones, except IL-10. The largestincrease was observed in cells transfected with actinregulatory elements (Fig. 5A). In the presence of iono-mycin/PMA, no statistically significant changes werefound but the trend was towards a decrease in fluores-cence at the highest concentration (Fig. 5B). For cellstransfected with a stable form of EGFP, similar resultswere found. DNCB failed to alter expression of IL-2or IFN-� in the presence of ionomycin/PMA, whilein the absence of these stimuli IFN-� showed an ap-parently dose-dependent increase reaching statisticalsignificance at the highest concentration (p< 0.001).

3.2.3. BenzocaineWe did not find benzocaine to be toxic in the LDH-

assay and used 1 mM as the highest concentration. Inthe absence of ionomycin/PMA, only actin at the high-est concentration was significantly increased (Fig. 6A).In the presence of ionomycin/PMA, a statistically sig-nificant inhibitory effect on IL-2 and IL-10 was seen atthe two highest concentrations. IFN-� was also inhib-ited, but only at the highest concentration (Fig. 6B).

3.2.4. Tolylene 2,4-diisocyanate (TDI)Cells were exposed to TDI at 500, 50 or 5�M. In the

absence of ionomycin/PMA, only IFN-� showed a sig-nificant induction and only at the highest concentration(Fig. 7A). However, in the presence of ionomycin/PMAa statistically significant increase was seen for IL-4 andagain only at the highest concentration (Fig. 7B).

3.2.5. Potassium tetrachloroplatinate (K2PtCl4)In experiments using IL-2 or IFN-� fused to a stabile

form of EGFP, cells were exposed to K2PtCl4 at 100,10 or 1�M. In the presence of ionomycin/PMA, expo-sure resulted in a dose-dependent reduction of IL-2 andIFN-� expression. Statistically significant values werefound at 100�M, with p< 0.001 for IL-2 andp< 0.05for IFN-�. In the absence of ionomycin/PMA, exposureeffects were not observed.

3.2.6. Penicillin GWe did not find penicillin G to be toxic. At the tested

concentrations (10, 1 and 0.1 mM), penicillin G expo-sure failed to alter expression of either IL-2 or IFN-� incells transfected with IL-2 or IFN-� fused to a stabileform of EGFP. This was found both in the presence andabsence of ionomycin/PMA.

266 T. Ringerike et al. / Toxicology 206 (2005) 257–272

Fig. 6. Benzocaine. Cells transfected with the regulatory elements from actin (black bars), IL-2 (grey bars), IL-4 (wide striped bars), IL-10(diamond bars) and IFN-� (narrow striped bars) fused to EGFP were exposed to 1 mM benzocaine as the highest concentration and 1:10 or1:100 dilution of this for 24 h in the absence (A) or presence (B) of ionomycin/PMA (1�M/10 ng/mL). In non-induced cells (A) at the highestconcentration, a statistical increase in fluorescence compared to the control for actin is apparent. In the presence of stimulation, IL-2, IL-10 andIFN-� are reduced at the highest concentration. IL-2 and IL-10 are also reduced at the 1:10 dilution (B). The graphs show the mean values withS.E.M. (n= 3). Statistically significant findings with*p< 0.05,** p< 0.01 and*** p< 0.001. The dashed line represents the control level.

3.2.7. Sodium dodecyl sulphate (SDS)Cells were exposed to SDS at 300, 30 or 3�M.

In cells transfected with IL-2 or IFN-� fused toa stabile form of EGFP, in the presence of ion-

omycin/PMA, exposure resulted in an apparentlydose-dependent reduction of IL-2 expression, withp< 0.05 at the highest concentration. IFN-� expres-sion was, however, not affected. In the absence

Fig. 7. TDI. Cells transfected with the regulatory elements from actin (black bars), IL-2 (grey bars), IL-4 (wide striped bars), IL-10 (diamondbars) and IFN-� (narrow striped bars) fused to EGFP were exposed to 500�M TDI, which led to 10% cytotoxicity, and 1:10 or 1:100 dilutionsof this for 24 h in the absence (A) or presence (B) of ionomycin/PMA (1�M/10 ng/mL). In non-induced cells (A) at the highest concentration,we found a statistically significant increase in fluorescence compared to the control for IFN-�. However, in stimulated cells (B) at the highestconcentration IL-4 was the only one that was statistically significantly different from the control. The graphs show the mean values with S.E.M.(n= 3 for actin, IL-4 and IL-10,n= 7 for IL-2 and IFN-�). Statistically significant findings with*p< 0.05. The dashed line represents the controllevel.

T. Ringerike et al. / Toxicology 206 (2005) 257–272 267

of ionomycin/PMA exposure effects were not ob-served.

3.2.8. Mercuric chloride (HgCl2)Cells transfected with IL-2 or IFN-� fused to a sta-

bile form of EGFP were exposed to mercuric chloride at6, 0.6 and 0.06�M. In the presence of ionomycin/PMAexposure resulted in an apparently dose-dependent re-duction of IFN-� expression reaching statistical signif-icance at 6�M (p< 0.05). IL-2 expression was, how-ever, not affected. In the absence of ionomycin/PMAexposure effects were not observed.

4. Discussion

We have shown that the prototype “Cell Chip”, com-posed of cytokine reporter cell lines on the basis ofthe murine T-cell lymphoma EL4, is able to detectthe immunomodulatory action of different model com-pounds. This current prototype works well for detectionof immunosuppressive potential. The suppressive ac-tivity can be determined in non-stimulated cells, but ismore readily detected in induced cells. However, the re-sults are less convincing for compounds with expectedsensitising potential.

4.1. Immunosuppressive substances

The immune system has very complex regulatorym cha-n e ac-t r T-c hi-b ctso ichc pro-m ulare im-m find aw ithd

4ns-

f t forI erec and

this suggests that the effects indeed are cytokine spe-cific. Our data are in agreement with other reportedfindings. IL-2 production in Jurkat cells, another T-cellline, required both PMA and an increased intracellu-lar Ca2+ concentration ([Ca2+] i) and this productioncould be blocked by Cs (Manger et al., 1986). Cs in-activates the protein phosphatase calcineurin. Conse-quently the nuclear factor of activated T-cells (NF-AT)remains phosphorylated and translocation to the nu-cleus is blocked, thereby failing to activate genes reg-ulated by NF-AT (Cai et al., 1996; Ho et al., 1996).Han et al. (1995)showed that Cs reduced mRNA ex-pression of IL-2, IL-3, IFN-γ, GM-CSF and TNF-α inconcanavalin A (Con A) stimulated cells. Con A exertsits action via CD3, whereas ionomycin/PMA pharma-cologically mimic complete stimulation through CD3and CD28 (Macian et al., 2002).

We also found reduced expression of actin, al-though this effect was much smaller than for the cy-tokines (seeFig. 1B). The rationale behind testing ofthe “Cell Chip” was to use the concentration that ledto 10% cytotoxicity as the highest concentration for alltested compounds. Apparently, the highest concentra-tion had a non-specific inhibitory action in addition tothe cytokine specific action. The concentrations usedin the present study (maximal 1µM or 1.2µg/mL) arehigher than the 1–100 ng/mL previously reported to re-sult in a dose-dependent inhibition of IL-2 expression(Meredith and Scott, 1994; Vandebriel et al., 1999) andthis could explain the non-specific inhibition.

4res-

s e att IL-4 andi 4 int ays ex-p

dif-f par-t ionat n( ells( sed[ no-

echanisms, and several different effector meisms. Many substances exert immunosuppressiv

ions. Many immunosuppressive compounds alteell activities. Their mode of action varies from inition prior to transcription, as for Cs, to later effen signalling, like for rapamycin. Depending on whells are affected, the immune function may be comised globally, or more or less restricted to particffector mechanisms. Therefore, testing putativeunosuppressive substances, one must expect toide range of diverse effects on cellular function wifferent substances.

.1.1. CyclosporineWe found that Cs reduced transcription in all tra

ected cell lines at all concentrations tested, excepL-4 at the lowest concentration. The findings wonfirmed in cells transfected with stabile EGFP

.1.2. RapamycinIn general, rapamycin suppressed cytokine exp

ion at the highest concentration, while exposurhe two lower concentrations resulted in reduced

expression in the absence of ionomycin/PMAncreased expression of all cytokines except IL-he presence of ionomycin/PMA. Collectively, this muggest that rapamycin has specific effects on IL-4ression.

Our observation that Cs and rapamycin haveerent effects on cytokine expression may be (ially) explained by the different modes of actnd impact on [Ca2+] i . Almawi et al. (1999)showed

hat Cs inhibited [Ca2+] i rise in phytohemagglutiniPHA) stimulated peripheral blood mononuclear cPBMCs), while rapamycin on the other hand increaCa2+] i . PHA exerts its action via CD3, whereas io

268 T. Ringerike et al. / Toxicology 206 (2005) 257–272

mycin/PMA exerts its action via both CD3 and CD28(Kruisbeek and Shevach, 1992; Macian et al., 2002).Almawi et al. (1999)also showed that both Cs andrapamycin inhibited ionomycin/PMA induced prolif-erative responses. Cs inhibits the early events of T-cell activation leading to cytokine expression, whilerapamycin targets a kinase involved in cytokine-drivensignals and T-cell expansion via cell cycle progression(Kirken and Wang, 2003) and thereby may not havean inhibitory effect on cytokine expression. This is incorrespondence with our findings.

4.1.3. PentamidineIn the absence of PMA/ionomycin at the two highest

concentrations expression of actin was increased, whileexpression of IL-10 was decreased. In the presence ofPMA/ionomycin we found a reduced expression of alltested cytokines (IL-2, IL-4, IL-10 and IFN-γ). Thissuppressive action is consistent with the reported inhi-bition of PHA and Con A induced mouse splenocyteproliferation (Ferrante et al., 1985). It differs, how-ever, from observations in PHA stimulated whole bloodwhere pentamidine, at similar concentrations as usedabove, selectively inhibited IL-8 production but did notaffect production of TNF-α, IL-1β, IL-2, IL-5 and IL-10 (Van Wauwe et al., 1996). These differences may(in part) be caused by differences in species, cell typesand stimuli.

In the stimulated cells, pentamidine did not alter theexpression of actin at any of the tested concentrations,w ibi-t bev ics.

4ion

w tableE nceo ndI ef-fe im-u sesw in-c -p llsh andt ac-

tivated in such a way are unlikely to respond to cos-timulatory agents, such as thalidomide.McHugh et al.(1995)have shown induction of IL-4 and IL-5 and in-hibition of IFN-γ production by PHA-stimulated hu-man PBMCs due to thalidomide exposure, in keepingwith the results obtained for the clones based on sta-ble EGFP. Similarly to pentamidine, differences may(in part) be caused by differences in species, cell typesand stimuli.

4.1.5. Bis(tri-n-butyltin)oxide (TBTO)The present study shows suppression of IL-2 but

not IFN-γ at 5 nM TBTO in ionomycin/PMA-activatedcells. RT-PCR showed that in vitro TBTO exposureof Con A activated rat splenocytes resulted in a dose-dependent decrease in IFN-γ mRNA expression at3 nM, while IL-2 and IL-10 expression were not af-fected (IL-4 was not tested;Vandebriel et al., 1999).Northern blot analysis of similarly treated cells did not,however, show any exposure effects on IFN-γ, IL-2,IL-4 and IL-10 mRNA expression. Again, differencesbetween species, cell types and stimuli could be re-sponsible for the differences observed.

4.2. Allergens and autoimmunity inducing agents

Allergens fall into different categories with regardto molecular weight and mechanisms whereby an aller-gic immune response is elicited. We included a proteinallergen preparation that causes IgE-mediated allergy( ns( ght“ teda l-l wei an-t ani

4dust

m singI thea ast oundf rr ofP 2

hile all cytokines showed a dose-dependent inhion. This suggests that the “Cell Chip” system canery (cytokine) specific in its response to xenobiot

.1.4. ThalidomideIn the absence of ionomycin/PMA IL-2 express

as decreased at the highest concentration with sGFP, but not with destabilised EGFP. In the presef ionomycin/PMA we found a decrease in IL-2 a

FN-γ expression in cells with stable EGFP and noect in cells with the destabilised form of EGFP.Haslettt al. (1998)showed that thalidomide is a potent costlator of primary human T-cells in vitro and synergiith stimulation via the T-cell receptor complex torease IL-2 mediated T-cell proliferation and IFNγroduction. Since ionomycin/PMA stimulated T-ceave undergone a productive proliferative response

herefore do not require further costimulation, cells

Der p), two low-molecular weight contact allergeDNCB and benzocaine) and low-molecular weichemical” allergens that cause either IgE-mediallergy (K2PtCl4 and penicillin G) or respiratory a

ergy with unknown mechanisms (TDI). Further,ncluded HgCl2 that causes polyclonal IgE and autoibody production, and SDS that is predominantlyrritant.

.2.1. Der p-dust mite allergenDer p, an allergen preparation of the house

ite, contains a number of protein allergens caugE-mediated respiratory allergy. Upon exposure inbsence of ionomycin/PMA the increase in IL-4 w

he largest, but increased expression was also for actin, IL-2 and IFN-�. IL-10 was not affected. Ouesults are in partial agreement with the findingshillips et al. (2003)with the human basophil KU81

T. Ringerike et al. / Toxicology 206 (2005) 257–272 269

cell line incubated with 1�g/mL Der p I. They foundincreased mRNA levels of IL-4, IL-5 and IL-13, butnot IFN-� and�-actin. The differences in effects may(in part) be explained by differences between speciesand cell types. The increase in IL-4 is as expected foran allergy-type immune response. This suggests thatthe prototypic cell chip may be further developed intoa pre-screen tool for protein allergens, although fur-ther testing using allergens and non-allergenic proteinsare required. An issue of relevance is the fact that theenzymatic activity of some mite allergens seems tobe required for the observed effects (Stewart, 2000;Hewitt et al., 1995; Schulz et al., 1998), suggestingthat this activity should be considered in evaluation ofthe test panel.

4.2.2. 1-Chloro-2,4-dinitrobenzene (DNCB)We found an increase in actin, IL-2, IL-4 and IFN-�

in the absence of ionomycin/PMA. DNCB is a pro-totypic contact allergen and in vivo DNCB induces aTh1 response. It has to be emphasised, however, thatin vivo DNCB is presented by dendritic cells to the T-cell receptor (Gorbachev and Fairchild, 2001). In vitro,DNCB may bind to various proteins of the T-cell itself.The absence of antigen presentation and costimulationby the dendritic cells makes the in vitro situation dif-ferent from the in vivo situation.

Only for the allergens DNCB and Der p did wefind a statistically significant increase in the expres-sion of cytokines in the absence of stimulation by ion-o theh ffectw us-i er pd hosei ant( ilityt eralle

4the

h cante r andh darda eo ex-p was

the only compound tested that showed a statisticallysignificant reduction of fluorescence in non-transfectedcells (data not shown). The reduction was about 20%at the highest concentration, while the reduction intransfected cells at the same concentration was larger(35–65% inhibition), suggesting that non-specific ef-fects form a part of the total effect.

4.2.4. Tolylene 2,4-diisocyanate (TDI)In the absence of ionomycin/PMA, we found an in-

crease in IFN-� at the highest concentration. In thepresence of stimulation IL-4 was induced at the sameconcentration. TDI is poorly soluble in aqueous so-lutions and at the highest concentration some precip-itation occurs. This may inhibit or change the toxicproperties of TDI in addition to alterations in the ac-cessibility to the cells.

TDI can cause occupational asthma (Liu andWisnewski, 2003). TDI has characteristics of both acontact and a respiratory allergen. In the local lymphnode assay (LLNA) it has been shown that at lowerconcentrations TDI acts as contact allergen while athigher concentrations it acts as a respiratory sensitizer,seen as a the predominant production of IFN-� and IL-4, respectively (Van Och et al., 2002). Although wefound IFN-� and IL-4 to be induced in non-stimulatedand stimulated cells, respectively, the relevance of thisfinding for the two-sided characteristic of TDI remainsto be established.

4tra-

c IL-2a 2-d here-f hats spi-r

4ad

t ineg eser eni-c o-v enic(

mycin/PMA. This was, however, only apparent atighest concentration and at least for DNCB the eas equally strong for actin. Control experiments

ng non-transfected cells exposed to DNCB and Did also result in an increase in fluorescence. T

ncreases were, however, not statistically significdata not shown). We cannot exclude the possibhat some non-specific effects contribute to the ovffects seen in DNCB or Der p treated cells.

.2.3. BenzocaineIn the absence of ionomycin/PMA, only actin at

ighest concentration showed a statistically signififfect (increase). Benzocaine is a weak sensitizeas been reported to give variable results in stannimal tests (Basketter et al., 1995). In the presencf ionomycin/PMA a dose-dependent inhibition ofression was found for all cytokines. Benzocaine

.2.5. Potassium tetrachloroplatinate (K2PtCl4)In the presence of ionomycin/PMA potassium te

hloroplatinate decreased the expression of bothnd IFN-�. This may support the generation of Thependent allergic responses. Our findings are t

ore in accordance with reports using the LLNA thowed ammonium tetrachloroplatinate to be a reatory sensitizer (Dearman et al., 1998).

.2.6. Penicillin GIn our test system, penicillin G evidently did not le

o the activation of cytokine genes, nor was cytokene activation by ionomycin/PMA suppressed. Thesults may be caused by lack of interaction of pillin with cell proteins, as penicillin needs to be calently coupled to proteins to become immunoghaptenisation;Padovan, 1998).

270 T. Ringerike et al. / Toxicology 206 (2005) 257–272

4.2.7. Sodium dodecyl sulphate (SDS)The irritant SDS showed a dose-dependent reduc-

tion in IL-2 in the presence of ionomycin/PMA atthe highest concentration (300�M). Effects of in vitroSDS exposure on cytokine production have been de-scribed for keratinocytes, where IL-8 was found to beinduced (Mohamadzadeh et al., 1994; Muller et al.,1994; Wilmer et al., 1994).

4.2.8. Mercuric chloride (HgCl2)In cells stimulated with ionomycin/PMA a de-

creased expression of IFN-� but not IL-2 was foundupon HgCl2 challenge. This corresponds to findingsby Du et al. (2000), who reported that in vitro expo-sure of the murine T-cell line CTLL-2 to HgCl2 didnot result in any detectable IL-2, IL-4, or IL-15 mRNAexpression.

Studies employing two murine T-cell hybridomasthat express IL-4 upon HgCl2 stimulation suggestedthat the effect was independent of antigen recogni-tion (Badou et al., 1997). Others, however, foundthat the proliferative response of T-cells required thepresence of antigen presenting cells and costimula-tory molecules, suggesting that it has properties ofan antigen-specific response (Pollard and Landberg,2001).

Since certain in vivo effects of HgCl2 exposure arestrain dependent, the genetic background of EL4 (be-ing C57BL/6) may be of interest (Abedi-Valugerdi andMoller, 2000). The reduction of IFN-� observed in oure rtainaV

4p

ngs eth-o PCRo isb con-c r de-t starp tom-e othfl de-c ages

of concurrent assessment of viability, the possibilityto measure fluorescence intensity per cell and possibleeffects on cell shape and size.

Several quality controls have been performed andare described in an accompanying paper (Ulleraset al., 2004). Firstly, the transfected cell lines and theparent cell line have similar gene expression, bothbasal and in response to stimulation. Secondly, thefluorescence intensity is correlated to EGFP gene ex-pression and parent gene expression (parent meaning,e.g. the IL-4 gene in IL-4/EGFP transfected) and fi-nally the cytokine levels correlate to the fluorescenceintensities.

The tested panel consisted of only one type of cells(EL4; T-cells). An increased selection of cell types andcytokines will most probably enhance the precision andsensitivity of the “Cell Chip”. One limitation with our invitro systems is the absence of antigen presenting cells.For a substance to give an effect in our system, it mustinteract directly with the cells. A high level of proteinin the medium might possibly reduce substance-cell in-teractions. Immunosuppressive compounds often exerttheir effects directly on T-cells, suggesting that the T-cell lymphoma used in the present study is a suitabletarget cell line to evaluate immunosuppressive poten-tial. Sensitizers, however, often exert their effects onother cell types such as keratinocytes (KC) and den-dritic cells (DC), suggesting that T-cells may not be asuitable target to assess sensitising potential. This no-tion is supported by the fact that the present study thate nti-f isingc uldt cellc

ec-t elli sile.T ion,p tot nt.

idedm s re-q ellC iden-t ri-g for af

xperiments is in agreement with the absence of ceutoimmune manifestations in C57BL/6 mice (Abedi-alugerdi and Moller, 2000; Kono et al., 1998).

.3. Methodological considerations and futureerspectives

This prototype in vitro immunotoxicity screeniystem has several important advantages. Other mds to measure cytokine expression, such as RT-r ELISA are more time-consuming. Our systemased on fluorescence technology, which enablesurrent viability assessment. Several methods foection of fluorescence are available, such as Fluo®

late reader, fluorescence microscopy and flow cytry. Although the project started off employing bow cytometry and plate-based assays, we soonided to use flow cytometry based on the advant

mploys a T-cell line was more successful in ideying immunosuppressive compounds than sensitompounds. Cell lines of KC and DC origin shohus be included in the panel of cells used in thehip approach.

Most in vitro exposure models lack organ architure, thereby diminishing the possibilities for cell–cnteraction, especially if one of the cell types is seshis lack of interaction often hampers cell maturatrecluding the evaluation of the sensitivity of cells

oxic compounds at different stages of developmeBoth stable and destabilised EGFP have prov

eaningful results. A choice between these typeuires additional testing. In conclusion, this “Chip” approach may be useful as a pre-screen to

ify immunotoxicity. Cell lines derived from other oins and additional compounds need to be tested

ull appreciation of this approach.

T. Ringerike et al. / Toxicology 206 (2005) 257–272 271

Acknowledgements

This work was mostly funded by the EU, con-tract #QLK4-CT-2000-00787, and with an additionalgrant from the Norwegian Institute of Public Healthto T. Ringerike. We thank all members of the CellChip project for useful discussions and suggestions.We are also grateful to Urszula Bialek-Wyrzykowskafor project management.

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