HSE Health & Safety

Executive

Development of a method for the in vitro identification of contact allergens

Prepared by Syngenta Central Toxicology Laboratory (CTL) for the Health and Safety Executive 2003

RESEARCH REPORT 142

HSE Health & Safety

Executive

Development of a method for the in vitro identification of contact allergens

CJ Betts, C Sellick, H Caddick, M Cumberbatch, JG Moggs, G Orphanides, RJ Dearman and I Kimber

Syngenta Central Toxicology Laboratory (CTL)

Alderley Park Macclesfield

Cheshire SK10 4TJ

Allergic contact dermatitis is an important occupational health issue and there is a range of methods available for the prospective identification of chemicals with the potential to cause skin sensitization. Improvements have been made recently with respect to the reduction and refinement of the use of experimental animals in such tests. The aim of this project was to examine the opportunities available for the development of in vitro approaches for the identification of contact sensitizers using transcript profiling by microarray. Experiments have been conducted initially using murine lymphoid tissue to ensure that gene changes identified by transcript profiling are robust and reproducible. Appropriate culture conditions have been developed for the isolation of homogenous populations of human blood derived dendritic cells (DC), cells which play pivotal roles in the acquisition of contact allergy. Transcript profiling of chemical allergen-activated human DC has generated a list of candidate genes whose altered expression may provide an in vitro correlate of contact allergenicity.

This report and the work it describes were funded by the Health and Safety Executive (HSE). Its contents, including any opinions and/or conclusions expressed, are those of the authors alone and do not necessarily reflect HSE policy.

HSE BOOKS

© Crown copyright 2003

First published 2003

ISBN 0 7176 2720 9

All rights reserved. No part of this publication may bereproduced, stored in a retrieval system, or transmitted inany form or by any means (electronic, mechanical,photocopying, recording or otherwise) without the priorwritten permission of the copyright owner.

Applications for reproduction should be made in writing to: Licensing Division, Her Majesty's Stationery Office, St Clements House, 2-16 Colegate, Norwich NR3 1BQ or by e-mail to hmsolicensing@cabinet-office.x.gsi.gov.uk

ii

CONTENTS

SUMMARY v

INTRODUCTION 1

PROJECT AIMS 5

METHODS 7Transcript profiling of murine lymph node cells 7 Transcript profiling of human dendritic cells 8

RESULTS 11 Transcript profiling of early gene changes in allergen-activated lymph node 11 cells Transcript profiling of allergen-activated human blood derived DC 14

DISCUSSION 19 Transcript profiling of early gene changes in allergen-activted lymph node 19 cells Transcript profiling of allergen-activated human blood derived DC 22

CONCLUSIONS 25

REFERENCES 27

BIBLIOGRAPHY 33 Publications 33

Publications (Abstracts) 34

APPENDIX A 35 Abbreviations 35

APPENDIX B 37 Primer sequences 37

APPENDIX C 38 Figure 1 38 Table 1 39 Table 2 40 Figure 2 41 Figure 3 42 Figure 4 43 Figure 5 44 Figure 6 45 Figure 7 46 Figure 8 47 Table 3 48 Figure 9 49 Table 4 50 Figure 10 51 Figure 11 52 Figure 12 53 Figure 13 54 Table 5 55

APPENDIX D 56 Submitted Paper

iii

iv

SUMMARY

Allergic contact dermatitis is an important occupational health issue and there is a range of methods available for the prospective identification of chemicals with the potential to cause skin sensitization. Many of these rely upon the assessment of challenge-induced skin reactions in sensitized animals, attempting to mimic the clinical manifestations of the disease. In contrast, a recently validated alternative method for contact allergenic hazard identification, the local lymph node assay (LLNA), measures skin sensitization potential as a function of lymph node cell proliferation in the lymph node draining the site of exposure. This assay represents a considerable improvement in animal welfare with respect to reduction and refinement of experimental usage, but there is still considerable interest in the possibility of replacing such tests with in vitro alternatives. The aim of this project was to examine the opportunities available for the development of in vitro approaches to the identification of contact sensitizers using transcript profiling by microarray to identify gene changes which could provide markers of contact allergenic potential. Experiments have been conducted initially using murine lymphoid tissue to ensure that gene changes identified by transcript profiling are robust and reproducible. These experiments demonstrated that provided rigorous measures are used to control for inter-filter differences and an appropriate level is set for significant changes, then changes in gene expression identified by microarray analysis could be confirmed by different analytical methods. Consistent allergen-induced changes in mRNA levels of three genes were demonstrated and it is possible that one or more of these genes may merit further investigation as potential alternative (non-isotopic) end points for the LLNA. In parallel with these studies, appropriate culture conditions have been developed for the isolation of homogenous populations of human blood derived dendritic cells (DC), cells which play pivotal roles in the acquisition of contact allergy. Transcript profiling of chemical allergen-activated human DC has generated a list of candidate genes whose altered expression may provide an in vitro correlate of contact allergenicity.

v

vi

INTRODUCTION

Skin sensitization, resulting in allergic contact dermatitis, is an important occupational health issue and is without doubt the most frequent manifestation of immunotoxicity among humans (Cronin, 1980; Kimber et al., 2002). In common with other forms of allergic disease contact allergy develops in two discrete phases. The first or induction phase is precipitated by topical exposure to the chemical allergen, and is associated with the initiation of a specific cutaneous immune response and the acquisition of sensitization. If the then sensitized subject is exposed subsequently to the same chemical allergen then the elicitation phase will occur wherein an accelerated and more aggressive secondary immune response is provoked at the site of encounter resulting in a local inflammatory reaction characterized clinically as allergic contact dermatitis (Basketter et al., 1999; Friedmann, 1996; Grabbe and Schwartz, 1998).

The acquisition of skin sensitization in a previously naïve individual requires the coordinated activation of cellular and molecular processes that act in concert to stimulate a cutaneous immune response of the necessary vigour and quality. Pivotal roles are played by epidermal Langerhans cells (LC) that in the normal epidermis form a semi-contiguous network and serve as sentinels of the adaptive immune system. During the induction phase of skin sensitization LC are known to internalize and transport antigen from the epidermis to draining lymph nodes via the afferent lymphatics (Kripke et al. 1990; Macatonia et al. 1987). Their migration from the skin is accompanied by differentiation such that they develop from antigen processing cells into mature immunostimulatory dendritic cells (DC) that have the potential to present antigen in an immunogenic form to responsive T lymphocytes (Furue et al. 1996; Steinman et al., 1995; Streilein and Grammer, 1989). The mobilization and maturation of LC and their localization within lymph nodes are processes that are initiated and orchestrated by chemokines and epidermal cytokines (Kimber et al., 1998; 2000; Sebastiani et al., 2002). The end result is the stimulation of an immune response in draining nodes that is associated with a number of changes, including increases in lymph node size and cellularity, the increased expression of a variety of cytokines and lymphocyte proliferation.

A range of methods is available for the prospective identification of chemicals with the capacity to cause contact sensitization. Many of these, including the guinea pig maximization test (Magnusson and Kligman, 1970), rely upon mimicking this elicitation phase measured as a function of challenge-induced erythema or oedema in previously sensitized animals. In contrast, the murine local lymph node assay (LLNA), a recently accepted alternative method for the assessment of contact allergenic hazard (Dean et al., 2001; Dearman et al., 1999; Gerberick et al., 2001) measures skin sensitization potential as a function of responses stimulated during the induction phase of contact allergy. The vigour of allergen-induced lymph node cell (LNC) proliferation in the lymph node draining the site of exposure correlates closely with the extent to which skin sensitization develops and this observation forms the basis of the LLNA (Kimber and Dearman, 1991). Lymphocyte proliferation is measured as a function of in vivo radiolabelled thymidine incorporation and this endpoint has been shown in a variety of inter-laboratory comparisons to be a sensitive and selective marker of contact allergenic potential (Gerberick et al., 2001).

All of the approaches summarized above have a requirement for experimental animals and there is consequently some interest in the development of alternative in vitro approaches for the identification of contact allergens. Activity in this area has been fuelled by an increasingly sophisticated appreciation of the immunobiology of skin sensitization and the availability of the appropriate cell and tissue culture models (Kimber et al., 1999b). It is

1

important to recognize however that the development of realistic in vitro methods is not a trivial exercise and poses substantial challenges for experimental toxicologists.

Among the strategies that have been explored recently is the analysis of allergen-induced changes in the phenotype and/or function of DC or LC-like cells. One approach which has generated some interest is the measurement of induced changes in interleukin (IL) 1b, a proinflammatory mediator which in the murine epidermis is produced exclusively by LCs Heufler et al., 1992; Matsue et al., 1992). This cytokine is known to be required for the induction by chemical allergen of LC migration and the acquisition of skin sensitization (Enk et al., 1993; Shornick et al., 1996; Cumberbatch et al., 1997). In addition, it was demonstrated that epidermal IL-1b mRNA expression was rapidly up-regulated following topical exposure of mice to contact allergen but was unaffected by treatment with skin irritants (Enk and Katz, 1992). This suggested that it might be possible to identify skin sensitizers as a function of their ability to induce increases in IL-1b expression. Initially this observation did not provide a realistic basis for the development of an in vitro method due to the difficulties associated with experiments requiring LC. Not only are these cells present in normal skin in low numbers, but they are also difficult to isolate and subject to rapid phenotypic and functional changes in culture. An attractive alternative to using native LC derived from demonstrations that human DC progenitors could be expanded in culture using an appropriate cocktail of cytokines and that cellular differentiation could be manipulated to generate and maintain cells with an LC-like/immature DC phenotype (Lenz et al., 1993; Romani et al., 1994; Sallusto and Lanzavecchia, 1994).

Using this approach, Reutter et al. (1997) reported that DC derived from human peripheral blood displayed increased IL-1b mRNA expression following treatment with some contact sensitizers, but not in response to a skin irritant (sodium lauryl sulphate; SLS). More recently we have performed similar studies in order to determine whether chemical allergen-induced changes in IL-1b mRNA expression in cultured human DC could provide a robust method for the identification of contact allergens in vitro (Kimber et al., 2001; Pichowski et al., 2000; 2001). Our experience to date has been that strong contact allergens, but not skin irritants, do indeed up-regulate the expression by human DC of this cytokine. We have shown, however, that there exist stable inter-individual differences between human blood donors with this respect and that even with DC derived from responsive individuals, only modest changes in IL-1b expression are observed with potent contact allergens (Kimber et al., 2001; Pichowski et al., 2000; 2001).

Given these limitations, the ability of contact allergens to regulate expression by cultured human DC of IL-1b does not represent a viable stand-alone method for the identification of skin sensitizing chemicals. It is of considerable interest, however, that contact allergens have been demonstrated to interact, probably directly, with cultured DC to induce changes in gene expression under conditions where skin irritants apparently do not. Indeed, other parameters of DC activation have been shown to be influenced by in vitro exposure to contact allergens including increases in phosphotyrosine (Kuhn et al., 1998) and changes in the expression of membrane determinants such as the chemokine receptor CCR7 and the adhesion molecule E­cadherin (Aiba et al., 2000). This suggests that there may be allergen-induced changes in DC phenotype or characteristics other than the up-regulation of IL-1b mRNA which may prove to have greater utility as the basis for an in vitro assessment of skin sensitizing activity.

The approach we have taken towards trying to identify genes which are regulated selectively by contact allergens and which display a greater dynamic range of expression than does IL­1b is the application of microarray transcript profiling (Pennie and Kimber, 2002). Thus,

2

the changes in the gene expression profile induced by chemical sensitization of DC have been characterized, allowing the selection of those genes where altered expression (either up- or down-regulation) provides the most specific, sensitive and robust correlate of contact allergenicity. Custom arrays comprising over 12,500 cDNA sequences selected to embrace holistically many diverse cellular pathways including those relevant to immune and inflammatory processes and other biological pathways including the regulation of cell division and differentiation, apoptosis, extracellular matrix interactions and stress responses have been utlized. In parallel with these investigations using human blood-derived DC, proof of principle experiments have been conducted to examine whether microarray technology can be applied to characterize in greater detail changes in gene expression by lymph node cells following local exposure of mice to contact allergen. A supplementary objective was to determine whether any such changes in gene expression might provide appropriate biomarkers of skin sensitization that could be used as adjuncts to, or replacements for, the current endpoint for hazard characterization in the LLNA, the in vivo incorporation of radiolabelled thymidine (Gerberick et al., 2001).

It is against this background that the work for the HSE grant “Development of a method for the in vitro identification of contact allergens” has been performed.

3

4

PROJECT AIMS

The overall aim of this programme of work was to examine chemical-allergen induced changes in gene expression profiles of cultured human DC (cells which play pivotal roles in the development of adaptive immune responses, including contact allergy). Transcript profiling (microarray technology) was applied to evaluate altered phenotypes associated with exposure of cultured DC to chemical contact allergens. Genes whose altered expression (up­or down-regulation) provide the most specific, sensitive and robust correlate of contact allergenicity have been selected. Proof of principle experiments were performed in parallel to investigate early changes in gene expression by lymph node cells following local exposure of mice to contact allergen were characterized by transcript profiling. A supplementary objective of these studies was to determine whether in principle any such changes in gene expression might provide appropriate biomarkers of skin sensitization that could be used as adjuncts to, or replacements for, current hazard characterization strategies.

5

6

METHODS

Transcript profiling of murine lymph node cells Female BALB/c strain mice (aged 8-12 weeks) were used throughout these studies. As described previously (Betts et al., 2002), animals were exposed on the dorsum of both ears to 25ml of various concentrations of 2,4-dinitrofluorobenzene (DNFB) in acetone:olive oil (AOO; 4:1) vehicle or to vehicle alone. Further control groups were untreated (naïve). In some experiments, mice received 50ml of 0.1% oxazolone in AOO vehicle or AOO alone bilaterally on the shaved flanks on day 0. Five days later, 25ml of 1% oxazolone in AOO or AOO vehicle alone were applied to the dorsum of both ears. At various times after the initiation of exposure, draining auricular lymph nodes were excised, pooled on an experimental group basis and a single cell suspension of lymph node cells (LNC) prepared by mechanical disaggregation under aseptic conditions as described previously (Dearman et al., 1996). Cell viability was assessed by trypan blue exclusion and total cellularity per lymph node recorded. Cells were cultured at 2.5 x106 cells/ml and proliferative responses were assessed by 24h incorporation of radiolabelled 3H-thymdine as described previously (Dearman et al., 1996). In some experiments, the sensitivity of induced changes in gene expression for the identification of contact allergens were compared with lymphocyte proliferative responses measured in vitro as described above or in vivo in a LLNA type protocol (Kimber and Dearman, 1991). In these experiments, mice received 25ml of chemical or vehicle alone on the dorsum of both ears daily for three consecutive days and proliferative responses were measured and LNC processed for RNA preparation 3 and 5 days after the initiation of exposure. Total RNA was prepared from freshly isolated murine LNC using TRIZOL (Life Technologies, Paisley, Scotland) according to the manufacturer’s instructions. In some experiments, polyA+ RNA was further purified using an oligo dT magnetic bead system (Dynal, Bromborough, UK) according to the manufacturer’s instructions. In house microarrays comprising 8734 murine cDNA sequences (3336 of which are assigned to known genes, the remainder of which are expressed sequence tags [ESTs]) were incubated with cDNA probes generated by reverse transcription of mRNA in the presence of 33P-labelled dCTP (Betts et al., 2002). Microarray filters were prehybridized before addition of the cDNA probe and hybridization for 18h at 65oC. Filters were washed and exposed to a storage phosphor screen for 2 days. Data were collected by phosphorimager and array analysis undertaken using ArrayVision 5.1 software. Rigorous controls are applied to the raw phosphorimager counts to control for filter differences, background and duplicate clone variation (Betts et al., 2002). The array membranes being compared were first adjusted for differences in probe labelling efficiency and hybridization by the summation of all the counts for each membrane and then any differences were adjusted with a correction factor to equalise the total counts for both membranes. Furthermore, any cDNA spot with a count value of less than five fold the background value obtained for the membrane was excluded. Similarly, any spots where there were discrepancies of greater than 1.5 fold in either of the duplicate spots representing each cDNA sequence from the average count for that pair were discounted. All genes which were identified as being regulated by allergen were verified visually. Finally, the intensity of each cDNA sequence on the control membrane was plotted against the intensity of the same cDNA on the treatment membrane to produce a scatter plot in which the majority of spots align along the 45o angle. If the scatter plot deviates from this characteristic ellipsoid alignment then the array analysis is rejected. Changes in gene expression were calculated as the ratio of DNFB-treated versus control animals (changes of over 1.5 fold were considered significant). Genes exhibiting significant changes in expression in response to DNFB as identified by microarray analysis were analyzed further by Northern blotting (Betts et al., 2002). Gene-specific primers were designed to amplify by polymerase chain reaction (PCR) the cDNA inserts corresponding to the selected genes and

7

the house keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). DNA probes were generated by random-prime labelling each PCR product with 32P-dCTP. Northern analysis was undertaken by standard methods (Sambrook et al., 1989) and hybridized blots were quantified using a phosphorimager. Gene expression changes were also confirmed by reverse transcription (RT)-PCR methods using total RNA (Wilson et al., 1996). The housekeeping gene hypoxanthine phosphoribosyl transferase (HPRT) was used as a control to normalize for any differences in template concentration. Product intensities were analyzed by a gel imaging system (Kodak Digital Science Electrophoresis Documentation Analysis System 120, Kodak, Hemel Hempstead, UK) consisting of a digital camera over an ultraviolet (UV) light box and calculated using imaging software (Betts et al., 2002). In some experiments PCR products were quantified by Southern blotting using standard methods (Sambrook et al., 1989) or slot blotting as per manufacturer’s instructions (Bio-Dot SF blotting apparatus, Biorad, UK). RT-PCR products were blotted onto nylon membrane (Zeta probe GT membrane, Biorad) and baked for 2 h at 80oC. Membranes were stored flat and dry until used. Short oligonucleotide probes specific for the sequence internal to each primer pair were end labelled by incubation with T4 polynucleotide kinase and 32P g-ATP using standard procedures (Sambrook et al., 1989). Following purification through G50 sephadex columns saturated with sodium chloride/Tris/ethylenediamine tetraacetic acid buffer, probes were hybridized overnight to blotted membranes (as per slot blot manufacturer’s instructions). Following washing at room temperature and 1oC below the temperature of dissociation for the oligonucleotide probe, membranes were wrapped in cling film and exposed to a phosphorstorage screen (Molecular Dynamics) for between 2 and 24 h dependent upon probe labelling intensity. Analysis was carried out by phosphorimager (Molecular Dynamics).

Transcript profiling of human dendritic cells Healthy male and female volunteers (age range 18-53) were recruited for these studies. Peripheral blood mononuclear cells were isolated by density gradient centrifugation (Pichowski et al., 2000; 2001). In initial experiments, DC progenitor cells were prepared by negative selection using magnetic beads labelled with anti-human CD2 and CD19 antibodies to deplete of T and B cells (Pichowski et al., 2000; 2001). Cells were cultured at 0.5 x 106

cells/ml in the presence of 1% autologous serum, 57.3 ng/ml human granulocyte/macrophage-colony stimulating factor (GM-CSF) and 31 ng/ml IL-4. Every second day cells were fed with fresh GM-CSF and IL-4. Alternatively, the DC progenitor population was prepared by negative selection using the Miltenyi MACS monocyte (CD14+ population) isolation kit which removes cells displaying the following cell surface markers; CD3, CD7, CD19, CD45RA, CD56 and IgE antibody. The purity of the isolation was confirmed by fluorescence activated cell sorter (FACS) analysis for CD14+ monocytes and contaminating B and T cells as described previously (Pichowski et al., 2000; 2001). CD14+ cells were cultured for 5 days in RPMI supplemented as above except that autologous serum was replaced by 10% FCS and recombinant cytokines were added to the culture medium as follows; 250ng/ml GM-CSF, 100ng/ml IL-4 and 10pg/ml transforming growth factor b (TGF­b). Following 5 days in culture, aliquots of cells were removed from the well and counted. Approximately 5x105 cells were incubated on ice for 30 min with 10 mg/ml mouse anti­human antibodies against the following cell surface markers: major histocompatibility complex (MHC) class II (HLA-DR), CD1a, CD83, CD86 , CD14, CD3, CD19, or with IgG2a and IgG2b isotype controls. Following incubation with fluorescein isothiocyanate (FITC)-conjugated goat F(ab)2 anti-mouse IgG, cells were washed and analyzed by a FACScaliber flow cytometer and associated Cell Quest™ software as described previously (Pichowski et al., 2000). On the same day of culture (day 5), cells were treated with contact allergen (DNFB) or vehicle (dimethyl sulphoxide; DMSO) alone. DNFB was diluted in 1% DMSO/RPMI to a concentration of 1.07 nM and pre-warmed for 30 min at 37oC. DNFB,

8

vehicle control (1% DMSO) and medium alone were diluted 1:100 into culture wells to provide final concentrations of 10.7 pM DNFB in 0.01% DMSO, 0.01% DMSO or medium alone. For experiments using cells isolated by the MACS system, a concentration of 0.5mM DNFB was used for sensitization (determined from dose response viability studies to be the maximum sub-toxic concentration). As a positive control for cytokine expression, cells were incubated for 1, 2 or for 24 h with the mitogen phorbol myristate acetate (PMA; 1mg/ml). Following sensitization, cells were resuspended at room temperature and pelleted in a bench top centrifuge at 400g. The cell pellet was lysed in 400ml of direct lysis buffer and mRNA isolated using the mRNA DIRECT kit (Dynal) as per manufacturer’s instructions. For experiments using cells isolated by the Miltenyi magnetic activated cell sorter (MACS) system, total RNA was prepared using TRIZOL. The RNA concentration in each sample was estimated spectrophotometrically. Radiolabelled cDNA probes were generated by reverse transcription of approximately 500ng of mRNA or 7-10mg of total RNA in the presence of 33P-dCTP as described for the mouse array. These cDNA probes were hybridized to nylon microarray filters comprising 12554 human genes arrayed in duplicate and the data collected onto a phosphorimager and analyzed as described above. Changes in gene expression were calculated as the ratio of normalized values derived for DNFB-treated DC compared with vehicle (DMSO) control-treated DC. Changes of 1.5-1.9 fold or greater were considered significant. For selected genes, RT-PCR and Southern blot analyses were carried out using standard techniques as described previously (Pichowski et al., 2000; 2001; Sambrook et al., 1989; Wilson et al., 1996). The cDNA generated by reverse transcription from 2ml mRNA was diluted 1:20 for the house keeping gene b-actin and used neat for all other primer pairs (5ml per PCR tube). Annealing temperatures were 62oC for IL-1b, b-actin, IL-13 and IL-18 and 55oC for IL-6 and IL-12p40. Cycle number varied between 23 and 31 dependent upon the amount of mRNA added into the RT reaction mix. PCR products were analyzed by image analysis over UV illumination (as described above) or by Southern or slot blotting followed by hybridization to 32P end-labelled oligonucleotides carried out using standard techniques (Sambrook et al., 1989).

9

10

RESULTS

TRANSCRIPT PROFILING OF EARLY GENE CHANGES IN ALLERGEN-ACTIVATED LYMPH NODE CELLS

Kinetics of allergen-induced lymph node activation In initial experiments, the kinetics of lymph node activation following topical application of a single sensitizing dose of the potent contact allergen DNFB were examined. At various times (18 to 120 h) following exposure to 0.5% DNFB, draining auricular lymph nodes were excised and activation assessed as a function of increases in total lymph node cellularity and in proliferation (Fig 1). Within 18 h of treatment with allergen, total cellularity in the lymph node increased approximately two-fold, compared with vehicle-treated controls. Numbers of LNC continued to increase with time, reaching maximal levels (approximately 6-fold compared with basal levels) after 96 h. Changes in cellularity preceded changes in lymphocyte proliferation, thus there was no detectable increase in 3H-thymidine incorporation observed 18 h of following exposure. It has been demonstrated previously that there is marked DC accumulation in draining lymph nodes within 18 h of treatment and there is therefore the potential for DC and T lymphocyte interactions within 18 h this time frame (Kinnaird et al., 1989). Given that the lymph node is relatively quiescent with respect to proliferation at this time point, 18 h was selected for transcript profiling of changes in gene expression relevant for contact allergens.

Transcript profiling of gene expression changes in lymph nodes 18h after allergen exposure Two independent experiments were performed to identify changes in gene expression 18 h following topical exposure to DNFB. Gene expression profiles of LNC derived from DNFB­treated mice were compared with naïve controls (experiment 1), or with vehicle (AOO) ­treated control mice (experiment 2). In both experiments the levels of expression of the majority of the 8734 gene sequences represented on the array were similar for tissue derived from control mice, compared with those which had been exposed to allergen. Treatment with DNFB resulted in no significant increases in gene expression compared with controls. However, a number of genes was consistently down-regulated in both experiments (Table 1). These included two expressed sequence tags (ESTs) and 3 genes of known function, one of which (coprophophrinogen oxidase) is represented 3 times on each array and was down­regulated 5 times out of 6 over the two experiments. Another gene found to be down­regulated consistently was the cellular adhesion molecule, glycosylation dependent cell adhesion molecule-1 (GlyCAM-1), with an average 2.2 fold decrease in the two experiments. Although the changes were relatively modest, the rigorous measures used to control for background, duplicate clone variation and inter-filter differences ensured that the changes observed were robust.

Transcript profiling of gene expression changes in lymph nodes 48 h after allergen exposure Due to the relatively modest changes in gene expression displayed 18 h after allergen exposure, further analyses were conducted using tissue isolated 48 h after treatment with DNFB. At this time point, marked elevations in cellularity and lymphocyte proliferation were observed (Fig. 1). As seen at 18 h, expression of the majority (approximately 95%) of the genes represented on the array remained unchanged following exposure to DNFB. However, in contrast to the small number of down-regulated genes recorded at 18 h, both up­and down-regulated genes compared with tissue isolated from vehicle-treated control mice were recorded. The levels of induced changes in gene expression were also more marked, ranging from 1.5 to 5.8 fold. The 18 most up-regulated genes (1.9 to 5.8 fold changes) and

11

the 25 most down-regulated genes (2.0 to 5.8 fold changes) are displayed in Table 2. The two most up-regulated genes were an EST, now designated as onzin (Sherwin et al., 2000), and guanylate binding protein 2 (GBP2) and the most down-regulated gene was GlyCAM-1. The fold changes in gene expression for these three genes at 18 and 48 h as they appear on the microarray membrane are represented visually in magnified form in Figure 2. Two gene sequences, signal transducer and activator of transcription (STAT) 5b and an EST of unknown function, which both remain at constitutive expression levels throughout the time course are shown for comparative purposes. The decrease in GlyCAM-1 transcripts at 18 and 48 h is clearly displayed, as is the increased expression of onzin and GBP2 at 48 h. The cDNA clones representing GlyCAM-1, onzin and GBP2 were sequence verified and changes in gene expression at 48 h confirmed by Northern blot analysis (data not shown).

Kinetic and dose response analyses of onzin, GBP2 and GlyCAM-1 gene expression Rather than verifying specific changes in gene expression using multiple repeat profiling, we elected to confirm changes in the expression of these three genes at 48 h by Northern blotting and for this purpose dose-response analyses were performed (Fig. 3). Groups of mice (n = 10) were exposed topically to concentrations of DNFB ranging from 0.5% to 0.05%, or to vehicle alone, and draining auricular lymph nodes excised after 48 h. Messenger RNA was isolated, and Northern blot analyses performed and quantitated by phosphorimager. Gene expression was normalized against the house keeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). At all concentrations examined, GlyCAM-1 expression was considerably lower than that observed for vehicle-treated control tissue. At the majority of concentrations tested (0.5% to 0.1% DNFB), GBP2 and onzin mRNA levels were up­regulated compared with control tissue. Maximal changes in expression for all three genes occurred at 0.5% DNFB. In subsequent experiments, kinetic analyses of gene expression changes induced by a single topical exposure to 0.5% DNFB were conducted using Northern blot analysis of mRNA. In addition, instead of verifying changes in the expression of the genes of interest by repeat microarrays, we elected to utilize a further method of gene expression analysis, RT-PCR of total RNA (Fig. 4). Furthermore, data derived from RT-PCR analyses conferred an additional level of specificity for GlyCAM-1 and GBP2 since the primer pairs were designed to lie outside the cDNA sequences represented on the array. Data obtained from Northern blotting and RT-PCR were normalized against the housekeeping gene GAPDH and hypoxanthine phosphoribosyltransferase (HRPT), respectively. Both methods provided similar kinetic profiles of gene expression for GlyCAM-1, onzin and GBP2 (Fig. 4). Consistent with previous analyses by microarray and Northern blotting, 48 h after allergen treatment GlyCAM-1 expression was down-regulated and onzin and GBP2 mRNA levels were up-regulated as assessed by both Northern blot and RT-PCR. GlyCAM-1 expression was down-regulated throughout the time course (18-120 h), with maximal down­regulation recorded by both methods at 72-96 h. Onzin expression was maximally increased 48 h after initiation of exposure and remained elevated at 120 h whereas GBP2 mRNA levels were also maximally up-regulated at 48 h but declined thereafter regardless of the method of analysis.

Induced changes in GlyCAM-1 expression : relationship to cellularity and proliferation The dose response experiments conducted as described above demonstrated that a reduced expression of GlyCAM-1 was associated with lymph node activation, with the dose­dependent increases in lymph node cellularity and proliferation being paralleled by concomitant decreases in GlyCAM-1 transcripts (Fig. 5). We wished to explore whether or not the observed changes in GlyCAM-1 were associated specifically with the induction of a vigorous proliferative response in draining nodes. To investigate this possibility, use was made of an experimental manipulation that in mice results in allergen-induced activation of

12

skin draining lymph nodes, together with increased cellularity, but with a very substantial reduction in lymph node cell proliferation. Thus, it has been reported previously that prior topical exposure of mice to the contact allergen oxazolone at a distant site results in a very marked inhibition of proliferative responses in draining auricular lymph nodes when days or weeks later mice are exposed on the ears to the same chemical sensitizer (Dearman et al., 1996; Kimber et al., 1989). This phenomenon is illustrated in Figure 6 where mice received 0.1% oxazolone on the shaved flanks 5 days prior to exposure to 1% of the same chemical on both ears. The results reveal that such pretreatment resulted in a marked reduction of proliferation in draining auricular nodes in the absence of a similar decrease in lymph node cellularity. Under these conditions of exposure it was observed that irrespective of prior treatment at a distant site, local sensitization with oxazolone was associated with a reduction in GlyCAM-1 mRNA levels. Two conclusions can be drawn from these data. The first is that the reduced expression of GlyCAM-1 mRNA in allergen activated lymph nodes is not restricted to DNFB; similar effects are observed with the chemically-unrelated sensitizer oxazolone. Second, that GlyCAM-1 expression is down-regulated in activated lymph nodes irrespective of the level of LNC proliferation induced.

Relative sensitivity of induced changes in onzin and GBP2 expression for identification of contact allergens : comparison with proliferation Given that up-regulated expression of both onzin and GBP2 mRNA was induced at relatively low concentrations of DNFB, in subsequent experiments the effect of treatment with other allergens on the expression of these molecules was compared with the induction of proliferative responses. The additional contact allergens selected for these comparisons were hexyl cinnamic aldehyde (HCA), a mild to moderate skin sensitizer which is a recommended positive control in tests for contact allergens and gives marked proliferative responses in the LLNA at concentrations of 10% and above (Dearman et al., 2001) and paraphenylene diamine (PPD), a relatively potent contact allergen which tests positive in the LLNA at doses of 0.25% and above (Warbrick et al., 1999). In these experiments, BALB/c strain mice received 25ml of 0.5% DNFB, 50% HCA, 1% PPD or vehicle (AOO) alone on the dorsum of both ears daily for 3 consecutive days; the dosing schedule used in the standard LLNA (Dearman et al., 1999). Lymph node tissue was isolated 3 or 5 days after the initiation of exposure, cellularity recorded and proliferative responses assessed in vitro (Fig. 7). Total RNA was isolated, and expression of onzin, GBP2 and the housekeeping gene HPRT determined by Southern blotting of PCR products (Fig. 8). In concurrent control animals, proliferative responses were determined in vivo using a standard LLNA protocol with 3H­thymidine incorporation assessed 5 days after initiation of exposure (Fig. 7). Consistent with the results of previous experiments using CBACa strain mice (Dearman et al., 1998; Warbrick et al., 1999), these concentrations of chemical tested positive in the LLNA with in each case an SI (stimulation index; relative to the concurrent vehicle-treated control) of greater than 3 achieved (Fig. 7). Exposure to DNFB, HCA and PPD also resulted in increases in LNC total cellularity and proliferation compared with vehicle-treated controls at both 3 and 5 days following initiation of exposure (Fig. 7). However, neither in vitro proliferation nor changes in LNC total cellularity were as sensitive for the detection of skin sensitizers as was the measurement of 3H-thymidine incorporation in vivo. Exposure to DNFB induced increased expression of transcripts for onzin at day 3, but not at day 5; up­regulation of onzin mRNA was also observed at 3 days, but not at 5 days, after initiation of exposure to HCA and PPD. More marked DNFB-induced effects were seen on GBP2 mRNA expression, particularly 3 days following treatment. Exposure to HCA also resulted in increased levels of GBP2 mRNA at both time points, whereas increases were only observed 3 days after exposure to PPD. In general, PPD and HCA exposure stimulated less profound increases than did DNFB in onzin and GBP2 mRNA levels. It is also of interest that RNA levels for both onzin and GBP2 were somewhat variable between AOO control groups, suggesting that further experience is required to determine the reliability of the induced changes in expression of these genes.

13

TRANSCRIPT PROFILING OF ALLERGEN-ACTIVATED HUMAN BLOOD DERIVED DC

Microarray analysis of DNFB-activated DC isolated using the Dynal system In parallel investigations, experiments have been conducted to examine the effect of contact allergen (DNFB) on the gene expression profile of cultured human DC. Peripheral blood mononuclear cells were isolated from a donor with a known stable responder phenotype with respect to DNFB-induced changes in IL-1b mRNA expression (2.9 and 1.7 fold up­regulation as determined by RT-PCR in two independent experiments). DC precursors were purified by negative selection using anti-CD2 and anti-CD19 Dynal beads. Cells were expanded in culture for 5 days and sensitized for 2 h at 37oC in the presence of a non-toxic concentration of DNFB (10.7 pM) demonstrated previously to cause increased IL-1b mRNA expression in responder donors or in the presence of vehicle (0.01% DMSO) alone. Prior to treatment with allergen at day 5, the phenotype of the cells was assessed by flow cytometry. Approximately 95% of the cells were MHC class II positive and there was some CD1a, CD14 and CD86 expression (data not shown). This phenotype is broadly consistent with that of immature DC and was observed following cell isolations from many individuals, although there was considerable inter-donor variation, particularly with respect to CD1a and CD14 expression as reported previously (Pichowski et al., 2000; 2001). Poly A+ RNA was isolated and subjected to microarray analysis using nylon microarray filters comprising approximately 12500 human genes arrayed in duplicate. Changes in gene expression were calculated as the ratio of normalized values obtained for DNFB-treated tissue compared with vehicle (DMSO) control treated tissue. Changes of 1.9 fold or greater were considered significant. The majority of detectable genes on the array was unchanged by DNFB treatment for 30 min or for 2 h. Examination of the identity of some of the high intensity spots revealed a pattern of gene expression consistent with what would be expected for blood-derived DC, with particularly high expression of genes encoding human MHC class II proteins. Treatment of DC with DNFB for 30 min resulted in very few changes in the transcript profile compared with control DMSO-treated cells, with 5 genes up-regulated (1.9 to 5.7-fold changes) and 2 genes down-regulated (1.9 to 2.2-fold changes, data not shown). Culture of DC from the same donor with allergen for 2 h resulted in more marked effects on mRNA expression with 42 genes identified as being significantly up-regulated, although no genes were down-regulated according to the criteria used (Table 3). A few of these up­regulated genes, including collagen-like factor, acetylcholinesterase and interleukin 13, are shown in magnified form as they appear on the array in Figure 9. Although the changes observed were relatively modest (maximum fold increase 2.4), the criteria for inclusion on the candidate gene list are relatively stringent and include visual confirmation of changes in mRNA expression. It is also important to note that in neither experiment was there a detectable signal on the microarray for IL-1b mRNA in either control (DMSO-treated) or DNFB-treated DC, illustrating the differential sensitivity of RT-PCR versus the microarray analysis which does not incorporate any amplification steps.

Donor specificity of the candidate genes identified by microarray analysis The identity of the genes represented in Figure 9, along with another up-regulated gene, leukotriene B4 omega-hydroxylase, was confirmed by sequencing of the clones and the gene annotation was verified by mining the gene database. Primers were designed to amplify segments of the gene sequence represented on the array for selected genes and have been used to develop and optimize RT-PCR assays to confirm changes in gene expression. However, additional experiments revealed that DC derived from the majority of donors under standard culture conditions do not express detectable levels of mRNA for IL-13, or any of the other genes of interest highlighted by the array, either constitutively or following sensitization with allergen (DNFB). In each case, the integrity of the culture system and of

14

the RT-PCR for the genes of interest has been confirmed by concurrent analysis of DC stimulated for 24 h with the mitogen phorbol myristate acetate (PMA; 1 mg/ml). Detectable changes in mRNA expression for the genes highlighted by array analysis was recorded only for PMA-activated cells and was limited to approximately 50% of donors. It seems that the high levels of mRNA for the up-regulated genes recorded for the donor subjected to microarray analysis were specific for that individual. Furthermore, it is possible that the allergen-induced IL-13 expression for this donor may derive from the presence of contaminating T cells since DC have not been reported previously to produce this cytokine.

Optimization of the MACS isolation system for human DC preparation The results of the experiments described above led us to examine the protocol utilized for the isolation of DC precursors in more detail. In this method, the DC precursor population is depleted of both B and T cells using anti-CD19 and anti-CD2 Dynal beads, and at this stage there are no detectable contaminating T or B cells. However, phenotypic examination of the DC populations isolated after culture with cytokines for 5 days revealed significant numbers of contaminating T and B cells. We have therefore investigated an alternative, more stringent, method for DC preparation using a magnetic activated cell sorter (MACS) microbead protocol which enriches for CD14+ cells (monocyte precursors of DC) by negative selection. This procedure depletes peripheral blood mononuclear cells of T and B cells, basophils, natural killer cells and mature DCs using a cocktail of anti-CD3, CD7, CD19, CD45RA, CD56 and anti-IgE antibodies. FACS analyses of the negatively selected population revealed that the majority of cells in this population were CD14+ with marked expression of CD86 and MHC class II, but little contamination from either T or B cells (no detectable CD3 or CD19) (Fig. 10a). The phenotype of the cells obtained following culture for 5 days in media supplemented with FCS, GM-CSF, IL-4 and TGF-b was consistently immature DC-like, with loss of CD14 expression and marked up-regulation of CD1a and MHC class II (HLA-DR) membrane markers (Fig. 10a). Indeed, the pattern of expression of these DC markers was indicative of a considerably more homogenous DC population than that obtained using the original methodology and there was considerably less inter-individual variation in DC phenotype. The antigen-internalization capacity of the LC-like population has been explored using uptake of fluorescently labelled dextran (FITC-dextran). The kinetics of FITC-dextran uptake by cultured LC has been investigated from days 3-10 of culture. Uptake occurred from day 3 and was maximal at day 5 (day 5 data are illustrated in Figure 10b), suggesting that the optimal time point for utilizing the cells in this system is day 5.

Inter-individual variation in DC phenotype Following optimization of the growth conditions for cells isolated using this procedure we have now analyzed 19 donors over a total of 23 isolations using the Miltenyi MACS system. A summary of the surface marker expression for these donors is displayed in Table 4. It is clear that the phenotype of the cell population after 5 days in culture with human cytokines is very consistent between subjects; in all but 2 cases (donors 2 and 4) CD14 expression is reduced dramatically and CD1a expression increases markedly as the cells differentiate from a monocytic to an immature DC-like phenotype. This cellular phenotype is an improvement on the previous Dynal system of cell isolation where expression of both CD1a and CD14 was extremely variable between donors. Immediately following isolation of the monocyte precursor population on day 0, the cells express high levels of CD14 and are positive for CD86 and MHC class II. After differentiation in vitro for five days the population phenotype is markedly different. CD14 expression has been lost, along with the majority of CD86 expression (both markers which are characteristic of a monocyte population). Instead the cells express high CD1a and a relatively immature MHC class II status associated with a LC-type phenotype. In addition, for all donors tested to date with the MACS system, the monocyte enriched precursor population obtained from the separation column has been free from contaminating T and B lymphocytes and remains so during the culture period.

15

Changes in cytokine mRNA expression provoked by treatment of DC with DNFB In a series of experiments, the ability of DC isolated and cultured as above to respond to exposure to contact allergen (0.5mM DNFB) by changes in cytokine mRNA expression was examined. The more sensitive detection system of Southern blotting of RT-PCR products was used to analyze expression of the cytokines under investigation; IL-1b and two other cytokines thought to be up-regulated upon LC activation, IL-18 and the inducible p40 subunit of IL-12. DC cultures were treated with DNFB or vehicle (0.01% DMSO) alone for 1 h after 3, 4 and 5 days of culture. Positive control mRNA for cytokine up-regulation was derived from day 5 cells stimulated for 24 h with the mitogen PMA (Figure 11a). In comparison with culture in medium alone, this donor responded to treatment with PMA with a very marked up-regulation in IL-1b and IL-18 gene expression and a somewhat more moderate increase in IL-12 p40 mRNA. Analysis of the cytokine gene expression in DNFB-exposed cells revealed that the donor under analysis up-regulated IL-1b expression in response to allergen at all time points tested. The increase was maximal at day 5 and in addition cells stimulated on day 5 of culture also responded with more marked increases in IL-18 and IL-12 p40 (Figure 11b).

Of interest was the observation that mRNA for IL-13 was up-regulated in DC isolated from donor 13 following exposure to PMA or DNFB. This cytokine, not generally thought to be expressed by DC, was highlighted as an up-regulated gene in our initial transcript profiling experiments. However, in these earlier experiments, expression of IL-13 was donor specific and appeared to correlate with T cell contamination of the cultures rather than exposure to allergen. These current data, showing clear expression of IL-13 in a LC-like population devoid of lymphocytes, also demonstrates that IL-13 is donor specific (Figure 12). Thus, constitutive and allergen-inducible IL-13 expression is observed for cells derived from donor 13, whereas in a second donor (No. 14) there is no detectable IL-13, despite these donors exhibiting similar cellular phenotypes (Table 4).

Other donors have also been screened for responder status with respect to IL-1b, IL-12p40 and IL-18 up-regulation and a further LC cytokine, IL-6, as detected by RT-PCR and Southern blotting. Donors 15 and 16 were assessed for up-regulation of IL-1b mRNA in response to stimulation of day 5 cultured DC with 0.5 mM DNFB. DC isolated from donor 15 up-regulated expression of IL-1b, IL-6 and IL-18 upon treatment with DNFB, although IL­12p40 was unaffected by allergen treatment. Positive control cells (PMA-treated) exhibited increased mRNA levels for all 4 cytokines compared with negative control cells (cultured with medium alone). In contrast, cells derived from donor 16 appear to be unresponsive, even to mitogenic (PMA) stimulation, in all but IL-12p40 expression (Fig. 13). Yet phenotypically these two cell populations are very similar with regards to cell surface marker expression (Table 4).

Microarray analysis of DNFB-activated DC isolated using the MACS system A large-scale preparation of human monocytes from donor 19 using the MACS system has been carried out and the differentiated cells have been treated on day 5 with 0.5mM DNFB or with vehicle alone (0.01% DMSO) for 2 h prior to RNA isolation. RNA was subjected to microarray analysis using nylon microarray filters comprising approximately 12500 human genes arrayed in duplicate. Changes in gene expression were calculated as the ratio of normalized values obtained for DNFB-treated tissue compared with vehicle (DMSO) control treated tissue. Changes of 1.5 fold or greater were considered significant. The majority of detectable genes on the array were unchanged by DNFB treatment for 2 h. As observed previously, the pattern of expression of the high intensity spots was consistent with what would be expected for blood-derived DC, with particularly high expression of genes encoding human MHC class II proteins and some cytokines and cytokine receptor subunits. The number of gene changes observed after stringent exclusion of any false positives was relatively small. The gene list obtained comprised fold changes ranging from 1.7 fold up­

16

regulation to 9.3 fold down-regulation encompassing changes in some 21 genes in total and is displayed in Table 5. The magnitude of these changes is considerably more marked than that observed for the microarray experiment conducted with DC isolated by Dynal bead separation, where the maximum fold change was 2.2. It is of interest that the majority of changes appear to be down-regulations of expression. Whether this observation is specific to this particular donor or the majority of changes in gene expression are genuinely decreased upon sensitization will be determined upon the analysis of further donors. One set of cDNAs identified as being down-regulated is a cluster of antigen processing and presentation genes, including the transporter associated with antigen processing (TAP) and RING (real interesting new gene) finger proteins contained within the human MHC gene complex (Beck et al., 1996), which may warrant further investigation.

17

18

DISCUSSION

TRANSCRIPT PROFILING OF EARLY GENE CHANGES IN ALLERGEN-ACTIVATED LYMPH NODE CELLS

We describe here proof of principle experiments in which micorarray analyses have been applied to the identification of novel genes associated with the induction of contact sensitization in mice. Allergen-induced changes in expression of more than 8500 genes 18 and 48 h after exposure to allergen have been examined. Relatively few gene changes were identified in lymph node tissue derived 18 h after allergen (DNFB) treatment, with no up­regulated genes and five genes observed to be consistently down-regulated across two independent array experiments. At this time point, although significant increases in proliferation were not recorded, lymph node cellularity was increased compared with control lymph nodes, which is probably due primarily to recruitment and retention of circulating lymphocytes in the lymph node. It has been demonstrated previously that within 18 h of exposure to allergen, LC (some of which bear allergen) have been induced to migrate from the skin and accumulate as DC in the draining lymph node where they interact with allergen­specific T cells (Cumberbatch et al., 1991; Kinnaird et al., 1989; Macatonia et al., 1987). Despite these DC and T cell interactions within the activated lymph node, no measurable elevations of gene expression were recorded at this time point. This is likely to reflect the fact that such interactions are occurring in a minority population only and are diluted out by mRNA derived from the majority of lymph node cells which are unaffected by allergen. Due to these rather modest changes in gene expression observed at 18 h, additional microarray analyses were conducted using tissue derived 48 h after allergen treatment. Consistent with the vigorous allergen-induced increases in cellularity and proliferation observed at this time point, a greater number of changes in gene expression, with increased magnitude, were recorded and both up- and down-regulations were observed.

Changes in mRNA levels of the two most up-regulated genes (onzin and GBP2), and the most down-regulated gene, GlyCAM-1, which was down-regulated at both 18 and 48 h after exposure to DNFB, were confirmed in subsequent experiments. Rather than perform repeat array analyses to confirm these changes in mRNA levels, we elected to use 2 different analytical methods to verify changes in gene expression; Northern blotting and RT-PCR. Dose response analyses using Northern blotting confirmed that exposure to 0.5% DNFB up­regulated onzin and GBP2 expression and down-regulated GlyCAM-1 mRNA levels. Maximal changes were observed following exposure to 0.5% DNFB, although changes in expression of GBP2 and GlyCAM-1 were detected after a single exposure to as little as 0.05% DNFB, and 0.1% DNFB for onzin. The kinetics of expression of the three genes of interest were also examined by Northern blot and RT-PCR following a single topical exposure to 0.5% DNFB. As well as these methods of analysis providing verification of the observed changes in mRNA levels, data derived from RT-PCR analyses conferred an additional level of specificity for GlyCAM-1 and GBP2 since the primer pairs were designed to lie outside the cDNA sequences represented on the array. Taking into account the differential sensitivities of these techniques, these analyses demonstrated a remarkable concordance between the two methods with respect to the kinetics of induced changes in expression, although not unexpectedly the fold changes in gene expression were not identical. This is in contrast to the observations of He et al (2001) who found that there was less than 50% concordance between genes identified by transcript profiling on microarrays as having altered expression following treatment with chemical allergen and the results of RT-PCR analyses. Transcript profiling has found various applications in immunology and allergy (reviewed in Schmidt-Weber et al., 2001), particularly for comparisons of tissue derived from patients with various forms of allergy with normal healthy control tissue, an approach which will identify primarily genes important in the clinical manifestations of the allergic response.

19

There are few reports of the application of this technology to understanding early events which occur in the induction phase of sensitization. He et al (2001) have also examined gene expression changes occurring in the draining lymph node after topical exposure to sensitizing chemicals using the same mouse strain as the experiments reported herein. A more aggressive sensitization protocol was employed (4 daily applications of 1% of the potent allergen oxazolone) and tissue was isolated 120 h after initiation of exposure. Under these conditions, very marked lymph node activation and proliferative responses will be observed. However, consistent with our data, few changes in gene expression compared with vehicle treated controls were identified (13 out of 6519) and the magnitude of changes is also comparable. There is no overlap between the genes identified as being regulated by allergen in the He et al (2001) studies and those which we have identified, which may be a reflection of the panels of genes represented on the two different arrays used and the kinetics of responses. For example, some of the genes identified in the former studies (cytochrome p450, napthalene hydroxylase and major urinary protein 4) are detected as being expressed constitutively on our arrays but are unchanged by allergen treatment. The lack of detection of various cytokine genes as being affected by allergen in both studies is also not surprising, given the relatively low expression of mRNA for these molecules and the general requirement for a more sensitive method such as RT-PCR for their measurement (He et al., 2001).

The most down-regulated gene at 48 h (which was also consistently down-regulated at 18 h) was the cellular adhesion molecule GlyCAM-1, a 50kD glycoprotein expressed by the high endothelial venules (HEV) which regulates the trafficking of L-selectin bearing lymphocytes (Lasky et al., 1992). In the resting lymph node, GlyCAM-1 protein is expressed constitutively and released into the lumen of the capillary vessels where it is thought to act as a soluble ligand for L-selectin on circulating lymphocytes, blocking lymphocyte attachment to the HEV and subsequent rolling and extravasation into the lymph node (Hoke et al., 1995). Upon activation, GlyCAM-1 expression and secretion is switched off, allowing lymphocytes to enter the lymph node and encounter and interact with antigen-bearing DC (Hoke et al., 1995; Mebius et al., 1993). Although this protein is secreted by a minority population only within the lymph node (the HEV), constitutive expression of GlyCAM-1 is detectable by both microarray and Northern blot analysis. Down-regulation of GlyCAM-1 expression in the peripheral lymph nodes following exposure of mice to the contact allergen oxazolone has been detected previously using Northern blotting (Hoke et al., 1995). In the current studies, dose response and kinetic experiments demonstrated that the down-regulation of GlyCAM-1 transcripts was inversely proportional to increases in cellularity and proliferation, observations in agreement with published studies detailing the inverse relationship of GlyCAM-1 mRNA expression and lymph node weight (Lasky et al., 1992; Mebius et al., 1993). Taken together with the inhibition of GlyCAM-1 expression following DNFB treatment reported herein, these data suggested that further examination of the relationship between reduced expression of this molecule and lymph node activation might provide an insight into the immune mechanisms of the induction of contact allergy. In order to explore the association between changes in GlyCAM-1 and lymphocyte proliferation, an experimental strategy was employed which in mice results in allergen-induced activation of draining lymph nodes and increased cellularity in the absence of marked proliferative responses. Thus, as demonstrated previously (Dearman et al., 1996; Kimber et al., 1989), prior exposure to the potent contact allergen oxazolone at a distant site resulted in substantial inhibition of proliferative responses in draining auricular lymph nodes following challenge with the same chemical on the dorsum of both ears. Marked increases in lymph node cellularity were recorded, however, and down-regulation of GlyCAM-1 transcripts correlated inversely with changes in lymph node cellularity. It would appear therefore that the reduction in GlyCAM-1 expression is associated primarily with changes in lymph node cell numbers or some other aspect of LN activation rather than proliferation. Allergen-induced

20

down-regulation of GlyCAM-1 expression at the protein level has been reported following immunohistochemical analyses with levels returning to the resting state within 7 to 10 days (Hoke et al., 1995). These data illustrate one of the potential confounding factors when a mixed cell population is examined by transcript profiling. GlyCAM-1 is expressed only by a minority population of cells within the total lymph node cell population, the HEVs, a population whose absolute cell numbers will remain relatively constant whilst the total cellularity of the lymph node increases, due to emigration of circulating lymphocytes and clonal expansion. The observed changes in GlyCAM-1 expression are therefore likely to be due to a combination of the active down-regulation of this molecule and the dilution effect of incoming lymphocytes on the contribution of the minority HEV to the lymph node mRNA pool.

The most highly up-regulated gene identified by transcript profiling was a novel mouse gene recently designated as “onzin” and identified by differential display analysis of mouse endometrium (Sherwin et al., 2000). Although the function of onzin is unknown, this gene was formerly identified (by microarray analysis) as being down-regulated in 32D myeloid cell line 32D by the oncogene c-myc (Nesbit et al., 2000), suggestive of a role in cell cycling. A human homologue (BM-004) of the protein displaying 79% homology to the mouse gene has been described (Sherwin et al., personal communication). The functional role of this gene in chemical sensitization and the immune response has yet to be determined. The second most up-regulated gene was GBP2, a 65kDa interferon (IFN) -g inducible GTPase identified in bone marrow-derived macrophages which is present at relatively low levels constitutively but is extremely abundant following IFN-g treatment (Vestal et al., 1998). Until recently little was known of the possible function of this gene, but it is now reported that fibroblasts generated to express GBP2 have constitutively higher growth rates (approximately 50% reduction in doubling time), a reduced need for serum-derived growth factors and achieve higher cell densities than do wild type cells (Gorbacheva et al., 2002). Given that topical exposure to chemical contact allergens induces the production of IFN-g by activated LNC (Dearman and Kimber, 2001), the up-regulation of GBP2 may play a role in the clonal expansion of T lymphocytes which is the central event in the induction of skin sensitization.

It is instructive to compare the relative sensitivity of the changes in expression of these three genes identified by microarray analysis with the standard endpoint of the LLNA, the incorporation of 3H-thymidine in vivo. In initial experiments, dose responses were performed with the potent contact allergen DNFB. The threshold for positivity for DNFB in a standard LLNA using BALB/c strain mice, the EC3 value (estimated concentration of chemical required to induce a 3 fold increase in thymidine incorporation), was calculated to be 0.05% (Basketter et al., 1997). In the dose response experiments, this concentration of DNFB also stimulated measurable changes in the expression of both GlyCAM-1 and GBP2, although the minimum concentration of DNFB to induce changes in mRNA for onzin was 0.1%. These data indicated that changes in expression of onzin, and particularly GBP2 and GlyCAM-1, may be relatively sensitive markers of the early gene changes occurring in the lymph node following activation by a contact allergen. In subsequent experiments, changes in expression of GBP2 and onzin induced by 0.5% DNFB and the additional sensitizers HCA (50%) and PPD (1%) were compared with proliferation measured in vitro and in vivo. These data confirmed that the original decision to measure contact allergenicity as a function of in situ 3H-thymidine incorporation does indeed provide for considerably greater sensitivity than does the parallel measurement in vitro. Thus SIs of 23, 17 and 10 were achieved for 0.5% DNFB, 50% HCA and 1% PPD, respectively, in the standard LLNA (measured at 5 days after the initiation of exposure). Measurement of proliferation in vitro at either day 3 or day 5 did not achieve a similar level of sensitivity, with PPD and HCA both failing to elicit a positive SI (3 or greater) at day 3 and SIs of 6, 6 and 3 recorded for 0.5% DNFB, 50% HCA and 1% PPD,

21

respectively, at day 5. The greater sensitivity of in vivo thymidine incorporation is a reflection presumably of the fact that this approach takes into account the increased cellularity of the lymph node as well as the increased proliferative response on a per cell basis. The measurement of induced changes in expression of onzin and GBP2 transcripts both show some promise as potential markers for contact allergens. However, particularly given the inter-experimental variation in basal levels of message for these two genes in vehicle-treated control cells, the reliability of allergen-induced changes in expression of GBP2 and onzin must be explored. In addition, experience with a wider range of sensitizers and non-sensitizers (including skin irritants) and a systematic exploration of the relative sensitivity of these changes compared with the current method would be necessary before any recommendations could be made as to the possibility that these observations may provide a basis for a non-isotopic endpoint for the LLNA.

TRANSCRIPT PROFILING OF ALLERGEN-ACTIVATED HUMAN BLOOD DERIVED DENDRITIC CELLS

During the initial stages of this project, human blood derived DC were isolated and cultured according to the protocol of Reutter et al. (1997). In this method, the DC precursor population is depleted of both B and T cells using anti-CD19 and anti-CD2 Dynal beads, and further purification of the monocytic precursors which differentiate in culture to become immature DC is forgone in favour of ensuring a high cell yield. Following culture for 5 days in vitro with the cytokines GM-CSF and IL-4, the resulting cell population did indeed express cell surface markers similar to immature DC, such as low CD86 and low MHC class II. However, expression of the LC markers indicative of the early stages of DC development, such as CD1a, were very variable between donors. Using this methodology, DCs were derived from a donor shown previously to respond to culture with DNFB with an up-regulation in IL-1b expression (measured by RT-PCR) and treated with DNFB or with vehicle (0.01% DMSO) alone for 30 min or for 2 h. Messenger RNA was isolated and subjected to array analysis. At both time points the majority of detectable genes on the array was unaffected by DNFB treatment. Modest changes (in terms of numbers of genes with changed expression and the magnitude of changes) were observed after 30 min of culture with DNFB. More marked changes were observed after 2 h treatment with DNFB, with 42 genes identified as being significantly up-regulated although there were no examples of down-regulated genes. The identity of a number of these genes (IL-13, collagenase, acetylcholinesterase, homeobox protein CDX4, collagen-like factor and leukotriene B4 omega-hydroxylase) was confirmed by sequencing the clones and the gene annotation was confirmed. Appropriate primers were designed and used to develop RT-PCR assays to confirm changes in gene expression. However, these analyses revealed that the changes identified by microarray were donor-specific. In addition, the identification of IL-13 as a gene expressed by DC from some donors and up-regulated by allergen was somewhat intriguing. Although IL-13 is often used in place of IL-4 in the generation of DC from peripheral blood mononuclear cell precursors (Cao et al., 2000; Coronel et al., 2001) and it has been demonstrated that DC express the IL-13 specific receptor chain IL-13Ra1 (Poudrier et al., 2000), this cytokine is not normally considered to be expressed by immature DC or LC (Kimber et al., 1999a). Indeed, IL-13 is more generally considered to be a T cell cytokine produced by T helper 2 type cells (Dearman and Kimber, 2001). It was therefore necessary to consider the homogeneity of the DC populations and to examine the protocol utilized for the isolation of DC precursors in further detail. Although no contaminating T or B lymphocytes were detectable upon initial cell isolation, phenotypic examination of the DC populations isolated after culture with cytokines for 5 days revealed significant numbers of these cell types. We therefore investigated an alternative, more stringent, method for DC preparation, using a magnetic activated cell sorter (MACS) microbead protocol, which

22

enriches for CD14 positive cells (monocyte precursors of DC) by negative selection, hence the cells of interest are unaffected by the binding of any purification-related antibodies or beads (Geissmann et al., 1998). In addition we increased the concentration of the human cytokines supplementing the media and included TGFb in our cultures, a cytokine thought to retain differentiating monocytes in a more immature DC/LC like phenotype (Geissmann et al., 1998; 1999; Jaksits et al., 1999). The resulting cells were CD14+, with little contamination from either T or B cells detectable at any point during the culture period. Comparisons of DC isolated over a total of 23 isolations from 19 donors have demonstrated that this protocol results in the precursor DC differentiating by day 5 into a more homogenous LC-like population with marked CD1a and MHC class II expression.

Subsequent studies focussed on the assessment of the response of this cell population to the chemical skin sensitizer DNFB. Expression of the cytokine genes IL-1b, IL-6, IL-12 (p40 subunit), IL-13 and IL-18 has been examined following treatment of DC with DNFB. Some of these cytokines, including IL-1b and IL-6, have been shown to be up-regulated in mouse skin following topical exposure to contact allergens (reviewed in Kimber et al., 2000). For DC isolated from some donors, DNFB induced marked up-regulation of expression of cytokines, including IL-1b, IL-13 and IL-18. For these donors, up-regulations in mRNA levels for cytokines such as IL-13 and IL-18 were more vigorous than the concurrent increases observed in IL-1b expression, suggesting that these alternative cytokines may provide a more sensitive measure of allergen-induced DC activation. However, despite the homogenous nature of the phenotype of the cells obtained from the donors, there was inherent variability between donors with regards to mRNA levels of cytokines, both in response to stimulation and constitutive expression. There are several reports on the suitability of human blood-derived DC for in vitro testing of chemical allergens using such end points as cytokine expression. Tuschl and Kovac (2001) described the difficulty of using IL-1b (as measured by intracellular cytokine staining) as an end point due to the lack of sensitivity observed without boosting protein levels by incubation with PMA. Instead they suggested that up-regulation of certain cell surface markers associated with DC maturation, such as the co-stimulatory molecule CD86, intercellular adhesion molecule-1 (ICAM-1; CD54) and MHC class II (HLA-DR), provide more consistent markers of contact sensitization potential in the majority of donors tested (DC derived from 10 out of 14 donors responding to stimulation with 2,4-dinitrochlorobenzene). We have failed to detect changes in the surface expression levels of CD86 or HLA-DR in our culture system, even following 24 h stimulation with 0.5mM DNFB and therefore focussed on the identification of candidate genes using microarray analysis.

Large scale preparations of DC isolated from several donors have been performed, the cells treated with DNFB or with vehicle (0.01% DMSO) alone or for 2 h and mRNA isolated. DC from one donor has been subjected to array analysis. As observed for DC isolated by Dynal bead separation, the majority of detectable genes on the array were unaffected by DNFB treatment. However, 19 genes were identified as being significantly down-regulated and 2 genes were up-regulated following culture with DNFB. The magnitude of these changes (maximum 9.3 fold down-regulation) was considerably more marked than those changes observed previously for tissue isolated using the Dynal bead separation method (maximum fold change 2.2), which is a reflection presumably of the more homogenous nature of the former DC population. The identity of these genes will be confirmed by sequencing the clones and the gene annotation will be checked. Repeat microarray analyses will be performed using the tissue isolated from the additional donors to explore the donor specificity and reproducibility of these changes. It is not possible to perform repeat array analyses using the same donor as the amount of RNA isolated per experiment is sufficient for one microarray analysis only and the amount of blood required per DC preparation precludes recall of the same individual within the appropriate timeframe. Following

23

confirmation of the reliability of these changes as described above, appropriate primers will be designed and used to develop RT-PCR assays which will be used to examine the sensitivity and selectivity of the changes in expression of selected genes in response to a wider range of contact allergens and non-allergens (including skin irritants). Although the results from the second microarray analysis are preliminary and require confirmation, some interesting genes have been identified. One set of cDNAs identified as being down­regulated represents a cluster of antigen processing and presentation genes, including the transporter associated with antigen processing (TAP) and real interesting new gene (RING) finger proteins contained within the human MHC gene complex (Beck et al., 1996), which may warrant further investigation. It is not possible at this stage to identify which of the genes within this cluster are being affected, but it is likely that this observation is a reflection of the fact that after 2 h treatment with DNFB, DC are starting to down-regulate genes associated with antigen processing mechanisms and up-regulate those genes which are associated with antigen presentation. The most down-regulated gene in these preliminary investigations was identified as transducin-like enhancer of split 2 (TLE2), a transcriptional repressor mammalian homologue of the Drosophilia transcriptional repressor groucho, a member of the Notch signalling pathway (Grbavec et al., 1998) which has been shown previously to be expressed during neuronal development. Expression of TLE2 has also been reported in immature epithelial cells and is elevated during metaplastic and neoplastic transformations suggestive of a role in the maintenance of the undifferentiated state in epithelial cells (Liu et al., 1996). There are no published reports of similar experiments using microarray technology to examine allergen-induced changes in the gene expression profile of human blood derived DC. Microarray analysis has been carried out on human blood derived DC in order to investigate the gene expression changes induced following differentiation during in vitro culture. Two such reports describe the transcript profiling of freshly isolated CD14+ monocyte precursors compared to cells differentiated for 7 or 14 days (Le Naour et al., 2001; Lapteva et al., 2001). Not surprisingly, results from these publications indicate that during differentiation it is the genes related to cell structure, migration, differentiation and growth control which show marked changes in expression. The data obtained from the in-house array analysis carried out during this project do not overlap with these published data, however, this is not unexpected given that we are analyzing allergen-induced gene changes between cells at the same developmental stage stimulated by and utilizing arrays containing a different set of gene sequences.

24

CONCLUSIONS

Proof of principle experiments have been conducted to examine whether early changes induced by in vivo exposure of mice to contact allergen DNFB can be identified using microarray technology. Repeat array analyses were conducted using lymphoid material derived 18 h after exposure to contact allergen; these demonstrated a consistent pattern of gene activation, with in each case the majority (95%) of the 8734 murine genes on the array remaining unchanged and a small number of genes, including GlyCAM-1, being down­regulated. Further analyses of tissue isolated 48 h following allergen treatment revealed additional up-regulated and down-regulated genes, with GlyCAM-1 remaining the most strongly down-regulated gene. Changes in expression of GlyCAM-1 and the two most strongly up-regulated genes (onzin and GBP2) have been confirmed using Northern blotting and RT-PCR and kinetic and dose response analyses have been conducted. These experiments have demonstrated that provided appropriate stringent criteria are applied, robust gene changes can be identified by microarray technology. Changes in expression of GlyCAM-1, onzin and GBP2 in appeared to be relatively sensitive and robust markers of lymph node cell activation in response to the contact allergen DNFB, although supplementary experiments have shown that decreases in mRNA levels of the adhesion molecule GlyCAM-1 which is expressed on the minority HEV population are related to the dilution effects of cellularity changes in the lymph node. In subsequent experiments up-regulated expression of GBP2 and onzin were demonstrated following exposure to the additional contact allergens PPD and HCA. It is possible therefore that both of these genes may merit further investigation as potential markers for contact allergens. However, particularly given the inter-experimental variation in basal levels of message for these two genes in vehicle-treated control cells, the reliability of allergen-induced changes in expression of GBP2 and onzin must be explored fully. In addition, experience with a wider range of sensitizers and non­sensitizers (including skin irritants) and a systematic exploration of the relative sensitivity of these changes compared with the current method would be necessary before any recommendations could be made as to the possibility that these observations may provide a basis for a non-isotopic endpoint for the LLNA.

In parallel with these experiments, assays have been conducted to profile the effect of the contact allergen DNFB on patterns of gene expression in cultured DC. Microarray analysis was conducted on DC derived from a responder donor (with respect to allergen-induced changes in IL-1b expression) using the Dynal bead separation method and a minority of up­regulated genes identified, including the cytokine IL-13. Using RT-PCR, the expression of IL-13 and other candidate genes by DC from other donors was examined, however, all candidate genes were shown to be donor-specific. Subsequent experiments were directed towards developing a protocol for isolating and culturing DC with a more consistent phenotype. Using this protocol a second large scale preparation of cells has been prepared and analyzed for allergen-induced gene changes by microarray. This has generated a candidate list of genes which must now be confirmed by microarray analysis of subsequent donors. The final list of genes will be assessed for selectivity and sensitivity of induced changes using a wider range of allergens and non-allergens. It is premature at present to make firm recommendations regarding the possible use of such analyses in the identification of contact allergens, but it is prudent to expect that if an appropriate candidate gene is identified with the necessary specificity and selectivity that such may only be applied as a prescreen for other more sensitive assays such as the LLNA.

25

26

REFERENCES

Aiba, S., Manome, H., Yoshino, Y., and Tagami, H. (2000). In vitro treatment of human transforming growth factor-b1-treated monocyte-derived dendritic cells with haptens can induce the phenotypic and functional changes similar to epidermal Langerhans cells in the induction phase of allergic contact sensitivity reaction. Immunology 101, 68-75.

Basketter, D.A., Dearman, R.J., Hilton, J., and Kimber, I. (1997). Dinitrohalobenzenes : evaluation of relative skin sensitization potential using the local lymph node assay. Contact Derm. 36, 97-100.

Basketter, D.A., Gerberick, G.F., Kimber, I., and Willis, C.M. (1999). Toxicology of Contact Dermatitis. Allergy, Irritancy and Urticaria. Wiley, Chichester.

Beck, S., Abdulla, S., Alderton, R.P., Glynne, R.J., Gut, I.I., Hosking, L.K., Jackson, A., Kelly, A., Newell, W.R., Sanseau, P., Radley, E., Thorpe, K.L., and Trowsdale, J. (1996). Evolutionary dynamics of non-coding sequences within the class II region of the human MHC. J. Mol. Biol. 255, 1-13.

Betts, C.J., Moggs, J.G., Caddick, H.T., Cumberbatch, M., Orphanides G., Dearman, R.J., Ryan, C.A., Gerberick, G.F., and Kimber, I. (2002). Assessment of Glycosylation Dependent Cell Adhesion Molecule 1 (GlyCAM-1) as a correlate of allergen-stimulated lymph node activation. (submitted for publication)

Cao, H., Verge, V., Baron, C., Martinache, C., Leon, A., Scholl, S., Gorin, N.C., Salamero, J., Assari, S., Bernard, J., and Lopez, M. (2000). In vitro generation of dendritic cells from human blood monocytes in experimental conditions compatible for in vivo cell therapy. J. Hematother. Stem Cell Res. 9, 183-194.

Coronel, A., Boyer, A., Franssen, J.D., Romet-Lemonne, J.L., Fridman, W.H., and Teilland, J.L. (2001). Cytokine production and T cell activation by macrophage-dendritic cells generated for therapeutic use. Br. J. Haematol. 114, 671-680.

Cronin, E. (1980). Contact Dermatitis. Churchill Livingstone, London.

Cumberbatch, M., Dearman, R.J., and Kimber, I. (1997). Langerhans cells require signals from both tumour necrosis factor-a and interleukin-1b for migration. Immunology 92, 388­395.

Cumberbatch, M., Illingworth, I., and Kimber, I. (1991). Antigen-bearing dendritic cells in the draining lymph nodes of contact-sensitized mice : cluster formation with lymphocytes. Immunology 74, 139-145.

Dean, J.H., Twerdok, L.E., Tice, R.R., Sailstad, D.E., Hatton, D.G., and Stokes, W.S. (2001). ICCVAM evaluation of the murine local lymph node assay. Conclusions and recommendations of an independent scientific review panel. Regul. Toxicol. Pharmacol. 34, 258-273.

Dearman, R.J., Basketter, D.A., and Kimber, I. (1999). Local lymph node assay : use in hazard and risk assessment. J. Appl. Toxicol. 19, 299-236

27

Dearman, R.J., Wright, Z.M., Basketter, D.A., Ryan, C.A., Gerberick, G.F., and Kimber, I. (2001). The suitability of hexyl cinnamic aldehyde as a calibrant for the local lymph node assay. Contact Derm. 44, 357-361.

Dearman, R.J., Hope, J.C., Hopkins, S.J., and Kimber, I. (1996). Antigen-induced unresponsiveness in contact sensitivity : association of depressed T lymphocyte proliferative responses with decreased interleukin 6 secretion. Immunol. Lett. 50, 29-34.

Dearman, R.J., and Kimber, I. (2001). Cytokine fingerprinting and hazard assessment of chemical respiratory allergy. J. Appl. Toxicol. 21, 153-163.

Enk, A.H., Angeloni, V.L., Udey, M.C., and Katz, S.I. (1993). An essential role for Langerhans cell-derived IL-1b in the initiation of primary immune responses in the skin. J. Immunol. 150, 3698-3704.

Enk, A.H., and Katz, S.I. (1992). Early molecular events in the induction phase of contact allergy. P.N.A.S. USA 89, 1398-1402.

Friedmann, P.S. (1996). Clinical aspects of allergic contact dermatitis. In Toxicology of Contact Hypersensitivity (I. Kimber, and T. Maurer, Eds), pp. 26-56. Taylor & Francis, London.

Furue, M., Chang, C.H., and Tamaki, K. (1996). Interleukin-1 but not tumour necrosis factor alpha synergistically up-regulates the granulocyte-macrophage colony-stimulating factor­induced B7-1 expression of murine Langerhans cells. Br. J. Dermatol. 135, 194-198.

Geissmann, F., Prost, C., Monnet, J.-P., Dy, M., Brousse, N., and Hermine, O. (1998). Transforming growth factor b1, in the presence of granulocyte/macrophage colony stimulating factor and interleukin 4, induces the differentiation of human peripheral blood monocytes into dendritic Langerhans cells. J. Exp. Med. 187, 961-966.

Geissmann, F., Revy, P., Regnault, A., Lepelletier, Y., Dy, M., Brousse, N., Amigorena, S., Hermine, O., and Durandy, A. (1999). TGF-b1 prevents the noncognate maturation of human dendritic Langerhans cells. J. Immunol. 162, 4567-4575.

Gerberick, G.F., Ryan, C.A., Kimber, I., Dearman, R.J., Lea, L.J., and Basketter, D.A. (2001). Local lymph node assay : validation for regulatory purposes. Am. J. Cont. Dermatol.11, 3-18.

Gorbacheva, V.Y., Lindner, D., Sen, G.C., and Vestal, D.J. (2002). The interferon (IFN)-induced GTPase, mGBP-2. Role in IFN-gamma-induced murine fibroblast proliferation. J. Biol. Chem. 277, 6080-6087.

Grabbe, S., and Schwartz, T. (1998). Immunoregulatory mechanisms involved in the elicitation of allergic contact hypersensitivity. Immunol. Today 19, 37-44.

Grbavec, D., Lo, R., Liu, Y., and Stifani, S. (1998). Transducin-like enhancer of split 2, a mammalian homologue of drosophila groucho, acts as a transcriptional repressor, interacts with hairy/enhancer of split proteins, and is expressed during neuronal development. Eur. J. Biochem. 258, 339-349.

28

He, B., Munson, A.E. and Meade, B.J. (2001) Analysis of gene expression induced by irritant and sensitising chemicals by oligonucleotide arrays. Int. Immunopharmacol. 1, 867­879.

Heufler, C., Topar, G., Koch, F., Trockenbacher, B., Kampgen, E., Romani, N., and Schuler, G. (1992) Cytokine gene expression in murine epidermal cell suspensions : interleukin 1b

and macrophage inflammatory protein 1 a are selectively expressed in Langerhans cells but differentially regulated in culture. J. Exp. Med. 176, 1221-1226.

Hoke, D., Mebius, R.E., Dybdal, N., Dowbenko, D., Gribling, P., Kyle, C., Baumhueter, S. and Watson, S.R. (1995) Selective modulation of the expression of L-selectin ligands by an immune response. Current Biology 5, 670-678

Jaksits, S., Kriehuber, E., Charbonnier, A.S., Rappersberger, K., Stingl, G., and Maurer, D. (1999). CD34+ cell-derived CD14+ precursor cells develop into Langerhans cells in a TGF-b1-dependent manner. J. Immunol. 163, 4869-4877.

Kimber, I., Basketter, D.A., Gerberick, G.F., and Dearman, R.J. (2002). Allergic contact dermatitis. Int. Immunopharm. 2, 201-211.

Kimber, I., Cumberbatch, M., Dearman, R.J., Bhushan, M., and Griffiths, C.E.M. (2000). Cytokines and chemokines in the initiation and regulation of epidermal Langerhans cell mobilization. Br. J. Dermatol. 142, 137-146.

Kimber, I., Cumberbatch, M., Dearman, R.J., and Knight, S.C. (1999a). Langerhans cell migration and cellular interactions. In : Dendritic Cells.Biology and Clinical Applications. Lotze, M.T. and Thomson, A.W. (Eds.). Academic Press, San Diego. pp 295-310.

Kimber, I., and Dearman, R.J. (1991). Investigation of lymph node cell proliferation as a possible immunological correlate of contact sensitizing potential. Food Chem. Toxicol. 29, 125-129.

Kimber, I., Dearman, R.J., Cumberbatch, M., and Huby, R.D.J. (1998). Langerhans cells and chemical allergy. Curr. Opin. Immunol. 10, 614-619.

Kimber, I., Pichowski, J.S., Basketter, D.A., and Dearman, R.J. (1999b). Immune responses to contact allergens : novel approaches to hazard evaluation. Toxicol. Lett. 106, 237-246.

Kimber, I., Pichowski, J.S., Betts, C.J., Cumberbatch, M., Basketter, D.A., and Dearman, R.J. (2001). Alternative approaches to the identification and characterization of chemical allergens. Toxicol. In Vitro 15, 307-312.

Kimber, I., Shepherd, C.J., Mitchell, J.A., Turk, J.L., and Baker, D. (1989). Regulation of lymphocyte proliferation in contact sensitivity : homeostatic mechanisms and a possible explanation of antigenic competition. Immunology 66, 577-582.

Kinnaird, A., Peters, S.W., Foster, J.R., and Kimber, I. (1989). Dendritic cell accumulation in draining lymph nodes during the induction phase of contact allergy in mice. Int. Arch. Allergy Appl. Immunol 89, 202-210.

Kripke, M.L., Munn, C.G., Jeevan, A., Tang, J.-M., and Bucana, C. (1990). Evidence that cutaneous antigen-presenting cells migrate to regional lymph nodes during contact sensitization. J. Immunol. 145, 2833-2838.

29

Kuhn, U., Brand, P., Wilemsen, J., Jonuleit, H., Enk, A.H., van Brandwijk-Peterhans, R., Saloga, J., Knop, J., and Backer, D. (1998). Induction of tyrosine phosphorylation in human MHC class II-positive antigen-presenting cells by stimulation with contact sensitizers. J. Immunol. 160, 667-673.

Lapteva, N., Ando, Y., Nieda, M., Hohjoh, H., Okai, M., Kikuchi, A., Dymshits, G., Ishikawa, Y., Juji, T., and Tokunaga, K. (2001). Profiling of genes expressed in human monocyte and monocyte-derived dendritic cells using cDNA expression array. Br. J. Haematol. 114, 191-197.

Lasky, L.A., Singer, M.S., Dowbenko, D., Imai, Y., Henzel, W.J., Grimley, C., Fennie, C., Gillett, N., Watson, S.R. and Rosen, S.D. (1992). An endothelial ligand for L-selectin is a novel mucin-like molecule. Cell 69, 927-938.

Le Naour, F., Hohenkirk, L., Grolleau, A., Misek, D.E., Lescure, P., Geiger, J.D., Hanash, S., and Beretta, L. (2001). Profiling changes in gene expression using differentiation and maturation of monocyte-derived dendritic cells using both oligonucleotide microarrays and proteomics. J. Biol. Chem. 276, 17920-17931.

Lennon, G.G., Auffray, C., Polymeropoulos, M., and Soares, M.B. (1996). The I.M.A.G.E. Consortium: An Integrated Molecular Analysis of Genomes and their Expression. Genomics 33, 151-152.

Lenz, A., Heine, M., Schuler, G., and Romani, N. (1993). Human and murine dendritic cells. Isolation by means of a novel method and phenotypic and functional characterisations. J. Clin. Invest. 95, 2587-2596.

Liu, Y., Dehni, G., Purcell, K.J., Sokolow, J., Carcangiu, M.L., Artavanis-Tsakonas, S., Stifani, S. (1996). Epithelial expression and chromosomal location of human TLE genes : implications for notch signalling and neoplasia. Genomics 31, 58-64.

Macatonia, S.E., Knight, S.C., Edwards, A.J., Griffiths, S., and Fryer, P. (1987). Localization of antigen on lymph node dendritic cells after exposure to the contact sensitizer fluorescein isothiocyanate. Functional and morphological studies. J. Exp. Med. 166, 1654-1667.

Magnusson, B., and Kligman, A.M. (1970). Allergic Contact Dermatitis in the Guinea Pig. Charles C Thomas, Springfield.

Matsue,H., Cruz, P.D. Jr., Bergstesser, P.R., and Takashima, A. (1992). Langerhans cells are the major source of mRNA for IL-1b and MIP-1a among unstimulated murine epidermal cells. J. Invest. Dermatol. 99, 537-541.

Mebius, R.E., Dowbenko, D., Willaims, A., Fennie, C., Lasky, L.A. and Watson, S.R. (1993) Expression of GlyCAM-1, and endothelial ligand for L-selectin, is affected by afferent lymphatic flow. J. Immunol. 151, 6769-6776.

Nesbit, C.E., Tersak, J.M., Grove, L.E., Drzal, A., Choi, H. and Prochownik, E.V. (2000). Genetic dissection of c-myc apoptotic pathways. Oncogene 19, 3200-3212.

Pennie, W.D., and Kimber, I. (2002). Toxicogenomics; transcript profiling and potential application to chemical allergy. Toxicol. In Vitro 16, 319-326.

30

Pichowski, J.S., Cumberbatch, M., Dearman, R.J., Basketter, D.A., and Kimber, I. (2000). Investigation of induced changes in interleukin 1b (IL-1b) mRNA expression by cultured human dendritic cells as an in vitro approach to skin sensitization testing. Toxicol. In Vitro 14, 351-360.

Pichowski, J.S., Cumberbatch, M., Dearman, R.J., Basketter, D.A., and Kimber, I. (2001). Allergen-induced changes in interleukin 1b (IL-1b) mRNA expression by human blood­derived dendritic cells : inter-individual differences and relevance for sensitization testing. J. Appl. Toxicol. 21, 115-121.

Poudrier, J., Graber, P., Herren, S., Berney, C., Gretener, D., Kosco-Vilbois, M.H., and Gauchat, J.F. (2000). A novel monoclonal antibody, C41, reveals IL-13Ralpha1 expression by murine germinal center B cells and follicular dendritic cells. Eur. J. Immunol. 30, 3157­3164.

Reutter, K., Jager, D., Degwert, J., and Hoppe, U. (1997). In vitro model for contact sensitization. II. Induction of IL-1b mRNA in human blood-derived dendritic cells by contact sensitizers. Toxicol. In Vitro 11, 619-626.

Romani, N., Gruner, S., Brang, D., Kampgen, E., Lenz, A., Trockenbacher, B., Konwalinka, G., Fritsch, P.O., Steinman, R.M., and Schuler, G. (1994). Proliferating dendritic cell progenitors in human blood. J. Exp. Med. 180, 83-93.

Sallusto, F., and Lanzavecchia, A. (1994). Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony stimulating factor plus interleukin-4 and down-regulated by tumor necrosis factor a. J. Exp. Med. 179, 1109-1118.

Sambrook, J., Fritsch, E.F and Maniatis, T. (1989). Extraction, purification and analysis of messenger RNA from eukaryotic cells. In Molecular Cloning: A laboratory manual (N. Ford, C. Nolan and M. Ferguson, Eds.) 2nd ed., pp Cold Spring Harbour Laboratory Press, New York.

Schmidt-Weber, C.B., Wohlfahrt, J.G., and Blaser, K. (2001) DNA arrays in allergy and immunology. Int. Arch. Allergy Immunol. 126, 1-10.

Sebastiani, S., Albanesi, C., De Pita, O., Puddu, P., Cavani, A., and Girolomoni, G. (2002). The roles of chemokines in allergic contact dermatitis. Arch. Dermatol. Res. 293, 552-559.

Sherwin, J.R.A., Sharkey, A.M. and Smith, S.K. (2000). Identification of LIF regulated genes in the mouse uterus (unpublished). Direct Submission to the NCBI nucleotide database. Accession number:AF263458.

Shornick, L.P., De Togni, P., Mariathasan, S., Goeliner, J., Strauss-Schoenberger, J., Carr, R.W., Ferguson, T.A., and Chaplin, D.D. (1996). Mice deficient in IL-1b manifest impaired contact hypersensitivity to trinitrochlorobenzene. J. Exp. Med. 183, 1427-1436.

Steinman, R.M., Hoffman, L., and Pope, M. (1995). Maturation and migration of cutaneous dendritic cells. J. Invest. Dermatol. 105, 2s-7s.

Streilein, J.W., and Grammer, S.E. (1989). In vitro evidence that Langerhans cells can adopt two functionally distinct forms capable of antigen presentation to T lymphocytes. J. Immunol. 143, 3925-3933.

31

Tuschl, H., and Kovac, R. (2001). Langerhans cells and immature dendritic cells as model systems for screening of skin sensitizers. Toxicol. In Vitro 15, 327-331.

Vestal, D.J., Buss, J.E., McKercher, S.R., Jenkins, N.A., Copeland, N.G., Kelner, G.S., Asundi, V.K., and Maki, R.A. (1998). Murine GBP-2: a new IFN-gamma-induced member of the GBP family of GTPases isolated from macrophages. J. Interferon Cytokine Res. 18, 977-85.

Warbrick, E.V., Dearman, R.J., Lea, L.J., Basketter, D.A., and Kimber, I. (1999). Local lymph node assay responses to paraphenylenediamine : intra- and inter-laboratory evaluations. J. Appl. Toxicol. 19, 255-260.

Wilson, R.A., Coulson, P.S., Betts, C., Dowling, M.-A. and Smythies, L.E. (1996). Impaired immunity and altered pulmonary responses in mice with a disrupted interferon-g receptor gene exposed to the irradiated Schistosoma mansoni vaccine. Immunology 87, 275-282.

32

BIBLIOGRAPHY

PUBLISHED PAPERS

1. Kimber, I., Pichowski, J.S., Basketter, D.A., and Dearman, R.J. (1999). Immune responses to contact allergens : novel approaches to hazard evaluation. Toxicol. Lett. 106, 237-246.

2. Kimber, I., Pichowski, J.S., Betts, C.J., Cumberbatch, M., Basketter, D.A., and Dearman, R.J. (2001). Alternative approaches to the identification and characterization of chemical allergens. Toxicol. In Vitro 15, 307-312.

3. Pennie, W.D., and Kimber, I. (2002). Toxicogenomics; transcript profiling and potential application to chemical allergy. Toxicol. In Vitro 16, 319-326.

4. Betts, C.J., Moggs, J.G., Caddick, H.T., Cumberbatch, M., Orphanides G., Dearman, R.J., Ryan, C.A., Gerberick, G.F., and Kimber, I. (2002). Assessment of Glycosylation Dependent Cell Adhesion Molecule 1 (GlyCAM-1) as a correlate of allergen-stimulated lymph node activation. (submitted for publication)

33

BIBLIOGRAPHY

PUBLISHED ABSTRACTS

1. Pichowski, J.S., Holden, P.R., Orphanides, G., Cumberbatch, M., Dearman, R.J., and Kimber, I. (2000). Transcript profiling of allergen-exposed human dendritic cells. Toxicol. Lett. 116, 101.

2. Kimber, I., Betts, C.J., Moggs, J.G., Cumberbatch, M., Orphanides, G., and Dearman, R.J. (2001). Transcript profiling of allergen-activated murine lymph node cells. Scand. J. Immunol. 54, 122.

3. Pichowski, J.S., Cumberbatch, M., Dearman, R.J., Basketter, D.A., and Kimber, I. (2001). Allergen-specific changes in interleukin 1b mRNA expression by human blood derived dendritic cells : donor variation. Toxicol. Sci. The Toxicologist 60, 305.

4. Kimber, I., Betts, C.J., Moggs, J.G., Cumberbatch, M., Orphanides, G., and Dearman, R.J. (2001). Allergen-induced changes in the gene expression profile of murine lymph node cells. Toxicol. Lett. 123, 25.

5. Betts, C.J., Sellick, C., Cumberbatch, M., Moggs, J.G., Orphanides, G., Dearman, R.J., and Kimber, I. (2001). Transcript profiling of allergen-activated murine lymph node cells (LNC) following topical exposure to a contact sensitizer. Toxicology 168, 66-67.

6. Betts, C.J., Moggs, J.G., Caddick, H., Cumberbatch, M., Orphanides, G., Dearman, R.J., and Kimber, I. (2001). Transcript profiling of allergen-activated murine lymph node cells. Immunology, 104, 81.

7. Betts, C.J., Moggs, J.G., Caddick, H., Cumberbatch, M., Orphanides, G., Dearman, R.J., Gerberick, G.F., Ryan, C.A., and Kimber, I. (2002). Transcript profiling of murine lymph node cells : allergen-induced early gene changes. Toxicol. Sci. The Toxicologist 66, 167-168.

8. Dearman, R.J., and Kimber, I. (2002). Induction of skin sensitization : gene expression profiles. Toxicol. Sci. The Toxicologist 66, 172-173.

9. Betts, C.J., Caddick, H., Cumberbatch, M., Moggs, J.G., Orphanides, G., Dearman, R.J., Ryan, C.A., Gerberick, G.F., and Kimber, I. (2002). Allergen-induced changes in glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1) in murine lymph node cells. Toxicology (in press).

34

APPENDIX A

ABBREVIATIONS

AOO, acetone:olive oil, 4:1; DC, dendritic cells; DMSO, dimethylsulphoxide; DNFB, 2,4-dinitrofluorobenzene; DPM, disintegrations per minute; EC3, estimated concentration of chemical necessary to give a stimulation index of 3 in the local lymph node assay; EST, expressed sequence tag; FITC, fluorescein isothiocyanate; FACS, fluorescence activated cell sorter; FCS, foetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GlyCAM-1, glycosylation-dependent cell adhesion molecule 1; GM-CSF, granulocyte macrophage colony stimulating factor; GBP2, guanylate binding protein 2; HCA, hexyl cinnamic aldehyde; HEV, high endothelial venules; HPRT, hypoxanthine phosphoribosyl transferase; ICAM-1, intercellular adhesion molecule-1; IL-, interleukin; LC, Langerhans cells; LNC, lymph node cells; LLNA, local lymph node assay; MACS, magnetic activated cell sorter; MHC, major histocompatability complex; OX, oxazolone; PPD, paraphenylene diamine; PMA, phorbol myristate acetate; RT-PCR, reverse transcription-polymerase chain reaction; RING, really interesting new gene; STAT, signal transducer and activator of transcription; SLS, sodium lauryl sulphate; SI, stimulation index; TLE2, transducin-like enhancer of split 2; TGF, transforming growth factor; TAP, transporter associated with antigen processing; UV, ultraviolet.

35

36

APPENDIX B

PRIMER SEQUENCES FOR RT-PCR OF HUMAN DC GENES

IL-1b sense 5’-GACACATGGG ATAACGAGGC-3’, antisense 5’-ACGCAGGACAGGTACAGATT-3’; IL-6 sense 5’-CCAGTTGCCTTCTCCCTGG-3’, antisense 5’-CTGCAGGAACTGGATCAGGA-3’; IL-12p40 sense 5’-GAAGATGGTATCACCTGGAC-3’, antisense 5’-TCTTGGCCTCGCATCT TAGA-3’; IL­13 sense 5’-GTGCCTCCCTCTACAGCCCTCAG-3’, 5’-TTCCCGCCTACCCA AGACATTTT-3’; IL-18 sense 5’-TTCGGGAAGAGGAAAGGAA-3’, antisense 5’-GATGTCACTTTTTGTATCCTT-3’; housekeeping gene b-actin sense 5’-GAGCGGAAATCGTGCGTGACATT-3’, antisense 5’-AAGCCATGCCAATCTCATCTTG-3’.

INTERNAL OLIGONUCLEOTIDE PROBE SEQUENCES FOR SOUTHERN BLOTTING OF HUMAN DC GENES

Housekeeping gene b-actin 5’-TACGCCAACACAGTGCTGTC-3’; IL-1b 5’-GATGTCTGGTCCATATGAAC-3’; IL-6 5’-GGAGACATGTAACAAGAGTA-3’; IL­12p40 5’-CATTCGCTCCTGCTGCTTCA-3’; IL-13 5’-TGAGCGGATTCTG CCCGCAC­3’; IL-18 5’-GAGATAATGCACCCCGGACC-3’.

37

APPENDIX C

a) 20

15

10

5

0 AOO 18 48 72 96 120

cellu

lari

ty(D

PM

x 1

0-3)

(cel

ls/n

ode

x 10

-6)

b) 14

12 10

8

6

4 2 0

AOO 18 48 72 96 120

Time (h)

prol

ifer

atio

n

Figure 1. Kinetic analysis of changes in lymph node cellularity and proliferation following a single topical exposure to 2,4-dinitrofluorobenzene (DNFB). Draining auricular lymph nodes were excised at various times after treatment with 0.5% DNFB in acetone:olive oil (AOO) vehicle, pooled per treatment group and a single cell suspension prepared by mechanical disaggregation. Lymph node cell viability was assessed by trypan blue exclusion and total cellularity per lymph node recorded (a). Proliferative responses were assessed following 24h culture of cells in the presence of 3H-thymidine; results are expressed as mean disintegrations per minute (DPM) and SD of cells cultured in quintuplicate (b). The cellularity and proliferation following exposure to vehicle (AOO) alone are represented as the mean of data obtained at 18 and 120 h. Results of a single representative experiment are shown.

38

Table 1. Fold decreases in gene expression observed in two independent array experiments conducted on mRNA tissue derived from auricular lymph nodes 18 h following exposure to 0.5% 2,4-dinitrofluorobenzene (DNFB). Messenger RNA was isolated and used as a template to generate radiolabelled cDNA probes which were hybridized to in-house array membranes comprising 8,734 cDNA sequences arrayed in duplicate. Hybridized blots were quantified by phosphorimager and analyzed by Array Vision software. In experiment 1 the comparator was naive cells whereas experiment 2 includes an acetone:olive oil (4:1; AOO) control. Expressed sequence tags are designated EST.

Gene annotation EMBL Expt. 1 Expt. 2 accession # fold decrease fold decrease

EST AA185432 2.2 1.9

Glycosylation-dependent cell adhesion molecule (GlyCAM)-1

AA288467 2.6 1.7

EST, down-regulated in metastasis (DRIM)-like

AA166336 2.5 1.6

Coproporphrinogen oxidase W53951 1.5 1.6 W87981 1.6 1.7

AA108600 1.6 <1.5 Apoptosis inhibitor 3 AA097598 1.8 1.8

39

Table 2. Up- (a) and down-regulations (b) in gene expression observed upon array analysis of auricular lymph node tissue taken at 48 h post topical exposure to 0.5% 2,4-dinitrofluorobenzene (DNFB). Messenger RNA was subjected to array analysis as described for Table 1. Fold changes are expressed relative to control mRNA derived from acetone:olive oil (AOO)-treated animals. Expressed sequence tags are designated EST.

a) Up-regulated array sequences

onzin 5.8 guanylate nucleotide binding protein 2 3.8 lymphocyte antigen 6 complex 3.4 small inducible cytokine B subfamily (Cys-X-Cys), member 9 2.9 ubiquitin specific protease 18 2.6 EST, Moderately similar to HEM45 [H.sapiens] 2.5 EST, Highly similar to endothelial actin-binding protein 2.5 small inducible cytokine A12 2.3 lymphocyte antigen 6 complex, locus C 2.1 histocompatibility 2, class II antigen E beta 2.1 EST, Weakly similar to lymphocyte antigen LY-6A.2 2.1 histocompatibility 2, complement component factor B 2.1 EST 2.0 CD20 antigen 2.0 peroxisomal membrane protein 20 2.0 regulatory protein, T lymphocyte 1 2.0 EST 2.0 lymphocyte antigen 6 complex, locus D 1.9

b) Down-regulated array sequences

EST 2.0 EST 2.0 EST 2.0 EST 2.0 EST 2.0 EST 2.0 staufen (RNA-binding protein) homolog 2 (Drosophila ) 2.0 EST 2.0 smoothened homolog (Drosophila) 2.0 thymus cell antigen 1, theta 2.0 EST 2.0 EST, weakly similar to x-linked inhibitor of apoptosis protein 2.0 EST 2.0 apoptosis inhibitor 3 2.0 zipcode-binding protein 1 2.0 EST 2.0 EST 2.5 nuclear receptor binding factor 1 2.5 EST, moderately similar to mitochondrial import inner 2.5 membrane translocase subunit peroxisome biogenesis factor 16 2.5 EST 2.5 EST, highly similar to KIAA0183 [H.sapiens] 2.5 phospholipase A2, activating protein 3.3 Glycosylation-dependent cell adhesion molecule -1 5.8

40

18h 48h

AOO DNFB AOO DNFB

GlyCAM-1

Onzin

STAT5b

GBP 2

EST

Figure 2. Visual changes in gene expression at 18 and 48 h post exposure to 2,4-dinitrofluorobenzene (DNFB). Tissue isolated 18 or 48 h after exposure to 0.5% DNFB in acetone:olive oil (AOO) vehicle, or to vehicle (AOO) alone was transcript profiled using DNA microarrays comprising 8734 murine genes arrayed in duplicate. The array membranes were analyzed by phosphorimager and selected areas of these digital images are displayed. Pairs of cDNA spots relating to the genes of interest (glycosylation-dependent cell adhesion molecule -1 [GlyCAM-1, EMBL accession number AA288467]; 5.9 fold down-regulation, onzin [EMBL accession number AA245029]; 5.8 fold up­regulation, guanylate binding protein 2 [GBP2, EMBL accession number AA153021]; 3.8 fold up­regulation) are marked by double black arrows at each time point. Two genes which remained at a constant expression level throughout all treatments and time points, signal transducer and activator of transcription (STAT) 5b and an unassigned expressed sequence tag (EST), are also highlighted for comparative purposes (white arrows).

41

DNFB concentration (%w/v) GlyCAM-1

AOO 0.05 0.1 0.25 0.5 150

100

50GlyCAM-1

0 AOO 0.05 0.10 0.25 0.50

Onzin60 50Onzin 40 30 20 10 0

AOO 0.05 0.10 0.25 0.50GBP-2GBP2100

80

60 40GAPDH20

0 AOO 0.05 0.10 0.25 0.50

% DNFB (%w/v)

Figure 3. Dose response analyses of glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), onzin and guanylate binding protein 2 (GBP2) gene expression. Draining auricular lymph nodes were isolated 48 h after exposure to various concentrations of 2,4-dinitrofluorobenzene (DNFB) in acetone: olive oil (AOO) vehicle or to vehicle (AOO) alone. Messenger RNA was prepared and expression of transcripts for GlyCAM-1, onzin and GBP2 was analyzed by Northern blotting. Bands were visualized by phosphorimager and data are shown as digitalized images and expressed graphically as phosphorimaging counts normalized against the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

phos

phor

imag

ing

coun

ts (x

10-5

)

42

a) b)a) b)

phos

phor

imag

ing

coun

ts

phos

phor

imag

ing

coun

tsph

osph

orim

agin

gco

unts

phos

phor

imag

ing

coun

tsph

osph

oim

agin

g co

unts

phos

phoi

mag

ing

coun

ts

(x 1

0-4)

(x 1

0-4)

(x 1

0-4)

(x 1

0-4)

GlyCAM-1GlyCAM-1

00AOO 18 24 48 72 96 120AOO 18 24 48 72 96 120 AOO 18 24 48 72 96 120AOO 18 24 48 72 96 120

OnzinOnzin OnzinOnzin

GlyCAM-1GlyCAM-110001000 2020

imag

ing

coun

tsim

agin

g co

unts

imag

ing

coun

tsim

agin

g co

unts

imag

ing

coun

tsim

agin

g co

unts

(X 1

0-4)

(X 1

0-4)

(X 1

0-4)

(X 1

0-4)

(X 1

0-4)

(X 1

0-4)800800

600600400400200200

00

1515

1010

55

5050404030302020101000

101088664422

(x 1

0 -4)

(x 1

0-4)

00AOO 18 24 48 72 96 120AOO 18 24 48 72 96 120 AOO 18 24 48 72 96 120AOO 18 24 48 72 96 120

GBP2GBP2GBP2 15GBP2 15

60605050404030302020101000

1010

55

00AOOAOO 1818 2424 4848 7272 9696 120120 AOOAOO 1818 2424 4848 7272 9696 120120

hours post-exposure tohours post-exposure to hours post-exposure tohours post-exposure toDNFBDNFB DNFBDNFB

Figure 4. The kinetics of allergen-induced changes in glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), onzin and guanylate binding protein 2 (GBP2) gene expression. At various times (18 to 120 h) after initiation of treatment with 0.5% 2,4-dinitrofluorobenzene (DNFB) in acetone: olive oil (AOO) vehicle or with vehicle (AOO) alone, draining auricular ly mph nodes were excised. Total RNA and mRNA were prepared and expression of transcripts for GlyCAM-1, onzin and GBP2 was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) (b) or Northern blotting (a), respectively. Expression was quantified by phosphorimager following Northern blot analysis or by Kodak Digital Imaging system for the detection and quantitation of RT-PCR products. Results are expressed as graphically as phosphorimaging counts normalized against the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for Northern blot analysis and as gel imaging counts normalized against the housekeeping gene hypoxanthine phosphoribosyl transferase (HPRT) for RT-PCR analysis. Vehicle (AOO) control gene expression data are expressed as the mean of results recorded for tissue derived 18 h and 120 h following exposure.

43

a)181614121086420

AOO 0.05 0.10 0.25 0.50 b)

20

15

10

5

0 AOO 0.05 0.10 0.25 0.50

c) 140120100806040200

AOO 0.05 0.1 0.25 0.5

DNFB (%w/v)

Figure 5. Allergen-induced lymph node activation and changes in glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) gene expression : dose response analyses. Draining auricular lymph nodes were isolated 48 h after exposure to various concentrations of 2,4-dinitrofluorobenzene (DNFB) in acetone: olive oil (AOO) vehicle or to vehicle (AOO) alone. Lymph nodes were pooled per treatment group and a single cell suspension prepared by mechanical disaggregation. Lymph node cell viability was assessed by trypan blue exclusion and total cellularity per lymph node recorded (a). Proliferative responses were assessed following 24h culture of cells in the presence of 3H-thymidine; results are expressed as mean disintegrations per minute (DPM) and SD of cells cultured in quintuplicate (b). Messenger RNA was prepared and GlyCAM-1 expression analyzed by Northern blotting (c). Bands were visualized by phosphorimager and data are expressed graphically as phosphorimaging counts normalized against the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

phos

phoi

mag

ing

prol

ifer

atio

nce

llula

rity

coun

ts (x

10-5

) (D

PM

x10

-3)

(cel

ls/n

ode

x10-6

)

44

a)

0

0.5

1

1.5

2

cells

/LN

x10

-6

b)

imag

ing

coun

tsD

PM

(x10

-4)

( x10

-4)

16 14 12 10

8 6 4 2 0

c) 12

10

8

6

4

2

0Pre-treatment (flank) AOO AOO OX

Challenge (ear) AOO OX OX

Figure 6. Allergen-induced lymph node activation and changes in glycosylation-dependent cell adhesion molecule (GlyCAM)-1 gene expression : influence of prior exposure. Animals were pre-treated on the shaved flank with 0.1% oxazolone (OX) in acetone:olive oil (AOO) vehicle or with vehicle alone five days prior to challenge on the dorsum of both ears with 1% OX in AOO. Additional control animals received AOO vehicle alone on the shaved flanks and the dorsum of both ears. Auricular lymph nodes were isolated 72 h after challenge on the dorsum of both ears, pooled per treatment group and a single cell suspension prepared by mechanical disaggregation. Lymph node cell viability was assessed by trypan blue exclusion and total cellularity per lymph node recorded (a). Proliferative responses were assessed following 24h culture of cells in the presence of 3H-thymidine; results are expressed as mean disintegrations per minute (DPM) and SD of cells cultured in quintuplicate (b). Total RNA was prepared and analyzed for expression of transcripts for GlyCAM-1 by reverse transcription-polymerase chain reaction (RT-PCR) (c). GlyCAM-1 expression data are displayed graphically as the mean and range of gel imaging counts obtained from analysis of repeated RT-PCR analysis (n=3) normalized against the housekeeping gene hypoxanthine phosphoribosyl transferase (HPRT).

45

a) Day 3 b) Day 5

cellularity cellularity

9 14

8 12 7

DP

M (

x10-2

)SI

ce

ll/no

de (

x10

–6) 10

DP

M (

x10-2

) ce

ll/no

de (x

10 –6

)

6 85

4 6 3

4 2

2

0

1

0

DNFB HCA PPD AOO DNFB HCA PPD AOO

in vitro proliferation in vitro proliferation

40 70

35 60 30

50 25

40 20

3015

2010

5 10

0 0

DNFB HCA PPD AOO DNFB HCA PPD AOO

in vivo proliferation (LLNA)

25

20

15

10

5

0

DNFB HCA PPD AOO

Figure 7. Changes in cellularity and in vitro proliferation on day 3 (a) and day 5 (b) following topical exposure to 0.5% 2,4-dinitrofluorobenzene (DNFB), 50% hexylcinnamaldehyde (HCA), 1% paraphenylenediamine (PPD) or vehicle (acetone:olive oil; AOO) alone. Mice received 25ml of chemical or vehicle alone on the dorsum of both ears on days 0, 1 and 2 prior to assay on days 3 (a) and 5 (b). Lymph node cell viability was assessed by trypan blue exclusion and total cellularity per lymph node recorded. In vitro proliferative responses were assessed following 24h culture of cells in the presence of 3H-thymidine. In vivo proliferation, as measured by use of the local lymph node assay protocol, was assayed on day 5 only and is expressed as the SI (stimulation index relative to vehicle treated control group).

46

a) Day 3 b) Day 5

DNFB HCA PPD AOO DNFB HCA PPD AOO

GBP2

Onzin

HPRT

phos

phor

imag

erco

unts

(x10

-7)

GBP2 GBP290

8090

8070 7060 6050 5040 4030 3020 2010 100 0

onzin 120 onzin120

100 100

80 80

60 60

40 40

20 20

0 0 DNFB HCA PPD AOO DNFB HCA PPD AOO

Figure 8. Allergen-induced lymph node expression of guanylate binding protein 2 (GBP) and onzin. Total RNA was prepared from auricular lymph node tissue excised on days 3 (a) and 5 (b) from mice exposed to 0.5% 2,4-dinitrofluorobenzene (DNFB), 50% hexylcinnamaldehyde (HCA), 1% paraphenylenediamine (PPD) or vehicle (acetone:olive oil; AOO) alone as described in the legend to Figure 7. Onzin and GBP2 expression was assessed by reverse transcription­polymerase chain reaction (RT-PCR) followed by slot blotting of PCR products to nylon membrane and hybridization with short oligonucleotide probes end-labelled with 32P. Hybridized blots were quantified by phosphorimager and the counts obtained normalized against expression of the housekeeping gene hypoxanthine phosphoribosyl transferase (HPRT).

47

Table 3. Gene changes observed following array analysis of human blood-derived dendritic cells cultured with 10.7pM 2,4-dinitrofluorobenzene (DNFB) or to 0.01% dimethyl sulphoxide (DMSO) vehicle alone for 2 hours. Messenger RNA was used to generate labelled cDNA probes which were hybridized to in-house cDNA arrays comprising of 12,554 human cDNA sequences arrayed in duplicate. Data was quantified by phosphorimager and analyzed using Array Vision software. Fold increases are shown relative to the vehicle control group and there were no significant down-regulations in gene expression.

Description Novel adipose-specific collagen-like factor Human homeobox protein CDX4 Human PKC alpha mRNA Human putative DNA methyltransferase Human collagenase and stromelysin genes Human genomic DNA of 8p21.3-p22 anti-oncogene Human leukocyte interferon Human PTGS2 gene Human neurogenic extracellular slit protein Slit2 mRNA Light chain 3 subunit of microtubule-associated proteins 1A and 1B Human myosin alkali light chain (ventricular) Human SH2-containing inositol 5-phosphatase (hSHIP) Human Ig rearranged H-chain, V region Human Zinc finger protein ZNF191 Human hyaluronan synthase mRNA Human CCR10 mRNA Human mRNA for 5-HT2B serotonin receptor Similar to C3HC4 zink finger Human kallikrein-like protein 5 Human acetylcholinesterase (ACHE) gene Protein phosphatase 2A 55kD regulatory subunit B Human DEAD-box protein p72 Human Ig heavy chain variable region from IgM rheumatoid factor Human leukotriene B4 omega-hydroxylase Human mRNA for Ig kappa light chain mPOU homeobox protein Human chromosome X region from filamin gene Human serine protease ovasin mRNA Human protease activated receptor 3 gene Human lambda DNA for Ig light chain Human activin receptor type IIB Human proteasome inhibitor hP131 subunit Keratin 6 beta Human genomic DNA of 8p21.3-p22 anti-oncogene High glycine tyrosine keratin type II.4 Similar to AAA family of ATPases Human genomic DNA of 8p21.3-p22 anti-oncogene Human Sec24 protein Human alpha SNAP mRNA Human histone H2B mRNA Zfp-29 Human IL-13

Fold increase 2.4 2.3 2.3 2.3 2.3 2.2 2.2 2.2 2.2 2.2 2.1 2.1 2.1 2.1 2.1 2.1 2.1 2.1 2.1 2.1 2.1 2.1 2.1 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 1.9 1.9 1.9 1.9 1.9

48

0.01% DMSO 10.7pM DNFB0.01% DMSO 10.7pM DNFB

Interleukin 13Interleukin 13(1.9(1.9 fold)fold)

CollagenaseCollagenase(2.2(2.2 fold)fold)

AcetylcholinesteraseAcetylcholinesterase(( 2.0 fold)2.0 fold)

Homeobox protein CDX4Homeobox protein CDX4(( 2.3 fold)2.3 fold)

Collagen-like factorCollagen-like factor(2.4(2.4 fold)fold)

Figure 9. Visual changes in areas of the human array membrane containing some of the genes up­regulated following treatment for 2 hours with 10.7pM 2,4-dinitrofluorobenzene (DNFB) or to 0.01% dimethyl sulphoxide (DMSO) control. Messenger RNA from blood-derived human dendritic cells was reverse transcribed to generate radiolabelled cDNA probes which were hybridised to human microarray membranes as described previously (Table 3). Gene expression changes are highlighted by arrows indicating the duplicate spots for that gene sequence and the fold increase in expression is given in brackets

49

Table 4. Percentage of human cells expressing CD14, CD1a or MHC class II immediately following monocyte isolation (day 0) and after 5 days in culture. CD14+ monocytic precursors were purified from fresh human blood by ficoll gradient and incubation with the monocyte isolation kit (Miltenyi Biotech). Cellular phenotype was analyzed immediately post-isolation and following differentiation in vitro with human interleukin-4, granulocyte-macrophage colony stimulating factor and transforming growth factor­b by flow cytometry. Values are expressed as the percentage of positive cells detectable above the relevant isotype control antibodies for donors 1 to 19.

Day 0 Day 5

MACS Donor Post-bead isolation number

%CD14 %CD1a %MHC II %CD14

1 1 88 68 89 0

2 2 73 19 97 0

3 3 63 83 98 0.5

4 4 71 15 92 13

5 5 54 57 90 0

6 6 76 97 98 3

7 1 83 70 90 0

8 7 57 80 97 0

9 8 58 85 98 0

10 9 35 65 95 0

11 10 87 93 97 0

12 1 78 84 96 0

13 11 90 95 98 0

14 12 54 93 96 0

15 13 96 83 98 1.5

16 14 86 67 99 0

17 11 85 96 99 0

18 15 56 88 96 0

19 16 89 90 95 0

20 17 70 79 88 0

21 18 57 91 93 0

22 19 65 86 98 0

23 11 81 94 91 0

50

Day 0 Day 5

b) 37oC4oC

FITC-dextran uptake

a)

MHC II

CD1a

CD14

CD86

E-cadherin

day 5

Figure 10. Cellular phenotype of peripheral blood monocytes at day 0, immediately after isolation, and on day 5, following differentiation in vitro into blood-derived dendritic cells. Cells were incubated with antibodies to detect the expression of the human cell surface markers displayed or with corresponding isotype controls. The conversion to a CD1a+ve, CD14-ve, CD86low population is clearly visible by day 5 (a). The ability of day 5 cells to internalize protein was demonstrated by culture with fluorescein isothiocyanate (FITC)-dextran for 30mins at 37oC, or 4oC. Internalization of the protein by active processes was assessed by flow cytometry (b) and is greatly reduced by incubation at 4oC which controls for the adherence of the dextran to the outer cell surface.

51

a) b) IL-1b

50300 2.0x24.7x250 40 1.3x

1.3x200 30150

20 100

1050

0 PMA Med

0 DNFB DMSO DNFB DMSO DNFB DMSO

day 3 day 4 day 5

IL-18 250 7.1x

140 5.2x200 120

100150

80100 60

40 1.7x50 1x0

20 0

PMA Med DNFB DMSO DNFB DMSO DNFB DMSO

day 3 day 4 day 5

IL-12 p40 30300

1.7x25 2.9x

20200 1.1x 15

4.8x10100

5 0 0

PMA Med DNFB DMSO DNFB DMSO DNFB DMSO

day 3 day 4 day 5

Figure 11. Expression of interleukin (IL)-1b, IL-18 and IL-12 p40 mRNA in donor 13 as detected by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blotting. RT-PCR products were hybridized to short radiolabelled oligonucleotide probes internal in sequence to that amplified by the PCR primers. Hybridized blots were quantified by phosphorimager and data normalized for b­actin (housekeeping gene) expression. Cytokine expression following treatment with phorbol myristate acetate (PMA [a] exposed on day 5 for 24h and assayed on day 6) and with 2,4-dinitrofluorobenzene (DNFB [b] exposed for 1h on days 3, 4 and 5) is up-regulated (fold increases as marked) with the maximal response observed on day 5 of in vitro differentiation. Data are displayed as phosphorimager counts with the fold change in expression compared with medium (for PMA) or with vehicle dimethyl sulphoxide (DMSO, for DNFB) control indicated above each bar.

52

PI

coun

ts x

10-5

P

I co

unts

x 1

0-5

PI

coun

ts x

10-5

a) donor 14 donor 13

DN

FB

DM

SO

Med

DN

FBD

MSO

Med

b

IL-13

-actin

b) 24 hours

0 5

10 15 20 25 30 35 40

DMSO

5.29x

0 5 10 15 20 25 30 35 40 56.5x

PI

coun

ts x

10-5

PMA Med DNFB

Figure 12. Expression of interleukin (IL)-13 mRNA in donor 13 as detected by reverse transcription­polymerase chain reaction (RT-PCR) and Southern blotting (as described for Fig. 11). The absence in donor 14, and presence in donor 13, of detectable message can be seen at the visual level on the Southern blots (a). Once normalized for b-actin (housekeeping gene) mRNA levels, expression of IL-13 message in donor 13 following 24h treatment (exposed on day 5 and assayed on day 6) is up-regulated in response to both phorbol myristae acetate (PMA) and 2,4-dinitrofluorobenzene (DNFB). Fold increases as marked (b).

53

Pho

spho

rim

agin

gco

unts

( x

105 )

‘Responder’ ‘Non-responder’ P

hosp

hori

mag

ing

Pho

spho

rim

agin

g P

hosp

hori

mag

ing

donor 15 donor 16 co

unts

( x

105 )

co

unts

( x

105 )

co

unts

( x

105 )

IL-1b IL-1b

10000 6.3x 16000 1.1x9000 140008000 12000 1.3x7000

1000060005000 3.4x 80004000 60003000 40002000

20001000 00

PMA DNFB DMSO Med PMA DNFB DMSO Med

IL-6 IL-6 3.6x 900120 1.1x

800100 700

2.2x 60080 0.9x50060 400

30040 200

20 1000 0

PMA DNFB DMSO Med PMA DNFB DMSO Med

IL-12p40 IL-12p40 2.3x180 1800

160 1.6x 0.9x 1600140 1400120 1200

1.2x100 1000 80 800 60 600 40 40020 200

0 0 PMA DNFB DMSO Med PMA DNFB DMSO Med

IL-18 IL-18

180 8003.5x160 700140 600120 2.5x 500100

400 0.9x 0.4x80 60 30040 20020 100

00 PMA DNFB DMSO Med PMA DNFB DMSO Med

Figure 13. Expression of interleukin (IL)-1b, IL-6, IL-18 and IL-12 p40 mRNA in donors 15 and 16 as detected by reverse transcription-polymerase chain reaction (RT-PCR) and slot blot analysis with radiolabelled oligonucleotide probes. Data was quantified by phosphorimager and normalized for housekeeping gene expression (b-actin). Fold increases in expression over dimethyl sulphoxide (DMSO, for 2,4-dinitrofluorobenzene [DNFB]) or medium (for phorbol myristate acetate [PMA]) controls following 2h treatment are shown above data bars.

54

Table 5. Gene changes observed following array analysis of human blood-derived dendritic cells from donor 19 treated with 0.5mM 2,4-dinitrofluorobenzene (DNFB) or with 0.01% dimethyl sulphoxide (DMSO) vehicle alone for 2h. Cells were treated following 5 days of culture with human cytokines and at a time when the cellular phenotype resembled that of immature dendritic cells. Total RNA was used to generate labelled cDNA probes which were hybridized to in-house cDNA arrays comprising of 12,554 human cDNA sequences arrayed in duplicate. Data was quantified by phosphorimager and analyzed using Array Vision software. Fold increases are shown relative to the vehicle control group.

Gene Description Fold Change Up-regulations ATP/ADP translocator (ANT1) gene 1.7 cAMP specific phosphodiesterase (PDE4A) gene 1.5 Down -regulations Transducin -like enhancer protein (TLE2) 9.3 Type -2 phosphatidic acid phosphatase -b (PAP2-b) 3.9 Antigen processing and presentation genes (LMP2, TAP1, RING8 etc) 3.6 Thrombin receptor 3.5 Germline DNA for Ig kappa variable region 3.5 C terminus of pol protein (reverse transcriptase) 3.5 Rolipram -sensitive 3’,5’ -cAMP phosphodiesterase 2.8 H3.3 histone, class B 2.4 G-septin a 1.9 Retinal glutamate transporter EAAT5 1.8 Sorting nexin 14 (SNX14) 1.8 Transcription factor RFX -B 1.7 Cellular proto -oncogene (c -mer) 1.6 Interferon -a-like gene 1.5 Germline T cell receptor b chain 1.5 Lamin B2 (LAMB2) 1.5 Ras-specific guanine nucleotide -releasing factor, CDC25 homolog 1.5 Protein trafficking protein (S31iii125) 1.5 Interleukin 1 RN 1.5

55

Printed and published by the Health and Safety ExecutiveC30 1/98

Printed and published by the Health and Safety Executive C1.10 0 9/03

ISBN 0-7176-2720-9

RR 142

9 78071 7 627202£10.00