Document 1800729

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 be
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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 [email protected]
ii
CONTENTS
SUMMARY
v
INTRODUCTION
1
PROJECT AIMS
5
METHODS
Transcript profiling of murine lymph node cells
Transcript profiling of human dendritic cells
7
7
8
RESULTS
Transcript profiling of early gene changes in allergen-activated lymph node
cells Transcript profiling of allergen-activated human blood derived DC
11 11 DISCUSSION
Transcript profiling of early gene changes in allergen-activted lymph node
cells Transcript profiling of allergen-activated human blood derived DC
19 19 CONCLUSIONS
25
REFERENCES
27
BIBLIOGRAPHY
Publications
Publications (Abstracts)
33 33 34
APPENDIX A
Abbreviations
35 35
APPENDIX B
Primer sequences
37 37
APPENDIX C
Figure 1
Table 1
Table 2
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Table 3
Figure 9
Table 4
Figure 10
Figure 11
Figure 12
Figure 13
Table 5
38 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 APPENDIX D
Submitted Paper 56 iii 14
22
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 E­
cadherin (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 IL­
1b 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 (up­
or 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 (TGF­
b). 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 anti­
human 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
33
P-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 ALLERGENACTIVATED 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 DNFB­
treated 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 down­
regulated 5 times out of 6 over the two experiments. Another gene found to be down­
regulated 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 up­
and 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 up­
regulated 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 down­
regulation 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 dose­
dependent 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 3H­
thymidine 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; up­
regulation 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 up­
regulation 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 up­
regulated 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 IL­
12p40 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 ALLERGENACTIVATED 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 up­
regulated 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 allergen­
specific 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 up­
regulated 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
3
H-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 down­
regulated 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 down­
regulated. 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 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.
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 up­
regulated 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.
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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, 2,4dinitrofluorobenzene; 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’; IL­
13 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’; IL­
12p40 5’-CATTCGCTCCTGCTGCTTCA-3’; IL-13 5’-TGAGCGGATTCTG CCCGCAC­
3’; IL-18 5’-GAGATAATGCACCCCGGACC-3’.
37 APPENDIX C
cellularity
(cells/node x 10-6 )
a)
20
15
10
5
0
AOO 18
48
72
96
120
b)
proliferation
(DPM x 10-3 )
14
12
10
8
6
4
2
0
AOO 18
48
72
96
120
Time (h)
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% 2,4dinitrofluorobenzene (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
accession #
Expt. 1
fold decrease
Expt. 2
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
W87981
AA108600
AA097598
1.5
1.6
1.6
1.8
1.6
1.7
<1.5
1.8
Apoptosis inhibitor 3
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
guanylate nucleotide binding protein 2
lymphocyte antigen 6 complex
small inducible cytokine B subfamily (Cys-X-Cys), member 9
ubiquitin specific protease 18
EST, Moderately similar to HEM45 [H.sapiens]
EST, Highly similar to endothelial actin-binding protein
small inducible cytokine A12
lymphocyte antigen 6 complex, locus C
histocompatibility 2, class II antigen E beta
EST, Weakly similar to lymphocyte antigen LY-6A.2
histocompatibility 2, complement component factor B
EST
CD20 antigen
peroxisomal membrane protein 20
regulatory protein, T lymphocyte 1
EST
lymphocyte antigen 6 complex, locus D
5.8
3.8
3.4
2.9
2.6
2.5
2.5
2.3
2.1
2.1
2.1
2.1
2.0
2.0
2.0
2.0
2.0
1.9
b) Down-regulated array sequences
EST
EST
EST
EST
EST
EST
staufen (RNA-binding protein) homolog 2 (Drosophila )
EST
smoothened homolog (Drosophila )
thymus cell antigen 1, theta
EST
EST, weakly similar to x-linked inhibitor of apoptosis protein
EST
apoptosis inhibitor 3
zipcode-binding protein 1
EST
EST
nuclear receptor binding factor 1
EST, moderately similar to mitochondrial import inner
membrane translocase subunit
peroxisome biogenesis factor 16
EST
EST, highly similar to KIAA0183 [H.sapiens]
phospholipase A2, activating protein
Glycosylation-dependent cell adhesion molecule -1
40
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
2.0
2.0
2.5
2.5
2.5
2.5
2.5
2.5
3.3
5.8
18h
AOO
48h
AOO
DNFB
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 up­
regulation, guanylate binding protein 2 [GBP2, EMBL accession number AA153021]; 3.8 fold up­
regulation) 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)
AOO 0.05
0.1
0.25
0.5
150
GlyCAM-1
100
50
phosphorimaging counts (x10-5 )
GlyCAM-1
Onzin
GBP-2
0
AOO
60
50
40
30
20
10
0
0.05
0.25
0.50
Onzin
AOO
100
0.10
0.05
0.10
0.25
0.50
GBP2
80
60
40
20
0
GAPDH
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 2,4dinitrofluorobenzene (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).
42
20
GlyCAM-1
15
10
5
0
AOO 18
24
48
72
96
120
AOO 18
Onzin
imaging counts
(X 10 -4 )
50
40
30
20
10
0
AOO
18
24
48
72
96
24
48
72
96
120
24
48
72
96
120
24
48
72
96
120
Onzin
AOO 18
GBP2
GBP2
15
60
50
40
30
20
10
0
AOO
10
8
6
4
2
0
120
imaging counts
(X 10 -4 )
phosphorimaging counts
(x 10 -4 )
b)
GlyCAM-1
imaging counts
(X 10 -4 )
1000
800
600
400
200
0
phosphorimaging counts
(x 10 -4 )
phosphoimaging counts
(x 10-4)
a)
18
24
48
72
96
120
10
5
0
AOO 18
hours post-exposure to
DNFB
hours post-exposure to
DNFB
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-3phosphate 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
cellularity
(cells/node x10-6 )
a)
18
16
14
12
10
8
6
4
2
0
AOO 0.05
0.10
0.25
0.50
AOO
0.05
0.10
0.25
0.50
AOO
0.05
0.1
0.25
0.5
b)
proliferation
(DPM x10-3 )
20
15
10
5
c)
phosphoimaging
counts (x10-5 )
0
140
120
100
80
60
40
20
0
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).
44
a)
cells/LN x10-6
2
1.5
1
0.5
0
DPM (x10-4 )
b)
16
14
12
10
8
6
4
2
0
c)
imaging counts
( x10-4 )
12
10
8
6
4
2
0
Pre-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 3 H-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
cell/node (x10 –6 )
cell/node (x10 –6 )
7
6
5
4
3
2
10
8
6
4
2
1
0
0
DNFB
HCA
PPD
AOO
DNFB
PPD
AOO
in vitro proliferation
in vitro proliferation
40
70
35
60
DPM (x10-2 )
DPM (x10-2 )
HCA
30
25
20
15
50
40
30
10
20
5
10
0
0
DNFB
HCA
PPD
AOO
DNFB
HCA
PPD
AOO
in vivo proliferation
(LLNA)
25
20
SI
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 3 H-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
DNFB
HCA
b) Day 5
PPD
AOO
DNFB
HCA
PPD
AOO
GBP2
Onzin
phosphorimagercounts (x10-7 )
HPRT
90
80
70
60
50
40
30
20
10
0
120
GBP2
90
80
70
60
50
40
30
20
10
0
onzin
120
100
100
80
80
60
60
40
40
20
20
0
0
GBP2
onzin
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 transcription­
polymerase chain reaction (RT-PCR) followed by slot blotting of PCR products to nylon
membrane and hybridization with short oligonucleotide probes end-labelled with 32 P. 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
48
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
0.01% DMSO 10.7pM DNFB
Interleukin 1 3
(1.9 fold)
Collagenase
(2.2 fold)
Acetylcholinesterase
(
2.0 fold)
Homeobox protein CDX4
(
2.3 fold)
Collagen-like factor
(2.4 fold)
Figure 9. Visual changes in areas of the human array membrane containing some of the genes up­
regulated 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 factor­
b 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
MACS
isolation
Donor
number
Day 5
Post-bead
%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
a)
Day 5
MHC II
CD1a
CD14
CD86
E-cadherin
b) FITC-dextran uptake
day 5
4o C
37o C
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 37o C, or 4o C. Internalization of the protein by active
processes was assessed by flow cytometry (b) and is greatly reduced by incubation at 4o C which controls
for the adherence of the dextran to the outer cell surface.
51
a)
b)
PI counts x 10-5
IL-1b
300
50
24.7x
250
40
200
30
150
2.0x
1.3x
1.3x
20
100
10
50
0
PMA
Med
0 DNFB DMSO DNFB DMSO DNFB DMSO
day 3
day 4
day 5
PI counts x 10-5
IL-18
7.1x
250
200
150
100
50
0
PMA
Med
140
120
100
80
60
40
20
0
5.2x
1x
1.7x
DNFB DMSO DNFB DMSO DNFB DMSO
day 3
day 4
day 5
PI counts x 10-5
IL-12 p40
30
300
25
2.9x
20
200
100
1.7x
1.1x
15
4.8x
10
5
0
PMA
Med
0
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 b­
actin (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 2,4dinitrofluorobenzene (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
donor 13
Med
Med
DMSO
DNFB
donor 14
DNFB
DMSO
a)
IL-13
b-actin
PI counts x 10-5
b)
24 hours
56.5x
40
35
30
25
20
15
10
5
40
35
30
25
20
15
10
5
0
0
PMA
Med
5.29x
DNFB
DMSO
Figure 12. Expression of interleukin (IL)-13 mRNA in donor 13 as detected by reverse transcription­
polymerase 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
‘Non-responder’
‘Responder’
donor 15
donor 16
IL-1b
Phosphorimaging
counts ( x105 )
IL-1b
10000
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
6.3x
16000
14000
12000
10000
8000
6000
4000
2000
0
3.4x
PMA
DNFB
DMSO
1.1x
1.3x
Med
PMA
Phosphorimaging
counts ( x105 )
IL-6
120
100
80
60
40
20
0
900
800
700
600
500
400
300
200
100
0
2.2x
DNFB
DMSO
Phosphorimaging
counts ( x105 )
1.6x
PMA
Phosphorimaging
counts ( x105 )
DNFB
DMSO
Med
DMSO
Med
DMSO
Med
IL-12p40
1800
1600
1400
1200
1000
800
600
400
200
0
0.9x
DNFB
DMSO
Med
2.3x
1.2x
PMA
DNFB
IL-18
800
700
600
500
400
300
200
100
0
3.5x
2.5x
PMA
0.9x
PMA
IL-18
180
160
140
120
100
80
60
40
20
0
Med
1.1x
Med
IL-12p40
180
160
140
120
100
80
60
40
20
0
DMSO
IL-6
3.6x
PMA
DNFB
DNFB
DMSO
Med
0.9x
0.4x
PMA
DNFB
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
Up-regulations
ATP/ADP translocator (ANT1) gene
cAMP specific phosphodiesterase (PDE4A) gene
Down -regulations
Transducin -like enhancer protein (TLE2)
Type -2 phosphatidic acid phosphatase -b (PAP2-b)
Antigen processing and presentation genes (LMP2, TAP1, RING8 etc)
Thrombin receptor
Germline DNA for Ig kappa variable region
C terminus of pol protein (reverse transcriptase)
Rolipram -sensitive 3’,5’ -cAMP phosphodiesterase
H3.3 histone, class B
G-septin a
Retinal glutamate transporter EAAT5
Sorting nexin 14 (SNX14)
Transcription factor RFX -B
Cellular proto -oncogene (c -mer)
Interferon -a -like gene
Germline T cell receptor b chain
Lamin B2 (LAMB2)
Ras-specific guanine nucleotide -releasing factor, CDC25 homolog
Protein trafficking protein (S31iii125)
Interleukin 1 RN
55
Fold Change
1.7
1.5
9.3
3.9
3.6
3.5
3.5
3.5
2.8
2.4
1.9
1.8
1.8
1.7
1.6
1.5
1.5
1.5
1.5
1.5
1.5
Printed and published by the Health and Safety Executive
C30 1/98
Printed and published by the Health and Safety Executive
C1.10 0 9/03
ISBN 0-7176-2720-9
RR 142
£10.00
9 78071 7 627202