Perinatal exposure of mice to TCDD decreases allergic sensitisation through inhibition of IL-4 production rather than T regulatory cell-mediated suppression.

IJOMEH 2010;23(1) 75
O R I G I N A L P A P E R S
International Journal of Occupational Medicine and Environmental Health 2010;23(1):75 – 83
DOI 10.2478/v.10001-010-0006-7
PERINATAL EXPOSURE OF MICE TO TCDD
DECREASES ALLERGIC SENSITISATION THROUGH
INHIBITION OF IL-4 PRODUCTION RATHER THAN
T REGULATORY CELL-MEDIATED SUPPRESSION
MACIEJ TARKOWSKI1, BARBARA KUR1, MAREK NOCUŃ1, and KRYSTYNA SITAREK2
1 Nofer Institute of Occupational Medicine, Łódź, Poland
Department of Immunotoxicology
2 Nofer Institute of Occupational Medicine, Łódź, Poland
Laboratory of Toxicity Assessment
Abstract
Objective: The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a widespread, man-made, persistent organic pollutant with
high immunotoxic potentials. It suppresses cell-mediated and humoral immune responses through mechanisms dependent
on aryl-hydrocarbon receptor expression and immunosuppressive activity of the cells. Most sensitive to TCDD are organ-
isms during fetal and infant life, mostly due to the developmental stage of many biological systems of the host, including
immune system. Recent data show that T regulatory cells that have the potential to suppress immune reactions and which
develop after TCDD exposure are also responsible for protection from allergy development. Our goal was to investigate if
perinatal exposure to TCDD can affect allergic sensitisation and if T reg cells participate in this phenomenon. Materials
and Methods: Mice, Balb/c, were perinatally exposed to TCDD or to the carrier. Six weeks old control or exposed mice
were sensitised with ovalbumin. Spleen cells of the animals were used to assess the constent of T reg cells by means of flow
cytometry. Levels of cytokines were assessed by ELISA technique in supernatants of the cells stimulated with anti-CD3
antibody. As a measure of sensitisation, total IgE and anti-OVA IgE were measured in serum of mice by ELISA method.
To assess the function of T reg cells isolated from OVA-sensitised control or TCDD exposed animals we performed transfer
studies. Results: Here we show that perinatal exposure to TCDD decreases allergic sensitisation and that this process is
related to inhibition of IL-4 synthesis rather than suppression mediated by T regulatory cells. Conclusion: We hypothesise
that dioxin exposure can be an important environmental modulator of immunological responses that participate in allergic
reactions.
Key words:
Dioxin, T regulatory cells, Allergic sensitization, Animal model, Perinatal exposure
Received: November 13, 2009. Accepted: December 4, 2009.
Address reprint request to M. Tarkowski, Department of Immunotoxicology, Nofer Institute of Occupational Medicine, św. Teresy 8, 91-348 Łódź, Poland
(e-mail: maciekt@imp.lodz.pl).
INTRODUCTION
The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is
a widespread, man-made, persistent organic pollutant.
This compound is very stable in environment and can ac-
cumulate in the tissues of organisms. Together with its al-
ready known, deleterious effects on diverse biological sys-
tems, including immune system, exposure to TCDD offers
possible risk to human health [1–6].
In adult animals, exposure to TCDD has been shown to
suppress cell-mediated and humoral immune responses
(for review, see [2,3]). However, perinatally exposed off-
spring is most susceptible to the toxic effects of TCDD.
Perinatally exposed animals showed, at the organ level, at-
rophy of the thymus [7], enhanced apoptosis of thymocytes
[8], and alterations in T-lymphopoiesis [9]. The outcome
of immunotoxic effects of TCDD on developing immune
system in animals was shown to result in exacerbation of

O R I G I N A L P A P E R S M. TARKOWSKI ET AL.
IJOMEH 2010;23(1)76
present at the very low levels that do not cause apparent
adverse health effects. But their accumulation over certain
amount of time, especially in fat tissue, may increase the
level of exposure during perinatal life, modulate develop-
ment of immune system and cause changes manifested
later in life. We tried to find out if perinatal exposure of
mice to TCDD could affect allergic sensitisation and what
immunological mechanisms might participate in these re-
actions.
MATERIALS AND METHODS
Animals
Inbred strain BALB/c mice (Harlan, Netherlands) were
used in the experiments. The animals were bred in ac-
cordance with the standard procedures and were kept in
a room with appropriate 12 h light/dark cycle. They re-
ceived water and pelleted food (Murigran) ad libitum.
Animal experiments were approved by the local Ethical
Committee (doc. no. L/BD/285). Analyses of perinatal
exposure use data collected from 6-week old male mice.
Experiments were repeated twice and the total number of
animals was 6. Spleens and blood were not pooled.
Reagents
The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD;
CAS 1746-01-6, AccuStandard, Inc. USA) was dissolved
in acetone and mixed with olive oil. Acetone was removed
by evaporation by heating the solution. Further dilutions
were done in olive oil. For sensitisation of animals we used
ovalbumin (OVA, CAS 9006-59-1, SIGMA). Ovalbumin
was dissolved in 1:1 mixture of PBS (Dulbecco’s Phos-
phate Buffered Saline, SIGMA) and aluminum hydroxide
(Alum, Al(OH)3, 13 mg/ml, CAS 1330-44-5, SIGMA) was
used at a final concentration of 20 μg OVA/1 mg Alum.
Perinatal exposure of mice to TCDD
Female and male mice were cohabited and the appear-
ance of a vaginal plug was considered to be the begin-
ning of pregnancy and gestational day (GD) 0. On GD15,
mice were given intraperitoneally a single dose of 100 μl
of TCDD at concentration of 10 μg/kg of body mass
postnatal autoimmune reactions [10,11] suppressed cell-
mediated and humoral immune responses [7,12–14]. In
studies on Dutch infants [15] prenatal PCB/dioxin expo-
sure was associated with changes in several immunological
parameters. At the preschool age of these children [16],
an association was found between PCB/dioxin exposure
and higher prevalence of infections of middle ear. This as-
sociation continued to be significant at their school age
[17]. Of other clinical effects observed in these children at
preschool age, but no more at school age, was decreased
susceptibility to allergic sensitisation that authors suggest
could be due to skewed immune response toward infec-
tious agents rather than direct effect of dioxin exposure.
However, experimental data from studies on adult animals
show that humoral immune responses, including antigen-
specific antibody production [18], synthesis of proallergic
Th2 derived cytokines such as IL-5, IL-4 [19,20] are sup-
pressed due to direct effect of TCDD exposure. Addition-
ally, experimental studies performed in rats showed that
exposure to TCDD can directly decrease allergic immune
response to house dust mite [21].
TCDD has been shown to induce immunosuppression in
mice through mechanisms dependent on the expression
of aryl-hydrocarbon receptor (AhR) [22,23] and devel-
opment of immunosuppressive T cells similar to natu-
ral T regulatory cells [24]. Analyses of the characteristics
of these cells [25] show that, like natural T reg cells, they
suppress responder cells in cell-contact dependent way,
do not produce IL-2, suppress early production of this cy-
tokine from responder cells, and exhibit high level of gran-
zyme b gene expression. However, these cells also produce
significant amounts of IL-10, a cytokine that is ascribed to
other T regulatory cell subpopulation known as Tr1. Natu-
ral, CD4CD25foxp3+ and inducible, IL10 (Tr1) T regu-
latory cells, as well as other subpopulations of suppres-
sor cells, including those which rely on TGF-β (reviewed
[26,27]) inhibit processes of allergic sensitisation and/or
suppress allergic inflammatory reactions by inhibiting Th2
cell–derived cytokines.
Environmental factors are important elements that affect
the development of allergy and asthma [28–30]. Dioxins
and PCBs are environmental contaminants, usually

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IJOMEH 2010;23(1) 77
manufacturer. Analyses were performed on flowcytom-
eter FACSCantoII (Becton Dickinson).
Total and anti-OVA specific IgE serum levels assessment
The concentrations of total IgE in serum were estimated
by ELISA (Becton Dickinson) according to the proto-
col supplied by manufacturer. The sensitivity of the test
was 2 ng/ml.
The concentrations of IgE specific to OVA in serum were
also assessed by ELISA (MD Biosciences, Switzerland)
according to the protocol supplied by manufacturer. The
sensitivity of the test was 3.8 ng/ml.
Cell transfer studies
CD4+CD25+ cells were isolated from spleens of 6-week
old mice perinatally exposed to TCDD or control animals.
Isolation of these cells was performed first by negative se-
lection of CD4+ T cells using antibody coated magnetic
beads (Invitrogen). In the next steps, CD25+ T cells were
isolated from CD4+ T cells using antibody coated beads
(Miltenyi Biotec) according to the protocol supplied by
manufacturer. The purity of isolated CD4+CD25+ T cells
was 99% as assessed by flow cytometry. Next, 0.4×106 of
these T cells, suspended in 0.2 ml of NaCl were trans-
ferred to untreated 6-week old Balb/c mice. Mice that
were transferred with CD4+CD25+ T cells were subject-
ed to the protocol of OVA sensitisation. First dose of OVA
was given on the day after the cell transfer. Blood isolated
on 30th day after the first dose of OVA was collected for
serum isolation and the use in assessment of total and spe-
cific anti-OVA IgE.
Flow cytometry analysis
Half a million of spleen T cells per sample were surface
stained with anti-CD3 (Alexa Fluor 488, clone 17A2,
Biolegend), CD4 (PE/Cy5, Clone GK 1.5, Biolegend) and
CD25 receptors (PE, clone PC 61, Biolegend). For assess-
ment of T regulatory cells, 1 x106 cells were surface stained
with CD4 and CD25 with antibodies indicated above, and
this was followed by intracellular labelling with anti-foxp3
antibodies (Alexa Fluor 488, clone 150D, Biolegend).
Intracellular staining was performed using Fix/perm and
or 100 μl of carrier (Olive Oil). Offspring was nursed
by their mothers and pups were weaned at 22nd day of
age, transferred to one cage and bred in regular condi-
tions. At 6 weeks of age, mice were sacrificed by intrap-
eritoneal injection of overdose of sodium pentobarbital
(100 mg/kg body mass) and spleens were removed for
further processing.
OVA sensitisation protocol
Sensitisation of 6-week old mice perinatally exposed to
TCDD or carrier, was performed by 3 intraperitoneal in-
jections of 150 μl of 20 μg OVA/1 mg on day 0, 14 and 21.
Nine days after the last dose, mice were sacrificed by in-
traperitoneal injection of overdose of sodium pentobarbi-
tal (100 mg/kg body mass) and blood was collected from
orbital sinus and spleens removed.
Preparation of spleen cells
Spleens after removal were kept in Petri dishes in cold
PBS until ready for cell isolation. Cells were removed
by tearing spleens over the surface of 100 μm pore cell
strainer (Becton-Dickinson) and washed with PBS. Eryth-
rocytes were removed by haemolysis using RBC Lysis Buf-
fer (BioLegend). Lysis was stopped after 5 min by add-
ing 10 ml PBS. Cell viability was estimated by trypan blue
exclusion method and the number of cells was estimated
by use of Neubauer haemocytometer.
In vitro stimulation of spleen cells
Spleen cells were seeded into 48 well plate at density
of 1.0×106 cells/well in 1 ml of culture media. Cells were
left unstimulated or stimulated with 0.1 mg of anti-CD3
monoclonal antibody (anti-mouse CD3 purified, BD
Pharmingen) for 3 days in incubator, at 37°C and in at-
mosphere containing 5% CO2. After that time plates were
centrifuged and the collected supernatant was stored
at –70°C till the time of cytokine analysis.
Assessment of cytokine concentration in supernatants
Cytokine supernatant levels were assessed with the use of
mouse Th1/Th2 Cytokine Cytometric Bead Array (Bec-
ton Dickinson) according to the protocol supplied by

O R I G I N A L P A P E R S M. TARKOWSKI ET AL.
IJOMEH 2010;23(1)78
The effect of perinatal exposure to TCDD on proallergic
inflammatory cytokines produced in vitro by spleen cells
of OVA-sensitised or non-sensitised mice
Allergic sensitisation depends on the release of IL-4, cytokine
that switches production of immunoglobulins to IgE class in
B cells. Allergic inflammatory processes include participation
of such cytokines as IL-5, which is major protein that affects
eosinophil activity, and TNF-α that is mainly released from
neutrophils and monocytes. We determined the levels of IL-4,
IL-5 and TNF-α in supernatants of cells stimulated in vitro
with anti-CD3 that were obtained from spleens of OVA sensi-
tised animals perinatally exposed to TCDD or to the carrier.
Concentrations of IL-4 in supernatans of anti-CD3 stimu-
lated cells were significantly lower in TCDD exposed mice,
whereas IL-5 and TNF-α were at similar levels (Figure 2).
The effect of perinatal exposure to TCDD on IFN-γ
and IL-2 cytokines produced in vitro by spleen cells
of mice sensitised to OVA
Allergic sensitisation is accompanied by preferential syn-
thesis of cytokines such as IL-4 that belongs to cytokines
produced by so called Th2 cells, and inhibition or no ef-
fect on cytokines such as IL-2 or IFN-γ that belong to cy-
tokines released by Th1 cells. We determined the levels
of IL-2 and IFN-γ in supernatants of cells stimulated in
vitro with anti-CD3 that were obtained from spleens of
Perm buffer (Biolegend) according to the protocol sup-
plied by manufacturer. Control samples were stained with
mouse IgG1 antibodies (Alexa Fluor 488, clone MOPC-21,
Biolegend) to control for foxp3 specific staining, and Rat
IgG1 antibodies (PE, clone RTK2071, Biolegend) to con-
trol for CD25 expression.
Statistical analysis
Statistical analyses were performed by T-test or nonpara-
metric test of Mann-Whitney using GraphPad Prism soft-
ware.
RESULTS
IgE levels in serum of OVA-sensitised,
perinatally TCDD-exposed or non-exposed mice
Six week old mice from perinatal exposure to TCDD or
control animals were subjected to the experimental, al-
lergic sensitisation by OVA and serum levels of total IgE
and IgE specific for OVA were evaluated. We found that
total levels of IgE in serum of mice perinatally exposed to
TCDD and OVA-sensitised were significantly higher than
in animals not exposed to dioxin (Figure 1). However, the
levels of serum IgE specific to OVA were higher in control
animals, although the difference was close to the limit of
statistical significance (p = 0.063).
* p < 0.05 ** p < 0.01.
Fig. 1. Serum total and anti-OVA IgE concentrations in mice
perinatally exposed to TCDD or carrier and sensitised to OVA.
* p < 0.05.
Fig. 2. Effect of TCDD on anti-CD3 stimulated production
of IL-4, IL-5 and TNF-α by spleen cells of OVA sensitised mice.

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IJOMEH 2010;23(1) 79
production of these cytokines in the same conditions and
the same animals in which production of other cytokines
was tested. We could show that mice exposed perinatally
to TCDD produced significantly (p < 0.05) lower levels
of IL-10 than control animals and the TGF-β concentra-
tions were not significantly different (Figure 4). Although
the non-stimulated cells did not show detectable levels
of IL-10 in any of the groups, TGF-β was present (TCDD
group 180±107; control 181±29.4) but not at the amounts
significantly different between the groups.
Analyses of CD4+CD25+foxp3+ T regulatory cells
in spleen of 6-week old mice with or without perinatal
exposure to TCDD
Perinatal exposure of mice to TCDD did not cause sig-
nificant changes in the percentage of T regulatory cells
among spleen cells as determined by expression of
CD4CD25foxp3 among all T cells or within CD4+ T cells
of spleen (Figure 5).
The effect of transfer of CD4+CD25+ T cells obtained
from spleens of mice exposed to TCDD or carrier on IgE
anti-OVA serum levels in OVA sensitised animals
To analyse whether T cells of mice perinatally exposed to
TCDD could affect the extent of allergic sensitisation we
performed cell transfer studies. T cells expressing CD4
and CD25 markers were isolated from spleens of 6 week
OVA sensitised mice perinatally exposed to TCDD or
to the carrier. Concentrations of IL-2 in supernatants of
anti-CD3 stimulated cells were significantly (p < 0.01)
higher in TCDD exposed mice, whereas IFN-γ release was
not significantly different between the groups (Figure 3).
The effect of perinatal exposure to TCDD
on in vitro production of IL-10 and TGF-β
by spleen cells of mice sensitised to OVA
IL-10 and TGF-β belong to regulatory factors that sup-
press immune responses. We estimated the levels of
** p < 0.01.
Fig. 3. Effect of perinatal exposure to TCDD on anti-CD3
stimulated in vitro production of IL-2 and IFN-γ by spleen cells
of mice sensitised to OVA.
Fig. 4. Effect of perinatal exposure to TCDD on anti-CD3
stimulated in vitro production of IL-10 and TGF-β by spleen
cells of mice sensitised to OVA.
Fig. 5. Effect of perinatal exposure to TCDD or carrier
(control) on the frequency of T regulatory cells in spleens.