The Toxicologist - Society of Toxicology

exposure resulted in splenomegaly. Mixed lymphocyte responses, and basal and
anti-CD3 antibody-mediated splenocyte proliferation were decreased following
treatment with DEX, DES, and CPS, but not TCDD. The most notable transcriptional
effect of exposure to DEX, DES, or CPS in thymus was modulation of the T-
cell Receptor Signaling pathway, which plays a role in the development and function
of T-cells. Ironically, only DES regulated this pathway in spleen. Upregulation
of genes associated with the Antigen Presentation and Dendritic Cell Maturation
pathways were the most distinctive effects of TCDD exposure in thymus. These elements,
which were also upregulated by DEX and DES, contribute to positive and
negative selection. While changes in gene expression in thymus showed many commonalities
between chemicals, in the spleen the gene expression profiles were quite
distinct for each chemical. DES altered expression in many pathways associated
with T- and B-cell function and immune cell signaling, but the effects of DEX,
CPS, and TCDD were more limited. These findings may provide insight into the
mechanisms invoked by each chemical and into the different action that a single
chemical has in two lymphoid organs.
71 TCDD-INDUCED MODULATION OF THE HUMAN
POLYMORPHIC HS1, 2 ENHANCER WITHIN THE
3’IgH REGULATORY REGION.
C. E. Sulentic, T. M. Fernando and S. Ochs. Pharmacology & Toxicology, Wright
State University, Dayton, OH.
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent environmental toxin
known to inhibit immunoglobulin (Ig) gene expression in animal studies.
Transcriptional regulation of the Ig heavy chain (IgH) involves the 3’IgH regulatory
region (3’IgHRR) and its enhancers (hs3, hs1,2, and hs4), which contain
DNA binding sites for several transcription factors, including NF-κB and dioxin
response elements (DRE). TCDD induces binding of the aryl hydrocarbon receptor
complex to a DRE in both the hs1,2 and hs4 enhancers and inhibits murine
3’IgHRR activation in a well characterized mouse B-cell line (CH12.LX). In humans,
a polymorphism of the hs1,2 enhancer, resulting in varying numbers of a 53
bp sequence tandemly repeated, has been correlated with autoimmune diseases like
IgA nephropathy and Celiac disease. The repeated sequence contains κB and DRE
binding sites. The objective of this study was to comparatively evaluate the effect of
TCDD and LPS on human and mouse hs1,2 enhancers in the CH12.LX model. In
transient luciferase studies, an increased number of repeats in the human hs1,2 enhancer
increased the sensitivity to LPS. Interestingly, TCDD also markedly enhanced
human hs1,2 activity and even augmented LPS-induced activation.
TCDD-induced activation positively correlated with the number of repeats. This
starkly contrasted with TCDD-induced inhibition of the mouse hs1,2 and
3’IgHRR in LPS-stimulated CH12.LX cells. Through sequence analyses, we verified
that the human hs1,2 enhancer retains the κB and DRE binding sites but lacks
binding sites for B-cell specific activator protein and NF-αP, which are both important
to mouse hs1,2 regulation. Mutational analyses are underway to evaluate the
significance of these binding sites in TCDD-induced modulation of both the
human and mouse hs1,2 enhancer. Since TCDD represents a large class of chemicals
found in the environment, diet, and pharmaceuticals, understanding chemicalinduced
modulation of the 3’IgHRR enhancers may provide a clue to the etiology
of certain autoimmune diseases. (Supported by NIEHS R01ES014676)
72 THE ROLE OF AHR IN MATURATION OF
DENDRITIC CELLS.
C. F. Vogel 1 , S. R. Goth 2 , D. Wu 1 , B. Yuen 2 , I. Pessah 2 and F. Matsumura 1 .
1
CHE, University of California Davis, Davis, CA and 2 Molecular Biosciences,
University of California Davis, Davis, CA.
Genes important for dendritic cell (DC) activation and maturation such as
chemokines and phenotypic surface markers were investigated in this study. In addition
to being functionally defined, many of these genes are highly expressed in
mature DC or show significant changes in expression during cell differentiation.
Using the AHR antagonist MNF and prototypic AHR agonist TCDD, we examined
whether AHR activity would affect the differentiation and activation of
human DC-derived from U937 cells into mature DC phenotype. Expression of
CD86 mRNA was clearly increased by TCDD. In contrast, MNF suppressed the
expression of CD86 in differentiating DC. In parallel, the expression of CD1a was
augmented by TCDD and suppressed by MNF. Further, we tested the effect of
MNF and TCDD on the expression of the DC-specific genes DC-CK1, IL-8 and
DC-STAMP. As described earlier in macrophages, TCDD increases IL-8 expression
about 20-fold in U937 derived DC. DC-STAMP was less but also significantly increased
by TCDD. In contrast, the upregulation of DC-CK1 was clearly blocked in
presence of TCDD. As in the case of DC surface markers, MNF has a contrary effect
compared to TCDD by increasing DC-CK1, but suppressing IL-8 and DC-
STAMP. Data from experiments with bone-marrow derived DC (BM-DC) from
mice confirm the results received from human DC models showing a significant increase
of CD86 and suppression of DC-CK1 in differentiating DC from wild type
but not from AHR null mice by TCDD. Furthermore, the constitutive expression
of chemokine receptor CCR6 was decreased in AHR null mice compared to wild
type mice and further increased by TCDD in DC only from wild type mice. The
expression of CCR6 is associated with a newly identified AHR-dependent IL-22-
producing helper T cell population. These data suggest a critical role of the AHR in
the regulation of DC-specific surface markers and chemokines which are critical for
regulation of T cell differentiation and support the hypothesis that the AHR exerts
a physiological role in DC differentiation and activation.
73 DIRECT REGULATION OF BACH2 BY 2, 3, 7, 8-
TETRACHLORODIBENZO-P-DIOXIN IN MURINE B
LYMPHOMA CH12.LX CELLS.
A. S. Phadnis 1, 2 , M. A. Manzan 2 , R. A. Thomas 4 and N. E. Kaminski 2, 3 .
1
Genetics Program, Michigan State University, East Lansing, MI, 2 Center for
Integrative Toxicology, Michigan State University, East Lansing, MI, 3 Pharmacology
and Toxicology, Michigan State University, East Lansing, MI and 4 Hamner Institutes
for Health Sciences, Research Triangle Park, NC.
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant
known to alter B cell function, resulting in marked suppression of the primary immune
response. The molecular mechanisms responsible for these immunotoxic effects
involve transcriptional regulation through the aryl hydrocarbon receptor
(AhR). To identify the novel genes that are directly modulated by the ligand-activated
AhR during B cell differentiation we performed a genome wide localization
analysis or ChIP on chip. For the ChIP on chip, DNA from the mouse B cell line,
CH12.LX was used, in which comparisons were made between CH12.LX cells activated
with lipopolysaccharide in the presence or absence of TCDD for 60 min.
One of the genes identified in this screen was Bach2, which is a B cell specific transcriptional
repressor. It is known to repress Blimp1 which is a master regulator of
the B cell differentiation program. To further elucidate the putative involvement of
Bach2 in TCDD-mediated impairment of B cell differentiation and function, a
time-course was performed to assess the effect of TCDD on Bach2 mRNA levels in
LPS-activated CH12.LX cells by quantitative Real-Time PCR. These studies
showed that in LPS-activated CH12.LX cells, Bach2 mRNA levels remained unchanged
in naïve cells at 2h, then decreased at 4h and remained decreased through
a 24h time period. In contrast, TCDD-treated cells exhibited a strong induction of
Bach2 at 2h, which then decreased to the same levels as that in LPS-activated cells.
In additional studies, TCDD induced a concentration-dependent increase in Bach2
mRNA levels at the 2h time point. Collectively, these studies suggest that Bach2 is
directly regulated by AhR and may be responsible for impaired Blimp-1 regulation
and altered B cell differentiation by TCDD. (Supported in part by NIH P42
ES04911 and R01 ES02520)
74 ALLERGEN-INDUCED CHANGES IN INTERLEUKIN-17
EXPRESSION IN MICE.
M. Hayes, I. Kimber and R. J. Dearman. Faculty of Life Sciences, Manchester
University, Manchester, United Kingdom.
Polarized subsets of T helper (Th) cells that are characterized by selective cytokine
secretion patterns orchestrate the development of immune and allergic responses. It
has been demonstrated previously that prolonged topical (13 day) exposure of
BALB/c strain mice to different classes of chemical allergen preferentially activates
divergent Th cell subsets. Thus, the contact allergen 2,4-dinitrochlorobenzene
(DNCB) and the respiratory sensitizer trimellitic anhydride (TMA) stimulate Th1
and Th2 cells, respectively. Recently a further subset of inflammatory Th cells that
secrete IL-17 has been shown to play important roles in some autoimmune and inflammatory
conditions. In the current investigations, the expression of IL-17 isoforms
has been examined in the skin and lymph nodes following topical exposure
to chemical allergen. BALB/c strain mice were exposed to a single topical dose of either
1% DNCB or 25% TMA, or to vehicle alone, for 30 min to 72 h. At various
times thereafter, cytokine production by skin explants (prepared from dorsal halves
of ear tissue) or by draining auricular lymph node cells (LNC) was measured by cytokine-specific
enzyme-linked immunosorbant assay (ELISA). Topical treatment
with DNCB, but not to TMA or vehicle alone, provoked a rapid increase in IL-
17A and IL-17F isoforms, peaking at 3 h. Levels of IL-17F were generally some 5-
fold higher than those observed for IL-17A. DNCB-activated LNC also produced
high levels of both IL-17A and IL-17F, reaching maximal levels after 6h of exposure.
The heterodimer (IL-17A/F) was also detected, but at considerably lower levels.
Treatment with TMA induced the same pattern of cytokines in the draining
lymph node, but with slightly delayed kinetics, peaking at 48 to 72h. These data
SOT 2010 ANNUAL MEETING 15