The Toxicologist - Society of Toxicology

689 ENHANCED SUPPRESSIVE FUNCTION ON
REGULATORY T CELL-LIKE CELL LINE, MT-2, BY
CONTINUOUS EXPOSURE TO ASBESTOS.
M. Maeda 1 , Y. Chen 1 , Y. Nishimura 1 , N. Kumagai 1 , H. Hayashi 1 , Y. Miura 2
and T. Otsuki 1 . 1 Hygiene, Kawasaki Medical School, Kurashiki, Okayama, Japan
and 2 Molecular Genetics, Kyushu University, Fukuoka, Japan.
[Introduction] Chronic exposure to asbestos occurs malignant mesothelioma. As
this disease has a long incubation period, CD4 + CD25 + regulatory T (Treg) cellsmediated
anti-tumor immune response may be impaired. To reveal whether Treg
cells exposed to asbestos attenuate an anti-tumor immune function in malignant
mesothelioma patients, six chrysotile-induced apoptosis-resistant sublines (MT-
2Rsts) were established by long-term (more than 8 months) and low-level (10
μg/ml) exposure to chrysotile-A or -B from the HTLV-1-immortalized human Tcell
line MT-2 (MT-2Org) having a Treg-like suppressive function, and their suppressive
functions were estimated by mixed lymphocyte reaction (MLR).
[Methods] The regulatory function in an allogeneic MLR was determined using
[ 3 H]-Thymidine incorporation. Expression of Treg markers (Foxp3, CTLA-4 and
GITR) was analyzed by flow cytometry. The mechanisms of the suppressive function
were clarified in MT-2Rst using a CFSE assay. Involvement of immunosuppressive
cytokines IL-10 and TGF-β1 in suppressive function was investigated
using an shRNA knockdown system. [Results and Discussions] Five MT-2Rst cell
lines suppressed proliferation of CD4 + CD25 - responder T-cells (Tresp) upon stimulation
with irradiated allogeneic PBMCs more strongly than MT-2Org. The results
of flow cytometry showed that MT-2Org and MT-2Rsts cells expressed high
levels of Foxp3 and GITR. When Tresp were stimulated with coated anti-CD3 antibody
and autologous induced-dendritic cells, the proliferation was suppressed by
MT-2Rst more strongly than MT-2Org. Furthermore, shRNA knockdown showed
that IL-10 and TGF-β1 produced from MT-2Rst suppressed the proliferation of
Tresp. In conclusion, these findings revealed that chronic exposure to asbestos enhanced
the suppressive function through cell contact-dependent mechanisms and
soluble factors in MT-2Rst, suggesting that asbestos exposure enhances Treg suppressive
function and lead to a decrease in anti-tumor immune function.
690 HISTONE DEACETYLASES PLAY A CRITICAL ROLE IN
REGULATING IFNα-MEDIATED ANTI-HCV GENE
EXPRESSION.
S. Mathews 1 , S. Joshi-Barve 1 , C. McClain 1, 2 and S. Barve 1 . 1 Department of
Medicine/GI, University of Louisville, Louisville, KY and 2 Robley Rex Veterans
Administration Medical Center, Louisville, KY.
Hepatitis C virus (HCV) is a major cause of chronic liver disease and is a huge burden
on the United States healthcare system. The FDA-approved standard of care is
pegylated interferon-alpha (IFNα) in combination with ribavirin; however up to
50% of patients do not respond to this therapy. Understanding the regulatory
mechanisms that play an essential role in the expression of IFNα anti-HCV genes
may lead to the development of therapeutic options that increase the effectiveness
of IFNα. The present work was carried out to examine the potential role of histone
deacetylases (HDACs) in the regulation of IFNα-stimulated anti-HCV genes.
Two class I HDAC inhibitors (HDACi), trichostatin A (TSA) and suberic bishydroxamate
(SBHA) were used to treat human hepatoma cells (Huh7 and HCVR;
Huh7 stably transfected with an HCV replicon) or primary human hepatocytes
prior to stimulation with IFNα. HDACi pretreatment inhibited induction of several
IFN-mediated antiviral genes, as analyzed by real-time PCR. Importantly, this
inhibition corresponded to decreased activation of the interferon stimulated response
element (ISRE) found in the promoters of IFNα antiviral genes.
Additionally, HDACi led to retention of phospho-STAT1 in the cytoplasm, potentially
due to altered acetylation of STAT1, and corresponded to impaired expression
of antiviral proteins. Immunoprecipitation analysis using lysates from IFNα-stimulated
cells identified HDAC1 binding to STAT1 in the nucleus and this interaction
was inhibited by HDACi. Since HDACi block the actions of class I HDACs,
siRNA was used to specifically identify HDAC1 and HDAC3 as critical regulators
of IFNα antiviral signaling. Taken together, the data from these experiments reveal
a unique requirement for HDACs 1 and 3 in regulating IFNα-mediated anti-HCV
gene expression in hepatocytes.
691 ZIRAM INDUCES APOPTOSIS IN HUMAN IMMUNE
CELLS.
Q. Li, M. Kobayashi and T. Kawada. Nippon Medical School, Tokyo, Japan.
Background: Ziram as a dithiocarbamate fungicide is widely used throughout the
world in agriculture and as an accelerating agent is used in latex production. It has
been reported that ziram shows immunotoxicity.
Purpose: To explore the mechanism of ziram-induced immunotoxicity, we investigated
ziram-induced apoptosis/necrosis and its underlying mechanism in human
immune cells.
Materials and Methods: U937, a human monocyte like cell line and NK-92MI, a
human NK cell line, were treated with ziram at 0.0312-2 μM for 2-24 h at 37°C in
a 5% CO2 incubator. Apoptosis/necrosis induced by ziram was determined by
analysis of FITC-Annexin-V/PI staining and the intracellular level of active caspase-3
by flow cytometry and DNA fragmentation analysis (1-3). Mitochondrial
cytochrome c release in U937 cells induced by ziram was also determined by flow
cytometry (3).
Results: We found that ziram induced apoptosis/necrosis in both U937 and NK-
92MI cells in a dose- and time-dependent manner, as shown by FITC-Annexin-
V/PI staining. DNA fragmentation was detected when U937 cells were treated with
0.5, 1 or 2 μM ziram for 24 h. Ziram also induced an increase of intracellular active
caspase-3 in U937 and NK-92MI cells in a dose-dependent manner, and a caspase-
3 inhibitor, Z-DEVD-FMK, significantly inhibited the ziram-induced apoptosis.
Moreover, it was found that ziram induced mitochondrial cytochrome c release in
U937 cells.
Conclusions: These findings indicate that ziram can induce apoptosis/necrosis in
both U937 and NK-92MI cells, and this effect is partially mediated by activation of
intracellular caspase-3 and mitochondrial cytochrome c release. From this perspective,
we speculate that ziram-induced immunotoxicity may be mediated by apoptosis
in immune cells.
Acknowledgements: This work was supported by a grant from the Ministry of
Education, Culture, Sports, Science and Technology (No.22590554).
References: 1. Li Q, et al. Toxicology 239:89-95, 2007.
2. Li Q, et al. Toxicology 255:53-57, 2009.
3. Li Q, et al. Arch Toxicol (in press)
692 ENHANCEMENT OF HUMORAL IMMUNITY BY THE
ENDOCRINE DISRUPTER PROPANIL IS NOT ALTERED
BY NEONATAL ANDROGEN TREATMENT.
R. Schafer 1 , S. Hileman 2 , R. Goodman 2 and I. Holaskova 1 . 1 Microbiology,
Immunology, and Cell Biology, West Virginia University, Morgantown, WV and
2 Physiology and Pharmacology, West Virginia University, Morgantown, WV.
Studies in our laboratory demonstrated that the pesticide propanil enhances the immune
response to a vaccine that is dependent on the gonads in females but is independent
of the gonads in males. This effect was independent of estrogen, progesterone
and testosterone. The dramatically different response of gonadectomized
male and female mice to propanil raised the possibility that the immune response is
sexually differentiated and accounts for the sexually-dimorphic effect of propanil in
adults. We hypothesized that androgen treatment of neonatal female mice would
normalize the enhanced antibody response after propanil exposure in ovariectomized
(OVX) adult females.
Neonatal C57Bl/6 pups were treated subcutaneously with testosterone propionate
(TP, 400 μg in corn oil) or corn oil within 24 hours of birth. Efficacy of TP treatment
in females was determined by vaginal smears and uterine weight. At 6 weeks
of age female mice were OVX or sham OVX. Mice were vaccinated at 10 weeks of
age with heat-killed Streptococcus pneumoniae and treated with propanil or vehicle.
One week later the spleens were collected and the antibody response to the vaccine
was determined by ELISPOT assay.
Testosterone treatment resulted in abrogation of the estrous cycle. Propanil significantly
increased IgM and IgG antibody producing cells in males treated with TP at
birth. In females given TP at birth, propanil significantly increased IgM, IgG, and
IgG3 responses. Ovariectomy abrogated the increase demonstrating that the enhanced
immune response after propanil exposure is still dependent on the ovaries in
TP treated females. Thus, neonatal androgen treatment produced the expected effects
on the reproductive system but did not alter the normal immune response to
S. pneumoniae vaccine in adults. This indicates that the immune system of females
was not masculinized during the neonatal time period and ovarian function is crucial
for the enhanced response.
693 ASBESTOS EXPOSURE CAUSES DECREASE IN
PRODUCTION OF CYTOKINES RELATED TO NK CELL
ACTIVATION.
Y. Nishimura, N. Kumagai, M. Maeda, H. Hayashi and T. Otsuki. Hygiene,
Kawasaki Medical School, Kurashiki, Japan.
Recently, we reported that peripheral blood NK cells in patients with malignant
mesothelioma (MM) showed low cytotoxicity with decrease in NKp46, one of receptors
utilized to recognize targets and transduce activation signal, which also decreased
in NK cells included in PBMCs cultured with chrysotile B (CB) asbestos.
Additionally, purified NK cells showed an increase in NKp46 when co-cultured
SOT 2011 ANNUAL MEETING 149