The Toxicologist - Society of Toxicology

model, ethanol was found to inhibit the innate immune response such as cytokine
productions in this model. The ethanol-treated mice sustained more bacteria
growth in their peritoneal cavity as compared to control septic mice. Much more
mortality occurs 18 hrs after E. coli challenge in the ethanol-treated mice. However,
the exact cellular and molecular mechanisms of such inhibition of innate immune
responses and the more lethality later are largely unknown. Thus, a microarray
study examining the global gene expression profiles of the peritoneal immune cells
at various time (1 hr, 2 hr and 18 hr) after E. coli challenge with or without ethanol
pretreatment was conducted. The data confirms that ethanol inhibits early innate
immune response to E. coli, including TLR signaling, cytokine/chemokines production,
macrophage mobilization and bactericidal activity. Functional assay suggests
that ethanol inhibits the leukocytes activation and the production of bactericidal
products (i. e., nitric oxide) rather than inhibits leukocytes accumulation in the
site of inflammation. This early inhibition leads to bacteria overgrowth and maybe
macrophage “hyper-reactive” stage at later time (18 hr). In addition, the gene expression
data here suggests novel molecular pathways, which may contribute to the
inhibition of the immune response, such as inhibition of an interferon amplification
loop, protein translation initiation, phosphlipase signaling and CDC42 activation.
This work was supported by NIH grant R01AA09595 from NIAAA.
680 METAL OXIDE NANOPARTICLES MODULATE
EXOSOMES.
B. Andersson 1 , T. Bürki-Thurnherr 2 , H. Krug 2 , B. Fadeel 3 , A. Scheynius 1 and S.
Gabrielsson 1 . 1 Clinical Allergy Research Unit, Karolinska Institutet, Stockholm,
Sweden, 2 Federal Laboratories for Material Science and Technology, St. Gallen,
Switzerland and 3 Institute of Environmental Medicine, Karolinska Institutet,
Stockholm, Sweden.
Metal oxide nanoparticles (MOnp) are widely used in the paint and coating industry
as well as in cosmetics, and they have been extensively studied from a toxicology
point of view. However, the interactions of MOnp with the immune system have
not yet been fully addressed. Dendritic cells (DC) are highly efficient antigen presenting
cells and are known to secrete membrane vesicles in the nano range (50-100
nm) referred to as exosomes. Exosomes have profound functions involving cell to
cell communication, delivery vehicles, and immune system activators or suppressors.
We hypothesise that MOnp can interact with DC and influence the exosome
production and function. Therefore, we exposed human monocyte-derived DC
(MDDC) to two commonly used and commercially available MOnp; TiO2 and
ZnO. MDDC viability was not affected by TiO2, whereas ZnO induced a dose dependent
increase in cell death (AnnexinV/PI staining) and caspase activity
(CaspAce). Non-toxic exposure of MOnp did not induce changes in DC surface
marker expression. The culture supernatant was collected and via several ultra centrifugation
steps, exosomes were isolated. Exosomes were coated on anti-MHC-II
conjugated Dynabeads® and subjected to TEM analysis and flow cytometry. TEM
images revealed that nanoparticles were not found inside the exosomes. Exosomes
from MDDC exposed to ZnO exhibited a tendency of decreased expression of the
tetraspanin CD81, the co-stimulatory marker CD40, and the antigen presenting
complex MHC-II. ZnO also induced a slight increase in apoptosis inducing FasL
expression, whereas TiO2 induced a decrease of FasL expression, compared to negative
control exposure. We conclude that exposure of MDDC to MOnp can influence
the exosome phenotype which might provoke differences in immune cell regulation
and cell death. Sponsored by the Seventh Framework Program of the
European Commission (FP7-NANOMMUNE, grant no. 214281).
681 ROLE OF NEUTROPHILS IN A MOUSE MODEL OF
CHEMICAL-INDUCED ASTHMA.
S. Smulders 1 , V. De Vooght 1 , G. Opdenakker 2 , B. Nemery 1 , P. Hoet 1 and J.
Vanoirbeek 1 . 1 Research Unit for Lung Toxicology, K.U. Leuven, Leuven, Belgium and
2 Rega Institute for Medical Research, Laboratory of Immunobiology, K.U. Leuven,
Leuven, Belgium.
Asthma is often characterized by a predominant neutrophilic inflammation. In this
study we tried to determine the specific role of neutrophils in the airway hyperreactivity
(AHR) and lung inflammation in a mouse model of chemical-induced
asthma by depleting the mice from neutrophils before pulmonary challenge. On
days 1 and 8, BALB/c received 0.3% toluene diisocyanate (TDI) or vehicle (acetone
olive oil) on each ear, followed by 2 intraperitoneal injections of cyclophosphamide
(CP) or saline on days 11 and 13, or 1 intravenous injection of anti-GR1
antibody (aGR1) or HBSS on day 13. On day 15, they received an oropharyngeal
challenge of 0.01% TDI or vehicle. On day 16, AHR to methacholine was measured.
Bronchoalveolar lavage differential cell count and total serum IgE were assessed.
Total lymphocyte count, lymphocyte subpopulations (CD4, CD8, CD25
and CD19) and release of IL4, IL10, IL13 and IFNγ by lymphocytes of the auricular
lymph nodes were assessed. CP and aGR1 induced a full neutrophil depletion.
CP led to a complete disappearance of the AHR upon methacholine provocation,
while aGR1 caused only a partial decrease in AHR. Furthermore, a full eosinophil
depletion was observed after CP- and aGR1-injections. CP-injected TDI-mice
showed an almost full depletion of the number of auricular lymphocytes, while this
was only slightly decreased in the aGR1-injected TDI-mice, mainly caused by depletion
of cytotoxic T-cells. A decrease of the concentration of IL-13 was detected
in the aGR1-injected TDI-mice compared to the HBSS-injected TDI-mice. In CPinjected
TDI-mice, decreased total serum IgE was observed compared to the salineinjected
TDI-mice. In conclusion, CP and aGR1 are potent neutrophil depletion
agents, which clearly influence AHR in our mouse model of chemical-induced
asthma, but due to side effects of the treatment on other cell-types the neutrophil
specific role could not yet be clarified. IAP: P6/35
682 EFFECTS OF PENICILLIUM MYCOTOXINS ON BOVINE
MACROPHAGES IN VITRO.
H. J. Boermans 2 , S. Oh 1 , N. A. Karrow 1 , B. Sharma 1 and S. Haladi 3 . 1 Animal
and Poultry Sciences, University of Guelph, Guelph, ON, Canada, 2 Biomedical
Sciences, University of Guelph, Guelph, ON, Canada and 3 Alltech, Guelph, ON,
Canada.
Penicillium fungal mycotoxins are often found in grains, crops, fruits, and fermented
products, especially during pre- and post- harvest, as well as storage periods.
Many studies have assessed the impact of oral exposure to mycotoxins from
Fusarium and Aspergillus species on livestock as well as human health. However,
little is known about the impact of Penicillium mycotoxins on these aspects, especially
their potential immunotoxicity. In this study, the potential immunotoxicity
of citrinin (CIT), ochratoxin A (OTA), patulin (PAT), mycophenolic acid (MPA)
and penicillic acid (PA) was evaluated using a bovine macrophage cell line
(BOMAC) by first assessing their potential cytotoxicity, and then their effects on
cell proliferation. BOMAC cells were exposed to a range of mycotoxin concentrations,
and then to different mycotoxin combinations for 48 hrs. Some cytotoxicity
was evident at concentrations greater than 2.4 uM for PAT, and 160 uM for PA,
however, at the IC50 (concentration that inhibits 50% cell proliferation) for these
mycotoxins, no cytotoxicity was observed. The mycotoxin IC50s from most potent
to least potent were 0.56 uM (PAT), 12.88 uM (OTA), 29.85 uM (PA), and 91.20
uM (CIT). Concentrations of MAP greater than 80 uM did not inhibit cell proliferation
enough to calculate an IC50. Ten different combinations of mycotoxins
were tested at concentrations equivalent to their IC25 without any cytotoxicity, and
proliferation was significantly reduced with the following seven mycotoxin combinations:
CITxOTA, CITxMPA, CITxPA, OTAxPAT, OTAxMPA, OTAxPA,
MPAxPA.
683 EFFECT OF ASBESTOS EXPOSURE ON PRODUCTION
OF CYTOKINES, APOPTOSIS, AND CELL
PROLIFERATION RELATED WITH DIFFERENTIATION
OF HUMAN CTL.
N. Kumagai, Y. Nishimura, M. Maeda, H. Hayashi and T. Otsuki. Hygiene,
Kawasaki Medical School, Kurashiki, Japan.
Asbestos exposure can cause malignant mesothelioma and lung cancer. In the antitumor
immunity, cytotoxic T lymphocytes (CTL) play a critical role. Previously, we
reported that asbestos exposure suppressed the induction of human CTL during
mixed lymphocyte reactions (MLR), accompanied with the decreases in IFN-γ and
TNF-α but not IL-2. However, the time of assay for TNF-α and IL-2, day 7 after
MLR, might not be optimal, because of no difference between control and allogenic
conditions. The present study examined the kinetics of TNF-α and IL-2 production
during 7-days MLR upon asbestos exposure, by using ELISA. In addition,
the proliferation and apoptosis of CD8 + cells were also examined by flow cytometry.
Human peripheral blood mononuclear cells (PBMCs) were cultured with irradiated
allogenic PBMCs with or without chrysotile B (CB) asbestos at 5 μg/ml.
The production of TNF-α did not differ between control and allogenic conditions
at day 2 and 4 as well as day 7 after MLR. However, the decrease in production of
TNF-α upon CB exposure was observed only at day 7, but not at day 2 or 4. The
production of IL-2, which had a peak at day 4 after MLR and no difference between
control and stimulating cultures, did not decrease upon CB exposure during
the entire period of culture. The percentage of annexin V + apoptotic cells in CD8 +
cells stimulated allogenically did not increase upon CB exposure. Additionally, the
percentage of CD25 + cells and CD45RO + cells in apoptotic CD8 + cells decreased
upon CB exposure. In contrast, CB exposure suppressed the increase in CFSE-negative
CD8 + cells, caused by cell division, upon allogenic stimulation. Thus, taken
together with our previous reports, these results indicate that CB exposure suppresses
the induction of CTL, accompanied with decreased productions of TNF-α
and IFN-γ and suppressed proliferation of CD8 + cells independent of IL-2 production.
These suppressive effects of asbestos exposure on induction of CTL might
promote the generation of tumor disease.
SOT 2011 ANNUAL MEETING 147