The Toxicologist - Society of Toxicology

sponds to the endosomal dsRNA that occurs in viral infections and because it’s signaling
pathway is mediated entirely by TRIF – an adaptor protein implicated in the
development of myocarditis that promotes an antiviral Th1 response. We hypothesize
that TLR3 is uniquely positioned to play a critical role in the innate response to
viral infection and that removing this receptor will result in a more severe viral infection
and produce a more intense myocarditis. To test the role of TLR3 signaling
in the development of myocarditis, TLR3-/- males and WT controls (N=10) were
infected with heart-passaged Coxsackie Virus B3 (CVB3) to induce autoimmune
myocarditis. Ten days later heart function was assessed by pressure-volume catheterization
of the heart. Heart tissue was harvested to quantify inflammatory cell infiltrate,
viral replication, and cytokine levels. Myocardial inflammatory cell infiltration
was significantly increased by 50% in TLR3-/- mice. Viral replication
increased from 10,000 PFU/g in WT to 900,000 PFU/g in TLR3-/-. Th 2 cytokines
IL-4, IL-10 and TGFb1 increased, while Th 1 IFNg decreased in TLR3-/vs
WT. Heart catheterization revealed left ventricular dysfunction in TLR3-/- as
characterized by: a decrease in systolic pressure from 106 mmHg in WT to 95
mmHg in TLR3-/-, a decrease in contractility (dPdT max) from 10,176 mmHg/sec
in WT to 8,304 mmHg/sec in TLR3-/-, a stroke volume-dependent decrease in
cardiac output from 6.2 mL/min in WT to 4.2 mL/min in TLR3-/-, and a 39%
drop in stroke work in TLR3-/-. TLR3 plays a protective role in CVB3-induced
myocarditis by promoting a Th 1 shift, decreasing the intensity of myocarditis, and
preventing systolic failure.
257 S-NITROSYLATION OF SURFACTANT PROTEIN-D
OCCURS IN BLEOMYCIN-INDUCED INJURY AND IS A
PRO-INFLAMMATORY MEDIATOR VIA ACTIVATION
OF NF-κB DEPENDENT PATHWAYS.
C. Guo and A. Gow. Pharmacology & Toxicology, Rutgers University, Piscataway, NJ.
Surfactant protein-D (SP-D) is a pulmonary collectin, which plays an important
role in innate immunity. Previously, we have demonstrated that SP-D is a target for
nitric oxide-mediated modification. S-nitrosylation of two key cysteines in the tail
domain of SP-D alters its multimeric structure. Our initial studies have shown that
the S-nitrosylated form of SP-D (SNO-SP-D) associates with CD91 to activate
macrophage chemotaxis. In this study, we investigated whether SNO-SP-D induces
a pro-inflammatory phenotype in macrophages via activation of NF-κB.
Acute lung injury model was induced by intratracheal administration of bleomycin
in iNOS-/- and C57/Bl6 (WT) mice and Bronchoalveolar lavage (BAL) was collected
at day 8-post injury. Bleomycin-induced lung injury was reduced in iNOS-/mice
relative to WT, as determined by a number of factors. The activity of NF-κB,
as assessed by TransAM ELISA, within cells of the lung lining was reduced, relative
to control, in bleomycin-injured iNOS-/- mice, as was the SNO-SP-D content.
BAL from bleomycin injured mice induced NF-κB activity in Raw 264.7 cells, and
this function was abrogated after removal of SNO-SP-D. SP-D over expressing
BAL (OE-BAL) was harvested from Swiss-black mice over-expressing rat SP-D. Snitrosylated
OE-BAL (SNO-OE-BAL) treatment of Raw264.7 cells resulted in upregulation
of IL-1β and iNOS mRNA, while OE-BAL had no effect. The SNO-
OE-BAL mediated gene expression of these pro-inflammatory mediators was
decreased upon SP-D depletion from the BAL. Pre-incubation of Raw264.7 with
caffeic acid phenethyl ester (CAPE), a potent NF-κB inhibitor, abrogated SNO-
OE-BAL mediated induction of proinflammatory genes. These findings provide a
mechanism for SNO-SP-D mediated inflammatory signaling within the lung and
demonstrate that this mechanism operates within an in vivo model of lung injury.
Supported by ES005022 and HL086621.
258 UTILIZATION OF EXTRACELLULAR GLUTATHIONE
BY PHAGOCYTOSIS REDUCES TUMOR NECROSIS
FACTOR α RELEASE IN MACROPHAGES.
N. S. Gould 1, 2 , E. Min 1 and B. J. Day 1, 2, 3 . 1 Medicine, National Jewish Health,
Denver, Co., 2 Pharmaceutical Sciences, University of Colorado AMC, Aurora, CO
and 3 Immunology, University of Colorado AMC, Aurora, CO.
The epithelial lining fluid (ELF) of the lung is an important protective barrier
against inhaled toxic chemicals or pathogens. The ELF maintains high levels of glutathione
(GSH) which can protect the lung by detoxifying oxidants. Additionally,
GSH has been shown to have a role in modulating inflammatory responses in epithelial
cells. One of the major cell types found in the ELF are alveolar
macrophages, which when stimulated can be a major source of oxidants and proinflammatory
cytokines. There is a positive correlation between ELF GSH and alveolar
macrophage intracellular GSH but it is unclear how GSH in the extracellular
environment is able to be utilized by macrophages and whether it attenuates cytokine
release. When macrophages are grown in media supplemented with 500 μM
GSH, there is a 2-3 times increase in intracellular GSH. This increase in intracellular
GSH corresponds to a significant reduction in NF-κB nuclear translocation and
tumor necrosis factor α (TNFα) release when stimulated with LPS. Furthermore,
treatment with acivicin or buthionine-sulfoximine, inhibitors of GSH breakdown
and synthesis, did not block GSH uptake. In contrast, cytochalasin D, an inhibitor
of phagocytosis, blocked the increase in intracellular GSH and blocked the attenuation
of TNFα release. Furthermore, in vivo, the activation of alveolar
macrophages is increased with cigarette smoke in aged mice with decreased GSH
levels leading to an increase in TNFα levels. These data suggest that macrophages
are able to utilize extracellular GSH through phagocytosis which can then modulate
inflammatory signaling in response to proinflammatory stimuli. Supported in
part by NIH grant HL084469 (BJD).
259 ACROLEIN INHIBITS THE CLEAVAGE OF ADHESION
MOLECULES IN CYTOKINE-STIMULATED
ENDOTHELIAL CELLS.
S. D. Sithu, A. Bhatnagar, S. Srivastava and S. E. D’Souza. School of Medicine,
University of Louisville, Louisville, KY.
Acrolein is an α,β-unsaturated aldehyde abundant in automobile exhaust, tobacco
smoke and industrial waste. It is also present in high quantities in several beverages
and food. Due to its α,β-unsaturation, acrolein is a strong electrophile and readily
reacts with cellular nucleophiles such as the -SH residues of reduced glutathione
(GSH). Depletion of GSH perturbs the redox state of the cells and has been implicated
in vascular dysfunctions. We observed that incubation of endothelial cells
(EC) with acrolein (0-10 μM; 1h) in serum-free medium followed by stimulation
with TNF-α (15ng/ml) or IL-1 after 24h, increased the cell surface expression of
ICAM-1 and VCAM-1 by 1.5-2 fold. However, the soluble ICAM-1 (sICAM-1)
and sVCAM-1 levels in the media were significantly lowered (20-90% for 2.5-
10μM acroline). The adhesion of monocytic cells to acrolein exposed EC as well as
the transmigration of the monocytic cells across acrolein exposed EC were significantly
increased as compared with untreated EC. GSH levels in acrolein-treated EC
was depleted by 50% at 1h and returned to normal levels by 6h. Similar to acrolein,
incubation of EC with the 9C α,β-unsaturated aldehyde 4, hydroxy trans-2-nonenal
(HNE), increased the cell surface expression of ICAM-1, increased monocyte
adhesion and transmigration, but decreased the cleavage of ICAM-1. Moreover,
tertiary butyl hydroperoxide (t-BHP, 0-100 μM), a well known oxidant, significantly
depleted GSH levels in EC and decreased the concentration of sICAM-1 in
the cell culture medium. sICAM-1 and sVCAM-1 levels in the plasma of acrolein
(5mg / kg for 24h by gavage) exposed mice challenged with LPS (1mg/kg) were decreased
by 15-20% as compared with the controls, gavage-fed with water. The
lower levels of sICAM-1 and sVCAM-1 seen upon exposure to α,β-unsaturated
aldehydes acrolein and HNE, and the non aldehydic oxidant t-BHP, in cytokine
stimulated EC suggest a relationship between GSH depletion and cytokine signaling
that regulate the processing of adhesion molecules affecting EC function.
260 INDUCTION OF PROINFLAMMATORY MEDIATORS
FACILITATES PCB-MEDIATED ENHANCEMENT OF
BRAIN METASTASIS FORMATION.
E. Bodone Sipos, L. Chen, I. Andras, B. Zhang, J. Wrobel, M. Park, J. Choi,
S. Eum and M. Toborek. Molecular Neuroscience and Vascular Laboratory,
Department of Neurosurgery, University of Kentucky, Lexington, KY.
Polychlorinated biphenyls (PCBs) are environmental toxicants that can cause vascular
inflammation. Our research has focus on a general hypothesis that exposure to
PCBs can promote the development of brain metastases by altering the blood brain
barrier (BBB) integrity. The crucial event in tumor cell extravasation is the arrest of
the blood-borne tumor cells in the capillary bed. This process is followed by adhesion
of tumor cells to the vascular endothelium and their transcapillary migration.
In the present study, we evaluated the hypothesis that PCB-induced inflammatory
processes at the BBB level can facilitate the formation of brain metastases. PCB118
was administered orally to mice at the dose of 150 μmol/kg. Forty eight hours later,
mice were injected with Lewis lung carcinoma cells tagged with luciferase into the
carotid artery. Brain tumors were detected by an in vivo imaging system. In addition,
expression of VCAM-1, ICAM-1, and MCP-1 was assessed by real-time PCR
(rtPCR) and immunofluorescence microscopy in isolated brain microvessels 6 h or
27 days after tumor cell injection. In vivo imaging demonstrated a potent development
of brain metastases during the one month monitoring period, with a marked
increase in metastasis formation in mice treated with PCB118 as compared to vehicle
treated animals. Our rtPCR and immunostaining results indicated that injection
of tumor cells induced the expression of VCAM-1, ICAM-1, and MCP-1 and
that these effects were potentiated by exposure to PCB118. The results of the present
study suggest that exposure to PCBs, such as PCB118, may induce the migration
of tumor cells across the BBB via a process facilitated by proinflammatory mediators
and leading to potentiation of brain metastasis formation. Supported by
P42 ES 07380.
SOT 2011 ANNUAL MEETING 55