The Toxicologist - Society of Toxicology

2326 ROLE OF SURFACTANT PROTEIN-D (SP-D) IN
REGULATING MACROPHAGE PHENOTYPE,
INFLAMMATION, AND LUNG FUNCTION IN ELDERLY
MICE FOLLOWING OZONE (O3) INHALATION.
A. M. Groves 1 , J. D. Laskin 2 , A. J. Gow 1 and D. L. Laskin 1 . 1 Rutgers University,
Piscataway, NJ and 2 UMDNJ-RWJ Medical School, Piscataway, NJ.
SP-D is a pulmonary collectin important in controlling inflammatory responses.
SP-D-/- mice develop progressive inflammation, characterized by accumulation of
enlarged, activated macrophages (MP)s and an emphysematous morphology.
Epidemiological data indicate that older individuals with emphysema are hypersensitive
to O3. Mechanisms underlying this response were analyzed using SP-D-/mice.
O3 inhalation (0.8 ppm, 3 hr) resulted in increased MPs in BAL from
younger (8 wk), but not older (27 wk) SP-D-/- mice 72 hr post exposure.
Inflammation was also increased in tissue sections from younger SP-D-/- mice. In
both younger and older mice, O3 effects on Newtonian resistance were greater in
SP-D-/- mice relative to WT mice. O3 also increased lung compliance in younger
SP-D-/- mice, but not younger WT mice. In contrast, in older mice, increases in
compliance were only noted in WT mice. We next determined if these differential
responses were correlated with MP phenotype. Activated MPs are classified into
proinflammatory/cytotoxic M1 MPs, and anti-inflammatory/profibrotic M2 MPs.
Immunohistochemical analysis of lungs from young WT mice showed low level
MP expression of the M1 marker, inducible nitric oxide synthase (iNOS), and the
M2 markers, arginase II (ArgII), YM-1 and mannose receptor (MR). Whereas in
younger mice, loss of SP-D resulted in increases in M2 MPs, in older SP-D-/- mice
both M1 and M2 MPs were seen. O3 inhalation caused a shift towards an M2 phenotype
in younger and older WT mice, as assessed by increased ArgII, YM-1 and
MR expression, with no change in iNOS. In contrast in SP-D-/- mice, O3 caused a
shift towards an M1 MP phenotype, as evident by increased iNOS, with little
change in ArgII, YM-1 or MR, and in older mice by decreased expression of MR.
These results suggest that SP-D plays a key role in regulating MP phenotype, pulmonary
inflammation and lung function in elderly mice following O3 inhalation
(NIH ES004738, GM034310, CA132624, AR055073, HL074115, ES005022).
2327 GLUTATHIONE DEPLETION POTENTIATES
CIGARETTE SMOKE INDUCED AIRWAY
INFLAMMATION.
B. J. Day 1, 2, 3 , E. Min 1 and N. S. Gould 1, 2 . 1 Medicine, National Jewish Health,
Denver, Co., 2 Pharmaceutical Sciences, University of Colorado AMC, Aurora, CO
and 3 Medicine, University of Colorado AMC, Aurora, CO.
Cigarette smoke (CS) is one of the main causes of respiratory disorders including
emphysema and chronic bronchitis. A common underlying factor in these diseases
is an exaggerated inflammatory response. CS causes an increase in inflammation
and oxidantion which triggers an upregulation of glutathione (GSH). The role and
effect of the upregulation of GSH on inflammatory pathways has not been completely
established. Recent data shows that GSH levels found in the epithelial lining
fluid (ELF) of the lung are inversely correlated to airway TNFα. Furthermore,
macrophage activation and TNFα release is exaggerated in low GSH environments.
The present study sought to determine if there was a direct link between GSH and
inflammation that may be potentiated by CS. L-Buthionine-sulfoxime (BSO) in
the drinking water was utilized as an inhibitor of GSH synthesis in vivo and mice
were administered either normal or BSO water for 7 d pretreatment and then exposed
to either air or CS for 5 d. Body weights were taken as an indicator of health
throughout the experiment. During the pretreatment phase, body weights did not
differ significantly between the groups, but during the CS exposure BSO administered
mice lost roughly 10% of their body weight from peak levels while there was
no significant change in other groups. An analysis of ELF GSH showed significant
increases in the CS only mice while BSO blunted the increase with the BAL cell
and lung tissue GSH showing similar trends while CS depleted GSH in the plasma
and liver. Myeloperoxidase (MPO) activity, a marker of neutrophil infiltration, was
three times higher in the lungs of BSO CS mice compared to BSO alone and
TNFα levels were elevated in both BSO air and CS exposed mice. In contrast, air
and CS only exposed mice did not have any increases in airway TNFα. These data
suggest that GSH plays a role in CS induced inflammation which may help to explain
why so many inflammatory diseases are associated with decreased GSH levels.
500 SOT 2011 ANNUAL MEETING
2328 THE GLUTATHIONE SYNTHESIS GENE GCLM
MODULATES PULMONARY INFLAMMATION
FOLLOWING INTRANASAL INSTILLATION OF DIESEL
EXHAUST PARTICULATE.
C. Weldy 1 , C. C. White 1 , D. P. Cox 1 , H. Wilkerson 1 , S. Gill 2 , W. Parks 2 and T.
J. Kavanagh 1 . 1 Environmental and Occupational Health Sciences, University of
Washington, Seattle, WA and 2 Pulmonary and Critical Care Medicine, University of
Washington, Seattle, WA.
Diesel exhaust particulate (DEP) is a major component of airborne particulate matter
(PM) and exposure to DEP has been associated with pulmonary irritation, inflammation
and exacerbation of asthma symptoms. DEP is a known pro-oxidant
and can produce inflammation in the lungs by producing reactive oxygen and nitrogen
species. Glutathione (GSH) is an important antioxidant thiol and the ratelimiting
enzyme in the synthesis of GSH is glutamate cysteine ligase (Gcl), composed
of catalytic (Gclc) and modifier (Gclm) subunits. As oxidative stress is
believed to play a role in DEP-induced inflammation, we hypothesized that mice
with compromised GSH synthesis (Gclm +/-, -/-) would be more susceptible to
DEP-induced inflammation. In the present study, we dosed C57BL/6 WT, Gclm
+/-, and Gclm -/- mice with DEP by intranasal instillation. Animals received a total
of 20μl of PBS or 20μl of 10mg/ml DEP solution in one 10μl instillation per nostril.
After 6 hours, animals were sacrificed and bronchial alveolar lavage fluid
(BALF) was collected. Gclm +/- mice had increased neutrophilia and IL6 and
TNFα in BALF than both WT and Gclm -/- mice. In contrast to this finding,
analysis of lung tissue gene expression by qPCR revealed that Gclm -/- mice had elevated
levels of mRNA transcripts for TNFα, IL6, GMCSF, IL1β, and MCP1 relative
to both WT and Gclm +/- mice after DEP treatment. These data suggest that
although Gclm -/- mice had a greater inflammatory gene expression response to
DEP than WT or Gclm +/-, they may be compromised in their ability to subsequently
synthesize or release inflammatory cytokines, and recruit neutrophils into
the alveolar space. These results indicate that GSH is an important factor in regulating
inflammatory response following DEP treatment. This work was supported
by NIEHS grants 1P50ES015915, P30ES07033 and T32ES07032.
2329 AIRBORNE ORGANIC PARTICULATE MATTER AS A
LUNG INFLAMMATION INDUCER: ROLE OF
PHOSPHOLIPASE A2.
S. R. Kotha 1 , M. G. Piper 1 , B. P. Caristophe 1 , N. Patric 1 , R. M. Uppu 2 , M. B.
Clay 1 and N. L. Parinandi 1 . 1 Division of Pulmonary, Allergy, Critical Care, and
Sleep Medicine, The Ohio Stae University College of Medicine, Columbus, OH and
2 Department of Environmental Toxicology, Southern University and A&M College,
Baton Rouge, LA.
Agricultural particulate matter (PM) has been linked to respiratory disorders including
occupational asthma in the workers at Central Ohio poultry farms. In
order to test the precise mechanism(s), human lung epithelial cells (A549), in culture,
were treated with varying concentrations of poultry PM (0.1-1.0 mg) for different
periods of time (0-60 min) following which Reactive oxygen species (ROS)
production, PLA2 activity, cPLA2 serine phosphorylation, ERK1/2 phosphorylation
(activation), and IL-8 release were determined. The results showed a significant
activation of PLA2 induced by poultry PM (1.0 mg/mL) at 1 h, which was significantly
attenuated by AACOCF3 (cPLA2 inhibitor) and PD98059 (ERK1/2 upstream
inhibitor), suggesting the modulation of cPLA2 activity by ERK1/2 in cells.
ERK1/2 phosphorylation was also induced by poultry PM which also paralleled
cPLA2 serine phosphorylation. cPLA2 immunoprecipitates from poultry PMtreated
cells exhibited enhanced association of ERK1/2 and cPLA2 in a time-dependent
fashion. ERK1/2 inhibition with PD98059 also attenuated the poultry
PM-induced serine phosphorylation of cPLA2 in cells. Furthermore, the poultry
PM-induced cPLA2 serine phosphorylation and IL-8 release were significantly inhibited
by AACOCF3 and PD98059, and by transfection with dominant-negative
ERK1/2 DNA in cells. Finally, the poultry-induced IL-8 release by the bone marrow-derived
macrophages of cPLA2 knockout mice was significantly lower. For the
first time, this study demonstrated that the agricultural PM-induced IL-8 secretion
by human lung epithelial cells was regulated by cPLA2 activation through ERKmediated
serine phosphorylation suggesting a probable mechanism of airway inflammation
among poultry farm workers.