J ALLERGY CLIN IMMUNOL

FEBRUARY 2008

S274 Abstracts

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1056 IL-13 Mediated Lung Effects Are Exclusively Dependent OnIL-13Ralpha1 (CD213a)

A. Munitz, E. Brandt, M. Mingler, F. D. Finkelman, M. E. Rothenberg;

Cincinati Children’s Hospital medical Center, Cincinnati, OH.

RATIONALE: Interleukin (IL) -13 is a potent activator of inflammation

and fibrosis during type-2 inflammation such as asthma, tissue remodeling

and parasite immunity. However, to date whether IL-13Ralpha1 is the only

(or main) IL-13 receptor in the lung has not been determined.

METHODS: To test the hypothesis that IL-13 receptor (R) alpha1

mediates IL-13-induced functions we report the generation of IL-

13Ralpha1 deficient mice by Velocigene technology and the characteriza-

tion of baseline immune phenotype of these mice. Furthermore, the effects

of intratracheal delivery of IL-13 to IL-13Ralpha1-/- mice were assessed.

In addition, the role of IL-13Ralpha1 was examined in an OVA-induced

model of experimental asthma that included global microarray analysis

(Affymetrix) of the allergen challenged lungs.

RESULTS: IL-13Ralpha1-/- mice displayed markedly decreased serum

IgE although IL-4 signaling components (i.e. serum IL-4, IL-4Ralpha)

were intact. Nevertheless, these mice generated a polarized Th2 reaction in

response to T cell dependent antigens (goat anti-mouse IgD and OVA). In

response to IL-13, IL-13Ralpha1-/- mice displayed no goblet cell hyper-

plasia, mucus production as well as no CCL2, CCL11, CCL17, CCL24 and

TGF-beta induction. In response to OVA challenge, IL-13Ralpha1-/-

mouse displayed no observable mucus production, and chemokine induc-

tion, yet they had preserved leukocyte recruitment. Global transcriptome

analysis identified IL-13Ralpha1-dependent and -independent genes in the

OVA-induced asthma model that mechanistically explained the observed

phenotype.

CONCLUSIONS: These results demonstrate a definite role for IL-

13Ralpha1 in mediating IL-13-induced lung pathology. Furthermore,

they provide the basis for generating IL-13Ralpha1-based blocking agents

for future therapeutic intervention.

Funding: NIH

1057 [Withdrawn]

1058 Poorly Controlled Childhood Asthma Is Associated WithIncreased Bacterial Lipopolysaccharide (LPS) AndExpression of Markers of Airway Remodeling InBronchoalveolar Lavage (BAL)

P. J. Hauk, M. Krawiec, J. Boguniewicz, A. M. Schiltz, E. Goleva,

A. H. Liu, D. Y. M. Leung; National Jewish Medical and Research Center,

Denver, CO.

RATIONALE: High exposure to environmental LPS has been associated

with increased asthma severity. To determine whether LPS might play a

role in childhood asthma, we studied bronchoalveolar lavage (BAL) fluid

from children with poorly controlled asthma who underwent clinically

indicated bronchoscopies for exposure to LPS and expression of pro-

inflammatory cytokines, chemokines and markers of airway remodeling.

METHODS: LPS levels in BAL were measured by the chromogenic

limulus amebocyte lysate test. TNF-alpha, IL-8, IL-13, MMP-9, TIMP-1,

PMN elastase and VEGF levels were measured by ELISA. Correlations

between BAL cell differentials, LPS, chemokine, and cytokine levels were

analyzed by Spearman Correlation.

RESULTS: Analysis of BAL from 18 poorly controlled asthmatic

children, ages 2.5 months to 11 years (median 12.5 months) showed LPS

levels between 2 and 216233 pg/mg BAL protein (mean 21024 pg/mg 6

12778 [SE]). High LPS levels correlated with increased MMP-9 levels (p

5 0.012) and elevated BAL neutrophils (p 5 0.044). The percentage of

BAL neutrophils correlated with increased levels of MMP-9 (p < 0.001),

TIMP-1 (p 5 0.002), and MMP-9/TIMP-1 ratio (p < 0.001). BAL levels of

the neutrophil marker PMN elastase also correlated with MMP-9 (p 5

0.001) and TIMP-1 (p 5 0.012) levels. No correlations were found between

LPS in BAL and levels of TNF-alpha, IL-8, IL-13, and VEGF.

CONCLUSIONS: Increased exposure to LPS may lead to recruitment of

neutrophils into the airways and early expression of genes related to airway

remodeling, including MMP-9, TIMP-1, and PMN elastase predisposing

some children to severe asthma.

Funding: NIH

1059 Characterization of the Glucocorticoid Response forTranscripts Associated with the RNA-binding ProteinTristetraprolin

F. Ishmael1, X. Fang1, U. Atasoy2, M. Gorospe3, C. Cheadle1, C. Stel-

lato1; 1Johns Hopkins University, Baltimore, MD, 2University of Colum-

bia-Missouri, Columbia, MO, 3Laboratory of Cellular and Molecular

Biology, National Institute of Aging, NIH, Baltimore, MD.

RATIONALE: Posttranscriptional regulation is emerging as a key factor

in the mechanism of glucocorticoid (GC) gene regulation. We hypothesize

that the GC-inducible, RNA-binding protein tristetraprolin (TTP) at least

partially mediates GC action. Our aim is to identify inflammatory

transcripts regulated by TTP and examine their response to GC treatment

in the presence or absence of TTP.

METHODS: Wild-type (WT) and TTP-deficient (TTP-/-) mouse embry-

onic fibroblasts (MEFs) were treated with budesonide (10-7M) or diluent

(DMSO) for 2 h. Total RNA was extracted, and the global effects of GC

were assessed with Illumina arrays. Expression of seven genes for which

the GC response was found dependent on TTP was validated by real-

time PCR. For these genes, binding of TTP to mRNA was determined by

immunoprecipitation (IP) of messenger ribonucleoprotein (mRNP) com-

plexes using a specific anti-TTP antibody and an isotype-matched IgG

control.

RESULTS: Array analysis revealed that at least 89% of GC-regulated

genes lost their response - either upregulation or inhibition - in TTP-/-

versus WT MEFs. Loss of GC regulation was validated by RT-PCR for:

CCL7, CXCL1, CCL2, CXCL5, CXCL7, MMP9, Serpina3n. As deter-

mined by enrichment of mRNA following mRNP-IP compared to control,

association of the following transcripts to endogenous TTP was identified

(fold increase over control IgG): CCL7 (58.4), CXCL1 (222.9), CXCL5

(21.2), IL-6 (51.1), MMP9 (11.8), [n 5 3, p < 0.05 for all data].

Association of TTP with transcripts of CXCL7, IL1rl1, and Serpin3n

was not observed.

CONCLUSIONS: TTP is a potentially critical determinant of

GC-mediated gene regulation that likely acts through both RNA-dependent

and independent mechanisms.

Funding: NIH

1060 IL-13 induced MUC5AC is regulated by 15-Lipoxygenase(LO) 1 Pathway in Human Bronchial Epithelial Cells

J. Zhao1, B. Maskrey2, V. O’Donnell2, K. Chibana1, J. Trudeau1,

S. Balzar1, S. Wenzel1; 1University of Pittsburgh, Pittsburgh, PA, 2Cardiff

University, Cardiff, UNITED KINGDOM.

RATIONALE: Th2 pathway activation is believed critical to asthma

pathogenesis. In response to IL-13, 15LO1 and MUC5AC are highly

expressed in epithelial cells in vitro, and in asthmatics in vivo. We hypoth-

esized that IL-13 induced 15 LO1 and its product 15 HETE regulate

MUC5AC gene expression in human bronchial epithelial cells.

METHODS: Primary human bronchial epithelial cells from bronchos-

copies of asthma and control subjects were cultured in air-liquid interface

(ALI) system. 100% confluent cells were exposed to IL-13 with/without a

15 LO1 chemical inhibitor (PD146176) or following specific 15 LO1

siRNA transfection. MUC5AC and 15 LO1 expressions were analyzed

by real time RT-PCR, immunohistochemistry (IHC) and Western

blot. 15HETE was analyzed by Liquid Chromatography/UV/Mass

Spectrometry.

RESULTS: IL-13 strongly induced both 15LO1 protein and mRNA in the

ALI system (n 5 10, p 5 0.002). LC/UV/MS results indicated that

endogenous 15HETE was induced by IL-13 stimulation, with nearly 100%

present intracellularly in a conjugated form with phosphotidyl ethanola-

mine (15 HETE-PE). No free 15 HETE was present intracellularly or