J ALLERGY CLIN IMMUNOL

FEBRUARY 2011

AB60 AbstractsSATURDAY

221 Tranglutaminase 2 Gene Deficiency Results In Decrease OfAirway Resistance And Eosinophilic Inflammation In MurineAsthma Model

H. Lee1, E. Shim1, J. Jung1,2, H. Kang1,2, H. Park1,2, S. Kim1,2, S. Cho1,2,

S. Chang3, K. Min1,2, Y. Kim4; 1Institute of Allergy and Clinical Immunol-

ogy, Seoul National University Medical Research Center, Seoul, KOREA,

REPUBLIC OF, 2Department of Internal Medicine, Seoul National Univer-

sity College of Medicine, Seoul, KOREA, REPUBLIC OF, 3Department of

Internal Medicine, Sung-Ae General Hospital, Bucheon, KOREA, REPUB-

LIC OF, 4Eulji Medical Center, Seoul, KOREA, REPUBLIC OF.

RATIONALE:Transglutaminase 2(TG2) is amultifunctional calcium-de-

pendent acyltransferase and ubiquitously expressed in mammalian tissue

existing both extracellularly and intracellulary. Recent investigation re-

ported TG2 was highly expressed in the asthmatic airways and augmented

the enzymatic activity of phospholipase A2 group 3 which hydrolyzed

phospholipid and release arachidonic acids. We hypothesized that TG2

would have some roles in pathogenesis of asthma.

METHODS:To evaluate expression of TG2 in asthma patients, we stained

lung tissue with anti-TG2 antibody and detected TG2mRNA expression in

induced sputum samples of asthmatic patients. To see the effect of TG2 de-

ficiency, wemade a murine ovalbumin (OVA) asthmamodel with TG2 null

mice and compared with wild type mice. Airway hyperresponsivenss

(AHR) to methacholine, airway inflammatory cells, and cytokines in ho-

mogenate of lung tissue were evaluated.

RESULTS: TG2 expression in bronchial epithelium and TG2mRNA level

in induced sputum samples were increased in asthmatics compared with

normal controls. TG2 null mice showed significant reduction in eosino-

philic airway inflammation, AHR, serum OVA-specific IgG1/G2a ratio

and Th2 cytokines (IL-4, IL-13) in lung homogenates compared with

WT mice after OVA challenge.

CONCLUSIONS: In human asthmatics, TG2 is highly expressed in air-

way epithelium and induced sputum. In murine asthma model, TG2 defi-

ciency effectively reverses airway hyperresponsiveness and eosinophilic

airway inflammation, Th2 immune responses. These findings suggest

that TG2 play important roles in the pathogenesis of asthma.

222 Airway Cytokine Pattern Predicts Severity of AsthmaSymptoms During Colds

B. R. Sabin1, A. T. Sabin2, P. McErlean1, T. Ward3, J. Liu3, H. Boushey3,

P. Avila1; 1NorthwesternUniversity, Chicago, IL, 2NorthwesternUniversity,

Evanston, IL, 3University of California San Francisco, San Francisco, CA.

RATIONALE: It is currently unknown whether patterns of cytokine ex-

pression in the airway following respiratory tract infections (RTIs) predict

severity of asthma symptoms.

METHODS: Patients with asthma (A), allergic rhinitis (AR), or neither

(N) with naturally acquired viral RTIs had induced sputum collected as

well as upper and lower respiratory symptoms recorded on days 2, 6,

and 42 of their illness. Induced sputumwas analyzed by multiplex bead as-

say for IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IFNg, TNFa, and GM-

CSF.

RESULTS: A multiple linear regression model using all ten analytes as-

sayed in 34 samples (16 A, 18 AR+N) was found to be significantly predic-

tive of actual asthma symptom scores (R250.65, p50.0004). This

correlation was primarily driven by IFNg, TNFa, and IL-5, as only these

three analytes were included in a reduced model that was created using

stepwise regression (R250.47, p<0.0001). In this reduced model, IFNg

had a negative coefficient, while the other two coefficients were positive.

Further, it appeared that the A, as opposed to the AR+N, subgroup was re-

sponsible for the correlation (R250.43 vs. R250.02). Induced sputum did

not predict upper airway symptom scores in any group.

CONCLUSIONS: Both a 10 cytokine model and a reduced 3 cytokine

model are predictive of a significant amount of variation of the asthma

symptom score. The negative correlation of IFNg and positive correlation

of IL-5 with asthma symptoms corroborate past experimental rhinovirus

inoculation studies. In addition, TNFa seems to be an important cytokine

in the pathogenesis of asthma symptoms during colds.

223 The Possible Role of Clusterin As a Biomarker RepresentingIncreased Oxidative Stress in Asthma

Y. Lee, T. Lee, T. Kim, H. Moon, Y. Cho; Asan Medical Center, Seoul,

KOREA, REPUBLIC OF.

RATIONALE: Asthma is a chronic inflammatory disease and oxidative

stress has been known to play an important role in the pathogenesis of

asthma. Clusterin, a ubiquitous glycoprotein, is closely linked to various

cellular functions to keep homeostasis. Clusterin has been also recognized

as a sensitive biosensor responding to increased oxidative stress in other in-

flammatory diseases. To date, the role of clusterin has not been investigated

in asthma.

METHODS: Serum levels of clusterin were measured by enzyme-linked

immunosorbent assays (ELISA) in 142 asthma patients and 33 age-

matched healthy controls. In 37 treatment na€ıve patients, the levels before

treatment were compared with those after treatment with ICS. Expression

of clusterin in peripheral blood mononuclear cells (PBMCs) and superna-

tants of induced sputum were assessed by western blot analysis in some of

the patients. In addition, expression of hyperoxidized peroxiredoxins in

PBMC was also evaluated as a cellular marker indicating increased oxida-

tive stress in cells.

RESULTS: Serum levels of clusterin were increased in the patients with

severe asthma showing lower pulmonary functions than those in normal

controls and the levels were reduced after ICS treatment in the treatment

na€ıve patients. The expression of clusterin in sputum and PBMCs were re-

markably enhanced in asthma patients and showed a significant correlation

with intracellular hyperoxidized peroxiredoxins of PBMCs.

CONCLUSIONS: Clusterin may be a useful biomarker indicating the de-

gree of oxidative stress in asthmatics and severity of the disease as well.

Further studies should be needed to investigate the precise role of clusterin

in the pathogenesis of asthma.

224 Munc13-2 And Munc13-4 Double Knock-out Attenuates TheDefect Of Airway Mucin Secretion Over 13-2 Null In MurineAirway Metaplasia

J. Lee1,2, Z. Azzegagh1, L. Piccotti1, P. Bathina1, M. J. Tuvim1, B. F.

Dickey1; 1MDAnderson Cancer Center, Houston, TX, 2Jeju National Uni-

versity, Jeju, KOREA, REPUBLIC OF.

RATIONALE: Mucin secretion into the airway is essential, whereas de-

fects may contribute to pathophysiologic conditions. Mucin is exocytosed

into the airway, controlled and regulated by exocytotic machinery. Ca2+

binding proteins such as rabphilin, Munc13, and synaptotagmin, play im-

portant roles in the final steps of exocytotic fusion. Among 4 isoforms of

Munc13, Munc13-2 and 13-4 are expressed in the lung. The defect in

Munc13-2 leads to significant accumulation of baseline mucins, mostly

Muc5b. In the metaplastic airway, Munc13-2 null shows significant defect

ofmucin secretion evenwith secretory agonist.Munc13-4, although partial

lacking of Ca2+ binding domains, may have regulatory roles in airwaymu-

cin exocytosis.

METHODS: Munc13-2 and 13-4 double knock-out mice were obtained

by crossing of Munc13-2+/- and 13-4+/- in C57/B6. Airway mucins are

quantified by mucin volume density in two separate experimental settings

of baseline accumulation and agonist stimulated secretion with ovalbumin

sensitization and challenge as an allergic inflammatory airway model.

RESULTS:Munc13-4 null mice revealed no phenotype in baseline mucin

accumulation and agonist stimulated mucin secretion, compared to the

wild type. However, double knock-outs of Munc13-2 and 13-4 showed sig-

nificant defect on the agonist stimulated mucin secretion over wild type.

The secretory defect of mucin is more than those of Munc13-2 nulls. No

difference was shown at baseline accumulation.

CONCLUSIONS: Munc13-4 plays a regulatory role in the exocytosis of

airway mucin. Lacking of Ca2+ binding domain, Munc13-4 alone may be

insufficient in functioning as Ca2+ sensor. Accompanied by Munc13-2, it

supplementally facilitates agonist stimulated mucin secretion in the meta-

plastic airway.