J ALLERGY CLIN IMMUNOL

VOLUME 131, NUMBER 2

Abstracts AB239

UESDAY

845 Epicutaneous Sensitization in Filaggrin Gene-DepletedMouse Induces Prolonged Airway Eosinophilia withoutObvious Dermatitis

Yusuke Suzuki1,2, Katsunori Masaki1, Shizuko Kagawa1,2, Hiroshi Kawa-

saki3,4, Keisuke Nagao3, Akiharu Kubo3,5, Tomoko Betsuyaku1, Masayuki

Amagai3, Koichiro Asano6; 1Division of Pulmonary Medicine, Department

of Medicine, Keio University School of Medicine, Tokyo, Japan, 2MSD

Endowed Program for Allergy Research, Tokyo, Japan, 3Department of

Dermatology, Keio University School of Medicine, Tokyo, Japan, 4The

Association for Preventive Medicine of Japan, Tokyo, Japan, 5Center of

Integrated Medical Research, Tokyo, Japan, 6Department of Pulmonary

Medicine, Tokai University School of Medicine, Isehara, Japan.

RATIONALE: Mutations in filaggrin gene, encoding a protein involved in

the epidermal barrier function, are associatedwith the susceptibility to atopic

dermatitis and asthma in human. To elucidate the role of filaggrin deficiency

in the pathogenesis of asthma, we have established an asthma model using

filaggrin knockout (KO) mice epicutaneously sensitized to allergen.

METHODS: House dust mite (HDM) ointment was repeatedly applied on

the ear of wild type (WT) and KO mice for 3 or 8 weeks, followed by

intranasal administration of the same allergen.

RESULTS: Four days after HDM challenge, WT and KO mice similarly

developed asthmatic features such as airway eosinophilic inflammation, epi-

thelialmucusproduction, serumtotal IgEandHDM-specific IgE. Interestingly,

neither WT nor KO mice showed obvious dermatitis. Eosinophil count of

bronchoalveolar lavagefluid remained high inKOmice until 7 days after nasal

challenge in contrast to reduced count in WT. Total and HDM-specific IgE

levels were also higher in KOmice at the time point. Significantly more PAS-

positive cells were observed in KO lungs 14 days after challenge than in WT

lungs. IL-17Aproduction in both spleen and lung cellswas greater inKOwhile

other cytokine levels were similar. IL-23 expression was significantly

increased by eight-week application of HDM only in ears of KO mice.

CONCLUSIONS: Filaggrin protects against the prolongation of asthmatic

phenotype. Skin IL-23, induced particularly in filaggrinKOmiceby epicutane-

ous sensitization,maymediate the effect through systemic IL-17A production.

846 Thymic Stromal Lymphopoietin Promotes Peripheral BloodCD34+ Cell Eosinophilic Lineage Commitment

Claudia C. K. Hui, MSc, Sina Rusta-Sallehy, BSc, Delia Heroux, BSC,

Judah Aryeh Denburg, MD, FRCPC, FAAAAI; McMaster University,

Hamilton, ON, Canada.

RATIONALE: Allergic diseases are often characterized by eosinophilic

inflammation. Both epithelial-derived TSLP and eosinophil/basophil

(Eo/B) lineage-committed progenitor cells are upregulated and found at

sites of allergic inflammation. We have previously shown that TSLP is

sufficient to induce the differentiation of cord blood (CB) CD34+ progen-

itor cells into eosinophils. However, the effects of TSLP on peripheral

blood (PB) CD34+ cell eosinophilic lineage commitment are unclear.

METHODS: PBCD34+ cells were stimulated with TSLP and assessed for

Eo/B colony forming units (CFU) by methylcellulose cultures and surface

expression of IL-3Ra, IL-5Ra, TSLPR and IL-7Ra by flow cytometry.

Neutralizing Abs were used to determine the dependence of TSLP on

Eo/B colony formation and surface receptor expression.

RESULTS: Stimulation with TSLP significantly increased the formation

of IL-3-responsive Eo/BCFU (p<0.001), which were significantly reducedby the addition of anti-TSLP mAb (p<0.01) and anti-TSLPR pAb

(p<0.05). Overnight stimulation of PB CD34+ cells with IL-3 and TSLP

significantly enhanced percent expression of IL-3Ra on CD34+ cells com-

pared to unstimulated control (p<0.001) and TSLP alone (p<0.05).Similarly, co-stimulation with IL-3 and TSLP upregulated TSLPR expres-

sion on CD34+ cells compared to unstimulated control (p<0.001).

CONCLUSIONS: We show for the first time that stimulation of human

PB CD34+ cells with TSLP promotes eosinophilic lineage commitment

through upregulation of IL-3Ra and TSLPR. Since TSLP is upregulated

in allergic disease, defining the influence of TSLP on eosinophilopoiesis

will allow for a better understanding of allergic disease development and

for identifying new therapeutic targets for atopic disorders.

847 Hypoxia Inducible Factor (HIF) Alpha Subunits ModulateEosinophil Migration, Oxidative Burst, and Degranulation

Laura E. Crotty Alexander, MD1,2, Shari Nicatia, BSc1,3, Elisa

McEachern, BS1, Randall Johnson, PhD4, David H. Broide, MB, ChB,

FAAAAI5, Victor Nizet, MD2; 1VA San Diego Healthcare System, La

Jolla, CA, 2University of California, San Diego, La Jolla, CA, 3Utrecht

University, Utrecht, Netherlands, 4University of Cambridge, United King-

dom, 5Division of Allergy, Immunology.

RATIONALE: HIF is a master regulator of inflammatory activities of

myeloid cells, including neutrophils, mast cells and macrophages. These

studies examine HIF-1a and HIF-2a regulation of eosinophil function,

namely chemotaxis, degranulation, and oxidative burst. The literature is

conflicting as to whether the two a-subunits have complementary versus

divergent gene regulatory roles. HIF-1a andHIF-2a levels are controlled by

prolyl hydroxylases and oxygen levels, in a subunit specific way, and are

potential therapeutic targets. We set out to tease apart the functional

differences in thea subunits, and determine their role in eosinophil function.

METHODS: Eosinophils were derived from C57BL/6 wild-type (WT),

HIF-1a and HIF-2a tie2cre knockout (KO) mouse bone-marrow.

Eosinophils were evaluated via transwell chemotaxis, degranulation,

oxidative burst, and bactericidal assays, utilizing eotaxin-1, MIP-1a, and

IL-5 as chemokines and stimulants.

RESULTS: Deletion of HIF-1a in eosinophils diminished their chemo-

taxis,while deletionofHIF-2a increased chemotaxis (WT58%,HIF-1aKO

22%*,HIF-2aKO84%*, *p<0.001).HIF-2aKOeosinophils had increased

HIF-1a levels by Western blot. HIF-1a KO eosinophils had increased de-

granulation in response to eotaxin-1 (29% greater than WT, p<0.0001).Deletion of either subunit led to increased oxidative burst (WT 2.7x105,

HIF-1aKO 8.2x105*, HIF-2aKO, 5.9x105*, *p<0.001). Therewere no dif-

ferences in bactericidal activity againstE. coliofWTversusKOeosinophils.

CONCLUSIONS: Thisworkdemonstrates thatHIF-1aandHIF-2a regulate

some eosinophil activities –migration in opposingways and oxidative burst in

analogous ways. Altering HIF-a subunit levels changes eosinophil function,

and thus suggests new therapeutic targets for allergic disease and asthma.

848 Functional Expression of the Novel C5a Receptor C5L2 inHuman Mast Cells

Priyanka Pundir, DVM1,2, Marianna Kulka, PhD2; 1Department of Bio-

medical Sciences, Atlantic Veterinary College, Charlottetown, PE,

Canada, 2National Research Council Canada, Charlottetown, PE, Canada.

RATIONALE: Anaphylatoxin C5a regulates diverse innate and adaptive

immune responses by chemoattracting and activating immune cells, such as

mast cells (MC). Its postulated thatC5a regulatesMC functionby activating

a G protein-coupled receptor (GPCR), however it can bind to C5aR, a

classical GPCR, or C5L2, not G protein coupled and thought to activate

distinct signaling. The presence and role of C5L2 in MC remain unknown.

We hypothesized that human MC express functional C5L2 receptor.

METHODS: C5aR and C5L2 expression in LAD2 MC was analyzed by

qPCR and flow cytometry. Degranulation, cytokine/chemokine produc-

tion, adhesion, and migration were measured using standard assays.

Lentivirus short hairpin (sh)RNA was utilized to create a stable C5L2-

deficient (LAD2-C5L2ko) and a control MC cell line (LAD2-cntr).

RESULTS: LAD2 expressed cell-surface C5L2 but not C5aR. C5a (100

ng/ml) stimulated LAD2 degranulation (25%), production of TNF (2261.3

pg/ml), GM-CSF (1560.4 pg/ml), MCP-1 (5363.6 pg/ml) and IP-10

(3264.3 pg/ml), adherence to fibronectin (466692%), and chemotaxis

(148.9360.94%). Pre-treatment with pertussis toxin, wortmanin,

U-73122, and Ro-31-8220 inhibited C5a-induced LAD2 adhesion.

Exposure to C5a reduced cell-surface C5L2 expression by ;45%.

LAD2-C5L2ko showed decreased expression of GM-CSF and MIP-1b

in response to C5a compared to LAD2-cntr. C5a-stimulated LAD2-

C5L2ko showed lesser adhesion to fibronectin compared to LAD2-cntr.

CONCLUSIONS: We demonstrated C5a activates LAD2 cytokine and

chemokine production, and adhesion via C5L2. MC activation by C5a was

sensitive to inhibitors of G protein signaling and these results provide the

first evidence of the crucial role of C5L2 in regulating MC functions.

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