Functional Expression of the Novel C5a Receptor C5L2 in Human Mast Cells
Transcript of Functional Expression of the Novel C5a Receptor C5L2 in Human Mast Cells
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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