J ALLERGY CLIN IMMUNOL
VOLUME 131, NUMBER 2
444 CLC3 Transcript Variants in the Activation and Migration of
Eosinophils in Allergic Asthmatics
Rohit Gaurav1, Min-Jung Kim1, Againdra Bewtra2, Devendra K.
Agrawal1; 1Center for Clinical and Translational Science and Department
of Biomedical Sciences, Creighton University School of Medicine,
Omaha, NE, 2Department of Medicine, Division of Allergy and Immunol-
ogy, Creighton University Medical Center, Omaha, NE.
RATIONALE: Migration and activation of eosinophils play a major role
in the pathophysiology of allergic asthma. Recently, we reported the role of
CLC3 in TGF-b-induced migration of eosinophils, increased expression of
CLC3b and CLC3e transcript variants on eosinophil membranes in
response to TGF-b and eotaxin. Here, we focused on the function of
CLC3 in eosinophils of allergic asthmatics.
METHODS: Human peripheral blood eosinophils were isolated (>99%
pure >98% viable) with negative selection from healthy and mild-to-
moderate allergic asthmatic donors. Cells were stimulated with TGF-b1,
eotaxin-1, eotaxin-3, DIDS and NPPB. QPCR, chemotaxis and whole-cell
patch were used to obtain outcome measures.
RESULTS: CLC3 and NOX2 (gp91phox) mRNA levels were 5-9 fold and
5-25 fold, respectively, higher in asthmatics compared to healthy individ-
uals (n54, p<0.05). Also, CLC3b and CLC3e transcript variants were 7-11
fold and 13-21 fold, respectively, higher in asthmatics than in controls
(n54, p<0.05). Significantly higher current due to CLC3 and greater mi-
gration of eosinophils were observed in the eosinophils of asthmatic com-
pared to healthy individuals.
CONCLUSIONS: Significantly greater increase in CLC3e transcript
variant compared to CLC3b in asthmatic eosinophils suggests it’s imper-
ative role in allergic asthma. Greater migratory potential of eosinophils and
higher activity of CLC3 channel in asthmatics support its importance in
allergic asthmatics. NADPH oxidase is vital for oxidative burst, and to
compensate the resultant charge imbalance, CLC3 works in conjunction
with NADPH oxidase. Higher level of NOX2 mRNA in asthmatics
suggests that the cells are in an activated state with active participation
of both CLC3 and NADPH oxidase.
445 Eosinophils Through the CXCR4 Chemokine Receptor
Christof Straub1, Konrad Pazdrak, MD, PhD2, Alexander
Kurosky, PhD3; 1University of Texas Medical Branch, Galveston, TX,
2
Univ. of TX Medical Branch, Galveston, TX, 3University of Texas Med-
ical Branch, League City, TX.
RATIONALE: HMGB1, a DAMP protein, has recently been implicated in
asthma. HMGB1 is secreted by necrosis or through active release following
stimulation, and it can subsequently act as a proinflammatory mediator
with cytokine-like properties. The protein is expressed by resident and
infiltrating airway cells and it can bind to any cell that expresses one of its
target receptors (e.g. RAGE, TLR4, CXCR4). We have investigated the
chemoattractant role of HMGB1 on eosinophils based on previous reports
that implicate HMGB1 in chemotaxis of other cell types.
METHODS: We purified bacterially-expressed, recombinant HMGB1 as
well as HMGB1 secreted by stimulated Eol-1 cells. The two HMGB1
proteins were assessed for chemotactic properties in assays with human
peripheral blood eosinophils (EosCD16-/CD3-/CD235a-) using a TranswellÒ
system (Corning; 5 mm pore size).
RESULTS: Both recombinant and secreted HMGB1 exert chemotactic
properties on human peripheral blood eosinophils, with stronger chemo-
tactic potencies exerted by the secreted form. Similar results were observed
with human neutrophils. Pretreatment of eosinophils with the CXCR4-
specific inhibitor AMD3100 blocked chemotaxis toward HMGB1.
Blockage of RAGE had no effects on eosinophil chemotaxis toward
HMGB1.
CONCLUSIONS: These results implicate HMGB1 as a novel, potent
chemotactic mediator for eosinophils and neutrophils. Since HMGB1 has
been found in the airways of asthmatics, this data points to an important
role of HMGB1 in asthma that is exerted via eosinophil- and neutrophil-
associated infiltration and inflammation.
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Abstracts AB123
446 Correlations Between Eosinophil Markers in Allergic
Disorders
Mahsheed Taeb, DO1, Katrina Elio, DO1, Oral Alpan, MD2,3; 1Inova
Fairfax Hospital, 2Amerimmune, LLC, VA, 3O&O ALPAN, LLC.
RATIONALE: CD69 is an activation marker present on eosinophils,
shown to be a marker of activation. On the other hand, FceRI at the surface
of eosinophils endow these cells with both effector and regulatory function
such as the production of IL-10. We describe 3 cases (ages 3, 12 and 28, all
males) with asthma and allergic rhinitis, presenting with a flare up of their
disease and peripheral blood eosinophilia in which we asked whether the
expression of CD69 correlates with the expression of high affinity IgE re-
ceptor (FceRI) on eosinophils.
METHODS: Whole blood was stained with antibodies to CD9, FceRI,
CD16, CD69. Samples were analyzed on a Accuri flow cytometer.
Eosinophils were separated from granulocytes by their characteristic
high side-scatter, dim staining for CD16 and CD9 positivity.
RESULTS: Eosinophil CD69 expression was 11.7, 8 and 5.5% and FceRI
expression was 3, 4.5 and 3.9%. To our surprise, eosinophils did not co-
express FceRI and CD69. All patients had IgE mediated allergies based on
SUNDAY
skin prick and serum allergen specific IgE levels.
CONCLUSIONS: We show for the first time, the distinct expression
profile of CD69 and FceRI on eosinophils. The non-coexisting expression
of CD69 and FceRI on eosinophils suggests distinct roles for each
requiring further work in this field.
447 Effect of Sensitization and Exposure to Mold On Asthma
Morbidity in Inner-City Children
Jean Curtin-Brosnan, MA1, Patrick J. Lenehan2, Patrick Breysse, Ph.D3,
Gregory B. Diette, MD, MHS1, Elizabeth Matsui, MD1,4; 1Johns Hopkins
University, Baltimore, MD, 2Johns Hopkins University, 3Johns Hopkins
School of Public Health, 4Johns Hopkins University School of Medicine,
Baltimore, MD.
RATIONALE: To examine the relationship between sensitization and
exposure to mold and asthma morbidity in inner-city children.
METHODS: 144 children (5-17 years old, 57% male and 91% black) with
persistent asthma were studied for one year. At baseline, subjects under-
went skin testing to Alternaria, Aspergillus, and Cladosporium. A net
wheal > _3mm was considered positive. Home assessments were conducted
every three months. Mold exposure was defined as any signs of mold: mois-
ture or water damage, musty smells or visible mildew. Symptoms and
asthma-related health care use were assessed by questionnaire every three
months. Participants were grouped into four categories: not sensitized to
either of the two indoor molds and not exposed; sensitized/not exposed; ex-
posed/not sensitized; and sensitized and exposed. Generalized linear
mixed-effects models and Wilcoxon rank-sum tests were run to determine
associations between group and measures of asthma morbidity.
RESULTS: At baseline, 51% of subjects were SPT+ to one or more of the
3 tested molds (Alternaria 34%, Aspergillus 29%, and Cladosporium
7%).Twenty percent had moisture or water damage, musty smells or visible
mildew in bedrooms/family rooms. Sensitization to mold was not associ-
ated with any asthma-related symptoms or acute care visits. After control-
ling for gender, age, and total IgE, sensitization and exposure to mold was
associated with emergency department visits (OR, 2.06;CI 1.10-3.87),
days of slowed activity (OR, 1.8;CI 0.96-3.56) and exercise-related symp-
toms (OR, 2.0;CI 0.98-3.95), although the symptoms associations were not
statistically significant.
CONCLUSIONS: Sensitization to mold was not associated with asthma-
related symptoms, but sensitization and exposure to mold was associated
with symptoms and ED visits.