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Jerome A. Sigua, MD, Becky Buelow, MD, Dorothy S

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1 CD49d-expressing neutrophils differentiate atopic from nonatopic individuals 
Jerome A. Sigua, MD, Becky Buelow, MD, Dorothy S. Cheung, MD, Erika Buell, BS, Desire Hunter, BS, Meribeth Klancnik, MLT, Mitchell H. Grayson, MD  Journal of Allergy and Clinical Immunology  Volume 133, Issue 3, Pages e5 (March 2014) DOI: /j.jaci Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 CD49d+ PMN are increased in blood and nasal lavage of subjects with atopy, and these cells can be increased in the nasal lavage with specific allergen challenge. Frequency of CD16+ PMN expressing CD49d in peripheral blood (A) and baseline nasal lavage (B). Subjects with atopy are indicated in blue (n = 18), and subjects without atopy (n = 10) are indicated in red. The large line represents the median, with the whiskers being the interquartile range. The Wilcoxon signed rank test was used to compare subjects with and without atopy. The frequency (C) and absolute number (D) of CD49d+ PMN were calculated in the nasal lavage before (“Pre”) and 6 hours after (“Post”) challenge with a relevant allergen (see text for details). Each subject is represented by a dot, with lines connecting their respective responses. The thick line marks the median values (for frequency, 1.94% pre vs 5.28% post; for absolute number, 45 pre vs 273 post). Frequency (E) and absolute number (F) of CD49d+ PMN following irrelevant allergen challenge (medians, for frequency, 1.88% pre vs 0.26% post; for absolute number, 200 pre vs 0 post). Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Relationship between CD49d+ PMN in the lavage and blood. In subjects with atopy (A), the frequency of blood CD49d+ PMN is inversely and significantly correlated with the frequency in the baseline nasal lavage. A similar phenomenon was not seen in subjects without atopy (B). C, Relationship between the change (Post-Pre) in CD49d+ PMN frequency in the nasal lavage versus the blood. There was a significant negative correlation. D, Similarly, the change in nasal lavage CD49d+ PMN frequency after relevant allergen challenge also was significantly related (but in a positive direction) to the baseline nasal lavage frequency of CD49d+ PMN. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig E1 Experimental design. Schematic of experimental plan (see text for full details). Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig E2 Identification of PMN from nasal lavage. A, Cytospin of nasal lavage reveals a significant quantity of multilobed neutrophils, as well as occasional eosinophils, macrophages, and epithelial cells. Representative Wright-Giemsa stained section shown (bar = 50 μm). B, After gating on appropriate granulocyte forward and side scatter characteristics, 2 distinct populations of cells could be outlined on the basis of CD16 expression, with PMN identified as granulocytes with high CD16 expression. C, By gating on CD16+ PMN, the frequency of these cells expressing CD49d could be determined by flow cytometry (black line = CD49d expression; gray line = isotype control). Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig E3 Fold mean fluorescence intensity (MFI) of CD49d expression on CD49d+ PMN in blood and nasal lavage. No significant differences in CD49d fold MFI expression on CD49d+ PMN was found between individuals with and without atopy in the peripheral blood (9.0 [7-13.4] vs 6.7 [ ], median [interquartile range], respectively) (A) or baseline nasal lavage (10.71 [ ] vs [ ], respectively) (B). Similarly, no significant changes in fold MFI of CD49d in these cells from “Pre” to “Post” relevant (6.2 [ ] to 7.8 [ ], respectively) (C) or irrelevant (9.5 [ ] to 11.7 [ ], respectively) allergen challenges (D). Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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