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David M. Pyle, BS, Victoria S. Yang, MD, Rebecca S

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1 IgE cross-linking critically impairs human monocyte function by blocking phagocytosis 
David M. Pyle, BS, Victoria S. Yang, MD, Rebecca S. Gruchalla, MD, PhD, J. David Farrar, PhD, Michelle A. Gill, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 131, Issue 2, Pages e5 (February 2013) DOI: /j.jaci Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 IgE cross-linking upregulates surface CD14 and downregulates CD64. Monocyte CD14 (A) and CD64 (B) expression after 48- or 96-hour culture in indicated conditions (N ≥ 9). C, CD14 expression after IgE cross-linking on monocytes from individuals with low or high serum IgE (left); Pearson correlation between CD14 expression and serum IgE (right; N ≥ 11). *P < .05, **P < .01 , and ***P < .001 for αIgE vs media and IgG within time points (Fig 1, A and B) or high IgE vs low IgE (Fig 1, C). MESF, Mean equivalent standard fluorescence. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 IgE cross-linking induces monocyte secretion of TNF-α, IL-6, and autoregulatory IL-10. A, TNF-α (left), IL-6 (middle), and IL-10 (right) concentrations in indicated monocyte culture conditions after 4, 24, or 48 hours (N ≥ 9). B, Similar data for monocytes cultured with αIgE in the presence of IL-10 and IL-10R neutralizing antibodies or isotype control (N = 3). C, IL-10 concentration after 48 hours of IgE cross-linking on monocytes from individuals with low (<100 U/mL) or high (>100 U/mL) serum IgE (left) and corresponding Pearson correlation (right; N = 12). D, Concentration of TNF-α after 4 hours (left); IL-6 after 48 hours (right) of IgE cross-linking on monocytes from individuals with low vs high serum IgE (N ≥ 10). *P < .05, **P < .01, ***P < .001 for αIgE vs media and IgG (Fig 2, A), blocking antibodies vs isotype (Fig 2, B), or high IgE vs low IgE (Fig 2, C and D). Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 IgE cross-linking impairs monocyte phagocytic function. Photomicrographs (A) (gray = DIC; blue = DAPI; green = BODIPY-FL) and particle count per cell (B) of BODIPY-labeled bacteria phagocytosed by monocytes after 96-hour culture in indicated conditions (N = 5). C, Fluorescence of internalized bacteria determined by flow cytometry for indicated conditions (N ≥ 9). D, Effect of IL-10 neutralization on phagocytosis in indicated conditions (N = 3). E, Internalization (CFU/cell; left) of live bacteria after 48-hour culture; percentage of internalized bacteria killed (right) after additional 16-hour incubation (N = 4). *P < .05, **P < .01, ***P < .001, NS P > .05 for αIgE vs media and IgG (Fig 3, C) or indicated comparisons (Fig 3, B, D, and E). MESF, Mean equivalent standard fluorescence. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 IgE cross-linking inhibits phagocytosis of apoptotic cells. A, CFSE fluorescence in monocytes (gated on CD14+) after phagocytosing CFSE-labeled apoptotic cells (Fig 4, A). B, Gating strategy for CFSElow and CFSEhigh monocytes. Photomicrographs (gray = DIC; blue = DAPI; green = CFSE) of sorted CFSElow monocytes (C) and CFSEhigh monocytes (D). CFSE fluorescence of CFSElow monocytes (E) and percentage of CFSEhigh of total monocytes (F) for indicated culture conditions (N = 4). *P < .05, **P < .01, ***P < .001, NS P > .05 for indicated comparisons (Fig 4, E and F). MESF, Mean equivalent standard fluorescence. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Inhibition of phagocytosis by IgE cross-linking is independent of serum IgE concentration. Fluorescence of internalized bacteria in monocytes from individuals with low (<100 U/mL) or high (>100 U/mL) serum IgE, cultured for 48 (A, left) or 96 (Fig 5, A, right) hours in indicated conditions; corresponding Pearson correlation (B) for αIgE condition (N = 12). NS P > .05 for indicated comparisons. MESF, Mean equivalent standard fluorescence. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 Proposed model of IgE cross-linking on human monocytes. IgE cross-linking drives (green arrow) increased CD14 expression and secretion of both proinflammatory cytokines (TNF-α and IL-6) and autoregulatory IL-10. These effects are enhanced in individuals with elevated serum IgE (blue lines). In contrast, phagocytosis and CD64 expression are suppressed by IgE cross-linking (red line). The IgE-mediated impairment of phagocytosis is independent of regulation by IL-10, suggesting that divergent pathways are activated by IgE cross-linking on monocytes. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E1 The effects of αIgE are not Fc-mediated. Expression of CD14 (A) and secretion of IL-6 (B) are depicted for monocytes cultured in the following: media alone, αIgE, control IgG, αIgE F(ab)’2 fragment, or IgG F(ab)’2 fragment. Data are expressed as triplicate mean ± SEM for 1 of 4 independent experiments. ***P < .001 for indicated comparisons. MESF, Mean equivalent standard fluorescence. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E2 TNF-α and IL-6 do not regulate secretion of IL-10. Concentration of IL-10 secreted by monocytes cultured for 4 or 48 hours with IgE cross-linking antibody in the presence of neutralizing antibodies for TNF-α and TNFRI (A), IL-6 and IL-6R (B), or appropriate isotype controls (N = 3). Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E3 Effects of IgE cross-linking are concentration dependent. A, Expression of CD14 (left) and CD64 (right) is depicted for monocytes cultured for 96 hours in the following: media alone, αIgE (1 or 10 μg/mL), or control IgG (1 or 10 μg/mL). B, Concentration of TNF-α (left), IL-6 (middle), and IL-10 (right) secreted by monocytes cultured as in Fig E3, A. **P < .01, ***P < .001, NS P > .05 for indicated comparisons (Fig E3, A) or for αIgE (1 or 10 μg/mL) vs media and IgG (1 or 10 μg/mL, respectively) (Fig E3, B). Data are expressed as triplicate means ± SEM for 1 of 5 independent experiments. MESF, Mean equivalent standard fluorescence. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Fig E4 IgE cross-linking impairs phagocytosis independently of TNF-α and IL-6. Fluorescence of internalized bacteria in monocytes cultured for 48 hours in media alone, IgE cross-linking antibody, or control IgG in the presence of neutralizing antibody for TNF-α and TNFRI (A), IL-6 and IL-6R (B), or appropriate isotype controls (N = 3). NS P > .05 for indicated comparisons. MESF, Mean equivalent standard fluorescence. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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