1. Mechanism of Siglec-8–mediated cell death in IL-5–activated eosinophils: Role for reactive oxygen species–enhanced MEK/ERK activation 
Gen Kano, MD, PhD, Maha Almanan, MD, Bruce S. Bochner, MD, Nives Zimmermann, MD 
Journal of Allergy and Clinical Immunology 
Volume 132, Issue 2, Pages (August 2013)
DOI: /j.jaci
Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2. Fig 1
Siglec-8 cross-linking induces a different mode of cell death in activated versus resting eosinophils. A, Representative morphology and the percentage of dead cells that have apoptotic or necrotic morphology in eosinophils cultured for 24 hours with indicated stimuli (n = 6 experiments and donors). B, Mean percentage of dead cells with necrotic morphology in multiple experiments (n = 6). #P = .055. C, Representative Annexin V/7AAD staining and the percentage of 7AAD+ and 7AAD− cells among all Annexin V+ cells. LR, Lower right quadrant; UR, upper right quadrant. D, Mean ratio of 7AAD+ cells among all Annexin V+ cells from multiple experiments (n = 25 experiments with 11 donors). *P < .01. E, Activity of released EPX after incubation with indicated stimuli for 4 hours (left) or 2-16 hours (right). **P < .02. PMA, Phorbol 12-myristate 13-acetate.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3. Fig 2
ROS generation is sufficient for eosinophil cell death and IL-5 enhancement of cell death. A, ROS accumulation induced by exogenous H2O2 with or without NaN3, cross-linked anti–Siglec-8, or isotype-matched control antibody was measured after 2 hours of incubation and 30 minutes of loading with dihydrorhodamine 123 (DHR). Results are shown as mean fluorescence intensity (MFI) values of DHR (left). Aliquots of the same cells were also incubated for 24 hours without DHR to determine Annexin V+ staining (right). Data are representative of 3 experiments. B, Representative experiment of eosinophils cultured with (solid bars) or without (open bars) IL-5 in conditions with H2O2 with or without NaN3 or anti–Siglec-8. The IL-5 effect was determined by dividing the percentage of Annexin V+ cells in IL-5[+] with IL-5[−] for each condition. Thus an IL-5 effect of less than 1.0 represents survival enhancement, whereas an IL-5 effect of greater than 1.0 implies cell death enhancement. C, Averaged results from 3 experiments and donors. *P < .05 and **P < .01.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4. Fig 3
Upregulation of the MEK/ERK pathway after anti–Siglec-8/IL-5 costimulation. A, A phospho-MAPK multiplex bead assay was performed on lysates of eosinophils (n = 4 donors) that were incubated for 10 minutes with indicated stimuli. ATF2, Activating transcription factor 2; HSP27, heat shock protein 27; MSK1, mitogen- and stress-activated protein kinase. **P < .01 compared with IL-5. B, Eosinophils incubated in the indicated conditions were analyzed by means of Western blotting with specific antibodies for phospho-ERK or total ERK (upper panel) and phospho-STAT5 and total STAT5. C, Time course of ERK phosphorylation was determined by means of flow cytometry. D, Western blotting of the nuclear fraction from cell lysates of cells treated for 1, 2, or 3 hours. E, Western blotting of nuclear fraction from lysates of cells stimulated for 2 hours with indicated stimuli. The ratio of densitometric value for the band of phosphokinase to that of total kinase in each lane is indicated (B, D, E). Data are representative of 3 experiments (B-E).
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5. Fig 4
Upregulation of the MEK/ERK pathway is required for enhanced cell death induced by anti–Siglec-8/IL-5 costimulation. Eosinophils were preincubated with U0126 (left panel) or PD (right panel) at the indicated doses and then cultured in media containing IL-5 with or without anti–Siglec-8. Annexin V+ cell numbers, 7AAD+ cell numbers, or both were determined after 24 hours. Shown are representative data from 3 experiments with each inhibitor. *P < .01 and §P < .01 comparing the percentage of Annexin V+ 7AAD+ cells and the percentage of Annexin V+ 7AAD− cells, respectively.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6. Fig 5
MEK/ERK upregulation is downstream of the ROS production induced by anti–Siglec-8/IL-5 costimulation. Eosinophils were preincubated with or without inhibitors and then cultured in media containing IL-5 and anti–Siglec-8. The lower panel shows Annexin V staining of cells from the same experiment at the 24-hour time point. A, Cells were stained with dihydrorhodamine 123 (DHR), and ROS levels were analyzed by means of flow cytometry. Data are representative of 3 experiments. MFI, Mean fluorescence intensity. B, Western blotting of lysates from cells harvested after 10 minutes of stimulation is shown. Data are representative of 3 experiments. The ratio of the densitometric value for band of phospho-ERK to that of total ERK in each lane is displayed. *P < .01 comparing the percentage of Annexin V+ 7AAD+ cells and §P < .01 and ¶P < .05 comparing the percentage of Annexin V+ 7AAD− cells.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions