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Inhibition of FcεRI-dependent mediator release and calcium flux from human mast cells by sialic acid–binding immunoglobulin-like lectin 8 engagement 

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Presentation on theme: "Inhibition of FcεRI-dependent mediator release and calcium flux from human mast cells by sialic acid–binding immunoglobulin-like lectin 8 engagement "— Presentation transcript:

1 Inhibition of FcεRI-dependent mediator release and calcium flux from human mast cells by sialic acid–binding immunoglobulin-like lectin 8 engagement  Hidenori Yokoi, MD, Oksoon H. Choi, PhD, Walter Hubbard, PhD, Hyun-Sil Lee, PhD, Brendan J. Canning, PhD, Hyun H. Lee, MD, Seung-Duk Ryu, PhD, Stephan von Gunten, MD, PhD, Carol A. Bickel, MS, Sherry A. Hudson, MSB, Donald W. MacGlashan, MD, PhD, Bruce S. Bochner, MD  Journal of Allergy and Clinical Immunology  Volume 121, Issue 2, Pages e1 (February 2008) DOI: /j.jaci Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Siglec-8 ligation inhibits release of histamine (A) and PGD2 (B) triggered through FcεRIα. Mast cells were preincubated with no antibody (diamonds), control CD51 mAb (0.05 μg/mL; squares), or Siglec-8 mAb 2C4 (0.05 μg/mL; triangles) for 30 minutes at 37°C. Cells were then activated with FcεRIα mAb CRA-1 for 10 minutes at the concentrations indicated, and supernatants were harvested and assayed. Values are means ± SD of n = 10. ∗P < .005, area under the curve analysis. Journal of Allergy and Clinical Immunology  , e1DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Siglec-8 engagement inhibits IgE-mediated contraction of airway smooth muscle. Anti-IgE (1 μg/mL) evoked contractions of human intrapulmonary bronchial rings that had been pretreated with either isotype-matched IgG control mAb or Siglec-8 mAb (both at 3 μg/mL) or a combination of an H1 antihistamine (H1) and cysteinyl leukotriene receptor type 1 antagonist (CysLT1; see the Methods section). Each bar represents the mean ± SEM of 3 experiments and is expressed as a percentage of the maximum attainable contraction evoked by 300 mmol/L BaCl2. ∗P < .05, 1-tailed paired t test. Journal of Allergy and Clinical Immunology  , e1DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Siglec-8 ligation inhibits Ca++ flux triggered through FcεRI. A, Mast cells were preincubated with or without CD51 mAb (0.5 μg/mL) or Siglec-8 mAb 2C4 (0.5 μg/mL) for 30 minutes on ice. Ca++ responses were then measured after addition of the FcεRIα mAb CRA-1. Responses are expressed as an increase in the fluorescence ratio measured at 510 nm when excited, alternating between 340 nm and 380 nm. Traces are from a single experiment representative of n = 3. B, Same as in Fig 3, A, except that results are the average for 3. The fluorescence ratio was quantified by determining the net increase in the ratio at 2.5 minutes after CRA-1 stimulation. Values are means ± SD. ∗P < .05, unpaired 1-tailed t test. Journal of Allergy and Clinical Immunology  , e1DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Siglec-8 ligation inhibits degranulation (A) and calcium flux (B and C) triggered through FcεRI in RBL-2H3 cells transfected with full-length human Siglec-8 (RBL-8WT) but not in cells transfected with Siglec-8 containing a tyrosine to phenylalanine point mutation in the membrane-proximal ITIM domain (RBL-8Y1F). Fig 4, A, Cells sensitized with DNP-specific IgE (50 ng/mL) overnight were preincubated with control OX18 mAb (red circle for RBL-8WT; green circle for RBL-8Y1F) or Siglec-8 mAb 2C4 (blue square for RBL-8WT; pink square for RBL-8Y1F) for 30 minutes on ice. Cells were then activated with goat anti-mouse IgG heavy and light chain (50 μg/mL) for 15 minutes (to simultaneously cross-link bound mouse mAb and DNP-IgE), and supernatants were harvested and assayed. Values are means ± SEM for n = 17. ∗P < .05, 1-tailed t test. Fig 4, B, Cells were sensitized with DNP-IgE (50 ng/mL) for 2 hours at 37°C, loaded with Fura-2AM, and preincubated with no antibody, Siglec-8 mAb 2C4, or OX18 mAb (each at 1 μg/mL) on ice for 30 minutes. Cells were then washed and stimulated with 50 μg/mL goat anti-mouse IgG heavy and light chains, and changes in cytosolic Ca++ levels were measured. Arrowheads indicate when anti-mouse IgG was added. Data shown are representative of n = 2. Fig 4, C, Changes in cytosolic Ca++ levels were quantified by calculating the net increase in cytosolic Ca++ levels 6 minutes after stimulation by using the data shown in Fig 4, B. Values are means ± SD (n = 2 except for RBL-8Y1F; no antibody [Ab] where only 1 experiment was performed). Journal of Allergy and Clinical Immunology  , e1DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Flow cytometry after sorting and expansion showing similar levels of Siglec-8 surface expression on RBL cells transfected with full-length wild-type Siglec-8 (RBL-8WT) or Siglec-8 in which the ITIM domain contains a substitution of phenylalanine for tyrosine (RBL-8Y1F). Region M1 incorporates 97% of IgG control signals and was identical to labeling of nontransfected RBL cells with Siglec-8 mAb (data not shown). Journal of Allergy and Clinical Immunology  , e1DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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