Distinct immune effector pathways contribute to the full expression of peanut-induced anaphylactic reactions in mice Katherine Arias, BHSc, Derek K.

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Distinct immune effector pathways contribute to the full expression of peanut-induced anaphylactic reactions in mice Katherine Arias, BHSc, Derek K. Chu, BHSc, Kristin Flader, BSc, Fernando Botelho, PhD, Tina Walker, BSc, Natalia Arias, Alison A. Humbles, PhD, Anthony J. Coyle, PhD, Hans C. Oettgen, MD, PhD, Hyun-Dong Chang, PhD, Nico Van Rooijen, PhD, Susan Waserman, MD, MSc, Manel Jordana, MD, PhD Journal of Allergy and Clinical Immunology Volume 127, Issue 6, Pages 1552-1561.e1 (June 2011) DOI: 10.1016/j.jaci.2011.03.044 Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Partial abrogation of PIA in MC-deficient KitW-sh/W-sh (Wsh) mice. A, Levels of peanut-specific IgE and IgG1 in serum. B, Peak anaphylactic symptom score of each mouse. C, Rectal temperature of naive (intraperitoneal: WT, ---●---; Wsh, ---o---; intravenous: WT, ---■---; Wsh, ---□---) and sensitized (intraperitoneal: WT, ―●―; Wsh, ―o―; intravenous: WT, ―■―; Wsh, ―□―) mice. ∗P < .05, Wsh intraperitoneal versus corresponding WT control mice. †P < .05, Wsh intravenous versus corresponding WT control mice. D and G, Percentage of hematocrit (Fig 1, D) and histamine levels (Fig 1, G). i.p., Intraperitoneal; i.v., intravenous. E and F, Representative pictures showing Evans blue extravasation in footpads (Fig 1, E) and intestinal bleeding (Fig 1, F). Data are presented as means ± SDs (4-11 mice per group from at least 3 independent experiments). Journal of Allergy and Clinical Immunology 2011 127, 1552-1561.e1DOI: (10.1016/j.jaci.2011.03.044) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Effect of basophil depletion on peanut-induced anaphylactic reactions. A, Percentage of basophils and MCs from sensitized mice after antibody administration. B, Peak anaphylactic symptom score of each mouse. C, Rectal temperature of naive (WT, ―▴―; KitW-sh/W-sh [Wsh], ―▵―) and sensitized (WT/rat IgG, ―■―; WT/Ba103, ―□―; Wsh/rat IgG, ―●―; Wsh/Ba103, ―o―) mice. ∗P < .05, Wsh/rat IgG versus corresponding WT control mice. †P < .05, Wsh/Ba103 versus corresponding WT control mice. D, Hematocrit percentage. Fig 2, C and D, Data are presented as means ± SDs (4-8 mice per group from at least 2 independent experiments). Journal of Allergy and Clinical Immunology 2011 127, 1552-1561.e1DOI: (10.1016/j.jaci.2011.03.044) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Dual contribution of MCs and phagocytes to PIA. A, Percentage of macrophages from sensitized mice after liposome delivery. B, Peak anaphylactic symptom score of each mouse. C, Rectal temperature of naive (WT, ―▴―; KitW-sh/W-sh [Wsh], ―▵―) and sensitized (WT/PBS liposomes, ―■―; WT/clodronate liposomes, ―□―; Wsh/PBS liposomes, ―●―; Wsh/clodronate liposomes, ―o―) mice. ∗P < .05, Wsh/PBS liposomes versus corresponding WT control mice. †P < .05, Wsh/clodronate liposomes versus corresponding WT control mice. D, Hematocrit percentage. E and F, Representative pictures showing Evans blue extravasation in footpads (Fig 3, E) and intestinal bleeding (Fig 3, F). Fig 3, C and D, Data are presented as means ± SDs (4-8 mice per group from at least 4 independent experiments). Clod, Clodronate. Journal of Allergy and Clinical Immunology 2011 127, 1552-1561.e1DOI: (10.1016/j.jaci.2011.03.044) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Double, but not single, absence of IgE and IgG1 signaling abolishes PIA. A, Peak anaphylactic symptom score of each mouse. B, Rectal temperature of naive (WT, ―o―; immunoglobulin-deficient, ―▵―) and sensitized (WT, ―●―; immunoglobulin-deficient, ―▴―) mice. ∗P < .05 relative to corresponding WT control mice. C and D, Hematocrit percentage (Fig 4, C) and histamine levels (Fig 4, D). Fig 4, B through D, Data are presented as means ± SDs (4-8 mice per group from at least 3 independent experiments). Journal of Allergy and Clinical Immunology 2011 127, 1552-1561.e1DOI: (10.1016/j.jaci.2011.03.044) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Blockade of IgG1 signaling in IgE-deficient mice abrogates PIA. A, Peak anaphylactic symptom score of each mouse. B, Rectal temperature of naive (---▴---) and sensitized (rat IgG, ―▴―; anti-FcγRII/III, ―▵―) mice. ∗P < .05 relative to corresponding WT control animals. C and D, Hematocrit percentage (Fig 5, C) and histamine levels (Fig 5, C). Fig 5, B through D, Data are presented as means ± SDs (4-8 mice per group from at least 3 independent experiments). Journal of Allergy and Clinical Immunology 2011 127, 1552-1561.e1DOI: (10.1016/j.jaci.2011.03.044) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 MC-dependent anaphylaxis to peanut occurs through both IgE and IgG1. A, Peak anaphylactic symptom score of each mouse. B, Rectal temperature of naive (WT, ―▴―; immunoglobulin deficient, ―▵―) and sensitized (WT/PBS liposomes, ―■―; WT/clodronate liposomes, ―□―; immunoglobulin-deficient/PBS liposomes, ―●―; immunoglobulin-deficient/clodronate liposomes, ―○―) mice. ∗P < .05, immunoglobulin-deficient/PBS liposomes versus corresponding WT control animals. †P < .05, immunoglobulin-deficient/clodronate liposomes versus corresponding WT control animals. C, Hematocrit percentage. Fig 6, B and C, Data are presented as means ± SDs (4-8 mice per group from at least 3 independent experiments). Clod, Clodronate. Journal of Allergy and Clinical Immunology 2011 127, 1552-1561.e1DOI: (10.1016/j.jaci.2011.03.044) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 Synergism between MCs and other FcγRIII-expressing cells in the elicitation of PIA. A, Peak anaphylactic symptom score of each mouse. B, Rectal temperature of naive (WT, ―▴―; KitW-sh/W-sh [Wsh], ―▵―) and sensitized (WT/rat IgG, ―■―; WT/ anti-FcγRII/III, ―□―; Wsh/rat IgG, ―●―; Wsh/anti-FcγRII/III, ―○―) mice. ∗P < .05, Wsh/anti-FcγRII/III versus Wsh/rat IgG. C, Hematocrit percentage. Fig 7, B and C, Data are presented as means ± SEMs (3-9 mice per group from at least 2 independent experiments). Journal of Allergy and Clinical Immunology 2011 127, 1552-1561.e1DOI: (10.1016/j.jaci.2011.03.044) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Partial abrogation of PIA in MC-deficient KitW/KitW-v (W-v) mice after intravenous but not intraperitoneal challenge. A, Peak anaphylactic symptom scores of each WT and W-v mouse. B, Rectal temperature of naive (intraperitoneal: WT, ---●---; W-v, ---○---; intravenous: WT, ---■---; W-v, ---□---) and sensitized (intraperitoneal: WT, ―●―; W-v, ―○―; intravenous: WT, ―■―; W-v, ―□―) mice. ∗P < .05 relative to corresponding naive mice. †P < .05 relative to corresponding WT control animals. C and D, Hematocrit percentage (Fig E1, C) and histamine levels (Fig E1, D). Fig E1, B through D, Data are presented as means ± SDs (4-11 mice per group from at least 2 independent experiments). Journal of Allergy and Clinical Immunology 2011 127, 1552-1561.e1DOI: (10.1016/j.jaci.2011.03.044) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions