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Platelets expressing IgG receptor FcγRIIA/CD32A determine the severity of experimental anaphylaxis

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Science Immunology  13 Apr 2018:
Vol. 3, Issue 22, eaan5997
DOI: 10.1126/sciimmunol.aan5997
  • Fig. 1 Platelets are required for hFcγRIIA-induced anaphylaxis.

    (A and B) Change in body temperature (left) and platelet count (right) during HA-hIgG–dependent anaphylaxis in hFcγRIIAtg (circles, n = 4) and FcγRnull (squares, n = 4) mice. (C) HA-hIgG–dependent anaphylaxis–induced changes in body temperature in hFcγRIIAtg (circles) and FcγRnull (squares) mice (n = 4), pretreated with anti-GPIbα antibody (red symbols) or isotype control (black symbols, n = 4). (D) Platelet counts in hFcγRIIAtg (circles) and FcγRnull (squares) mice, pretreated (green symbols, n = 5) or not (open symbols, n = 4 or 5) with romiplostim. (E) Change in body temperature (left) and survival (right) during HA-hIgG–dependent anaphylaxis in hFcγRIIAtg (circles) and FcγRnull (squares) mice pretreated (green symbols) or not (open symbols) with romiplostim (n = 3). ↓: Death of all romiplostim-treated hFcγRIIAtg mice before the first temperature readout. (A to E) Temperature data are means ± SEM, and platelet counts from individual mice are indicated together with the means ± SEM. Data in (A) to (E) are representative of at least two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 2 hFcγRIIA-expressing platelets are sufficient to induce anaphylaxis in resistant mice.

    (A) Maximal aggregation (%) of washed platelets from hFcγRIIAtg mice compared with baseline and (B) expression of platelet activation markers after incubation with indicated agonist. Data are pooled from three independent experiments. NS, not stimulated. (C to F) hFcγRIIAtg (circles) and FcγRnull (squares) mice were challenged or not with HA-hIgG (n = 4). (C and D) ΔGeoMean CD62P expression (C) on single platelets and (D) within platelet-neutrophil aggregates, (E) percentage of neutrophils associated to CD41+ platelets, and (F) ΔGeoMean CD62L expression on single neutrophils. PSA, passive systemic anaphylaxis. (G and H) Indicated groups of mice were pretreated with anti-CD62P mAb or isotype control (black circles, n = 4) and challenged or not with HA-hIgG (all groups, n = 4). (G) Percentage of neutrophils associated to CD41+ platelets 30 min after challenge and (H) changes in body temperature over time. (I) Washed platelets from hFcγRIIAtg mice (red squares, left) or from FcγRnull mice (gray squares, right), or Tyrode’s albumin buffer (TyAlb) alone (open squares), were transferred intravenously into FcγRnull mice before challenge with HA-hIgG (all groups, n = 3). hFcγRIIAtg mice receiving intravenous injection of TyAlb, followed by challenge with HA-hIgG, served as a positive control (open circles, n = 5). wPLA, washed platelets. Changes in body temperature were recorded. (C to I) Data are representative of at least two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 3 Platelet-released serotonin contributes to hypothermia in mice undergoing HA-hIgG–dependent anaphylaxis.

    (A) Releasates (Rel.) from HA-hIgG–activated washed platelets from hFcγRIIAtg mice left untreated (black squares, n = 6) or treated with PCPA (red squares, n = 8) or HA-hIgG–containing TyAlb alone (open squares, n = 9) were transferred intravenously into FcγRnull mice, and changes in body temperature were recorded. (B) Changes in body temperature during HA-hIgG–dependent anaphylaxis in hFcγRIIAtg (circles) and FcγRnull (squares) mice, pretreated with PCPA (black symbols, n ≥ 8) or vehicle control (open symbols, n ≥ 8). (C) Platelet counts from the experiment represented in (B) 70 min after anaphylaxis induction. (D) Changes in body temperature in WT mice injected intravenously with 0 (n = 4), 20 (n = 3), or 100 μg (n = 3) of serotonin. Data are representative of three independent experiments. (A to C) Data are pooled from three (A) or two (B and C) independent experiments. *P < 0.05, **P < 0.01, and ****P < 0.0001.

  • Fig. 4 Platelets contribute to hIgG-dependent anaphylaxis in mouse models reproducing human FcγR complexity.

    (A to C) Change in body temperature (left) and platelet count (right) during HA-hIgG–dependent anaphylaxis in (A) hFcγRKI (triangles, n = 4) and FcγRnull (squares, n = 4) mice. (B) hFcγRKI mice pretreated with anti-hFcγRIIA mAb (red triangles, n = 4) or isotype control (black triangles, n = 4). (C) hFcγRKI pretreated with anti-GPIbα antibody (red triangles, n = 4) or isotype control (black triangles, n = 4) and FcγRnull mice pretreated with anti-GPIbα antibody (red squares, n = 4). (A to C) Temperature data are means ± SEM, and platelet counts from individual mice are indicated together with the means ± SEM. Data are representative of at least two independent experiments. *P < 0.05, ***P < 0.001, and ****P < 0.0001.

  • Fig. 5 Releasate from HA-hIgG–activated human platelets induces hypothermia in WT mice.

    (A) Direct activation of washed human platelets by HA-hIgG in vitro. Maximal aggregation (%) of stimulated washed human platelets compared with baseline and (B and C) percentage of platelets expressing the activation markers CD62P (B) and activated αIIbβ3 integrin (C) after in vitro incubation with indicated stimuli. Data are pooled from five independent experiments. (D) Transfer of releasate from activated washed human platelets induces hypothermia. Releasate from washed human platelets activated with HA-hIgG (black diamonds, n = 12) or HA-hIgG–containing TyAlb alone (open diamonds, n = 8) was transferred intravenously into WT mice, and changes in body temperature were immediately recorded. Data are pooled from three independent experiments. (E) Serotonin (n = 4) and histamine (n = 5) content in the releasate of HA-hIgG–stimulated washed human platelets. (A to E) Data are means ± SEM and are representative of at least three independent experiments. *P < 0.05, ** P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 6 Evidence for platelet activation during human drug-induced anaphylaxis.

    (A) Platelet counts in blood of individuals from the NASA study presenting no (n = 67), mild (n = 37), or severe (n = 30) anesthesia-induced anaphylaxis and the same severe patients during follow-up visit (n = 30). (B) Platelet and (C) leukocyte counts of four cardiac surgery cases (black symbols) and their matched control (open symbols) pairs (identical symbols within a pair) from the NASA cohort indicated as percentage of baseline (left) and absolute counts (right). A smoothed trend line has been added to indicate average behavior of each group (second-order polynomic, three neighbors: red, patients; green, controls). *P < 0.05, unpaired t test of cases versus controls, considering all values in the 3- to 4-hour interval (gray area). (D) CD62P-expressing platelets (left, percentage; right, geometric mean CD62P minus isotype staining) from the NASA study patients presenting no (n = 73), mild (n = 35), or severe (n = 29) anesthesia-induced anaphylaxis or the same mild (n = 35) or severe (n = 29) patients during follow-up visit. (A and D) Statistically significant differences between groups of anesthetized subjects ($P < 0.05, $$$P < 0.001, and $$$$P < 0.0001) and within a group of subjects comparing two different time points (##P < 0.01, ###P < 0.001, and ####P < 0.0001.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/22/eaan5997/DC1

    NASA study group

    Fig. S1. Clinical signs of HA-hIgG–dependent anaphylaxis and platelet depletion using anti-GPIbα antibodies.

    Fig. S2. Platelets associate with Ly6Chi monocytes during HA-hIgG–dependent anaphylaxis and require FcγRIIA expression for activation.

    Fig. S3. Serotonin but not histamine concentration is augmented during HA-hIgG–dependent anaphylaxis.

    Fig. S4. Generation of hFcγRKI mice.

    Fig. S5. Expression of hFcγRs in blood of humans or transgenic mice.

    Fig. S6. Formation of platelet/neutrophil aggregates is dispensable for HA-hIgG–dependent anaphylaxis in hFcγRKI mice.

    Fig. S7. Platelet gating in NASA study.

    Fig. S8. Platelet-bound hIgG and hFcγRIIA expression on platelets ex vivo after HA-hIgG injection into hFcγRIIAtg mice.

  • Supplementary Materials

    Supplementary Material for:

    Platelets expressing IgG receptor FcγRIIA/CD32A determine the severity of experimental anaphylaxis

    Héloïse Beutier, Béatrice Hechler, Ophélie Godon, Yu Wang, Caitlin M. Gillis, Luc de Chaisemartin, Aurélie Gouel-Ch?ron, Stéphanie Magnenat, Lynn E. Macdonald, Andrew J. Murphy, NASA study group, Sylvie Chollet-Martin, Dan Longrois, Christian Gachet, Pierre Bruhns,* Friederike Jönsson*

    *Corresponding author. Email: joensson{at}pasteur.fr (F.J.); bruhns{at}pasteur.fr (P.B.)

    Published 13 April 2018, Sci. Immunol. 3, eaan5997 (2018)
    DOI: 10.1126/sciimmunol.aan5997

    This PDF file includes:

    • NASA study group
    • Fig. S1. Clinical signs of HA-hIgG–dependent anaphylaxis and platelet depletion using anti-GPIbα antibodies.
    • Fig. S2. Platelets associate with Ly6Chi monocytes during HA-hIgG–dependent anaphylaxis and require FcγRIIA expression for activation.
    • Fig. S3. Serotonin but not histamine concentration is augmented during HAhIgG–dependent anaphylaxis.
    • Fig. S4. Generation of hFcγRKI mice.
    • Fig. S5. Expression of hFcγRs in blood of humans or transgenic mice.
    • Fig. S6. Formation of platelet/neutrophil aggregates is dispensable for HA-hIgG–dependent anaphylaxis in hFcγRKI mice.
    • Fig. S7. Platelet gating in NASA study.
    • Fig. S8. Platelet-bound hIgG and hFcγRIIA expression on platelets ex vivo after HA-hIgG injection into hFcγRIIAtg mice.

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