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An IgG-induced neutrophil activation pathway contributes to human drug-induced anaphylaxis

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Science Translational Medicine  10 Jul 2019:
Vol. 11, Issue 500, eaat1479
DOI: 10.1126/scitranslmed.aat1479
  • Fig. 1 NMBA-specific antibodies concentrations are elevated in severe AHR.

    (A to D) Schematic representation of (A) quaternary ammonium morphine (QAM) antigen, (B) suxamethonium, (C) atracurium, and (D) rocuronium. (E and F) Measurements of (E) IgE and (F) IgG against QAM in patients presenting with mild AHR (n = 43), severe AHR (n = 43), and matched controls (n = 86). Wilcoxon signed-rank test, ****P < 0.0001, **P < 0.01, *P < 0.05. Mann-Whitney U test, ####P < 0.0001, ###P < 0.001. (D and E) Results are represented as boxplots; whiskers indicate the 10th and 90th percentiles. (G) Plotting of individual data of anti-QAM IgE and IgG concentrations for controls (orange triangles) and patients (blue diamonds). Inset: Correlation plot including only patients and controls with anti-QAM IgE of >0.3 kU/liter. Spearman’s correlation, ***P < 0.001.

  • Fig. 2 Modulation of IgE and IgG receptor expression on myeloid cells during AHR.

    (A and B) FcεRI, (C and D) CD64 (FcγRI), (E to H) CD32A (FcγRIIA), (I and J) CD32B (FcγRIIB), and (K and L) CD16 (FcγRIII) expression on circulating basophils (A, G, and I), eosinophils (B and F), neutrophils (C, E, and K), and monocytes (D, H, J, and L) in the blood from patients 30 min after onset of AHR during the follow-up visit (V2) or from matched controls. Values represent the delta geometric mean (ΔGeoMean) between specific staining and corresponding isotype or fluorescence-minus-one control. Wilcoxon signed-rank test, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Mann-Whitney U test, ####P < 0.0001, ###P < 0.001, #P < 0.05. All boxplot whiskers represent 5th and 95th percentiles.

  • Fig. 3 Circulating neutrophils are activated during AHR.

    (A to C) Surface expression on circulating neutrophils of activation markers (A) CD11b, (B) CD18, and (C) CD66b expressed as geometric mean of fluorescence intensity after subtraction of isotype control. (D) Neutrophil elastase in the plasma of patients 30 min after AHR occurrence in patients with mild (n = 37) or severe (n = 32) AHR, at V2 (n = 69) or in healthy controls (n = 30). (E) Circulating NETs measured as DNA-MPO complexes in the serum of patients 30 min after AHR occurrence in patients with mild (n = 43) or severe (n = 43) AHR, at V2 (n = 86) or in matched controls (n = 86). OD, optical density. (F) Activity of PAF-AH in the plasma of patients 30 min after AHR occurrence in patients with mild (n = 42) or severe (n = 42) AHR or of healthy controls (n = 33). Wilcoxon signed-rank test, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Mann-Whitney U test, ####P < 0.0001, ###P < 0.001, ##P < 0.01, #P < 0.05. All boxplot whiskers represent 10th and 90th percentiles.

  • Fig. 4 Evidence of an IgG-neutrophil-PAF pathway in the presence or absence of IgE-mediated anaphylaxis markers.

    Evidence for IgE-dependent anaphylaxis was classified as absent (n = 27), equivocal (n = 19), or strong (n = 40) for each patient of the NASA cohort. Data from Figs. 1 to 3 were reanalyzed on the basis of these criteria, and (A) FcεRI expression on basophils, (B) anti-NMBA IgG, (C) CD32B expression on basophils, (D) CD32A expression on monocytes, (E) CD32A, (F) CD16 expression on neutrophils, circulating (G) neutrophil elastase, (H) NETs (DNA-MPO complexes), (I) CD11b, (J) CD18, (K) CD66b expression on neutrophils, and (L) plasma PAF-AH activity were compared between controls and patients with absent, equivocal, or strong evidence for IgE-dependent anaphylaxis. Wilcoxon signed-rank test, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Mann-Whitney U test, ####P < 0.0001, ###P < 0.001, ##P < 0.01, #P < 0.05. All boxplot whiskers represent 10th and 90th percentiles.

  • Fig. 5 NMBA–anti-NMBA IgG complexes activate neutrophils ex vivo.

    (A) Anti-rocuronium ELISA; four patients with high rocuronium-specific IgG antibody indicated with colored lines. (B) Specificity of purified anti-rocuronium IgG from patient #7016 measured by ELISA against indicated protein antigens and their morphine (Morph), suxamethonium (Suxa), and rocuronium (Roc) derivatives or against common allergens: amoxicillin, peanut extract, Bet v 1 (the major birch pollen antigen, Betula verrucosa) and ovalbumin (OVA). One of two independent experiments is shown. (C) Flow cytometry analysis of the binding of (anti-rocuronium IgG + rocuronium-HSA) ICs to CHO cell transfectants expressing human CD16A/FcγRIIIA variant V176 (blue line), compared to rocuronium-HSA alone (black line), and unstained cells (gray-shaded histogram), one of three independent experiments. (D and E) Fluorescent-based measurement of (D) ROS or (E) cell-free DNA (NETs) released by purified neutrophils from healthy donors (n = 4), either left untreated (media) or incubated with control ICs (OVA IC), rocuronium-HSA, or (anti-rocuronium IgG + rocuronium-HSA) complexes (Roc IC). Error bars represent SD. Mann-Whitney U test, *P < 0.01. AU, arbitrary units.

  • Table 1 Characteristics of the patients.

    Values are expressed as percentages or as median [25th to 75th percentile]. ACEI, angiotensin-converting enzyme inhibitor; ARA, angiotensin II receptor antagonist; BB, beta blocker; CI, calcium channel inhibitor.

    VariableCases (n = 86)Controls (n = 86)P
    Female gender62%62%1*
    Age (years)57 [40;66]58 [42;66]0.66†
    Previous general
    anesthesia
    84%87%0.66*
    Medication (ACEI/
    ARA/BB/CI)
    38%29%<0.0001*
    History of allergy
      Drugs13%13%1*
      Latex2%8%0.17*
      Food7%0%0.03*
      Hymenoptera venom3%0%0.09*
      Pollen/moth/
    animals/mold
    15%13%0.83*
    Asthma12%5%0.16*
    Atopy21%19%0.85*
    Type of surgery
      Cardiothoracic,
    vascular
    20%20%1*
      Maxillofacial8%8%1*
      Orthopedic
    neurosurgery
    20%20%1*
      Visceral, urologic,
    gynecologic
    52%52%1*
    Scheduled surgery92%98%0.17*
    Surgery with a context
    of infection
    2.3%2.3%1*
    NMBA used during surgery
      Suxamethonium56%56%1*
      Atracurium51%51%1*
      Rocuronium3%3%1*

    *Fisher’s exact test.

    †Student’s t test.

    • Table 2 Clinical signs and therapeutic interventions during AHRs, according to severity grade.

      Values are expressed as percentages or as median [25th to 75th percentile] when appropriate. The numbers of observations and missing values varied according to the numeric value analyzed: maximum heart rate (HR) (43/17), minimum HR (14/6), minimum PAS (70/21), minimum SpO2 (24/9), and minimum end-tidal CO2 (etCO2) (33/7). ICU, intensive care unit; IV, intravenous; NA, not available; SAP, systolic arterial pressure; SpO2, photoplethysmographic oxygen saturation.

      Clinical/hemodynamic signsSevere (n = 43)Mild (n = 43)P
      Erythema (%)65790.2*
      Tachycardia (%)58440.3*
      Maximum HR (min−1)130[125;133]127[111;136]0.3
      Bradycardia (%)2670.04*
      Minimum HR (min−1)42[40;49]52[51;54]0.1
      Arrhythmia (%)2320.007*
      Arterial hypotension (%)10063<0.0001*
      Minimum SAP value (mmHg)50[43;60]65[58;73]0.01
      Bronchospasm (%)37280.5*
      Hypoxemia (%)497<0.0001*
      Minimum SpO2 (%)82[82;88]91[90;91]0.1
      Hypocapnia (%)6314<0.0001*
      Minimum etCO2 (mmHg)18[13;21]26[24;29]0.002
      Cardiac arrest (%)2300.001*
      Delay of resuscitation
      Delay between anesthesia induction and first signs of the reaction (min)5[5;16]5.5[5;10]0.6
      Delay between first signs of the reaction and treatment initiation (min)1[0;2]0[0;0.75]0.008
      Therapeutic interventions
      Epinephrine IV alone (%)350<0.0001*
      Cumulative doses of epinephrine (mg)2.5[1;6.9]NANA
      Norepinephrine IV alone (%)500.5*
      Cumulative doses of norepinephrine IV (mg)1.75[1.62;1.87]NANA
      Epinephrine IV and norepinephrine IV (%)1200.06*
      Cumulative doses of epinephrine IV (mg)5[2;5.4]NANA
      Ephedrine and/or phenylephrine IV alone (%)044<0.0001*
      Ephedrine and/or phenylephrine IV, then epinephrine, and/or norepinephrine IV (%)492<0.0001*
      Cumulative doses of epinephrine IV (mg)0.6[0.3;4.1]0.1NA
      Atropine IV (%)1250.4*
      Cumulative doses of atropine IV (mg)1[1;1]1[1;1]NA
      b2-Adrenergic agonist IV/inhaled (%)14121*
      Glucocorticoids (%)23231*
      Histamine receptor antagonists (%)021*
      Fluid resuscitation with crystalloids (%)70420.02*
      Cumulative volume of crystalloids (liters)1[1;2]0.87[0.5;1.37]0.03
      Fluid resuscitation with colloids (%)8390.008*
      Cumulative volume of colloids (liters)0.5[0.5;1]0.65[0.5;0.85]0.8
      Evolution
      Surgery canceled (%)8119<0.0001*
      Admission to ICU (%)7626<0.0001*

      *Fisher’s exact test.

      †Mann-Whitney U test.

      Supplementary Materials

      • stm.sciencemag.org/cgi/content/full/11/500/eaat1479/DC1

        Materials and Methods

        Fig. S1. Anti-QAM, anti-suxamethonium, anti-atracurium, and anti-rocuronium IgE and IgG ImmunoCAP.

        Fig. S2. NMBA-dependent analyses of FcR expression.

        Fig. S3. Complement pathway CH50, C3, and C4 during AHR versus V2.

        Fig. S4. NMBA-dependent analyses of neutrophil CD11b and CD18 expression and circulating NETs.

        Fig. S5. Effect of resuscitation fluids and drugs used during AHR on neutrophil activation.

        Fig. S6. IgG markers in patients with strong evidence of IgE pathway.

        Fig. S7. MST analyses of rocuronium and suxamethonium protein partners.

        Fig. S8. Characterization of patient-derived anti-rocuronium IgG.

        Fig. S9. Human neutrophil activation by soluble NMBA–anti-NMBA IgG ICs.

        Table S1. Individual values of QAM IgE (kilounits per liter) and QAM IgG (micrograms per milliliter) for case-control pairs.

        Table S2. Tryptase and histamine measurements.

        Table S3. Flow cytometry antibodies.

        Table S4. Nuclear magnetic resonance data of carboxylated rocuronium in D2O.

        Data file S1. Primary data.

        Reference (80)

      • The PDF file includes:

        • Materials and Methods
        • Fig. S1. Anti-QAM, anti-suxamethonium, anti-atracurium, and anti-rocuronium IgE and IgG ImmunoCAP.
        • Fig. S2. NMBA-dependent analyses of FcR expression.
        • Fig. S3. Complement pathway CH50, C3, and C4 during AHR versus V2.
        • Fig. S4. NMBA-dependent analyses of neutrophil CD11b and CD18 expression and circulating NETs.
        • Fig. S5. Effect of resuscitation fluids and drugs used during AHR on neutrophil activation.
        • Fig. S6. IgG markers in patients with strong evidence of IgE pathway.
        • Fig. S7. MST analyses of rocuronium and suxamethonium protein partners.
        • Fig. S8. Characterization of patient-derived anti-rocuronium IgG.
        • Fig. S9. Human neutrophil activation by soluble NMBA–anti-NMBA IgG ICs.
        • Table S1. Individual values of QAM IgE (kilounits per liter) and QAM IgG (micrograms per milliliter) for case-control pairs.
        • Table S2. Tryptase and histamine measurements.
        • Table S3. Flow cytometry antibodies.
        • Table S4. Nuclear magnetic resonance data of carboxylated rocuronium in D2O.
        • Reference (80)

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        Other Supplementary Material for this manuscript includes the following:

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