Allergology International 69 (2020) 78e83 Contents lists available at ScienceDirect Allergology International journal homepage: http://www.elsevier.com/locate/alit Original Article Decreased intracellular histamine concentration and basophil activation in anaphylaxis Satoshi Yamaga a, Yuhki Yanase b, Kaori Ishii b, Shinichiro Ohshimo a, Nobuaki Shime a, * Michihiro Hide b, a Department of Emergency and Critical Care Medicine, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan b Department of Dermatology, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan article info abstract Article history: Background: Histamine is a crucial mediator in the development of anaphylaxis. Although histamine is Received 9 February 2019 promptly degraded because of its short half-life in plasma, basophils, which release histamine, remain in Received in revised form the blood for days. To explore basophils as a potential marker and their involvement in the pathogenesis 18 May 2019 of anaphylaxis, we evaluated the intracellular histamine concentration and the degree of basophil Accepted 21 May 2019 activation in anaphylaxis patients. Available online 24 June 2019 Methods: We conducted a caseecontrol study enrolling anaphylaxis patients and healthy controls. Basophil activation was evaluated by flow cytometry using up-regulation of CD203c expression. Keywords: Anaphylaxis Results: We enrolled 23 patients and measured their blood histamine concentration. Basophil activation Basophil activation was analyzed in seven of 23 patients. The median intracellular histamine concentrations at admission CD203c were significantly lower in patients compared with controls (16.4 ng/mL [interquartile range {IQR}, 2.70 Intracellular histamine to 34.0] vs. 62.3 ng/mL [IQR, 46.0 to 85.1]; p < 0.0001). The median basophil number at admission was Plasma histamine also significantly lower in patients compared with controls (2.21 cell/mL [IQR, 0.75 to 12.3] vs. 21.0 cell/mL [IQR, 19.5 to 28.9]; p ¼ 0.027). CD203c expression was not up-regulated in any of the seven patients Abbreviations: in vitro, but it was up-regulated in response to anti-IgE stimulation in vitro in two patients at admission AUC, area under the curve; CI, confidence and four patients at follow-up. interval; EDTA, ethylenediaminetetraacetic Conclusions: Anaphylaxis is associated with a decrease in intracellular histamine, and a reduced number fl acid; FITC, uorescein isothiocyanate; and reactivity of peripheral basophils. Impaired basophil function and a decrease in their number and HPLC, high performance liquid intracellular histamine levels in the circulation may reflect the underlying mechanism, suggesting that chromatography; ICU, intensive care unit; basophils may be a marker of anaphylaxis. IQR, interquartile ranges; PBS, phosphate- © buffered saline; PC7, phycoerythrin Copyright 2019, Japanese Society of Allergology. Production and hosting by Elsevier B.V. This is an open access covalently linked to cyanine 7; article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). PE, phycoerythrin; ROC, receiver operating characteristic; SD, standard deviation Introduction the mediators, histamine induces vasodilatation, increases vascular permeability,4,5 causes tachycardia, and decreases blood pressure.6 Anaphylaxis is an acute life-threatening allergic response.1 It Moreover, the plasma concentration of histamine may reflect the may be caused either by immune mechanisms including type I severity of anaphylaxis.7 However, histamine released into the allergy, which is mediated by IgE, or a non-immune mechanism, peripheral blood circulation is rapidly hydrolyzed by histamine which directly activates mast cells or basophils.2 Regardless of the methyltransferase and its half-life in plasma is only a few minutes.8 trigger or mechanisms, chemical mediators released from mast Laroche et al.8 demonstrated that the increase in plasma hista- cells in tissues and basophils in the bloodstream are suspected to mine level can be detected in 10 out of 19 patients with anaphy- play important roles in the development of anaphylaxis.3 Among laxis. Robert et al.9 showed that the increase in plasma histamine level was observed in 47% of patients who presented to the emer- gency department with acute allergic reactions. However, the half- * Corresponding author. Department of Dermatology, Graduate School of life of basophils is 2e3 days, and the decrease in the amount of & Biomedical Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku intracellular histamine caused by basophil degranulation is not Hiroshima 734-8551, Japan. E-mail address: [email protected] (M. Hide). restored immediately. Thus, the amount of intracellular histamine Peer review under responsibility of Japanese Society of Allergology. may reflect basophil degranulation in the blood for a few days after https://doi.org/10.1016/j.alit.2019.05.009 1323-8930/Copyright © 2019, Japanese Society of Allergology. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/). S. Yamaga et al. / Allergology International 69 (2020) 78e83 79 anaphylaxis occurs. Moreover, basophil activation may be evalu- Fisher Scientific, Waltham, MA, USA). Basophils can be identified by ated by the level of cell surface protein, such as CD203c and CD63. CRTH2 and CD203c double-positive and CD3 negative cells. The In this study, we investigated the level of intracellular histamine basophil activation level was evaluated by analyzing the increase in and cell surface CD203c in basophils and their reactivity compared CD203c expression. To evaluate the capacity of basophils to with clinical characteristics of basophil donors, to explore the po- respond, we stimulated basophils with anti-human IgE reagent, tential of basophils as a marker and their involvement in the which was prepared using the Allergenicity® kit. We also measured pathogenesis of anaphylaxis. the number of basophils in healthy controls using the same pro- tocols. The measurement of plasma and whole blood histamine and Methods flow cytometry analysis were conducted at admission and the next morning as a follow-up. Study design Statistical analysis We conducted a caseecontrol study in the emergency and critical care center and intensive care unit (ICU) of Hiroshima We reported continuous variables as the mean and standard University Hospital. We recruited patients with anaphylaxis whose deviation (SD) or the median and interquartile range (IQR), and plasma and total histamine concentrations could be measured. The categorical variables as proportions. We used the Wilcoxon rank- criteria for anaphylaxis was adopted from the 2006 National sum test to compare continuous variables. Analysis of the Institute of Allergy and Infectious Disease/Food Allergy and receiver operating characteristic (ROC) curve and calculation of the Anaphylaxis Network meeting.1 The severity of anaphylaxis was area under the curve (AUC) was used to assess the diagnostic ac- classified based on the classification recommended by Brown curacy of plasma and intracellular histamine levels for anaphylaxis. et al.10 Patient information including causes of anaphylaxis, medical All analyses were conducted using JMP 12 software (SAS Institute, history, clinical symptoms, plasma and total histamine concentra- Cary, NC, USA). A two-sided p-value <0.05 was considered to tions in blood, disease severity, medications, and outcomes were indicate statistical significance. collected and analyzed together with the number, activity, and activation capacity of basophils in blood. We also collected blood Results samples from age and sex-matched controls, who visited outpa- tient clinic or worked in the hospital for the levels of plasma and Study subjects whole blood histamine. This study was approved by the ethics committee for epidemiology of Hiroshima University. All the par- We enrolled 23 patients with anaphylaxis who had their levels ticipants provided written informed consent to enroll in this study of plasma and whole blood histamine were measured. Table 1 and to obtain blood samples. shows the baseline characteristics of the patients enrolled. Drugs are the most frequent cause of anaphylaxis (74%). The top two Measurement of histamine concentrations in whole blood and drugs that were suspected to cause anaphylaxis were radio- plasma contrast media in seven cases, and anticancer drugs in four cases. Twenty-one (91%) patients were classified with severe anaphy- Blood samples were collected from patients with anaphylaxis laxis (Table 2). and healthy controls at admission and on the next day (follow up). Ethylenediaminetetraacetic acid (EDTA)-treated peripheral whole blood was immediately stored in a refrigerator (4 C) until analysis. Table 1 The samples of whole blood and plasma were mixed with HClO4 to Clinical characteristics of the enrolled patients. fi a nal concentration of 2.5% HClO4 to denature excess protein, Variables Anaphylaxis Control followed by incubation for 30 min, and then histamine was Number of patients 23 23 extracted by centrifugation. The amounts of histamine were Age, yr 56.5 ± 16.4 56.9 ± 17.3 measured using high performance liquid chromatography (HPLC), Male sex, n (%) 16 (70) 16 (70) as described previously.11 The HPLC system consisted of an LC-4A Out of hospital/In hospital 9/14 (Shimadzu, Kyoto, Japan) with an RF-535 fluorescence monitor