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Chemokines during anaphylaxis: the importance of CCL2 and CCL2-dependent chemotactic activity for basophils

Chemokines during anaphylaxis: the importance of CCL2 and CCL2-dependent chemotactic activity for... Background: The role of chemokines in anaphylaxis is unclear. Methods: We prospectively recruited 49 patients presenting to the emergency department with an acute episode of anaphylaxis and 28 healthy subjects. We measured serum levels of the chemokines CCL2, CCL5, CCL7, CCL8, CCL11, CCL13, CCL17, CCL21, CCL22, CCL24, and CCL26, tryptase, the absolute number of circulating basophils, monocytes, lymphocytes, and PMNs, and whole blood FCER1A, CPA3 and HDC gene expression at two time points: during the ana‑ phylactic episode and in convalescent samples collected approximately 3 months later. We then investigated the in vitro chemotactic activity of chemokines induced during anaphylaxis for the in vitro migration of the corresponding cells. Results: Only CCL2 chemokine levels were significantly increased in anaphylaxis samples (median 514 pg/ml) compared to convalescent samples (284 pg/ml, P < 0.0001) and healthy subjects (279 pg/ml, P < 0.0001); there was no significant difference in any of the other chemokines. There was a significant positive correlation between the rates of increase of serum CCL2 (median [range]: 106.0% [− 44.7% to 557.4%]) and tryptase (133.8% [− 6.6% to 893.4%]; r = 0.68, P < 0.0001) and between the acute concentration of serum CCL2 and the acute concentration of serum tryptase (r = 0.77, P < 0.0001). The number of circulating basophils, but not other blood cells, significantly decreased during anaphylaxis (median 5.0 vs. 19.1 cells/µl in convalescent samples; P < 0.0001); a decrease in whole‑blood gene expression of basophil markers (P ≤ 0.0018) confirmed these changes. Anaphylactic serum enhances the in vitro migration of basophils via CCL2‑ dependent chemotactic activity; in contrast, no CCL2‑ dependent chemotactic activ‑ ity was observed for convalescent samples. Conclusions: Our findings imply an important and specific role for CCL2‑mediated chemotactic activity in the pathophysiology of human anaphylaxis. Keywords: Anaphylaxis, Chemokines, CCL2, Tryptase, Basophils, Chemotaxis, Migration Background insect venom, foods, and medications [1]. The major Anaphylaxis is a potentially life-threatening, rapidly pro- immunopathogenesis and pathophysiological mecha- gressing systemic hypersensitivity reaction, often follow- nisms of anaphylaxis involve IgE antibodies, effector mast ing exposure to a small amount of allergen, including cells, basophils and the mediators histamine, platelet- activating factor, and cysteinyl leukotrienes [2–4]. How- ever, anaphylaxis also induces changes in other mediators, *Correspondence: peter.korosec@klinika‑golnik.si University Clinic of Respiratory and Allergic Diseases Golnik, Golnik 36, including tryptase [5–7], prostaglandins [8], cytokines [6, 4204 Golnik, Slovenia 9], and chemokines [7]. A recent study demonstrated an Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Vantur et al. Clin Transl Allergy (2020) 10:63 Page 2 of 11 increase in CCL2 (C-C Motif Chemokine Ligand 2) levels in 26 patients, by medication in 14 patients, by food in during human anaphylaxis [7], with no changes demon- 5 patients and by other triggers in 4 patients (idiopathic strated for the chemokines CCL5 [7] and CCL11 [6, 7]. in 2 patients and infection in 2 patients); 21 experienced The chemokines CCL2, CCL5, and CCL11 are involved Mueller [26] grade IV, 16 experienced grade III, 6 expe- in basophil migration, with the CCR2 ligand CCL2 and rienced grade II, and 6 experienced grade I reactions the CCR3 ligand CCL11 eliciting the most potent migra- (Table  1). We collected blood samples during the reac- tory responses [10]. Chemokines that diffuse out from tion (at presentation to the emergency department; in the site of release and form a concentration gradient to all patients we routinely measured acute serum tryptase, which leukocytes respond and migrate might be involved Table  1) and convalescent samples after the anaphy- in the recruitment of basophils [4] or other potential lactic episode. None of those patients were included in effector cells of anaphylaxis, including monocytes/mac - our previous study of anaphylaxis [7]. The median time rophages [11], neutrophils [12, 13], and platelets [14], to from the onset of symptoms to sample collection was the inflamed tissue sites. 100  min (range 30 to 240  min); convalescent samples In addition to CCL2 [7], several other chemokines were collected a median of 80  days after the anaphylac- might be involved in different allergic diseases. For tic episode (Table 1). In 30 patients, we measured serum monocyte chemoattractant proteins (MCPs), an increase chemokines and tryptase, and in 26 patients, we meas- in CCL7 levels in allergic conjunctivitis [15], an increase ured blood absolute cell counts and gene expression (of in CCL8 levels in chronic/eosinophilic atopic dermatitis those 41 patients, 15 were in both subgroups); for com- [16], and an increase in CCL13 levels in allergic rhinitis parison, we recruited 20 healthy subjects (13 females; [17] were demonstrated. For eotaxin chemokines, CCL11 age, 20–76 years) (Additional file  1: Table S1). The serum and CCL24 levels were increased in asthma and allergic of 8 anaphylactic patients and isolated basophils of 8 rhinitis [18], and CCL26 levels were increased in allergic healthy subjects (4 females; age, 25–44  years) were used rhinitis [17] and atopic skin inflammation [19]. CCL5, for in  vitro migration experiments (Additional file  1: CCL17, CCL21, and CCL22 were also reported to be Table S1). involved in asthma, atopic dermatitis, allergic rhinitis and Ethical approval was obtained from the Slovenian drug hypersensitivity syndrome [20–25]. National Medical Ethics Committee. All subjects pro- Given that different chemokines might be important vided written informed consent. in allergic diseases, we performed a series of interlinked studies to better understand the role of chemokines and Serum chemokines and tryptase, absolute cell counts, their chemotactic activity in anaphylaxis. In a prospec- and gene expression tive study during and after anaphylaxis caused by Hyme- We measured serum concentrations of the MCP noptera venom, medication, food, or other (idiopathic), chemokines CCL2/MCP-1, CCL7/MCP-3, CCL8/MCP- we investigated the serum concentration of a large panel 2, and CCL13/MCP-4; the eotaxin chemokines CCL11/ of different chemokines (CCL2, CCL5, CCL7, CCL8, Eotaxin, CCL24/Eotaxin-2 and CCL26/Eotaxin-3; and CCL11, CCL13, CCL17, CCL21, CCL22, CCL24, and the chemokines CCL5/Rantes, CCL17/TARC, CCL21/ CCL26), which were previously described in allergic dis- SLC and CCL22/MDC using ELISAs according to the eases [6, 7, 15–25]. In whole blood samples, we measured manufacturers’ instructions (Quantikine R&D Systems, the absolute numbers of circulating basophils, mono- Minneapolis, MN, USA; Abnova, Taipei, Taiwan; and cytes, lymphocytes, and polymorphonuclear leukocytes Biolegend, San Diego, California, CA, USA). We meas- (PMNs) and the gene expression of the basophil markers ured serum tryptase (ImmunoCAP, Thermo Fisher, Upp - FCER1A, carboxypeptidase A3 (CPA3) and L-histidine sala, Sweden), and concentrations that exceeded 11.4 µg/l decarboxylase (HDC). We then proceeded to analyse were considered increased. Enumeration of circulating the chemotactic activity of chemokines that are induced basophils (CD123+HLA-DR− cells), monocytes, lym- during anaphylaxis in the controlled setting of a cellular phocytes, and PMNs in fresh whole blood was performed in vitro migration assay. via flow cytometry as previously described [7, 27]. FCER1A, CPA3, and HDC gene expression was analysed Methods in whole blood samples (PAXgene, PreAnalytiX, Hom- Study participants brechtikon, Switzerland) as previously described [7, 27]. We prospectively recruited 49 patients (24 females; Detailed methods are provided in Additional file 1. age, 19–82  years; Table  1) presenting to the Emergency Department of University Hospital Golnik, Slovenia, with Cell separation and in vitro migration assays an acute episode of anaphylaxis (January 2017 to Octo- CCR2 surface expression on basophils of healthy donors ber 2019). The reaction was caused by an insect sting was determined by anti-CCR2 mAb (CD192; Miltenyi V antur et al. Clin Transl Allergy (2020) 10:63 Page 3 of 11 Table 1 Characteristics of the patients Patients with an acute episode of anaphylaxis (n = 49) Sex, n (%) 24 female (49.0) 25 male (51.0) Age years, average (range) 50 (19–82) Trigger, n (%) Hymenoptera venom 26 (53.1) Medication 14 (28.6) Food 5 (10.2) Other 4 (8.2) Time from the onset of anaphylactic symptoms to sample collection at presentation to the emergency department, 100 (69) median (IQR) in minutes Convalescent sampling after anaphylactic episode, median (IQR) in days 80 (93) Emergency treatment, n (%) Epinephrine 25 (51.0) Corticosteroids 43 (87.8) Grade of reaction, n (%) Mueller I 6 (12.2) Mueller II 6 (14.6) Mueller III 16 (32.7) Mueller IV 21 (42.9) Symptoms, n (%) Any skin feature 44 (89.8) Any gastrointestinal feature 15 (30.6) Any respiratory feature 26 (53.1) Hypoxemia (SpO2 ≤ 92%) 7 (14.3) Any cardiovascular feature 24 (49.0) Hypotension (SBP < 90 mmHg) 19 (38.8) Acute tryptase > 11.4 µg/l, n (%) 32 (65.3) SBP systolic blood pressure Biotec, Bergisch Gladbach, Germany) staining, and For control experiments, we used recombinant CCL2 basophils were isolated from peripheral whole blood by (Thermo Fisher Scientific, Massachusetts, USA). To negative immunomagnetic selection (Miltenyi Biotec, block CCL2, we used anti-CCL2 neutralizing antibodies Bergisch Gladbach, Germany) and exactly quantified by (Sigma-Aldrich, Missouri, USA). All experiments were absolute flow cytometry basophil counts as previously independently performed in triplicate. Detailed methods described (CD123+HLA-DR− cells and microbeads) [7, are described in Additional file 1. 27]. For the migration assay, we used modified Boyden chamber and polycarbonate membrane cell culture Statistical analysis inserts (Corning Inc., New York, USA). A total of 20,000 The distribution of the data was assessed using the basophils were added to the upper wells, and the samples D’Agostino test. As the majority of the data were nonpara- to be tested were placed in the lower wells. After incuba- metric, we used the Wilcoxon or Mann–Whitney test. P tion for 30 to 150 min at 37 °C, we collected the cells that values were Bonferroni-corrected for the complete set of 19 had migrated to the lower wells and quantified them by variables, and a P-value < 0.0026 was accepted as significant. absolute basophil counts [7, 27]. Basophil migration was calculated by using the following equation: Basophil migration(%) = absolute number of migrated basophils/absolute number of seeded basophils ×100. Vantur et al. Clin Transl Allergy (2020) 10:63 Page 4 of 11 For the cellular in  vitro migration assay, all experiments 70 pg/ml) and CCL13 (113, 104 and 123 pg/ml), showed were independently performed in triplicate, and the results no differences during anaphylaxis or later compared to were compared with a t-test; a P-value < 0.05 was accepted healthy subjects (Fig. 1 and Table 2). as significant. To quantify associations between variables, we used Spearman or Pearson correlation. All analyses were Eotaxin chemokines CCL11/eotaxin, CCL24/eotaxin‑2 performed using GraphPad Prism (GraphPad Software, La and CCL26/eotaxin‑3 Jolla, CA, USA). There were no differences in CCL11 (median 200, 211 and 160 pg/ml), CCL24 (1124, 946 and 1159 pg/ml) and CCL26 Results (1903, 1898 and 1877  pg/ml) between the acute and con- The chemokine CCL2, but not other chemokines, valescent time points and the healthy subjects (Fig.  1 and is significantly increased during anaphylactic episodes Table 2). MCP chemokines CCL2/MCP‑1, CCL7/MCP‑3, CCL8/MCP‑2, and CCL13/MCP‑4 Chemokines CCL5/Rantes, CCL17/TARC, CCL21/SLC CCL2 concentrations measured during the anaphylac- and CCL22/MDC tic episode (median 514 pg/ml) were significantly higher There were no differences in CCL5 (median 40.9, 41.8 than those measured in convalescent serum samples col- and 47.0  ng/ml), CCL17 (342, 303 and 344  pg/ml), lected later (median 80 days; 284 pg/ml; P < 0.0001; Fig. 1 CCL21 (1179, 1099 and 938 pg/ml) and CCL22 (666, 717 and Table  2). This marked increase (median 106.0%; and 766 pg/ml) between the acute and convalescent time range: − 44.7% to 557.4%) was evident in 28/30 patients points and the healthy subjects (Fig. 1 and Table 2). (Fig.  2). Serum CCL2 levels during the acute episode were also significantly higher than those observed in Increase in serum CCL2 levels highly correlates healthy subjects (median 279 pg/ml, P < 0.0001); however, with an increase in serum tryptase CCL2 levels in convalescent samples showed no differ - The median concentration of serum tryptase was signifi - ence in comparison to CCL2 levels in healthy subjects cantly higher during the anaphylactic episode (12.9 µg/l) (Fig.  1 and Table  2). Other MCP chemokines, including than later or in healthy subjects (5.4 and 3.3 µg/l, respec- CCL7 (median 8.9, 8.5 and 8.1 pg/ml), CCL8 (73, 69 and tively; P ≤ 0.0005; Fig.  1 and Table  2). The median rate Fig. 1 Serum CCL2, CCL5, CCL7, CCL8, CCL11, CCL13, CCL17, CCL21, CCL22, CCL24, CCL26, and tryptase levels in 30 patients during an acute anaphylactic episode and in convalescent samples collected later V antur et al. Clin Transl Allergy (2020) 10:63 Page 5 of 11 Table 2 Laboratory data of anaphylactic patients and healthy subjects Variables median (IQR) Patients with an acute episode Healthy subjects P-value a b of anaphylaxis (n = 49) (n = 28) Acute sampling Convalescent sampling Acute vs. Acute vs. Convalescent vs. convalescent healthy healthy subjects subjects Serum chemokines (pg/ml) CCL2/MCP‑1 514 (561) 284 (128) 279 (147) < 0.0001 < 0.0001 0.9726 CCL7/MCP‑3 8.9 (2.9) 8.5 (2.1) 8.1 (1.4) 0.7549 0.1636 0.1038 CCL8/MCP‑2 73 (56) 69 (35) 70 (19) 0.1579 0.1425 0.4226 CCL13/MCP‑4 113 (81) 104 (88) 123 (103) 0.9032 0.3367 0.7648 CCL11/Eotaxin 200 (174) 211 (174) 160 (60) 0.2710 0.1641 0.0555 CCL24/Eotaxin‑2 1124 (1377) 946 (1036) 1159 (1561) 0.0940 0.8665 0.5919 CCL26/Eotaxin‑3 1903 (88) 1898 (181) 1877 (155) 0.0412 0.1746 0.4640 CCL5/Rantes 40,907 (34,588) 41,795 (27,011) 46,993 (33,574) 0.6669 0.7485 0.9659 CCL17/TARC 342 (194) 303 (203) 344 (180) 0.0148 0.7093 0.1931 CCL21/SLC 1179 (1193) 1099 (645) 938 (417) 0.2206 0.3376 0.4175 CCL22/MDC 666 (321) 717 (251) 766 (260) 0.7045 0.8014 0.5408 Serum tryptase µg/l 12.9 (10.6) 5.3 (2.0) 3.3 (2.0) < 0.0001 < 0.0001 0.0005 > 11.4 µg/l, n (%) 18 (60) 1 (3.3) 0 (0) < 0.0001 < 0.0001 1 Absolute cell count (cells/µl) Basophils 5.0 (7.3) 19.1 (10.9) – < 0.0001 – – Monocytes 396 (163) 441 (108) – 0.8417 – – Lymphocytes 1378 (1219) 1461 (470) – 0.3904 – – PMNs 3881 (2873) 2875 (1144) – 0.0388 – – Gene expression in whole blood (fold change) FCER1A 0.11 (0.24) 0.40 (0.64) – – – 0.0002 CPA3 0.22 (0.51) 0.72 (1.16) – 0.0002 – – HDC 0.11 (0.30) 0.38 (0.57) – – – 0.0018 Serum of patients used for in vitro migration assays Tryptase (µg/l) 22.6 (16.5) 6.4 (6.2) 4.3 (5.8) 0.0002 0.0003 0.5358 > 11.4 µg/l, n (%) 8 (100) 0 (0) 0 (0) 0.0002 0.0002 1 CCL2 (pg/ml) 1964 (1114) 278 (175) 302 (76) 0.0002 0.0003 0.3969 Basophils of healthy donors used for the in vitro migration assays Absolute count (cells/ – – 18.3 (6.3) – – – µl) CCR2 expression MFI – – 353 (422) – – – % CCR2 + basophils – – 94 (27) – – – PMNs polymorphonuclear leukocytes, FCER1A α subunit of the high-affinity IgE receptor, CPA3 carboxypeptidase A3, HDC histidine decarboxylase, CCR2 C-C motif chemokine receptor 2, MFI mean fluorescence intensity P values were Bonferroni-corrected, and P-value < 0.0026 was accepted as significant. Statistically significant P values are presented in boldface In 30 patients, we measured serum chemokines and tryptase, and in 26 patients, we measured blood absolute cell counts and gene expression (of those 41 patients, 15 were in both subgroups); serum samples from 8 patients were used for in vitro migration experiments In 20 healthy subjects, we measured serum chemokines and tryptase; basophils from 8 healthy subjects were used for in vitro migration experiments of increase of serum tryptase was 133.8% (range: − 6.6% rates of increase of serum CCL2 and tryptase (r = 0.68, to 893.4%; increase in 28/30 patients), which was com- P < 0.0001) and between the acute concentration of serum parable to the increase in serum CCL2 levels (Fig.  2). CCL2 and the acute concentration of serum tryptase There was a significant positive correlation between the (r = 0.77, P < 0.0001; Fig. 2). Vantur et al. Clin Transl Allergy (2020) 10:63 Page 6 of 11 Fig. 2 Comparison and correlation between serum CCL2 and tryptase levels in 30 patients with acute anaphylactic episodes Anaphylactic episodes involve a substantial reduction compared to convalescent blood samples collected later in circulating basophils (Fig. 3). Basophils, monocytes, lymphocytes, and PMNs The absolute number of circulating basophils was sig - Gene expression of basophil markers nificantly lower during the anaphylactic episode (median We observed significantly lower whole blood gene 5.0 cells/µl) than in convalescent blood samples collected expression of FCER1A, CPA3, and HDC during the ana- later (19.1  cells/µl, P < 0.0001; Fig.  3 and Table  2). This phylactic episode than in convalescent samples (respec- decrease (median 65.6%, range 30.0–98.6%) was evident tively; P ≤ 0.0018; Fig. 3 and Table 2). Acute whole blood in all patients. There were no significant differences in gene expression of FCER1A, CPA3, and HDC significantly the monocyte (median 396 and 441 cells/µl), lymphocyte correlated with the acute absolute number of circulating (1378 and 1461 cells/µl), and PMN (3881 and 2875 cells/ basophils (r = 0.53, 0.79 and 0.81, respectively; P ≤ 0.005; µl) absolute cell counts during the acute episode Additional file 1: Fig. S1). Fig. 3 Absolute basophil, monocyte, lymphocyte and PMN counts and whole blood FCER1A, CPA3, and HDC gene expression in 26 patients during the acute anaphylactic episode and in convalescent samples collected later. PMNs polymorphonuclear leukocytes, FCER1A α subunit of the high‑affinity IgE receptor, CPA3 carboxypeptidase A3, HDC histidine decarboxylase V antur et al. Clin Transl Allergy (2020) 10:63 Page 7 of 11 Anaphylactic serum enhances the in vitro migration observed for other chemokines, which were previously of basophils via CCL2-dependent chemotactic activity implicated in different allergic diseases or during aller - A basophil migration assay was performed using MACS- gic inflammation [15–25]. Thus, the chemokine patterns separated human basophils and a modified Boyden of CCL5, CCL7, CCL8, CCL11, CCL13, CCL17, CCL21, chamber, with quantification by absolute flow cytom - CCL22, CCL24, and CCL26 might be of limited impor- etry basophil counts. Basophils from all healthy donors tance for anaphylaxis. While activation of the pathways showed high surface expression of CCR2 (Table  2). All associated with multiple inflammatory cytokines, includ - experiments were performed independently in triplicate ing IL-2, IL-6, IL-10, and TNFRI, were observed during (detailed information is provided in Additional file 1). anaphylaxis [6, 9], the selective CCL2 chemokine cas- cade might be important for chemotactic activity during Basophil migration induced by HBSS and rCCL2 anaphylactic reactions. It was recently reported that the As shown in Fig. 4a, during a 150-min incubation time with CCL2-FcεRI-histone deacetylase 3 signalling axis medi- Hank’s balanced salt solution (HBSS), only a small number ates passive anaphylaxis by increasing the expression of of basophils (mean, SD), 0.8 ± 0.7%) transmigrated across CCL2 in mast cells [28, 29]. After allergen stimulation, the polycarbonate membrane. Basophil migration was sig- histone deacetylase 3 binds to FcεRI, and histone deacet- nificantly enhanced by the presence of 10  nM rCCL2 and ylase 3 increases the expression of SP1 and C-Jun, which 50 nM rCCL2 (8.1 ± 4.0% and 14.0 ± 8.1%; 150 min of incu- bind to the promoter sequences of CCL2 to increase the bation); however, after blocking with anti-CCL2 mAbs, expression of CCL2 [28, 29]. Conversely, in the absence rCCL2-induced migration was almost completely inhibited of allergen stimulation, histone deacetylase 3 binds to the (0.8 ± 0.6%, P = 0.036 and 0.8 ± 0.1%, P = 0.047, blocking at promoter sequences of CCL2 to suppress the expression 10 nM and 50 nM of rCCL2, respectively). of CCL2 [28]. Our study design and the median time between symp- Basophil migration induced by anaphylactic tom onset and sample collection were highly comparable and convalescent serum with those of previous studies of anaphylaxis [3, 6, 7, 9, We compared the effects of acute serum collected during 30]. This suggests that this is an unbiased comparison, the anaphylactic episode and convalescent serum collected including the confirmation that the chemokines CCL5 later (Table 2) on basophil migration. After 150 min of incu- and CCL11, which may affect other important effector bation, the number of transmigrated basophils was signifi - cells of allergic inflammation such as eosinophils, are cantly higher (twofold) in the presence of acute serum in the not induced during anaphylaxis [6, 7]. The nature of the lower wells than in the presence of convalescent serum in management of anaphylaxis (including administration of the lower wells (mean, SD: 54.0 ± 19.3% vs. 28.3 ± 11.7%, high-dose corticosteroids) makes it difficult to exclude respectively; P = 0.01) (Fig.  4b). The number of transmi - the potential confounding effects of treatment. How - grated cells increased linearly until 150  min of incubation ever, it was previously demonstrated that serum CCL2, and then plateaued (Fig. 4c). A neutralizing antibody against CCL5, and CCL11 levels are not affected by corticoster - CCL2 significantly blocked basophil transmigration induced oid treatment [7]. Nevertheless, corticosteroid treatment by anaphylactic serum (37.7 ± 20.8% vs. 26.1 ± 19.7%; or the time between symptom onset and sample collec- P = 0.001). However, a neutralizing antibody against CCL2 tion may have affected the levels of the other measured did not show any blocking effect on the chemotactic chemokines. response induced by convalescent serum (32.5 ± 12.5% vs. In the present study, we could not determine the cel- 30.7 ± 18.3%) (Fig. 4b). lular sources of CCL2 during anaphylaxis. However, we showed a highly significant positive correlation between Discussion elevated blood levels of acute CCL2 and tryptase and Our study demonstrated that there is a selective and sig- strong similarities between the percentage increases nificant increase in serum CCL2 chemokine levels dur - of CCL2 and tryptase from convalescent serum sam- ing anaphylactic reactions. No significant changes were ples. Tryptase is largely mast cell-derived, and mature (See figure on next page.) Fig. 4 a–c Eec ff ts of CCL2 and acute and convalescent serum from patients with an anaphylactic episode on basophil in vitro migration. Basophil migration after 150 min of incubation: a in the presence of rCCL2 in the lower wells and after CCL2 blocking and b in the presence of acute or convalescent serum in the lower wells and after CCL2 blocking. c Time course of basophil migration (after 30, 60, 90, 120, and 150 min of incubation) in the presence of acute and convalescent serum in the lower wells. All experiments were independently performed in triplicate, with a basophils from 5 healthy donors; b basophils from the same 5 healthy donors and serum samples from 5 patients and c basophils from 3 healthy donors and serum samples from 3 patients (details in Additional file 1: Table S1). HBSS Hank’s balanced salt solution Vantur et al. Clin Transl Allergy (2020) 10:63 Page 8 of 11 V antur et al. Clin Transl Allergy (2020) 10:63 Page 9 of 11 β-tryptase is stored in mast cell granules and released where the activation and degranulation of mast cells upon mast cell activation during acute anaphylaxis [2, occur, thereby contributing to the clinical presentation of 5–7]. Therefore, it is tempting to speculate that the the sites of allergic reaction. This is consistent with clini - blood CCL2 increase that occurs during anaphylaxis is cal observations of different severities and end-organ pat - IgE related and mainly mast cell derived. In vitro activa- terns of anaphylaxis, which suggest that local rather than tion of human mast cells by anti-IgE induces secretion generalized mast cell and/or basophil degranulation may of CCL2 after 30 min of stimulation, and this increase is predominate in some individuals [41]. However, a sub- observed earlier than TNF-α, IL-13, or GM-CSF mast cell stantially broader assessment is needed to explore and secretion, peaking 90 min after anti-IgE stimulation [31]. confirm CCL2-mediated cellular crosstalk during acute Furthermore, mast cell CCL2 secretion is induced at low allergic reactions. allergen concentrations or in the context of low receptor Although CCL2 displays major chemotactic activity for occupancy with IgE [32], and due to the short time frame monocytes and interferes with the egress of monocytes within which the increase in serum CCL2 was observed from the bone marrow to the circulation during homeo- in the current and previous studies [7], rapid induction stasis and inflammation [42], no changes in circulating of CCL2 production by the allergen-IgE axis might be monocytes were observed during anaphylaxis. However, feasible. Nevertheless, CCL2 is produced by a variety of studies in mice have shown that depletion of monocytes/ cell types, including endothelial cells and smooth muscle macrophages can reduce anaphylaxis in both IgG-medi- cells [33], astrocytes as a major source of CCL2 during ated passive models and active models [2, 11]. central nervous system inflammation [34] and cancer and stromal cells as a major source of CCL2 in tumour micro- Conclusion environments [35]; thus, the cellular sources of CCL2 In conclusion, our data suggest that during anaphylaxis, during anaphylaxis are currently unknown. Additional an increase in the chemokine CCL2 occurs, which corre- and substantially studies of mast cell CCL2 expression lates with CCL2-mediated chemotactic activity in baso- and secretion are required to confirm this speculation. phils and substantial migration of circulating basophils. The results of experimental allergen challenge in the Our findings imply an important and specific role for nose, airways, and skin have demonstrated that there CCL2 in the pathophysiology of human anaphylaxis. is an influx of basophils to inflammatory sites several hours after allergen exposure [36–39]. Furthermore, it Supplementary information was recently demonstrated that airway allergen chal- Supplementary information accompanies this paper at https ://doi. org/10.1186/s1360 1‑020‑00367 ‑2. lenge induces TLR-dependent CCL2 production in the lungs and that circulating basophils are recruited to the Additional file 1. Additional methods and results. Table S1. Detailed lungs by CCL2-mediated transendothelial migration and information on the number of participants for whom we assessed dif‑ a chemotactic gradient [40]. Our study demonstrated sig- ferent laboratory parameters. Additional results. Figure S1. Correlation nificantly enhanced CCL2-mediated chemotactic activity between absolute basophil counts and whole‑blood FCER1A, CPA3, and HDC gene expression in 26 anaphylactic patients of anaphylactic serum for the migration of isolated baso- phils. Conversely, no CCL2-mediated basophil chemo- tactic activity was observed for the convalescent serum Abbreviations CCL: C‑ C Motif Chemokine Ligand; CCR: C ‑ C motif chemokine receptor; CPA: collected later after the reaction. The CCL2-mediated Carboxypeptidase A3; FCER1A: α subunit of the high‑affinity IgE receptor; basophil chemotactic activity of anaphylactic serum and HBSS: Hank’s balanced salt solution; HDC: l ‑Histidine decarboxylase; MCP: the reduction in circulating basophils during anaphylaxis, Monocyte chemoattractant protein; MDC: Macrophage‑Derived Chemokine; PMNs: Polymorphonuclear leukocytes; SLC: Secondary Lymphoid Tissue coupled with the previous finding that basophils are the Chemokine; TARC : Thymus and Activation Regulated Chemokine. granulocytes most resistant to apoptosis [41], suggest that anaphylaxis induces rapid CCL2-mediated basophil Acknowledgements Not applicable. migration rather than elimination by apoptosis. Moreo- ver, anaphylaxis-related basophil migration appears to be Authors’ contributions selective because no significant changes were observed PKorosec, RV and AK designed the study. PKopac and RE evaluated the patients and revised the manuscript. RV, MRihar, AK, MRijavec, and UBS for monocytes, lymphocytes, PMNs, or chemokines performed laboratory tests and experiments and revised the manuscript. that may affect other effector cells, and the decreased PKorosec and RV wrote the manuscript. All authors read and approved the gene expression of basophil markers FCER1A, CPA3 and final manuscript. HDC corroborates the flow cytometry data. Therefore, Funding it is tempting to speculate that CCL2 might be mast cell The research was supported by the Slovenian Research Agency (Grant Nos. derived and that circulating basophils influx to the sites P3‑0360 and 39238). Vantur et al. Clin Transl Allergy (2020) 10:63 Page 10 of 11 Availability of data and materials 15. Kuo C‑H, Collins AM, Boettner DR, Yang Y, Ono SJ. Role of CCL7 in type I Raw data were generated at the University Clinic of Respiratory and Allergic hypersensitivity reactions in murine experimental allergic conjunctivitis. J Diseases, Golnik, Slovenia. 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The chemokine receptor CCR3 participates in tissue remodeling during Competing interests atopic skin inflammation. J Dermatol Sci. 2013;71:12–21. The authors declare that they have no competing interests. 20. Esaki H, Takeuchi S, Furusyo N, Yamamura K, Hayashida S, Tsuji G, et al. Levels of immunoglobulin E specific to the major food allergen and Author details chemokine (C‑ C motif ) ligand (CCL)17/thymus and activation regulated University Clinic of Respiratory and Allergic Diseases Golnik, Golnik 36, chemokine and CCL22/macrophage‑ derived chemokine in infantile 4204 Golnik, Slovenia. Biotechnical Faculty, University of Ljubljana, Ljubljana, atopic dermatitis on Ishigaki Island. J Dermatol. 2016;43:1278–82. Slovenia. Medical Faculty, University of Ljubljana, Ljubljana, Slovenia. 21. Ahrens B, Schulz G, Bellach J, Niggemann B, Beyer K. 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Airway epithelial TSLP production of TLR2 drives type 2 immunity in allergic airway inflammation. Eur J Immunol. 2018;48(11):1838–50. 41. Simons FER, Frew AJ, Ansotegui IJ, Bochner BS, Golden DBK, Finkelman FD, et al. Risk assessment in anaphylaxis: current and future approaches. J Allergy Clin Immunol. 2007;120:S2–24. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Clinical and Translational Allergy Springer Journals

Chemokines during anaphylaxis: the importance of CCL2 and CCL2-dependent chemotactic activity for basophils

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10.1186/s13601-020-00367-2
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Abstract

Background: The role of chemokines in anaphylaxis is unclear. Methods: We prospectively recruited 49 patients presenting to the emergency department with an acute episode of anaphylaxis and 28 healthy subjects. We measured serum levels of the chemokines CCL2, CCL5, CCL7, CCL8, CCL11, CCL13, CCL17, CCL21, CCL22, CCL24, and CCL26, tryptase, the absolute number of circulating basophils, monocytes, lymphocytes, and PMNs, and whole blood FCER1A, CPA3 and HDC gene expression at two time points: during the ana‑ phylactic episode and in convalescent samples collected approximately 3 months later. We then investigated the in vitro chemotactic activity of chemokines induced during anaphylaxis for the in vitro migration of the corresponding cells. Results: Only CCL2 chemokine levels were significantly increased in anaphylaxis samples (median 514 pg/ml) compared to convalescent samples (284 pg/ml, P < 0.0001) and healthy subjects (279 pg/ml, P < 0.0001); there was no significant difference in any of the other chemokines. There was a significant positive correlation between the rates of increase of serum CCL2 (median [range]: 106.0% [− 44.7% to 557.4%]) and tryptase (133.8% [− 6.6% to 893.4%]; r = 0.68, P < 0.0001) and between the acute concentration of serum CCL2 and the acute concentration of serum tryptase (r = 0.77, P < 0.0001). The number of circulating basophils, but not other blood cells, significantly decreased during anaphylaxis (median 5.0 vs. 19.1 cells/µl in convalescent samples; P < 0.0001); a decrease in whole‑blood gene expression of basophil markers (P ≤ 0.0018) confirmed these changes. Anaphylactic serum enhances the in vitro migration of basophils via CCL2‑ dependent chemotactic activity; in contrast, no CCL2‑ dependent chemotactic activ‑ ity was observed for convalescent samples. Conclusions: Our findings imply an important and specific role for CCL2‑mediated chemotactic activity in the pathophysiology of human anaphylaxis. Keywords: Anaphylaxis, Chemokines, CCL2, Tryptase, Basophils, Chemotaxis, Migration Background insect venom, foods, and medications [1]. The major Anaphylaxis is a potentially life-threatening, rapidly pro- immunopathogenesis and pathophysiological mecha- gressing systemic hypersensitivity reaction, often follow- nisms of anaphylaxis involve IgE antibodies, effector mast ing exposure to a small amount of allergen, including cells, basophils and the mediators histamine, platelet- activating factor, and cysteinyl leukotrienes [2–4]. How- ever, anaphylaxis also induces changes in other mediators, *Correspondence: peter.korosec@klinika‑golnik.si University Clinic of Respiratory and Allergic Diseases Golnik, Golnik 36, including tryptase [5–7], prostaglandins [8], cytokines [6, 4204 Golnik, Slovenia 9], and chemokines [7]. A recent study demonstrated an Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Vantur et al. Clin Transl Allergy (2020) 10:63 Page 2 of 11 increase in CCL2 (C-C Motif Chemokine Ligand 2) levels in 26 patients, by medication in 14 patients, by food in during human anaphylaxis [7], with no changes demon- 5 patients and by other triggers in 4 patients (idiopathic strated for the chemokines CCL5 [7] and CCL11 [6, 7]. in 2 patients and infection in 2 patients); 21 experienced The chemokines CCL2, CCL5, and CCL11 are involved Mueller [26] grade IV, 16 experienced grade III, 6 expe- in basophil migration, with the CCR2 ligand CCL2 and rienced grade II, and 6 experienced grade I reactions the CCR3 ligand CCL11 eliciting the most potent migra- (Table  1). We collected blood samples during the reac- tory responses [10]. Chemokines that diffuse out from tion (at presentation to the emergency department; in the site of release and form a concentration gradient to all patients we routinely measured acute serum tryptase, which leukocytes respond and migrate might be involved Table  1) and convalescent samples after the anaphy- in the recruitment of basophils [4] or other potential lactic episode. None of those patients were included in effector cells of anaphylaxis, including monocytes/mac - our previous study of anaphylaxis [7]. The median time rophages [11], neutrophils [12, 13], and platelets [14], to from the onset of symptoms to sample collection was the inflamed tissue sites. 100  min (range 30 to 240  min); convalescent samples In addition to CCL2 [7], several other chemokines were collected a median of 80  days after the anaphylac- might be involved in different allergic diseases. For tic episode (Table 1). In 30 patients, we measured serum monocyte chemoattractant proteins (MCPs), an increase chemokines and tryptase, and in 26 patients, we meas- in CCL7 levels in allergic conjunctivitis [15], an increase ured blood absolute cell counts and gene expression (of in CCL8 levels in chronic/eosinophilic atopic dermatitis those 41 patients, 15 were in both subgroups); for com- [16], and an increase in CCL13 levels in allergic rhinitis parison, we recruited 20 healthy subjects (13 females; [17] were demonstrated. For eotaxin chemokines, CCL11 age, 20–76 years) (Additional file  1: Table S1). The serum and CCL24 levels were increased in asthma and allergic of 8 anaphylactic patients and isolated basophils of 8 rhinitis [18], and CCL26 levels were increased in allergic healthy subjects (4 females; age, 25–44  years) were used rhinitis [17] and atopic skin inflammation [19]. CCL5, for in  vitro migration experiments (Additional file  1: CCL17, CCL21, and CCL22 were also reported to be Table S1). involved in asthma, atopic dermatitis, allergic rhinitis and Ethical approval was obtained from the Slovenian drug hypersensitivity syndrome [20–25]. National Medical Ethics Committee. All subjects pro- Given that different chemokines might be important vided written informed consent. in allergic diseases, we performed a series of interlinked studies to better understand the role of chemokines and Serum chemokines and tryptase, absolute cell counts, their chemotactic activity in anaphylaxis. In a prospec- and gene expression tive study during and after anaphylaxis caused by Hyme- We measured serum concentrations of the MCP noptera venom, medication, food, or other (idiopathic), chemokines CCL2/MCP-1, CCL7/MCP-3, CCL8/MCP- we investigated the serum concentration of a large panel 2, and CCL13/MCP-4; the eotaxin chemokines CCL11/ of different chemokines (CCL2, CCL5, CCL7, CCL8, Eotaxin, CCL24/Eotaxin-2 and CCL26/Eotaxin-3; and CCL11, CCL13, CCL17, CCL21, CCL22, CCL24, and the chemokines CCL5/Rantes, CCL17/TARC, CCL21/ CCL26), which were previously described in allergic dis- SLC and CCL22/MDC using ELISAs according to the eases [6, 7, 15–25]. In whole blood samples, we measured manufacturers’ instructions (Quantikine R&D Systems, the absolute numbers of circulating basophils, mono- Minneapolis, MN, USA; Abnova, Taipei, Taiwan; and cytes, lymphocytes, and polymorphonuclear leukocytes Biolegend, San Diego, California, CA, USA). We meas- (PMNs) and the gene expression of the basophil markers ured serum tryptase (ImmunoCAP, Thermo Fisher, Upp - FCER1A, carboxypeptidase A3 (CPA3) and L-histidine sala, Sweden), and concentrations that exceeded 11.4 µg/l decarboxylase (HDC). We then proceeded to analyse were considered increased. Enumeration of circulating the chemotactic activity of chemokines that are induced basophils (CD123+HLA-DR− cells), monocytes, lym- during anaphylaxis in the controlled setting of a cellular phocytes, and PMNs in fresh whole blood was performed in vitro migration assay. via flow cytometry as previously described [7, 27]. FCER1A, CPA3, and HDC gene expression was analysed Methods in whole blood samples (PAXgene, PreAnalytiX, Hom- Study participants brechtikon, Switzerland) as previously described [7, 27]. We prospectively recruited 49 patients (24 females; Detailed methods are provided in Additional file 1. age, 19–82  years; Table  1) presenting to the Emergency Department of University Hospital Golnik, Slovenia, with Cell separation and in vitro migration assays an acute episode of anaphylaxis (January 2017 to Octo- CCR2 surface expression on basophils of healthy donors ber 2019). The reaction was caused by an insect sting was determined by anti-CCR2 mAb (CD192; Miltenyi V antur et al. Clin Transl Allergy (2020) 10:63 Page 3 of 11 Table 1 Characteristics of the patients Patients with an acute episode of anaphylaxis (n = 49) Sex, n (%) 24 female (49.0) 25 male (51.0) Age years, average (range) 50 (19–82) Trigger, n (%) Hymenoptera venom 26 (53.1) Medication 14 (28.6) Food 5 (10.2) Other 4 (8.2) Time from the onset of anaphylactic symptoms to sample collection at presentation to the emergency department, 100 (69) median (IQR) in minutes Convalescent sampling after anaphylactic episode, median (IQR) in days 80 (93) Emergency treatment, n (%) Epinephrine 25 (51.0) Corticosteroids 43 (87.8) Grade of reaction, n (%) Mueller I 6 (12.2) Mueller II 6 (14.6) Mueller III 16 (32.7) Mueller IV 21 (42.9) Symptoms, n (%) Any skin feature 44 (89.8) Any gastrointestinal feature 15 (30.6) Any respiratory feature 26 (53.1) Hypoxemia (SpO2 ≤ 92%) 7 (14.3) Any cardiovascular feature 24 (49.0) Hypotension (SBP < 90 mmHg) 19 (38.8) Acute tryptase > 11.4 µg/l, n (%) 32 (65.3) SBP systolic blood pressure Biotec, Bergisch Gladbach, Germany) staining, and For control experiments, we used recombinant CCL2 basophils were isolated from peripheral whole blood by (Thermo Fisher Scientific, Massachusetts, USA). To negative immunomagnetic selection (Miltenyi Biotec, block CCL2, we used anti-CCL2 neutralizing antibodies Bergisch Gladbach, Germany) and exactly quantified by (Sigma-Aldrich, Missouri, USA). All experiments were absolute flow cytometry basophil counts as previously independently performed in triplicate. Detailed methods described (CD123+HLA-DR− cells and microbeads) [7, are described in Additional file 1. 27]. For the migration assay, we used modified Boyden chamber and polycarbonate membrane cell culture Statistical analysis inserts (Corning Inc., New York, USA). A total of 20,000 The distribution of the data was assessed using the basophils were added to the upper wells, and the samples D’Agostino test. As the majority of the data were nonpara- to be tested were placed in the lower wells. After incuba- metric, we used the Wilcoxon or Mann–Whitney test. P tion for 30 to 150 min at 37 °C, we collected the cells that values were Bonferroni-corrected for the complete set of 19 had migrated to the lower wells and quantified them by variables, and a P-value < 0.0026 was accepted as significant. absolute basophil counts [7, 27]. Basophil migration was calculated by using the following equation: Basophil migration(%) = absolute number of migrated basophils/absolute number of seeded basophils ×100. Vantur et al. Clin Transl Allergy (2020) 10:63 Page 4 of 11 For the cellular in  vitro migration assay, all experiments 70 pg/ml) and CCL13 (113, 104 and 123 pg/ml), showed were independently performed in triplicate, and the results no differences during anaphylaxis or later compared to were compared with a t-test; a P-value < 0.05 was accepted healthy subjects (Fig. 1 and Table 2). as significant. To quantify associations between variables, we used Spearman or Pearson correlation. All analyses were Eotaxin chemokines CCL11/eotaxin, CCL24/eotaxin‑2 performed using GraphPad Prism (GraphPad Software, La and CCL26/eotaxin‑3 Jolla, CA, USA). There were no differences in CCL11 (median 200, 211 and 160 pg/ml), CCL24 (1124, 946 and 1159 pg/ml) and CCL26 Results (1903, 1898 and 1877  pg/ml) between the acute and con- The chemokine CCL2, but not other chemokines, valescent time points and the healthy subjects (Fig.  1 and is significantly increased during anaphylactic episodes Table 2). MCP chemokines CCL2/MCP‑1, CCL7/MCP‑3, CCL8/MCP‑2, and CCL13/MCP‑4 Chemokines CCL5/Rantes, CCL17/TARC, CCL21/SLC CCL2 concentrations measured during the anaphylac- and CCL22/MDC tic episode (median 514 pg/ml) were significantly higher There were no differences in CCL5 (median 40.9, 41.8 than those measured in convalescent serum samples col- and 47.0  ng/ml), CCL17 (342, 303 and 344  pg/ml), lected later (median 80 days; 284 pg/ml; P < 0.0001; Fig. 1 CCL21 (1179, 1099 and 938 pg/ml) and CCL22 (666, 717 and Table  2). This marked increase (median 106.0%; and 766 pg/ml) between the acute and convalescent time range: − 44.7% to 557.4%) was evident in 28/30 patients points and the healthy subjects (Fig. 1 and Table 2). (Fig.  2). Serum CCL2 levels during the acute episode were also significantly higher than those observed in Increase in serum CCL2 levels highly correlates healthy subjects (median 279 pg/ml, P < 0.0001); however, with an increase in serum tryptase CCL2 levels in convalescent samples showed no differ - The median concentration of serum tryptase was signifi - ence in comparison to CCL2 levels in healthy subjects cantly higher during the anaphylactic episode (12.9 µg/l) (Fig.  1 and Table  2). Other MCP chemokines, including than later or in healthy subjects (5.4 and 3.3 µg/l, respec- CCL7 (median 8.9, 8.5 and 8.1 pg/ml), CCL8 (73, 69 and tively; P ≤ 0.0005; Fig.  1 and Table  2). The median rate Fig. 1 Serum CCL2, CCL5, CCL7, CCL8, CCL11, CCL13, CCL17, CCL21, CCL22, CCL24, CCL26, and tryptase levels in 30 patients during an acute anaphylactic episode and in convalescent samples collected later V antur et al. Clin Transl Allergy (2020) 10:63 Page 5 of 11 Table 2 Laboratory data of anaphylactic patients and healthy subjects Variables median (IQR) Patients with an acute episode Healthy subjects P-value a b of anaphylaxis (n = 49) (n = 28) Acute sampling Convalescent sampling Acute vs. Acute vs. Convalescent vs. convalescent healthy healthy subjects subjects Serum chemokines (pg/ml) CCL2/MCP‑1 514 (561) 284 (128) 279 (147) < 0.0001 < 0.0001 0.9726 CCL7/MCP‑3 8.9 (2.9) 8.5 (2.1) 8.1 (1.4) 0.7549 0.1636 0.1038 CCL8/MCP‑2 73 (56) 69 (35) 70 (19) 0.1579 0.1425 0.4226 CCL13/MCP‑4 113 (81) 104 (88) 123 (103) 0.9032 0.3367 0.7648 CCL11/Eotaxin 200 (174) 211 (174) 160 (60) 0.2710 0.1641 0.0555 CCL24/Eotaxin‑2 1124 (1377) 946 (1036) 1159 (1561) 0.0940 0.8665 0.5919 CCL26/Eotaxin‑3 1903 (88) 1898 (181) 1877 (155) 0.0412 0.1746 0.4640 CCL5/Rantes 40,907 (34,588) 41,795 (27,011) 46,993 (33,574) 0.6669 0.7485 0.9659 CCL17/TARC 342 (194) 303 (203) 344 (180) 0.0148 0.7093 0.1931 CCL21/SLC 1179 (1193) 1099 (645) 938 (417) 0.2206 0.3376 0.4175 CCL22/MDC 666 (321) 717 (251) 766 (260) 0.7045 0.8014 0.5408 Serum tryptase µg/l 12.9 (10.6) 5.3 (2.0) 3.3 (2.0) < 0.0001 < 0.0001 0.0005 > 11.4 µg/l, n (%) 18 (60) 1 (3.3) 0 (0) < 0.0001 < 0.0001 1 Absolute cell count (cells/µl) Basophils 5.0 (7.3) 19.1 (10.9) – < 0.0001 – – Monocytes 396 (163) 441 (108) – 0.8417 – – Lymphocytes 1378 (1219) 1461 (470) – 0.3904 – – PMNs 3881 (2873) 2875 (1144) – 0.0388 – – Gene expression in whole blood (fold change) FCER1A 0.11 (0.24) 0.40 (0.64) – – – 0.0002 CPA3 0.22 (0.51) 0.72 (1.16) – 0.0002 – – HDC 0.11 (0.30) 0.38 (0.57) – – – 0.0018 Serum of patients used for in vitro migration assays Tryptase (µg/l) 22.6 (16.5) 6.4 (6.2) 4.3 (5.8) 0.0002 0.0003 0.5358 > 11.4 µg/l, n (%) 8 (100) 0 (0) 0 (0) 0.0002 0.0002 1 CCL2 (pg/ml) 1964 (1114) 278 (175) 302 (76) 0.0002 0.0003 0.3969 Basophils of healthy donors used for the in vitro migration assays Absolute count (cells/ – – 18.3 (6.3) – – – µl) CCR2 expression MFI – – 353 (422) – – – % CCR2 + basophils – – 94 (27) – – – PMNs polymorphonuclear leukocytes, FCER1A α subunit of the high-affinity IgE receptor, CPA3 carboxypeptidase A3, HDC histidine decarboxylase, CCR2 C-C motif chemokine receptor 2, MFI mean fluorescence intensity P values were Bonferroni-corrected, and P-value < 0.0026 was accepted as significant. Statistically significant P values are presented in boldface In 30 patients, we measured serum chemokines and tryptase, and in 26 patients, we measured blood absolute cell counts and gene expression (of those 41 patients, 15 were in both subgroups); serum samples from 8 patients were used for in vitro migration experiments In 20 healthy subjects, we measured serum chemokines and tryptase; basophils from 8 healthy subjects were used for in vitro migration experiments of increase of serum tryptase was 133.8% (range: − 6.6% rates of increase of serum CCL2 and tryptase (r = 0.68, to 893.4%; increase in 28/30 patients), which was com- P < 0.0001) and between the acute concentration of serum parable to the increase in serum CCL2 levels (Fig.  2). CCL2 and the acute concentration of serum tryptase There was a significant positive correlation between the (r = 0.77, P < 0.0001; Fig. 2). Vantur et al. Clin Transl Allergy (2020) 10:63 Page 6 of 11 Fig. 2 Comparison and correlation between serum CCL2 and tryptase levels in 30 patients with acute anaphylactic episodes Anaphylactic episodes involve a substantial reduction compared to convalescent blood samples collected later in circulating basophils (Fig. 3). Basophils, monocytes, lymphocytes, and PMNs The absolute number of circulating basophils was sig - Gene expression of basophil markers nificantly lower during the anaphylactic episode (median We observed significantly lower whole blood gene 5.0 cells/µl) than in convalescent blood samples collected expression of FCER1A, CPA3, and HDC during the ana- later (19.1  cells/µl, P < 0.0001; Fig.  3 and Table  2). This phylactic episode than in convalescent samples (respec- decrease (median 65.6%, range 30.0–98.6%) was evident tively; P ≤ 0.0018; Fig. 3 and Table 2). Acute whole blood in all patients. There were no significant differences in gene expression of FCER1A, CPA3, and HDC significantly the monocyte (median 396 and 441 cells/µl), lymphocyte correlated with the acute absolute number of circulating (1378 and 1461 cells/µl), and PMN (3881 and 2875 cells/ basophils (r = 0.53, 0.79 and 0.81, respectively; P ≤ 0.005; µl) absolute cell counts during the acute episode Additional file 1: Fig. S1). Fig. 3 Absolute basophil, monocyte, lymphocyte and PMN counts and whole blood FCER1A, CPA3, and HDC gene expression in 26 patients during the acute anaphylactic episode and in convalescent samples collected later. PMNs polymorphonuclear leukocytes, FCER1A α subunit of the high‑affinity IgE receptor, CPA3 carboxypeptidase A3, HDC histidine decarboxylase V antur et al. Clin Transl Allergy (2020) 10:63 Page 7 of 11 Anaphylactic serum enhances the in vitro migration observed for other chemokines, which were previously of basophils via CCL2-dependent chemotactic activity implicated in different allergic diseases or during aller - A basophil migration assay was performed using MACS- gic inflammation [15–25]. Thus, the chemokine patterns separated human basophils and a modified Boyden of CCL5, CCL7, CCL8, CCL11, CCL13, CCL17, CCL21, chamber, with quantification by absolute flow cytom - CCL22, CCL24, and CCL26 might be of limited impor- etry basophil counts. Basophils from all healthy donors tance for anaphylaxis. While activation of the pathways showed high surface expression of CCR2 (Table  2). All associated with multiple inflammatory cytokines, includ - experiments were performed independently in triplicate ing IL-2, IL-6, IL-10, and TNFRI, were observed during (detailed information is provided in Additional file 1). anaphylaxis [6, 9], the selective CCL2 chemokine cas- cade might be important for chemotactic activity during Basophil migration induced by HBSS and rCCL2 anaphylactic reactions. It was recently reported that the As shown in Fig. 4a, during a 150-min incubation time with CCL2-FcεRI-histone deacetylase 3 signalling axis medi- Hank’s balanced salt solution (HBSS), only a small number ates passive anaphylaxis by increasing the expression of of basophils (mean, SD), 0.8 ± 0.7%) transmigrated across CCL2 in mast cells [28, 29]. After allergen stimulation, the polycarbonate membrane. Basophil migration was sig- histone deacetylase 3 binds to FcεRI, and histone deacet- nificantly enhanced by the presence of 10  nM rCCL2 and ylase 3 increases the expression of SP1 and C-Jun, which 50 nM rCCL2 (8.1 ± 4.0% and 14.0 ± 8.1%; 150 min of incu- bind to the promoter sequences of CCL2 to increase the bation); however, after blocking with anti-CCL2 mAbs, expression of CCL2 [28, 29]. Conversely, in the absence rCCL2-induced migration was almost completely inhibited of allergen stimulation, histone deacetylase 3 binds to the (0.8 ± 0.6%, P = 0.036 and 0.8 ± 0.1%, P = 0.047, blocking at promoter sequences of CCL2 to suppress the expression 10 nM and 50 nM of rCCL2, respectively). of CCL2 [28]. Our study design and the median time between symp- Basophil migration induced by anaphylactic tom onset and sample collection were highly comparable and convalescent serum with those of previous studies of anaphylaxis [3, 6, 7, 9, We compared the effects of acute serum collected during 30]. This suggests that this is an unbiased comparison, the anaphylactic episode and convalescent serum collected including the confirmation that the chemokines CCL5 later (Table 2) on basophil migration. After 150 min of incu- and CCL11, which may affect other important effector bation, the number of transmigrated basophils was signifi - cells of allergic inflammation such as eosinophils, are cantly higher (twofold) in the presence of acute serum in the not induced during anaphylaxis [6, 7]. The nature of the lower wells than in the presence of convalescent serum in management of anaphylaxis (including administration of the lower wells (mean, SD: 54.0 ± 19.3% vs. 28.3 ± 11.7%, high-dose corticosteroids) makes it difficult to exclude respectively; P = 0.01) (Fig.  4b). The number of transmi - the potential confounding effects of treatment. How - grated cells increased linearly until 150  min of incubation ever, it was previously demonstrated that serum CCL2, and then plateaued (Fig. 4c). A neutralizing antibody against CCL5, and CCL11 levels are not affected by corticoster - CCL2 significantly blocked basophil transmigration induced oid treatment [7]. Nevertheless, corticosteroid treatment by anaphylactic serum (37.7 ± 20.8% vs. 26.1 ± 19.7%; or the time between symptom onset and sample collec- P = 0.001). However, a neutralizing antibody against CCL2 tion may have affected the levels of the other measured did not show any blocking effect on the chemotactic chemokines. response induced by convalescent serum (32.5 ± 12.5% vs. In the present study, we could not determine the cel- 30.7 ± 18.3%) (Fig. 4b). lular sources of CCL2 during anaphylaxis. However, we showed a highly significant positive correlation between Discussion elevated blood levels of acute CCL2 and tryptase and Our study demonstrated that there is a selective and sig- strong similarities between the percentage increases nificant increase in serum CCL2 chemokine levels dur - of CCL2 and tryptase from convalescent serum sam- ing anaphylactic reactions. No significant changes were ples. Tryptase is largely mast cell-derived, and mature (See figure on next page.) Fig. 4 a–c Eec ff ts of CCL2 and acute and convalescent serum from patients with an anaphylactic episode on basophil in vitro migration. Basophil migration after 150 min of incubation: a in the presence of rCCL2 in the lower wells and after CCL2 blocking and b in the presence of acute or convalescent serum in the lower wells and after CCL2 blocking. c Time course of basophil migration (after 30, 60, 90, 120, and 150 min of incubation) in the presence of acute and convalescent serum in the lower wells. All experiments were independently performed in triplicate, with a basophils from 5 healthy donors; b basophils from the same 5 healthy donors and serum samples from 5 patients and c basophils from 3 healthy donors and serum samples from 3 patients (details in Additional file 1: Table S1). HBSS Hank’s balanced salt solution Vantur et al. Clin Transl Allergy (2020) 10:63 Page 8 of 11 V antur et al. Clin Transl Allergy (2020) 10:63 Page 9 of 11 β-tryptase is stored in mast cell granules and released where the activation and degranulation of mast cells upon mast cell activation during acute anaphylaxis [2, occur, thereby contributing to the clinical presentation of 5–7]. Therefore, it is tempting to speculate that the the sites of allergic reaction. This is consistent with clini - blood CCL2 increase that occurs during anaphylaxis is cal observations of different severities and end-organ pat - IgE related and mainly mast cell derived. In vitro activa- terns of anaphylaxis, which suggest that local rather than tion of human mast cells by anti-IgE induces secretion generalized mast cell and/or basophil degranulation may of CCL2 after 30 min of stimulation, and this increase is predominate in some individuals [41]. However, a sub- observed earlier than TNF-α, IL-13, or GM-CSF mast cell stantially broader assessment is needed to explore and secretion, peaking 90 min after anti-IgE stimulation [31]. confirm CCL2-mediated cellular crosstalk during acute Furthermore, mast cell CCL2 secretion is induced at low allergic reactions. allergen concentrations or in the context of low receptor Although CCL2 displays major chemotactic activity for occupancy with IgE [32], and due to the short time frame monocytes and interferes with the egress of monocytes within which the increase in serum CCL2 was observed from the bone marrow to the circulation during homeo- in the current and previous studies [7], rapid induction stasis and inflammation [42], no changes in circulating of CCL2 production by the allergen-IgE axis might be monocytes were observed during anaphylaxis. However, feasible. Nevertheless, CCL2 is produced by a variety of studies in mice have shown that depletion of monocytes/ cell types, including endothelial cells and smooth muscle macrophages can reduce anaphylaxis in both IgG-medi- cells [33], astrocytes as a major source of CCL2 during ated passive models and active models [2, 11]. central nervous system inflammation [34] and cancer and stromal cells as a major source of CCL2 in tumour micro- Conclusion environments [35]; thus, the cellular sources of CCL2 In conclusion, our data suggest that during anaphylaxis, during anaphylaxis are currently unknown. Additional an increase in the chemokine CCL2 occurs, which corre- and substantially studies of mast cell CCL2 expression lates with CCL2-mediated chemotactic activity in baso- and secretion are required to confirm this speculation. phils and substantial migration of circulating basophils. The results of experimental allergen challenge in the Our findings imply an important and specific role for nose, airways, and skin have demonstrated that there CCL2 in the pathophysiology of human anaphylaxis. is an influx of basophils to inflammatory sites several hours after allergen exposure [36–39]. Furthermore, it Supplementary information was recently demonstrated that airway allergen chal- Supplementary information accompanies this paper at https ://doi. org/10.1186/s1360 1‑020‑00367 ‑2. lenge induces TLR-dependent CCL2 production in the lungs and that circulating basophils are recruited to the Additional file 1. Additional methods and results. Table S1. Detailed lungs by CCL2-mediated transendothelial migration and information on the number of participants for whom we assessed dif‑ a chemotactic gradient [40]. Our study demonstrated sig- ferent laboratory parameters. Additional results. Figure S1. Correlation nificantly enhanced CCL2-mediated chemotactic activity between absolute basophil counts and whole‑blood FCER1A, CPA3, and HDC gene expression in 26 anaphylactic patients of anaphylactic serum for the migration of isolated baso- phils. Conversely, no CCL2-mediated basophil chemo- tactic activity was observed for the convalescent serum Abbreviations CCL: C‑ C Motif Chemokine Ligand; CCR: C ‑ C motif chemokine receptor; CPA: collected later after the reaction. The CCL2-mediated Carboxypeptidase A3; FCER1A: α subunit of the high‑affinity IgE receptor; basophil chemotactic activity of anaphylactic serum and HBSS: Hank’s balanced salt solution; HDC: l ‑Histidine decarboxylase; MCP: the reduction in circulating basophils during anaphylaxis, Monocyte chemoattractant protein; MDC: Macrophage‑Derived Chemokine; PMNs: Polymorphonuclear leukocytes; SLC: Secondary Lymphoid Tissue coupled with the previous finding that basophils are the Chemokine; TARC : Thymus and Activation Regulated Chemokine. granulocytes most resistant to apoptosis [41], suggest that anaphylaxis induces rapid CCL2-mediated basophil Acknowledgements Not applicable. migration rather than elimination by apoptosis. Moreo- ver, anaphylaxis-related basophil migration appears to be Authors’ contributions selective because no significant changes were observed PKorosec, RV and AK designed the study. PKopac and RE evaluated the patients and revised the manuscript. RV, MRihar, AK, MRijavec, and UBS for monocytes, lymphocytes, PMNs, or chemokines performed laboratory tests and experiments and revised the manuscript. that may affect other effector cells, and the decreased PKorosec and RV wrote the manuscript. All authors read and approved the gene expression of basophil markers FCER1A, CPA3 and final manuscript. HDC corroborates the flow cytometry data. Therefore, Funding it is tempting to speculate that CCL2 might be mast cell The research was supported by the Slovenian Research Agency (Grant Nos. derived and that circulating basophils influx to the sites P3‑0360 and 39238). Vantur et al. Clin Transl Allergy (2020) 10:63 Page 10 of 11 Availability of data and materials 15. Kuo C‑H, Collins AM, Boettner DR, Yang Y, Ono SJ. Role of CCL7 in type I Raw data were generated at the University Clinic of Respiratory and Allergic hypersensitivity reactions in murine experimental allergic conjunctivitis. J Diseases, Golnik, Slovenia. 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