Synergistic Activation of Src, ERK and STAT Pathways in Pbmcs for Staphylococcal Enterotoxin a Induced Production of Cytokines and Chemokines

ORIGINAL ARTICLE Asian Pacific Journal of Allergy and Immunology

Synergistic activation of Src, ERK and STAT pathways in PBMCs for Staphylococcal enterotoxin A induced production of cytokines and chemokines

Xueting Liu,1 Yuhuan Wen,1 De Wang, 1 Zhongqiu Zhao,2,3 Joseph Jeffry,2 Liping Zeng,1 Zehong Zou,1 Huifang Chen,1 Ailin Tao1

Abstract

Background: Staphylococcal enterotoxin A (SEA) is a well-known superantigen and stimulates human peripheral blood mononuclear cells (PBMCs) involving in the pathogenesis of inflammatory disorders and cancer.

Objective: To better understand the biological activities of SEA and the possible intracellular mechanisms by which SEA plays its roles in conditions like staphylococcal inflammatory and/or autoimmune disorders and immunotherapy.

Methods: Recombinant SEA (rSEA) was expressed in a prokaryotic expression system and its effects on the cytokine and chemokine production was examined by Enzyme-linked Immunospot (ELISpot) Assay and ELISA analysis.

Results: In vitro experiments showed rSEA could significantly enhance secretion of a broad spectrum of cytokines and chemokines from PBMCs dose-dependently. Increased secretion of cytokines and chemokines from rSEA stimulated PB- MCs was barely affected by C-C motif chemokine receptor 2 (CCR2) antagonist INCB3344. However, Src, ERK and STAT pathway inhibitors were able to successfully block the enhanced secretion of most of cytokines and chemokines produced by rSEA stimulated PBMCs.

Conclusions: Our work suggested that rSEA serves as a potent stimulant of PBMCs, and induces the release of cytokines and chemokines through Src, ERK and STAT pathways upon a relatively independent network. Our work also strongly supported that Src, ERK and STAT signaling inhibitors could be effective therapeutic agents against diseases like toxic shock syndrome or infection by microbes resistant to antibiotics.

Key words: superantigen, staphylococcal enterotoxin A, PBMCs , cytokines and chemokines, Src, ERK and STAT pathways.

From: Corresponding author: 1 The Second Affiliated Hospital, The State Key Laboratory of Ailin Tao Respiratory Disease, Guangdong Provincial Key Laboratory of Allergy The Second Affiliated Hospital, The State Key Laboratory of & Clinical Immunology, Sino-French Hoffmann Institute, Center for Respiratory Disease, Guangdong Provincial Key Laboratory of Allergy Immunology, Inflammation, & Immune-mediated disease, Guangzhou & Clinical Immunology, Sino-French Hoffmann Institute, Center for Medical University, Guangzhou 510260, China. Immunology, Inflammation, & Immune-mediated disease, Guangzhou 2 Center for the Study of Itch, Department of Anesthesiology, Medical University, Guangzhou 510260, China. Washington University School of Medicine, E-mail: [email protected] 3 Barnes-Jewish Hospital, St. Louis, MO 63110, USA

Introduction Staphylococcal enterotoxins (SEs) include more than 20 stimulation of antigen presenting cells and T-cells.1,2 As a re- distinct members, such as Staphylococcal enterotoxin A (SEA) sult, these activated immune cells produce and release mas- and Staphylococcal enterotoxin B (SEB). The SEA is able to sive T cell mediators, cytokines and chemokines, etc,1-3 in- activate a large population of both CD4+ and CD8+ T cells at volving in the pathogenesis of a variety of human diseases, very low concentrations.1,2 It is commonly accepted that SEs ranging from food poisoning,4 inflammatory and/or autoim- bind to major histocompatibility complex (MHC) class II on mune disorders,5,6 to toxic shock,7 and even cancer.8 Activation antigen presenting cells and the variable region of specific Vβ of protein tyrosine kiases (PTKs) is a common step of T cell regions of T-cell receptors, sequentially triggering excessive response to superantigen stimulation.

52 SEA activate PBMC via Src, ERK and STAT

Systemic cytokine and chemokine responses induced by Methods and Materials SEs have been demonstrated by numerous clinical and animal For detecting Human IL-1β, IL-2, IL-4, IL-6, IL-8, IFN-γ, studies, however, whether these cytokines and chemokines can TNF-α, CCL-2, MIP1A, and IP-10, their ELISA kits were pur- interact through a form of cross-talk, and thereafter influence chased from R&D Systems (Minneapolis, Minnesota, USA). the course of the diseases remained inconclusive. For exam- ELISpot kits for human IFN-γ and IL-4 were purchased from ple, medically relevant staphylococci, especially hospital- or Mabtech AB (Sweden). Src-family kinases inhibitor PP2 and community-associated methicillin resistant Staphylococcus au- MEK/ERK inhibitor U0126 were obtained from Selleck Chem- reus (MRSA), would lead to or aggravate septic shock, with a ical Company (Houston, Texas, USA). Pyridone 6 (P6), a pan- dysregulated response called “cytokine storm” or “cytokine re- JAK inhibitor, was purchased from Merck Millipore (Billerica, lease syndrome”. Despite its pivotal role in causing sepsis, the Massachusetts, USA). CCR2 antagonist INCB3344 was ob- pathophysiological role of “cytokine storm” in regulating the tained from MedChem Express (New Jersey, USA). Peripheral expression of chemokines or vice versa, is poorly understood.9 blood samples were from 12 healthy adult blood donors (6 Secondly, it has been reported that SEs stimulation of human female and 6 male, ranging in age from 18 to 60 years) at the peripheral blood monocyte cells (PBMCs) results in a rapid and Second Affiliated Hospital of Guangzhou Medical University. dose-dependent down-regulation of chemokine receptor (i.e. All donors’ health condition was evaluated by the medical of- CCR1, CCR2, and CCR5),10 mediated by TNF-α,10 in a chemo- ficials. The study protocol was reviewed and approved by the kine ligand-independent mechanism.11 It is unclear whether Ethical Committee of the Guangzhou Medical University and such a chemokine receptor down-regulation would alter the written informed consent was obtained from each participant subsequent cytokine and chemokine release and activities. before being included in this study. Lastly, there are at least three intracellular signaling pathways of T-cell activation upon superantigen binding to exert their Expression, puriﬁcation and identiﬁcation of recombinant potent effects, the phosphoinositide 3 kinase (PI3K)/mam- SEA. malian target of rapamycin (mTOR), NFκB and MAPK path- The procedure of construction, expression, and character- ways.2,12-14 Whether the global cytokine and chemokine respons- ization of the recombinant SEA was similar to our recent re- es induced by SEs share the same or differential intracellular port.19 Staphylococcus aureus strain ATCC-13565 was used to signaling pathways has remained poorly defined. amplify the SEA gene for cloning and expression as previous- The recombinant SEs were able to provide well character- ly described.20 Genomic DNA was extracted from the bacte- ized and quantified stimulants, and significantly higher and ria by using GenElute bacterial genomic DNA kit (Sigma, St invariable levels of cytokine and chemokine release than those Louis, USA). The mature peptide of the SEA coding region challenged or vaccinated by native SEs.4,15,16 The larger magni- was decided according to Swiss-Prot Accession No. H6URY2 tude of expression of cytokines and chemokines induced by and amplified with the following pair of primers: 5’-AGCGA recombinant SEs should provide more insights for the toxin- GAAAAGCGAAGAAAT-3’ and 5’-ACTTGTATATAAATATAT induced cascades. Superantigens can induce immune cell ac- AT-3’. The PCR were purified and further modified by adding tivation and subsequent cellular changes, the same as conven- the NdeI and XhoI restriction sites and Strep-tag II sequence tional antigens. Cytokines, growth factors, chemokines released with the following PCR primers: F2 5’-GGAATTCCATATGA from immune cells would subsequently activate intracellular GCGAGAAAAGCGAAGAAAT-3’ (with NdeI site underlined); signaling pathways, resulting in inflammation and tissue dam- R2, 5’-CCGCTCGAGTTATTATTTTTCGAACTGCGGGTGG ages. Our previous work found that SEA could stimulate hu- CTCCAACTTGTATATAAATATATAT-3’ (with XhoI site un- man PBMCs to release a wide range of cytokines, and immune derlined and Strep-tag II sequence in bold italic). The modi- cell cross-talk-dependent activation of ERK and STATs in PB- fied products were inserted into the pET44a protein expression MCs.3 Other researches demonstrated that Src family kinases vector. The rSEA was expressed in the Escherichia coli strain mediate the phosphorylation of STATs and play a critical role Rosetta-gami 2 and purified by using Strep-Tactin affinity in signal transduction pathways16 and that Src regulates the chromatography and its sequences were identified by liquid expression of IL-1β, IL-6 and IL-8 and these effects of Src are chromatography with mass spectrometry analysis (LC-MS/ mediated through activation of ERK-MAPK pathway.17 Our re- MS Analysis). To remove any contaminating LPS, rSEA protein cent work on allergen rMal f 1 proved that single antigen can was purified by High-Capacity Endotoxin Removal Resin simultaneously predispose DCs bias toward different pathways (Thermo Fisher Scientific). The LPS content was analyzed by to inflammation.18 IL-1β and TNF-α are relating to cell death the Tachypleus Amebocyte Lysate (TAL) method. Samples that and inflammasome activation; IL-2 is part of the body’s natu- contained less than 0.5 EU/mg LPS were deemed to be suitable ral response in discriminating between “self” and “non-self” for use in the following assays. via its direct effects on T cells. IL-8, CCL2 (MCP1) and MI- P1A (CCL3) recruit immune cells to the sites of inflammation Cytokines Analysis by Enzyme-linked Immunospot (ELISpot) as response to tissue injury or infection. IL-6 and TNF-α have Assay potential bias towards TH22/TH17 while IL-4 towards TH2 and The procedure was carried out similar to our previous 3 IFN-γ towards TH1 (IP-10 is secreted by monocytes in response work. Briefly, PBMCs were separated from heparinized blood to IFN-γ), respectively. In this work, we therefore aimed to obtained from healthy donors by Ficoll-Paque PLUS (GE decipher all the possible inflammatory pathways triggered by Healthcare, USA) density gradient centrifugation. Generally in SEA by detecting the above-mentioned cytokines and chemo- this study PBMC were seeded in triplicates into wells for each kines. test and each experiment was repeated three times.

53 Asian Pac J Allergy Immunol 2020;38:52-63 DOI 10.12932/AP-220818-0396

Human IFN-γ and IL-4 secreting cell numbers were de- LC-MS/MS analysis. The Full-scan mass spectrum of a repre- termined using ELISpot kits. Briefly, 96-well nitrocellulose- sentative peptide fragment in the tryptic digests derived from bottomed plates were coated with murine anti-human IFN-γ rSEA (T3) was shown in Figure 1B. (1-D1K) or IL-4 monoclonal antibody (IL-4I) at a concentration of 15 μg/mL in PBS and incubated at 4°C. After 24 h, the SEA increases IFN-γ and IL-4-secreting cell numbers in human plates were washed 5 times with sterile PBS and blocked with PBMCs by different concentrations RPMI containing 10% fetal bovine serum (FBS). Then PBMCs ELISpot assay is a widely used method for quantification were distributed into 96-well nitrocellulose-bottomed plates of cytokine-secreting cell numbers and monitoring cellular (3 × 105 cells/well) with addition of rSEA at different concen- immune responses.21 In the present study, human PBMC (3 × trations from 1 pg/mL to1 μg/mL and we used anti-CD3mAb 105 cells/well) were incubated for 18 h in the absence (served (100ng/mL) as a positive control. as controls) or presence of rSEA (at concentrations from 1 pg/ After an 18 h incubation and being washed five times with mL to 1 μg/mL) and the number of spot forming cells (SFC) se- PBST (10 mmol/L PBS pH7.4, 0.1% Tween-20), the plates creting IFN-γ (Figure 1C) and IL-4 (Figure 1D) were analyzed were incubated with biotinylated anti-human IFN-γ (7-B6-1) by ELISpot assays. The results demonstrated that significantly or IL-4 mAb (IL-4II) at a concentration of 1 μg/mL at room higher numbers of IFN-γ (Figure 1C) and IL-4 (Figure 1D) temperature for 1h, followed by streptavidin-horseradish per- spots were detected in PBMCs incubated with rSEA than that oxidase (1:1000 in dilution buffer) incubation at room tem- in controls. Also the number of spots secreting IFN-γ and IL-4 perature for 1h and washed as previously. Finally, the color was were seen in a dose-dependent manner stimulated by rSEA with developed by TMB solution, after the spots were developed PBMCs (Figure 1C-D). for 10 to 15 min, the plates were washed with distilled water and air-dried. The number of antigen-specific IFN-γ-secreting SEA induces the secretion of cytokines and chemokines in hu- (or IL-4-secreting) spot forming cells was determined with a man PBMCs at different time points computer-assisted video imaging ELISpot reader (AID, Ger- As the ELISpot data shown in Figure 1C-D, 100 ng/mL man). of rSEA was the intermediate concentration to induce cytokines release by PBMCs. This concentration was decided as the Cytokines Analysis by ELISA dose for further rSEA and PBMCs co-culture assay and time- Freshly isolated PBMCs in 24-well plates (5 × 105 cells/ course experiments to investigate more kinds of cytokines and well) were stimulated by rSEA at 100 ng/mL for 15 min and chemokines secreted from PBMCs. Briefly, PBMCs (5 × 105 1, 3, 6 and 24 h. Then cell culture supernatants were harvest- cells/well) were stimulated with 100 ng/mL rSEA for 15 min ed and cytokine levels were measured by ELISA. IL-1β, IL-2, and 1, 3, 6 and 24 h. A more automated assay,21 ELISA was IL-4, IL-6, IL-8, IFN-γ, TNF-α, CCL-2, MIP1A and IP-10 used to measure the levels of cytokines IL-1β, IL-2, IL-4, IL- concentrations in cell culture supernatants were measured us- 6, IFN-γ, TNF-α and chemokines CCL-2, IL-8, MIP1A, IP-10 ing ELISA assay following the manufacturer’s instructions. in cell culture supernatants. The results showed all cytokines For experiments with CCR2 antagonist INCB3344, Src-fam- and chemokines detected increased gradually over time, and ily kinases inhibitor PP2, MEK/ERK inhibitor U0126 or pan- peaked at 24 h. (Figure 2A, B). JAK inhibitor P6 treatment, PBMCs were pre-incubated with INCB3344 (10 nM, 100 nM, 1 μM, 10μM), PP2 (0.4, 2, 10μM), A kDa U0126 (0.4, 2, 10, 20 μM) or P6 (5 nM, 50 nM, 500 nM, 5 μM) for 2 h and then stimulated with 100 ng/mL rSEA for 24 h still 97.4 in the presence of INCB3344, PP2, U0126 or P6. 66.2 Statistical Analysis GraphPad Prism Software was used for statistical compar- isons with one-way ANOVA followed by post hoc analysis. All 45 quantitative data was presented as mean ± SEM, which were from three independent experiments. Statistical significance was concluded if P value < 0.05. rSEA 31 Results Production of recombinant SEA In the present study, to obtain a large quantity and high 21 purity of materials, the size of 31 kDa SEA protein was produced in the Escherichia coli and purified by using Strep-Tac- 1 M tin affinity chromatographyFigure ( 1A). After purification by High-Capacity Endotoxin Removal Resin, the concentration Figure 1. In vitro activation of human PBMCs stimulated by of LPS was 0.061 EU/mg as detected by the Tachypleus Ame- different concentrations of rSEA. bocyte Lysate (TAL) method. To identify the peptide sequenc- A, SDS-PAGE analysis of purified rSEA. es, rSEA was processed with tryptic digestion and subsequent

54 SEA activate PBMC via Src, ERK and STAT

B 821.09 100 T3: 15KSELQGTALGNLKQIYYYNEK35 90 (MW = 2459.24)

70 1055.58 1491.67 879.38 1120.52 60 992.46 553.26 716.32 50 390.20 1212.65 40 373.17 641.88 1282.75 Relative Abundance Relative 186.12 30 528.29

20 1532.77 1340.75 10 1646.87 1875.97 2375.92 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 m/z C rSEA (μg/mL) 0 10-6 10-5 10-4

IFN-γ 10-3 10-2 10-1 1 anti-CD3

400

300 * ** ** ** 200 ** **

100 ** IFN-γ SFC/10000 PBMC

0 0 10-6 10-5 10-4 10-3 10-2 10-1 1 anti-CD3 rSEA (μg/mL) Figure 1. (Continued) B, Full-scan ion evaporation mass spectra of T3 peptide fragments derived from the tryptic digests of rSEA. C-D, Measurement of IFN-γ (C) and IL-4 (D) secretion by ELISpot assay. Representative images of ELISpot wells were shown on the top and histograms of IFN-γ and IL-4 secreting spot forming cells (SFC) per 104 PBMC were shown on the bottom. rSEA were used at concentrations from 1 pg/mL to 1 μg/mL. In C-D, * P < 0.05, ** P < 0.01 compared to PBMCs stimulated with PBS (one-way ANOVA followed by post hoc analysis, referred as “ANOVA” in following figure legends).

55 Asian Pac J Allergy Immunol 2020;38:52-63 DOI 10.12932/AP-220818-0396

D rSEA (μg/mL) 0 10-6 10-5 10-4

IL-4 10-3 10-2 10-1 1 anti-CD3

250 ** * 200 * * 150 * 100 * * 50 * IL-4 SFC/10000 PBMC IL-4

0 0 10-6 10-5 10-4 10-3 10-2 10-1 1 anti-CD3 rSEA (μg/mL) Figure 1. (Continued)

1400 500 4000 A *** *** *** 1200 *** 400 ** 1000 3000 800 300 2000 ** 600 *** 200 IL-2 (pg/mL) IL-2 IFN-γ (pg/mL)

** TNF-α (pg/mL) 400 1000 100 200 ** * 0 0 0 0 15min 1h 3h 6h 24h 0 15min 1h 3h 6h 24h 0 15min 1h 3h 6h 24h

600 160 ** ** 160 *** 500 *** 120 400 120 ***

300 80 ** 80

200 (pg/mL) IL-4 (pg/mL) IL-6 IL-1β (pg/mL) IL-1β 40 40 * 100 ** * 0 0 0 0 15min 1h 3h 6h 24h 0 15min 1h 3h 6h 24h 0 15min 1h 3h 6h 24h Figure 2. rSEA induces the secretion of cytokines and chemokines in human PBMCs at different time points. A, rSEA induced secretion of cytokines (IL-2, IFN-γ, TNF-α, IL-1β, IL-4 and IL-6) in human PBMCs.

56 SEA activate PBMC via Src, ERK and STAT

B 60 *** 600 50 500 *** *** ** 40 400 * 30 ** 300

20 (pg/mL) IL-8 200 CCL-2 (ng/mL) CCL-2

10 * 100 0 0 0 15min 1h 3h 6h 24h 0 15min 1h 3h 6h 24h

90 300 * * *** * 250 80 *** 200

70 * 150

100 IP-10 (pg/mL) IP-10 MIP-1α (pg/mL) MIP-1α 60 50 *

50 0 0 15min 1h 3h 6h 24h 0 15min 1h 3h 6h 24h Figure 2. (Continued) B, rSEA induced the secretion of chemokines (CCL-2, IL-8, MIP1A, IP-10) in human PBMCs. * P < 0.05, ** P < 0.01 , ***P < 0.001 compared with PBMCs stimulated without rSEA (ANOVA).

A 800 400 3000 * 600 * 300 2000 400 200 IL-2 (pg/mL) IL-2 IFN-γ (pg/mL) TNF-α (pg/mL) 1000 200 100

rSEA 0 rSEA 0 rSEA 0 (100ng/mL) - + + + + + (100ng/mL) - + + + + + (100ng/mL) - + + + + + INCB3344 INCB3344 INCB3344 (nM) - - 10 100 1000 10000 (nM) - - 10 100 1000 10000 (nM) - - 10 100 1000 10000

12 80 160

10 * 60 * 120 8

6 40 80

4 (pg/mL) IL-4 (pg/mL) IL-6 IL-1β (pg/mL) IL-1β 20 40 2

rSEA 0 rSEA 0 rSEA 0 (100ng/mL) - + + + + + (100ng/mL) - + + + + + (100ng/mL) - + + + + + INCB3344 INCB3344 INCB3344 (nM) - - 10 100 1000 10000 (nM) - - 10 100 1000 10000 (nM) - - 10 100 1000 10000 Figure 3. Effect of CCR2 antagonist INCB3344 on the productionof rSEA-induced cytokines and chemokines. PBMCs were pre-incubated with INCB3344 (10 nM, 100 nM, 1 μM and 10 μM) for 2 h and then stimulated with 100 ng/mL rSEA for 24 h still in the presence of INCB3344. Determination the concentrations of detected cytokines (IL-1β, IL-2, IL-4, IL-6, IFN-γ, TNF-α) (A) and chemokines (CCL-2, IL-8, MIP1A, IP-10)

57 Asian Pac J Allergy Immunol 2020;38:52-63 DOI 10.12932/AP-220818-0396

B 50 3000 40 * 2000 30

IL-8 (pg/mL) IL-8 1000 CCL-2 (ng/mL) CCL-2 10

rSEA 0 rSEA 0 (100ng/mL) - + + + + + (100ng/mL) - + + + + + INCB3344 INCB3344 (nM) - - 10 100 1000 10000 (nM) - - 10 100 1000 10000

120 300

80 200

40 100 IP-10 (pg/mL) IP-10 MIP-1α (pg/mL) MIP-1α

rSEA 0 rSEA 0 (100ng/mL) - + + + + + (100ng/mL) - + + + + + INCB3344 INCB3344 (nM) - - 10 100 1000 10000 (nM) - - 10 100 1000 10000 Figure 3. (Continued) (B) in cell culture supernatants by ELISA. * P < 0.05 compared with PBMCs stimulated with 100 ng/mL rSEA without INCB3344 (ANOVA).

A 800 4000 400 * 600 3000 *** 300

400 2000 200 IL-2 (pg/mL) IL-2 IFN-γ (pg/mL) TNF-α (pg/mL) 200 100 1000

*** *** *** rSEA 0 rSEA 0 rSEA 0 (100ng/mL) - + + + + (100ng/mL) - + + + + (100ng/mL) - + + + + PP2 (μM) - - 0.4 2 10 PP2 (μM) - - 0.4 2 10 PP2 (μM) - - 0.4 2 10

30 200 80 25 160

20 * 60 120 15 * * 40 ** 80

10 (pg/mL) IL-4 (pg/mL) IL-6 IL-1β (pg/mL) IL-1β 20 5 40 *** rSEA 0 rSEA 0 rSEA 0 (100ng/mL) - + + + + (100ng/mL) - + + + + (100ng/mL) - + + + + PP2 (μM) - - 0.4 2 10 PP2 (μM) - - 0.4 2 10 PP2 (μM) - - 0.4 2 10

Figure 4. Effect of Src-family kinases inhibitor PP2 on rSEA-induced cytokine and chemokine production. PBMCs were pre-incubated with PP2 (0.4, 2, 10 μM) for 2 h and then stimulated with 100 ng/mL rSEA for 24 h still in the presence of PP2. Determination the concentrations of detected cytokines (IL-1β, IL-2, IL-4, IL-6, IFN-γ, TNF-α) (A) and chemokines (CCL-2, IL-8, MIP1A, IP-10)

58 SEA activate PBMC via Src, ERK and STAT

B 30 3000 25 * 20 2000

15 * **

10 (pg/mL) IL-8 1000 CCL-2 (ng/mL) CCL-2 5

rSEA 0 rSEA 0 (100ng/mL) - + + + + (100ng/mL) - + + + + PP2 (μM) - - 0.4 2 10 PP2 (μM) - - 0.4 2 10

120 300

80 200

40 ** IP-10 (pg/mL) IP-10 100 MIP-1α (pg/mL) MIP-1α ***

rSEA 0 rSEA 0 (100ng/mL) - + + + + (100ng/mL) - + + + + PP2 (μM) - - 0.4 2 10 PP2 (μM) - - 0.4 2 10 Figure 4. (Continued) (B) in cell culture supernatants by ELISA. * P < 0.05, ** P < 0.01, ***P < 0.001 compared with PBMCs stimulated with 100 ng/mL SEA without PP2 (ANOVA).

Effect of CCR2 antagonist INCB3344 on rSEA-induced cyto- Effect of Src-family kinases inhibitor PP2 on the production of kine and chemokine production rSEA-induced cytokines and chemokines A previous study reported INCB3344 as a novel, potent and Next, we used antagonists or inhibitors of intracellular selective CCR2 antagonist; it inhibited the binding of CCL2 signaling pathways to further characterize the pathway of to mouse PBMCs in a dose-dependent manner.22 Here we rSEA-induced cytokine release. A Src-family kinases inhibitor also used INCB3344 to examine whether blocking the CCR2 (PP2)23 was used and the results of ELISA assays indicated that would significantly influence the production of rSEA-induced Src-family kinases inhibitor PP2 (2-10 μM) could inhibit the cytokines and chemokines. The results of the ELISA assays in- secretion of all detected rSEA-induced cytokines (IL-1β, IL-2, dicated the CCR2 antagonist INCB3344 was barely able to sig- IL-4, IL-6, IFN-γ, TNF-α) and chemokines (CCL-2, IL-8, MI- nificantly change the production of all cytokines and chemok- P1A, IP-10), significantlyat the concentrations of 10 μM of PP2 ines (Figure 3A, B). The only exception is at the highest dose (Figure 4A and 4B). (10 μM), INCB3344 can inhibit secretion of some rSEA-induced cytokines and chemokines, including IL-2, TNF-α, IL-4, IL-6 and IL-8 slightly (Figure 3A and 3B).

A 80 60 80 * * 60 60 * 40 40 40 *** ** IL-4 (pg/mL) IL-4 (pg/mL) IL-6 IL-1β (pg/mL) IL-1β 20 *** *** 20 *** *** *** 20 *** *** rSEA 0 rSEA 0 rSEA 0 (100ng/mL) - + + + + + (100ng/mL) - + + + + + (100ng/mL) - + + + + + U0126 (μM) - - 0.4 2 10 20 U0126 (μM) - - 0.4 2 10 20 U0126 (μM) - - 0.4 2 10 20 Figure 5. Effect of MEK inhibitor U0126 on rSEA-induced cytokine and chemokine production. PBMCs were pre-incubated with U0126 (0.4, 2, 10, 20 μM) for 2 h and then stimulated with 100 ng/mL rSEA for 24 h still in the presence of U0126. Determina- tion the concentrations of detected cytokines (IL-1β, IL-4, IL-6) (A) and chemokines (CCL-2, IL-8, MIP1A, IP-10)

59 Asian Pac J Allergy Immunol 2020;38:52-63 DOI 10.12932/AP-220818-0396

B 8000 3000 6000 * 2000 ** 4000 ** ** *** IL-8 (pg/mL) IL-8 CCL-2 (ng/mL) CCL-2 2000 1000 *** ***

rSEA 0 rSEA 0 (100ng/mL) - + + + + + (100ng/mL) - + + + + + U0126 (μM) - - 0.4 2 10 20 U0126 (μM) - - 0.4 2 10 20

120 300 * * * 80 200

** 40 (pg/mL) IP-10 100 MIP-1α (pg/mL) MIP-1α ***

rSEA 0 rSEA 0 (100ng/mL) - + + + + + (100ng/mL) - + + + + + U0126 (μM) - - 0.4 2 10 20 U0126 (μM) - - 0.4 2 10 20 Figure 5. (Continued) (B) in cell culture supernatants by ELISA. * P < 0.05, ** P < 0.01, ***P < 0.001 compared with PBMCs stimulated with 100 ng/mL rSEA without U0126 (ANOVA).

Effect of MEK inhibitor U0126 on rSEA-induced cytokine and Effect of pan-JAK inhibitor P6 on rSEA-induced cytokine and chemokine production chemokine production Our previous work reported rSEA acted through ERK and We also applied pan-JAK inhibitor P6 to check its possible STAT pathways to exert anti-tumor activity.3 Similarly, we used role. Our data showed that P6 significantly reduced rSEA-in- the MEK inhibitor U0126 and the results of the ELISA assays duced cytokine and chemokine secretion in a dose dependent indicated that U0126 (0.4-20 μM) could inhibit rSEA-induced manner detected by ELISA assays (Figure 6). Again, our pre- secretion of cytokines (IL-1β, IL-4, IL-6) and chemokines viously published data showed that P6 significantly reduced (CCL-2, IL-8, MIP1A, IP-10) in a dose-dependent manner rSEA-induced TNF-α and IFN-γ secretion dose dependently (Figure 5). Notably, our previous published data showed that as detected by ELISA assays, but it inhibited IL-2 secretion only when PBMCs were treated with rSEA in the presence of 20 at the highest dose.3 μM U0126, the production of TNF-α, IFN-γ and IL-2 was completely abolished by the MEK inhibitor.3

A 25 16 80 20 12 * 60 15 ** * 8 40 ** 10

IL-4 (pg/mL) IL-4 ** (pg/mL) IL-6 IL-1β (pg/mL) IL-1β ** 4 ** 20 5 ** *** *** *** rSEA 0 rSEA 0 rSEA 0 (100ng/mL) - + + + + + (100ng/mL) - + + + + + (100ng/mL) - + + + + + P6 (nm) - - 5 50 500 5000 P6 (nm) - - 5 50 500 5000 P6 (nm) - - 5 50 500 5000 Figure 6. Effect of pan-JAK inhibitor P6 on rSEA-induced cytokine and chemokine production. PBMCs were pre-incubated with P6 (5 nM, 50 nM, 500 nM, 5 μM) for 2 h and then stimulated with 100 ng/mL rSEA for 24 h still in the presence of P6. Determination the concentrations of detected cytokines (IL-1β, IL-4, IL-6) (A) and chemokines (CCL-2, IL-8, MIP1A, IP-10)

60 SEA activate PBMC via Src, ERK and STAT

B 3000 2500 2000 * 2000 * 1500 * ** ** 1000 **

1000 (pg/mL) IL-8 CCL-2 (ng/mL) CCL-2 *** 500

rSEA 0 rSEA 0 (100ng/mL) - + + + + + (100ng/mL) - + + + + + P6 (nm) - - 5 50 500 5000 P6 (nm) - - 5 50 500 5000

250 300 200 * * 200 150 **

100

100 (pg/mL) IP-10 MIP-1α (pg/mL) MIP-1α ** 50 *** *** *** rSEA 0 rSEA 0 (100ng/mL) - + + + + + (100ng/mL) - + + + + + P6 (nm) - - 5 50 500 20 P6 (nm) - - 5 50 500 5000 Figure 6. (Continued) (B) in cell culture supernatants by ELISA. * P < 0.05, ** P < 0.01, ***P < 0.001 compared with PBMCs stimulated with 100 ng/mL rSEA without P6 (ANOVA).

Discussion Staphylococcal enterotoxin A (SEA), as a biomarker of and lasted a long term (> 24 h in vitro). Moreover, it was re- Staphylococcus aureus, is among the most potent T cell acti- vealed by previous studies that at least several proinflamma- vators ever known.1,2 Recently, SEA protein fused to Fab frag- tory factors, i.e., IFN-γ,25 TNF-α,27 and IL-826 played a leading ments of monoclonal antibodies, or recombinant SEA by en- role in the production of other cytokines and chemokines. gineering technology, have been applied to in vitro or animal Collectively, these studies supported the notion that the cyto- studies. Such attempts helped us better understand the role of kines induced by superantigen would further potentiate the SEA in the pathogenesis of multiple clinical syndromes rang- production of chemokines. ing from acute food poisoning,4 atopic dermatitis,5 to highly Chemokines, master regulators in a number of microenvi- lethal toxic shock,7 and even cancer.8 To obtain SEA in a fast, ronment responses, are small polypeptides produced by vari- accurate and low cost way, we produced recombinant SEA ous cell types. Previous studies reported that superantigens (rSEA) by cloning the coding sequence into a pET44a vec- caused a rapid, dose-dependent down-regulation of chemok- tor, and transforming the coding sequence into E. coli strain ine receptor (i.e., CCR1, CCR2, and CCR5) binding sites while Rosetta-gami 2. The SEA sequences were confirmed by the stimulating PBMCs.10 INCB3344, a novel, potent and selective LC-MS/MS analysis. Such purified and identified rSEA could CCR2 antagonist, inhibited the binding of CCL2 to mouse provide an efficient tool for us to study research projects in PBMCs in a dose-dependent manner.22 Our results indicated immunotherapy and atopic dermatitis, etc. that, even though blocking a certain chemokine receptor (i.e., Increasing evidence has shown that T cells release exces- CCR2), the rSEA induced systemic cytokine and chemokine sive cytokines and chemokines in response to bacterial exo- responses were largely unaffected. Therefore, it is less like- toxins, resulting in conditions like streptococcal toxic shock ly that one or two chemokines production or their receptors syndrome.7 SEA and SEB are particularly capable of inducing down-regulation would alter other cytokine and chemokine cytokine/chemokine release from T-cells.7,24-27 For example, SEB production and activities induced by superantigens. In all, our could robustly activate CD4+ and CD8+ T cells contributing to work strongly support the notion that a cognate interaction the cytokine storm and toxic shock.7,27 Previously Rajagopal- of cytokine and chemokine network still presents and rep- an et al reported that 28 of 30 cytokines in total or 27 of 30 resents one mechanism of communication between PBMCs chemokines in total as examined were significantly elevated and superantigens stimulation. at 3 h after SEB challenge in mice.27 Consistent with previous There are at least three intracellular signaling mediators work,8,16,18 the present study also indicated that SEA was able for T-cell activation upon superantigens binding; the phos- to inaduced a prompt, non-specific and profound activation phoinositide 3 kinase (PI3K)/mammalian target of rapamycin of PBMCs and secretion of cytokines and chemokines from (mTOR), NFκB and MAPK pathways.2,12-14 The cytokines and PBMCs, and the latter occurred in a dose-dependent manner chemokines produced by superantigen stimulated PBMCs act

61 Asian Pac J Allergy Immunol 2020;38:52-63 DOI 10.12932/AP-220818-0396

through their individual receptors and distinct receptor me- cancer, would be interesting to work with. diated signaling pathways. However, there was considerable In conclusion, we obtained a recombinant SEA (rSEA) in evidence to support the involvement of cross-talk between a fast, accurate and low cost way and confirmed this purified multiple signaling pathways. For example, the MAPK family rSEA could significantly promote proliferation of PBMCs and includes JNK, ERK, and p38. Inhibiting p38 and ERK path- enhance secretion of different cytokines and chemokines. Our way almost completely blocked the effect of SEB in regulating studies indicated blocking CCR2 cannot alter other cytokine the production of proinflammatory cytokines.28 Exposure to and chemokine production from PMBCs induced by rSEA in Staphylococcal enterotoxins-PBMC-conditioned medium re- vitro. However, Src, ERK and STAT pathway inhibitors all suc- sulted in STAT and the Janus protein-tyrosine kinase (Jak) cessfully blocked the enhanced secretion of most of cytokines activation.29,30 Fyn, a member of Src family, in the T-cells also and chemokines produced by rSEA stimulated PBMCs. activated by SEs for the signaling process,31 and SEA induced activation of STAT3 in human CD4+ T cell lines could be al- 23 Acknowledgments most completely inhibited by a Src kinase inhibitor (PP1). This work was supported by the National Natural Science Our previous work reported rSEA could exert its potent an- Foundation of China under Grants 81301948 and 30640033. ti-tumor activity via the cooperation of ERK and STAT pathways.3 To further elucidate the cellular mechanisms contributing to T-cells activation and sequential immune response, Declaration of conflicting interests in this study, we applied a few antagonists or inhibitors to the The authors report no conflict of interest. signaling pathways and their effects on the production of the cytokines and chemokines was examined. Our data showed References that rSEA-induced release of cytokines and chemokines from 1. Pinchuk IV, Beswick EJ, Reyes VE. Staphylococcal enterotoxins. Toxins PBMCs could be dose-dependently inhibited by any of these (Basel). 2010;2:2177-97. 2. Krakauer T. Update on staphylococcal superantigen-induced signaling three intracellular signaling pathways. Combined with pre- pathways and therapeutic interventions. Toxins (Basel). 2013;5:1629-54. 3,4,8,32 vious findings, our data support the hypothesis that Src, 3. Liu X, Zeng L, Zhao Z, He J, Xie Y, Xiao L, et al. PBMC activation via the ERK and STAT pathways convey signaling responses in an ERK and STAT signaling pathways enhances the anti-tumor activity of independent but also synergistic manner. There were only a Staphylococcal enterotoxin A. Mol Cell Biochem. 2017. 4. Agheli R, Emkanian B, Halabian R, Fallah Mehrabadi J, Imani Fooladi AA. few similar studies checking network cross-talk among triple Recombinant Staphylococcal Enterotoxin Type A Stimulate Antitumoral 15,33 or even more pathways in a study, however, to our knowl- Cytokines. Technol Cancer Res Treat. 2017;16:125-32. edge there is no such literature ever used SEA or SEB. Thus, our 5. Kim BS, Choi JK, Jung HJ, Park KH, Jang YH, Lee WJ, et al. 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Antibiotic treatment cytokine and chemokine responses in immunized mice challenged with has been complicated by the development of resistant strain staphylococcal enterotoxin B. Toxicon. 2017;133:82-90. (such as methicillin-resistant S. aureus (MRSA)) infection, 8. Yousefi F, Siadat SD, Saraji AA, Hesaraki S, Aslani MM, Mousavi SF, et al. 2,12,34 Tagging staphylococcal enterotoxin B (SEB) with TGFaL3 for breast cancer which represents one of the most costly diseases in USA. therapy. Tumour Biol. 2016;37:5305-16. There are numerous experimental approaches attempting to 9. Kang SS, Kim HJ, Jang MS, Moon S, In Lee S, Jeon JH, et al. Gene block cytokine storm including trying to interfere with the expression profile of human peripheral blood mononuclear cells induced earliest events, or more directly, to reduce the synthesis of cy- by Staphylococcus aureus lipoteichoic acid. Int Immunopharmacol. 2012; 13:454-60. tokines. One “old” approach is the use of low-dose steroids. 10. Rahimpour R, Mitchell G, Khandaker MH, Kong C, Singh B, Xu L, et For example, a FDA-approved immunosuppressive corticos- al. Bacterial superantigens induce down-modulation of CC chemokine teriod, dexamethasone, has been used clinically for various responsiveness in human monocytes via an alternative chemokine inflammatory diseases.12,35 Dexamethasone potently inhibited ligand-independent mechanism. J Immunol. 1999;162:2299-307. 11. Tikhonov I, Doroshenko T, Chaly Y, Smolnikova V, Pauza CD, Voitenok N. SEA- and SEB-induced cytokine release and T-cell prolifer- Down-regulation of CXCR1 and CXCR2 expression on human neutrophils 12,35 ation in human PBMCs. Another important way is to use upon activation of whole blood by S. aureus is mediated by TNF-alpha. Clin inhibitors to block signal transduction pathways activated by Exp Immunol. 2001;125:414-22. super antigens.2,12,34 The obvious advantage is that many differ- 12. Krakauer T. PI3K/Akt/mTOR, a pathway less recognized for staphylococcal superantigen-induced toxicity. Toxins (Basel). 2012;4:1343-66. ent superantigens or even pathogens eliciting similar host re- 13. Zeng YQ, Lu C, Dai Z. Editorial: Memory T Cells: Effectors, Regulators, and sponses or pathways could be effectively inhibited. Our work Implications for Transplant Tolerance. Front Immunol. 2016;7:7. indicated, therapeutic inhibitors of Src, ERK and STAT sig- 14. Krakauer T. FDA-approved immunosuppressants targeting staphylococcal nal transduction might be potential therapeutic approaches superantigens: mechanisms and insights. Immunotargets Ther. 2017;6: 17-29. against inflammatory diseases and carcinoma, especially to 15. Aksamitiene E, Achanta S, Kolch W, Kholodenko BN, Hoek JB, Kiyatkin treat those cases with microbial resistance against antibiot- A. Prolactin-stimulated activation of ERK1/2 mitogen-activated protein ics or to spare steroid usage. 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16. Chaturvedi P, Reddy MVR, Reddy EP. Src kinases and not JAKs activate 27. Rajagopalan G, Tilahun AY, Asmann YW, David CS. Early gene expression STATs during IL-3 induced myeloid cell proliferation. Oncogene. 1998;16: changes induced by the bacterial superantigen staphylococcal enterotoxin 1749. B and its modulation by a proteasome inhibitor. Physiol Genomics. 17. Singer CA, Lontay B, Unruh H, Halayko AJ, Gerthoffer WT. Src mediates 2009;37:279-93. cytokine-stimulated gene expression in airway myocytes through ERK 28. Kearney DE, Wang W, Redmond HP, Wang JH. Bacterial superantigens MAPK. Cell Commun Signal. 2011;9:14. enhance the in vitro proinflammatory response and in vivo lethality of the 18. Li L, Wang S, Zou Z, Tao A, Ai Y. Activation profile of THP-1 derived TLR2 agonist bacterial lipoprotein. J Immunol. 2011;187:5363-9. dendritic cells stimulated by allergen Mal f 1 beyond its IgE-binding ability. 29. McKay DM, Botelho F, Ceponis PJ, Richards CD. Superantigen immune Int Immunopharmacol. 2018;62:139-46. stimulation activates epithelial STAT-1 and PI 3-K: PI 3-K regulation 19. Liu X, Zeng L, Zhao Z, Xie Y, Wang S, Zhang J, et al. Construction, of permeability. Am J Physiol Gastrointest Liver Physiol. 2000;279: Expression, and Characterization of rSEA-EGF and In Vitro Evaluation of G1094-103. its Antitumor Activity Against Nasopharyngeal Cancer. Technol Cancer 30. Nielsen M, Svejgaard A, Ropke C, Nordahl M, Odum N. Staphylococcal Res Treat. 2018;17:1533033818762910. enterotoxins modulate interleukin 2 receptor expression and ligand 20. Ling C, Zhang J, Chen H, Zou Z, Lai H, Lin D, et al. Expression and -induced tyrosine phosphorylation of the Janus protein-tyrosine kinase 3 refolding of mite allergen pro-Der f1 from inclusion bodies in Escherichia (Jak3) and signal transducers and activators of transcription (Stat proteins). coli. Protein Expr Purif. 2015;109:93-8. Proc Natl Acad Sci U S A. 1995;92:10995-9. 21. Slota M, Lim JB, Dang Y, Disis ML. ELISpot for measuring human immune 31. Yamasaki S, Tachibana M, Shinohara N, Iwashima M. Lck-independent responses to vaccines. Expert Rev Vaccines. 2011;10:299-306. triggering of T-cell antigen receptor signal transduction by staphylococcal 22. Brodmerkel CM, Huber R, Covington M, Diamond S, Hall L, Collins enterotoxins. J Biol Chem. 1997;272:14787-91. R, et al. Discovery and pharmacological characterization of a novel 32. Si SY, Liu JL, Liu JL, Xu BX, Li JZ, Qin YY, et al. Recombinant adenovirus of rodent-active CCR2 antagonist, INCB3344. J Immunol. 2005;175:5370-8. SEA and CD80 genes driven by MMRE and mouse TERT promoter induce 23. Dai X, Wang LJ, Wu J, Shi YX, Li GP, Yang XQ. Src kinase inhibitor PP2 effective antitumor immune responses against different types of tumor cells regulates the biological characteristics of A549 cells via the PI3K/Akt in vitro and in vivo. Oncol Rep. 2017;37:3037-45. signaling pathway. Oncol Lett. 2018;16:5059-65. 33. Lo RK, Cheung H, Wong YH. Constitutively active Galpha16 stimulates 24. Pinchuk IV, Beswick EJ, Saada JI, Suarez G, Winston J, Mifflin RC, STAT3 via a c-Src/JAK- and ERK-dependent mechanism. J Biol Chem. et al. Monocyte chemoattractant protein-1 production by intestinal 2003;278:52154-65. myofibroblasts in response to staphylococcal enterotoxin a: relevance to 34. Gerlach H. Agents to reduce cytokine storm. F1000Res. 2016;5:2909. staphylococcal enterotoxigenic disease. J Immunol. 2007;178:8097-106. 35. Krakauer T. Differential inhibitory effects of interleukin-10, interleukin-4, 25. Muralimohan G, Vella AT. A role for IFNgamma in differential and dexamethasone on staphylococcal enterotoxin-induced cytokine superantigen stimulation of conventional versus plasmacytoid DCs. Cell production and T cell activation. J Leukoc Biol. 1995;57:450-4. Immunol. 2006;242:9-22. 26. Krakauer T. Interleukin-8 production by human monocytic cells in response to staphylococcal exotoxins is direct and independent of interleukin-1 and tumor necrosis factor-alpha. J Infect Dis. 1998;178:573-7.

63 CASE REPORT Asian Pacific Journal of Allergy and Immunology

Schnitzler’s syndrome: A female elderly case presenting intractable non-pruritic febrile urticarial rush

Nanae Kimura,1,2 Hiromichi Takeshita,2 Tatsuya Kai,3 Yasushi Inoue,3 Masutaka Furue1

Abstract

Schnitzler’s syndrome is an acquired autoinflammatory disease characterized by chronic urticarial rash and monoclonal gammopathy (predominantly IgM type). A 75-year-old Japanese woman complained of high fever and non-pruritic urticarial rash appearing almost every day for 3 years. Her abnormal laboratory data included leukocytosis and neutrophilia with elevated erythrocyte sedimentation rate and C-reactive protein level. Hyperglobulinemia of IgA and IgM was also noted. Histological analysis revealed perivascular and interstitial neutrophilic infiltration without any signs of vasculitis. Immunofixation analysis confirmed IgM-kappa-type monoclonal gammopathy. Oral prednisolone initially improved her symptoms, but recurrence was observed upon its tapering. The addition of colchicine successfully controlled her symptoms and allowed a reduction in the dose of systemic steroid.

Key words: Schnitzler’s syndrome, IL-1β, acquired autoinflammatory disease, urticarial rash, monoclonal gammopathy

From: Corresponding author: 1 Graduate School of Medical Sciences, Department of Dermatology, Nanae Kimura Kyushu University, Fukuoka, Japan Graduate School of Medical Sciences, Department of Dermatology, 2 Dermatology Unit, Japanese Red Cross Fukuoka Hospital, Fukuoka, Kyushu University, Japan 3-1-1, Maidashi, Higashi-ku, Fukuoka city, Fukuoka, 812-8582, Japan 3 Collagen Diseases Unit, Japanese Red Cross Fukuoka Hospital, E-mail: [email protected] Fukuoka, Japan

Introduction Schnitzler’s syndrome is an acquired autoinflammatory Antinuclear antibody, anti-neutrophil cytoplasmic antibodies, disease characterized by chronic “non-pruritic” urticarial rash and anti-human T-cell leukemia virus antibody were all neg- and monoclonal gammopathy (predominantly IgM type), ative. CT scan revealed only reactive axillary and inguinal which was first reported by the French dermatologist Liliane lymphadenopathy. Bone marrow aspiration showed no hema- Schnitzler in 1972.1 Recently, much attention has focused on the topoietic malignancies. role of interleukin-1 beta (IL-1β) as a pathogenetic trigger in Histological analysis revealed perivascular and interstitial this disease.2,3 neutrophilic infiltration without any signs of vasculitis. Dermal edema was very mild (Figure 1b). These features are classified Report of Case as neutrophilic urticarial dermatosis and urticarial vasculitis A 75-year-old Japanese woman complained of high fever was denied.2 and urticarial rash appearing almost every day for 3 years. Ex- Regarding hyperglobulinemia, we further performed se- amination revealed an intermittent fever up to 39°C and dis- rum electrophoresis and immunofixation. While serum elec- seminated non-pruritic urticarial rash, which had lasted for sev- trophoresis indicated no particular abnormality, we identified eral days (Figure 1a), with fatigue, sweating, and sacroiliac joint abnormal bands for both IgM and kappa chain by immunofix- pain. Treatments for diabetes had been undergoing for several ation (Figure 1c). Furthermore, an abnormal band for kappa years. chain was also detected by urine immunofixation (data not Laboratory evaluation showed leukocytosis (22.5 × 103/µL) shown), indicating a diagnosis of Schnitzler’s syndrome with and neutrophilia (18.3 × 103/µL) with elevated erythrocyte sed- IgM-kappa-type monoclonal gammopathy.1 imentation rate of 70 mm/h and C-reactive protein (CRP) level Systemic prednisolone at an initial dose of 30 mg/day suc- of 21.22 mg/dl. Slight elevation of ferritin (334.9; normal range, cessfully improved the patient’s fever and skin rash with nor- 3.6 to 114.0 ng/ml) was also observed. In addition, hyperglob- malization of leukocytosis and CRP level. Tapering the dose of ulinemia of IgA (525 mg/dl) and IgM (253 mg/dl) was noted. prednisolone led to recurrence of her skin rash and additional

64 A case of Schnitzler’s syndrome

a b

Figure 1. a. Disseminated urticarial eruptions on the trunk. They were non-pruritic and persisted for several days. b. Perivascular and interstitial neutrophilic infiltration was seen without any vasculitis. Dermal edema was mild. c. Abnormal bands for both IgM and kappa chain were identified by immunofixation. intake of azathioprine at 50 mg/day did not help to inhibit the IgM gammopathy. Other autoinflammatory diseases such as leukocytosis and elevation of CRP. After the cessation of aza- familial cold autoinflammatory syndrome, Muckle-Wells syn- thioprine, oral colchicine was initiated at 0.5 mg/day, which drome, neonatal-onset multisystem inflammatory disease and led to dramatic subsidence of the symptoms and improvement familial Mediterranean fever were also excluded by negative of the laboratory findings. Good control of the patient’s symp- familial or infantile history and neurological or ophthalmolog- toms was achieved with 12.5 mg/day prednisolone. Serum ical symptoms. Urticarial vasculitis was denied by histological IgM levels kept high, 253 mg/dl to 561 mg/dl, during 5-month analysis. We also ruled out Sweet syndrome based on clinical follow up period. and histological manifestation of her eruption.3 Since it was proposed that IL-1 signaling plays a critical Discussion role in Schnitzler’s syndrome, the anti-IL-1 antibodies anakin- For making the correct diagnosis of Schnitzler’s syndrome, ra and canakinumab are currently recommended as first-line differential diagnoses were considered and excluded follow- treatment.1 However, they are still off-label treatments in Japan. ing Strasbourg diagnostic criteria.1 Adult-onset Still’s disease Systemic steroids are instead the mainstay in Japan, and col- (AOSD) has similar clinical characteristics such as intermittent chicine may work as a second-line medicine, as demonstrated fever, pruritic urticarial rash and leukocytosis. We ruled out in the present case. Simons et al. recommended colchicine as AOSD due to lack of significant increase of transaminases and one of the main treatments for none-severe cases without sig- ferritin. But her urticaria was “non-pruritic” and she presented nificant alterations in quality of life or persistent elevation of

65 ERRATUM Asian Pacific Journal of Allergy and Immunology

Erratum: Correction for affiliation ofrd 3 author Randomized comparison of caregivers’ ability to use epinephrine autoinjectors and prefilled syringes for anaphylaxis

Panadda Suwan,1 Phatcharee Praphaiphin,2 Pantipa Chatchatee3 1 Department of Pediatrics, Faculty of Medicine Vajira Hospital, Navamindradhiraj University, Bangkok, Thailand 2 Branch of Pediatric Nursing, Nursing Department, Faculty of Medicine Vajira Hospital, Navamindradhiraj University, Bangkok, Thailand 3 Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, King Chulalongkorn Memorial Hospital, Bangkok, Thailand

DOI: 10.12932/AP-020318-0275 Asian Pac J Allergy Immunol 2018;36:248-256

Corrected affiliation ofrd 3 author 3 Pediatric Allergy & Clinical Immunology Research Unit, Division of Allergy and Immunology, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, King Chulalongkorn Memorial Hospital, the Thai Red Cross Society, Bangkok, Thailand