Cross-sectional study of cholinergic urticaria subtypes and bronchial hyperresponsiveness

Cholinergic urticaria (CholU) is classified into several subtypes: (1) conventional sweat allergy-type CholU (conventional SAT-CholU), (2) CholU with palpebral angioedema (CholU-PA), 3) CholU with acquired anhidrosis and/or hypohidrosis (CholU-Anhd); 1) and 2) include SAT based on pathogenesis. There have been no studies on differences in the prevalence of bronchial asthma among the subtypes. We analyzed bronchial responsiveness using the methacholine dose indicator Dmin, respiratory symptoms, and exhaled nitric oxide (FeNO). Median log10 Dmin (interquartile range) of patients with conventional SAT-CholU (n = 11), CholU-PA (n = 11), and CholU-Anhd (n = 11) was 0.381 (− 0.829, 1.079), 0.717 (0.249, 0.787), and 1.318 (0.121, 1.699), respectively (p = 0.516). Respiratory symptoms were less frequently observed in CholU-Anhd than in conventional SAT-CholU or CholU-PA. FeNO of patients with conventional SAT-CholU, CholU-PA, and CholU-Anhd was 23 (18.5, 65.0), 39 (32.0, 59.5), and 25 (19.0, 33.0) ppb, respectively (p = 0.237). Nine% of conventional SAT-CholU patients and more than half of CholU-PA patients required treatment for asthma. Log Dmin tended to be lower in patients with SAT-CholU than in those with CholU-Anhd. CholU-PA might be associated with asthma.

Cholinergic urticaria (CholU) is characterized by pruritic wheals with surrounding erythema triggered by an increase in core body temperature that is caused by exercise, high environmental temperature, or emotional stress 1 . Patients' complaints of stinging, tingling pain, or itching usually resolve within 1 h. Respiratory and other severe symptoms such as angioedema and anaphylaxis have been reported to accompany CholU 2,3 , which is most common in young adults with an estimated prevalence of 4-11% 4,5 . Although the precise underlying mechanism is unclear, histamine, cholinergic agents, sweat allergy, serum factors, poral occlusion, and anhidrosis are associated with symptom onset. CholU can be classified into the following several subtypes based on dermatologic characteristics: (1) conventional sweat allergy type (conventional SAT-CholU), (2) CholU with palpebral angioedema (CholU-PA), (3) CholU with acquired anhidrosis and/or hypohidrosis (CholU-Anhd), and other rare subtypes such as follicular-type CholU with a positive autologous serum skin test result 6 . Conventional SAT-CholU is associated with sweat allergy; the same is true of CholU-PA, which has more severe symptoms than conventional SAT-CholU and is accompanied by palpebral angioedema around the eyelids and is strongly associated with atopic diseases such as atopic dermatitis, asthma, and allergic rhinitis 7 . Almost all patients with CholU-PA are female. As conventional SAT-CholU and CholU-PA are both associated with type I allergy to sweat and atopic diseases, they can be grouped as SAT. CholU-Anhd is characterized by acquired generalized hypohidrosis or anhidrosis without sweat allergy. In contrast to conventional SAT-CholU and CholU-PA, CholU-Anhd is not associated with atopic diseases. In CholU-Anhd patients, reduction of acetylcholine receptor M3 on the epithelial cells of sweat glands and Ach-degrading enzyme acetylcholine esterase are seen in hypohidrotic area, and it is thought that the overflow of acetylcholine leaks into mast cells and causes wheal 8,9 .
A previous study showed that 13% of patients with CholU have asthma 10 . And another study reported that 40% of patients with CholU-PA had current or a history of asthma 7 , which is characterized by chronic airway inflammation and bronchial hyperresponsiveness 11 . In a previous study, bronchial hyperresponsiveness was more frequently observed in CholU patients without history of asthma (43%) than in chronic urticaria patients and healthy adults (7%) 12 . However, there have been no studies on differences in the prevalence of asthma among CholU subtypes. This was investigated in the present study by evaluating bronchial responsiveness in each

Discussion
This is the first study investigating differences in bronchial hyperresponsiveness among subtypes of CholU. We showed that D min was lower in patients with SAT (conventional SAT-CholU and CholU-PA) than in those with CholU-Anhd, although it did not differ significantly among the 3 subtypes. Respiratory symptoms were more frequently observed in patients with SAT and FeNO was elevated in patients with CholU-PA. Although the differences among the 3 subtypes were nonsignificant, this result may reflect the distinct pathogenesis of conventional SAT-CholU, CholU-PA, and CholU-Anhd. Namely, SAT is associated with sweat allergy and CholU-PA is closely related to atopic diseases 7 . A previous study investigating bronchial responsiveness in patients with CholU did not stratify the results based on CholU subtype 12 . Our study provides insight into the respiratory features of each CholU subtype based on a manifestation other than skin symptoms. Nine % of conventional SAT-CholU patients and more than half of CholU-PA patients required treatment for asthma based on the decision of attending pulmonologist after physical examination and interview. It is important to identify the subtype of CholU as this can determine the disease management strategy. A previous study showed that symptom duration and intensity were associated with bronchial hyperresponsiveness 12 , although we did not observe any association between the severity of urticaria symptoms and bronchial hyperresponsiveness. The study by Petalas et al. excluded patients with history of asthma, atopy, and smoking; under this study setting, the authors demonstrated that the respiratory symptoms of CholU resulted from bronchial hyperresponsiveness 12 ; however, it is possible that they had fewer patients with SAT subtypes (conventional SAT-CholU and CholU-PA) than our study because they excluded patients with a history of atopic diseases. Not all of our patients with bronchial hyperresponsiveness required asthma treatment. We did not include normal subjects as a control group but in a previous report, log D min was > 50 U in subjects with no history of asthma or other respiratory diseases and who had no current respiratory symptoms 14 . Bronchial hyperresponsiveness may be caused by CholU itself in some patients. In order to detect asthma, it is important to pay attention to respiratory symptoms, FeNO, and history of asthma as well as bronchial hyperresponsiveness. Our results also suggest that continuous methacholine inhalation is useful  www.nature.com/scientificreports/ for evaluating bronchial responsiveness in CholU, which can reveal the underlying pathogenic mechanism in each subtype. Our study had some limitations. Firstly, it was conducted at a single institution and had a limited sample size, which may have contributed to the lack of significant differences among the 3 CholU subtypes. Secondly, we did not exclude all confounding factors such as smoking history and history of asthma that can affect bronchial responsiveness. Although the proportion of smokers was similar across subtypes and there were no differences in baseline Rrs, these confounding factors may influence the result of bronchial responsiveness. Therefore, A multicenter study with a large sample size is needed to validate our findings. Additionally, future studies should address the prevalence of CholU in patients with asthma as a comorbidity.
In conclusion, log D min tended to be lower in patients with SAT (conventional SAT-CholU and CholU-PA) than in those with CholU-Anhd. Distinguishing between subtypes of CholU may reveal different degrees of bronchial responsiveness based on differences in pathogenesis. And CholU-PA might be associated with asthma.

Materials and methods
Patients. Patients 16-80 years of age with CholU were prospectively enrolled. CholU was diagnosed and classified into subtypes by a dermatologist according to previously reported criteria 15 . Briefly, patients were diagnosed as cholinergic urticaria with provocation test such as exercise-induced test or acetylcholine intradermal injection. Sweat allergy was assessed by autologous sweat test. Patients who were contraindicated for methacholine inhalation challenge (e.g., severe airflow obstruction, recent asthma attack, or uncontrolled hypertension) or had uncontrolled asthma were excluded. The patients were divided into the following 3 groups: (1) conventional SAT-CholU, (2) CholU-PA, and (3) CholU-Anhd and follicular-type CholU. SAT was defined as (1) and (2), as both subtypes are associated with type I allergy to sweat.
This study was approved by the ethics committee of Kobe University (no. 160114) and was conducted in accordance with the Helsinki declaration. All patients provided written, informed consent before enrollment. If patients were under 20 years of age, their guardians also signed the agreement form. The study was registered with the University Medical Hospital Information Network of Japan (UMIN 000025669; https:// upload. umin. ac. jp/ cgi-open-bin/ ctr/ ctr_ view. cgi? recpt no= R0000 27550).
Bronchial responsiveness. Bronchial responsiveness was evaluated by continuous methacholine inhalation challenge with simultaneous measurement of respiratory resistance (Rrs) using a previously developed device (Astograph; Chest, Tokyo, Japan) 14  www.nature.com/scientificreports/ D min of 50 was recorded, but this was considered as no bronchial responsiveness in the analyses. According to previous studies 14 , we analyzed D min using the log10-transformed value, which has been validated in tests for bronchial responsiveness in clinical practice 16 . All participants stopped taking oral antihistamines, leukotriene receptor antagonists, theophylline, systemic, or inhaled corticosteroids, and inhaled long-acting β2 agonists for at least 72 h prior to assessment. The use of short-acting β2 agonists were permitted if participants have dyspnea from asthma exacerbation. The pulmonary function test was performed using a spirometer (Auto Spirometer SYSTEM21; Minato Medical Science Co, Osaka, Japan). Exhaled nitric oxide (FeNO) was measured using an electrochemical NO analyzer (NIOX VERO; Aerocrine AB, Solna, Sweden).
Endpoints. The study was designed as a prospective, single-center observational study. The primary endpoint was log D min in the 3 subtypes of CholU, and the secondary endpoints were respiratory symptoms, FeNO, and forced expiratory volume in 1 s (FEV1, % predicted value). We also compared D min , respiratory symptoms, FeNO, and FEV1 (% predicted) between patients with SAT (conventional SAT-CholU and CholU-PA) and those with CholU-Anhd.
Statistical analysis. Differences between groups were evaluated with the chi-squared test or Fisher's exact test for qualitative data and the Kruskal-Wallis test for quantitative data. All statistical analyses were performed using EZR v1.38 (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R v 3.3.2 software (R Foundation for Statistical Computing, Vienna, Austria) 19 .

Ethics declarations.
The study was performed in accordance with the Helsinki declaration and was approved by the ethics committee of Kobe University (No. 160114). And all the study's participants signed a written informed consent.

Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.