The levels of soluble ST2 in sera and bullous fluid from patients with bullous pemphigoid

ARTICLE

ejd.2012.1706

Auteur(s) : Koji Wakatabi¹, Mayumi Komine^1,2 mkomine12@jichi.ac.jp, Jitlada Meephansan¹, Yasushi Matsuyama^2,3, Hidetoshi Tsuda¹, Shin-ichi Tominaga², Mamitaro Ohtsuki¹

¹ Department of Dermatology,

² Department of Biochemistry,

³ Department of Allergy and Rheumatology, Jichi Medical University, 3311-1 Yakushiji Shimotsuke, Tochigi 329-0498, Japan

Reprints: M. Komine

Bullous pemphigoid (BP) is an autoimmune bullous dermatosis characterized by subepidermal blistering and the presence of an autoantibody against the hemidesmosomal protein bullous pemphigoid antigen 180 (BP180) [1]. The affected tissue usually shows prominent eosinophil and neutrophil infiltration. BP patients usually have elevated peripheral blood eosinophil counts, which correlate well with the disease activity. Th2 cytokines and chemokines, such as interleukin (IL)-4, IL-5, eotaxin, monocyte chemoattractant protein (MCP)-4/CCL13, and thymus- and activation-regulated chemokine (TARC)/CCL17 have been reported to be elevated in the sera and bullous fluid of BP patients [2].

Soluble ST2 (sST2) and membrane-bound ST2L are members of the Toll-like/IL-1 receptor family, and sST2 possesses the extracellular domain structure of ST2L [3]. The ST2 gene was originally found to be one of the primary response genes in the G₀/G₁ transition phase of BALB/c-3T3 cells [4]. Alternative processing of the primary transcript generates 2 messenger RNAs (mRNA) of different sizes. The short mRNA encodes sST2, and the long mRNA encodes ST2L, which is selectively expressed on Th2-type cells and mast cells [5]. ST2L was recently revealed as the receptor for IL-33 [6].

IL-33 is a member of the IL-1 family [7]. It can promote Th2 and mast cell activation, leading to a Th2-type inflammation. Administration of IL-33 in mice induces the production of Th2 cytokines and severe pathological changes in the mucosal organs [8].

sST2 has been shown to antagonize IL-33-ST2L signaling by competing with ST2L as a decoy receptor for IL-33 [9]. Serum sST2 levels are elevated in patients with bronchial asthma [10], pulmonary fibrosis [11], myocardial infarction [12], and sepsis [13]. Recently, sST2 has been shown to inhibit signals from TLR4 [14]. The significance of sST2 in the clinical setting is obscure; however, we suggest that a balance between IL-33 and sST2 is important for inflammatory responses. Thus, we decided to investigate the sST2 concentration in sera and bullous fluid of BP patients, together with IL-33 levels.

Materials and methods

Samples

Informed consent was obtained from all participants. Sera were collected from 18 BP patients and 16 normal controls. Bullous fluid was collected from the bullae of 11 BP patients and 5 burn patients. The clinical backgrounds of these patients are summarized in Table 1. Burn patients and normal controls were not under any systemic medication at the time of sample collection. Suction blisters were induced on 2 healthy volunteers, and bullous fluid was collected. The institutional ethical committee of Jichi Medical University approved this project.

Table 1 BP patients and their clinical backgrounds.

F, female; M, male; AH, anti-histamine; PSL, prednisolone; BMS, beta-methasone; NA, nicotinamide; TS, topical steroids.

Enzyme-linked immunosorbent assay (ELISA)

The sST2 and IL-33 levels in the sera and bullous fluid, and TARC/CCL17 levels in bullous fluid were evaluated by ELISA with an sST2 ELISA kit from MBL (Nagoya, Japan), IL-33 DuoSet^® ELISA Development System and Quantikine^® TARC/CCL17 ELISA kit from R&D Systems (Minneapolis, MN, USA), respectively, according to the manufacturers’ instructions. The lowest standard is 23 pg/ml, which allows the detection of 0.34 pg/ml of IL-33 [14]. For sST2, the sera and bullous fluids were directly applied to a pre-coated 96-well plate. For IL-33, the sera and bullous fluid were diluted 1:1 with fetal bovine serum before the procedure because of the occasional non-specific high value that decreases to below the detectable limit with the 1:1 dilution [15]. The optical density was determined with a spectrophotometer (Model 3550; Bio-Rad, Hercules, CA, USA) set to 450 nm.

Clinical laboratory tests

The white blood cell (WBC) count, eosinophil number, and anti-BP180 antibody and lactate dehydrogenase (LDH) levels of 18 BP patients were routinely investigated.

The area of skin involvement of the patients was evaluated on the basis of the ratio of the involved area to the total body surface area; patients were grouped into 3 categories according to the extent of involvement:

• –. I: less than 20%,
• –. II: 20-80%,
• –. III: above 80%.

Statistical analysis

The Mann-Whitney U test was used for comparison of sST2 levels, Spearman's correlation coefficient by rank test was used for determining the correlation between sST2 levels and clinical laboratory test results and the Kruskal-Wallis test was used for determining the correlation between sST2 levels and clinical severity. A p value of less than 0.05 was considered statistically significant.

Results

The sST2 levels in the sera and bullous fluid were elevated in BP patients as compared to those in normal controls and burn patients, respectively

The serum sST2 levels in BP patients were significantly higher than those in healthy volunteers (figure 1A). The sST2 levels in the bullous fluid from BP patients were significantly higher than those from burn patients (figure 1B). The sST2 levels in the bullous fluid from suction blisters of normal healthy controls were below the detectable limits.

The sST2 levels in the sera correlated with WBC count, serum LDH levels, and skin involvement area but did not correlate with sST2 levels in the bullous fluid or with other laboratory test results

A significant correlation was found between the levels of sST2 and the WBC count and serum LDH levels (figure 2A) (correlation coefficients=0.648 and 0.692, respectively). However, there was no significant correlation between serum sST2 levels and peripheral blood eosinophil number, anti-BP180 antibody level, or sST2 levels in bullous fluid. The correlation coefficients were -0.034, 0.258, and 0.214, respectively.

BP patients were divided into 3 groups according to the extent of skin involvement. The Kruskal-Wallis test revealed a statistically significant correlation between serum sST2 level and the area of skin involved (figure 2B).

The IL-33 level estimated by ELISA was below detectable limits in both the sera and bullous fluid in all the samples

IL-33 was below detectable limits in the sera from BP patients and normal healthy volunteers as well as in bullous fluid from BP patients, burn patients, and contact dermatitis patients (data not shown).

The sST2 levels in the bullous fluid from BP patients did not show any correlation with the WBC count, eosinophil number, serum BP180 or LDH levels, skin involvement area, or bullous fluid TARC/CCL17 level

We investigated the correlation of sST2 levels in bullous fluid with the WBC count, eosinophil number, serum levels of BP180, serum levels of LDH, and the area of skin involvement. The levels of sST2 in the bullous fluid showed no correlation with any of the first 4 factors above (correlation coefficients=-0.172, -0.109, 0.493, and 0.245, respectively). The Kruskal-Wallis test revealed a p-value of 0.26 for the correlation between sST2 and skin involvement area. We also measured TARC/CCL17 levels in the bullous fluid, which did not correlate with sST2 levels in the bullous fluid (correlation coefficient=0.297).

Discussion

In this study, we detected sST2 in the sera and bullous fluid of BP patients, and the serum sST2 levels correlated with the WBC count, serum LDH level, and involved skin area. ELISA did not detect IL-33, the ligand for sST2, in the same samples. We diluted the sera 1:1 with fetal bovine serum because if the sera are not diluted, the gamma globulin in the sample sera greatly affects the results of ELISA in this system [15]. Western blotting with goat polyclonal anti-IL-33 antibody (R&D Systems) did not show any evidence of the presence of IL-33 in the sera or bullous fluid.

We assume that excessive amounts of sST2 are required to inhibit IL-33 activity, and it appears reasonable that a minimum amount of IL-33 existed in the sera and bullous fluid of BP patients. The possibility of sST2 playing a role other than blocking IL-33 signaling could also be considered.

The correlation between serum sST2 levels and overall clinical severity, including the involved skin area, WBC count, and serum LDH level, suggests that sST2 may reflect the systemic inflammatory reaction caused by skin inflammation. In contrast, sST2 levels in the bullous fluid may reflect the local inflammatory level rather than systemic inflammation. We speculate that if the level of local inflammation is high, the level of sST2 in the bullous fluid on that site may be high, regardless of the area involved. In accordance with this, very high sST2 levels were detected in the bullous fluid from 2 contact dermatitis patients (data not shown). The positive correlation of the serum sST2 level and the WBC count may reflect that it was derived from a certain population of peripheral WBC. CD4-positive T cells, especially Th2 cells, express ST2, suggesting that Th2 cells are the major source of sST2; however, this needs further investigation. Serum levels of LDH reflect the degree of skin involvement in several skin disorders [16-18]. The positive correlation between serum sST2 and serum LDH may suggest that serum sST2 levels reflect the degree of whole-body skin inflammation. sST2 has been reported not only as a decoy receptor for IL-33 but also an inhibitor of the signaling induced by lipopolysaccharide (LPS) [19]. The role of IL-33 is also reported in innate immune responses [20]. IL-33 is released when cells undergo necrosis, which may occur in the epidermal roof of the blister, and the IL-33 released may be caught by sST2 and inactivated. LPS released from bacteria that grow on erosive skin lesions could induce local inflammation, which may be inactivated by the existing sST2. Hence, sST2 may function as a suppressor of inflammation in innate immune responses as well as in acquired Th2 reactions in BP per se.

Substantial reports exist on the detection of cytokines in the bullous fluid of BP patients. Eotaxin and TARC/CCL17 levels have been reported to correlate with the number of skin-infiltrating eosinophils [2, 21]. In bullous fluid, the levels of TARC/CCL17 did not correlate with the levels of sST2, suggesting that infiltrating eosinophils are not involved in increasing the levels of sST2.

The pathophysiological significance of elevated sST2 levels in the sera and bullous fluid from BP patients is unknown. The fact that serum sST2 levels are correlated with the area of skin involvement, LDH level, and WBC count suggests that it may reflect the severity of the disease or the severity of skin involvement and that it may be useful as a marker for disease severity. In the pathophysiology of BP, sST2 may serve as a negative feedback signal because it could inhibit inflammatory signals through the IL-33/ST2L and LPS/TLR4 systems; however, their precise mechanisms need to be elucidated.

Acknowledgement

Financial support: This work was supported by a grant from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and a grant from the Ministry of Health, Labor, and Welfare (Research for Intractable Diseases). Conflict of interest: none.

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