Diacron-Reactive Oxygen Metabolites Levels Are Initially Elevated in Patients with Bullous Pemphigoid

Abstract

ROS are involved in the pathogenesis of bullous pemphigoid (BP), but this involvement has not been fully elucidated. In this study, to further elucidate the pathogenic role of ROS in BP, we examined the results of the diacron-reactive oxygen metabolite test and the biological antioxidant potential test for 16 patients with BP who visited our hospital before being treated with systemic corticosteroids. In the patients with BP, the average diacron-reactive oxygen metabolite levels, expressed in Carratelli units, were significantly reduced at 1 month of treatment (from 335.6 ± 40.3 Carratelli units to 224.7 ± 61.6 Carratelli units, P < .001). Bullous Pemphigoid Disease Area Index (erosions/blisters) scores correlated with diacron-reactive oxygen metabolite levels (r = 0.51), suggesting that those levels reflect the disease severity. We also performed staining of 3,5-dibromotyrosine in skin tissues. The 3,5-dibromotyrosine is expected to be a marker of tissue damage related to inflammation and allergies. The 3,5-dibromotyrosine was stained in infiltrated cells around the dermis, throughout the blister fluid, and at the basement membrane within the blister. It is considered that tissue destruction caused by the myeloperoxidase released from neutrophils and by eosinophil peroxidase released from eosinophils is involved in blister formation. The results suggest that ROS play a role in BP.

Keywords: Bullous pemphigoid; Diacron-reactive oxygen metabolites; Dibromotyrosine; Oxidative stress; ROS.

Figure 1

Oxidative stress in patients with pemphigoid. (a) Box plot of d-ROMs levels for 16 patients with BP. The d-ROM levels are for before treatment and at 1 month of treatment (from 335.6 ± 40.3 U.CARR to 224.7 ± 61.6 U.CARR, P < .001, paired t-test). (b) Box plot of BAP levels for the 16 patients with BP. The BAP levels are for before treatment and at 1 month of treatment (from 2102.8 ± 340.9 μmol/l to 1869.8 ± 426.1 μmol/l, P = .0923, paired t-test). The box shows the first and third quartiles, the median (line), and the arithmetic mean (x-mark). The whiskers represent values below the first quartile and above the third quartile within the 1.5-fold interquartile range, respectively. Outliers beyond the whiskers are shown as squares. (c) Changes in d-ROM levels in 3 patients with BP at 1 month of treatment with systemic corticosteroids. (d–g) Direct immunofluorescent staining of DiBrY. Two-color wide-field fluorescence microscopy images of BP erythema for DiBrY (green) and DAPI (blue) are shown. Bar = 50 μm. (d) Normal human skin. BAP, biological antioxidant potential; BP, bullous pemphigoid; DiBrY, 3,5-dibromotyrosine; d-ROM, diacron-reactive oxygen metabolite; U.CARR, Carratelli unit.

Figure 2

Oxidative stress in patients with pemphigoid. (a–f) IHC staining of DiBrY. The upper row is a low magnification of the blister formation area (×40). Bar = 500 μm. The lower row is a high magnification of the blister area (×400). Bar = 50 μm. (g) Normal human skin with DiBrY staining (×40). Bar = 500 μm. (h) Normal human skin with DiBrY staining (×400). Bar = 50 μm. (i) Positive rate of DiBrY in immunostaining (DIF and IHC). (j) Scatterplot of BPDAI scores (total) and d-ROM levels. (k) Scatterplot of BPDAI scores (erosions/blisters) and d-ROM levels. Scatterplot showing a moderate positive correlation (r = 0.51; Pearson’s correlation coefficient). The dotted line is the trend line. BPDAI, Bullous Pemphigoid Disease Area Index; DiBrY, 3,5-dibromotyrosine; DIF, direct immunofluorescence; d-ROM, diacron-reactive oxygen metabolite; IHC, immunohistochemistry.