Elevated levels of interleukin-9 in the serum of bullous pemphigoid: possible association with the pathogenicity of bullous pemphigoid

Abstract

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease (sAIBD). In addition to disease causing autoantibodies, several leukocyte subsets, including mast cells and eosinophils, play key roles in mediating skin inflammation. Detailed immunophenotyping and, more recently, the therapeutic effects of interleukin-4 (IL-4) receptor alpha inhibition in BP pointed to a prominent role of T helper 2 (Th2) cells. Among other cell types, IL-9 is expressed by Th2 and mast cells and potentially drives allergic, Th2-dominated inflammation. Although cytokines in BP have been relatively well investigated, the role of IL-9 has remained enigmatic. This study aimed to evaluate the effect of IL-9 in BP. Serum IL-9 levels were significantly elevated in patients with BP and decreased upon induction of remission. Serum IL-9 levels were not elevated in epidermolysis bullosa acquisita, another sAIBD. The time-course analysis using serum sets from four patients with BP revealed that serum IL-9 was a sensitive biomarker of BP. IL-9-positive cells infiltrated dominantly in BP lesions, especially in the blister fluid, and Th9 cells were abundant. Therefore, IL-9 was elevated in the serum and lesions of BP, which could be a biomarker of BP.

Keywords: autoantibody; autoimmune bullous disease; basement membrane; cytokine; pemphigoid.

Figure 1

Serum IL-9 in patients with bullous pemphigoid (BP) and epidermolysis bullosa acquisita (EBA). (A) Serum IL-9 concentrations in normal control (NC; n = 30), BP (n = 22), and EBA (n = 20). One-way ANOVA followed by Kruskal–Wallis test was performed. (B) Comparison of IL-9 concentrations between patients in the active stage and in the remission stage of BP (n = 22). The Mann–Whitney test was used. The Pearson correlation test was performed with serum total IgE (C; n = 20) or serum IgE anti-BP180 (D; n = 22) and serum IL-9.

Figure 2

Time-course analysis of serum IL-9 levels during treatment for bullous pemphigoid (BP). In four patients with BP (A–D), serum IL-9, anti-BP180 titer (ELISA or CLEIA), and the absolute count of peripheral eosinophils were measured at the initiation of the treatment every week (except case C). The treatments were informed on the upper section of each figure. PSL, prednisolone; mPSL, methylprednisolone; mPSL pulse, mPSL pulse therapy (1,000 mg of mPSL for 3 days); CyA, cyclosporin A; AZP, azathioprine.

Figure 3

IL-9 expressions in the lesional skins of bullous pemphigoid (BP), atopic dermatitis (AD), and psoriasis vulgaris (Pso). Biopsied skin sections from BP (n = 8), AD (n = 7), Pso (n = 8), and normal control (NC; n = 7) were stained with anti-human IL-9 antibodies by immunohistochemistry. (A) Representative pictures with low magnification (upper panels) and high magnification in the dermis (middle panels) or blisters (lower panel) (bar = 100 µm). (B) IL-9–positive cells were counted at ×200 magnification. The average of counts from three fields at ×200 magnification was evaluated in each section. Data were analyzed by one-way ANOVA followed by Tukey’s test.

Figure 4

Double staining of IL-9 and transcription factors for Th2, Th17, Th9, or tryptase. A biopsied specimen from lesional BP skin was stained with combinations of anti–IL-9 (red) and the following antibodies (green): anti-PU.1 for Th9 cells (upper and middle panels), anti-GATA3 for Th2 cells, anti-RORγt for Th17 cells, anti-Foxp3 for regulatory T cells, and anti-tryptase for mast cells (lower panels), followed by 4', 6-diamidino-2-phenylindole (DAPI) to counterstain the nucleus (blue). Double-positive cells are highlighted with arrows.