464 INVESTIGATIVE REPORT
ActaDV ActaDV Advances in dermatology and venereology Acta Dermato-Venereologica
Increased Activity and Apoptosis of Eosinophils in Blister Fluids, Skin and Peripheral Blood of Patients with Bullous Pemphigoid
Judith ENGMANN 1, Urda RÜDRICH 1, Georg BEHRENS 2, Eleni PAPAKONSTANTINOU 1, Manuela GEHRING 1, Alexander KAPP 1 and Ulrike RAAP 3 Departments of 1 Dermatology and Allergy and 2 Clinical Immunology and Rheumatology, Hannover Medical School, Hannover, and 3 University- Department of Dermatology and Allergy, Klinikum Oldenburg, Oldenburg, Germany Germany
Bullous pemphigoid( BP) is an autoimmune blistering skin disease that is more common in elderly individuals. The aim of this study was to determine the functional activity of eosinophils in patients with BP compared with healthy donors. Blood, skin and blisterderived eosinophils were strongly activated in patients with BP, seen by increased surface expression of CD69 compared with controls. CD11b was also increased in BP blood eosinophils, which may explain the striking accumulation of eosinophils in BP( 1 × 10 6 per ml blister fluid). Furthermore, CCL26 was expressed by activated eosinophils in BP skin and in blister fluid. BP eosinophils also released IL-6, IL-8 and IL-1α in BP blister fluids. Apoptosis in cultivated BP eosinophils was increased and accompanied by enhanced surface externalization of CD95. Caspase 3 positive eosinophils in lesional BP skin and blister fluid also showed the initiation of apoptosis. These results reveal novel pathophysiological aspects of BP, with a strong activation pattern and increased apoptosis of eosinophils in the peripheral blood, skin and blister fluids.
Key words: apoptosis; bullous pemphigoid; CD69; eosinophils, eotaxin.
Accepted Nov 17, 2016; Epub ahead of print Nov 21, 2016 Acta Derm Venereol 2017; 97: 464 – 471.
Corr: Ulrike Raap, Director University-Department of Dermatology and Allergy, Klinikum Oldenburg, Rahel-Strauss-Str. 10, DE-26133 Oldenburg, Germany. E-mail: Raap. ulrike @ klinikum-oldenburg. de
Bullous pemphigoid( BP) is an autoimmune blistering skin disease that more commonly affects individuals from the age of 60 years. In general, more women than men are affected by BP( 1, 2). Clinically, the early phases of BP often show flares, and sometimes additionally wheals with strong pruritus, while in late phases flares are also accompanied by tense blisters( 3). The diagnosis of BP is confirmed by positive direct immunofluorescence with linear expression of C3 and IgG at the dermoepidermal junction zone, as well as through positive indirect immunofluorescence with autoantibodies against the hemidesmosomal proteins BP180 and BP230. With regard to the extracellular membrane of BP180, a genetic predisposition has been detected in HLA-DQB1 * 0301( 4). Seventy percent of patients with untreated BP display elevated total IgE levels. In addition, specific IgE antibodies, reacting with the NC16A domain of BP180, have been described( 5).
Due to the formation of antigen-antibody complexes a variety of different cytokines, such as IL-6 and IL-8, are released( 6). Eotaxin is also secreted by epidermal keratinocytes( 7), which has strong chemotactic effects on eosinophils. In patients with BP, characteristic findings are high rates of peripheral blood eosinophils, eosinophil-rich infiltrates in subepidermal blisters( 8, 9), and increased numbers of tissue-resident eosinophils in skin lesions( 10). This eosinophil accumulation is of particular interest, since eosinophils may contribute to the inflammation associated with BP through their release of chemokines, cytokines, and cytotoxic granule proteins. In other eosinophil-associated skin diseases, including atopic dermatitis and allergic contact eczema, the eosinophil-derived cytotoxic granule protein ECP correlates with disease severity( 11 – 13). MCP-4, ECP, EPO and IL-5( 14, 15) have been detected in blister fluids of BP patients and support the inflammatory response. It has been demonstrated that MMP9( 16), which is released by eosinophils, plays a role in the detachment of the dermal – epidermal junction in BP( 17, 18).
This study assessed the activation pattern of eosinophils in patients with BP compared with controls. Furthermore the study explored the cytokine profile of BP eosinophils and compared the apoptotic responses of BP eosinophils with those of healthy controls.
METHODS Subject groups and patient samples
BP was defined by specific histopathological observation of subepidermal blistering, a positive indirect and direct immunofluorescence, and detection of specific auto-antibodies related to BP180 and / or BP230( 19). Patients with BP( n = 25, Table SI 1) did not receive systemic immunosuppressive treatment with glucocorticosteroids or diaminodiphenylsulfone or local therapy with glucocorticosteroids for the preceding 4 weeks. Blisters of the skin from patients with BP were punctured immediately after admission, analysed for eosinophil counts( Neubauer chamber) and subsequently processed to perform cytospins( 1 × 10 5 cells / spot). As controls, eosinophils were obtained from the peripheral blood of 10 healthy individuals who were not atopic and had normal total IgE levels and no allergen-specific IgE-antibodies against 10 common aeroallergens. Furthermore, control blister fluids of inpatients with diseases such as bullous erysipel and phototoxic dermatitis were included( Table SII 1). Serum and blister fluids
1 https:// www. medicaljournals. se / acta / content / abstract / 10.2340 / 00015555-2581 doi: 10.2340 / 00015555-2581 Acta Derm Venereol 2017; 97: 464 – 471
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