Eosinophil localization to the basement membrane zone is autoantibody‐ and complement‐dependent in a human cryosection model of bullous pemphigoid

Abstract

Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by antibodies (IgG and IgE) targeting cell-substrate adhesion proteins. A variety of BP models suggest that autoantibody-dependent neutrophil degranulation is essential for blister formation. However, lesional biopsies reveal a predominance of eosinophils and few neutrophils. Our goal was to evaluate the role of antibodies and complement in eosinophil localization, degranulation, and split formation at the dermo-epidermal junction (DEJ) utilizing a human skin cryosection model of BP paired with a human eosinophilic cell line, 15HL-60. Expression of receptors for IgG (FcγRII), IgE (FcεRI), and complement (CR1 and CR3) was confirmed on 15HL-60 cells using flow cytometry. 15HL-60 expression of granule protein (eosinophil derived neurotoxin (EDN) and eosinophil peroxidase (EPO)) mRNA and their degranulation in vitro was confirmed using RT-PCR and ELISA, respectively. For cryosection experiments, BP or control sera or IgG and IgE antibodies purified from BP sera were utilized in combination with 15HL-60 cells ± fresh complement. Both BP serum and fresh complement were required for localization of 15-HL60 cells to the DEJ. Interestingly, eosinophil localization to the DEJ was dependent on IgG, but not IgE, and complement. However, no subepidermal split was observed. Additionally, the 15HL-60 cells did not degranulate under any experimental conditions and direct application of cell lysate to cryosections did not result in a split. Our observation that eosinophil localization to the DEJ is dependent on IgG mediated complement fixation provides additional insight into the sequence of events during the development of BP lesions.

Evidence for a role of eosinophils in blister formation in bullous pemphigoid

“…In agreement with a recent study, we observed that eosinophils activated solely with BP antibodies were not able to induce DES . Noteworthy, also similar to our study, IL‐5‐activated eosinophils have previously been demonstrated to bind throughout the dermis and not specifically to the basement membrane .…”

Eosinophils as putative therapeutic targets in bullous pemphigoid

“…Eosinophilic cells derived from a human myeloid leukaemia cell line were shown to localize at the dermal‐epidermal junction in the presence of BP serum, but not upon incubation with control serum . The attachment to the DEJ was mediated by IgG and complement, whereas IgE had no effect . However, under these conditions, a subepithelial blistering could not be observed, presumably owing to a lack of eosinophil degranulation .…”

“…Eosinophilic cells derived from a human myeloid leukaemia cell line were shown to localize at the dermal‐epidermal junction in the presence of BP serum, but not upon incubation with control serum . The attachment to the DEJ was mediated by IgG and complement, whereas IgE had no effect .…”

“…Anti‐inflammatory therapies such as interferon‐gamma or prednisolone plus intravenous immunoglobulins for patients with severe BP resulted in a decrease in IL‐5 levels and blood eosinophil numbers in parallel with the clinical improvement . Serum EDN levels strongly correlated with NC16A‐specific IgG . It has been suggested that by releasing tissue factor (TF), eosinophils might activate the coagulation cascade in BP, subsequently resulting in inflammation, tissue damage, blister formation and possibly thrombotic risk …”

023 Eosinophil Cationic Protein (ECP), a predictive marker of bullous pemphigoid severity and outcome

“…In this line, BP is clinically characterized by the presence of tense blisters along with intense pruritus, occurring preferentially on inflammatory, erythematous plaques. The use of mouse models allowed the demonstration that binding of autoantibodies onto their antigenic proteins is associated with an inflammatory cascade, including mast cell degranulation, and the recruitment and activation of neutrophils leading to dermal-epidermal separation 1,[11][12][13] . However, the role of eosinophils, which are the main tissue-infiltrated inflammatory cell type in the lesional skin of BP patient 14 , and especially how their granule proteins could impact BP disease severity and outcome still remains unclear in human.…”