This novel ELISA will facilitate the diagnosis of anti-p200 pemphigoid.

The cell stress chaperone heat shock protein 90 (Hsp90) has been implicated in inflammatory responses and its inhibition has proven successful in different mouse models of autoimmune diseases, including epidermolysis bullosa acquisita. Here, we investigated expression levels and secretory responses of Hsp90 in patients with bullous pemphigoid (BP), the most common subepidermal autoimmune blistering skin disease. In comparison to healthy controls, the following observations were made: (i) Hsp90 was highly expressed in the skin of BP patients, whereas its serum levels were decreased and inversely associated with IgG autoantibody levels against the NC16A immunodominant region of the BP180 autoantigen, (ii) in contrast, neither aberrant levels of circulating Hsp90 nor any correlation of this protein with serum autoantibodies was found in a control cohort of autoimmune bullous disease patients with pemphigus vulgaris, (iii) Hsp90 was highly expressed in and restrictedly released from peripheral blood mononuclear cells of BP patients, and (iv) Hsp90 was potently induced in and restrictedly secreted from human keratinocyte (HaCaT) cells by BP serum and isolated anti-BP180 NC16A IgG autoantibodies, respectively. Our results reveal an upregulated Hsp90 expression at the site of inflammation and an autoantibody-mediated dysregulation of the intracellular and extracellular distribution of this chaperone in BP patients. These findings suggest that Hsp90 may play a pathophysiological role and represent a novel potential treatment target in BP.

Autoantibody-mediated diseases comprise a heterogeneous group of disorders in which the pathogenic potential of autoantibodies has been clearly demonstrated. In general, their treatment relies on the long-term use of systemic corticosteroids and other immunosuppressants that are associated with considerable adverse reactions. EndoS, an endoglycosidase derived from Streptococcus pyogenes, specifically hydrolyzes the N-linked glycan of native IgG and has previously been shown to modulate the interaction between the Fc portion of autoantibody and Fcγ receptors on leukocytes. Here, different models of autoimmunity to type VII collagen, a structural protein of the dermal-epidermal junction (DEJ), were employed to explore the therapeutic potential of EndoS. First, pretreatment of otherwise pathogenic anti-murine type VII collagen (mCOL7) IgG with EndoS significantly reduced split formation at the DEJ in cryosections of murine skin and abrogated clinical disease in mice. Next, the effect of EndoS was also seen when the enzyme was injected into mice after pathogenic anti-mCOL7 IgG had been administered. Finally, to mimic the patient situation even closer, EndoS was applied in mice that had already developed clinical disease after immunization with mCOL7. In all EndoS-treated mice, disease progression was stopped, and in the majority of mice, clinical disease even regressed. Of note, EndoS was shown to hydrolyze already in vivo-bound pathogenic autoantibodies. In addition, EndoS treatment decreased lesional expression of activating FcγRs while increasing FcγRIIB expression.

Although reports documented aberrant cytokine expression in autoimmune bullous dermatoses (AIBDs), cytokine-targeting therapies have not been established in these disorders. We showed previously that IL-6 treatment protected against tissue destruction in experimental epidermolysis bullosa acquisita (EBA), an AIBD caused by autoantibodies to type VII collagen (COL7). The anti-inflammatory effects of IL-6 were mediated by induction of IL-1ra, and prophylactic IL-1ra administration prevented blistering. In this article, we demonstrate elevated serum concentrations of IL-1β in both mice with experimental EBA induced by injection of anti-COL7 IgG and in EBA patients. Increased IL-1α and IL-1β expression also was observed in the skin of anti-COL7 IgG-injected wild-type mice compared with the significantly less diseased IL-1R–deficient or wild-type mice treated with the IL-1R antagonist anakinra or anti–IL-1β. These findings suggested that IL-1 contributed to recruitment of inflammatory cells into the skin. Accordingly, the expression of ICAM-1 was decreased in IL-1R–deficient and anakinra-treated mice injected with anti-COL7. This effect appeared to be specifically attributable to IL-1 because anakinra blocked the upregulation of different endothelial adhesion molecules on IL-1–stimulated, but not on TNF-α–stimulated, cultured endothelial cells. Interestingly, injection of caspase-1/11–deficient mice with anti-COL7 IgG led to the same extent of skin lesions as in wild-type mice. Collectively, our data suggest that IL-1, independently of caspase-1, contributes to the pathogenesis of EBA. Because anti–IL-1β in a prophylactic setting and anakinra in a quasi-therapeutic setting (i.e., when skin lesions had already developed) improved experimental EBA, IL-1 appears to be a potential therapeutic target for EBA and related AIBDs.

Recently, the C-terminus of laminin γ1 has been identified as target antigen in anti-p200 pemphigoid and the disease was renamed as anti-laminin γ1 pemphigoid. However, the pathogenic relevance of these autoantibodies has not yet been demonstrated. Therefore, we employed an ex vivo model of autoantibody-mediated leukocyte-dependent neutrophil activation and dermal-epidermal separation (DES) using cryosections of human skin. We showed that anti-p200 pemphigoid sera (n = 7) induced DES in a time-dependent manner, in contrast to sera from healthy controls. Furthermore, laminin γ1-specific IgG and serum depleted from anti-laminin γ1 reactivity were generated using the recombinant C-terminus of laminin γ1 (LAMC1-term; amino acids 1364 to 1609). Interestingly, both fractions labeled the dermal-epidermal-junction (DEJ) by indirect immunofluorescence microscopy on human foreskin and recognized a 200 kDa protein by immunoblotting with dermal extract. Human and rabbit IgG against LAMC1-cterm failed to attract neutrophils at the DEJ and to induce DES. In contrast, patient serum depleted from LAMC1-cterm reactivity led to the same extent of DES as non-depleted IgG. Repeated injection of rabbit anti-murine LAMC1-cterm IgG into both neonatal and adult C57BL/6mice as well as repetitive immunization of various mouse strains with murine LAMC1-cterm failed to induce macro- and microscopic lesions. In all mice, circulating anti-LAMC1-cterm antibodies were present, but only in some mice, IgG deposits were seen at the DEJ. We conclude that autoantibodies in anti-p200 pemphigoid sera are pathogenic while pathogenicity is not mediated by autoantibodies against laminin γ1. Further studies are needed to identify the pathogenically relevant autoantigen in anti-p200 pemphigoid.