S2k guidelines (consensus statement) for diagnosis and therapy of dermatitis herpetiformis initiated by the European Academy of Dermatology and Venereology (EADV).

INTRODUCTION: Dermatitis herpetiformis (DH) is a chronic, pruritic, gluten-induced skin disorder characterized by subepidermal granular IgA deposition and a variable degree of enteropathy identical to that seen in coeliac disease. So far, there has been no European consensus about the management of DH. METHODS: The guidelines were created by small subgroups of a guideline committee consisting of 26 specialists from various medical fields and one patients' representative. The members of the committee then discussed the guidelines and voted for the final version at two consensus meetings. The guidelines were developed under the support of the European Academy of Dermatology and Venereology (EADV) and in collaboration with the European Dermatology Forum (EDF). RESULTS: The guidelines summarize evidence-based and expert-based recommendations (S2 level) for the management of DH (see Appendix). CONCLUSION: These guidelines will improve the quality of management of DH and support dermatologists in their diagnostic and therapeutic decisions.

Monocytes enhance neutrophil-induced blister formation in an ex vivo model of bullous pemphigoid.

BACKGROUND: Lesions of bullous pemphigoid (BP), an autoimmune subepidermal blistering disease characterized by the presence of tissue-bound and circulating autoantibodies to hemidesmosomal antigens, harbor a mixed inflammatory cellular infiltrate. In various models, neutrophils, eosinophils, mast cells, monocytes as well as B and T cells have been shown to be involved in the pathogenesis of BP. However, their interactions with and effective role in blister formation remain uncertain. This study was aimed at investigating the effect of monocyte/neutrophil interaction on blister formation in an ex vivo BP model. METHODS: Skin cryosections were incubated with purified human neutrophils and monocytes, in the presence or absence of BP autoantibodies. Production of reactive oxygen species (ROS), degranulation, mediator release (neutrophil elastase [NE], myeloperoxidase [MPO], matrix metalloproteinase-9 [MMP-9]), binding of Fcγ receptor (CD16, CD32, CD64), and cell adhesion (CD18, ICAM-1) was investigated using appropriate inhibitors. Dermal-epidermal separation (DES) was assessed by light microscopy and quantified by Fiji software. RESULTS: Monocytes and neutrophils synergistically interact resulting in a significantly higher DES compared to either monocytes or neutrophils separately (P < .0001). Monocyte/neutrophil-induced DES was associated with increased ROS production and was dependent on adhesion and FcγRIII binding. Upon stimulation by the granule-poor fraction of monocyte supernatants, neutrophils increased their release of MMP-9, thereby also DES at the dermal-epidermal junction of skin cryosections. CONCLUSION: Our observations suggest that the interaction of cells, as shown here for monocytes and neutrophils, enhances mediator release resulting in an increased subepidermal blister formation. Thus, blocking intercellular cross talk promises a new therapeutic approach for blocking tissue damage in BP.

Serological diagnostics in the detection of IgG autoantibodies against human collagen VII in epidermolysis bullosa acquisita: a multicentre analysis.

BACKGROUND: Epidermolysis bullosa acquisita (EBA) is a rare, potentially devastating autoimmune disease of the skin. IgG autoantibodies directed against type VII collagen (Col7), the major component of anchoring fibrils, induce skin fragility leading to cutaneous and mucocutaneous blister formation, which is mostly of a scarring phenotype. Thus, powerful and reproducible diagnostic assays are critical to establish the diagnosis of EBA early to avoid irreversible sequelae. OBJECTIVES: The present international, retrospective multicentre study included a large cohort of patients with EBA and evaluated the diagnostic power of four different diagnostic assays for the detection of anti-Col7 IgG autoantibodies. METHODS: Overall, 95 EBA sera and 200 control sera consisting of 100 bullous pemphigoid sera, 50 pemphigus vulgaris sera and 50 sera of healthy controls were tested for anti-Col7 IgG autoantibodies using indirect immunofluorescence (IIF), two commercial enzyme-linked immunosorbent assay (ELISA) systems and Western blot (WB) analysis. EBA sera were taken from patients with positive direct immunofluorescence and IgG reactivity in at least one of the immunoserological assays (IIF, ELISA, WB). RESULTS: A Col7-NC1/NC2 ELISA (MBL, Nagoya, Japan) showed the highest sensitivity (97·9%), followed by a Col7-NC1 ELISA (Euroimmun, Lübeck, Germany) (89·5%), WB with Col7-NC1 (85·3%), and IIF on saline-split human skin (74·7%). The specificities of both ELISA systems were comparable (NC1 98·7%, NC1/NC2 99·3%). Furthermore, WB was more sensitive than IIF, which was more specific. CONCLUSIONS: The two commercially available ELISA systems allow for a highly sensitive and specific diagnosis of EBA. The sensitivity of the Col7-NC1/NC2 ELISA is significantly higher compared with the ELISA based on the Col7-NC1 domain only.

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

BACKGROUND: Bullous pemphigoid (BP) is an autoimmune bullous disease of the skin characterized by subepidermal blister formation due to tissue-bound and circulating autoantibodies to the hemidesmosomal antigens BP180 and BP230. Although eosinophils and their toxic mediators are found abundantly in BP lesions, their role in blister formation has remained unclear. OBJECTIVE: To investigate the role of eosinophils in the pathogenesis of BP with a specific focus on blister formation and to define conditions inducing dermal-epidermal separation (DES). METHODS: In an ex vivo human model of BP, normal human skin cryosections were incubated with purified human peripheral blood eosinophils with or without activation in the presence or absence of BP autoantibodies, brefeldin A, diphenyleneiodonium, DNase or blocking F(ab')2 fragments to CD16, CD18, CD32 and CD64. Dermal-epidermal separation was assessed by light microscopy studies and quantified using Fiji software. RESULTS: Following activation with IL-5 and in the presence of BP autoantibodies, eosinophils induced separation along the dermal-epidermal junction of ex vivo skin. Dermal-epidermal separation was significantly reduced by blocking any of the following: Fcγ receptor binding (P = 0.048), eosinophil adhesion (P = 0.046), reactive oxygen species (ROS) production (P = 0.002), degranulation (P < 0.0001) or eosinophil extracellular trap (EET) formation (P = 0.048). CONCLUSIONS: Our results provide evidence that IL-5-activated eosinophils directly contribute to BP blister formation in the presence of BP autoantibodies. Dermal-epidermal separation by IL-5-activated eosinophils depends on adhesion and Fcγ receptor activation, requires elevated ROS production and degranulation and involves EET formation. Thus, targeting eosinophils may be a promising therapeutic approach for BP.

[Molecular diagnosis of autoimmune dermatoses]. / Molekulare Diagnostik der blasenbildenden Autoimmundermatosen.

Bullous autoimmune diseases are organ-specific disorders characterized by an autoantibody-mediated blistering of skin and mucous membranes. The detection of tissue-bound and serum autoantibodies is prerequisite for the diagnosis of autoimmune blistering diseases. The individual entities of this group may be difficult to differentiate on clinical grounds alone. An accurate diagnosis is however important for prognosis and therapy. A preliminary diagnostic step includes direct and indirect immunofluorescence microscopy, which provide information about the binding pattern and isotype of autoantibodies and allow the diagnosis of the autoimmune blistering disease. Subsequent characterization of the molecular specificity of autoantibodies is necessary for the exact classification of autoimmune bullous dermatoses. The quantitative measurement of autoantibodies against structural proteins of the skin may be often used to assess disease severity at follow-up.

[Molecular diagnosis of collagen vascular diseases and vasculitides]. / Molekulare Diagnostik der Kollagenosen und Vaskulitiden.

Collagen vascular diseases and vasculitides comprise various diseases, which may affect virtually every organ system. Therefore, their diagnosis and management is often an interdisciplinary challenge. Because of the heterogeneous symptoms, these diseases have significant overlap, which interferes with the clinical diagnosis and may require additional investigation. Therefore, a rational and comprehensive diagnostic work-up should be performed at the initial presentation before initiation of therapy. The detection of antinuclear (ANA) or anticell antibodies by indirect immunofluorescence microscopy on Hep2 cells is used to screen for autoantibodies in collagen vascular diseases. The molecular specificity of autoantibodies should be further characterized using immunoassays with recombinant or purified protein. When systemic autoimmune disease is suspected, the function of the frequently affected organs should be evaluated. The immunopathological findings should always be interpreted in the context of clinical, histological, and imaging data. The detection of autoantibodies is helpful for the initial diagnosis, provides prognostic information, may indicate involvement of organs or systems and some parameters may also be used for disease monitoring. The clinical significance of autoantibodies is emphasized by the fact that their detection constitutes diagnostic criteria for most collagen vascular diseases and several vasculitides. The screening for ANCA may be performed using immunoassays with recombinant myeloperoxidase and proteinase 3 or by indirect immunofluorescence microscopy on granulocytes. In this article, the current diagnostic tools and their relevance for the diagnosis and monitoring of systemic autoimmune diseases with primary skin involvement are reviewed.

BP180- and BP230-specific IgG autoantibodies in pruritic disorders of the elderly: a preclinical stage of bullous pemphigoid?

Pruritus increasingly occurs in the elderly population and is associated with a variety of dermatoses of mixed aetiology. Clinical and experimental evidence suggests that senile pruritus may be linked to autoimmune events initiated by loss of self-tolerance against cutaneous autoantigens, which is facilitated by immune ageing processes. T-cell immunity, which underpins the production of pathogenic autoantibodies in autoimmune diseases, is deregulated by immune senescence thereby leading to autoimmune disorders such as bullous pemphigoid (BP). High mortality rates of BP combined with steadily increasing incidence emphasize the need for an effective diagnostic strategy at an early stage. We summarize here the current understanding of immunological alterations during the ageing process, thereby focusing on aberrant T-cell responses against the basement membrane antigens BP180 and BP230, which may eventually lead to the clinical outcome of BP.

Molecular diagnosis in autoimmune skin blistering conditions.

Blister formation in skin and mucous membranes results from a loss of cell-cell or cell-matrix adhesion and is a common outcome of pathological events in a variety of conditions, including autoimmune and genetic diseases, viral and bacterial infections, or injury by physical and chemical factors. Autoantibodies against structural components maintaining cell-cell and cell-matrix adhesion induce tissue damage in autoimmune blistering diseases. Detection of these autoantibodies either tissue-bound or circulating in serum is essential to diagnose the autoimmune nature of disease. Various immunofluorescence methods as well as molecular immunoassays, including enzyme-linked immunosorbent assay and immunoblotting, belong to the modern diagnostic algorithms for these disorders. There is still a considerable need to increase awareness of the rare autoimmune blistering diseases, which often show a severe, chronic-relapsing course, among physicians and the public. This review article describes the immunopathological features of autoimmune bullous diseases and the molecular immunoassays currently available for their diagnosis and monitoring.

IgE autoantibodies against the intracellular domain of BP180.

BACKGROUND: Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease characterized by autoantibodies against the hemidesmosomal proteins BP180 (type XVII collagen) and BP230. BP not only involves IgG-mediated neutrophil activation, leading to blistering, but also IgE-dependent activation of mast cells and basophils. While IgG and IgE autoantibodies target the extracellular noncollagenous (NC) 16A domain of BP180, little is known whether other BP180 regions are targeted by these antibody classes. OBJECTIVES: To characterize IgE and IgG autoantibody binding to antigenic sites on the intracellular domain (ICD) of BP180 compared with BP180 NC16A. METHODS: IgE/IgG autoreactivity against recombinant BP180 ICD and NC16A was determined by immunoblotting of sera from 18 patients with BP and 10 controls. RESULTS: Total serum IgE was elevated in 16 of 18 BP sera. Most BP sera tested positive (15 of 18) to NC16A with both immunoglobulin classes. Additionally, 14 of 18 sera showed IgE reactivity with an epitope mapped to the ICD of BP180 (amino acid residues 103-266). Mapping of ICD antigenic sites revealed similar IgE and IgG reactivities for most regions except for greater IgE reactivity to amino acid residues 234-398 (11 of 18 BP sera) than IgG (five of 18). Control sera failed to display IgE reactivity to these antigens. CONCLUSIONS: The results indicate that BP180 NC16A is not the only antigenic determinant of IgE autoantibodies in BP and that additional, novel epitopes exist on different regions of the ICD of BP180. The heterogeneous autoimmune response against BP180 suggests intramolecular epitope spreading during disease progression.

Cicatrising conjunctivitis with anti-basement membrane autoantibodies in ectodermal dysplasia.

AIMS: To report circulating and mucosa-deposited anti-basement membrane zone autoantibodies in a series of six ectodermal dysplasia patients with severe bilateral cicatrising conjunctivitis and blindness due to both corneal disease and intractable surface inflammation. We also report clinical improvement with steroid-sparing systemic immunosuppression combined with clearance of bacterial colonisation. METHODS: Conjunctival and buccal immunohistopathology, and serological analysis using a panel of epithelial basement membrane zone proteins including the bullous pemphigoid antigen 180 (BP180) were carried out as part of an ocular pemphigoid work-up in each patient. The degree of photophobia, conjunctival inflammation and visual acuity were monitored to evaluate the response to immunosuppression. The mean duration of follow-up was 31 (SD 6) months. RESULTS: Four of the six patients showed positive immunopathology: direct immunofluorescence testing of the conjunctiva in one patient demonstrated linear IgA deposition along the basement membrane zone, and IgG and IgM in the buccal mucosa of another patient. Circulating autoantibodies to BP180 were detected in two other patients. Treatment with systemic immunosuppression, combined with clearance of bacterial colonisation, reduced the severity of photophobia and degree of conjunctival inflammation in 5/6 (83%) patients. CONCLUSIONS: Systemic immunosuppression, used as steroid-sparing therapy, combined with clearance of bacterial colonisation can control inflammation and disabling photophobia, and allow improvement in vision, in a subgroup of ectodermal dysplasia patients who have severe cicatrising conjunctivitis which shares clinical and immunopathological features with ocular mucous membrane pemphigoid.

Binding of avian IgY to type VII collagen does not activate complement and leucocytes and fails to induce subepidermal blistering in mice.

BACKGROUND: Epidermolysis bullosa acquisita (EBA) is a severe autoimmune skin disease characterized by tissue-bound and circulating autoantibodies to type VII collagen, the major component of anchoring fibrils. When passively transferred into mice, rabbit IgG against type VII collagen induces Fc-dependent activation of complement, the recruitment of leucocytes into the skin, and subepidermal blistering. In addition to these inflammatory mechanisms, clinical and experimental evidence suggests that antibodies against type VII collagen might induce blisters by disrupting the ligand function of type VII collagen by an Fc-independent mechanism. OBJECTIVES: To study noninflammatory mechanisms of blister formation in experimental EBA. METHODS: We generated chicken IgY antibodies directed to recombinant type VII collagen and examined their pathogenic activity using ex vivo and animal models. RESULTS: Mice injected with these chicken IgY antibodies showed binding of the antibodies to the dermal-epidermal junction of skin sections. However, IgY antibodies did not fix complement C3 in enzyme-linked immunosorbent assay and immunofluorescence complement-binding assays. In addition, IgY antibodies did not induce dermal-epidermal separation ex vivo. Following their passive transfer into Balb/c mice, chicken IgY antibodies against type VII collagen bound at the dermal-epidermal junction, but, in contrast to rabbit IgG, did not fix complement C3, recruit granulocytes, or induce skin blisters. CONCLUSIONS: These findings demonstrate that binding of avian IgY to type VII collagen is not pathogenic in vivo and strongly suggest that in experimental EBA, antibodies to type VII collagen induce blisters not by direct disruption of the ligand function of type VII collagen, but rather by an Fc-dependent engagement of humoral and cellular inflammatory factors.

NADPH oxidase is required for neutrophil-dependent autoantibody-induced tissue damage.

The contribution of phagocyte-derived reactive oxygen species to tissue injury in autoimmune inflammatory diseases is unclear. Here we report that granulocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase crucially contributes to tissue injury in experimental models of the antibody-mediated autoimmune disease epidermolysis bullosa acquisita. Neutrophil cytosolic factor 1-deficient mice lacking functional NADPH oxidase were resistant to skin blistering by the passive transfer of antibodies against type VII collagen. Pharmacological inhibition or deficiency of human NADPH oxidase abolished dermal-epidermal separation caused by autoantibodies and granulocytes ex vivo. In addition, recruitment of granulocytes into the skin was required for tissue injury, as demonstrated by the resistance to experimental blistering of wild-type mice depleted of neutrophils and of CD18-deficient mice. Transfer of neutrophil cytosolic factor 1-sufficient granulocytes into neutrophil cytosolic factor 1-deficient mice demonstrated that granulocytes provide the NADPH oxidase required for tissue damage. Our findings identify granulocyte-derived NADPH oxidase as a key molecular effector engaged by pathogenic autoantibodies and provide relevant targets for prevention of tissue damage in granulocyte-mediated autoimmune diseases.

Successful adjuvant treatment of severe bullous pemphigoid by tryptophan immunoadsorption.

Bullous pemphigoid (BP) is an autoimmune blistering skin disease associated with circulating autoantibodies to the hemidesmosomal antigens BP180 and BP230. We report two cases of therapy-refractory BP adjuvantly treated by tryptophan immunoadsorption. In both patients, this treatment was associated with rapid clinical improvement and reduction in the required corticosteroid dosage. In addition, levels of circulating anti-BP180 autoantibodies decreased markedly. Antibodies that were eluted from the tryptophan matrix bound to BP180 and induced dermal-epidermal separation in cryosections of human skin. Our observations suggest that immunoadsorption may be a helpful adjuvant treatment in severe BP.