Sidonia Mihai

Immunopathology and molecular diagnosis of autoimmune bullous diseases

•  Introduction •  Pemphigus diseases ‐  Pemphigus vulgaris ‐  Pemphigus foliaceus •  Pemphigus variants ‐  Pemphigus herpetiformis ‐  Pemphigus erythematosus ‐  Drug‐induced pemphigus ‐  Paraneoplastic pemphigus ‐  IgA pemphigus •  Pemphigoid diseases ‐  Bullous pemphigoid ‐  Pemphigoid gestationis ‐  Linear IgA disease ‐  Mucous membrane pemphigoid •  Anti‐p200 pemphigoid •  Epidermolysis bullosa acquisita •  Dermatitis herpetiformis Duhring

Induction of Complement-Fixing Autoantibodies against Type VII Collagen Results in Subepidermal Blistering in Mice

Experimental models reproducing an autoimmune response resulting in skin blistering in immunocompetent animals are lacking. Epidermolysis bullosa acquisita (EBA) is a bullous skin disease caused by autoantibodies to type VII collagen. In this study, we describe an active disease model of EBA by immunizing mice of different strains with murine type VII collagen. All mice developed circulating IgG autoantibodies that recognized type VII collagen and bound to the lamina densa of the dermal-epidermal junction. Importantly, subepidermal blisters developed in 82% of SJL-1, 56% of BALB/c mice, and 45% of FcγRIIb-deficient mice, but not in SKH-1 mice. In susceptible animals, deposits of IgG1, IgG2, and complement C3 were detected at the dermal-epidermal junction. In contrast, in the nondiseased mice, tissue-bound autoantibodies were predominantly of the IgG1 subclass and complement activation was weak or absent. This active disease model reproduces in mice the clinical, histopathological, and immunopathological findings in EBA patients. This robust experimental system should greatly facilitate further studies on the pathogenesis of EBA and the development of novel immunomodulatory therapies for this and other autoimmune diseases.

The relevance of the IgG subclass of autoantibodies for blister induction in autoimmune bullous skin diseases

Autoimmune bullous skin diseases are characterized by autoantibodies and T cells specific to structural proteins maintaining cell–cell and cell–matrix adhesion in the skin. Existing clinical and experimental evidence generally supports a pathogenic role of autoantibodies for blister formation. These autoantibodies belong to several IgG subclasses, which associate with different functional properties and may thus determine the pathogenic potential of IgG antibodies. In pemphigus diseases, binding of IgG to keratinocytes is sufficient to cause intraepidermal blisters without engaging innate immune effectors and IgG4 autoantibodies seem to mainly mediate acantholysis. In contrast, in most subepidermal autoimmune blistering diseases, complement activation and recruitment and activation of leukocytes by autoantibodies are required for blister induction. In these conditions, tissue damage is thought to be mainly mediated by IgG1, but not IgG4 autoantibodies. This review summarizes the current knowledge on the pathogenic relevance of the IgG subclass of autoantibodies for blister formation. Characterization of the pathogenically relevant subclass(es) of autoantibodies not only provides mechanistic insights, but should greatly facilitate the development of improved therapeutic modalities of autoimmune blistering diseases.

The Alternative Pathway of Complement Activation Is Critical for Blister Induction in Experimental Epidermolysis Bullosa Acquisita

Epidermolysis bullosa acquisita is a subepidermal blistering disease associated with tissue-bound and circulating autoantibodies against type VII collagen, a major constituent of the dermal-epidermal junction. The passive transfer of Abs against type VII collagen into mice induces a subepidermal blistering disease dependent upon activation of terminal complement components. To further dissect the role of the different complement activation pathways in this model, we injected C1q-deficient, mannan-binding lectin-deficient, and factor B-deficient mice with rabbit Abs against murine type VII collagen. The development and evolution of blistering had a similar pattern in mannan-binding lectin-deficient and control mice and was initially only marginally less extensive in C1q-deficient mice compared with controls. Importantly, factor B-deficient mice developed a delayed and significantly less severe blistering disease compared with factor B-sufficient mice. A significantly lower neutrophilic infiltration was observed in factor B-deficient mice compared with controls and local reconstitution with granulocytes restored the blistering disease in factor B-deficient mice. Our study provides the first direct evidence for the involvement of the alternative pathway in an autoantibody-induced blistering disease and should facilitate the development of new therapeutic strategies for epidermolysis bullosa acquisita and related autoimmune diseases.

Broad requirement for terminal sialic acid residues and FcγRIIB for the preventive and therapeutic activity of intravenous immunoglobulins in vivo

Intravenous immunoglobulin (IVIg) therapy is widely used to treat a variety of autoimmune diseases including immunothrombocytopenia, chronic inflammatory demyelinating polyneuropathy, and more recently autoimmune skin blistering diseases. Despite this well‐documented clinical success, the precise molecular and cellular mechanisms underlying this immunomodulatory activity are discussed controversially. In particular, the clinically relevant therapeutic pathway of IVIg‐mediated immune modulation has not been studied in detail. In the present study, we use four independent in vivo model systems of auto‐Ab‐mediated autoimmune disease to identify a common pathway explaining IVIg activity under therapeutic conditions in vivo. We show that irrespective of the in vivo model system, IVIg activity is strictly dependent on the presence of terminal sialic acid residues and the inhibitory FcγRIIB under preventive as well as therapeutic treatment conditions. In contrast, specific ICAM3 grabbing nonintegrin related 1, previously demonstrated to be essential under preventative treatment conditions, showed a disease‐specific impact on IVIg‐mediated resolution of established autoimmune disease.

IgG4 autoantibodies induce dermal–epidermal separation

Bullous pemphigoid (BP) is a sub‐epidermal autoimmune blistering disease associated with autoantibodies to the dermal–epidermal junction (DEJ). Patients’ autoantibodies induce dermal–epidermal separation when co‐incubated with cryosections of human skin and leucocytes from healthy volunteers. IgG autoantibodies trigger complement and/or leucocyte activation resulting in specific pathology in several autoimmune conditions. In these diseases, IgG1 and IgG3 isotypes, but not the IgG4 subclass, are thought to trigger inflammatory pathways resulting in tissue damage. The capacity of IgG4 autoantibodies to mediate tissue damage has not yet been demonstrated. In this study, we isolated IgG1 and IgG4 autoantibodies from bullous pemhigoid patientsserum and analysed their blister‐inducing potential in our cryosection assay. As expected, complement‐fixing IgG1 autoantibodies induced sub‐epidermal splits in this experimental model. Purified IgG4 did not fix complement, but, interestingly, like IgG1, activated leucocytes and induced dermal–epidermal separation. The potential of IgG4 autoantibodies to induce Fc‐dependent dermal–epidermal separation was significantly lower compared to IgG1. Our results demonstrate that IgG4 autoantibodies are able to activate leucocytes and point to a hitherto less recognized function of IgG4. Moreover, for the first time, we clearly demonstrate that BP IgG4 autoantibodies have the capacity to induce leucocyte‐dependent tissue damage.

T Cells Are Required for the Production of Blister-Inducing Autoantibodies in Experimental Epidermolysis Bullosa Acquisita

Epidermolysis bullosa acquisita is a prototypical organ-specific autoimmune disease caused by autoantibodies against type VII collagen of the dermal-epidermal junction. Although mechanisms of autoantibody-induced blister formation were extensively characterized, the initiation of autoantibody production in autoimmune blistering diseases is still poorly defined. In the current study, we addressed the role of T cells for the production of blister-inducing autoantibodies in mice immunized with type VII collagen. To detect autoreactive type VII collagen-specific T cells, lymph node cells from immunized SJL mice were stimulated in vitro with recombinant Ag, and their proliferation was measured by radioactive thymidine incorporation and flow cytometry analysis of CFSE-labeled cells. Interestingly, using synthetic peptides of the immunogen, partly different T and B cell epitopes in mice immunized with type VII collagen were demonstrated. In contrast to wild-type mice, immunization with type VII collagen of SJL athymic nude mice lacking T cells did not induce an autoimmune response and blistering phenotype. Importantly, SJL nude mice repleted with T cells from immunized wild-type mice showed a robust and durable autoantibody production resulting in subepidermal blistering disease in the recipients. Our present results demonstrate that T cells are required for the initiation of autoimmunity against type VII collagen in experimental epidermolysis bullosa acquisita and provide a basis for developing T cell-directed immunomodulatory strategies for this and related autoimmune diseases.

The role of Fc receptors and complement in autoimmunity

Autoantibodies interact with the innate immune system, including the complement network and Fc receptors (FcRs) bearing effector cells, resulting in the induction of tissue injury. It was suggested that these two pro-inflammatory pathways might mediate distinct effector responses, and that only one or the other effector arm may usually dominate an inflammatory response. Recent studies, however, support the notion that autoantibody-induced tissue injury may depend on both, FcRs and selected pathways of the complement network. This review summarizes our current knowledge on the interactions between autoantibodies, FcRs and complement components as essential triggers of tissue injury in autoimmune diseases like rheumatoid arthritis, anti-glomerular basement membrane glomerulonephritis and subepidermal blistering diseases. Manipulation of these connective pathways might be of therapeutic use to control antibody-mediated autoimmune diseases.

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.

Autoantibody-induced intestinal inflammation and weight loss in experimental epidermolysis bullosa acquisita.

Type VII collagen (COL7) is a major constituent of the cutaneous basement membrane. Loss of tolerance to COL7 leads to the blistering skin disease epidermolysis bullosa acquisita (EBA). Antibodies to COL7 have also been detected in patients with inflammatory bowel disease (IBD), yet reports on the expression of COL7 in the gut are controversial and a pathogenic relevance of anti‐COL7 autoantibodies in IBD has not been demonstrated. We therefore characterized the expression patterns of COL7 in murine gastrointestinal organs and investigated if anti‐COL7 antibodies induce an inflammatory response in the gut. COL7 expression was analysed on the mRNA and protein levels. Mice were injected with rabbit anti‐murine COL7 IgG (passive EBA) or immunized with a fragment of murine COL7 (active EBA). COL7 was found to be expressed in buccal mucosa, oesophagus, stomach, small intestine, and colon. In addition to skin blistering, in both passive and active EBA, autoantibodies bound to the gastrointestinal basement membrane, fixed complement, and led to recruitment of leukocytes. Furthermore, blister formation was observed in the oesophagus (40%/38% of mice in passive/active model), stomach (40%/63%), small intestine (20%/13%), and colon (20%/13%). Compared to control animals, we found a significantly reduced body weight in diseased mice, suggesting that autoantibody‐induced gastrointestinal inflammation is clinically relevant. Those observations may help us to understand the co‐incidence of IBD with EBA, and vice versa: The inflammatory response in IBD might expose novel antigens (COL7), which leads to the formation of anti‐COL7 antibodies. On the contrary, anti‐COL7 antibody‐induced gastrointestinal inflammation might pave the way for IBD pathogenesis. In summary, our results provide strong evidence that COL7 is expressed in different portions of the gut and that anti‐COL7 antibodies induce distinct gastrointestinal tissue damage. Copyright