Cassian Sitaru

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

Mechanisms of blister induction by autoantibodies

Abstract:  Autoimmune diseases are characterized by defined self‐antigens, organ specificity, autoreactive T cells and/or autoantibodies that can transfer disease. Autoimmune blistering diseases are organ‐specific autoimmune diseases associated with an immune response directed to structural proteins mediating cell–cell and cell–matrix adhesion in the skin. While both autoreactive T and B cells have been detected and characterized in patients with autoimmune blistering diseases, current evidence generally supports a pathogenic role of autoantibodies for blister formation. The immunopathology associated with blisters induced by autoantibodies relies on several mechanisms of action. Autoantibodies from patients with pemphigus diseases can exert a direct effect just by binding to their target mediated by steric hindrance and/or by triggering the transduction of a signal to the cell. In most subepidermal autoimmune blistering conditions, in addition to the binding to their target antigen, autoantibodies need to interact with factors of the innate immune system, including the complement system and inflammatory cells, in order to induce blisters. Generally, decisive progress has been made in the characterization of the mechanisms of blister formation in autoimmune skin diseases. However, various aspects, including the exact contribution of steric hindrance and signal transduction for pemphigus IgG‐induced acantholysis or the fine tuning of the inflammatory cascade triggered by autoantibodies in some subepidermal blistering diseases, still need to be addressed. Understanding the mechanisms by which autoantibodies induce blisters should facilitate the development of more specific therapeutic strategies of autoimmune blistering diseases.

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.

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. Copyright

PI3Kβ Plays a Critical Role in Neutrophil Activation by Immune Complexes

The β isoform of phosphoinositide 3-kinase may be an effective therapeutic target in inflammatory diseases. The Integrating Isoform The class I phosphoinositide 3-kinases (PI3Ks) are implicated in processes such as growth factor signaling and inflammation. PI3Kγ is activated by G protein–coupled receptors (GPCRs), whereas PI3Kα and PI3Kδ are activated by protein tyrosine kinase–coupled receptors. PI3Kβ is unusual in that it appears to respond to signals from both types of receptors, depending on the cellular context. Kulkarni et al. investigated the responses of mouse neutrophils to immune complexes of antibody and antigen, which trigger chronic inflammation in conditions such as autoimmune arthritis. Genetic and pharmacological evidence suggested that immune complexes stimulated PI3Kβ in a process involving activation of FcγR, a tyrosine kinase–coupled low-affinity antibody receptor, and autocrine signaling by a proinflammatory lipid (LTB4) through its GPCR. Mice deficient in PI3Kβ fared better than did controls in models of arthritis and inflammatory skin disease. These data implicate PI3Kβ in the integration of signals from tyrosine kinase–coupled receptors and GPCRs—and suggest that this isoform may be an effective therapeutic target in inflammatory diseases. Neutrophils are activated by immunoglobulin G (IgG)–containing immune complexes through receptors that recognize the Fc portion of IgG (FcγRs). Here, we used genetic and pharmacological approaches to define a selective role for the β isoform of phosphoinositide 3-kinase (PI3Kβ) in FcγR-dependent activation of mouse neutrophils by immune complexes of IgG and antigen immobilized on a plate surface. At low concentrations of immune complexes, loss of PI3Kβ alone substantially inhibited the production of reactive oxygen species (ROS) by neutrophils, whereas at higher doses, similar suppression of ROS production was achieved only by targeting both PI3Kβ and PI3Kδ, suggesting that this pathway displays stimulus strength–dependent redundancy. Activation of PI3Kβ by immune complexes involved cooperation between FcγRs and BLT1, the receptor for the endogenous proinflammatory lipid leukotriene B4. Coincident activation by a tyrosine kinase–coupled receptor (FcγR) and a heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptor (BLT1) may provide a rationale for the preferential activation of the β isoform of PI3K. PI3Kβ-deficient mice were highly protected in an FcγR-dependent model of autoantibody-induced skin blistering and were partially protected in an FcγR-dependent model of inflammatory arthritis, whereas combined deficiency of PI3Kβ and PI3Kδ resulted in near-complete protection in the latter case. These results define PI3Kβ as a potential therapeutic target in inflammatory disease.

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.

Enzyme-linked immunosorbent assay using multimers of the 16th non-collagenous domain of the BP180 antigen for sensitive and specific detection of pemphigoid autoantibodies.

Abstract:  Bullous pemphigoid (BP) and pemphigoid gestationis (PG) are acquired autoimmune subepidermal blistering diseases characterized by autoantibodies against the hemidesmosomal proteins BP180/type XVII collagen and BP230. In the vast majority of BP and PG patients, these autoantibodies bind to epitopes clustered within the 16th non‐collagenous domain of BP180. An ELISA system for the detection of these autoantibodies was developed and evaluated using 16th non‐collagenous domain (NC16A) tetramers instead of monomers. In contrast to antigens fused to large proteins used in the past for the detection of autoantibodies against type XVII collagen, tetrameric antigen fragments bearing a small hexahistidine tag allow for high expression levels without the need to cleave off the fusion partner. Using tetrameric BP180 NC16A, positive reactions were found in 106 (89.8%) of 118 randomly selected BP sera and in all of 20 (100%) randomly selected PG sera, whereas only 2.2% of a large cohort of control subjects were positive in this assay, including patients with rheumatoid arthritis (two of 107), progressive systemic sclerosis (two of 50), systemic lupus erythematosus (one of 72), and healthy blood donors (10 of 494). Thus, the sensitivity and specificity of the new anti‐tetrameric NC16A ELISA were 89.9% and 97.8% respectively. Levels of circulating autoantibodies against BP180 paralleled disease activity in the pemphigoid patients. In conclusion, the use of tetrameric NC16A in ELISA results in a sensitive and specific tool for diagnosis and monitoring of BP and PG.

Protein A immunoadsorption: a novel and effective adjuvant treatment of severe pemphigus

Summary Background Pemphigus foliaceus (PF) and pemphigus vulgaris (PV) are autoimmune blistering skin diseases usually treated with high‐dose systemic corticosteroids and other immunosuppressants that may cause severe side‐effects. Plasmapheresis also has been demonstrated to be of benefit in the treatment of pemphigus. In contrast to plasmapheresis, staphylococcal protein A immunoadsorption (PA–IA) specifically removes immunoglobulin from the circulation, allows treatment of larger plasma volumes, and does not require the substitution of plasma components.

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.

Immunoblotting and enzyme-linked immunosorbent assay for the diagnosis of pemphigoid gestationis

OBJECTIVES: To investigate the sensitivity of immunoblotting and enzyme-linked immunosorbent assay (ELISA) to detect autoantibodies to bullous pemphigoid antigen 180 in patients with pemphigoid gestationis and to correlate autoantibody serum levels with disease activity. METHODS: In serum samples obtained from 44 pregnant patients before initiation of therapy and from the same number of healthy blood donors, the autoantibody reactivity was assayed by immunofluorescence microscopy on human skin sections as well as Western blot analysis and 2 different ELISAs by using recombinant forms of the immunodominant domain of BP180. In addition, ELISA reactivity with this autoantigen was assayed in 6 patients during the course of the disease, and its correlation with the clinical disease activity was estimated by applying the Spearman rank correlation test. RESULTS: By indirect immunofluorescence microscopy, complement-fixing autoantibodies to the dermal-epidermal junction were found in 93% of patients’ sera. By immunoblotting and ELISA, autoantibodies to bullous pemphigoid antigen 180 were detected in 93% and 86.3% of pemphigoid gestationis patients, respectively, but in none of the healthy controls. Serum levels of autoantibodies as detected by ELISA paralleled the patients’ disease activity. CONCLUSIONS: Our study shows that immunoblotting and ELISA are sensitive tools for the detection of autoantibodies to bullous pemphigoid antigen 180 in patients with pemphigoid gestationis. In addition, the ELISA is useful to monitor autoantibody serum levels. LEVEL OF EVIDENCE: II-2