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.     

The alpha 5 chain of type IV collagen is the target of IgG autoantibodies in a novel autoimmune disease with subepidermal blisters and renal insufficiency

We describe a novel autoimmune disease characterized by severe subepidermal bullous eruptions and renal insufficiency with IgG autoantibodies directed against the NC1 domain of the alpha5(IV) collagen chain. In vivo deposits of IgG and C3 were found along the dermal-epidermal junction of skin lesions. The identity of the target antigen was determined by immunochemical analyses of candidate antigens using the patients' autoantibodies. The patients' IgG autoantibodies reacted with a 185-kDa polypeptide that was distinguished from the known autoantigens of the extracellular matrix including type XVII collagen, type VII collagen, or the alpha3, beta3, and gamma2 chains of laminin 5. Preincubation of the serum with recombinant alpha5(IV)NC1 domain of type IV collagen abolished immunoreactivity with the 185-kDa antigen. The serum reacted specifically with the alpha5(IV)NC1, among the six NC1 domains of type IV collagen, by Western blot and enzyme-linked immunosorbent assay analyses. The patients' autoantibodies reacted with normal skin and renal glomerulus but not with skin and glomerulus of a patient with Alport syndrome in which the basement membranes are devoid of the alpha5(IV) collagen chain. This study provided for the first time unambiguous evidence for the alpha5(IV) collagen chain as the target antigen in a novel autoimmune disease characterized by skin and renal involvement.     

Blister‐inducing antibodies target multiple epitopes on collagen VII in mice

Epidermolysis bullosa acquisita (EBA) is an autoimmune subepidermal blistering disease of mucous membranes and the skin caused by autoantibodies against collagen VII. In silico and wet laboratory epitope mapping studies revealed numerous distinct epitopes recognized by EBA patients' autoantibodies within the non‐collagenous (NC)1 and NC2 domains of collagen VII. However, the distribution of pathogenic epitopes on collagen VII has not yet been described. In this study, we therefore performed an in vivo functional epitope mapping of pathogenic autoantibodies in experimental EBA. Animals (n = 10/group) immunized against fragments of the NC1 and NC2 domains of collagen VII or injected with antibodies generated against the same fragments developed to different extent experimental EBA. Our results demonstrate that antibodies targeting multiple, distinct epitopes distributed over the entire NC1, but not NC2 domain of collagen VII induce blistering skin disease in vivo. Our present findings have crucial implications for the development of antigen‐specific B‐ and T cell‐targeted therapies in EBA.     

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 patients'serum 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.     

The flavonoid luteolin inhibits Fcγ-dependent respiratory burst in granulocytes, but not skin blistering in a new model of pemphigoid in adult mice

Bullous pemphigoid is an autoimmune blistering skin disease associated with autoantibodies against the dermal-epidermal junction. Passive transfer of antibodies against BP180/collagen (C) XVII, a major hemidesmosomal pemphigoid antigen, into neonatal mice results in dermal-epidermal separation upon applying gentle pressure to their skin, but not in spontaneous skin blistering. In addition, this neonatal mouse model precludes treatment and observation of diseased animals beyond 2–3 days. Therefore, in the present study we have developed a new disease model in mice reproducing the spontaneous blistering and the chronic course characteristic of the human condition. Adult mice were pre-immunized with rabbit IgG followed by injection of BP180/CXVII rabbit IgG. Mice pre-immunized against rabbit IgG and injected 6 times every second day with the BP180/CXVII-specific antibodies (n = 35) developed spontaneous sustained blistering of the skin, while mice pre-immunized and then treated with normal rabbit IgG (n = 5) did not. Blistering was associated with IgG and complement C3 deposits at the epidermal basement membrane and recruitment of inflammatory cells, and was partly dependent on Ly-6G-positive cells. We further used this new experimental model to investigate the therapeutic potential of luteolin, a plant flavonoid with potent anti-inflammatory and anti-oxidative properties and good safety profile, in experimental BP. Luteolin inhibited the Fcγ-dependent respiratory burst in immune complex-stimulated granulocytes and the autoantibody-induced dermal-epidermal separation in skin cryosections, but was not effective in suppressing the skin blistering in vivo. These studies establish a robust animal model that will be a useful tool for dissecting the mechanisms of blister formation and will facilitate the development of more effective therapeutic strategies for managing pemphigoid diseases.     

Granulocyte-dependent Autoantibody-induced Skin Blistering

Autoimmune phenomena occur in healthy individuals, but when self-tolerance fails, the autoimmune response may result in specific pathology. According to Witebsky''s postulates, one of the criteria in diagnosing a disease as autoimmune is the reproduction of the disease in experimental animals by the passive transfer of autoantibodies. For epidermolysis bullosa acquisita (EBA), a prototypic organ-specific autoimmune disease of skin and mucous membranes, several experimental models were recently established. In the animal model described in our present work, purified IgG antibodies against a stretch of 200 amino acids (aa 757-967) of collagen VII are injected repeatedly into mice reproducing the blistering phenotype as well as the histo- and immunopathological features characteristic to human EBA ¹. Full-blown widespread disease is usually seen 5-6 days after the first injection and the extent of the disease correlates with the dose of the administered collagen VII-specific IgG. The tissue damage (blister formation) in the experimental EBA is depending on the recruitment and activation of granulocytes by tissue-bound autoantibodies ^2,-4. Therefore, this model allows for the dissection of the granulocyte-dependent inflammatory pathway involved in the autoimmune tissue damage, as the model reproduces only the T cell-independent phase of the efferent autoimmune response. Furthermore, its value is underlined by a number of studies demonstrating the blister-inducing potential of autoantibodies in vivo and investigating the mechanism of the blister formation in EBA ^1,3,-6. Finally, this model will greatly facilitate the development of new anti-inflammatory therapies in autoantibody-induced diseases. Overall, the passive transfer animal model of EBA is an accessible and instructive disease model and will help researchers to analyze not only EBA pathogenesis but to answer fundamental biologically and clinically essential autoimmunity questions.     

Complement-fixing anti-type VII collagen antibodies are induced in Th1-polarized lymph nodes of epidermolysis bullosa acquisita-susceptible mice

The environment encountered in secondary lymphoid organs (e.g., lymph nodes) influences the outcome of immune responses. Immunization of mice with type VII collagen, an adhesion protein expressed at the cutaneous basement membrane, induces experimental epidermolysis bullosa acquisita (EBA). In this model, clinical disease is associated with the H2s haplotype of the MHC found in SJL/J mice. Most other strains (e.g., BALB/c, C57BL/6, NZM2410/J) are resistant to clinical disease, despite autoantibody production. Comparison of autoantibody response in EBA-resistant and -susceptible mice showed an IgG2-dominated response in the latter. We hypothesized that EBA susceptibility is due to specific cytokine gene expression in draining lymph nodes (dLN). To challenge this hypothesis, EBA-susceptible (SJL/J) and -resistant (BALB/c, C57BL/6) mice were immunized with type VII collagen, followed by analysis of clinical phenotype, subclasses of circulating and tissue-bound autoantibodies, complement activation, and cytokine gene expression in dLN. Disease manifestation was associated with induction of complement-fixing autoantibodies, confirming previous observations. Furthermore, however, IFN-γ/IL-4 ratio in dLN of EBA-susceptible mice was significantly increased compared with EBA-resistant strains, suggesting a Th1 polarization. Immunization of H2s-congenic C57BL/6 mice (B6.SJL-H2s) led to Th1 polarization in dLN and clinical disease. In addition to their cytokine milieu, EBA-susceptible and -resistant mice also differed regarding the expression of FcγR on peripheral leukocytes, in which a higher FcγRIV expression in SJL/J and B6.SJL-H2s mice, compared with C57BL/6, was associated with skin lesions. In summary, blistering in experimental EBA is regulated by both adaptive (divergent class switch recombination due to polarized cytokine expression) and innate (FcγR expression) immune mechanisms.     

Repetitive immunization breaks tolerance to type XVII collagen and leads to bullous pemphigoid in mice

Bullous pemphigoid (BP) is a subepidermal autoimmune blistering disease of the elderly associated with considerable morbidity and mortality. As unspecific immunosuppressants are still the mainstay of BP therapy, several animal models, based on the passive transfer of autoantibodies or immune cells, have been developed to obtain a better understanding of the pathogenesis of BP and evaluate novel therapeutic interventions. We describe in this study an experimental model inducing BP by immunization of immunocompetent mice with a recombinant form of the immunodominant 15th noncollagenous domain of murine BP180 (type XVII collagen). The homologous noncollagenous 16A domain of human BP180 has previously been identified as an immunodominant region in human BP. Immunization of female SJL/J mice with the murine peptide led to clinical disease within 14 wk in 56% of mice. In contrast, none of the other strains developed blisters despite the presence of autoantibodies. The clinical disease manifested for at least 8 wk without further manipulation. This novel immunization-induced model reflects key immunopathological characteristics of human BP, including binding of complement-fixing autoantibodies along the dermal-epidermal junction, elevated total IgE serum levels, and infiltration of skin lesions with eosinophilic granulocytes. The use of immunocompetent mice and the induction of sustained clinical disease not requiring additional interventions make this immunization-induced mouse model most suitable to further explore the pathogenesis of BP and novel therapeutic interventions for this and other autoantibody-mediated diseases.     

Autoimmunity against type VII collagen in inflammatory bowel disease

Autoimmunity against type VII collagen, an adhesion molecule of the extracellular matrix in epithelial basement membranes, is causing the rare organ-specific epidermolysis bullosa acquisita (EBA). An intriguing association between EBA and inflammatory bowel disease (IBD) has been extensively documented over the last decades, but, because of the very low incidence of EBA, received little attention from physicians involved in the care of patients with IBD. More recently, autoantibodies against type VII collagen have been detected in up to 68% of IBD patients. Although these findings suggest that chronic intestinal inflammation in IBD predisposes for autoimmunity against type VII collagen, their relevance for the pathogenesis of both IBD and EBA is still unclear. In this review article, the main features of the association between IBD and EBA are presented and pathomechanistic hypotheses as well as future lines of investigation in this area are discussed. Future research should provide new pathomechanistic insights and will likely facilitate the development of more specific and effective immunotherapeutic strategies for both conditions.     

Human IgG1 monoclonal antibody against human collagen 17 noncollagenous 16A domain induces blisters via complement activation in experimental bullous pemphigoid model

Bullous pemphigoid (BP) is an autoimmune blistering disease caused by IgG autoantibodies targeting the noncollagenous 16A (NC16A) domain of human collagen 17 (hCOL17), which triggers blister formation via complement activation. Previous in vitro analysis demonstrated that IgG1 autoantibodies showed much stronger pathogenic activity than IgG4 autoantibodies; however, the exact pathogenic role of IgG1 autoantibodies has not been fully demonstrated in vivo. We constructed a recombinant IgG1 mAb against hCOL17 NC16A from BP patients. In COL17-humanized mice, this mAb effectively reproduced a BP phenotype that included subepidermal blisters, deposition of IgG1, C1q and C3, neutrophil infiltration, and mast cell degranulation. Subsequently, alanine substitutions at various C1q binding sites were separately introduced to the Fc region of the IgG1 mAb. Among these mutated mAbs, the one that was mutated at the P331 residue completely failed to activate the complement in vitro and drastically lost pathogenic activity in COL17-humanized mice. These findings indicate that P331 is a key residue required for complement activation and that IgG1-dependent complement activation is essential for blister formation in BP. This study is, to our knowledge, the first direct evidence that IgG1 Abs to hCOL17 NC16A can induce blister formation in vivo, and it raises the possibility that IgG1 mAbs with Fc modification may be used to block pathogenic epitopes in autoimmune diseases.     

Laminin 5 can promote assembly of the lamina densa in the skin equivalent model

Skin equivalents were prepared by culturing human keratinocytes on the surface of type I collagen gel contracted by human skin fibroblasts (dermal equivalents) and by raising the gel to an air-liquid interface. A stratified squamous epithelium was formed with a well-differentiated cornified layer at the top of keratinocyte layers within 7 days after plating of the keratinocytes on the dermal equivalents. Although major basement membrane components such as collagens IV and VII and laminin 5 were detected immunohistochemically at the dermal-epidermal junction, a lamina densa was rarely observed by electron microscopy even in 14-day skin equivalents. When laminin 5 (1, 5 or 20 μg/ml) was added to the culture medium on day 7 through day 14, types IV and VII collagens at the dermal-epidermal junction stained more strongly by immunohistochemistry compared with the control. Patches of lamina densa were present along the epidermal-dermal junction, and vesicles containing electron-opaque sheets approximately 0.6 μm in diameter that reacted with anti-collagen IV antibody were also observed in basal keratinocytes in 14-day skin equivalents by electron microscopy. Morphometric analysis showed that the total length of lamina densa along the dermal-epidermal junction as well as in the vesicles increased up to 180%, 230% or 520% of control cultures by the addition of laminin 5 (1, 5 or 20 μg/ml, respectively). These results suggest that laminin 5 accelerates formation of the lamina densa along dermal-epidermal junction of the skin equivalents, depending on the concentration of laminin 5 supplemented exogenously.     

Proteinases of the bone morphogenetic protein-1 family convert procollagen VII to mature anchoring fibril collagen

Collagen VII is the major structural component of the anchoring fibrils at the dermal-epidermal junction in the skin. It is secreted by keratinocytes as a precursor, procollagen VII, and processed into mature collagen during polymerization of the anchoring fibrils. We show that bone morphogenetic protein-1 (BMP-1), which exhibits procollagen C-proteinase activity, cleaves the C-terminal propeptide from human procollagen VII. The cleavage occurs at the BMP-1 consensus cleavage site SYAA/DTAG within the NC-2 domain. Mammalian tolloid-like (mTLL)-1 and -2, two other proteases of the astacin enzyme family, were able to process procollagen VII at the same site in vitro. Immunohistochemical and genetic evidence supported the involvement of these enzymes in cleaving type VII procollagen in vivo. Both BMP-1 and mTLL-1 are expressed in the skin and in cultured cutaneous cells. A naturally occurring deletion in the human COL7A1 gene, 8523del14, which is associated with dystrophic epidermolysis bullosa and eliminates the BMP-1 consensus sequence, abolished processing of procollagen VII, and in mutant skin procollagen VII accumulated at the dermal-epidermal junction. On the other hand, deficiency of BMP-1 in the skin of knockout mouse embryos did not prevent processing of procollagen VII to mature collagen, suggesting that mTLL-1 and/or mTLL-2 can substitute for BMP-1 in the processing of procollagen VII in situ.     

Antibodies to pathogenic epitopes on type XVII collagen cause skin fragility in a complement-dependent and -independent manner

In bullous pemphigoid (BP), the most prevalent autoimmune blistering disease, type XVII collagen (COL17) is targeted by circulating autoantibodies. BP is thought to be an autoantibody-mediated complement-fixing blistering disease, and a juxtamembranous noncollagenous 16A (NC16A) domain spanning Glu(490) to Arg(566) was proved to be the main pathogenic region on COL17, although precise pathogenic epitopes within NC16A have not been elucidated. In this study, we showed that injection of rabbit IgG Abs targeting Asp(522) to Gln(545) induced skin fragility associated with in vivo deposition of IgG and complement in neonatal COL17-humanized mice. Notably, immunoadsorption of rabbit anti-NC16A IgG Ab with this epitope (Asp(522) to Gln(545)) or the anti-NC16A IgG administered together with the peptides of this epitope as a decoy ameliorated skin fragility in the injected neonatal COL17-humanized mice compared with the anti-NC16A IgG alone even though all of the mice showed both IgG and complement deposition. These results led us to investigate an additional, complement-independent mechanism of skin fragility in the mice injected with anti-COL17 Abs. The rabbit anti-NC16A IgG depleted the expression of COL17 in cultured normal human keratinocytes, whereas immunoadsorption of the same IgG with this epitope significantly suppressed the depletion effect. Moreover, passive transfer of F(ab')(2) fragments of the human BP or rabbit IgG Abs against COL17 demonstrated skin fragility in neonatal COL17-humanized mice. In summary, this study reveals the importance of Abs directed against distinct epitopes on COL17, which induce skin fragility in complement-dependent as well as complement-independent ways.     

A role for complement in antibody-mediated inflammation: C5-deficient DBA/1 mice are resistant to collagen-induced arthritis

Collagen-induced arthritis (CIA) represents an animal model of autoimmune polyarthritis with significant similarities to human rheumatoid arthritis that can be induced upon immunization with native type II collagen. As in rheumatoid arthritis, both cellular and humoral immune mechanisms contribute to disease pathogenesis. Genotypic studies have identified at least six genetic loci contributing to arthritis susceptibility, including the class II MHC. We have examined the mechanism of Ab-mediated inflammation in CIA joints, specifically the role of complement activation, by deriving a line of mice from the highly CIA-susceptible DBA/1LacJ strain that are congenic for deficiency of the C5 complement component. We show that such C5-deficient DBA/1LacJ animals mount normal cellular and humoral immune responses to native type II collagen, with the activation of collagen-specific TNF-alpha-producing T cells in the periphery and substantial intra-articular deposition of complement-fixing IgG Abs. Nevertheless, these C5-deficient mice are highly resistant to the induction of CIA. These data provide evidence for an important role of complement in Ab-triggered inflammation and in the pathogenesis of autoimmune arthritis.     

Lysyl Hydroxylase 3 Localizes to Epidermal Basement Membrane and Is Reduced in Patients with Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in COL7A1 resulting in reduced or absent type VII collagen, aberrant anchoring fibril formation and subsequent dermal-epidermal fragility. Here, we identify a significant decrease in PLOD3 expression and its encoded protein, the collagen modifying enzyme lysyl hydroxylase 3 (LH3), in RDEB. We show abundant LH3 localising to the basement membrane in normal skin which is severely depleted in RDEB patient skin. We demonstrate expression is in-part regulated by endogenous type VII collagen and that, in agreement with previous studies, even small reductions in LH3 expression lead to significantly less secreted LH3 protein. Exogenous type VII collagen did not alter LH3 expression in cultured RDEB keratinocytes and we show that RDEB patients receiving bone marrow transplantation who demonstrate significant increase in type VII collagen do not show increased levels of LH3 at the basement membrane. Our data report a direct link between LH3 and endogenous type VII collagen expression concluding that reduction of LH3 at the basement membrane in patients with RDEB will likely have significant implications for disease progression and therapeutic intervention.     

Humanization of autoantigen

Transmissibility of characteristic lesions to experimental animals may help us understand the pathomechanism of human autoimmune disease. Here we show that human autoimmune disease can be reproduced using genetically engineered model mice. Bullous pemphigoid (BP) is the most common serious autoimmune blistering skin disease, with a considerable body of indirect evidence indicating that the underlying autoantigen is collagen XVII (COL17). Passive transfer of human BP autoantibodies into mice does not induce skin lesions, probably because of differences between humans and mice in the amino acid sequence of the COL17 pathogenic epitope. We injected human BP autoantibody into Col17-knockout mice rescued by the human ortholog. This resulted in BP-like skin lesions and a human disease phenotype. Humanization of autoantigens is a new approach to the study of human autoimmune diseases.     

Importance of Balance between Extracellular Matrix Synthesis and Degradation in Basement Membrane Formation

The epidermal basement membrane (BM) plays important roles in adhesion between epidermis and dermis and in controlling epidermal differentiation. In a skin-equivalent (SE), components of the epidermal BM such as laminin 5 and type IV and VII collagens were detected in conditioned media and in basal keratinocytes. Despite production of these BM components, however, BM was rarely observed at the dermal-epidermal junction. One possible explanation for the absence of BM in SEs is that matrix metalloproteinases (MMPs) degrade newly synthesized extracellular matrices. In fact, several MMPs, such as MMPs-1, 2, 3, and 9, were observed to be present in conditioned media and some of them were in active forms. Tissue inhibitor of metalloproteinase (TIMP)-2 was not detected, although TIMP-1 was present. BM degradation activity presumably exceeds BM formation activity in the SE, resulting in the absence of lamina densa at the dermal-epidermal junction. Synthetic MMP inhibitors CGS27023A and MMP inhibitor I, which inhibit MMPs 1, 2, 3, and 9, markedly augmented deposition of laminin 5 and type IV and VII collagens at the dermal-epidermal junction, resulting in formation of continuous epidermal BM. These results suggest that the balance between synthesis and degradation of BM components is important for BM formation.     

The recombinant expression of full-length type VII collagen and characterization of molecular mechanisms underlying dystrophic epidermolysis bullosa.

Type VII collagen is a major component of anchoring fibrils, attachment structures that mediate dermal-epidermal adherence in human skin. Dystrophic epidermolysis bullosa (DEB) is an inherited mechano-bullous disorder caused by mutations in the type VII collagen gene and perturbations in anchoring fibrils. In this study, we produced recombinant human type VII collagen in stably transfected human 293 cell clones and purified large quantities of the recombinant protein from culture media. The recombinant type VII collagen was secreted as a correctly folded, disulfide-bonded, helical trimer resistant to protease degradation. Purified type VII collagen bound to fibronectin, laminin-5, type I collagen, and type IV collagen and also supported human dermal fibroblast adhesion. In an attempt to establish genotype-phenotype relationships, we generated two individual substitution mutations that have been associated with recessive DEB, R2008G and G2749R, and purified the recombinant mutant proteins. The G2749R mutation resulted in mutant type VII collagen with increased sensitivity to protease degradation and decreased ability to form trimers. The R2008G mutation caused the intracellular accumulation of type VII collagen. We conclude that structural and functional studies of in vitro generated type VII collagen mutant proteins will aid in correlating genetic mutations with the clinical phenotypes of DEB patients.     

Systemic autoimmune disease caused by autoreactive B cells that receive chronic help from Ig V region-specific T cells

B cells present BCR V region-derived Id-peptides on their MHC class II molecules to Id-specific CD4+ T cells. Prolonged Id-driven T-B collaboration could cause autoimmune disease, but this possibility is difficult to test in normal individuals. We have investigated whether mice doubly transgenic for an Id+ Ig L chain and an Id-specific TCR develop autoimmune disease. Surprisingly, T cell tolerance was not complete in these mice because a low frequency of weakly Id-reactive CD4+ T cells accumulated with age. These escapee Id-specific T cells provided chronic help for Id+ B cells, resulting in a lethal systemic autoimmune disease including germinal center reactions, hypergammaglobulinemia, IgG autoantibodies, glomerulonephritis, arthritis, skin affection, and inflammatory bowel disease. Inflamed tissues contained foci of Id-driven T-B collaboration, with deposition of IgG and complement. The disease could be transferred with B and T cells. The results demonstrate a novel mechanism for development of autoimmune disease in which self-reactive Id+ B cells receive prolonged help from Id-specific T cells, thus bypassing the need for help from T cells recognizing conventional Ag.     

The plant lectin wheat germ agglutinin inhibits the binding of pemphigus foliaceus autoantibodies to desmoglein 1 in a majority of patients and prevents pathomechanisms of pemphigus foliaceus in vitro and in vivo

Pemphigus foliaceus (PF) is a life-threatening autoimmune blistering skin disease caused by pathogenic IgG autoantibodies against desmoglein 1 (dg1), a desmosomal cadherin-type adhesion glycoprotein. Using lectins and glycosidases, we have shown that dg1 displays an N-glycosylation pattern of the complex triantennary type. We have found that lectins and glycosidases interfere with N-bound sugar residues on the amino-terminal ectodomain of dg1 and completely abolish, in vitro, the antigenicity of dg1 in most of the patients' sera. Moreover, in an ex vivo model using punch biopsies from normal human skin, we demonstrate that preincubation of the epidermis in wheat germ agglutinin (WGA) prevents PF autoantibody binding, acantholysis, and subcorneal blistering. In addition, we show that topical treatment with WGA inhibits PF autoantibody binding to keratinocytes in both newborn BALB/c mice and in organotypic human epidermis grafted onto the back of SCID mice. The epidermis of these pretreated animals displays a regular morphology, whereas control animals develop the immunopathologic phenotype of PF. These findings suggest that WGA may interfere with autoantibody binding to dg1, preventing experimental PF without affecting the adhesive function of dg1. Our observations may provide a new approach to the therapy of PF.