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Tight junctions (TJs) firmly seal epithelial cells and are key players in the epithelial barrier. TJs consist of several proteins, including those of the transmembrane claudin family and the scaffold zonula occludens (ZO) family. Epithelial tissues are exposed to different conditions: to air in the stratified epithelium of the skin and to liquids in the monolayer of the intestine. The TJs in stratified oral mucosal epithelium have remained insufficiently elucidated in terms of distributions, appearances and barrier functions of TJ proteins in normal buccal mucosa. We investigated these and ZO-1 and claudin-1 were found to be expressed in the top third and in the bottom three quarters of the mucosal epithelium. ZO-1 in the buccal mucosa was found to have an irregular linear appearance. ZO-1 in the buccal mucosa continuously existed in several layers. Electron microscopy revealed the buccal mucosa to have kissing points. In a biotin permeation assay that sought to investigate inside-outside barrier function, the biotin tracer penetrated several ZO-1 layers but did not pass through all the ZO-1 layers. We found that the oral mucosal cell knockdown of TJP1 or CLDN1 resulted in decreases of TER but no significant change in FITC-dextran leakage. Our results suggest that the distribution and appearance of ZO-1 in the buccal mucosa differ from those in the skin. We were unable to prove barrier function in this study but we did show barrier function against small molecules in vivo and against ions in vitro.

Background and purpose Pemphigus is caused by autoantibodies against desmoglein (Dsg) 1, Dsg3 and/or non-Dsg antigens. Pemphigus vulgaris (PV) is the most common manifestation of pemphigus, with painful erosions on mucous membranes. In most cases, blistering also occurs on the skin, leading to areas of extensive denudation. Despite improvements in pemphigus treatment, time to achieve remission is long, severe adverse events are frequent, and 20% of patients do not respond adequately. Current clinical developments focus exclusively on modulating B cell function or autoantibody half-life. However, topical modulation of PV autoantibody-induced blistering is an attractive target because it could promptly relieve symptoms. Experimental approach To address this issue, we performed an unbiased screen in a complex biological system using 141 small molecule inhibitors from a chemical library. Specifically, we evaluated PV IgG-induced Dsg3 internalization in HaCaT keratinocytes. Validation of the 20 identified compounds was performed using keratinocyte fragmentation assays, as well as a human skin organ culture (HSOC) model. Key results Overall, this approach led to the identification of 4 molecules involved in PV IgG-induced skin pathology: MEK1, TrkA, PI3Kα, and VEGFR2. Conclusion and implications This unbiased screen revealed novel mechanisms by which PV autoantibodies induce blistering in keratinocytes and identified new treatment targets for this severe and potentially life-threatening skin disease.

Mucous membrane pemphigoid (MMP) is a rare group of heterogeneous chronic autoimmune diseases that predominantly manifest as blistering of the mucous membranes. MMP lesions often heal with scarring, which may result in impaired organ function and significant morbidity. The exact pathogenic mechanisms of MMP are still largely unknown while the diagnosis is based on a combination of clinical, histological and immuno-pathological findings. Several treatment modalities of MMP are available and are reported in the literature, however, such treatment options are principally guided by expert opinions and descriptive reports. Non-specific immunosuppression, especially corticosteroids, remains the mainstay of treatment, which often leads to severe adverse effects. Therefore, safer and more effective therapeutic modalities are required. This comprehensive literature review outlines the current knowledge and recent advances in the field of MMP management, with particular emphasis on the oral variant of MMP.

Cutaneous lupus erythematosus (CLE) is a rare, potentially disfiguring, chronic autoimmune disease with extremely variable skin and mucosal membrane manifestations. Hydroxychloroquine (HCQ) is an antimalarial drug that has been used in various countries to treat autoimmune diseases including CLE. HCQ was banned for a long time in Japan because of severe chloroquine retinopathy and was reapproved as a first-line treatment for CLE in 2015. There are no case reports describing the effectiveness of HCQ for CLE with oral mucosal lesions in the dental field. We present a case of CLE whose oral lesions were successfully treated with HCQ.

Immunoglobulins (Igs) consist of two antigen-binding regions (Fab) and one constant region (Fc). Protein A and protein G are bacterial proteins used for the purification of IgG by virtue of their high affinities for the Fc fragment. Rheumatoid factors are autoantibodies against IgG Fc fragments, which are present in the body under physiological conditions. Little is known about the influence of Fc-binding proteins on the pathogenicity of antibody-induced autoimmune diseases. Pemphigoid diseases are a group of autoimmune subepidermal blistering disorders that includes bullous pemphigoid and mucous membrane pemphigoid. IgGs targeting the non-collagenous NC16A domain of the 180-kDa bullous pemphigoid antigen (BP180) are known to induce skin fragility in mice and the depletion of BP180 in keratinocytes. In this study, mAb against NC16A in combination with Fc-binding proteins was found to enhance BP180 depletion. Although mAb against the C-terminus of BP180 does not show pathogenicity in vivo or in vitro, mAb treatment with Fc-binding proteins clearly induced skin fragility in mice and BP180 depletion in keratinocytes. Anti-BP180 mAbs and Fc-binding proteins were colocalized in the cytoplasm and at the basement membrane zone. Cell adhesion strengths were decreased in parallel with BP180 amounts. Clinically, bullous pemphigoid patients had higher rheumatoid factor titers than controls. Anti-BP180 mAb in combination with high-titer rheumatoid factor serum was found to enhance BP180 depletion. Furthermore, saliva from mucous membrane pemphigoid patients contained larger quantities of bacteria and Fc-binding proteins than controls. Our results suggest that Fc-binding proteins (rheumatoid factor or protein G) may enhance the pathogenicity of autoantibodies in pemphigoid diseases. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Background Pemphigoid diseases are a heterogeneous group of autoimmune blistering disorders characterized by predominant deposition of immunoglobulin G or immunoglobulin A autoantibodies against structural proteins of the dermoepidermal junction (DEJ). Sole linear immunoglobulin M (IgM) deposits at the DEJ in pemphigoid diseases have been observed; however, IgM-specific target antigens have not been identified. Objective Characterization of patients with IgM pemphigoid. Methods Skin biopsy specimens and sera from IgM-positive patients were assessed using histopathology, direct and indirect immunofluorescence microscopy, enzyme-linked immunosorbent assays, immunoblotting, cryosection assay, complement fixation test, and internalization assays. Results Tissue-bound linear IgM deposits along the DEJ and circulating IgM autoantibodies against type XVII collagen (Col17) were detected. These circulating IgM autoantibodies showed no complement activating or blister inducing capacity, but the ability of Col17 internalization ex vivo. Limitations Limited number of patients. Conclusion This study provides further evidence for the role of IgM autoantibodies in pemphigoid disease and highlights Col17 as a target antigen in IgM pemphigoid.

Background Mucous membrane pemphigoid (MMP) is a rare chronic autoimmune subepithelial blistering disorder, targeting multiple basement membrane zone (BMZ) proteins including collagen XVII (COL17). Circulating autoantibodies of MMP are often undetected due to their lower titers. The oral mucosa is a valuable substrate for the detection of autoantibodies in MMP patients. However, obtaining normal human oral mucosa is more difficult than obtaining normal human skin. We established immortalized normal human oral mucosal keratinocytes (OMKs) and performed immunoblotting using immortalized OMK lysate for detecting autoantigens in MMP. Methods Immortalized OMKs were generated from primary OMKs using E6/E7 proteins of HPV. We compared the protein expression levels of major BMZ proteins between primary OMKs and immortalized OMKs. We performed immunoblotting to detect autoantigens using cell lysates from immortalized OMKs in 30 MMP patients. Results There were no significant differences between primary OMKs and immortalized OMKs in terms of protein expression levels of the BMZ proteins, including COL17, laminin 332, integrin α6/β4, collagen VII, and collagen IV. Cell lysates of immortalized OMKs effectively identified MMP autoantigens in 60% (18/30) of MMP sera. We found an interesting case of MMP whose autoantibodies preferentially reacted to the 120-kD protein that is an ectodomain of COL17. Conclusion We demonstrated that a cell lysate of immortalized OMKs is a reliable substrate for the detection of MMP autoantigens. This newly developed immunoblotting analysis method promises to contribute to the diagnosis of MMP.

The basement membrane zone consists of multiple components, including collagen XVII (COL17), which is the target of bullous pemphigoid. To our knowledge, no research has addressed the differences in basement membrane zone components between the skin and oral mucosa; therefore, we investigated the basement membrane zone proteins, with a focus on COL17. The mRNA and protein expression levels of COL17 were significantly higher in oral keratinocytes than in skin keratinocytes. Hemidesmosomal COL17 expression was markedly higher in oral keratinocytes than in skin keratinocytes, and its level was associated with adhesion strength. Oral keratinocytes adhered to the extracellular matrix more tightly than did skin keratinocytes in vitro. Based on these results, we attempt to explain the clinical diversity of bullous pemphigoid. COL17 depletion was more prominent in skin keratinocytes than in oral keratinocytes after treatment with COL17-NC16A mAbs, which have in vivo pathogenicity. COL17 C-terminus mAbs, which are not pathogenic, facilitated COL17 depletion in combination treatment with COL17-NC16A mAbs in both types of keratinocytes. In summary, the greater amount of COL17 in oral keratinocytes than in skin keratinocytes is associated with the higher strength of oral keratinocyte hemidesmosomal adhesion at the basement membrane zone. Our results may explain why bullous pemphigoid blistering tends to be more prevalent in the skin than in the oral mucosa.

Special Issue: Thematic issue: New insights into pemphigoid diseases, Type XVII collagen (COL17) and the non-collagenous 16A (NC16A) domain is regarded as the major pathogenic domains for bullous pemphigoid (BP). Some patients with BP have autoantibodies against parts of COL17 outside the NC16A domain (hereinafter the non-NC16A domain) and show less inflammatory manifestations. There were no significant differences in titres and IgG subclasses between NC16A-BP and non-NC16A-BP as determined by indirect immunofluorescent microscopy. The neutrophil activation capacities determined by ROS release did not differ between NC16A-BP and non-NC16A-BP. However, NC16A-BP IgG depleted COL17 in a dose-dependent manner. Treatment with NC16A-BP IgG, but not with non-NC16A-BP IgG, significantly decreased the adhesion strength. We speculate that the differences in clinical severity between NC16A-BP and non-NC16A-BP relate to the degree of COL17 depletion.

Macropinocytosis is an endocytic pathway that is involved in the nonselective fluid uptake of extracellular fluid. Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease associated with autoantibodies to type XVII collagen (COL17), which is a component of hemidesmosome. When keratinocytes are treated with BP-IgG, COL17 internalizes into cells by way of the macropinocytosis. We investigated the mechanism of COL17 macropinocytosis using DJM-1 cells, a cutaneous squamous cell carcinoma cell line. First, non-hemidesmosomal COL17 was preferentially depleted by stimulation with the BP-IgG in the DJM-1 cells. To investigate the signaling involved in COL17-macropinocytosis, the inhibition of small GTPase family members Rac1 and Cdc42 was found to strongly repress COL17 internalization; in addition, the Rho inhibitor also partially blocked that internalization, suggesting these small GTPases are involved in signaling to mediate COL17-macropinocytosis. Western blotting using Phostag-SDS-PAGE demonstrated high levels of COL17 phosphorylation in DJM-1 cells under steady-state condition. Treatment with BP-IgG increased the intracellular calcium level within a minute, and induced the overabundant phosphorylation of COL17. The overabundant phosphorylation of COL17 was suppressed by a protein kinase C (PKC) inhibitor. In addition, PKC inhibitor repressed COL17 endocytosis using cell culture and organ culture systems. Finally, the depletion of COL17 was not observed in the HEK293 cells transfected COL17 without intracellular domain. These results suggest that COL17 internalization induced by BP-IgG may be mediated by a PKC pathway. In summary, BP-IgG initially binds to COL17 distributed on the plasma membrane, and COL17 may be internalized by means of a macropinocytic pathway related to the phosphorylation of the intracellular domain by PKC.

Mucous membrane pemphigoid (MMP) is a rare autoimmune blistering disease targeting various autoantigens, including the C-terminus of collagen XVII (COL17) and laminin332. Bullous pemphigoid (BP) is the most common autoimmune blistering disease, affecting the mucosae in 10-20% of cases. COL17, particularly, the non-collagenous (NC)16A domain is the pathogenic epitope for BP. This article is protected by copyright. All rights reserved.

Tight junctions (TJs) firmly seal epithelial cells and are key players in the epithelial barrier. TJs consist of several proteins, including those of the transmembrane claudin family and the scaffold zonula occludens (ZO) family. Epithelial tissues are exposed to different conditions: to air in the stratified epithelium of the skin and to liquids in the monolayer of the intestine. The TJs in stratified oral mucosal epithelium have remained insufficiently elucidated in terms of distributions, appearances and barrier functions of TJ proteins in normal buccal mucosa. We investigated these and ZO-1 and claudin-1 were found to be expressed in the top third and in the bottom three quarters of the mucosal epithelium. ZO-1 in the buccal mucosa was found to have an irregular linear appearance. ZO-1 in the buccal mucosa continuously existed in several layers. Electron microscopy revealed the buccal mucosa to have kissing points. In a biotin permeation assay that sought to investigate inside-outside barrier function, the biotin tracer penetrated several ZO-1 layers but did not pass through all the ZO-1 layers. We found that the oral mucosal cell knockdown of TJP1 or CLDN1 resulted in decreases of TER but no significant change in FITC-dextran leakage. Our results suggest that the distribution and appearance of ZO-1 in the buccal mucosa differ from those in the skin. We were unable to prove barrier function in this study but we did show barrier function against small molecules in vivo and against ions in vitro.

The basement membrane zone (BMZ) is framed by hemidesmosomes and extracellular matrix (ECM) including collagen IV (COL4). Hemidesmosomes are multiprotein complexes that include collagen XVII (COL17). BMZ proteins can be targeted in autoimmune subepidermal blistering diseases, e.g., pemphigoid targeting COL17. The blistering mechanisms in pemphigoid have not been fully elucidated, especially in mucous membrane pemphigoid (MMP), which mainly affects the mucosa. In this study, we showed that oral lesions in pemphigoid may be attributed to the inhibition of protein-protein interactions by autoantibodies. Using immunoprecipitation, we revealed that COL17 directly binds to COL4 in normal human keratinocytes and normal human oral keratinocytes. In particular, the C-terminus of COL17 is binding site to COL4 in oral keratinocytes. The precise COL4-binding region on COL17 was determined by protein-protein binding assay to be from amino acid Gly1175 to Asp1340 on the C-terminus. MMP-IgG or mAb recognizing the C-terminus hindered the interaction of COL17 with COL4 in oral keratinocytes. Furthermore, keratinocyte adhesion strength to COL4-coated plates was significantly reduced by the treatment of mAb against the C-terminus. In addition, the inflammatory infiltrates around perilesions were significantly less in MMP compared to BP. These results indicate that pemphigoid IgG targeting the C-terminus plays a pathogenic role in blister formation in the oral mucosa to inhibit protein interactions with less inflammation.