Mariko Yokouchi

Langerhans cell antigen capture through tight junctions confers preemptive immunity in experimental staphylococcal scalded skin syndrome.

Epidermal LCs but not dermal DCs take up skin surface protein through intact tight junctions and mediate IgG1 antibody responses to bacterial antigen, conferring protective immunization.

Pathogenic epitopes of autoantibodies in pemphigus reside in the amino-terminal adhesive region of desmogleins which are unmasked by proteolytic processing of prosequence.

Pemphigus targets desmogleins (Dsgs), which are thought to be synthesized as inactive precursor proteins with prosequences that are cleaved by substilisin-like proprotein convertases, such as furin, to yield mature adhesive molecules. We hypothesized that some pemphigus pathogenic antibodies (Abs), which presumably interfere with adhesion, only bind the mature form. A pathogenic and three non-pathogenic anti-Dsg1 monoclonal Abs (mAbs) isolated from a pemphigus foliaceus (PF) patient, were used for immunoprecipitation and ELISA of recombinant precursor and mature Dsg1. The pathogenic Ab binds mature Dsg1, whereas non-pathogenic Abs bind either only the precursor or both the precursor and mature Dsg1. Competition ELISA showed that the majority of PF sera target the same or nearby epitopes defined by the pathogenic anti-Dsg1 mAb that blocked >20% binding of 29 out of 40 PF sera. Furthermore, the immunoreactivity of 45 PF sera against the mature Dsg1 was 3.2 fold stronger than that against the precursor Dsg1 by ELISA. Similar results were observed in anti-Dsg3 Abs in 47 pemphigus vulgaris sera, suggesting that most pemphigus sera target epitopes that are unmasked by proteolytic processing. These findings support the idea that at least some pathogenic pemphigus autoantibodies induce the loss of cell adhesion by directly binding the trans-interaction site of Dsgs.

Functional tight junction barrier localizes in the second layer of the stratum granulosum of human epidermis

BACKGROUND Mammalian epidermis has two diffusion barriers, the stratum corneum (SC) and tight junctions (TJs). We reported previously that a single living cell layer exists between the SC and TJ-forming keratinocytes in mice; however, the exact location of the TJ barrier in human epidermis has not been defined. OBJECTIVE To investigate the precise distribution of epidermal TJs in relation to various cell-cell junction proteins and the SC and to clarify the barrier function of TJs against macromolecules in human skin. METHODS The localization of various junctional proteins was investigated in human skin sections and in the roofs of bullae formed by ex vivo exfoliative toxin (ET) treatment in three dimensions. ET and single-chain variable fragments (scFv) against desmoglein 1 were used as large diffusion probes. RESULTS Human stratum granulosum (SG) cells have a distinct distribution of TJ, adherens junction, and desmosome proteins in the uppermost three layers (SG1-SG3 from the surface inward). Ex vivo injection of ET or scFv demonstrated that only SG2-SG2 junctions function as a TJ barrier, limiting the inside-out diffusion of these proteins. The roofs of bullae formed by ex vivo ET treatment consisted of SC, SG1 cells, and TJ-forming SG2 cells, probably mimicking bulla formation in bullous impetigo. CONCLUSION Human epidermis has three SG cell layers with distinct properties just beneath the SC, of which only SG2 cells have functional TJs. Our results suggest that human epidermal TJs between SG2 cells form a paracellular diffusion barrier against soluble proteins, including immunoglobulins and bacterial toxins.

Epidermal tight junction barrier function is altered by skin inflammation, but not by filaggrin-deficient stratum corneum

BACKGROUND The tight junction (TJ) barrier is located in the granular layer of the epidermis. Filaggrin deficiency predisposes patients to atopic dermatitis (AD) by impairing stratum corneum (SC) barrier function. Altered TJ barrier function has been observed in the skin of patients with AD; however, it remains unclear whether TJ function is influenced by filaggrin deficiency directly or secondarily via skin inflammation. OBJECTIVE To investigate the in vivo effects of filaggrin deficiency and skin inflammation on epidermal TJ function. METHODS Morphological changes in the TJ were investigated in filaggrin knockout mice and mice with hapten-induced dermatitis using en face visualization of epidermal sheets, and functional changes in the TJ were assessed with an in vivo permeation assay using tracers of various sizes. RESULTS In filaggrin knockout mice, there was no apparent change in the honeycomb morphology of the TJ, TJ component mRNA expression, or TJ barrier function in neonates and adults, indicating that filaggrin-deficiency had no direct effects on the TJ. By contrast, in mice with hapten-induced dermatitis, the mRNA expression of TJ components was decreased markedly and the TJ barrier function was size-dependently impaired: the TJ leaked small tracers (<5 kDa), but not large tracers (>30 kDa). CONCLUSION Filaggrin deficiency did not affect the epidermal TJ barrier directly, but once dermatitis occurred, the skin inflammation induced TJ dysfunction. Since TJ dysfunction induces the SC barrier impairment, skin inflammation will enhance skin permeability to external antigens and result in a vicious cycle of barrier dysfunction and skin inflammation.

Non-pathogenic pemphigus foliaceus (PF) IgG acts synergistically with a directly pathogenic PF IgG to increase blistering by p38MAPK-dependent desmoglein 1 clustering

BACKGROUND Pemphigus foliaceus (PF) is an autoimmune blistering disease caused by autoantibodies (Abs) against desmoglein 1 (Dsg1). PF sera contain polyclonal Abs which are heterogeneous mixture of both pathogenic and non-pathogenic Abs, as shown by isolation of monoclonal Abs (mAbs). OBJECTIVE To investigate how pathogenic and non-pathogenic anti-Dsg1 Abs contribute to blister formation in PF. METHODS Using organ-cultured human skin, we compared the effect of a single pathogenic anti-Dsg1 IgG mAb, a single non-pathogenic anti-Dsg1 IgG mAb, and their mixture on blister formation as analyzed by histology, subcellular localization of IgG deposits and desmosomal proteins by confocal microscopy, and desmosomal structure by electron microscopy. In addition, we measured keratinocyte adhesion by an in vitro dissociation assay. RESULTS 24h after injection, a single pathogenic anti-Dsg1 IgG caused a subcorneal blister with IgG and Dsg1 localized linearly on the cell surface of keratinocytes. A single non-pathogenic anti-Dsg1 IgG bound linearly on the keratinocytes but did not induce blisters. A pathogenic and a non-pathogenic IgG mAb injected together caused an aberrant granular pattern of IgG and Dsg1 in the lower epidermis with blister formation in the superficial epidermis. Electron microscopy demonstrated that the mixture of mAbs shortened desmosomal lengths more than a single mAb in the basal and spinous layers. Furthermore, although Dsg1 clustering required both cross-linking of Dsg1 molecules by the non-pathogenic IgG plus a pathogenic antibody, the latter could be in the form of a monovalent single chain variable fragment, suggesting that loss of trans-interaction of Dsg1 is required for clustering. Finally, a p38MAPK inhibitor blocked Dsg1 clustering. When pathogenic strength was measured by the dissociation assay, a mixture of pathogenic and non-pathogenic IgG mAbs disrupted keratinocyte adhesion more than a single pathogenic mAb. This pathogenic effect was only partially suppressed by the p38MAPK inhibitor. CONCLUSION These findings indicate that a polyclonal mixture of anti-Dsg1 IgG antibodies enhances pathogenic activity for blister formation associated with p38MAPK-dependent Dsg1 clustering and that not only pathogenic antibodies but also non-pathogenic antibodies coordinately contribute to blister formation in PF.

Maintenance of tight junction barrier integrity in cell turnover and skin diseases

The skin forms a life‐sustaining barrier between the organism and the physical environment. The physical barrier of the skin is mainly comprised of the stratum corneum (SC) and tight junctions (TJs). In recent years, there have been significant advances in our understanding of the epidermal TJ function, composition and regulation. In contrast to the SC, TJs are highly dynamic structures. It was discovered that spatiotemporal regulation of dynamic TJ replacement from cell to cell maintains the TJ barrier homeostasis of the skin, despite continuous cellular turnover. This review summarizes current knowledge about how TJ barrier homeostasis is maintained in simple and stratified epithelia, and how diseases and other conditions affect the TJ barrier in the skin.