Shin Morizane

The antimicrobial protein REG3A regulates keratinocyte proliferation and differentiation after skin injury.

Epithelial keratinocyte proliferation is an essential element of wound repair, and abnormal epithelial proliferation is an intrinsic element in the skin disorder psoriasis. The factors that trigger epithelial proliferation in these inflammatory processes are incompletely understood. Here we have shown that regenerating islet-derived protein 3-alpha (REG3A) is highly expressed in keratinocytes during psoriasis and wound repair and in imiquimod-induced psoriatic skin lesions. The expression of REG3A by keratinocytes is induced by interleukin-17 (IL-17) via activation of keratinocyte-encoded IL-17 receptor A (IL-17RA) and feeds back on keratinocytes to inhibit terminal differentiation and increase cell proliferation by binding to exostosin-like 3 (EXTL3) followed by activation of phosphatidylinositol 3 kinase (PI3K) and the kinase AKT. These findings reveal that REG3A, a secreted intestinal antimicrobial protein, can promote skin keratinocyte proliferation and can be induced by IL-17. This observation suggests that REG3A may mediate the epidermal hyperproliferation observed in normal wound repair and in psoriasis.

Antimicrobial peptides in the pathogenesis of psoriasis

One characteristic abnormality of lesional skin in psoriasis is the excessive production of antimicrobial peptides and proteins (AMPs). AMPs typically are small (12–50 amino acids), have positive charge and amphipathic structure, and are found in all living organisms including mammals, insects, plants and invertebrates. These peptides are best known for their integral role in killing pathogenic microorganisms; however, in vertebrates, they are also capable of modifying host inflammatory responses by a variety of mechanisms. In psoriatic lesions, many AMPs are highly expressed, and especially the associations between psoriasis and cathelicidin, β‐defensins or S100 proteins have been well studied. Among them, a cathelicidin peptide, LL‐37, has been highlighted as a modulator of psoriasis development in recent years. AMPs had been thought to worsen psoriatic lesions but recent evidence has also suggested the possibility that the induction of AMPs expression might improve aspects of the disease. Further investigations are needed to uncover a previously underappreciated role for AMPs in modulating the immune response in psoriasis, and to improve disease without the risks of systemic immunosuppressive approaches.

Assessment of Streptococcus pyogenes microcolony formation in infected skin by confocal laser scanning microscopy

BACKGROUND Streptococcus pyogenes and Staphylococcus aureus are often simultaneously detected from many cases of non-bullous impetigo with atopic dermatitis. OBJECTIVES Using confocal laser scanning microscopy (CLSM), to investigate formation of S. pyogenes microcolonies in skin lesions. METHODS The S. pyogenes cells in the stationary growth phase alone were strongly stained with fluorescein isothiocyanate-concanavalin A (FITC-ConA), and this staining was reduced by pretreatment with amylase. Although the components of sugars in glycocalyx produced by S. pyogenes cells are unknown, we suggested that the materials stained by FITC-ConA were consistent with the presence of ConA-reactive sugars in glycocalyx produced by S. pyogenes cells. RESULTS S. pyogenes cells associated with streptococcal impetigo skin and croton-oil inflamed mouse skin formed microcolonies encircled by materials (glycocalyx) that stained strongly with FITC-ConA, and these findings were consistent with those in biofilms. In croton-oil inflamed mouse skin, polymorphonuclear leukocytes (PMNs) infiltrated to just below the epidermis in the cefdinir-treated group but only to the middle dermis in the cefdinir-non-treated group. In this case S. pyogenes and S. aureus cells formed separate microcolonies and existed independently in the outer walls of pustule lesions of streptococcal impetigo. CONCLUSION In skin infections, S. pyogenes and S. aureus formed aggregates of microcolonies (similar to that in biofilms) encircled by glycocalyx, which can make the infection hard to eradicate using an antimicrobial agent alone. The effect of conventional antimicrobial agents against biofilm is mainly due to the increase of the invasion of PMNs into the biofilm.

TH2 cytokines increase kallikrein 7 expression and function in patients with atopic dermatitis.

Here we report that Th2 cytokines increase kallikrein 7 expression and function in keratinocytes. Our finding describes a link between the Th2 environment and epidermal barrier dysfunction in atopic dermatitis.

The role of CD4 and CD8 cytotoxic T lymphocytes in the formation of viral vesicles.

Background  Herpetic vesicles caused by herpes simplex virus and varicella zoster virus, and hydroa vacciniforme (HV) are characterized by umbilicated vesicule formation.

Detection of antibodies against the non-calcium-dependent epitopes of desmoglein 3 in pemphigus vulgaris and their pathogenic significance

Background  Antidesmoglein (anti‐Dsg) 3 serum antibody titres are usually correlated with the disease activity of pemphigus vulgaris (PV), but some patients retain high titres even in remission.

Pemphigus vegetans with IgG and IgA antidesmoglein 3 antibodies

SIR, Pemphigus vegetans is an uncommon variant of pemphigus that is characterized by vegetating erosions on the intertriginous area. Direct immunofluorescence (DIF) reveals IgG deposition on keratinocyte cell surfaces in the epidermis in all cases, usually associated with C3 deposition. A few case reports of pemphigus vegetans demonstrated IgA deposition on keratinocyte cell surfaces, although the nature of the IgAclass antibodies remains to be determined. We describe a patient with pemphigus vegetans who had both IgG and IgA antidesmoglein (Dsg) 3 antibodies. A 67-year-old Japanese man visited our hospital in August 2003 because of a 2-month history of pustules and erosions appearing on his face, axillae and groins. The skin lesions extended to the abdomen, buttocks and scalp. He had had a gastric ulcer and cerebral infarction at the age of 58 and 61 years, respectively, and he was treated with an antihypertensive drug. Cutaneous examination revealed pustules and erosions on the verrucous plaques in the above-mentioned areas (Fig. 1). Aphthae and white plaques were seen in the oral mucosa and soft palate. Laboratory tests showed anaemia and hypoproteinaemia. The patient’s C-reactive protein level was 3Æ3 mg dL (normal < 0Æ3), and his serum IgA level was 411 mg dL (normal 110–424). Other routine tests were within normal limits. Skin biopsy from the inguinal lesion showed marked acanthosis, suprabasal acantholysis in focal areas and large intraepidermal abscesses composed of many eosinophils (Fig. 2). A dense infiltrate with mixed cells of eosinophils, neutrophils and lymphocytes was observed in the upper dermis. DIF revealed the deposition of IgG, IgA and C3 on keratinocyte cell surfaces in the epidermis. Indirect immunofluorescence for IgG, IgA, IgM and C3 was negative in each case. Immunoblotting using human epidermal extracts showed no specific binding of IgG or IgA from the patient’s serum. The IgG enzyme-linked immunosorbent assay (ELISA) index values of anti-Dsg1 and anti-Dsg3 antibodies were 5Æ2 and 51Æ2 (cut-off value 20), respectively, determined by MESACUP desmoglein test ‘Dsg1’ and ‘Dsg3’ (MBL, Nagoya, Japan). We further performed ELISA for detecting IgA anti-Dsg1 or anti-Dsg3 antibodies using the same plates as described above and horseradish peroxidase-conjugated rabbit polyclonal antihuman IgA antibody (Dako, Glostrup, Denmark). We prepared 20 serum samples from normal individuals (age 59 ± 16Æ6 years) following informed consent to set a cut-off value for the ELISA. The mean ± SD of the normal control samples on ELISA for IgA anti-Dsg1 and anti-Dsg3 antibodies was 0Æ095 ± 0Æ014 and 0Æ106 ± 0Æ029, respectively. To exclude the background levels of normal sera, we set the cutoff value of 0Æ200 for Dsg1 and 0Æ250 for Dsg3. The values in the patient’s sera before therapy were 0Æ123 (negative) for Dsg1 and 0Æ900 (positive) for Dsg3. We also performed a novel ELISA using baculovirusexpressed human desmocollins 1–3, as reported previously. Neither IgG nor IgA was detected in the desmocollin ELISAs. The patient was hospitalized, and treated with oral prednisolone 20 mg daily, but no improvement was observed after 10 days of treatment. Therefore, treatment with a combination of oral prednisolone 30 mg daily and dapsone 75 mg daily was started. This regimen resulted in healing of the verrucous lesions within 3 weeks. The doses of prednisolone and dapsone were tapered to 20 mg and 50 mg daily, A

Cathelicidin antimicrobial peptide LL‐37 augments interferon‐β expression and antiviral activity induced by double‐stranded RNA in keratinocytes

Cathelicidin antimicrobial peptide LL‐37 has the capacity to kill a wide range of microbes and to modify host immunity. Recently, our group observed that the activation of keratinocytes by LL‐37 and DNA greatly increases interferon (IFN)‐β through Toll‐like receptor (TLR)9. However, the effect of LL‐37 on the induction of IFN‐β through TLR3, a sensor of double‐stranded (ds) RNA, in keratinocytes is not well known.

Dexamethasone but not tacrolimus suppresses TNF-α-induced thymic stromal lymphopoietin expression in lesional keratinocytes of atopic dermatitis model.

BACKGROUND Thymic stromal lymphopoietin (TSLP) initiates the Th2-type allergic inflammation, and is thought to play an important role in the pathogenesis of atopic dermatitis (AD). TNF-α is a key cytokine which is involved in the pathophysiology of various inflammatory diseases, and the expression level is elevated in the sera and skin of patients with AD. In addition, TNF-α has been reported to induce TSLP expression in epidermal keratinocytes. Topical glucocorticoids and calcineurin inhibitors are safe and effective agents for AD, but the effects of these agents on TNF-α-induced TSLP expression are not fully understood. OBJECTIVE To investigate whether the glucocorticosteroid dexamethasone and the calcineurin inhibitor tacrolimus could affect TSLP expression induced by TNF-α in lesional keratinocytes of AD. METHODS The effects of topical dexamethasone and tacrolimus on TSLP expression were evaluated in an AD mouse model induced by repeated 2,4,6-trinitro-1-chlorobenzene application. Co-immunostaining for TSLP and TNF-α was performed using skin samples from AD patients and the mouse model. Normal human epidermal keratinocytes (NHEKs) were cultured with dexamethasone or tacrolimus in the presence of TNF-α to analyze TSLP expression. RESULTS Topical application of dexamethasone but not tacrolimus repressed TSLP expression in the mouse model. TSLP and TNF-α showed similar distribution pattern in epidermal keratinocytes of AD lesions and the mouse model. TSLP expression was induced by TNF-α via NF-κB in a dose-dependent and an autocrine and/or paracrine manner in NHEKs, which was significantly suppressed by dexamethasone but not by tacrolimus. Similarly to TSLP expression, IL-6, TNF-α, IL-8, and IL-36γ expression induced by TNF-α were significantly suppressed by dexamethasone but not by tacrolimus in NHEKs. CONCLUSION Dexamethasone but not tacrolimus suppresses the TSLP expression induced by TNF-α in lesional keratinocytes of AD model. Our observations uncover the unreported functional difference between topical glucocorticosteroids and calcineurin inhibitors in cutaneous inflammatory diseases.

Involvement of granulysin-producing T cells in the development of superficial microbial folliculitis

Background  Granulysin is a recently identified antimicrobial protein expressed on cytotoxic T cells, natural killer (NK) cells and NKT cells. It has been shown that granulysin contributes to the defence mechanisms against mycobacterial infection. Superficial microbial folliculitis is a common skin disease. In a previous report, we showed that, as a first line of defence, α‐defensin, a human neutrophil peptide, and β‐defensin (human β‐defensin‐2) were expressed in infiltrating neutrophils and in lesional epidermal keratinocytes, respectively, in superficial folliculitis. As we also observed many infiltrating lymphocytes in lesional dermis, we hypothesized that infiltrating lymphocytes may possess antimicrobial substances, such as granulysin, and play a role in the defence mechanism as a second line of defence.