Hidenori Okazaki

IL-17 and IL-22 mediate IL-20 subfamily cytokine production in cultured keratinocytes via increased IL-22 receptor expression

IL‐20 cytokine subfamily members, including IL‐19, IL‐20, and IL‐24, are highly expressed in psoriatic skin lesions. Here, we demonstrate that psoriasis mediators IL‐17 and IL‐22 synergistically induce the production of IL‐20 subfamily proteins in cultured human keratinocytes. Interestingly, expression of the IL‐22 receptor (IL‐22R) also increased in epidermal lesions versus normal skin. IL‐22R over‐expression using an adenoviral vector to mimic psoriatic conditions in cultured keratinocytes significantly enhanced IL‐17‐ and IL‐22‐induced production of IL‐20 subfamily cytokines. Furthermore, IL‐17 and IL‐22 coordinately enhanced MIP‐3α, IL‐8, and heparin‐binding EGF‐like growth factor (HB‐EGF) production, depending on the amount of IL‐22R expression. Additionally, because IL‐20 and IL‐24 share the IL‐22R with IL‐22, the function of IL‐20 and IL‐24 was also increased. IL‐20 and IL‐24 have effects similar to that of IL‐22; IL‐24 showed more potent expression than IL‐20. A combination of IL‐24 and IL‐17 increased the production of MIP‐3α, IL‐8, and HB‐EGF, as did a combination of IL‐22 and IL‐17. These data indicate that increased IL‐22R expression in epidermal keratinocytes contributes to the pathogenesis of psoriasis through enhancing the coordinated effects of IL‐22 and IL‐17, inducing the production of the IL‐20 subfamily, chemokines, and growth factors.

Nodal Lymphangiogenesis and Metastasis Role of Tumor-Induced Lymphatic Vessel Activation in Extramammary Paget's Disease

Nodal lymphangiogenesis promotes distant lymph node (LN) metastasis in experimental cancer models. However, the role of nodal lymphangiogenesis in distant metastasis and in the overall survival of cancer patients remains unknown. Therefore, we investigated mechanisms that might facilitate regional and distant LN metastasis in extramammary Pagets disease (EMPD). We retrospectively analyzed the impact of tumor-induced lymphatic vessel activation on the survival of 116 patients, the largest cohort with EMPD studied to date. Nodal lymphangiogenesis was significantly increased in metastatic, compared with tumor-free, LNs (P = 0.022). Increased lymphatic invasion within regional LNs was significantly associated with distant metastasis in LN (P = 0.047) and organs (P = 0.003). Thus, invasion within regional LNs is a powerful indicator of systemic tumor spread and reduced patient survival in EMPD (P = 0.0004). Lymphatic vessels associated with tumors expressed stromal cell-derived factor-1 (SDF-1), whereas CXCR4 was expressed on invasive Paget cells undergoing epithelial-mesenchymal transition (EMT)-like process. A431 cells overexpressing Snail expressed increased levels of CXCR4 in the presence of transforming growth factor-beta1. Haptotactic migration assays confirmed that Snail-induced EMT-like process promotes tumor cell motility via the CXCR4-SDF-1 axis. Sinusoidal lymphatic endothelial cells and macrophages expressed SDF-1 in subcapsular sinuses of lymph nodes before Paget cell arrival. Our findings reveal that EMT-related features likely promote lymphatic metastasis of EMPD by activating the CXCR4-SDF-1 axis.

Eccrine Sweat Contains IL-1α, IL-1β and IL-31 and Activates Epidermal Keratinocytes as a Danger Signal

Eccrine sweat is secreted onto the skins surface and is not harmful to normal skin, but can exacerbate eczematous lesions in atopic dermatitis. Although eccrine sweat contains a number of minerals, proteins, and proteolytic enzymes, how it causes skin inflammation is not clear. We hypothesized that it stimulates keratinocytes directly, as a danger signal. Eccrine sweat was collected from the arms of healthy volunteers after exercise, and levels of proinflammatory cytokines in the sweat were quantified by ELISA. We detected the presence of IL-1α, IL-1β, and high levels of IL-31 in sweat samples. To investigate whether sweat activates keratinocytes, normal human keratinocytes were stimulated with concentrated sweat. Western blot analysis demonstrated the activation of NF-κB, ERK, and JNK signaling in sweat-stimulated keratinocytes. Real-time PCR using total RNA and ELISA analysis of supernatants showed the upregulation of IL-8 and IL-1β by sweat. Furthermore, pretreatment with IL-1R antagonist blocked sweat-stimulated cytokine production and signal activation, indicating that bioactive IL-1 is a major factor in the activation of keratinocytes by sweat. Moreover, IL-31 seems to be another sweat stimulator that activates keratinocytes to produce inflammatory cytokine, CCL2. Sweat is secreted onto the skins surface and does not come into contact with keratinocytes in normal skin. However, in skin with a defective cutaneous barrier, such as atopic dermatitis-affected skin, sweat cytokines can directly act on epidermal keratinocytes, resulting in their activation. In conclusion, eccrine sweat contains proinflammatory cytokines, IL-1 and IL-31, and activates epidermal keratinocytes as a danger signal.

Interactions between myofibroblast differentiation and epidermogenesis in constructing human living skin equivalents.

BACKGROUND During skin wounding and healing, skin homeostasis is interrupted. How the altered epithelial-mesenchymal interactions influence scar formation and epidermogenesis should be investigated using three-dimensional models that are similar to in vivo structures. OBJECTIVE In this study, we assessed the effects of epithelial-mesenchymal interactions on myofibroblast differentiation and how myofibroblasts influence epidermogenesis using a human living skin equivalent (LSE) model. METHODS We constructed a fibroblast-populated type I collagen gel upon which LSEs were formed by seeding with normal human keratinocytes. Samples of the collagen gel and LSEs were collected at different time points. Myofibroblast differentiation, epidermal differentiation, and proliferation status were investigated immunohistochemically. Several measures were taken to suppress α-smooth muscle actin (α-SMA) expression to determine the effects of myofibroblasts on epidermogenesis, including the addition of basic fibroblast growth factor or a transformation growth factor-β (TGF-β) kinase inhibitor to the culture medium and the inclusion of an amniotic membrane (AM) in the dermal matrix. RESULTS The myofibroblast/fibroblast ratio in the fibroblast-populated collagen gel kept rising during culture. In the LSEs, most fibroblasts were α-SMA-negative, except for those along the dermal-epidermal junction. The suppression of α-SMA expression enhanced epidermal differentiation and decreased TGF-β1 expression in the epidermis. The inhibition of TGF-β kinase completely suppressed α-SMA expression in the dermal matrix. CONCLUSIONS Epidermogenesis suppressed α-SMA expression in the fibroblast-rich dermal matrix, except near the dermal-epidermal junction. The α-SMA-positive cells at the dermal-epidermal junction contributed to the hyperproliferative phenotype of the epidermis. In contrast, the hyperproliferative epidermis expressed more TGF-β1, which is responsible for myofibroblast differentiation.

Targeted overexpression of Angptl6/angiopoietin-related growth factor in the skin promotes angiogenesis and lymphatic vessel enlargement in response to ultraviolet B

Angiogenesis is required for physiological tissue repair processes, such as cutaneous wound healing. However, recent studies indicate that endogenous angiogenic factors may enhance photo‐induced skin alterations in response to experimental ultraviolet (UV)‐B exposure. Angiopoietin‐related growth factor (AGF), also known as angiopoietin‐like protein 6 (Angptl6), is known to promote new blood vessel formation and vascular hyperpermeability. Importantly, epidermal overexpression of Angptl6/AGF in mice promotes wound healing in the skin. However, it remains unclear whether overexpression of Angptl6/AGF facilitates tissue repair processes in response to UV‐B irradiation. To test this hypothesis, we subjected Angptl6/AGF transgenic mice to acute or chronic UV‐B exposure. Surprisingly, transgenic mice showed enhanced photosensitivity to subthreshold doses of UV‐B that did not induce skin alterations in wild‐type littermates. Marked enlargement of blood vessels was observed after a single exposure to UV‐B in Angptl6/AGF transgenic mice, although no epidermal changes were observed. Chronic UV‐B exposure over 14 weeks promoted cutaneous skin damage in Angptl6/AGF transgenic mice, whereas wild‐type mice showed little or no macroscopic skin alteration. In addition to pronounced angiogenesis and epidermal hyperplasia, marked enlargement of dermal lymphatic vessels was observed in UV‐B‐exposed Angptl6/AGF transgenic mice. Electron microscopy analysis further revealed that the number and size of collagen bundles in the dermis was markedly reduced after chronic UV‐B exposure in Angptl6/AGF transgenic mice. Taken together, these results indicate that ectopic expression of Angptl6/AGF in mice likely promotes UV‐B‐induced skin alterations, and that angiogenesis could be a therapeutic target in prevention of skin photo‐aging.

Ectodomain Shedding of Lymphatic Vessel Endothelial Hyaluronan Receptor 1 (LYVE-1) Is Induced by Vascular Endothelial Growth Factor A (VEGF-A).

Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), a type I transmembrane glycoprotein, is known as one of the most specific lymphatic vessel markers in the skin. In this study, we found that the ectodomain of LYVE-1 undergoes proteolytic cleavage, and this process produces soluble LYVE-1. We further identified the cleavage site for ectodomain shedding and generated an uncleavable mutant of LYVE-1. In lymphatic endothelial cells, ectodomain shedding of LYVE-1 was induced by vascular endothelial growth factor (VEGF)-A, an important factor for angiogenesis and lymphangiogenesis under pathological conditions. VEGF-A-induced LYVE-1 ectodomain shedding was mediated via the extracellular signal-regulated kinase (ERK) and a disintegrin and metalloproteinase (ADAM) 17. Wild-type LYVE-1, but not uncleavable LYVE-1, promoted migration of lymphatic endothelial cells in response to VEGF-A. Immunostaining analyses in human psoriasis skin lesions and VEGF-A transgenic mouse skin suggested that the ectodomain shedding of LYVE-1 occurred in lymphatic vessels undergoing chronic inflammation. These results indicate that the ectodomain shedding of LYVE-1 might be involved in promoting pathological lymphangiogenesis.

Vesicular LL-37 contributes to inflammation of the lesional skin of palmoplantar pustulosis.

“Pustulosis palmaris et plantaris”, or palmoplantar pustulosis (PPP), is a chronic pustular dermatitis characterized by intraepidermal palmoplantar pustules. Although early stage vesicles (preceding the pustular phase) formed in the acrosyringium contain the antimicrobial peptides cathelicidin (hCAP-18/LL-37) and dermcidin, the details of hCAP-18/LL-37 expression in such vesicles remain unclear. The principal aim of the present study was to clarify the manner of hCAP-18/LL-37 expression in PPP vesicles and to determine whether this material contributed to subsequent inflammation of lesional skin. PPP vesicle fluid (PPP-VF) induced the expression of mRNAs encoding IL-17C, IL-8, IL-1α, and IL-1β in living skin equivalents, but the level of only IL-8 mRNA decreased significantly upon stimulation of PPP vesicle with depletion of endogenous hCAP-18/LL-37 by affinity chromatography (dep-PPP-VF). Semi-quantitative dot-blot analysis revealed higher concentrations of hCAP-18/LL-37 in PPP-VF compared to healthy sweat (2.87±0.93 µM vs. 0.09±0.09 µM). This concentration of hCAP-18/LL-37 in PPP-VF could upregulate expression of IL-17C, IL-8, IL-1α, and IL-1β at both the mRNA and protein levels. Recombinant hCAP-18 was incubated with dep-PPP-VF. Proteinase 3, which converts hCAP-18 to the active form (LL-37), was present in PPP-VF. Histopathological and immunohistochemical examination revealed that early stage vesicles contained many mononuclear cells but no polymorphonuclear cells, and the mononuclear cells were CD68-positive. The epidermis surrounding the vesicle expresses monocyte chemotactic chemokine, CCL2. In conclusion, PPP-VF contains the proteinase required for LL-37 processing and also may directly upregulate IL-8 in lesional keratinocytes, in turn contributing to the subsequent inflammation of PPP lesional skin.

Possible association of vascular endothelial growth factor with the development of edema in drug-induced hypersensitivity syndrome

Figure 1. (a) Marked edema in the face and neck 11 days after onset. Generalized maculopapular lesions were observed on the head and trunk. (b) Edema and skin eruption were dramatically reduced 17 days after onset. Dear Editor, Drug-induced hypersensitivity syndrome (DIHS ⁄ DRESS), a systemic reaction to causative drugs, is characterized by fever, hepatitis, lymphadenopathy and a maculopapular eruption associated with reactivation of human herpes virus (HHV)-6. Prominent facial edema represents a characteristic feature of DIHS ⁄DRESS, indicating that the cutaneous vasculature might be altered during the systemic reaction. However, it remains unclear whether inflammatory mediators are involved in the initial development of DIHS ⁄DRESS, leading to increased vascular permeability and formation of edema in the head and neck area. Vascular endothelial growth factor (VEGF), a major angiogenesis factor, promotes vascular permeability in the skin. VEGF is highly induced in several skin diseases such as psoriasis. In fact, the induction of VEGF in ischemic limbs may cause increased vascular leakage, resulting in the formation of edema. Therefore, VEGF potently induces the development of edema in the skin. As such, we present a case of DIHS ⁄DRESS with prominent edema in the face and neck associated with elevated serum levels of VEGF. A 44-year-old man with a history of mania was commenced on carbamazepine. Twenty-five days after initiation of the drug, the patient developed marked erythema on the back. Nine days after the onset of erythema, the patient developed a highgrade fever, generalized maculopapular lesions and prominent edema of the face and laryngeal area (Fig. 1a). Carbamazepine was discontinued at this time. Laboratory findings showed leukocytosis (2.2 · 10 ⁄L), eosinophilia (5.7 · 10 ⁄L) and hepatitis (alanine aminotransferase [ALT] 250 IU ⁄mL). In particular, serum VEGF levels were markedly increased at

Heparinoid suppresses Der p-induced IL-1β production by inhibiting ERK and p38 MAPK pathways in keratinocytes

Epidermal keratinocytes initiate skin inflammation by activating immune cells. The skin barrier is disrupted in atopic dermatitis (AD) and epidermal keratinocytes can be exposed to environmental stimuli, such as house dust mite (HDM) allergens. We showed previously that HDM allergens activate the NLRP3 inflammasome of keratinocytes, thereby releasing pro‐inflammatory cytokines. Heparinoid is an effective moisturizer for atopic dry skin. However, a recent report showed that heparinoid treatment can improve inflammation of lichen planus. Therefore, we hypothesized that it acts on epidermal keratinocytes not only as a moisturizer, but also as a suppressant of the triggers of skin inflammation. We found that HDM allergen‐induced interleukin (IL)‐1β release from keratinocytes was inhibited significantly by heparinoid pretreatment without affecting cell viability. However, heparinoid did not affect caspase‐1 release, suggesting that heparinoid did not affect HDM allergen‐induced inflammasome activation. Heparinoid treatment not only decreased intracellular levels of pro‐IL‐1β, but also suppressed IL‐1β messenger RNA (mRNA) expression in keratinocytes. Among the intracellular signalling pathways, the activation of extracellular signal‐regulated kinase and p38 pathways, which are required for IL‐1β expression in keratinocytes, was inhibited by heparinoid treatment. The inhibitory effect of heparinoid on IL‐1β mRNA expression was also confirmed with living skin equivalents. Our results demonstrated that heparinoid suppresses the initiation of keratinocyte‐mediated skin inflammation.