Relationships among plasma granzyme B level, pruritus and dermatitis in patients with atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a multifactorial inflammatory skin disease characterized by skin barrier dysfunction, allergic inflammation and intractable pruritus resistant to conventional antipruritic treatments, including H1-antihistamines. Granzymes (Gzms) are a family of serine proteases expressed by cytotoxic T lymphocytes and natural killer cells that have been shown to modulate inflammation. However, the relationship between Gzms and pathology in AD remains unclear. OBJECTIVE: This study assessed the correlation between plasma GzmB levels and severity of pruritus and dermatitis, in AD patients. METHODS: Plasma was collected from 46 patients with AD, 24 patients with psoriasis, and 30 healthy controls. AD severity was assessed with the scoring atopic dermatitis (SCORAD) index, psoriasis severity with the psoriasis area and severity index (PASI), and degree of pruritus by visual analogue scale (VAS) score. GzmA, GzmB and gastrin releasing peptide (GRP) levels were measured by enzyme-linked immunosorbent assays. RESULTS: Plasma GzmB concentrations were significantly higher in patients with AD and psoriasis than in healthy controls. Correlation analyses showed that plasma GzmB concentrations positively correlated with SCORAD and serum levels of severity markers such as thymus and activation-regulated chemokine, and lactate dehydrogenase in AD patients. Moreover, plasma levels of GRP, an itch-related peptide, were higher in patients with AD, positively correlating with VAS score and plasma GzmB level. In addition, plasma GzmB concentration was significantly lower in the treatment group than the untreated group with AD. Meanwhile, there were no correlations among GzmB levels, VAS score and PASI score in patients with psoriasis. In contrast to the results of plasma GzmB, plasma GzmA levels were unchanged among AD, psoriasis and healthy groups, and showed no correlations with VAS score and SCORAD index in patients with AD. CONCLUSION: Plasma GzmB levels may reflect the degree of pruritus and dermatitis in patients with AD.

Topically applied semaphorin 3A ointment inhibits scratching behavior and improves skin inflammation in NC/Nga mice with atopic dermatitis.

BACKGROUND: Epidermal hyperinnervation in atopic dermatitis (AD) is activated directly by various external stimuli, causing enhanced itching. Nerve density is regulated by the nerve repulsion factor semaphorin 3A (Sema3A), along with nerve elongation factors. OBJECTIVE: To investigate the effects of Sema3A ointment in the NC/Nga mouse model of AD. METHODS: An AD-like phenotype was induced by repeated application of Dermatophagoides farinae body (Dfb) ointment to the dorsal skin of NC/Nga mice. Vaseline, heparinoid, betamethasone, tacrolimus and recombinant Sema3A ointments were applied to the lesional skin once a day for 4 days. Transepidermal water loss (TEWL) was measured before and after each treatment. We also scored the degree of dermatitis and recorded videos to observe scratching behavior. Subsequently, we collected skin samples from these mice for histological analyses. RESULTS: Topical application of Sema3A, betamethasone and tacrolimus ointments significantly inhibited scratching behavior and improved dermatitis scores in Dfb-treated mice compared with control mice, whereas vaseline and heparinoid had no effects. A significant improvement of TEWL was observed only in Sema3A ointment-treated mice. Moreover, Sema3A ointment reduced the densities of PGP9.5- and substance P-immunoreactive nerve fibers in the epidermis and the numbers of inflammatory cells, such as CD4 immunoreactive T cells and eosinophils, and improved acanthosis in the Dfb-treated mice compared with controls. CONCLUSION: Sem3A ointment may have therapeutic efficacy in patients with pruritus and dermatitis of AD.

Matrix metalloproteinase-8 is involved in dermal nerve growth: implications for possible application to pruritus from in vitro models.

Cutaneous nerve density is related to abnormal itch perception in dermatoses, such as atopic dermatitis and xerosis. However, the mechanisms underlying the elongation of dermal nerve fibers within the interstitial collagen (CoL) matrix are poorly understood. In this study, a culture system of rat dorsal root ganglion neurons consisting of type I CoL and a Boyden chamber containing a nerve growth factor (NGF) concentration gradient was used. Nerve fibers penetrating into type I CoL gel were observed in the presence of the NGF concentration gradient. Levels of matrix metalloproteinase-8 (MMP-8) mRNA and protein were increased in the cultured neurons and the conditioned medium, respectively. The nerve fiber penetration was dose dependently inhibited by MMP-8 blockers. Moreover, MMP-8 immunoreactivity was partially localized at growth cones in NGF-responsive nerve fibers. Semaphorin 3A stimulation also showed the opposite effects on these NGF-dependent events. Intriguingly, MMP-8 expression was upregulated by type I and III CoLs, which are substrates for this enzyme. These results suggested that MMP-8 is involved in sensory nerve growth within the interstitial CoL matrix through modulation by the axonal guidance molecules and/or extracellular matrix components. These findings provide insight into the development of pruritus involving skin nerve density.

Evaluation of epidermal nerve density and opioid receptor levels in psoriatic itch.

BACKGROUND: Psoriasis is a complex, multifactorial inflammatory skin disease with genetic and environmental interactions. Patients with psoriasis exhibit erythematous plaques with itch, but the mechanisms of psoriatic itch are poorly understood. OBJECTIVES: This study was performed to investigate epidermal nerve density and opioid receptor levels in psoriatic skin with or without itch. METHODS: Twenty-four patients with psoriasis aged between 39 and 82 years were included in this study. The number of epidermal nerve fibres, the levels of semaphorin 3A (Sema3A) and the expression patterns of µ- and κ-opioid systems were examined immunohistologically in skin biopsies from psoriatic patients with or without itch and healthy volunteers as controls. RESULTS: The number of epidermal nerve fibres tended to increase in approximately 40% of psoriatic patients with itch compared with healthy controls, while such intraepidermal nerves were not observed in other itchy patients. In comparison with healthy controls, Sema3A levels also tended to decrease in the epidermis of psoriatic patients with itch. However, no relationship was found between nerve density and Sema3A levels in the epidermis of psoriatic patients with itch. The levels of µ-opioid receptor and ß-endorphin in the epidermis were the same in healthy controls and psoriatic patients with or without itch. The levels of κ-opioid receptor and dynorphin A were significantly decreased in the epidermis of psoriatic patients with itch compared with healthy controls. CONCLUSIONS: Based on Sema3A levels in the epidermis, epidermal opioid systems, rather than hyperinnervation, may be involved in the pathogenesis of psoriatic itch.

Inhibitory effects of UV-based therapy on dry skin-inducible nerve growth in acetone-treated mice.

BACKGROUND: UV-based therapy has anti-pruritic effects in inflammatory skin diseases, such as atopic dermatitis and psoriasis. These anti-pruritic effects may be partly due to inhibition of intraepidermal nerve growth, but they have not been fully characterized. OBJECTIVE: This study was performed to characterize the anti-nerve growth effects of UV-based therapies in acetone-treated mice as an acute dry skin model. METHODS: Nerve fibers penetrate into the epidermis 24h after acetone treatment in mice, and nerve growth peaks 48h after acetone treatment. To investigate the effects of UV-based therapies on the epidermal nerve fibers, including combination treatment with corticosteroid ointment, the mice were treated with psoralen ultraviolet A (PUVA), PUVA and betamethasone valerate ointment (PUVA+BV), narrowband ultraviolet B (NB-UVB), or an excimer lamp. Each therapy was provided 24h after acetone treatment, and skin samples were taken 48h later. Nerve fiber densities and expression levels of nerve growth factor (NGF) and semaphorin 3A (Sema3A) in the epidermis were examined by immunohistochemistry. RESULTS: Penetration of nerve fibers into the epidermis was observed in the acetone-treated mice, concomitant with increased NGF and decreased Sema3A levels in the epidermis. The acetone-induced intraepidermal nerve growth was significantly decreased by PUVA, PUVA+BV, NB-UVB, and excimer lamp treatments compared with controls. In addition, PUVA+BV and NB-UVB normalized the abnormal expression of NGF and Sema3A in the epidermis, but no such normalization was observed with excimer lamp treatment. CONCLUSION: UV-based therapies, especially NB-UVB and excimer lamp treatments, may be effective therapeutic methods for pruritus involving epidermal hyperinnervation.

Keratinocyte-derived anosmin-1, an extracellular glycoprotein encoded by the X-linked Kallmann syndrome gene, is involved in modulation of epidermal nerve density in atopic dermatitis.

BACKGROUND: Epidermal nerve density is increased in atopic dermatitis (AD), suggesting that the hyperinnervation is partly responsible for abnormal itch perception. It is probably controlled by axonal guidance molecules produced by keratinocytes. An extracellular matrix glycoprotein anosmin-1 encoded by KAL1 has chemoattractive or chemorepulsive effects on different neuronal types. OBJECTIVE: This study was performed to investigate the roles of anosmin-1 in skin innervation. METHODS: Rat dorsal root ganglion (DRG) neurones were cultured in conditioned medium from control or KAL1-overexpressing cells for neurite outgrowth assay. KAL1 expression in cultured epidermal keratinocytes or human skin was examined by quantitative RT-PCR (qRT-PCR). Anosmin-1 distribution in normal and atopic skin was examined immunohistochemically. The effects of calcium concentrations and cytokines on KAL1 expression in cultured normal human epidermal keratinocytes (NHEK) were analysed by qRT-PCR. RESULTS: Neurite outgrowth in cultured DRG neurones was inhibited by conditioned medium from KAL1-overexpressing cells, while it was rescued by addition of recombinant fibroblast growth factor receptor 1 for capturing anosmin-1. KAL1 transcripts were expressed in cultured keratinocytes or in normal skin. Anosmin-1 was strongly expressed in the basal cell layer of normal skin, but decreased in atopic skin, concomitant with increases of epidermal nerve fibres. KAL1 expression was downregulated during keratinocyte differentiation. The expression was also upregulated by interleukin-4 (IL-4), IL-13 or transforming growth factor (TGF)-beta1. TGF-beta1 acted synergistically with IL-13 to enhance KAL1 expression, while interferon-gamma inhibited its expression. CONCLUSION: Anosmin-1 produced by epidermal keratinocytes in response to calcium concentrations or cytokines may modulate epidermal nerve density in AD.

Psoralen-ultraviolet A therapy alters epidermal Sema3A and NGF levels and modulates epidermal innervation in atopic dermatitis.

BACKGROUND: Epidermal nerve densities are increased in patients with atopic dermatitis (AD), suggesting that it is partly responsible for the intense itching in the skin. Epidermal hyperinnervation in AD patients is decreased by ultraviolet (UV) phototherapy, although the underlying mechanisms are poorly understood. Interestingly, abnormal expression of axonal guidance molecules, such as nerve growth factor (NGF) and semaphorin 3A (Sema3A), is found in the epidermis of AD patients. Therefore, UV phototherapy may alter levels of axonal guidance molecule expression in atopic skin. OBJECTIVE: This study was performed to investigate whether epidermal Sema3A and NGF levels in AD are influenced by psoralen-UVA (PUVA) therapy. METHODS: Skin biopsies obtained from chronic AD patients before and after PUVA therapy were used. Both Sema3A and NGF in the skin were examined at mRNA and protein levels by quantitative RT-PCR and immunohistochemistry, respectively. Nerve fibers in the skin were stained with anti-PGP9.5 antibody, and the number of epidermal nerve fibers was counted. RESULTS: PUVA therapy decreased epidermal nerve densities in AD patients, concomitant with decreases in both visual analog scale (VAS) scores for pruritus and clinical severity scores. Increased fluorescence intensity of Sema3A and decreased fluorescence intensity of NGF were observed in the epidermis of PUVA-treated group. Moreover, Sema3A mRNA levels were upregulated in the PUVA-treated skins compared with untreated controls, while NGF mRNA levels in the skin were downregulated by the treatment. CONCLUSION: PUVA therapy may reduce epidermal hyperinnervation of AD by normalization of abnormal Sema3A and NGF expression in the epidermis.

Intraepidermal nerve fibers increase in dry skin of acetone-treated mice.

BACKGROUND: Skin dryness is apparent in several pruritic skin diseases, such as xerosis and atopic dermatitis. Our previous study has demonstrated an increase of nerve fibers in the epidermis of patients with xerosis, suggesting the contribution of nerve fibers to itching. OBJECTIVE: This study was conducted to reveal a direct linkage between dry skin and intraepidermal nerve growth. METHODS: ICR mice treated with acetone were used as a dry skin model. Time-dependent measurement of transepidermal water loss (TEWL) and stratum corneum (SC) hydration was performed on the treated areas. Moreover, both the distribution of intraepidermal nerve fibers and the expression of epidermal nerve growth factor (NGF) and amphiregulin (AR) were examined sequentially with immunohistochemistry and quantitative RT-PCR. The same experiments were carried out in control mice treated with sterile water. RESULTS: Enhanced TEWL and decreased SC hydration were observed in the acetone-treated skins during the first hour after the treatment. These parameters gradually returned to the normal range within 48 h. In the acetone-treated mice, we found that there were many nerve fibers in the epidermis between 16 and 48 h after the treatment. No changes of the parameters for barrier disruption and intraepidermal nerve growth were observed in the control skins. Moreover, the expression of epidermal NGF and AR at the protein and mRNA levels was increased before the penetration of nerve fibers into the epidermis. CONCLUSIONS: These results suggest that increases of epidermal NGF and AR levels are associated with intraepidermal nerve growth in acetone-treated mice.