Atsuko Kamo

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.

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.

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.

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.

In vitro model for penetration of sensory nerve fibres on a Matrigel basement membrane: implications for possible application to intractable pruritus

Background  Epidermal hyperinnervation occurs in dermatoses with intractable pruritus, such as atopic dermatitis, suggesting that the hyperinnervation is partly responsible for abnormal itch perception.

Topical application of emollients prevents dry skin-inducible intraepidermal nerve growth in acetone-treated mice

Abstract In common dermatoses, such as atopic dermatitis (AD), a decline in skin barrier function often accompanies an increased severity of clinical symptomatology, including pruritus [1]. Skin barrier disruption alters epidermal innervation and increases nerve density in the skin [2]. These findings are indicative of increases in sensory receptors responsive to exogenous trigger factors, suggesting that hyperinnervation is partly responsible for intense itch sensations [2]. Therefore, the abnormal innervation associated with skin barrier dysfunction such as dry skin has been considered as a target of antipruritic therapy. Moreover, recent studies have demonstrated that epidermal innervation is probably regulated by a fine balance of nerve elongation factors (e.g., nerve growth factor (NGF), amphiregulin, gelatinase) [2] and nerve repulsion factors (e.g., semaphorin 3A (Sema3A), anosmin-1) [2,3]. Although many people use emollients daily to alleviate symptoms of clinically and subjectively dry skin [4], the effects of emollients on nerve fiber density and nerve growth activity in dry skin remain unclear. We therefore examined the anti-nerve growth effects of petrolatum and heparinoid cream in the epidermis of acetone-treated mice, an animal model of acute dry skin [5].

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.

Neurotropin inhibits the increase in intraepidermal nerve density in the acetone-treated dry-skin mouse model.

Epidermal hyperinnervation is considered one cause of sensitization to itch, and is thought to regulated by keratinocyte‐derived axonal guidance molecules, including nerve growth factor (NGF) and semaphorin (Sema)3A. Neurotropin (NTP) shows antipruritic effects in allergic disease and is also used for pain relief. Using cultured rat dorsal root ganglion neurones, we previously found that NTP inhibited NGF‐induced neurite outgrowth. However, no such inhibitory effect has been shown in vivo. We therefore assessed the effects of intraperitoneal administration of NTP on nerve density and expression of NGF and Sema3A mRNAs in the epidermis of acetone‐treated mice showing epidermal hyperinnervation. We found that NTP significantly reduced intraepidermal nerve growth in these acetone‐treated mice. NTP significantly upregulated epidermal Sema3A mRNA, but had no effect on expression of epidermal NGF mRNA. These findings indicate that NTP may reduce intraepidermal nerve density by inducing expression of Sema3A in the epidermis.

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.

Histamine H4 Receptor Antagonists Ineffective against Itch and Skin Inflammation in Atopic Dermatitis Mouse Model

TO THE EDITOR Histamine is the best known pruritogen in humans and the most commonly used experimental itch-causing substance. It induces increased itch responses in the lesional skin of atopic dermatitis (AD) patients compared with normal skin (Ikoma et al., 2006). However, histamine H1 receptor (H1R) antagonists frequently fail to relieve the itch in AD patients as well as it does in patients with systemic diseases such as kidney and liver diseases. The lack of amelioration by high-potency H1R antagonists of different types in patients with itch suggests that other systems are involved (Ikoma et al., 2006; Ständer and Weisshaar, 2012). Involvement of histamine H4 receptor (H4R) in histamine-evoked itch in animal models has been reported (Dunford et al., 2007; Thurmond et al., 2008). However, the therapeutic efficacy of H4R antagonists on the H1R antagonistresistant itch in AD is poorly understood. We therefore examined the therapeutic effects of H4R antagonists on itch and skin inflammation in AD using NC/Nga mice, a mouse model of AD that has been previously described (Tanaka et al., 2012). Male NC/Nga mice (Charles River Japan, Yokohama, Japan), 10 weeks old, were maintained in clean condition. All animal procedures were approved by the institutional Animal Care and Use Committee of Juntendo University Graduate School of Medicine. It is generally accepted worldwide that AD patients are highly sensitized to house dust mite allergens (Sanda et al., 1992); and that house dust mite Dermatophagoides farinae body (Dfb) and feces are wellknown major environmental allergens (Matsuoka et al., 1995). We used a Dfb ointment-induced AD-like mouse model (Dfb-NC/Nga) to evaluate the therapeutic efficacy of H4R antagonists against itch-related behavior (scratching) and dermatitis in a mouse model of AD. Dermatitis was induced by application of Dfb ointment (Biostir, Kobe, Japan) twice a week for 3 weeks as described (Yamamoto et al., 2009). Severity of skin lesion was graded according to the criteria as described (Matsuda et al., 1997). Animals that received repeated application of Dfb ointment to their skin (Figure 1a) showed higher dermatitis scores than controls after 3 weeks (data not shown). After the induction, transepidermal water loss was measured using a Tewameter TM210 (Courage and Khazawa, Cologne, Germany) and scratching behavior was observed for 2 hours using a MicroAct (Neuroscience, Tokyo, Japan) as described (Inagaki et al., 2003). Dfb-NC/Nga mice showed significant loss of transepidermal water and more scratching bouts (data not shown). We examined effects of H4R antagonists, JNJ7777120 and JNJ28307474, on dermatitis and scratching behavior in Dfb-NC/Nga mice. In mice, antagonist JNJ28307474 shows a longer plasma half-life than JNJ7777120 (Thurmod et al., unpublished observations). Mice that scored over 5 for dermatitis severity were treated by either intraperitoneal injection with a vehicle (20% dimethylsulphoxide and 80% 2-hydroxypropyl-b-cyclodextrin in saline) or H4R antagonists (10 or 30 mg kg ) three times per week for 3 weeks (Figure 1a). Dermatitis score was assessed after each Dfb application, and the data were expressed as fold change values over score of dermatitis before H4R antagonist treatment (baseline) in each group. No significant amelioration of dermatitis followed treatment by either of these H4R antagonists (Figure 1b and c). In addition, scratching behavior was recorded before and after H4R antagonist treatment (# shown in Figure 1a). The data were expressed as fold change values over number of scratching bouts before the treatment (baseline) in each group. Behavior analyses revealed neither treatment inhibited scratching behavior (Figure 1d and e). Moreover, the fold change value of scratching bouts significantly increased by treatment of 30 mg kg 1 JNJ28307474 (Figure 1e). Treatment with JNJ7777120 or JNJ28307474 had no effect on locomotion activity (Kamo et al., unpublished observations). In this study, we found that treatment with H4R antagonist (JNJ7777120 or Accepted article preview online 20 August 2013; published online 19 September 2013 Abbreviations: AD, atopic dermatitis;; Dfb, Dermatophagoides farinae body; H1R, histamine H1 receptor; H4R, histamine H4 receptor A Kamo et al. Effects of H4R Antagonist on Dfb-AD Model