Mitsutoshi Tominaga

Antimicrobial Peptides Human β-Defensins and Cathelicidin LL-37 Induce the Secretion of a Pruritogenic Cytokine IL-31 by Human Mast Cells

In addition to their microbiocidal properties, human β-defensins (hBDs) and cathelicidin LL-37 stimulate a number of mammalian cell activities, including migration, proliferation, and cytokine/chemokine production. Because hBDs and LL-37 cause mast cells to release pruritogens such as histamine and PGs, we hypothesized that these peptides would stimulate the secretion of a novel pruritogenic mediator IL-31, predominantly produced by T cells. hBDs and LL-37 enhanced IL-31 gene expression and IL-31 protein production and release in the human mast cell line LAD2, as well as in peripheral blood-derived cultured mast cells, suggesting that mast cells are another source of IL-31. Moreover, the expression of IL-31 was elevated in psoriatic skin mast cells, and hBD-2–4 and LL-37, but not hBD-1, enhanced its expression in vivo in rat skin mast cells. hBDs and LL-37 also induced the release of other pruritogenic mediators, including IL-2, IL-4, IL-6, GM-CSF, nerve growth factor, PGE2, and leukotriene C4, and increased mRNA expression of substance P. hBD– and LL-37–mediated IL-31 production/release was markedly reduced by pertussis toxin and wortmannin, inhibitors of G-protein and PI3K, respectively. As evidenced by the inhibitory effects of MAPK-specific inhibitors, hBD-2–4 and LL-37 activated the phosphorylation of MAPKs p38, ERK, and JNK that were required for IL-31 production and release. The ability of hBDs and LL-37 to stimulate the production and release of IL-31 by human mast cells provides a novel mechanism by which skin-derived antimicrobial peptides/proteins may contribute to inflammatory reactions and suggests a central role of these peptides in the pathogenesis of skin disorders.

Decreased production of semaphorin 3A in the lesional skin of atopic dermatitis

transduction pathway plays a critical role in the process of tumour formation, growth and metastasis. Phosphorylation of the EGFR leads to activation of a signal transduction cascade of biochemical and physiological changes. Five sites of in vivo autophosphorylation have been identified in the EGFR: three major (Try1068, Tyr1148 and Tyr1173) and two minor (Tyr992 and Tyr1086). When hyperplastic sebaceous glands were stained with antiphospho-EGFR (Tyr1068) antibody, strong expression of phospho-EGFR was observed in sebaceous hyperplasia (Fig. 2b). Phospho-Tyr1068 is a direct binding site for Grb2. This site is involved in activation of MAP kinase signalling. MAP kinase signalling may be related to the sebaceous hyperplasia overlying dermatofibroma. The Drosophila segment polarity gene hedgehog (HH) encodes a precursor protein that undergoes autocleavage to generate Nand C-terminal peptides of 19 and 25 kDa, respectively. Both proteins are secreted and appear to function in embryonic and imaginal disc patterning. Vertebrate homologues of Drosophila HH include sonic hedgehog (SHH), alternatively designated VHH-1, desert hedgehog (DHH) and IHH. Each contains N-terminal signal peptides that apparently function as secreted proteins involved in the mediation of various cell–cell interactions. HH is a secreted protein and Patched and Smoothened are its receptors. Niemann et al. reported that IHH promotes proliferation of sebocyte precursors. In contrast, SHH promotes proliferation of hair follicle precursor cells. Hence, we checked the expression of IHH in sebaceous hyperplasia over dermatofibroma. Strong staining with antibody against IHH was detected in sebaceous hyperplasia. This result may show that components of the HH– Patched signalling pathway may be upregulated at this area. In summary, upregulation of the EGF–EGFR signalling pathway and the HH–Patched signalling pathway may be involved in this unusual phenotype of sebaceous hyperplasia overlying dermatofibroma.

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.

Itch and nerve fibers with special reference to atopic dermatitis: Therapeutic implications

Nerve density in the epidermis is partly involved in itch sensitization in pruritic skin diseases, such as atopic dermatitis (AD). Epidermal innervation is thought to be regulated by the balance between nerve elongation factors (e.g. nerve growth factor) and nerve repulsion factors (e.g. semaphorin 3A). Semaphorin 3A (Sema3A) has been shown to inhibit nerve growth factor (NGF)‐induced sprouting of sensory nerves, and epidermal Sema3A levels are lower in AD patients, concomitant with an increase in epidermal nerve density. In addition, treatment with anti‐NGF, Sema3A replacement, and several existing treatments, such as ultraviolet‐based therapies, normalized the hyperinnervation in AD, resulting in suppression of itching. This review expands knowledge regarding potential therapeutic strategies for ameliorating intractable pruritus in AD.

Histological Characterization of Cutaneous Nerve Fibers Containing Gastrin-Releasing Peptide in NC/Nga Mice: An Atopic Dermatitis Model

TO THE EDITOR Histological observations have shown that unmyelinated nerve fibers originating from dorsal root ganglions are present at higher densities in the epidermis in several skin diseases such as atopic dermatitis (AD), suggesting that hyperinnervation is partly responsible for abnormal itch perception (Steinhoff et al., 2003b; Ikoma et al., 2006; Paus et al., 2006). However, it has been difficult to histologically identify itch-specific fibers in the skin because there have been no itch-specific markers available. Recently, a study of gastrin-releasing peptide receptor (GRPR)-deficient mice has shown that the GRP/GRPR system is specifically involved in itch but not pain perception (Sun and Chen, 2007). Therefore, this study was conducted to immunohistochemically identify and characterize cutaneous nerve fibers containing GRP in NC/ Nga mice using an AD model (Matsuda et al., 1997). Male NC/Nga mice (SLC Japan, Shizuoka, Japan) were maintained in specific pathogen-free (SPF) or airuncontrolled conventional (Cnv) conditions. We used Cnv-NC/Nga mice of 8–12 weeks of age that manifested mild to severe skin lesions similar to AD, and SPF-NC/Nga mice with no AD as a control. All animal procedures were approved by the institutional Animal Care and Use Committee at Juntendo University Graduate School of Medicine. The immunohistochemical protocol for cutaneous nerve fibers was performed as described previously (Tominaga et al., 2007a). Cryosections (thickness 20 mm) from dorsal neck skin were double-stained with antiGRP and anti-product gene protein 9.5 (PGP9.5). The sections were observed with a confocal laser-scanning microscope DMIRE2 (Leica, Wetzlar, Germany). In Cnv-NC/Nga mice, GRPþPGP9.5þ fibers were observed in both the epidermis (Figure 1a) and the dermis (Figure 1b). They were mainly found in the dermis of SPF-NC/Nga mice, especially at the dermoepidermal junction (Figure 1c). Although the number of nerve fibers was significantly high in the epidermis and the dermis of Cnv-NC/Nga mice compared with that in SPF-NC/Nga mice (Figure 1d), the percentage of GRPþ fibers in PGP9.5þ fibers was approximately 2.4-fold higher in the epidermis of Cnv-NC/ Nga mice, although this was not the case in the dermis (Figure 1e). To further examine whether GRPþ fibers contain neuropeptides such as substance P (SP) and calcitonin-generelated peptide (CGRP), which are involved in neurogenic inflammation and/or hypersensitivity of itch sensation (Stander and Steinhoff, 2002; Steinhoff et al., 2003b), skin samples of NC/Nga mice were double-stained with antibodies against GRP, SP, and CGRP. In the epidermis and the dermis of Cnv-NC/ Nga mice, almost all GRPþ fibers contained SP (Figure 1f) or CGRP (Figure 1i), as did those in the dermis of SPF-NC/Nga mice (Figure 1g and j). Many CGRPþ fibers in the skin of Cnv-NC/Nga (Supplementary Figure S1a) and SPF-NC/Nga (Supplementary Figure S1b) mice also contained SP. To estimate the percentage of GRPþSPþ or GRPþCGRPþ fibers in PGP9.5þ fibers, the skin samples of NC/Nga mice were triple-stained with antibodies against GRP, PGP9.5, SP, and CGRP (Figure 1h and k). In comparison with SPF-NC/Nga mice, the percentages of GRPþSPþ and GRPþCGRPþ fibers in PGP9.5þ fibers were approximately 3.5-fold and 3.8-fold higher in the epidermis of Cnv-NC/Nga mice, respectively, although this was not the case in the dermis. GRP SPþ or GRP CGRPþ fibers were also observed in the skin of these mice (Supplementary Table S1), but they remained as uncharacterized nerve fibers in this study. The cutaneous GRPþ fibers in Cnv-NC/Nga mice were immunohistochemically characterized using antibodies against itch-related molecules such as transient receptor potential vanilloid 1 (TRPV1), protease-activated receptor 2 (PAR-2), mu-opioid receptor (MOR), and tropomyosin-receptor-kinase A (Paus et al., 2006). The GRPþ fibers expressed TRPV1, but some GRP TRPV1þ fibers were also observed in the atopic skin (Figure 2a). The GRPþ fibers also expressed PAR-2, MOR, or tropomyosin-receptor-kinase A in the atopic skin (Figure 2b–d). In this study, we found that GRPþ fibers were present in mouse skin, and that the percentage of GRPþ fibers in PGP9.5þ fibers was markedly high only in the epidermis of atopic NC/ Nga mice. Smallto medium-sized adult dorsal root ganglions neurons express GRP, and its receptor is present in the superficial dorsal horn.

Roles of glutamate, substance P, and gastrin-releasing peptide as spinal neurotransmitters of histaminergic and nonhistaminergic itch

Summary Histamine‐independent itch was mediated by the combined action of glutamate, substance P, and gastrin‐releasing peptide while histamine‐dependent itch relied primarily on glutamate. Co‐administration of antagonists may prove useful to treat antihistamine‐resistant chronic itch. ABSTRACT We investigated roles for substance P (SP), gastrin‐releasing peptide (GRP), and glutamate in the spinal neurotransmission of histamine‐dependent and ‐independent itch. In anesthetized mice, responses of single superficial dorsal horn neurons to intradermal (i.d.) injection of chloroquine were partially reduced by spinal application of the &agr;‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole proprionate acid (AMPA)/kainate antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX). Co‐application of CNQX plus a neurokinin‐1 (NK‐1) antagonist produced stronger inhibition, while co‐application of CNQX, NK‐1, and GRP receptor (GRPR) antagonists completely inhibited firing. Nociceptive‐specific and wide dynamic range‐type neurons exhibited differential suppression by CNQX plus either the GRPR or NK‐1 antagonist, respectively. Neuronal responses elicited by i.d. histamine were abolished by CNQX alone. In behavioral studies, individual intrathecal administration of a GRPR, NK‐1, or AMPA antagonist each significantly attenuated chloroquine‐evoked scratching behavior. Co‐administration of the NK‐1 and AMPA antagonists was more effective, and administration of all 3 antagonists abolished scratching. Intrathecal CNQX alone prevented histamine‐evoked scratching behavior. We additionally employed a double‐label strategy to investigate molecular markers of pruritogen‐sensitive dorsal root ganglion (DRG) cells. DRG cells responsive to histamine and/or chloroquine, identified by calcium imaging, were then processed for co‐expression of SP, GRP, or vesicular glutamate transporter type 2 (VGLUT2) immunofluorescence. Subpopulations of chloroquine‐ and/or histamine‐sensitive DRG cells were immunopositive for SP and/or GRP, with >80% immunopositive for VGLUT2. These results indicate that SP, GRP, and glutamate each partially contribute to histamine‐independent itch. Histamine‐evoked itch is mediated primarily by glutamate, with GRP playing a lesser role. Co‐application of NK‐1, GRP, and AMPA receptor antagonists may prove beneficial in treating chronic itch.

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.

Roles for substance P and gastrin-releasing peptide as neurotransmitters released by primary afferent pruriceptors

Recent studies support roles for neurokinin-1 (NK-1) and gastrin-releasing peptide (GRP) receptor-expressing spinal neurons in itch. We presently investigated expression of substance P (SP) and GRP in pruritogen-responsive primary sensory neurons and roles for these neuropeptides in itch signaling. Responses of dorsal root ganglion (DRG) cells to various pruritogens were observed by calcium imaging. DRG cells were then processed for SP, GRP, and isolectin B-4 (IB4; a marker for nonpeptidergic neurons) immunofluorescence. Of pruritogen-responsive DRG cells, 11.8-26.8%, 21.8-40.0%, and 21.4-26.8% were immunopositive for SP, GRP, and IB4, respectively. In behavioral studies, both systemic and intrathecal administration of a NK-1 receptor antagonist significantly attenuated scratching evoked by chloroquine and a protease-activated receptor 2 agonist, SLIGRL, but not histamine, bovine adrenal medulla peptide 8-22 (BAM8-22), or serotonin. Systemic or intrathecal administration of a GRP receptor antagonist attenuated scratching evoked by chloroquine and SLIGRL but not BAM8-22 or histamine. The GRP receptor antagonist enhanced scratching evoked by serotonin. These results indicate that SP and GRP expressed in primary sensory neurons are partially involved as neurotransmitters in histamine-independent itch signaling from the skin to the spinal cord.

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.