Prurigo nodularis (PN) has multiple hyperkeratotic nodules and provokes intense itching. Itches are conducted by afferent C-fibers, which are unmyelinated nerve fibers originating from dorsal root ganglia neurons. Normally, C-fibers possess free nerve endings at the boundary between the epidermis and the dermis, and some nerve fibers invade the epidermis. The innervation of C-fibers is regulated, to some extent, by nerve growth factor (NGF)-induced nerve elongation and semaphorin 3A (Sema3A) repulsion. Increased NGF expression in the epidermis has been reported in atopic dermatitis (AD) lesions and psoriasis vulgaris (PV). Neuropeptides released from peripheral nerve fibers are closely related to the pathogenesis of pruritus. Sema3A is an axon-guidance molecule that inhibits neurite outgrowth of C-fibers. Recent studies indicated that Sema3A expression levels are decreased in the epidermis of patients with AD and PV, however, little is known about Sema3A expression in PN. The present study therefore aimed to investigate the expression of Sema3A in the epidermis of PN in relation to itch intensity and to compare the expression of Sema3A and NGF between subjects with PN, AD and PV, and healthy controls.
Experimental protocols for this research were approved by the Ethical Committee of the Yokohama City University Graduate School of Medicine. Fourteen PN patients, five AD patients, 15 PV patients and 15 healthy volunteers were recruited for the study. The average of ages were 53.1 ± 13.9, 52.2 ± 23.4, 59.0 ± 20.1 and 61.4 ± 13.3 years, respectively. The degree of pruritus was evaluated using a visual analog scale in the patients, the average was 56.4 ± 23.4, 45.0 ± 13.5 and 44.3 ± 30.7, respectively. Primary antibodies used for immunohistochemical staining were as follows: anti-PGP 9.5 (1:800 dilution; UltraClone, Isle of Wight, UK), anti-NGF (1:20 dilution; sc-548; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-Sema3A (1:200 dilution; ab23393; Abcam, Cambridge, UK). The number of epidermal PGP 9.5-immunoreactive fibers in 10 random fields of 1 × 103 μm² of each specimen was independently evaluated by two researchers. Quantitative reverse transcription polymerase chain reaction (qRT–PCR) analysis was performed with TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA). The levels of mRNA expression for each gene were calculated by the comparative ΔΔ Ct-method relative to β-actin. To detect differences between the itch intensity in each condition and mRNA expression levels or the number of nerve fibers, correlations were analyzed by Student's t-test and Spearman's rank correlation coefficient analysis.
Immunohistochemistry (Figs 1,2a) and qRT-PCR analysis (Fig. 2b,c) demonstrated hyperproliferation of cutaneous nerves in samples taken from the patients compared with healthy skin (HS) (P < 0.01) and increases in NGF expression as well as decreases in Sema3A expression in these patients compared with HS. These aberrant expressions of NGF were prominent in PN by immunostaining compared with those in AD and PV, although significant difference was not observed by qRT–PCR between these three diseases. The expression of NGF was positively correlated with the itching sensation (r = 0.757, P < 0.05) and the number of nerve fibers in the epidermis (r = 0.569, P < 0.05). In contrast, the expression of Sema3A was negatively correlated with the itching sensation (r = −0.604, P < 0.01) and the number of nerve fibers (r = −0.833, P < 0.01). The expression of Sema3A was negatively correlated with the NGF expression in PN (r = −0.424, P = 0.131).
Recently, it was reported that lesional and uninvolved PN skin biopsies showed significantly decreased intraepidermal nerve fiber density regardless of patient age, origin of PN, intensity or quality of pruritus. However, our data showed nerve fiber elongation in epidermis of PN. This discrepancy may be explained by the difference in different sampling standards and immunostaining methods. We speculate that downregulation of Sema3A may contribute to the itching sensation in association with nerve fiber elongation in PN as well as upregulation of NGF. Damage to the skin barrier caused by scratching may induce upregulation of NGF, which could further facilitate hyperinnervation in the epidermis. Sema3A has also been known to directly modulate the immune system through its action as a negative-feedback regulator of dendritic cell-induced T-cell proliferation. Reduced Sema3A expression in the epidermis may therefore facilitate the infiltration of immune cells in PN.
Further study is needed to clarify the role of aberrant expression of Sema3A and NGF in the development and aggravation of PN.