Immunocytochemical studies of cutaneous nerves in human normal and prurigo nodularis skin

Abstract

Cutaneous nerves may regulate the immune system and be involved in inflammatory events of the skin. Prurigo nodularis is a skin disease which histologically reveals a massive neural hyperplasia and a local inflammation in the lesional skin. The aim of the thesis is to study normal cutaneous nerves and changes in such nerve fibers in the mechanism of the cutaneous inflammation of prurigo nodularis, by the investigation of certain bioactive substances and their receptors in nerve fibers, and other cells, of normal and diseased skin.

The intraepidermal nerves of normal adult human skin were demonstrated by protein gene product 9.5 (PGP 9.5) immunoreactivity. The free nerve endings distributed in the strata basale, spinosum and granulosum, appeared as thin and varicose fibers, and branched straight or bent. They existed at every site of the human body, including the face, trunk and extremities, with different densities. The low affinity nerve growth factor receptor (p75 NGFr) immunoreactivity was found mainly in the peripheral part of cutaneous nerve trunks and fibers, whereas the neuron-specific enolase immunoreactivity was mainly seen within the axons. Ultrastructurally, the Schwann cell membrane was strongly p75 NGFr-immunoreactive (IR). The Schwann cell membrane facing the connective tissue was more strongly p75 NGFr-IR than that close to the axon. However, the axon seldom showed any such immunoreactivity.

For the prurigo nodularis lesional skin, the distribution of calcitonin gene-related peptide (CGRP), galanin, neuropeptide tyrosine (NPY), peptide histidine isoleucine amide (PHI), peptide histidine methionine (PHM), substance P (SP), vasoactive intestinal polypeptide (VIP), p75 NGFr and PGP 9.5, was investigated. In summary, the CGRP, p75 NGFr- and PGP 9.5-IR structures in prurigo nodularis were stronger and more densely present than in the controls. Regarding NPY, PHI, PHM and VIP, there were no differences found between patient and control groups. The galanin, and SP staining was very weak. Ultrastructurally, a lot of nerve fibers were found clustered together in the dermal tissue. The axons were enlarged and seldom showed any NGFr immunoreactivity. The Schwann cell membrane showed a strong p75 NGFr immunoreactivity. The Schwann cell bodies were extended and irregularly shaped, and tended to separate into many branches en-wrapping the axons. The perineurium cells also revealed a strong p75 NGFr immunoreactivity. The Schwann cells inside the perineurium were less p75 NGFr-IR than those outside of the perineurium.

In histamine and NGFr double-staining, mast cells were seen in close vicinity to NGFr-positive nerves and sometimes seemingly to contact single nerve fiber. The histamine-containing mast cell number was significantly increased in the lesional dermis. At the ultrastructural level, the mast cell bodies become larger, having more abundant cytoplasm and mitochondria, but comparatively fewer characteristic granules. The mast cells were also frequently seen to contact other cell types and a mast cell infiltration into the epidermis was also found. Mast cells were often observed to sprout long dendrites, with or without granules. By a human histocompatibility complex class II (subregion DR) (HLA-DR) and protein S-100 (S-100) double-staining, dermal HLA-DR- and S-100-IR cells were shown to apparently be more numerous than in the controls. In the involved skin there were also more S-100-IR coarse nerve fibres in the dermis as compared to controls. By eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin/eosinophil protein X (EDN/EPX) labeling on lesional skin, the eosinophils were significantly increased in number compared to the uninvolved skin and the normal skin.

By ECP and PGP 9.5 double-labelling, the PGP 9.5-IR nerves were found increased in the areas where there were increased numbers of eosinophils. The nerves were in close vicinity to eosinophils, and occasionally contacted each other. By ultrastructural immunogold labelling, CGRP immunoreactivity was demonstrated to be increased in the dense-core vesicles of the axons of lesional skin, the axons which contain CGRP were more enlarged and had more dense-core vesicles than the axons which did not contained CGRP. By CGRP and tryptase (or ECP) double-labelling, the tryptase-containing mast cells and ECP-containing eosinophils were found to be increased in the lesional skin at locations where CGRP-containing nerve fibers were increased as well.

The studies in the thesis indicate that normal intraepidermal nerve terminals may have area-specific functions. The cutaneous nerves are maintained and regulated by the surrounding Schwann cells, which mainly express p75 NGFr. In prurigo nodularis, the over-expression of p75 NGFr in Schwann cells and the perineurium cells could contribute to the neurohyperplasia. The increased CGRP- or p75 NGFr-IR nerve fibers may interact with inflammatory cells, e.g. mast cells, eosinophils and the Langerhans cells, which might make the disease persist and result in strong pruritus.