The ceruminous gland is a modified sweat gland and plays a major role in formation of cerumen (earwax) in the external auditory canal (EAC) [1]. The sweat glands of EAC skin secrete a wide variety of antimicrobial proteins and peptides, such as lysozyme, lactoferrin, and β-defensins [2-6]. Gland secretion is neurally regulated by acetylcholine, substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide, and other components [7]. Among them, SP and CGRP play a major role in the gland secretion induced by harmful stimuli [8]. In preliminary studies, we have published reports about the expression of antimicrobial proteins and peptides in the cerumen and the ceruminous glands. Therefore, it is conceivable that the interrelationship of antimicrobial factors and secretagogues such as SP and CGRP has clinical significance in their essential protective roles for the harmful states. However, the mechanism involved in the secretion has not been elucidated. Moreover, little is known about the expression of SP and CGRP in the EAC skin.

In the present study, we examined distribution patterns of SP and CGRP in human EAC skin, especially its relation with ceruminous glands using antibodies and the avidin-biotin peroxidase complex method.

On the H&E stain, the well-developed large ceruminous glands (Fig. 1A) and small sebaceous glands near hair follicles (Fig. 1B) were found in the dermis. The secretory cell of a ceruminous gland was usually lined with high columnar cells resting on myoepithelial cells. High active columnar cells had secretory buds extending to the lumen of the tubule (Fig. 1A).

Results of the immunostaining for SP and CGRP in the epidermis and dermis of the EAC skin are summarized in Table 1. The degree of immunohistochemical positive density was determined using a 5-point scoring scale (reaction intensities: -, no staining; +, mild; ++, moderate; +++, strong; ++++, very strong).

SP immunoreactivity was not detected in the epithelium of EAC skin (Fig. 2A, B) and sebaceous glands (Fig. 2D). In the ceruminous glands, SP-positive staining was found in both acini and myoepithelial cells (Fig. 2C).

In the epithelium, CGRP immunoreactivity was detected in the suprabasal region, which contained granular and prickle cell layers (Fig. 3A, B). CGRP positive staining was located in the dermis, ceruminous glands, sebaceous glands, and to a lesser extent around hair follicles. Furthermore, in the ceruminous glands, perinuclear region of secretory cells was positively stained (Fig. 3C, D) and more extensive in the cytoplasm of myoepithelial cells (Fig. 3D).

The density of SP and CGRP staining was highest in myoepithelial cells (Figs. 2C, 3C, 3D) of the ceruminous glands.

The appendages of the skin are sweat glands, sebaceous glands, and hair. Glands of EAC skin are responsible for secreting lipids, peptides, and proteins, which are the components of cerumen and are important for maintaining the innate immunity of EAC [1,5,6]. Interestingly, the ceruminous glands are known to secrete several important antimicrobial proteins and peptides [2-6]. The ceruminous glands (modified sweat) in EAC skin are similar to the serous glands in the respiratory submucosa in terms of the biochemical protection. They afford and share several common properties. In particular, both contain numerous electron-dense secretory vesicles in the cytoplasm [9-11].

Ceruminous glands are located in the dermis deep to the sebaceous glands. They are simple coiled tubular glands. The glandular epithelium is cuboidal or columnar and has protrusions extending to the lumen of the tubule. In the active form, secretory cells are tall, with apical protrusions that were pinched off into the lumen of the gland. The ceruminous glands were found in a single layer of secretory cells resting upon one layer of myoepithelial cells, which surround the secretory cells as an outer layer of the tubule. Generally, the myoepithelial cells are found in the sweat glands, mammary glands, lacrimal glands, and salivary glands. Myoepithelial cells can function to squeeze-out the contents of the secretory cells into the duct.

Recently, some authors have suggested that ceruminous glands play a role in the protection of EAC skin since these glands contain antimicrobial factors [5,6]. The family of antimicrobial peptides secreted from these cells has been suggested to contribute to skin defenses, but the EAC skin has been in little attention [5].

In general, it has been known that skin gland secretion is under neural control. The secretion of fluids and electrolytes is regulated by the parasympathetic system, whereas protein secretion is regulated by the sympathetic system. SP and CGRP have been known as a potent glandular fluid secretagogue [12] and to play a role in the gland secretion induced by harmful stimuli [8]. However, little is known about the secretagogues of EAC skin glands. It is suspected that the interrelation between antimicrobial materials and secretagugues has clinical significance in various EAC skin diseases. Moreover, it remains unclear whether SP and/or CGRP is expressed in the cells of EAC skin.

In the present study, the results of immunohistochemical analysis revealed that SP and CGRP were present in the cytoplasm of ceruminous gland cells, but not or sparse in sebaceous gland cells. Myoepithelial cells were intensely immunostained for both peptides. These findings indicated that SP and CGRP are expressed in the gland cells of EAC skin and suggest that these relate to the ceruminous gland secretion, in turn, those act to nullify pathogenic insults under normal conditions.

This is the first report of the presence of SP and CGRP in the gland cells of normal human EAC skin. The SP and CGRP immunoreactivity patterns observed in our study indicate that these secretary factors may participate in the regulation of secretion of cerumen components. These observations may provide important implications for EAC skin disorders. Further research into the secretion of antimicrobial proteins and peptides using these secretagogues is needed for increased understanding of the innate immune protection in the EAC skin.