In the present study, the morphology and distribution of epithelial MCs in the rat upper airways were demonstrated by immunohistochemistry for 5-HT. Previous studies on the rat larynx reported 5-HT immunoreactivity in glottic epithelial MCs, but not in epiglottic epithelial MCs, and it has been suggested that there are two distinct subtypes of epithelial MCs in the rat larynx based on 5-HT immunoreactivity (Domeij et al. 1991; Lidegram et al. 1996). In the present study, by injecting rats with the precursor of 5-HT we were able to intensely visualize epithelial MCs in both the glottic and epiglottic regions of the rat larynx. In addition, 5-HT immunoreactivity was present in the majority of c-kit-immunoreactive epithelial MCs in the upper airways. Therefore, a subtype of epithelial MCs lacking 5-HT immunoreactivity was less likely to be present in the larynx and all laryngeal c-kit-immunoreactive MCs contained 5-HT, indicating that epithelial MCs in the airways may be characterized by immunoreactivity for 5-HT. Furthermore, c-kit is a marker for intestinal MMCs (Vogal et al. 2018). Since c-kit immunoreactivity was detected in both epithelial MCs and CTMCs in the present study, c-kit may be another general marker for airway MCs.
The double immunofluorescence analysis revealed that HDC and tryptase were expressed in CTMCs, but not in epithelial MCs in the rat upper airways, and the results obtained on epithelial MCs without HDC immunoreactivity were consistent with previous findings suggesting the absence of or the markedly lower histamine content in epithelial MCs in the rat respiratory and digestive systems (Fan and Iseki 1999). Furthermore, tryptase was not detected in the epithelial MMCs of the rat intestines; therefore, they may have expressed other serine protease proteins, namely, rat MC protease 2 (Chen et al. 1993; Friend et al. 1996). These findings suggest that the features of epithelial MCs lacking mediators, such as histamine and tryptase, are similar to those of MMCs in the rat intestine.
The electron microscopic analysis revealed the presence of a few granules of a heterogenous size and electron density in the epithelial MCs of the rat larynx and trachea, similar to intestinal MMCs (Akpavie and Pirie 1989; Fan and Iseki 1999). Furthermore, the rough endoplasmic reticulum, Golgi bodies, and mitochondria were evident in the epithelial MCs of the rat larynx and trachea, which is consistent with intestinal MMCs (Akpavie and Pirie 1989; Huntley et al. 1984; Kent 1966). On the other hand, CTMCs in the rat larynx and trachea contained a higher number of electron-dense granules of a similar size, which was identical to CTMCs in other organs, including the rat intestines (Nagata et al. 2001). In addition, cytoplasmic projections and organelles were evident in the cytoplasm of CTMCs in the rat larynx and trachea which was identical to intestinal CTMCs (Kent 1966). The ultrastructural features and immunohistochemical properties of epithelial MCs and CTMCs in the rat larynx and trachea suggest that these cells are the counterparts of MMCs and CTMCs, respectively, in the rat intestines.
The distribution of epithelial MCs appeared to be concentrated in the cranial segment of the upper respiratory tract, particularly in the glottis and cranial parts of the trachea. These regions are known for their chemosensory functions because they have adapted several chemosensory structures (Bradley 2000; Masuda et al. 2019). The topographical arrangement of intraepithelial MCs may be suitable for detecting antigens in inhaled air. Since rat MCs express high-affinity receptors for immunoglobulin E and toll-like receptors on their cell surface (Pietrzak et al. 2011; Swieter et al. 1989), they may be activated by a wide range of stimuli, particularly bacteria and viruses (Dileepan et al. 2023; Moon et al. 2009). Three-dimensional reconstructed views of epithelial MCs in whole-mount preparations revealed that the cytoplasmic processes of these cells were in contact with epithelial cells, suggesting that these cells release 5-HT from both the perinuclear cytoplasm and cytoplasmic processes in the vicinity of epithelial cells when activated. Furthermore, epithelial MCs made contacted with each other through their cytoplasmic processes, suggesting that these cells propagate activation signals to enhance their responses to stimuli. Although previous studies demonstrated that 5-HT from rat tracheal CTMCs caused smooth muscle contractions (Ikawati et al. 2001; Liu et al. 2005), the functional significance of 5-HT released from epithelial MCs remains elusive. In the mouse trachea, 5-HT increased ciliary beating and particle transport velocity, and the effects of 5-HT were completely blocked by the 5-HT receptor antagonists, methysergide and cyproheptadine (König et al. 2009; Weiterer et al. 2014). These findings suggest that 5-HT released from epithelial MCs regulates epithelial cell functions by inducing ciliary beating and mucus secretion in the rat larynx and trachea.
In conclusion, the present study revealed the topographic distribution, cellular morphology, and immunohistochemical properties of epithelial MCs in the rat larynx and trachea. Epithelial MCs with numerous cytoplasmic processes may release 5-HT in the vicinity of epithelial cells, which regulates innate immune responses by modulating epithelial cell functions at the entrance gate of the upper airways.