Marco Tizzano

Cholinergic neurotransmission links solitary chemosensory cells to nasal inflammation

Significance Millions of people worldwide suffer from chronic nasal inflammation involving obstructed airflow and nasal discharge. Although nasal inflammation is often considered to be a reaction to allergens, approximately one-quarter of all cases are nonallergic rhinitis. The causes of this disease are unknown, but symptoms may be triggered or exacerbated by a variety of inhaled irritants or even seemingly innocuous odors. We report here that specialized chemosensory cells of the nasal epithelium of mice detect potential irritants and transmit this information to pain-sensing nerve terminals, which then release bioactive peptides to trigger an inflammatory response—all without the necessity for activity of the adaptive immune system. This previously unidentified pathway may offer therapeutic targets for intervention in nonallergic rhinitis. Solitary chemosensory cells (SCCs) of the nasal cavity are specialized epithelial chemosensors that respond to irritants through the canonical taste transduction cascade involving Gα-gustducin and transient receptor potential melastatin 5. When stimulated, SCCs trigger peptidergic nociceptive (or pain) nerve fibers, causing an alteration of the respiratory rate indicative of trigeminal activation. Direct chemical excitation of trigeminal pain fibers by capsaicin evokes neurogenic inflammation in the surrounding epithelium. In the current study, we test whether activation of nasal SCCs can trigger similar local inflammatory responses, specifically mast cell degranulation and plasma leakage. The prototypical bitter compound, denatonium, a well-established activator of SCCs, caused significant inflammatory responses in WT mice but not mice with a genetic deletion of elements of the canonical taste transduction cascade, showing that activation of taste signaling components is sufficient to trigger local inflammation. Chemical ablation of peptidergic trigeminal fibers prevented the SCC-induced nasal inflammation, indicating that SCCs evoke inflammation only by neural activity and not by release of local inflammatory mediators. Additionally, blocking nicotinic, but not muscarinic, acetylcholine receptors prevents SCC-mediated neurogenic inflammation for both denatonium and the bacterial signaling molecule 3-oxo-C12-homoserine lactone, showing the necessity for cholinergic transmission. Finally, we show that the neurokinin 1 receptor for substance P is required for SCC-mediated inflammation, suggesting that release of substance P from nerve fibers triggers the inflammatory events. Taken together, these results show that SCCs use cholinergic neurotransmission to trigger peptidergic trigeminal nociceptors, which link SCCs to the neurogenic inflammatory pathway.

α-Gustducin immunoreactivity in the airways

The G-protein subunit α-gustducin is a marker of chemoreceptive cells. In the present study, we examined the immunohistochemical localization of α-gustducin in rat airway epithelium both by light and electron microscopy. α-Gustducin immunoreactivity was found in solitary cells that presented ultrastructural features of chemoreceptor cells, i.e. flask-shaped or pear-shaped, with an apical process with thin microvilli protruding into the lumen. The immunostaining was mainly concentrated in the apical process and along the basolateral cell surface. To investigate whether α-gustducin-immunoreactive cells represented a distinct cell subset in rat airways, we performed double-label immunocytochemistry with antibodies to protein gene groduct (PGP) 9.5, a marker of neuroendocrine cells, and to phospholipase C beta2 (PLCβ2), a component of the bitter signalling pathway. α-Gustducin-immunoreactive cells were present in a subset of PGP-9.5-immunoreactive elements, although not all α-gustducin-positive cells expressed PGP 9.5 labelling. In addition, a subset of α-gustducin-expressing cells colocalized PLCβ2. This work thus demonstrates that solitary α-gustducin-immunoreactive cells exist throughout the airways and represent a specialized cell type with morphological and immunohistochemical characteristics of chemoreceptor cells.

Identification and characterization of a specific sensory epithelium in the rat larynx.

A specific laryngeal sensory epithelium (SLSE), which includes arrays of solitary chemoreceptor cells, is described in the supraglottic region of the rat. Two plates of SLSE were found, one on each side of the larynx. The first plate was located in the ventrolateral wall of the larynx, and the second was located in the interarytenoidal region. In SLSE, immunoblotting showed the presence of α‐gustducin and phospholipase C β2 (PLCβ2), which are two markers of chemoreceptor cells. At immunocytochemistry, laryngeal immunoreactivity for α‐gustducin was localized mainly in solitary chemosensory cells. Double‐label immunocytochemistry using confocal microscopy demonstrated that α‐gustducin–expressing cells in large part colocalize type III IP3 receptor (IP3R3), another key molecule in bitter taste perception. However, some IP3R3–expressing cells do not colocalize α‐gustducin. At ultrastructural immunocytochemistry, these cells showed packed apical microvilli, clear cytoplasmic vesicles, and cytoneural junctions. SLSE was characterized by high permeability to a tracer due to poorly developed junctional contacts between superficial cells. Junctions were short in length and showed little contact with the terminal web. Ultrastructural analysis showed deep pits among the superficial cells. In SLSE, high density of intraepithelial nerve fibers was found. The lamina propria of the SLSE appeared thicker than that in other supraglottic regions. It was characterized by the presence of a well‐developed subepithelial nerve plexus. The immunocytochemical and ultrastructural data suggested that SLSE is a chemoreceptor located in an optimal position for detecting substances entering the larynx from the pharynx or the trachea. J. Comp. Neurol. 475:188–201, 2004.

Evidence of solitary chemosensory cells in a large mammal: the diffuse chemosensory system in Bos taurus airways

The diffuse chemosensory system (DCS) of the respiratory apparatus is composed of solitary chemosensory cells (SCCs) that resemble taste cells but are not organized in end organs. The discovery of the DCS may open up new approaches to respiratory diseases. However, available data on mammalian SCCs have so far been collected from rodents, the airways of which display some differences from those of large mammals. Here we investigated the presence of the DCS and of SCCs in cows and bulls (Bos taurus), in which the airway cytology is similar to that in humans, focusing our attention on detection in the airways of molecules involved in the transduction cascade of taste [i.e. α‐gustducin and phospholipase C of the β2 subtype (PLCβ2)]. The aim of the research was to extend our understanding of airway chemoreceptors and to compare the organization of the DCS in a large mammal with that in rodents. Using immunocytochemistry for α‐gustducin, the taste buds of the tongue and arytenoid were visualized. In the trachea and bronchi, α‐gustducin‐immunoreactive SCCs were frequently found. Using immunocytochemistry for PLCβ2, the staining pattern was generally similar to those seen for α‐gustducin. Immunoblotting confirmed the expression of α‐gustducin in the tongue and in all the airway regions tested. The study demonstrated the presence of SCCs in cows and bulls, suggesting that DCSs are present in many mammalian species. The description of areas with a high density of SCCs in bovine bronchi seems to indicate that the view of the DCS as made up of isolated cells totally devoid of ancillary elements is probably an oversimplification.

Acyl Homoserine Lactones Induce Early Response in the Airway

Acyl homoserine lactones (AHLs) are intercellular signaling molecules used in quorum sensing by Gram‐negative bacteria. We studied the early effects on the rat airway of in vivo intratracheal administration of AHLs (i.e., P. aeruginosa and B. cepacia) to test the hypothesis that AHLs also act on the airway cells, modifying secretory mechanisms which are important in mucosal defense. One hour after treatment, N‐butyryl‐homoserine lactone (C4‐HL) had caused dilated extracellular spaces, loss of cilia, reduction of secretory material, and the presence of prenecrotic elements in the epithelium, while N‐octanoyl‐homoserine lactone (C8‐HL) caused a mild lesion in the epithelium. After treatment with either C4‐ or C8‐HL, reduced immunoreactivity was found using CC10 antibody. At ultrastructural examination, dilatation of the mitochondria was evident in ciliate and secretory cells, while solitary chemosensory cells appeared better preserved, showing aspects of nucleocytoplasmic activation. Using microarray analysis, we found down‐regulation of early gene Fos and Egr1 in all AHL‐treated specimens. In vivo pharmacological magnetic resonance imaging after C4‐ or C8‐HL treatment showed a slight increase in tracheal secretion at a first evaluation 5 min after administration, with no increase in the following minutes. In conclusion, AHLs induce an early mucosal response, and the chondriomas of ciliate and secretory cells are the main cytological target of AHL action. Our results show that AHL action is not limited to activation of conspecific bacteria, but also modifies innate airway defense mechanisms. Anat Rec, 292:439–448, 2009.