Neurotransmitters in airway parasympathetic neurons altered by neurotrophin-3 and repeated allergen challenge

Abstract

Changes in airway nerves associated with chronic inflammation may underlie the pathogenesis and symptoms of lower airway diseases, such as asthma. The molecules most likely causing such alterations are neurotrophins (NTs) and/or related neurokines. In several species, including humans, lower airway parasympathetic postganglionic neurons that project axons to airway smooth muscle are either cholinergic or nonadrenergic noncholinergic (NANC), the latter synthesizing vasoactive intestinal peptide and nitric oxide, but not acetylcholine. In guinea pig trachealis smooth muscle, cholinergic nerve terminals arise from ganglionic neurons located near the tracheal smooth muscle, whereas the source of NANC nerve fibers is from neurons in ganglia located in the adjacent myenteric plexus of the esophagus, making this an ideal species to study regulation of parasympathetic neurotransmitter phenotypes. In the present study, we determined that, 48 hours after repeated allergen challenge, the NANC phenotype of airway parasympathetic ganglionic neurons changed to a cholinergic phenotype, and NT-3 mimicked this change. Nerve growth factor, brain-derived neurotrophic factor, leukemia inhibitory factor, or IL-1β had no effect on either phenotype, and they did not induce these neurons to synthesize substance P or tyrosine hydroxylase. These results indicate a role for inflammation and NT-3 in regulating biochemical and anatomical characteristics of principal neurons in adult airway parasympathetic ganglia.

Figure 1.

Allergen challenge of passively sensitized guinea pigs elicits airway inflammation and remodeling. Compared with control tracheas (A), eosinophils (arrows) were increased in the trachea 24 (B, arrows) and 48 hours (data not shown) after a single allergen challenge. (C) Summary of the effects of single allergen challenges (groups A and B) on eosinophil density. Eosinophils were also increased 24 hours (data not shown) and 48 hours (D, arrows) after a second allergen challenge. Also common with chronic allergen challenge, there was disruption of the epithelial layer lining the lower airways 48 hours after a second allergen challenge (arrowheads in D). Scale bar in D is for images in A, B, and D. Asterisks in C signify differences in the number of eosinophils in treated tissue as compared with control.

Figure 2.

Neurons in esophageal parasympathetic ganglia change to a cholinergic phenotype 48 hours after repeated allergen challenge. (A) All control neurons in tracheal ganglia are immunoreactive for choline acetyltransferase (ChAT), but, in the same section, as shown in Ai, not for markers associated with nonadrenergic, noncholinergic (NANC) innervation, such as vasoactive intestinal peptide (VIP) or for nitric oxide synthase (NOS; data not shown). (B) All control neurons in esophageal ganglia that project axons to the trachealis muscle express VIP (arrow) and NOS (data not shown), but not ChAT, as shown in the same section (Bi). (C) Esophageal neurons from animals exposed to allergen on 2 days and analyzed 48 hours later (group D) were no longer VIP immunoreactive and, in Ci, the same neurons were immunoreactive for ChAT. (D) A tracheal ganglion from an animal exposed to allergen on 2 days and analyzed 48 hours later (group D) remained cholinergic (three others showed similar ChAT immunoreactivity. Neither tracheal nor esophageal ganglia neurons became substance P (SP)–, tyrosine hydroxylase (TH)–, or neuropeptide Y (NPY)–immunoreactive for any group (data not shown). Scale bar on right image (A-C) is for corresponding image on the left.

Figure 3.

Summary of the effects of different allergen challenges on neurotransmitter immunoreactivity in neurons located in esophageal ganglia. (A) VIP and ChAT-ir in group C esophageal neurons were unaffected 24 hours after a repeated allergen challenge. (B) VIP and ChAT-ir in group D esophageal neurons were decreased and increased, respectively, 48 hours after a repeated allergen challenge.

Figure 4.

Neurotrophin (NT)-3 mimics the effect of allergen challenge. Individual NTs nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT-3, or neurokines, leukemia inhibitory factor (LIF) or IL-1β, were injected into the trachealis muscle and, after 48 hours, the neurotransmitter phenotype was determined immunohistologically in tracheal and esophageal parasympathetic ganglia. (A) Neurons that normally express VIP immunoreactivity (ir) in esophageal ganglionic neurons. (B) NT-3 (100 ng/ml), 48 hours after exposure, esophageal neurons were ChAT immunoreactive. (C) NT-3 (100 ng/ml), 48 hours after exposure, had no effect on ChAT-ir in tracheal ganglionic neurons. These data for esophageal neurons are summarized in D. NGF, BDNF, LIF, or IL-1β (all 100 ng/ml, 48 hours; n = 4 animals for each exposure) had no effect on the cholinergic or NANC neurotransmitter phenotype 48 hours after exposure, nor did they induce immunoreactivity for SP, TH, or NPY (data not shown). Scale bar in C is for images in A–C.

Figure 5.

Immunofluorescent staining for NT-3 in a frozen section of guinea pig trachea 2 days after a single allergen challenge. The staining appeared to be in small, inflammatory-like cells (arrows, green) located in and below the epithelium; the size and location of these cells was very similar to eosinophil-like cells observed after single allergen challenge (Figure 1). Fewer cells were immunoreactive in animals 1 day after a single allergen exposure (data not shown). ChAT-ir (red) near airway smooth muscle shows proximity of mucosal inflammatory cells near ChAT-ir nerves.