Mast cell-cholinergic nerve interaction in mouse airways

Abstract

We addressed the mechanism by which antigen contracts trachea isolated from actively sensitized mice. Trachea were isolated from mice (C57BL/6J) that had been actively sensitized to ovalbumin (OVA). OVA (10 microg ml(-1)) caused histamine release (approximately total tissue content), and smooth muscle contraction that was rapid in onset and short-lived (t(1/2) < 1 min), reaching approximately 25% of the maximum tissue response. OVA contraction was mimicked by 5-HT, and responses to both OVA and 5-HT were sensitive to 10 microm-ketanserin (5-HT(2) receptor antagonist) and strongly inhibited by atropine (1microm). Epithelial denudation had no effect on the OVA-induced contraction. Histological assessment revealed about five mast cells/tracheal section the vast majority of which contained 5-HT. There were virtually no mast cells in the mast cell-deficient (sash -/-) mouse trachea. OVA failed to elicit histamine release or contractile responses in trachea isolated from sensitized mast cell-deficient (sash -/-) mice. Intracellular recordings of the membrane potential of parasympathetic neurons in mouse tracheal ganglia revealed a ketanserin-sensitive 5-HT-induced depolarization and similar depolarization in response to OVA challenge. These data support the hypothesis that antigen-induced contraction of mouse trachea is epithelium-independent, and requires mast cell-derived 5-HT to activate 5-HT(2) receptors on parasympathetic cholinergic neurons. This leads to acetylcholine release from nerve terminals, and airway smooth muscle contraction.

Figure 1. Ovalbumin-induced contractions of trachea isolated from actively sensitized mice in the absence (control) and presence of atropine (1 μm, n= 4) or ketanserin (10 μm, n= 5)

The bar graph depicts mean ±s.e.m. contraction as a percentage of the maximal obtainable contraction. An asterisk (*) denotes P < 0.01. Inset, representative trace of airway smooth muscle contraction in response to 10 μg ml⁻¹ ovalbumin (Con) in the presence and absence of atropine (Atro) or ketanserin (Ket). Arrows indicate the addition of OVA to the tissue bath.

Figure 2. Cumulative concentration–response curves for 5-HT in the absence (filled squares) and presence (open squares) of atropine (100 nm)

Data are means ±s.e.m., n= 5.

Figure 3. Immunohistochemical localization of mast cells in a tracheal section taken from the airway of a C57BL/6J mouse at 20× magnification (L = lumen, SM = airway smooth muscle; white bar = 50 μm)

Shown is a section stained independently with antibodies against mast cell tryptase (red) and 5-HT (green), and a photomicrograph of the two images overlayed (yellow), demonstrating the localization of 5-HT within mast cells. All 5-HT-positive mast cells identified in 33 tracheal sections from 4 mice were mast cell tryptase-positive. Mast cells were undetectable in 22 sections stained for 5-HT and mast cell tryptase from the trachea of 3 sash−/− mice (data not shown). The arrowheads indicate one of two labelled mast cells localized to the basement membrane (left), and one of several associated with the airway smooth muscle (right). In all histological sections examined, the majority of mast cells were concentrated within the smooth muscle layer, facilitating their interaction with tracheal ganglia and nerve terminals. Mast cells were virtually absent from the cartilaginous ventral portion of the airway.

Figure 4. The maximum contractile response to ovalbumin (OVA) and 5-HT in trachea isolated trachea from OVA-sensitized wild type and mast cell-deficient sash−/− mice

The trachea from wild type mice (n= 18) contracted to OVA, whereas trachea isolated from sash−/− mice (n= 3) did not. The trachea obtained from wild type (n= 11) and sash−/− mice (n= 3) contracted in response to 5-HT (100 μm). The bar graphs represent means ±s.e.m., *P < 0.01.

Figure 5. OVA (10 μg ml⁻¹)-induced contractions of trachea isolated from actively sensitized mice

The contractile responses were compared in trachea in which the epithelium was intact (control) vs. trachea in which the epithelium was mechanically removed (denuded). In each case histological assessment was obtained to showing the status of the epithelium. In denuded tissues approximately 80–100% of the basement membrane was free of any epithelial cells (not shown). The bar graphs represent the means ±s.e.m. of the peak contraction as a percentage of the maximum obtainable contraction induced by methacholine (Mch), n= 7–8. Inset, representative trace of OVA-induced contractions in control and epithelium denuded trachea showing that the time course of the contraction is not influenced by the absence of epithelium. Arrows indicate the addition of OVA to the tissue bath.

Figure 6. 5-HT and OVA cause membrane depolarization in airway parasympathetic neurons

A, photomicrograph of a parasympathetic ganglion within the trachea of a C57BL/6J mouse. An asterisk (*) indicates one neuron in the centre of one ganglion composed of approximately 40–60 neurons. This ganglion was located on the serosal aspect of the smooth muscle layer. P1 and P2 respectively are pre- and post-ganglionic nerve fibres; C indicates a capillary; and SM indicates smooth muscle fibres beneath the ganglion. Inset, example of membrane potential depolarization obtained with an intracellular recording of a neuron within a parasympathetic ganglion after exposure to the nicotinic agonist DMPP (30 μm for 10 s); the depolarization reached action potential threshold as evidenced by 2 spikes (the action potentials are not readily resolved at this time scale but can be seen as a thin vertical line. B, mean membrane depolarization of airway neurons after exposure to OVA (10 μg ml⁻¹ for 1 min) and 5-HT (10 μm for 1 min); mean resting membrane potential =−49 ± 4.5 mV in 4 neurons and −53 ± 2.4 mV in 9 neurons respectively. 5-HT-induced depolarization is sensitive to 1 μm-ketanserin (Ket). Data are means ±s.e.m., *P < 0.01, n= 3.