Airway Neurons Research Articles

Conformational rearrangements associated with Panx1 channel gating.

Pannexins (Panx1-3) are ATP permeable membrane channels that are widely expressed in the human body. It has been demonstrated that pannexins play central roles in ATP release from a wide range of cell types including airway epithelia, blood cells, glial cells, and neurons. Pannexin mediated ATP release contributes to blood pressure regulation, neurotransmission, and apoptotic cell clearance. Our group and others determined structures of Panx1 using cryo-EM, which uncovered that Panx1 assembles into a unique heptameric channel with a potential pathway for the ATP molecule. However, how Panx1 controls ATP migration through the pore remains unclear. In fact, it is still uncertain whether the currently available Panx1 structures represent open or closed conformations. We recently discovered a cell metabolite as a bona fide Panx1 agonist. We performed cryo-EM single particle reconstructions in the presence or absence of this agonist to shed light on the Panx1 channel gating mechanism. We found a major conformational rearrangement in a region surrounding the ATP permeation pathway. Cysteine accessibility studies using whole cell patch clamp electrophysiology support that the conformational rearrangement in this region is important for Panx1 channel gating. This is in contrast to the proposed-gating mechanism in apoptotic cells, in which caspase-dependent C-terminal cleavage unplugs the pore for permeant molecules. Based on the current study, we provide a novel mechanism underlying Panx1 channel gating in non-apoptotic cells.

Healthy Human Laryngopharyngeal Sensory Innervation Density Correlates with Age.

Somatosensory feedback from upper airway structures is essential for swallowing and airway defense but little is known about the identities and distributions of human upper airway neurons. Furthermore, whether sensory innervation modifies with aging is unknown. In this study, we quantify neuronal and chemosensory cell density in upper airway structures and correlate with age. Participants underwent biopsies from base of tongue, lateral and midline pharyngeal wall, epiglottis, and arytenoids (N=25 13 female/12 male; 20-80 years, mean 51.4 years without clinical diagnosis of dysphagia or clinical indication for biopsy). Tissue sections were labeled with antibodies for all neurons, myelinated neurons, and chemosensory cells. Densities of lamina propria innervation, epithelial innervation, solitary chemosensory cells, and taste buds were calculated and correlated with age. Arytenoid had the highest density of innervation and chemosensory cells across all measures compared to other sites. Taste buds were frequently observed in arytenoid and epiglottis. Base of tongue, lateral pharynx, and midline posterior pharynx had minimal innervation and few chemosensory cells. Epithelial innervation was present primarily in close proximity to chemosensory cells and taste buds. Overall innervation and myelinated fibers in the arytenoid lamina propria decline with aging. Findings establish the architecture of healthy adult sensory innervation and demonstrate the varied distribution of laryngopharyngeal innervation, necessary steps toward understanding the sensory basis for swallowing and airway defense. We also document age-related decline in arytenoid innervation density. These findings suggest that sensory afferent denervation of the upper airway may be a contributing factor to presbyphagia. NA Laryngoscope, 133:773-784, 2023.

Multicolor labeling of airway neurons and analysis of parasympathetic heterogeneity

We report subpopulations of airway parasympathetic neurons expressing substance P, neuronal nitric oxide synthase, and tyrosine hydroxylase, highlighting unexplored heterogeneity in this population. These neurotransmitter-specific subpopulations did not form intraganglionic interneurons, but rather, extended outside the ganglia, into the airways, to distant innervation targets. Our experiments demonstrate the utility of multicolor labeling to characterize airway innervation, allowing us to confirm the extensive heterogeneity of postganglionic parasympathetic neurons. These methods will facilitate future investigations of neurophysiology and neural contributions to airway disease.

Nicotinic α7 acetylcholine receptor (α7nAChR) in human airway smooth muscle

Diseases such as asthma are exacerbated by inflammation, cigarette smoke and even nicotine delivery devices such as e-cigarettes. However, there is currently little information on how nicotine affects airways, particularly in humans, and changes in the context of inflammation or asthma. Here, a longstanding assumption is that airway smooth muscle (ASM) that is key to bronchoconstriction has muscarinic receptors while nicotinic receptors (nAChRs) are only on airway neurons. In this study, we tested the hypothesis that human ASM expresses α7nAChR and explored its profile in inflammation and asthma using ASM of non-asthmatics vs. mild-moderate asthmatics. mRNA and western analysis showed the α7 subunit is most expressed in ASM cells and further increased in asthmatics and smokers, or by exposure to nicotine, cigarette smoke or pro-inflammatory cytokines TNFα and IL-13. In these effects, signaling pathways relevant to asthma such as NFκB, AP-1 and CREB are involved. These novel data demonstrate the expression of α7nAChR in human ASM and suggest their potential role in asthma pathophysiology in the context of nicotine exposure.

The Emerging Role of Ion Channels in the Pathophysiology and Treatments of Asthma

Asthma is one of the most common chronic respiratory diseases in the world. Although treatments and medications that can control the symptoms of asthma are available, no cure has been developed. Ion channels are transmembrane proteins that facilitate the diffusion of ions across membranes. In the airways, ion channels are involved in signalling between cells and regulation of intracellular ion concentration, which may lead to the activation of airway cells, immune cells, and neurons. This review mainly focuses on discoveries made so far in animal models and clinical trials regarding ion channels and asthma and gives some insight into developing treatments targeting ion channels.

Consideration of Pannexin 1 channels in COVID-19 pathology and treatment.

Pannexin1 (PANX1) is a ubiquitously expressed, channel-forming protein found in a number of tissues thoughout the body (e.g. lung, vasculature, liver, central nervous system, immune system) that is important in many key physiological and immune responses. PANX1 channels passively flux ATP (predominantly), multiple metabolites, and likely other small anions. PANX1 channels regulate inflammation and host responses to several pathogens, including viruses. While there is currently no evidence suggesting novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and PANX1 directly interact, there is an urgent need for therapeutic strategies, especially those targeting the hyper-inflammation and cytokine storm that occurs in severe cases of COVID-19. Here we argue that PANX1, and drugs known to target PANX1 (including the FDA-approved drug probenecid), should be the focus of further investigation in the context of SARS-CoV-2 infection and its associated pathology in COVID-19 patients.

Optogenetic Control of Airway Cholinergic Neurons In Vivo.

Dysregulation of airway nerves leads to airway hyperreactivity, a hallmark of asthma. Although changes to nerve density and phenotype have been described in asthma, the relevance of these changes to nerve function has not been investigated due to anatomical limitations where afferent and efferent nerves run in the same nerve trunk, making it difficult to assess their independent contributions. We developed a unique and accessible system to activate specific airway nerves to investigate their function in mouse models of airway disease. We describe a method to specifically activate cholinergic neurons using light, resulting in immediate, measurable increases in airway inflation pressure and decreases in heart rate. Expression of light-activated channelrhodopsin 2 in these neurons is governed by Cre expression under the endogenous choline acetyltransferase promoter, and we describe a method to decrease variability in channelrhodopsin expression in future experiments. Optogenetic activation of specific subsets of airway neurons will be useful for studying the functional relevance of other observed changes, such as changes to nerve morphology and protein expression, across many airway diseases, and may be used to study the function of subpopulations of autonomic neurons in lungs and other organs.

Palatal Sensory Function Worsens in Untreated Snorers but not in CPAP-Treated Patients With Sleep Apnea, Indicating Vibration-Induced Nervous Lesions

Signs of both motor and sensory nervous lesions have previously been shown in the upper airway of patients with OSA and habitual snorers. Snoring per se may damage all upper airway neurons over time, thereby causing progression to manifest sleep apnea. To test this hypothesis, nonsnoring subjects, untreated snorers, and CPAP-treated patients underwent repeated sensory testing of the soft palate in a prospective long-term study. Cold detection threshold (CDT) testing at the soft palate and lip with a thermode and nocturnal respiratory recordings were performed in 2008 to 2009 with retesting 6 to 7 years later. In 25 untreated snorers, palatal CDT worsened from a median (25th-75th percentile range) 4.2°C (3.2-5.9) to 11.0°C (7.0-17.4) (P< .001). The apnea-hypopnea index increased from a median 7.0 to 14.0 events/h (P< .05). There was no significant correlation between changes in CDT and the apnea-hypopnea index. In 21 nonsnoring control subjects, palatal CDT increased from a median 3.2°C to 5.6°C (P< .005). In 19 CPAP-treated patients, palatal CDT did not significantly change; eight patients had improved values. CDTs worsened significantly more in the snorers group than in the control subjects (P< .05) and the CPAP-treated patients (P< .001). There was no significant difference between control subjects and CPAP-treated patients. CDT worsened considerably over time in untreated snorers, significantly more than in nonsnoring control subjects and CPAP-treated patients. Untreated snorers therefore risk developing poor sensitivity in the upper airway. In contrast, efficient treatment of OSA seems to protect the sensory innervation, as the CPAP-treated group maintained their sensitivity to cold and, in some cases, the sensitivity even improved.

A HEATED SITUATION: THERMAL INJURY INDUCING AN IDIOPATHIC PULMONARY FIBROSIS EXACERBATION

A HEATED SITUATION: THERMAL INJURY INDUCING AN IDIOPATHIC PULMONARY FIBROSIS EXACERBATION

Nebulized hypertonic saline triggers nervous system-mediated active liquid secretion in cystic fibrosis swine trachea

Inhaled hypertonic saline (HTS) treatment is used to improve lung health in patients with cystic fibrosis (CF). The current consensus is that the treatment generates an osmotic gradient that draws water into the airways and increases airway surface liquid (ASL) volume. However, there is evidence that HTS may also stimulate active secretion of ASL by airway epithelia through the activation of sensory neurons. We tested the contribution of the nervous system and airway epithelia on HTS-stimulated ASL height increase in CF and wild-type swine airway. We used synchrotron-based imaging to investigate whether airway neurons and epithelia are involved in HTS treatment-triggered ASL secretion in CFTR−/− and wild-type swine. We showed that blocking parasympathetic and sensory neurons in airway resulted in ~50% reduction of the effect of HTS treatment on ASL volume in vivo. Incubating tracheal preparations with inhibitors of epithelial ion transport across airway decreased secretory responses to HTS treatment. CFTR−/− swine ex-vivo tracheal preparations showed substantially decreased secretory response to HTS treatment after blockage of neuronal activity. Our results indicated that HTS-triggered ASL secretion is partially mediated by the stimulation of airway neurons and the subsequent activation of active epithelia secretion; osmosis accounts for only ~50% of the effect.

Substance P represents a novel first-line defense mechanism in the nose

Neuropeptides, such as substance P (SP), have long been seen as mediators of widespread continuous airway inflammation, a process known as neurogenic inflammation. However, this has been difficult to demonstrate clinically, suggesting an alternative role for these signaling molecules. We sought to examine the role of SP in nasal infection by assessing the release of SP in response to viral stimulation and characterizing the effects of SP on innate immunity, with the latter reflected in changes in local Toll-like receptor (TLR) expression. The distribution of SP and TLRs in the nasal mucosa and local airway neurons was assessed with immunohistochemistry. The TLR7 agonists R-837 and R-848 were used to mimic a viral insult in the upper airways represented by primary human nasal epithelial cells (HNECs) and murine nasal epithelial cells (MNECs) and isolated murine trigeminal ganglial neurons. SP release from HNECs, MNECs, and trigeminal ganglial neurons was quantified with EIA. The effects of SP on TLR expression on HNECs were determined by using flow cytometry and confocal microscopy. SP was released from the sensory neurons, MNECs, and HNECs within 15minutes of local TLR7 stimulation. Subsequently, stimulation with SP induced upregulation of TLR expression in HNECs within 30minutes through induction of TLR movement within HNECs. Upregulation of TLR expression was not evident when cells were treated with the neurokinin 1 receptor antagonist aprepitant before SP stimulation. This highlights a novel role for sensory neuropeptides as acute and local mediators of pathogen-driven inflammation, rapidly priming innate immune defenses in the airway.

Ozone Exposure Initiates a Sequential Signaling Cascade in Airways Involving Interleukin-1Beta Release, Nerve Growth Factor Secretion, and Substance P Upregulation

Previous studies demonstrated that interleukin-1β (IL-1β) and nerve growth factor (NGF) increase synthesis of substance P (SP) in airway neurons both after ozone (O3) exposure and by direct application. It was postulated that NGF mediates O3-induced IL-1β effects on SP. The current study specifically focused on the influence of O3 on IL-1β, NGF, and SP levels in mice bronchoalveolar lavage fluid (BALF) and whether these mediators may be linked in an inflammatory-neuronal cascade in vivo. The findings showed that in vivo O3 exposure induced an increase of all three proteins in mouse BALF and that O3-induced elevations in both NGF and SP are mediated by the inflammatory cytokine IL-1β. Further, inhibition of NGF reduced O3 induced increases of SP in both the lung BALF and lung tissue, demonstrating NGF serves as a mediator of IL-1β effects on SP. These data indicate that IL-1β is an early mediator of O3-induced rise in NGF and subsequent SP release in mice in vivo.

P4 Establishing A Role For Trpv1 On Sensory Nerves In Copd Associated Chronic Cough

Background An increase in cough reflex sensitivity to capsaicin in COPD has been described in some studies, suggesting a role for TRPV1 in the disease phenotype. We utilised a guinea-pig cigarette-smoke (CS) exposure COPD model to investigate an enhanced cough phenotype, and evaluate the role of TRPV1 using a novel clinical-ready inhibitor, XEN-D0501. 1 Furthermore, we confirmed enhanced cough responses in COPD patients using a dose-response capsaicin challenge to determine E Max . Methods Guinea-pigs were exposed to air/cigarette smoke (CS) for 1 h, twice daily, for 8 days. Coughs evoked by aerosolised capsaicin (30 μM), depolarisation of isolated vagus nerve tissue induced by capsaicin (1 μM), or increases in intracellular calcium [Ca 2+ ] i to capsaicin (1 μM) in airway-terminating (i.n. DiI stained) jugular and nodose neurons were evaluated. Vagus nerve was obtained from human non-smoker/smoker subjects for similar assessment. Coughs evoked by capsaicin (4 inhalations, 0.49–1000 µM) were recorded in COPD and compared with healthy controls. Results Capsaicin-evoked cough was increased in COPD patients (Fig.1A) and in CS-exposed guinea-pigs (Fig.1B) compared to controls. Capsaicin induced greater depolarisation in nerve tissue from CS-exposed guinea-pigs, and in human vagus nerves from smokers, compared to controls. Capsaicin also induced greater [Ca 2+ ] i increases in airway-terminating jugular and nodose (which are normally capsaicin-unresponsive) neurons from CS-exposed guinea-pigs. XEN-D0501 (i.p. 1 h before cough recording) almost completely inhibited the cough response to capsaicin in both air- and CS-exposed guinea-pigs (Fig.1B). Conclusions CS-exposure evoked increased cough to capsaicin in guinea-pigs, mimicking the enhanced cough phenotype observed in COPD patients. This was paralleled by enhanced capsaicin responses in isolated vagus nerves and airway neurons from CS-exposed guinea-pigs and in human vagus from smokers suggesting the enhanced cough phenotype is due to increased TRPV1-mediated sensory nerve responsiveness. Inhibition of the CS-enhanced cough response by XEN-D0501 further implicated a role for TRPV1. This data, together with the finding that TRPV1 KO mice display less inflammation in a similar pre-clinical model of CS-exposure 2 , indicates the potential utility of TRPV1 antagonists in the treatment of COPD, which is currently being evaluated in an ongoing COPD clinical trial. References Round. Br J Clin Pharmacol. 2011;72:921–931 Baxter ATS. 2014;A2079

CADM1 Controls Actin Cytoskeleton Assembly and Regulates Extracellular Matrix Adhesion in Human Mast Cells

CADM1 is a major receptor for the adhesion of mast cells (MCs) to fibroblasts, human airway smooth muscle cells (HASMCs) and neurons. It also regulates E-cadherin and alpha6beta4 integrin in other cell types. Here we investigated a role for CADM1 in MC adhesion to both cells and extracellular matrix (ECM). Downregulation of CADM1 in the human MC line HMC-1 resulted not only in reduced adhesion to HASMCs, but also reduced adhesion to their ECM. Time-course studies in the presence of EDTA to inhibit integrins demonstrated that CADM1 provided fast initial adhesion to HASMCs and assisted with slower adhesion to ECM. CADM1 downregulation, but not antibody-dependent CADM1 inhibition, reduced MC adhesion to ECM, suggesting indirect regulation of ECM adhesion. To investigate potential mechanisms, phosphotyrosine signalling and polymerisation of actin filaments, essential for integrin-mediated adhesion, were examined. Modulation of CADM1 expression positively correlated with surface KIT levels and polymerisation of cortical F-actin in HMC-1 cells. It also influenced phosphotyrosine signalling and KIT tyrosine autophosphorylation. CADM1 accounted for 46% of surface KIT levels and 31% of F-actin in HMC-1 cells. CADM1 downregulation resulted in elongation of cortical actin filaments in both HMC-1 cells and human lung MCs and increased cell rigidity of HMC-1 cells. Collectively these data suggest that CADM1 is a key adhesion receptor, which regulates MC net adhesion, both directly through CADM1-dependent adhesion, and indirectly through the regulation of other adhesion receptors. The latter is likely to occur via docking of KIT and polymerisation of cortical F-actin. Here we propose a stepwise model of adhesion with CADM1 as a driving force for net MC adhesion.

Diacetyl Increases Sensory Innervation and Substance P Production in Rat Trachea

Inhalation of diacetyl, a butter flavoring, causes airway responses potentially mediated by sensory nerves. This study examines diacetyl-induced changes in sensory nerves of tracheal epithelium. Rats (n = 6/group) inhaled 0-, 25-, 249-, or 346-ppm diacetyl for 6 hr. Tracheas and vagal ganglia were removed 1-day postexposure and labeled for substance P (SP) or protein gene product 9.5 (PGP9.5). Vagal ganglia neurons projecting to airway epithelium were identified by axonal transport of fluorescent microspheres intratracheally instilled 14 days before diacetyl inhalation. End points were SP and PGP9.5 nerve fiber density (NFD) in tracheal epithelium and SP-positive neurons projecting to the trachea. PGP9.5-immunoreactive NFD decreased in foci with denuded epithelium, suggesting loss of airway sensory innervation. However, in the intact epithelium adjacent to denuded foci, SP-immunoreactive NFD increased from 0.01 ± 0.002 in controls to 0.05 ± 0.01 after exposure to 346-ppm diacetyl. In vagal ganglia, SP-positive airway neurons increased from 3.3 ± 3.0% in controls to 25.5 ± 6.6% after inhaling 346-ppm diacetyl. Thus, diacetyl inhalation increases SP levels in sensory nerves of airway epithelium. Because SP release in airways promotes inflammation and activation of sensory nerves mediates reflexes, neural changes may contribute to flavorings-related lung disease pathogenesis.

66. Evidence for neuroplastic changes in the brainstem following TH2 lung inflammation: A role for the central nervous system in contributing to the symptoms of asthma and investigating a potential target for therapy

Inflammatory events in the lung, such as atopic asthma, induce changes in neuronal activity of afferent airway vagal fibres and neurons in the brainstem, as well as changes in cough, anxiety and depression. Here, we investigated the effect of acute and chronic Th2 lung inflammation on behaviour an expression of neuroplasticity and inflammatory genes in the brainstem. Allergic lung inflammation was induced using the well-established Ovalbumin (OVA) model of asthma. Open field activity and the elevated plus maze were used to asses behavioural changes; brainstem tissue including the nucleus of the solitary tract was collected for analysis of gene expression; bronchial lavage fluid (BALF) and serum was collected for cytokine measurement. We found increased levels of serum and BALF IL-5 and IL-4 and increased numbers of eosinophils, confirming robust Th2 mediated inflammation. Despite this, we did not find significant changes in open field or anxiety behaviour nor in central inflammatory gene expression. In contrast, significant increases were found in the expression of c-fos, as well as a subset of glutamate and neurotrophin receptors in the brainstem, changes most pronounced following chronic lung inflammation. Our results show that robust, lung inflammation does not result in behavioural changes, despite evidence of immune-to-brain communication. We are currently investigating if immunomodulation of lung inflammation with TLR agonists induces changes in behaviour and central neuronal and inflammatory gene expression.

Early postnatal ozone exposure alters rat nodose and jugular sensory neuron development

Sensory neurons originating in nodose and jugular ganglia that innervate airway epithelium (airway neurons) play a role in inflammation observed following exposure to inhaled environmental irritants such as ozone (O3). Airway neurons can mediate airway inflammation through the release of the neuropeptide substance P (SP). While susceptibility to airway irritants is increased in early life, the developmental dynamics of afferent airway neurons are not well characterized. The hypothesis of this study was that airway neuron number might increase with increasing age, and that an acute, early postnatal O3 exposure might increase both the number of sensory airway neurons as well as the number SP-containing airway neurons. Studies using Fischer 344 rat pups were conducted to determine if age or acute O3 exposure might alter airway neuron number. Airway neurons in nodose and jugular ganglia were retrogradely labeled, removed, dissociated, and counted by means of a novel technique employing flow cytometry. In Study 1, neuron counts were conducted on postnatal days (PD) 6, 10, 15, 21, and 28. Numbers of total and airway neurons increased significantly between PD6 and PD10, then generally stabilized. In Study 2, animals were exposed to O3 (2 ppm) or filtered air (FA) on PD5 and neurons were counted on PD10, 15, 21, and 28. O3-exposed animals displayed significantly less total neurons on PD21 than FA controls. This study shows that age-related changes in neuron number occur, and that an acute, early postnatal O3 exposure significantly alters sensory neuron development.

Role of Nerve Growth Factor in Ozone-Induced Neural Responses in Early Postnatal Airway Development

Airway neural plasticity contributes to the process of airway remodeling in response to airway irritants. However, the mechanisms of neural remodeling in the airways during the early postnatal period, when responses to airway irritation may be most sensitive, have not been characterized. This study used a rat model to examine a possible mechanism of ozone (O(3))-induced neural hyperresponsiveness during a critical period of developmental, postnatal day (PD) 6, that may be mediated by the neurotrophin nerve growth factor (NGF), resulting in an enhanced release of inflammatory neuropeptide substance P (SP) from airway nerves. Rat pups between PD6-PD28 were killed 24 hours after exposure to O(3) (2 ppm, 3 hours) or filtered air (FA), to establish a timeline of NGF synthesis, or else they were exposed to O(3) or NGF on PD6 or PD21 and re-exposed to O(3) on PD28, and killed on PD29. Measurement endpoints included NGF mRNA in tracheal epithelial cells, NGF protein in bronchoalveolar lavage fluid, airway SP-nerve fiber density (NFD), and SP-positive airway neurons in vagal ganglia. Acute exposure to O(3) increased NGF in bronchoalveolar lavage fluid on PD10 and PD15, and mRNA expression in epithelial cells on PD6, compared with FA controls. NGF protein and mRNA expression in the O(3)-PD6/O(3)-PD28 groups were significantly higher than in the O(3)-PD21/O(3)-PD28 and O(3)-PD6/FA-PD28 groups. NGF-PD6/O(3)-PD28 increased the SP innervation of airway smooth muscle and SP-positive sensory neurons, compared with the NGF-PD21/O(3)-PD28 or NGF-PD6/FA-PD28 groups. NGF enhanced sensory innervation, which may mediate acute responses or prolong sensitivity to O(3) during early life. The model may be relevant in O(3) responses during early childhood.

Secondhand smoke exposure alters K+ channel function and intrinsic cell excitability in a subset of second‐order airway neurons in the nucleus tractus solitarius of young guinea pigs

Extended exposure to secondhand smoke (SHS) in infants and young children increases the incidence of cough, wheeze, airway hyper-reactivity and the prevalence and earlier onset of asthma. The adverse effects may result from environmentally-induced plasticity in the neural network regulating cough and airway function. Using whole-cell patch-clamp recordings in brainstem slices containing anatomically identified second-order lung afferent neurons in the nucleus tractus solitarius (NTS), we determined the effects of extended SHS exposure in young guinea pigs for a duration equivalent to human childhood on the intrinsic excitability of NTS neurons. SHS exposure resulted in marked decreases in the intrinsic excitability of a subset of lung afferent second-order NTS neurons. The neurons exhibited a decreased spiking capacity, prolonged action potential duration, reduced afterhyperpolarization, decrease in peak and steady-state outward currents, and membrane depolarization. SHS exposure effects were mimicked by low concentrations of the K+ channel blockers 4-aminopyridine and/or tetraethyl ammonium. The data suggest that SHS exposure downregulates K+ channel function in a subset of NTS neurons, resulting in reduced cell excitability. The changes may help to explain the exaggerated neural reflex responses in children exposed to SHS.

Distribution of major basic protein on human airway following in vitro eosinophil incubation.

Major basic protein (MBP) released from activated eosinophils may influence airway hyperresponsiveness (AHR) by either direct effects on airway myocytes or by an indirect effect. In this study, human bronchi, freshly isolated human eosinophils, or MBP purified from human eosinophil granules were incubated for studying eosinophil infiltration and MBP localization. Eosinophils immediately adhered to intact human airway as well as to cultured human airway myocytes and epithelium. Following incubation 18–24 h, eosinophils migrated into the airway media, including the smooth muscle layer, but had no specific recruitment to airway neurons. Eosinophils released significant amounts of MBP within the airway media, including areas comprising the smooth muscle layer. Most deposits of MBP were focally discrete and restricted by immunologic detection to a maximum volume of ∼300 μm3 about the eosinophil. Native MBP applied exogenously was immediately deposited on the surface of the airway, but required at least 1 h to become detected within the media of the airway wall. Tissue MBP infiltration and deposition increased in a time- and concentration-dependent manner. Taken together, these findings suggest that eosinophil-derived cationic proteins may alter airway hyperresponsiveness (AHR) in vivo by an effect that is not limited to the bronchial epithelium.