Three days after a single exposure to ozone, the mechanism of airway hyperreactivity is dependent on substance P and nerve growth factor

Ozone causes persistent airway hyperreactivity in humans and animals. One day after ozone exposure, airway hyperreactivity is mediated by release of eosinophil major basic protein that inhibits neuronal M(2) muscarinic receptors, resulting in increased acetylcholine release and increased smooth muscle contraction in guinea pigs. Three days after ozone, IL-1β, not eosinophils, mediates ozone-induced airway hyperreactivity, but the mechanism at this time point is largely unknown. IL-1β increases NGF and the tachykinin substance P, both of which are involved in neural plasticity. These experiments were designed to test whether there is a role for NGF and tachykinins in sustained airway hyperreactivity following a single ozone exposure. Guinea pigs were exposed to filtered air or ozone (2 parts per million, 4 h). In anesthetized and vagotomized animals, ozone potentiated vagally mediated airway hyperreactivity 24 h later, an effect that was sustained over 3 days. Pretreatment with antibody to NGF completely prevented ozone-induced airway hyperreactivity 3 days, but not 1 day, after ozone and significantly reduced the number of substance P-positive airway nerve bundles. Three days after ozone, NK(1) and NK(2) receptor antagonists also blocked this sustained hyperreactivity. Although the effect of inhibiting NK(2) receptors was independent of ozone, the NK(1) receptor antagonist selectively blocked vagal hyperreactivity 3 days after ozone. These data confirm mechanisms of ozone-induced airway hyperreactivity change over time and demonstrate 3 days after ozone that there is an NGF-mediated role for substance P, or another NK(1) receptor agonist, that enhances acetylcholine release and was not present 1 day after ozone.     

Atropine pretreatment enhances airway hyperreactivity in antigen-challenged guinea pigs through an eosinophil-dependent mechanism

Airway hyperreactivity in antigen-challenged animals is mediated by eosinophil major basic protein (MBP) that blocks inhibitory M(2) muscarinic receptors on parasympathetic nerves, increasing acetylcholine release onto M(3) muscarinic receptors on airway smooth muscle. Acutely, anticholinergics block hyperreactivity in antigen-challenged animals and reverse asthma exacerbations in the human, but are less effective in chronic asthma. We tested whether atropine, given before antigen challenge, affected hyperreactivity, M(2) receptor function, eosinophil accumulation, and activation. Sensitized guinea pigs received atropine (1 mg/kg ip) 1 h before challenge and 6 h later. Twenty-four hours after challenge, animals were anesthetized, vagotomized, paralyzed, and ventilated. Airway reactivity to electrical stimulation of the vagi and to intravenous acetylcholine was not altered by atropine pretreatment in nonsensitized animals, indicating that atropine was no longer blocking postjunctional muscarinic receptors. Antigen challenge induced airway hyperreactivity to vagal stimulation that was significantly potentiated by atropine pretreatment. Bronchoconstriction induced by acetylcholine was not changed by antigen challenge or by atropine pretreatment. M(2) receptor function was lost in challenged animals but protected by atropine pretreatment. Eosinophils in bronchoalveolar lavage and within airway tissues were significantly increased by challenge but significantly reduced by atropine pretreatment. However, extracellular MBP in challenged airways was significantly increased by atropine pretreatment, which may account for reduced eosinophils. Depleting eosinophils with antibody to IL-5 before challenge prevented hyperreactivity and significantly reduced MBP in airways of atropine-pretreated animals. Thus atropine pretreatment potentiated airway hyperreactivity by increasing eosinophil activation and degranulation. These data suggest that anticholinergics enhance eosinophil interactions with airway nerves.     

Ozone-induced hyperresponsiveness and blockade of M2 muscarinic receptors by eosinophil major basic protein.

Control of airway smooth muscle is provided by parasympathetic nerves that release acetylcholine onto M(3) muscarinic receptors. Acetylcholine release is limited by inhibitory M(2) muscarinic receptors. In antigen-challenged guinea pigs, hyperresponsiveness is due to blockade of neuronal M(2) receptors by eosinophil major basic protein (MBP). Because exposure of guinea pigs to ozone also causes M(2) dysfunction and airway hyperresponsiveness, the role of eosinophils in ozone-induced hyperresponsiveness was tested. Animals were exposed to filtered air or to 2 parts/million ozone for 4 h. Twenty-four hours later, the muscarinic agonist pilocarpine no longer inhibited vagally induced bronchoconstriction in ozone-exposed animals, indicating M(2) dysfunction. M(2) receptor function in ozone-exposed animals was protected by depletion of eosinophils with antibody to interleukin-5 and by pretreatment with antibody to guinea pig MBP. M(2) function was acutely restored by removal of MBP with heparin. Ozone-induced hyperreactivity was also prevented by antibody to MBP and was reversed by heparin. These data show that loss of neuronal M(2) receptor function after ozone is due to release of eosinophil MBP.     

Substance P-induced airway hyperreactivity is mediated by neuronal M(2) receptor dysfunction

Neuronal muscarinic (M(2)) receptors inhibit release of acetylcholine from the vagus nerves. Hyperreactivity in antigen-challenged guinea pigs is due to blockade of these M(2) autoreceptors by eosinophil major basic protein (MBP) increasing the release of acetylcholine. In vivo, substance P-induced hyperactivity is vagally mediated. Because substance P induces eosinophil degranulation, we tested whether substance P-induced hyperreactivity is mediated by release of MBP and neuronal M(2) receptor dysfunction. Pathogen-free guinea pigs were anesthetized and ventilated. Thirty minutes after intravenous administration of [Sar(9),Met(O(2))(11)]- substance P, guinea pigs were hyperreactive to vagal stimulation and M(2) receptors were dysfunctional. The depletion of inflammatory cells with cyclophosphamide or the administration of an MBP antibody or a neurokinin-1 (NK(1)) receptor antagonist (SR-140333) all prevented substance P-induced M(2) dysfunction and hyperreactivity. Intravenous heparin acutely reversed M(2) receptor dysfunction and hyperreactivity. Thus substance P releases MBP from eosinophils resident in the lungs by stimulating NK(1) receptors. Substance P-induced hyperreactivity is mediated by blockade of inhibitory neuronal M(2) receptors by MBP, resulting in increased release of acetylcholine.     

Eosinophils and airway nerves in asthma

In the lungs, neuronal M2 muscarinic receptors limit the release of acetylcholine from postganglionic cholinergic nerves. However, these receptors are not functional under certain circumstances in animal models of hyperreactivity such as occurs after exposure of sensitised animals to an allergen or during a respiratory tract virus infection. This loss of M2 receptor function leads to an increase in acetylcholine release from cholinergic nerves and thus is a mechanism for the vagally mediated hyperreactivity seen in these animals. Studies in animal models of hyperreactivity have shown that eosinophils localise to the airway nerves of sensitised animals after antigen challenge. Inhibiting this localisation of eosinophils either with an antibody to the eosinophil survival cytokine IL-5 or the eosinophil adhesion molecule VLA-4 prevents loss of M2 muscarinic receptor function. It is likely that eosinophil MBP is responsible for the loss of M2 receptor function, since inhibiting eosinophil MBP with an antibody or neutralising MBP with heparin prevents this loss of function. These data are also supported by ligand binding studies where it has been shown that eosinophil MBP is an allosteric antagonist at neuronal M2 muscarinic receptors. Loss of function of lung neuronal M2 muscarinic receptors may also occur under certain circumstances in patients with asthma, although the mechanisms are not yet established.     

Effects of inflammatory cells on neuronal M2 muscarinic receptor function in the lung

In the lungs, acetylcholine released from the parasympathetic nerves stimulates M3 muscarinic receptors on airway smooth muscle inducing contraction and bronchoconstriction. The amount of acetylcholine released from these nerves is limited locally by neuronal M2 muscarinic receptors. These neuronal receptors are dysfunctional in asthma and in animal models of asthma. Decreased M2 muscarinic receptor function results in increased release of acetylcholine and in airway hyperreactivity. Inflammation has long been associated with hyperreactivity and the role of inflammatory cells in loss of neuronal M2 receptor function has been examined. There are several different mechanisms for loss of neuronal M2 receptor function. These include blockade by endogenous antagonists such as eosinophil major basic protein, decreased expression of M2 receptors following infection with viruses or exposure to pro inflammatory cytokines such as gamma interferon. Finally, the affinity of acetylcholine for these receptors can be decreased by exposure to neuraminidase.     

Double-stranded RNA causes airway hyperreactivity and neuronal M2 muscarinic receptor dysfunction.

Viral infection causes dysfunction of inhibitory M2 muscarinic receptors (M2Rs) on parasympathetic nerves, leading to airway hyperreactivity. The mechanisms of M2R dysfunction are incompletely understood. Double-stranded RNA (dsRNA), a product of viral replication, promotes the expression of interferons. Interferon-gamma decreases M2R gene expression in cultured airway parasympathetic neurons. In this study, guinea pigs were treated with dsRNA (1 mg/kg ip) on 2 consecutive days. Twenty-four hours later, anesthetized guinea pigs had dysfunctional M2Rs and were hyperresponsive to electrical stimulation of the vagus nerves, in the absence of inflammation. DsRNA did not affect either cholinesterase or the function of postjunctional M3 muscarinic receptors on smooth muscle. M2Rs on the nerves supplying the heart were also dysfunctional, but M2Rs on the heart muscle itself functioned normally. Thus dsRNA causes increased bronchoconstriction and bradycardia via increased release of ACh from the vagus nerves because of loss of M2R function on parasympathetic nerves in the lungs and heart. Production of dsRNA may be a mechanism by which viruses cause dysfunction of neuronal M2Rs and airway hyperreactivity.     

Eosinophil adhesion to cholinergic nerves via ICAM-1 and VCAM-1 and associated eosinophil degranulation

In vivo, eosinophils localize to airway cholinergic nerves in antigen-challenged animals, and inhibition of this localization prevents antigen-induced hyperreactivity. In this study, the mechanism of eosinophil localization to nerves was investigated by examining adhesion molecule expression by cholinergic nerves. Immunohistochemical and functional studies demonstrated that primary cultures of parasympathetic nerves express vascular cell adhesion molecule-1 (VCAM-1) and after cytokine pretreatment with tumor necrosis factor-alpha and interferon-gamma intercellular adhesion molecule-1 (ICAM-1). Eosinophils adhere to these parasympathetic neurones after cytokine pretreatment via a CD11/18-dependent pathway. Immunohistochemistry and Western blotting showed that a human cholinergic nerve cell line (IMR-32) expressed VCAM-1 and ICAM-1. Inhibitory experiments using monoclonal blocking antibodies to ICAM-1, VCAM-1, or CD11/18 and with the very late antigen-4 peptide inhibitor ZD-7349 showed that eosinophils adhered to IMR-32 cells via these adhesion molecules. The protein kinase C signaling pathway is involved in this process as a specific inhibitor-attenuated adhesion. Eosinophil adhesion to IMR-32 cells was associated with the release of eosinophil peroxidase and leukotriene C(4). Thus eosinophils adhere to cholinergic nerves via specific adhesion molecules, and this leads to eosinophil activation and degranulation; this may be part of the mechanism of eosinophil-induced vagal hyperreactivity.     

Mechanisms of organophosphate insecticide-induced airway hyperreactivity

It has been suggested that pesticide exposure may be a contributing factor underlying the increased incidence of asthma in the United States and other industrialized nations. To test this hypothesis, airway hyperreactivity was measured in guinea pigs exposed to chlorpyrifos, a widely used organophosphate pesticide. Electrical stimulation of the vagus nerves caused frequency-dependent bronchoconstriction that was significantly potentiated in animals 24 h or 7 days after a single subcutaneous injection of either 390 mg/kg or 70 mg/kg of chlorpyrifos, respectively. Mechanisms by which chlorpyrifos may cause airway hyperreactivity include inhibition of acetylcholinesterase (AChE) or dysfunction of M3 muscarinic receptors on airway smooth muscle or of autoinhibitory M2 muscarinic receptors on parasympathetic nerves in the lung. AChE activity in the lung was significantly inhibited 24 h after treatment with 390 mg/kg of chlorpyrifos, but not 7 days after injection of 70 mg/kg of chlorpyrifos. Acute exposure to eserine (250 microg/ml) also significantly inhibited lung AChE but did not potentiate vagally induced bronchoconstriction. Neuronal M2 receptor function was tested using the M2 agonist pilocarpine, which inhibits vagally induced bronchoconstriction in control animals. In chlorpyrifos-treated animals, pilocarpine dose-response curves were shifted significantly to the right, demonstrating decreased responsiveness of neuronal M2 receptors. In contrast, chlorpyrifos treatment did not alter methacholine-induced bronchoconstriction, suggesting that chlorpyrifos does not alter M3 muscarinic receptor function on airway smooth muscle. These data demonstrate that organophosphate insecticides can cause airway hyperreactivity in the absence of AChE inhibition by decreasing neuronal M2 receptor function.     

Differential expression of TRAIL and TRAIL receptors in allergic asthmatics following segmental antigen challenge: evidence for a role of TRAIL in eosinophil survival

Asthma is a chronic lung disease exhibiting airway obstruction, hyperresponsiveness, and inflammation, characterized by the infiltration of eosinophils into the airways and the underlying tissue. Prolonged eosinophilic inflammation depends on the balance between the cell's inherent tendency to undergo apoptosis and the local eosinophil-viability enhancing activity. TRAIL, a member of the TNF family, induces apoptosis in most transformed cells; however, its role in health and disease remains unknown. To test the hypothesis that Ag-induced inflammation is associated with TRAIL/TRAIL-R interactions, we used a segmental Ag challenge (SAC) model in ragweed-allergic asthmatics and nonasthmatic patients and analyzed bronchoalveolar lavage (BAL) material for 2 wk. In asthmatic patients, the level of TRAIL in BAL fluid dramatically increased 24 h after SAC, which significantly correlated with BAL eosinophil counts. Immunohistochemical analysis of bronchial biopsies from asthmatic patients demonstrated that TRAIL staining was increased in epithelial, airway smooth muscle, and vascular smooth muscle cells and throughout the interstitial tissue after SAC. This was confirmed by quantitative immunocytochemical image analysis of BAL eosinophils and alveolar macrophages, which demonstrated that expression levels of TRAIL and DcR2 increased, whereas expression levels of the TRAIL-Rs DR4 and DR5 decreased in asthmatic subjects after SAC. We also determined that TRAIL prolongs eosinophil survival ex vivo. These data provide the first in vivo evidence that TRAIL expression is increased in asthmatics following Ag provocation and suggest that modulation of TRAIL and TRAIL-R interactions may play a crucial role in promoting eosinophil survival in asthma.     

Up-regulation and activation of eosinophil integrins in blood and airway after segmental lung antigen challenge

We hypothesized that there are clinically relevant differences in eosinophil integrin expression and activation in patients with asthma. To evaluate this, surface densities and activation states of integrins on eosinophils in blood and bronchoalveolar lavage (BAL) of 19 asthmatic subjects were studied before and 48 h after segmental Ag challenge. At 48 h, there was increased expression of alpha(D) and the N29 epitope of activated beta(1) integrins on blood eosinophils and of alpha(M), beta(2), and the mAb24 epitope of activated beta(2) integrins on airway eosinophils. Changes correlated with the late-phase fall in forced expiratory volume in 1 s (FEV(1)) after whole-lung inhalation of the Ag that was subsequently used in segmental challenge and were greater in subjects defined as dual responders. Increased surface densities of alpha(M) and beta(2) and activation of beta(2) on airway eosinophils correlated with the concentration of IL-5 in BAL fluid. Activation of beta(1) and beta(2) on airway eosinophils correlated with eosinophil percentage in BAL. Thus, eosinophils respond to an allergic stimulus by activation of integrins in a sequence that likely promotes eosinophilic inflammation of the airway. Before challenge, beta(1) and beta(2) integrins of circulating eosinophils are in low-activation conformations and alpha(D)beta(2) surface expression is low. After Ag challenge, circulating eosinophils adopt a phenotype with activated beta(1) integrins and up-regulated alpha(D)beta(2), changes that are predicted to facilitate eosinophil arrest on VCAM-1 in bronchial vessels. Finally, eosinophils present in IL-5-rich airway fluid have a hyperadhesive phenotype associated with increased surface expression of alpha(M)beta(2) and activation of beta(2) integrins.     

Decreased expression of membrane IL-5 receptor alpha on human eosinophils: I. Loss of membrane IL-5 receptor alpha on airway eosinophils and increased soluble IL-5 receptor alpha in the airway after allergen challenge

IL-5 is a key cytokine for eosinophil maturation, recruitment, activation, and possibly the development of inflammation in asthma. High concentrations of IL-5 are present in the airway after Ag challenge, but the responsiveness of airway eosinophils to IL-5 is not well characterized. The objectives of this study were to establish, following airway Ag challenge: 1) the expression of membrane (m)IL-5Ralpha on bronchoalveolar lavage (BAL) eosinophils; 2) the responsiveness of these cells to exogenous IL-5; and 3) the presence of soluble (s)IL-5Ralpha in BAL fluid. To accomplish these goals, blood and BAL eosinophils were obtained from atopic subjects 48 h after segmental bronchoprovocation with Ag. There was a striking reduction in mIL-5Ralpha on airway eosinophils compared with circulating cells. Furthermore, sIL-5Ralpha concentrations were elevated in BAL fluid, but steady state levels of sIL-5Ralpha mRNA were not increased in BAL compared with blood eosinophils. Finally, BAL eosinophils were refractory to IL-5 for ex vivo degranulation, suggesting that the reduction in mIL-5Ralpha on BAL eosinophils may regulate IL-5-mediated eosinophil functions. Together, the loss of mIL-5Ralpha, the presence of sIL-5Ralpha, and the blunted functional response (degranulation) of eosinophils to IL-5 suggest that when eosinophils are recruited to the airway, regulation of their functions becomes IL-5 independent. These observations provide a potential explanation for the inability of anti-IL-5 therapy to suppress airway hyperresponsiveness to inhaled Ag, despite a reduction in eosinophil recruitment.     

Role of insulin in antigen-induced airway eosinophilia and neuronal M2 muscarinic receptor dysfunction

In the lungs, neuronalM₂ muscarinic receptors limit AChrelease from parasympathetic nerves. In antigen-challenged animals, eosinophil proteins block these receptors, resulting in increased AChrelease and vagally mediated hyperresponsiveness. In contrast, diabeticrats are hyporesponsive and have increasedM₂ receptor function. Becausethere is a low incidence of asthma among diabetic patients, weinvestigated whether diabetes protects neuronalM₂ receptor function inantigen-challenged rats. Antigen challenge of sensitized rats decreasedM₂ receptor function, increasedvagally mediated hyperreactivity by 75%, and caused a 10-fold increase in eosinophil accumulation around airway nerves. In antigen-challenged diabetic rats, neuronal M₂receptor function was preserved and there was no eosinophilaccumulation around airway nerves. Insulin treatment of diabetic ratscompletely restored loss of M₂receptor function, vagally mediated hyperresponsiveness, andeosinophilia after antigen challenge. These data demonstrate thatinsulin is required for development of airway inflammation, loss ofneuronal M₂ muscarinic receptorfunction, and subsequent hyperresponsiveness in antigen-challenged ratsand may explain decreased incidence of asthma among diabetic humans.

     

Eosinophil Granule Proteins: Form and Function

Experimental and clinical data strongly support a role for the eosinophil in the pathogenesis of asthma, allergic and parasitic diseases, and hypereosinophilic syndromes, in addition to more recently identified immunomodulatory roles in shaping innate host defense, adaptive immunity, tissue repair/remodeling, and maintenance of normal tissue homeostasis. A seminal finding was the dependence of allergic airway inflammation on eosinophil-induced recruitment of Th2-polarized effector T-cells to the lung, providing a missing link between these innate immune effectors (eosinophils) and adaptive T-cell responses. Eosinophils come equipped with preformed enzymatic and nonenzymatic cationic proteins, stored in and selectively secreted from their large secondary (specific) granules. These proteins contribute to the functions of the eosinophil in airway inflammation, tissue damage, and remodeling in the asthmatic diathesis. Studies using eosinophil-deficient mouse models, including eosinophil-derived granule protein double knock-out mice (major basic protein-1/eosinophil peroxidase dual gene deletion) show that eosinophils are required for all major hallmarks of asthma pathophysiology: airway epithelial damage and hyperreactivity, and airway remodeling including smooth muscle hyperplasia and subepithelial fibrosis. Here we review key molecular aspects of these eosinophil-derived granule proteins in terms of structure-function relationships to advance understanding of their roles in eosinophil cell biology, molecular biology, and immunobiology in health and disease.     

Cigarette smoke-induced bronchoconstriction in dogs: vagal and extravagal mechanisms

We reassessed the severity of cigarette smoke-induced bronchoconstriction and the mechanisms involved in anesthetized dogs. To evaluate the severity of smoke-induced bronchoconstriction, we measured airway pressure and airflow resistance (Rrs, forced oscillation method). We studied the mechanisms in other dogs by measuring airway pressure, central airway smooth muscle tone in tracheal segments in situ, and respiratory center drive by monitoring phrenic motor nerve output, including the role of vagal and extravagal nerves vs. the role of blood-borne materials during inhalation of cigarette smoke. Rrs increased more than fourfold with smoke from one cigarette delivered in two tidal volumes. About half the airway response was due to local effects of smoke in the lungs. The remainder was due to stimulation of the respiratory center, which activated vagal motor efferents to the airway smooth muscle. Of this central stimulation, about half was due to blood-borne materials and the rest to vagal pulmonary afferents from the lungs. We conclude that inhalation of cigarette smoke in dogs causes severe bronchoconstriction which is mediated mainly by extravagal mechanisms.     

Eotaxin represents the principal eosinophil chemoattractant in a novel murine asthma model induced by house dust containing cockroach allergens

Asthma represents a serious health problem particularly for inner city children, and recent studies have identified that cockroach allergens trigger many of these asthmatic attacks. This study tested the concept that asthma-like pulmonary inflammation may be induced by house dust containing cockroach allergens. An aqueous extract was prepared from a house dust sample containing endotoxin and high levels of cockroach allergens. BALB/c mice were immunized with the house dust extract (HDE) and received two additional pulmonary challenges. Bronchoalveolar lavage (BAL) eosinophil counts and eotaxin levels were significantly increased in immunized mice exposed to the HDE, whereas neutrophils were the predominant BAL inflammatory cell in the unimmunized mice. Kinetics studies in immunized mice demonstrated a peak pulmonary inflammatory response 48 h after the last challenge. The allergic response in this model was further confirmed by histological and physiological studies demonstrating a significant influx of eosinophils and lymphocytes in the peribronchial area, and severe airway hyperreactivity through whole-body plethysmography. The specificity of the response was established by immunizing with HDE and challenging with purified cockroach allergen, which induced pulmonary eosinophilia and airway hyperreactivity. Ab inhibition of eotaxin significantly inhibited the number of BAL eosinophils. These data describe a novel murine model of asthma-like pulmonary inflammation induced by house dust containing endotoxin and cockroach allergens and further demonstrate that eotaxin represents the principal chemoattractant for the recruitment of the pulmonary eosinophils.     

Interaction between histamine and vagal stimulation on tracheal smooth muscle in dogs

We examined the interaction between histamine and vagal efferent activity on airway smooth muscle reactivity in 11 anesthetized vagotomized dogs using an isolated closed segment of the intrathoracic trachea filled with Tyrode solution under an isovolumetric condition. Intratracheal pressure change was measured as an index of tracheal smooth muscle tone. The administration into the tracheal segment of histamine (0.1 or 1.0 mg/ml) in six dogs and methacholine chloride (0.001 or 0.01 mg/ml) in the other five dogs elevated intratracheal pressure by about 5 cmH2O. The electrical stimulation of the peripheral ends of both of the cut cervical vagus nerves in the presence of histamine produced significantly greater responses than the additive responses of these two stimuli applied individually (two-way analysis of variance, P less than 0.025). However, the combined effects of vagal stimulation and methacholine were not significantly different from the additive responses of these two stimuli applied individually. The average values of intratracheal pressure elevated by the combined effects of vagal stimulation and histamine were significantly higher than those obtained by the combination of vagal stimulation and methacholine (two-way analysis of variance, P less than 0.01). This suggests that histamine potentiates tracheal smooth muscle reactivity to electrical vagal stimulation, which may contribute to the hyperreactivity observed in patients with asthma.     

Time course study for antigen-induced airway hyperreactivity and the effect of soluble IL-5 receptor

To study the role of IL-5 in allergic airway hyperreactivity, the time course for the production of cytokines, the infiltration of inflammatory cells and the onset of airway hyperreactivity after three inhalations of antigens were studied in mice. The effect of the soluble α-chain of murine recombinant interleukin-5 receptor (sIL-5Rα) on these phenomena was also examined. Whereas IL-5 and IL-4 were produced in significant amounts, IL-1, IL-2 and γ-interferon (γ-IFN) were not detected even after three antigen inhalations. Monocytes and eosinophils but not neutrophils increased significantly after the third antigen exposure. The airway responsiveness to acetylcholine increased after the third aeroantigen-challenge. sIL-5Rα, administered after each antigen-challenge, suppressed BAL eosinophilia with little effect on airway hyperreactivity.     

Ozone-induced changes in muscarinic bronchial reactivity by different testing methods

We examined the effect of ozone (O3) on muscarinic bronchial reactivity in the guinea pig and compared reactivity determined by two different routes of agonist delivery. Reactivity before and from 4 h to 2 days after O3 exposure (3.0 ppm, 2 h) was determined by measuring specific airway resistance upon administration of intravenous acetylcholine and/or aerosolized methacholine challenge in 34 unanesthetized, spontaneously breathing animals. Before exposure, we observed more gradual and reproducible results to intravenous agonist. After exposure, hyperreactivity to parenteral agonist occurred consistently, but not to inhaled agonist. Hyperreactivity demonstrable by either route was similar in magnitude and time course within 14 h of exposure. Two days later, hyperreactivity to inhaled agonist had remitted; that to intravenous drug persisted. Our results indicate that variability in the occurrence and time course of O3-induced hyperreactivity to inhaled agonist may be a consequence of the technique employed. The consistent occurrence of hyperreactivity after O3 to parenteral agonist suggests mechanisms other than airway mucosal hyperpermeability are responsible for this hyperreactivity.     

Responsiveness, inflammation, and effects of deep breaths on obstruction in mild asthma

Using cellular and biochemical characteristics of bronchoalveolar lavage (BAL) liquid as an index of inflammation, we examined the relationships between change of airway caliber after a deep inhalation (DI), degree of base-line airway hyperresponsiveness, and peripheral airway inflammation in a group of 16 atopic asymptomatic mild asthmatics and 6 normal subjects. Compared with normal subjects, asthmatics demonstrated 1) significantly higher BAL concentrations of histamine, total protein, the sulfidopeptide leukotrienes (SRS-A), and leukotiene B4; 2) a decrease in specific airway conductance (sGaw) with a DI at base line vs. an increase in normal subjects (before vs. after percent change in sGaw, -10 vs. 12, P less than 0.05); and 3) no significant difference in BAL total cell count or leukocyte differential. Significant correlations were demonstrated between 1) percent of BAL eosinophils vs. degree of airway hyperresponsiveness; 2) base-line level of airway obstruction vs. degree of hyperresponsiveness; 3) effects of a DI vs. BAL concentrations of eosinophils, total protein, and histamine; 4) base-line forced expired volume in 1 s vs. BAL concentrations of total protein and histamine; and 5) BAL concentrations of the various mediators with each other. These data support the notion that 1) the response to a DI in mild, stable asthmatics represents a physiological indicator of peripheral obstruction because of inflammation and 2) this inflammation is associated with increases in several known mediators of airway inflammation and hyperreactivity.