A.D. Fryer

Disruption of Nrf2 enhances susceptibility to severe airway inflammation and asthma in mice

Oxidative stress has been postulated to play an important role in the pathogenesis of asthma; although a defect in antioxidant responses has been speculated to exacerbate asthma severity, this has been difficult to demonstrate with certainty. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a redox-sensitive basic leucine zipper transcription factor that is involved in the transcriptional regulation of many antioxidant genes. We show that disruption of the Nrf2 gene leads to severe allergen-driven airway inflammation and hyperresponsiveness in mice. Enhanced asthmatic response as a result of ovalbumin sensitization and challenge in Nrf2-disrupted mice was associated with more pronounced mucus cell hyperplasia and infiltration of eosinophils into the lungs than seen in wild-type littermates. Nrf2 disruption resulted in an increased expression of the T helper type 2 cytokines interleukin (IL)-4 and IL-13 in bronchoalveolar lavage fluid and in splenocytes after allergen challenge. The enhanced severity of the asthmatic response from disruption of the Nrf2 pathway was a result of a lowered antioxidant status of the lungs caused by lower basal expression, as well as marked attenuation, of the transcriptional induction of multiple antioxidant genes. Our studies suggest that the responsiveness of Nrf2-directed antioxidant pathways may act as a major determinant of susceptibility to allergen-mediated asthma.

Muscarinic inhibitory receptors in pulmonary parasympathetic nerves in the guinea-pig.

1 In anaesthetized guinea‐pigs, gallamine produced a dose‐related potentiation of the bronchoconstriction induced by electrical stimulation of the cervical vagus nerves; (+)‐tubocurarine and suxamethonium lacked this effect. 2 The bronchoconstriction produced by intravenous injection of acetylcholine or histamine, however, was not potentiated by gallamine. 3 Vagally‐induced bradycardia was abolished by gallamine, confirming antagonism of the effect of acetylcholine on muscarinic receptors in the heart. 4 The muscarinic receptor agonist pilocarpine, had the opposite effect to gallamine in the lung as it inhibited vagally‐mediated bronchoconstriction. 5 Pretreatment of guinea‐pigs with either guanethidine or propranolol did not affect the gallamine‐induced potentiation of vagally‐mediated bronchoconstriction. 6 The potentiating effect of gallamine in the lung can be explained by blockade of inhibitory, muscarinic receptors located in the parasympathetic nerves supplying the lungs.

Human eosinophil major basic protein is an endogenous allosteric antagonist at the inhibitory muscarinic M2 receptor.

The effect of human eosinophil major basic protein (MBP) as well as other eosinophil proteins, on binding of [3H]N-methyl-scopolamine ([3H]NMS: 1 x 10(-10) M) to muscarinic M2 receptors in heart membranes and M3 receptors in submandibular gland membranes was studied. MBP inhibited specific binding of [3H]NMS to M2 receptors but not to M3 receptors. MBP also inhibited atropine-induced dissociation of [3H]NMS-receptor complexes in a dose-dependent fashion, demonstrating that the interaction of MBP with the M2 muscarinic receptor is allosteric. This effect of MBP suggests that it may function as an endogenous allosteric inhibitor of agonist binding to the M2 muscarinic receptor. Inhibition of [3H]NMS binding by MBP was reversible by treatment with heparin, which binds and neutralizes MBP. Eosinophil peroxidase (EPO) also inhibited specific binding of [3H]NMS to M2 receptors but not to M3 receptors and inhibited atropine-induced dissociation of [3H]NMS-receptor complexes. On a molar basis, EPO is less potent than MBP. Neither eosinophil cationic protein nor eosinophil-derived neurotoxin affected binding of [3H]NMS to M2 receptors. Thus both MBP and EPO are selective allosteric antagonists at M2 receptors. The effects of these proteins may be important causes of M2 receptor dysfunction and enhanced vagally mediated bronchoconstriction in asthma.

Pretreatment with antibody to eosinophil major basic protein prevents hyperresponsiveness by protecting neuronal M2 muscarinic receptors in antigen-challenged guinea pigs.

In antigen-challenged guinea pigs there is recruitment of eosinophils into the lungs and to airway nerves, decreased function of inhibitory M2 muscarinic autoreceptors on parasympathetic nerves in the lungs, and airway hyperresponsiveness. A rabbit antibody to guinea pig eosinophil major basic protein was used to determine whether M2 muscarinic receptor dysfunction, and the subsequent hyperresponsiveness, are due to antagonism of the M2 receptor by eosinophil major basic protein. Guinea pigs were sensitized, challenged with ovalbumin and hyperresponsiveness, and M2 receptor function tested 24 h later with the muscarinic agonist pilocarpine. Antigen-challenged guinea pigs were hyperresponsive to electrical stimulation of the vagus nerves compared with controls. Likewise, loss of M2 receptor function was demonstrated since the agonist pilocarpine inhibited vagally-induced bronchoconstriction in control but not challenged animals. Pretreatment with rabbit antibody to guinea pig eosinophil major basic protein prevented hyperresponsiveness, and protected M2 receptor function in the antigen-challenged animals without inhibiting eosinophil accumulation in the lungs or around the nerves. Thus, hyperresponsiveness is a result of inhibition of neuronal M2 muscarinic receptor function by eosinophil major basic protein in antigen-challenged guinea pigs.

Parainfluenza virus infection damages inhibitory M2 muscarinic receptors on pulmonary parasympathetic nerves in the guinea-pig.

1 The effect of viral infection on the function of neuronal M2 muscarinic autoreceptors in the lungs was studied in anaesthetized guinea‐pigs. 2 Guinea‐pigs were inoculated intranasally with either parainfluenza type 3 or with a vehicle control. Four days later the animals were anaesthetized, paralysed and artificially ventilated. Pulmonary inflation pressure, tidal volume, blood pressure, and heart rate were recorded. Both vagus nerves were cut and electrical stimulation of the distal portions caused bronchoconstriction (measured as an increase in pulmonary inflation pressure) and bradycardia. 3 In control animals, pilocarpine (1–100 μg kg−1, i.v.) attenuated vagally‐induced bronchoconstriction by stimulating inhibitory M2 muscarinic receptors on parasympathetic nerves in the lungs. Conversely, blockade of these receptors with the antagonist gallamine (0.1–10 mg kg−1, i.v.) produced a marked potentiation of vagally‐induced bronchoconstriction. These results confirm previous findings. 4 In guinea‐pigs infected with parainfluenza virus, pilocarpine did not inhibit vagally‐induced bronchoconstriction. Furthermore, gallamine did not potentiate vagally‐induced bronchoconstriction to the same degree as in uninfected controls. 5 There was no increase in baseline pulmonary inflation pressure in the infected animals over the controls. Receptors on airway smooth muscle were unchanged by viral infection since large doses of pilocarpine caused equivalent bronchoconstriction in both groups of animals. Gallamine inhibited the vagally‐induced fall in heart rate equally in both groups of animals indicating that virus‐induced changes in M2 receptor function on pulmonary parasympathetic nerves are not part of a generalized decrease in M2 receptor function. 6 These results demonstrate that the M2 muscarinic receptor‐mediated inhibition of acetylcholine release from parasympathetic nerves in the lungs is decreased in animals infected with parainfluenza virus. Loss of this inhibition would result in increased release of acetylcholine from the parasympathetic nerves and may explain virus‐induced airway hyperresponsiveness.

Muscarinic acetylcholine receptors and airway diseases

Parasympathetic nerves provide the dominant autonomic innervation of the airways. Release of acetylcholine from parasympathetic nerves activates postjunctional muscarinic receptors present on airway smooth muscle, submucosal glands, and blood vessels to cause bronchoconstriction, mucus secretion, and vasodilatation, respectively. Acetylcholine also feeds back onto prejunctional muscarinic receptors to enhance or inhibit further acetylcholine release. In asthma and chronic obstructive pulmonary disease, bronchoconstriction and mucus secretion is increased and the airways are hyperresponsive to contractile agents. These changes are due to increased parasympathetic nerve activity. The number and function of postjunctional muscarinic receptors in the airways are unchanged in animal models of asthma. Rather, it is the supply of acetylcholine to the postjunctional cells (smooth muscle and submucosal gland) that is increased. The increase in acetylcholine release occurs because prejunctional, inhibitory M(2) muscarinic receptors on the parasympathetic nerves are dysfunctional. M(2) muscarinic receptor dysfunction and subsequent airway hyperreactivity have been demonstrated to occur in animals in response to a variety of triggers, including antigen challenge, virus infection, ozone exposure, and vitamin A deficiency. In humans, there is evidence that loss of M(2) muscarinic receptor function is related to asthma. The mechanisms by which neuronal M(2) muscarinic receptor function is lost and its relevance to human airway disease are discussed in this review.

Neuronal muscarinic receptors attenuate vagally‐induced contraction of feline bronchial smooth muscle

1 In anaesthetized cats, stimulation of the vagus nerves produced bradycardia and a bronchoconstriction which was measured as an increase in lung resistance (RL) and a fall in dynamic lung compliance (Cdyn); these effects were abolished by atropine. 2 Gallamine potentiated vagally‐mediated changes in RL and Cdyn at doses that blocked muscarinic receptors in the heart and inhibited neuromuscular transmission. (+)‐Tubocurarine and suxamethonium did not affect the response of the lung or the heart to vagal stimulation. 3 Bronchoconstriction induced by intravenous acetylcholine was not potentiated by gallamine, indicating that postsynaptic muscarinic receptors in the lung and changes in muscle tone were not involved. 4 Potentiation of vagally‐induced bronchoconstriction appears to be due to blockade of inhibitory muscarinic receptors located in the pulmonary parasympathetic nerves innervating both central and peripheral airways. 5 Pilocarpine was an agonist for these neuronal receptors as it inhibited vagally‐induced bronchoconstriction at low doses (10 ng to 1 μg kg−1). 6 The results demonstrate that gallamine is an antagonist and pilocarpine an agonist at neuronal muscarinic receptors which attenuate parasympathetic nerve activity in feline lung.

Function of pulmonary M2 muscarinic receptors in antigen-challenged guinea pigs is restored by heparin and poly-L-glutamate

The effect of heparin and poly-L-glutamate on the function of inhibitory M2 muscarinic autoreceptors on parasympathetic nerves in the lung was tested in antigen-challenged guinea pigs. After antigen challenge, M2 receptor function is decreased, thus increasing release of acetylcholine from the vagus and potentiating vagally induced bronchoconstriction. Guinea pigs were anesthetized, tracheostomized, vagotomized, paralyzed, and ventilated. Electrical stimulation of the vagi caused bronchoconstriction and bradycardia. In controls, pilocarpine attenuated vagally induced bronchoconstriction by stimulating neuronal M2 muscarinic receptors. Conversely, blocking these autoreceptors with gallamine potentiated vagally induced bronchoconstriction. In challenged animals the effects of both drugs were markedly reduced, confirming M2 receptor dysfunction. 20 min after heparin or poly-L-glutamate, the effects of both pilocarpine and gallamine on vagally induced bronchoconstriction were restored, demonstrating recovery of M2 receptor function. Neither heparin nor poly-L-glutamate affected vagally induced responses in control animals. Thus antigen-induced dysfunction of M2 receptors can be reversed by polyanionic polysaccharides (heparin) or polyanionic peptides (poly-L-glutamate). This suggests that a polycationic substance such as eosinophil major basic protein, cationic protein, or peroxidase may be responsible for antigen-induced pulmonary M2 receptor dysfunction.

Neuronal eotaxin and the effects of ccr3 antagonist on airway hyperreactivity and M2 receptor dysfunction

Eosinophils cluster around airway nerves in patients with fatal asthma and in antigen-challenged animals. Activated eosinophils release major basic protein, which blocks inhibitory M2 muscarinic receptors (M2Rs) on nerves, increasing acetylcholine release and potentiating vagally mediated bronchoconstriction. We tested whether GW701897B, an antagonist of CCR3 (the receptor for eotaxin as well as a group of eosinophil active chemokines), affected vagal reactivity and M2R function in ovalbumin-challenged guinea pigs. Sensitized animals were treated with the CCR3 antagonist before inhaling ovalbumin. Antigen-challenged animals were hyperresponsive to vagal stimulation, but those that received the CCR3 antagonist were not. M2R function was lost in antigen-challenged animals, but not in those that received the CCR3 antagonist. Although the CCR3 antagonist did not decrease the number of eosinophils in lung tissues as assessed histologically, CCR3 antagonist prevented antigen-induced clustering of eosinophils along the nerves. Immunostaining revealed eotaxin in airway nerves and in cultured airway parasympathetic neurons from both guinea pigs and humans. Both IL-4 and IL-13 increased expression of eotaxin in cultured airway parasympathetic neurons as well as in human neuroblastoma cells. Thus, signaling via CCR3 mediates eosinophil recruitment to airway nerves and may be a prerequisite to blockade of inhibitory M2Rs by eosinophil major basic protein.

Antibody to VLA-4, but not to L-selectin, protects neuronal M2 muscarinic receptors in antigen-challenged guinea pig airways.

Antigen challenge of sensitized guinea pigs decreases the function of inhibitory M2 muscarinic autoreceptors on parasympathetic nerves in the lung, potentiating vagally induced bronchoconstriction. Loss of M2 receptor function is associated with the accumulation of eosinophils around airway nerves. To determine whether recruitment of eosinophils via expression of VLA-4 and L-selectin is critical for loss of M2 receptor function, guinea pigs were pretreated with monoclonal antibodies to VLA-4 (HP1/2) or L-selectin (LAM1-116). Guinea pigs were sensitized and challenged with ovalbumin, and M2 receptor function was tested. In controls, blockade of neuronal M2 muscarinic receptors by gallamine potentiated vagally induced bronchoconstriction, while in challenged animals this effect was markedly reduced, confirming M2 receptor dysfunction. Pretreatment with HP1/2, but not with LAM1-116, protected M2 receptor function in the antigen-challenged animals. HP1/2 also inhibited the development of hyperresponsiveness, and selectively inhibited accumulation of eosinophils in the lungs as measured by lavage and histology. Thus, inhibition of eosinophil influx into the lungs protects the function of M2 muscarinic receptors, and in so doing, prevents hyperresponsiveness in antigen-challenged guinea pigs.