Non-bronchodilating mechanisms of tiotropium prevent airway hyperreactivity in a guinea-pig model of allergic asthma.

BACKGROUND AND PURPOSE: Asthma is characterized by reversible bronchoconstriction and airway hyperreactivity. Although M(3) muscarinic receptors mediate bronchoconstriction, non-selective muscarinic receptor antagonists are not currently recommended for chronic control of asthma. We tested whether selective blockade of M(3) receptors, at the time of antigen challenge, blocks subsequent development of airway hyperreactivity in antigen-challenged guinea-pigs. EXPERIMENTAL APPROACH: Ovalbumin-sensitized guinea-pigs were pretreated with 1 µg·kg(-1) of a kinetically selective M(3) receptor antagonist, tiotropium, or 1 mg·kg(-1) of a non-selective muscarinic receptor antagonist, atropine, and challenged with inhaled ovalbumin. Animals were anaesthetized, paralyzed, ventilated and vagotomized 24 h later. We measured vagally mediated bronchoconstriction and i.v. ACh-induced bronchoconstriction. KEY RESULTS: Electrical stimulation of both vagus nerves induced frequency-dependent bronchoconstriction in sensitized animals that was significantly increased after antigen challenge. Antigen-induced hyperreactivity was completely blocked by tiotropium pretreatment but only partially blocked by atropine pretreatment. Surprisingly, although tiotropium blocked bronchoconstriction induced by i.v. ACh, it did not inhibit vagally-induced bronchoconstriction in sensitized controls, suggesting that tiotropium does not block hyperreactivity by blocking receptors for vagally released ACh. Rather, tiotropium may have worked through an anti-inflammatory mechanism, since it inhibited eosinophil accumulation in the lungs and around nerves. CONCLUSIONS AND IMPLICATIONS: These data confirm that testing M(3) receptor blockade with exogenous ACh does not predict vagal blockade. Our data also suggest that selective blockade of M(3) receptors may be effective in asthma via mechanisms that are separate from inhibition of bronchoconstriction.

Upping the antedrug: is a novel anti-inflammatory Toll-like receptor 7 agonist also a bronchodilator?

In this issue of British Journal of Pharmacology, Biffen and colleagues present a novel Toll-like receptor 7 (TLR7) antedrug to treat allergic disease that is rapidly metabolized in the lung to limit side effects due to systemic exposure. Asthma is characterized as an allergic disease of the lung, and TLR7 agonists are proposed to ameliorate allergic inflammation in the lung, a characteristic of prophylactic medications. We have previously shown that TLR7 agonists of multiple structural classes are acute bronchodilators, characteristic of rescue medication for asthma attacks. It will be interesting to determine whether the bronchodilating effect extends to the novel class of TLR7 agonists described here for a prophylactic and rescue therapy in one drug. Combined with the antedrug approach, this would further limit side effects improving on current combination therapies. LINKED ARTICLE This article is a commentary on Biffen et al., pp. 573-586 of this issue. To view this paper visit http://dx.doi.org/10.1111/j.1476-5381.2011.01790.x.

Cigarette smoke induces genetic instability in airway epithelial cells by suppressing FANCD2 expression.

Chromosomal abnormalities are commonly found in bronchogenic carcinoma cells, but the molecular causes of chromosomal instability (CIN) and their relationship to cigarette smoke has not been defined. Because the Fanconi anaemia (FA)/BRCA pathway is essential for maintenance of chromosomal stability, we tested the hypothesis that cigarette smoke suppresses that activity of this pathway. Here, we show that cigarette smoke condensate (CSC) inhibited translation of FANCD2 mRNA (but not FANCC or FANCG) in normal airway epithelial cells and that this suppression of FANCD2 expression was sufficient to induce both genetic instability and programmed cell death in the exposed cell population. Cigarette smoke condensate also suppressed FANCD2 function and induced CIN in bronchogenic carcinoma cells, but these cells were resistant to CSC-induced apoptosis relative to normal airway epithelial cells. We, therefore, suggest that CSC exerts pressure on airway epithelial cells that results in selection and emergence of genetically unstable somatic mutant clones that may have lost the capacity to effectively execute an apoptotic programme. Carcinogen-mediated suppression of FANCD2 gene expression provides a plausible molecular mechanism for CIN in bronchogenic carcinogenesis.

Selective muscarinic receptor antagonists for airway diseases.

Airway tone and airway hyperreactivity are mediated by the parasympathetic nerves that release acetylcholine onto muscarinic receptors (M1-M5). Stimulation of M1 and M3 muscarinic receptors causes bronchoconstriction. The M1 muscarinic receptor is excitatory, and facilitates neuronal transmission at the parasympathetic ganglion. The M2 receptor is an inhibitory prejunctional autoreceptor. The discovery of discrete muscarinic receptor subtypes prompted development of selective muscarinic receptor antagonists. Selective M3 receptor antagonists and antagonists selective for M1 and M3 receptors have recently entered clinical trials and offer much promise for the treatment of airways diseases.

Bone morphogenetic protein-5 (BMP-5) promotes dendritic growth in cultured sympathetic neurons.

BACKGROUND: BMP-5 is expressed in the nervous system throughout development and into adulthood. However its effects on neural tissues are not well defined. BMP-5 is a member of the 60A subgroup of BMPs, other members of which have been shown to stimulate dendritic growth in central and peripheral neurons. We therefore examined the possibility that BMP-5 similarly enhances dendritic growth in cultured sympathetic neurons. RESULTS: Sympathetic neurons cultured in the absence of serum or glial cells do not form dendrites; however, addition of BMP-5 causes these neurons to extend multiple dendritic processes, which is preceded by an increase in phosphorylation of the Smad-1 transcription factor. The dendrite-promoting activity of BMP-5 is significantly inhibited by the BMP antagonists noggin and follistatin and by a BMPR-IA-Fc chimeric protein. RT-PCR and immunocytochemical analyses indicate that BMP-5 mRNA and protein are expressed in the superior cervical ganglia (SCG) during times of initial growth and rapid expansion of the dendritic arbor. CONCLUSIONS: These data suggest a role for BMP-5 in regulating dendritic growth in sympathetic neurons. The signaling pathway that mediates the dendrite-promoting activity of BMP-5 may involve binding to BMPR-IA and activation of Smad-1, and relative levels of BMP antagonists such as noggin and follistatin may modulate BMP-5 signaling. Since BMP-5 is expressed at relatively high levels not only in the developing but also the adult nervous system, these findings suggest the possibility that BMP-5 regulates dendritic morphology not only in the developing, but also the adult nervous system.

Anticholinergic therapy for airway diseases.

Anticholinergics are commonly used in the treatment of airway diseases. While their effectiveness in chronic asthma offers no advantage over beta-agonists, evidence continues to accumulate suggesting substantial additional benefit in acute asthma attacks. This increased response to anticholinergics suggest that cholinergic bronchoconstriction is increased in acute asthma. Multiple mechanisms related to changes in expression and function of inhibitory M2 muscarinic receptors on the airway parasympathetic nerves may be involved, and are discussed. The use of anticholinergics in chronic obstructive pulmonary disease and in rhinitis are also considered.

Structure of the human M(2) muscarinic acetylcholine receptor gene and its promoter.

The M(2) muscarinic receptor inhibits the release of acetylcholine from cholinergic fibers in the lungs and elsewhere. In airway parasympathetic neurons, M(2) receptor expression is decreased by viral infections and by interferon-gamma, increasing actylcholine release. Dexamethasone increases M(2) receptor expression, decreasing acetylcholine release. We carried out 5' rapid amplification of cDNA ends beginning with mRNA from human heart and IMR32 human neuroblastoma cells. This demonstrated a 5' UTR of 100 BP, corresponding to two sequences on chromosome 7, separated by a 22.6 kB intron. The splice acceptor site is at -45 relative to the initiating atg. The 3000 BP upstream of 5' RACE product were subcloned into a pGL3 luciferase reporter vector. Deletional constructs were expressed in IMR32 cells. These demonstrated that 412 BP provided full expression of the reporter gene, and suggested a repressor element between -1848 and -1510.

Effects of dexamethasone on antigen-induced airway eosinophilia and M(2) receptor dysfunction.

In antigen-challenged guinea pigs, airway hyperreactivity is due to recruitment of eosinophils to the airway nerves and dysfunction of M(2) muscarinic receptors. M(2) receptor dysfunction is caused by eosinophil major basic protein, which is an allosteric antagonist at the receptor. Because glucocorticoids inhibit airway hyperreactivity in humans and in animal models of asthma, we tested whether dexamethasone treatment (6 microg. kg(-)(1). d(-)(1) for 3 d, intraperitoneal) before antigen challenge prevents M(2) receptor dysfunction and airway hyperreactivity. Guinea pigs were sensitized to ovalbumin via intraperitoneal injections, and were challenged with ovalbumin via inhalation. Twenty-four hours later, hyperreactivity and M(2) receptor function were tested. Antigen-challenged animals were hyperreactive to vagal stimulation, and demonstrated loss of M(2) receptor function. Dexamethasone pretreatment prevented hyperreactivity and M(2) receptor dysfunction in antigen-challenged guinea pigs. Antigen challenge resulted in recruitment of eosinophils to the airways and to the airway nerves. Dexamethasone prevented recruitment of eosinophils to the airway nerves but did not affect total eosinophil influx into the airways. These results demonstrate that dexamethasone prevents antigen-induced hyperreactivity by protecting neuronal M(2) muscarinic receptors from antagonism by eosinophil major basic protein, and this protective mechanism appears to be by specifically inhibiting eosinophil recruitment to the airway nerves.

Glucocorticoid treatment increases inhibitory m(2) muscarinic receptor expression and function in the airways.

M(2) muscarinic receptors on parasympathetic nerve endings inhibit acetylcholine release in the airways. In this study, the effects of dexamethasone on M(2) receptors in vivo and in primary cultures of airway parasympathetic neurons were tested. Treating guinea pigs with dexamethasone (0.1 mg/kg, daily for 2 d) substantially increased inhibitory M(2) muscarinic receptor function, decreasing airway responsiveness to electrical stimulation of the vagi. At the same time, dexamethasone decreased the response to acetylcholine but not to methacholine, suggesting that cholinesterase activity was increased. When both cholinesterase and M(2) receptors were blocked (using physostigmine and gallamine, respectively) vagally induced bronchoconstriction was increased to control values. In primary cultures of airway parasympathetic neurons, dexamethasone significantly decreased the release of acetylcholine in response to electrical stimulation. Blocking inhibitory M(2) receptors using atropine (10(-5) M) increased acetylcholine release. After the M(2) receptors were blocked there was no difference in acetylcholine release between control and dexamethasone-treated cultures. M(2) receptor gene expression was increased by more than fivefold in dexamethasone-treated cultures. Immunostaining of dexamethasone-treated neurons demonstrated more intense staining. Thus, decreased vagally mediated reflex bronchoconstriction after glucocorticoid treatment may be the result on increased M(2) receptor expression and function as well as increased degradation of acetylcholine by cholinesterase.

Eosinophil recruitment to the airway nerves.

Increased vagal reflexes contribute to bronchoconstriction in asthma. Antigen challenge of sensitized animals induces vagal hyperresponsiveness. This review will discuss the evidence that eosinophils increase release of acetylcholine from the parasympathetic nerves. After antigen challenge, eosinophils are actively recruited to the airway nerves, possibly through expression of chemotactic substances and adhesion molecules by the nerves. Tachykinins acting on neurokinin 1 receptors activate the eosinophils. Activated eosinophils release eosinophil major basic protein (MBP), which is an endogenous antagonist for M2 muscarinic receptors. The M2 muscarinic receptors on the parasympathetic nerves in the lungs normally inhibit release of acetylcholine. When M2 receptors are blocked by MBP, acetylcholine release is increased, resulting in hyperresponsiveness. Neutralization of MBP with polyanionic substances restores M2 receptor function and eliminates hyperresponsiveness. Antibodies to MBP prevent M2 receptor dysfunction and hyperresponsiveness, as do antibodies to the adhesion molecule very late antigen 4, which prevent eosinophil migration. A low dose of dexamethasone, which does not affect total eosinophil influx into the lungs and airways, prevents eosinophils from clustering around the nerves and prevents antigen-induced M2 dysfunction and hyperresponsiveness. Furthermore, animal studies show that viral infections, which are important precipitants of asthma attacks, and exposure to air pollutants such as ozone can also activate airway eosinophils, leading to a chain of events similar to that seen after antigen challenge. Finally, a similar clustering of eosinophils around airway nerves, as well as release of MBP onto the nerves, is seen in fatal asthma, suggesting that similar mechanisms may be involved in human airway hyperresponsiveness.

No evidence for infection of human cells with porcine endogenous retrovirus (PERV) after exposure to porcine fetal neuronal cells.

BACKGROUND: Recent demonstration of human cell infection in vitro with porcine endogenous retrovirus (PERV) has raised safety concerns for new therapies that involve transplantation of pig cells or organs to humans. To assess better the specific risk that may be associated with the transplantation of fetal pig neuronal cells to the central nervous system of patients suffering from intractable neurologic disorders (Parkinson's disease, Huntington's disease, and epilepsy), we have performed studies to determine whether there is evidence for in vivo or in vitro transmission of PERV from fetal pig neuronal cells to human cells. METHODS: Ventral mesencephalon (VM) and lateral ganglionic eminence cells were isolated from fetal pigs and transplanted into patients with neurological conditions as part of clinical studies. Blood samples taken from patients at various time points posttransplant were tested for evidence of PERV. In vitro studies to test for PERV infection of human cells after cocultivation with either fetal porcine ventral mesencephalon or porcine fetal lateral ganglionic eminence cells were also performed. RESULTS: We found no evidence of PERV provirus integration in the DNA from PBMC of 24 neuronal transplant recipients. In addition, no PERV was released from cultured fetal porcine neuronal cultures, and there was no transfer of PERV from fetal pig neuronal cells to human cells in vitro. CONCLUSIONS: Our results demonstrate by both examination of transplant patient blood samples and in vitro studies that there is no evidence for transmission of PERV from porcine fetal neural cells to human cells.

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.

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.

Effects of neurokinin receptor antagonists in virus-infected airways.

We investigated the effects of a neurokinin-1 (NK(1)) receptor antagonist (SR-140333) and a NK(2) receptor antagonist (SR-48968) on airway responsiveness and on the function of neuronal M(2) muscarinic receptors, which normally inhibit vagal acetylcholine release, in guinea pigs infected with parainfluenza virus. Antagonists were given 1 h before infection and daily thereafter. Four days later, bronchoconstriction induced by either intravenous histamine (which is partly vagally mediated) or electrical stimulation of the vagus nerves was increased by viral infection compared with control. In addition, the ability of the muscarinic agonist pilocarpine to inhibit vagally induced bronchoconstriction was lost in virus-infected animals, demonstrating loss of neuronal M(2) receptor function. Macrophage influx into the lungs was inhibited by pretreatment with both antagonists. However, only the NK(1) receptor antagonist prevented M(2) receptor dysfunction and inhibited hyperresponsiveness (measured as an increase in either vagally induced or histamine-induced bronchoconstriction). Thus virus-induced M(2) receptor dysfunction and hyperresponsiveness are prevented by a NK(1) receptor antagonist, but not by a NK(2) receptor antagonist, whereas both antagonists had similar anti-inflammatory effects.

Ovalbumin sensitization changes the inflammatory response to subsequent parainfluenza infection. Eosinophils mediate airway hyperresponsiveness, m(2) muscarinic receptor dysfunction, and antiviral effects.

Asthma exacerbations, many of which are virus induced, are associated with airway eosinophilia. This may reflect altered inflammatory response to viruses in atopic individuals. Inhibitory M(2) muscarinic receptors (M(2)Rs) on the airway parasympathetic nerves limit acetylcholine release. Both viral infection and inhalational antigen challenge cause M(2)R dysfunction, leading to airway hyperresponsiveness. In antigen-challenged, but not virus-infected guinea pigs, M(2)R dysfunction is due to blockade of the receptors by the endogenous antagonist eosinophil major basic protein (MBP). We hypothesized that sensitization to a nonviral antigen before viral infection alters the inflammatory response to viral infection, so that M(2)R dysfunction and hyperreactivity are eosinophil mediated. Guinea pigs were sensitized to ovalbumin intraperitoneally, and 3 wk later were infected with parainfluenza. In sensitized, but not in nonsensitized animals, virus-induced hyperresponsiveness and M(2)R dysfunction were blocked by depletion of eosinophils with antibody to interleukin (IL)-5 or treatment with antibody to MBP. An additional and unexpected finding was that sensitization to ovalbumin caused a marked (80%) reduction in the viral content of the lungs. This was reversed by the antibody to IL-5, implicating a role for eosinophils in viral immunity.

Interaction of viral infections with muscarinic receptors.

Both in humans and in experimental animals, much of the airway hyperresponsiveness that accompanies viral infections is the result of increased reflex bronchoconstriction. The M3 muscarinic receptors on the airway smooth muscle function normally during viral infections so that the direct effects of acetylcholine on the smooth muscle are not altered. In contrast, the M2 muscarinic receptors on the vagal nerve endings, which normally inhibit acetycholine release, are markedly dysfunctional during viral infections. This leads to substantial increases in acetylcholine release and potentiated reflex bronchoconstriction. Multiple mechanisms account for virus-induced M2 receptor dysfunction. Viral neuraminidase may deglycosylate the M2 receptor, decreasing acetylcholine affinity. Furthermore, both viruses and interferon-gamma decrease M2 receptor gene expression. Finally, in atopic hosts, viral infection causes M2 receptor dysfunction by activating eosinophils, causing them to release major basic protein which binds to the M2 receptor, functioning as an endogenous antagonist.

Long-term survival of fetal porcine lateral ganglionic eminence cells in the hippocampus of rats.

Embryonic porcine brain tissue from the lateral ganglionic eminence was transplanted into the adult rat hippocampus to determine whether fetal striatal cells could survive, differentiate, and integrate in a heterotopic site. The hippocampus, a common site of epileptic seizure activity, was chosen to determine if fetal striatal cells could supply inhibitory GABAergic neurons that may serve to block seizures. Cells were either implanted with a single deposit using a standard metal cannula or by five smaller disseminated deposits with a glass micropipette. At 20-24 weeks, animals immunosuppressed with cyclosporin showed long-term survival of porcine cells in the adult hippocampus. Analysis by immunohistochemistry and in situ hybridization showed that the grafts contained glial and neuronal cell types, including GABAergic neurons within graft core and networks of porcine neuronal fibers extending from the graft into the host parenchyma. In addition, a marker of porcine presynaptic terminals, synaptobrevin, was abundant within the grafts and was found associated with hippocampal structures and cell layers suggesting functional integration of grafted cells within the host. The survival of xenografts in the hippocampus and potential integration of inhibitory components provides evidence that these grafts may serve as an internal negative feedback mechanism to quench epileptiform activity.

Antigen-induced hyperreactivity to histamine: role of the vagus nerves and eosinophils.

M2 muscarinic receptors limit acetylcholine release from the pulmonary parasympathetic nerves. M2 receptors are dysfunctional in antigen-challenged guinea pigs, causing increased vagally mediated bronchoconstriction. Dysfunction of these M2 receptors is due to eosinophil major basic protein, which is an antagonist for M2 receptors. Histamine-induced bronchoconstriction is composed of a vagal reflex in addition to its direct effect on airway smooth muscle. Because hyperreactivity to histamine is seen in antigen-challenged animals, we hypothesized that hyperreactivity to histamine may be due to increased vagally mediated bronchoconstriction caused by dysfunction of M2 receptors. In anesthetized, antigen-challenged guinea pigs, histamine-induced bronchoconstriction was greater than that in control guinea pigs. After vagotomy or atropine treatment, the response to histamine in antigen-challenged animals was the same as that in control animals. In antigen-challenged animals, blockade of eosinophil influx into the airways or neutralization of eosinophil major basic protein prevented the development of hyperreactivity to histamine. Thus hyperreactivity to histamine in antigen-challenged guinea pigs is vagally mediated and dependent on eosinophil major basic protein.

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.