Allison D. Fryer - US grants

Viral infections such as influenza are characterized by an increased bronchoconstriction to a variety of stimuli in vivo. Although this hyperreactivity is usually temporary, viral infections in childhood are associated with development of asthma. The hyperreactivity to any specific agonist could result from direct effects on smooth muscle or effects on cells that control smooth muscle contraction. A major indirect route of hyperreactivity Is via the vagus nerve. The vagus nerve releases acetylcholine that binds to muscarinic receptors on the smooth muscle resulting in bronchoconstriction. In preliminary data I have shown that the amount of acetylcholine released by the nerve terminals is regulated by muscarinic receptors which are located on the nerve terminals, and that these neuronal receptors are a different subtype than the muscarinic receptors on the smooth muscle. The neuronal muscarinic receptors are inhibitory in that when stimulated, they limit the amount of acetylcholine released from the nerves. Damage to these neuronal receptors would remove both the tonic inhibition and the negative feedback that occurs with all stimuli which increase vagal activity. It is my hypothesis that the pathologic absence of this normal negative feedback will result in airways hyperreactivity. Viral infections such as influenza and parainfluenza, temporarily increases the bronchoconstriction produced by a variety of stimuli. The mechanism of viral-induced airway hyperreactivity is poorly understood. However, there is evidence to suggest that part of the defect lies in the vagus nerves. Viruses contain enzymes on their surface which can damage cells and receptors on cells. One of these enzymes, neuraminidase, can damage receptors like the muscarinic receptor which inhibits acetylcholine release from the vagus nerves in the lung. Neuraminidase may be produced in large quantities during viral infection as new viruses grow in the airways. I have shown that during viral infection neuronal inhibitory muscarinic receptors are not functioning. The experiments in this proposal are designed to test whether damage to these receptors on the vagus nerves play an important role in the reactivity of the lungs, whether this effect is due specifically to inhibition of neuronal muscarinic receptors by neuraminidase, and what role neuraminidase plays in other models of airways disease, especially antigen challenge. This grant proposes studying muscarinic receptor function in vivo and in vitro as well as studying the receptors at a genetic level. It is expected that results from this study will provide important new insights into the mechanism of airway hyperreactivity induced by viral infection and may yield results which further our understanding of other models of airway disease.

Viral infections such as influenza are characterized by an increased bronchoconstriction to a variety of stimuli in vivo. Although this hyperreactivity is usually temporary, viral infections in childhood are associated with development of asthma. The hyperreactivity to any specific agonist could result from direct effects on smooth muscle or effects on cells that control smooth muscle contraction. A major indirect route of hyperreactivity Is via the vagus nerve. The vagus nerve releases acetylcholine that binds to muscarinic receptors on the smooth muscle resulting in bronchoconstriction. In preliminary data I have shown that the amount of acetylcholine released by the nerve terminals is regulated by muscarinic receptors which are located on the nerve terminals, and that these neuronal receptors are a different subtype than the muscarinic receptors on the smooth muscle. The neuronal muscarinic receptors are inhibitory in that when stimulated, they limit the amount of acetylcholine released from the nerves. Damage to these neuronal receptors would remove both the tonic inhibition and the negative feedback that occurs with all stimuli which increase vagal activity. It is my hypothesis that the pathologic absence of this normal negative feedback will result in airways hyperreactivity. Viral infections such as influenza and parainfluenza, temporarily increases the bronchoconstriction produced by a variety of stimuli. The mechanism of viral-induced airway hyperreactivity is poorly understood. However, there is evidence to suggest that part of the defect lies in the vagus nerves. Viruses contain enzymes on their surface which can damage cells and receptors on cells. One of these enzymes, neuraminidase, can damage receptors like the muscarinic receptor which inhibits acetylcholine release from the vagus nerves in the lung. Neuraminidase may be produced in large quantities during viral infection as new viruses grow in the airways. I have shown that during viral infection neuronal inhibitory muscarinic receptors are not functioning. The experiments in this proposal are designed to test whether damage to these receptors on the vagus nerves play an important role in the reactivity of the lungs, whether this effect is due specifically to inhibition of neuronal muscarinic receptors by neuraminidase, and what role neuraminidase plays in other models of airways disease, especially antigen challenge. This grant proposes studying muscarinic receptor function in vivo and in vitro as well as studying the receptors at a genetic level. It is expected that results from this study will provide important new insights into the mechanism of airway hyperreactivity induced by viral infection and may yield results which further our understanding of other models of airway disease.

DESCRIPTION: (Adapted from the Applicant's Abstract): Exposure to ozone and to antigen causes airway hyperresponsiveness, which is due to increased release of acetylcholine from the vagus nerves. Increased release is due to dysfunction of neuronal M2 muscarinic receptors, which normally limit acetylcholine release, thus limiting bronchoconstriction. Loss of M2 receptor function and the subsequent hyperactivity is dependent upon eosinophils. Steroids are commonly used in the treatment of asthma on the assumption that they are anti-inflammatory. This proposal will address whether steroids prevent hyperreactivity by protecting neuronal M2 muscarinic receptor function. The effects of dexamethasone on hyperreactivity and M2 receptor function in vivo before and after exposure to ozone and antigen will be tested. The applicant will determine the specific mechanisms by which dexamethasone protects neuronal M2 receptor function; including whether dexamethasone inhibits eosinophil migration to nerves in the lungs by interfering with ICAM and VCAM expression. The applicant will also determine whether dexamethasone directly affects production of neuronal M2 receptors (measuring function, immunocytochemistry, M2 mRNA) in primary cultures of parasympathetic nerves. The applicant will also test whether steroids affect the activity of the human M2 receptor promoter (which the applicant has cloned). Finally the applicant will test whether dexamethasone also prevents hyperreactivity in a model that does not involve M2 receptor dysfunction (3 days post ozone). It is anticipated that these studies will lead to a greater understanding of the mechanisms by which hyperreactivity occurs, and the effects of steroids in countering hyperreactivity. Since the applicant has demonstrated that the neuronal M2 receptors are dysfunctional in man following exposure to ozone, and others have demonstrated M2 receptor dysfunction in asthmatic humans, these data may be applicable to the hyperreactivity characteristic of asthma and of exposure to pollutants in man.

Viral infections are known to exacerbate asthma in both adults and children. While a variety of mechanisms are likely to contribute to this effect, neural control of the airways is markedly abnormal in both humans and experimental animals with viral airway infections. Under normal circumstances, the release of acetylcholine from airway vagal fibers is limited by inhibitory M2 muscarinic receptors on the nerve endings. The negative feedback normally provided by these receptors is lost during viral infections. Viral infections can affect M2 receptors via both indirect (inflammatory cell-mediated) and direct (inflammatory cell-independent) mechanisms. Both mechanisms will be further investigated in this project. Specific aims of this project are: Specific aim #1: To determine the mechanisms of virus-induced inflammation of the airway nerves and its role in virus-induced M2 receptor dysfunction. We will investigate the role of interleukin-5, tachykinins, and the interaction of VCAM-1 and VLA-4 in both inflammation of the airway nerves and loss of M2 receptor function. Histology of the airway nerves and functional studies of the M2 receptor will be used as endpoints. Specific aim #2: To determine the effects of inflammatory cells and their products on M2 receptor expression and function. Supernatants from activated eosinophils will be added to airway parasympathetic nerves, and the effects on M2 receptor function (determined by measuring stimulated acetylcholine release) and expression will be measured. Interleukin-2 (a cytokine known to affect neurons), interferon-gamma (a product of CD8+ cells responding to viral infections), interleukin-5 (also produced by CD8+ cells in response to virus under certain circumstances), and interleukin-6 (produced by Schwann cells in response to injury; also with effects on neurons) will be tested for effects on similar endpoints. The effects of dexamethasone (which we have shown increases M2 receptor function in vivo), will also be determined. Specific aim #3: To determine the direct effects of viral infection on M2 receptor expression and function. Cultured nerve cells will be infected with parainfluenza virus and the effects on effects on M2 receptor function and expression will be measured. Because oxygen radicals can be important intermediates in virus-induced gene expression, we will study the effects of antioxidants on virus-induced changes in M2 receptor expression and function. The direct effects of neuraminidase on M2 receptor function will also be tested, as we have shown that viral neuraminidase decreases M2 receptor agonist affinity.

Under normal circumstances, the release of acetylcholine from airway parasympathetic nerves is limited by inhibitory M2 muscarinic receptors. The negative feedback normally provided by these receptors is decreased or lost in some humans with asthma and in animal models of asthma, leading to hyper-responsiveness. In antigen challenged guinea pigs, we have demonstrated that eosinophils causes M2 receptor dysfunction by releasing major basic protein, which is an allosteric antagonist at the M2 receptor. Hyper-responsiveness can be blocked by an antibody to major basic protein, and can be acutely characterized by airway eosinophilia. Hyper-responsiveness 2 and 3 days after ozone exposure is not blocked by eosinophil depletion, or reversed by heparin, unless the animals are previously sensitized to an antigen. It has recently been demonstrated that the inflammatory response of the lungs may vary depending on the atopic status of the host. It is our hypothesis that the inflammatory response of the lungs to injury, and the mechanisms by which the inflammation causes airway hyper-responsiveness and dysfunction of inhibitory M2 muscarinic receptors, depends upon whether the host is atopic. We postulate that in an animal model of atopy (sensitization to ovalbumin by intraperitoneal injection without inhalational challenge), subsequent ozone exposure will cause an influx of eosinophils. This will be accompanied by M2 receptor dysfunction and by hyper- responsiveness lasting a minimum of 3 days with characteristics more similar to those seen after antigen challenge (i.e., will be blocked by depletion of eosinophils, and will be reversed by heparin). In this grant we will also examine whether development of hyperresponsiveness to ozone in humans is dependent upon atopic status. These data will identify the mechanism behind the different responses of humans to ozone and possibly other air pollutants, thus allowing for better prediction of which individuals are at risk and the development of interventions.

DESCRIPTION (provided by applicant): Ozone mediated hyperresponsiveness is mediated by the vagus nerves. However, the mechanisms of ozone induced hyperreaactivity change over 3 days after a single exposure to ozone. Acutely, hyperreactivity is due to loss of inhibitory neuronal M2 receptor function and subsequent increased acetylcholine release. Chronically, hyperreactivity is mediated by substance P, also from parasympathetic nerves. Eosinophils mediate neuronal M2 dysfunction immediately after ozone, and 2 days later still mediate increased acetylcholine release but now by a mechanism separate from M2 receptor loss. 3 days after a single ozone exposure, eosinophils stimulate parasympathetic nerves to express and release substance P, a neurotransmitter normally limited to sensory nerves. Eosinophils mediate all of these effects, as eosinophil depletion or blockade of migration into the lungs prevents ozone induced changes in nerve function. It is our hypothesis that ozone induced hyperreactivity requires an interaction between eosinophils and airway nerves that leads to acute and chronic changes in expression and release of ACh, substance P and their receptors by the parasympathetic nerves. To address this hypothesis we will 1) determine in isolated parasympathetic nerves what mechanisms (eotaxin, ICAM , VCAM) mediate eosinophil recruitment to airway-nerves and how TNF( and IL-1(, cytokines increased by ozone exposure, affect eosinopihl recruitment. 2) Determine how eosinophils induce expression of substance P in parasympathetic nerves. 3) These studies include examining signalling mechanisms initiated by eosinophils adhesion or release of cytokines such as nerve growth factor and LIF from eosinophils that induce substance P expression. 4) Using blocking antibodies and receptor antagonists to determine mechanisms by which ozone, via eosinophiils, induces substance P in parasympathetic nerves in vivo. It is anticipated that these studies will lead to a greater understanding of the interaction of eosinophils with nerves and the multiple mechanisms underlying hyperreactivity to ozone.

We have published papers demonstrating that organophosphates (OPs) induce airway hyperreactivity that is dose related, associated with loss of inhibitory neuronal M2 receptor function that normally limit acetylcholine release, and occurs at doses significantly lower than those that inhibit acetylcholinesterase. Here we show that sensitized animals (sensitized to an antigen but never challenged with antigen) are significantly more sensitive to OPs than non sensitized controls. 0.001mg/kg of the OP parathion did not cause hyperreactivity in non-sensitized guinea pigs but it doubled vagally-induced bronchoconstriction in sensitized animals. Higher doses of OPs, that did affect non sensitized animals, had a significantly greater effect in sensitized animals. In addition, we show that the mechanism for OP induced hyperreactivity does not depend on eosinophils in non sensitized animals, but is switched to require eosinophils after sensitization. We have developed a model for eosinophil-nerve interactions that includes active recruitment and adhesion of eosinophils to parasymapthetic nerves followed by activation and release of eosinophil major basic protein that is an endogenous antagonist for the M2 receptors. It is our hypothesis that OP-induced hyperreactivity in sensitized animals is mediated by OPs affecting chemotactic factors and adhesion molecules that enhance eosinophil recruitement to nerves, and also OP induced eosinophil activation. We will test this in vitro inhuman and guinea pig parasympathetic nerves and in vivo in guinea pigs. There are 4 specific aims: We will test whether increased sensitivity to OPs extends to the OP class and will include other non OP insecticides as controls (aim1). Aim 2 will examine the ability of OPs to alter expression of chemotactic factors, their receptors and adhesion molecules and alter eosinophil-nerve interactions at a cellular level. Aim 3 will examine how OPs activate eosinophils and aim 4 will determine the physiological relevance pathways identified in aims 2 and 3 in vivo. Human exposures to OPs is great in the United States and worldwide, given that more than 80% of children with asthma are also sensitized to antigen, these studies could directly impact levels of OP exposure considered safe and provide targets for intervention after exposure.

[unreadable] DESCRIPTION (provided by applicant): This conference will be the tenth gathering of basic and clinical scientists from around the world focused exclusively on muscarinic receptor pharmacology and physiology, biochemistry and molecular biology. Muscarinic receptor expression function and dysfunction are critical in disorders of the central nervous system as well as in a wide range of autonomic and inflammatory diseases. Recently many new drugs targeting muscarinic receptors have been approved by the FDA and there are more in development. This conference will include sessions on the structure and function of muscarinic receptors, their physiological role and contribution to disease pathophysiology, and the development of new therapeutics targeting muscarinic receptors. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): We have demonstrated that in a guinea pig model, organophosphorus pesticides (OPs) cause airway hyperreactivity that is dose-related and associated with the functional loss of autoinhibitory muscarinic M2 receptors that normally limit acetylcholine release from parasympathetic nerves that innervate airway smooth muscle. We recently reported that sensitization to antigen alters the mechanisms underlying OP-induced airway hyperreactivity to involve IL-5-dependent mechanisms in the sensitized but not the non-sensitized guinea pig. How OPs cause neuronal M2 dysfunction in the non-sensitized animal is not known but our preliminary data indicate that this effect is not mediated by cholinesterase inhibition or direct antagonistic interactions with neuronal M2 receptors. Rather, OPs appear to influence neuronal M2 receptor function indirectly via effects on macrophages since depletion of macrophages using liposome-encapsulated clodronate protects against OP-induced airway hyperreactivity. It is our hypothesis that OPs activate macrophages to upregulate expression and release of inflammatory cytokines previously shown to cause M2 receptor dysfunction in various models of airway hyperreactivity. We propose four Aims to test this hypothesis. In Aim 1, we will use in vivo physiological measurements to confirm that macrophages mediate airway hyperreactivity caused by OPs and determine whether their role changes over time, as has been observed for eosinophils in ozone-induced airway hyperreactivity. Aim 2 will utilize macrophages isolated from bronchoalveolar lavage collected from OP-treated versus untreated guinea pigs guinea pigs to examine the effect of OPs on macrophage expression and release of inflammatory cytokines implicated in airway hyperreactivity. In Aim 3, we will use primary nerve cell cultures to determine whether OP- induced macrophage mediators interact with nerves directly to alter M2 receptor expression or function or the structural plasticity of nerves. Aim 4 will confirm the in vivo physiological relevance of OP-induced macrophage mediators identified in aims 2 and 3. Mechanistic studies are critical to developing preventive and therapeutic approaches for OP-induced airway hyperreactivity, which are likely to differ between sensitized (allergic) and non-sensitized individuals, and for determining the risks to human health posed by OP exposures. The public health implications of these studies are significant in light of the increasing prevalence of asthma, the wide spread exposure of humans and especially children to OPs and the credible threat of terrorist use of OP pesticides and nerve agents. PUBLIC HEALTH RELEVANCE: Recent epidemiological studies suggest a link between exposure to organophosphorus pesticides (OPs) and asthma. Our previous work confirms this link by demonstrating that OPs cause airway hyperreactivity, a major symptom of asthma, in a guinea pig model. The goal of this project is to elucidate the mechanism(s) by which OPs cause airway hyperreactivity, which will be critical to developing effective preventive and therapeutic approaches to OP-induced airway hyperreactivity. Given the documented widespread exposure to OPs not only in the U.S. but worldwide, and the credible threat of terrorist use of OPs, the proposed work is of significant public health relevance.

Project Summary: Obesity increases the incidence and severity of asthma, but an incomplete understanding of the molecular mechanisms underlying obesity-related asthma make it difficult prevent and treat this phenotype. Parasympathetic nerves mediate one mechanism of airway hyperreactivity. These nerves provide dominant autonomic control of airway tone and release acetylcholine (ACh), which activates M3 muscarinic receptors on airway smooth muscle, causing contraction and bronchoconstriction. ACh release is controlled by inhibitory M2 muscarinic receptors on these nerves. Thus, airway hyperreactivity results from decreased neuronal M2 receptor function and subsequent increased ACh release. Our preliminary data show that obesity is associated with increased bronchoconstriction in response to parasympathetic nerve stimulation, with reduced neuronal M2 receptor function, and that these effects are mediated by insulin even in the absence of inflammation. Thus, our hypothesis is that increased insulin, as seen in obesity, binds to insulin receptors on airway parasympathetic nerves resulting in airway hyperreactivity by reducing M2 muscarinic function on parasympathetic nerves (thus increasing ACh release). We propose to test how insulin reduces M2 receptor expression and function, identify which insulin receptor and signaling pathways mediate neuronal M2 dysfunction and test whether manipulating insulin (with diet, oral anti-glycemic drugs and with an insulin binding antibody) protects M2 receptor function and inhibits obesity induced airway hyperreactivity. This research is significant because it has the potential to explain why obese patients with increased insulin, are more prone to asthma and identify novel strategies, including control of insulin, and M3 selective muscarinic antagonists, that may treat obesity-related asthma.

The Oregon Students Learn and Experience Research (OSLER) TL1 program is created to train transdisciplinary clinical and translational researchers. To maximize a transdisciplinary environment, we will provide training in clinical and translational science to graduate PhD students (basic science, behavioral, social science) as well as MD and DMD students. We will build upon our successful TL1 program by recruiting PhD students from OHSU basic science programs, as well as from Portland State University, University of Oregon, and Oregon State University. Graduate students will participate after satisfactorily completing their qualifying exam; MD and DMD students will most likely participate between their third and fourth year of medical or dental school. Graduate and professional students will design an individual plan for this fellowship that is specific to career plans as well as to their proposed research. MD and DMD students will enroll in the Master of Clinical Research (MCR) program and will complete all requirements for the degree within the training period. This didactic program is specifically constructed around the ?case? of the trainee's mentored research; as trainees progress in the curriculum, they develop research skills through class exercises such as writing a research proposal and IRB form, designing the data management system, and calculating power and sample size. Graduate students will be encouraged to participate in the MCR, but at a minimum must complete competencies in quantitative methods, data management, and research leadership. All students will participate in the FOLIO curriculum, which is designed to provide practical skills of critical thinking, time management, team science, design science, and problem solving. TL1 trainees will also be trained in science communication and writing. Two key experiences will enhance the transdisciplinary training: a clinical and translational science journal club, and participation in the MD/PhD grant rounds. Through participating in common activities, students from different disciplines will gain an understanding of the benefits of team science and will enhance their ability to understand the clinical setting of their research. PhD students will be provided with enhancement experiences such as attending clinics and rounds with clinicians in their research focus. This program will create clinicians and scientists who will pursue careers in transdisciplinary clinical and translational science.