Mast Cell Uptake of Particulate Allergens

The most common form of asthma is allergic asthma, which is developed by inhalation of airborne allergenic particles such as pollen, animal hair, or fur. One of the major effector cells of asthma are mast cells which are positioned in the asthmatic airways so that they immediately interact with inhaled allergens via IgE antibodies bound on their surfaces and respond by the release mediators that initiate the early phase of allergic asthma. Recently, we discovered that pathogenic bacteria can co-opt the endocytic activities of mast cells and gain entry via a route that avoids intracellular degradation (Shin et al Science 289:785-788, 2000). This process involved distinct cellular entities comprising of caveolink cholesterol and glycolipids called caveolae (or rafts). Moreover, infected MC exhibited a remarkably high level of sustained mediator release.

We examined the involvement of caveolae in the uptake of particulate allergens and discovered remarkable similarities between bacterial entry and IgE-mediated entry of allergens into mast cells. We plan to confirm and extend our observations by (i) determining whether caveolae are involved in the uptake of particulate allergens, (ii) identifying the specific role(s) of caveolin in the uptake of allergens and (iii) determining the fate of the internalized allergens in the mast cell and how it affects cellular secretion of inflammatory mediators. These studies could reveal remarkable convergence in the entry of allergens and pathogenic bacteria in mast cells and should help us develop effective strategies to prevent asthma as well as infectious diseases.

H. Leonardo Aguila, Ph.D.

NK Cells in Allergic Asthma

NK cells are one of the main effector arms of innate immunity, they express molecules present in various hematopoietic lineages, as well as NK restricted markers.  Through their cytotoxic functions, they are able to clear intracellular pathogens and tumors.  Their ability to produce cytokines, makes them attractive candidates to modulate the outcome of adaptive immune responses.  The understanding of the development, heterogeneity and function of this compartment is important in order to design protocols aimed to modulate or alter NK cell functions.  I propose to study the role of NK cells on priming, and induction of asthma.  Many cellular and molecular mediators have been identified during the process of presentation, sensitization, and in the effector phase of acute and chronic asthma.  NK cells have the ability to express many of the soluble mediators involved in the asthma process; however, their contribution in modulating the disease have not been studied extensively.  I will study the phenotypic and functional heterogeneity of the NK cell compartment in a murine model of allergic asthma; comparing peripheral vs. organ specific NK cell populations.  I will evaluate the role of NK cell subpopulations by antibody depletion with antibodies of known and novel specificities, defining their direct or indirect participation in the pathogenic process.  Also, the use of mutant mice with alterations in their NK cell compartment will be examined.  Finally, we will attempt to generate murine NK cell lines with differential functional abilities to explore their potential to modulate immune responses by adoptive transfer protocols.

John P. Atkinson, M.D.

A Role for Complement-induced T-Regulatory Cells in Human Asthma

Asthma is a syndrome of unknown etiology featuring a Th2 dominant immune-mediated inflammatory response to environmental antigens. The basic immune defects predisposing to asthma are unknown as are the regulatory mechanisms that prevent such responses in non-asthmatics. Regulatory T cells (Tregs) have emerged as a central player in the control of immune responses. We have described a novel means to generate Tregs from naive human peripheral blood CD4⁺ lymphocytes. It requires the cross-linking of CD3 and the complement regulatory protein CD46. These cells possess properties of Tregs as they are dependent on IL-2, suppress proliferation of naive T cells through soluble IL-10 and granzyme B. They also facilitate dendritic cell maturation through secretion of GM-CSF and CD40L. We propose that such cells home to and reside in the airway, gut and skin. In such locations symbiotic as well dangerous microbes coexist along with numerous potential environmental antigens

In Specific Aim 1 we will characterize a human Treg population relative to their ability to modulate Th1, Th2 and B cell responses. In Specific Aim 2 we will compare these Tregs in normal and asthmatic individuals. These studies will define the homing pattern, cytokine profile and surface markers of CD46 generated Tregs. We will ask if CD46 generated Tregs reside in lung tissue and regulate immune cells essential to the development of asthma. Our hypothesis is that an aberrant response and a failure to rectify the error in innate immune sensing lie at the core of asthma pathology

Jonathan M. Backer, M.D.

Role of PI 3-Kinases in Mast Cell Degranulation

Mast cells are critical mediators of physiological responses to allergens, and are also strongly implicated in the development of asthma. Mast cells function through the release of both pre-formed mediators, which are stored in secretory granules, as well as the de novo synthesis of bioactive lipids. Mast cell degranulation is mediated primarily by the crosslinking of IgE-bound FceRI receptors on the cell surface. However, coincident stimulation with adenosine leads to an increase in the magnitude of FceRI-mediated degranulation. This synergistic regulation of degranulation by allergens and adenosine is likely to be an important component of the pathophysiology of asthma. This proposal examines the role of Class I and Class III PI 3-kinases in mast cell degranulation. Aims I and II are based on preliminary data showing that the Class III VPS34 PI 3-kinase is required for degranulation mediated by G-protein-coupled receptors (GPCRs), and for adenosine-mediated enhancement of IgE-stimulated degranulation. Aim I studies the subcellular localization of VPS34 in basal and stimulated mast cells, and the effects of overexpression of wild-type and mutant VPS34. Aim II tests whether VPS34 activity is regulated by GPCRs. Finally, Aim III examines the role of PKCb and PKCd as downstream mediators of Class I PI 3 -kinases in IgE-stimulated degranulation, and tests whether constitutively active PKC mutants can rescue degranulation in cells treated with PI 3-kinase inhibitors. These studies should yield new insights into mast cell physiology. Moreover, our focus on distinct isoforms of PI 3-inase may identify new targets for the pharmacological treatment of asthma.

Albert Bendelac, M.D., Ph.D.

NKT Cell Regulation of Human Asthma

A central pathogenic component of asthmatic disease in humans is the bronchioalveolar infiltration by hemopoietic cell types producing TH2 cytokines and chemokines, which recruit and coordinate the various effector mechanisms involved in asthma pathogenesis.

These Th2 cytokineichemokine producing cell types include CD4 T cells as well as eosinophils and basophils. While the causative agents of disease, CD4 Th2 cells are generally thought to include MHC class II-restricted, allergen peptide-specific cells, IL-4/IL-13 producing CD1d-restricted lipidspecific NKT cells have also been considered. Studies in a mouse model of OVA-induced airway hyperreactivity have suggested a crucial role for NKT cells. In humans with asthma, emerging studies have revealed a depletion of NKT cells from peripheral blood and an extraordinary level of NKT cell infiltration among the CD4 T cells recovered from bronchioalveolar lavage (BAL) as well as in situ in bronchial biopsies. These findings combined with the conspicuous lipid transfer properties of many prominent allergenic proteins and their structural homology with lipid transfer proteins involved in CD1d mediated presentation of lipids, suggest the intriguing possibility that lipid disturbances may contribute to asthma through NKT cell recruitment and activation.

In this project, we propose to explore the hypothesis that NKT cells are involved in the pathogenesis of human asthma. We have assembled collaborators within the University of Chicago Asthma Research Center and have already obtained preliminary data demonstrating massive infiltration of NKT cells in the human asthmatic airway. We will first perform a general study of asthma patients and controls to determine the degree of airway infiltration by NKT cells, their functional properties and antigenic specificities. Because we have shown that NKT cells recognize the self glycosphingolipid iGb3 we will study the regulation of this NKT cell trigger in vivo and in vitro and the contribution of lung and allergen lipid transfer proteins. Finally, we will study the contribution of NKT cells in the mouse model of OVA-induced airway hyperreactivity. These studies will provide original insights into the pathogenesis of asthma and into the functions of NKT cells.

Michael R. Blackburn, Ph.D.

Role of the Adenosine A3 Receptor in Experimental Asthma

Adenosine is a signaling nucleoside that elicits physiological effects by engaging G-protein coupled receptors.   Adenosine signaling has been implicated in inflammatory lung diseases such as asthma, however, the mechanisms involved are unclear.  We have generated mice deficient in the enzyme adenosine deaminase (ADA).  ADA controls the levels of adenosine in tissues and cells, and consequently, adenosine accumulates in the lungs of ADA-deficient mice.  ADA-deficient mice develop features seen in asthmatics, inlcuding lung eosinophilia and mucus hypersecretion.  These features appear dependent on increases in lung adenosine; suggesting adenosine signaling plays an important role in lung eosinophilia and mucus hypersecretion.   Recent findings show that A3 adenosine receptor transcripts are elevated in the mucus producing cells of the bronchial airways, leading to the hypothesis that adenosine signaling through the A3 receptor plays an important role in mucus hypersecretion in the airways of inflamed lungs.  The goal of this proposal is to address this hypothesis by conducting studies in various genetically modified mice.  Three specific aims are proposed: 1)  Determine if elevated adenosine and A3 receptor expression are general features of mucus hypersecretion in inflamed airways; 2) Determine if A3 receptor expression is necessary and sufficient for mucus hypersecretion; and 3) Examine the influence of A3 receptor signaling on second messenger production in in vitro models of mucus hypersecretion.  Through these experiments we will learn more about the role of adenosine signaling in asthma, and how signaling through the A3 adenosine receptor influences the production of mucus, which is a more pathogenic component of this disease.

Michael R. Blackburn, Ph.D. (Extension Award)

Adenosine Deaminase Enzyme Therapy and Asthma Exacerbations 

Asthma is an inflammatory disease of the airways that is associated with acute and resolvable bronchonstriction, as well as chronic airway remodeling.  Whereas pathways that lead to initial inflammatory cascades in asthma have been elucidated, little is known about the pathways associated with asthma exacerbations or chronic airway remodeling.  Similarly, current asthma treatments are effective at controlling initial inflammatory insults in the lung; however, treatment strategies to control aspects of ongoing disease are lacking.  Adenosine is a signaling nucleoside that is generated during cellular stress and damage.  Accordingly, adenosine levels are elevated in the lungs of asthma patients.  Numerous studies have implicated adenosine as a pro-inflammatory signal in the asthmatic lung.  Our studies in models of adenosine-induced and Th2 cytokine induced airway injury have demonstrated that adenosine deaminase (ADA) enzyme therapy is effective in lowering adenosine levels in the injured lung and can improve the status of airway inflammation and remodeling.  However, the efficacy of ADA enzyme therapy in the treatment of allergen-induced airway disease has not been examined.  The specific hypothesis that will be addressed in this project is that ADA enzyme therapy can prevent and reverse exacerbations of allergen-induced airway inflammation and remodeling.  To address this hypothesis, we will utilize a chronic model of ovalbumin-induced airway inflammation in the mouse to test the benefit of ADA enzyme therapy in response to allergen in the lung.  Given that adenosine is elevated in the asthmatic lung and that lowering lung adenosine can improve airway inflammation and remodeling in adenosine-dependent lung disease in mice, it seems reasonable to pursue the development of ADA enzyme therapy for the treatment of asthma.  Testing the efficacy of this therapy in allergen-based models will greatly facilitate this goal.

Richard Bond, Ph.D.

Effects of Beta-Adrenoceptor Inverse Agonists Treatment on Murine Models of Asthma

We propose to investigate whether the chronic use of ß-adrenoceptor (ßAR) inverse agonists is a viable strategy for the treatment of asthma. Traditionally, ßAR agonists are used to increase signaling and induce bronchodilation.  While ßAR agonist therapy is effective for acute treatment, with chronic use their effectiveness is significantly reduced. Very recently, a large clinical trial using a long-acting ßAR agonist, salmeterol, was stopped due to possible increased mortality and adverse events. 

Recently, a paradigm shift has occurred in the therapeutic use of ßAR drugs in congestive heart failure (CHF).  As in asthma, ßAR agonists are used in CHF to acutely increase signaling. The chronic use of ßAR agonists in CHF leads to increased mortality. By contrast, some ßAR inverse agonists, once contraindicated in heart failure, are now the most successful drugs ever used to decrease mortality in CHF. ßAR inverse agonists are currently contraindicated in asthma. We suggest there are many parallels between the treatment of asthma and CHF with ßAR ligands, and that ßAR inverse agonists may be useful in the treatment of asthma.

We have obtained preliminary data consistent with this hypothesis in mouse model of asthma where chronic treatment with ß2AR inverse agonists significantly reduces the effects of a bronchoconstrictor. The long-term goal of these studies is to provide a mechanistic basis for the potential use of ß AR inverse agonists in treating asthma.  Our central hypothesis is that agonists and inverse agonists have reciprocal effects on cellular signaling and therefore chronic use inverse agonists increases signaling.

Richard A. Bond, Ph.D. (Extension Award)

Efficacy and Tolerability of Oral Nadolol in the Treatment of Mild Asthma

Study Objectives:  To avoid the potential decrease in FEV1 associated with the commencement of asthma treatment with the beta-adrenoceptor inverse agonist, nadolol, by beginning with a sub-therapeutic dose and using a gradual titration to achieve the target dose.

Rationale:  Previous studies in an allergen-driven murine model of asthma have shown that the effect of the beta-adrenoceptor inverse agonist, nadolol, on airway hyperresponsiveness (AHR) is dependent upon the duration of treatment.  Acute administration of nadolol produces an increase in AHR, while with chronic administration there is a decrease in AHR to methacholine (Callaerts-Vegh et al., PNAS, 2004).

These data, along with the analogies of these results to the results obtained with certain 'beta-blockers' in the treatment of congestive heart failure (CHF), led to an FDA-approved Phase IIa pilot study to determine the safety and efficacy of nadolol treatment of mild asthmatics.  The results of that trial showed that chronic treatment (9 weeks) with nadolol produced a dose-dependent beneficial effect on the PC20 methacholine in subjects with mild asthma.  However, as was the case in the murine studies, and in CHF, acute dosing with nadolol produced a deterioration of some pulmonary function tests. For example, 3 of the 10 subjects completing the initial trial experienced a >12% fall in FEV1 following administration of the first dose of nadolol.  This first study used a starting dose of 10 mg.  While this fall in FEV1 was asymptomatic in these patients due to the mild status of their asthma (their average FEV1 was >90% predicted), this fall in FEV1 could prevent expanding the therapeutic strategy into patients with moderate or severe asthma.  Therefore, this second study is designed to determine whether the initial adverse effects of nadolol can be avoided by beginning at a very low dose (1.25 mg) and slowly increasing the dose (every 2 weeks, rather than weekly as in the first sudy), until either the target dose (160 mg) or a maximal tolerated dose is achieved.

Mark R. Boothby, M.D., Ph.D.

Memory Regulation and Allergic Airway Responses

The first encounter of an atopic individual with an antigen can lead to TH2-dominated allergic process whereas a non-atopic person would not develop this problem. Microbial exposures are linked with susceptibility to atopic asthma. Similarly, virus infections are a major cause of asthma flares but immunologic mechanisms whereby a virus would reactivate a Th2-dominated allergic response are lacking. Airway obstruction in asthma is intermittent and reversible; patients experience periods of clinical quiescence during which inflammation is attenuated but some allergen-specific T helper (Th)2 cells must become memory cells. However, little is known about contributions of allergen-specific memory T cells to asthma. We have developed a mouse model that permits us to investigate rigorously the potential for memory T cells to influence disease susceptibility. Using this model, we will investigate cellular and molecular mechanisms by which viruses and memory T cells can affect allergic disease. Our hypothesis is that virus infections can influence the development, maintenance, and reactivation of pools of allergen-specific memory Th1 and Th2 cells and thereby influence allergic susceptibility. We will use single- and dual-specificity T cells to establish how virus infections can be programmed to affect allergic airway inflammation. Further, we will investigate whether signals induced during virus infection influence memory Th2 compartments. Together, these studies will yield crucial insights into the role of viruses and memory in asthma.

Michael B. Brenner, M.D.

New Class of Allergens in Asthma

A major paradigm for T cell biology is based on the recognition of peptide antigens presented by MHC class I and class II molecules to activate specific T cells.  Similarly, the current dogma indicates that the allergens in allergic asthma are proteins. Yet, recent advances in basic immunology reveal that T cells and Th2 polarized responses may also be mediated by CD1 restricted T cells that recognize lipid antigens.  Further, recent evidence points to a key role for CD1 reactive NKT cells in IL-4 and IL-13 driven airway hyperreactivity.  Since major allergen sources are rich in lipids, we hypothesize that CD1-presented non-protein antigens may be important in asthma.  We will focus on molds and environmental hydrocarbon contaminants such as plasticizers as these sources are implicated in asthma and contain CD1 presented antigens.  Thus, we propose to extract and identify lipids from molds that stimulate CD1 restricted T cell responses in asthmatics (Aim 1).  Since exogenous lipids in vivo are transported in lipoprotein particles, we will fractionate and assay VLDL derived lipids from asthmatics to identify antigens that may have come from unknown exogenous sources (Aim 2).  In addition, we will determine if environmental hydrocarbons like plasticizers elicit enhanced CD1 restricted T cell responses that are relevant in asthma (Aim 3).  These studies may open the door to understanding a new class and range of antigen specificities in atopic and/or non atopic asthma that would have far reaching impact and could lead to new therapeutic opportunities. >

Robert Brenner, Ph.D.

Knockout Studies of the BK K+ Channel Beta1 Subunit in Airway Smooth Muscle

Although airway smooth muscle (ASM) contraction is initiated by calcium release from intracellular stores, there is considerable evidence that the control of membrane potential by potassium channels is an important factor in regulating ASM constriction. It is hypothesized that depolarization or block of potassium channels allows recruitment of voltage-dependent calcium channels (VDCCs) and increased contraction. The large conductance calcium-activated (BK type) potassium channel is activated by calcium and voltage, and so this channel is an ideal negative-feedback regulator of VDCCs. BK channel opening hyperpolarizes membranes, deactivates VDCCs and thereby opposes contraction. Previously, we had shown that the BK channel accessory beta1 subunit is required for BK channel function in vascular smooth muscle and bladder smooth muscle. Knockout of the beta1 subunit results in BK channels that fail to open, increased calcium influx and increased tone. Although potassium channels have been implicated in controlling ASM constriction, as of yet no mouse model has been utilized to evaluate their roles. BK channels are abundantly expressed in ASM, and have been implicated in mediating airway relaxation by b-adrenergic agonists. Our preliminary data demonstrate that BK channel beta1 subunits are expressed in ASM. Patch clamp recordings of BK channels in ASM of beta1 knockout mice have a dramatically reduced open probability. We propose to utilize the beta1 knockout to determine how BK channels regulate calcium signaling in ASM cells and determine the relevance of BK channel function in physiological studies of tracheal contraction in vitro and airway responsiveness of unrestrained mice.

Alec M. Cheng, Ph.D.

Regulation of Mast Cell Function by the Signaling Adaptor GADS

Mast cells play a critical role in allergic response by virtue of the high affinity FceR which binds to the Fc portion of the IgE.  Anitigen binding to IgE on FceR activates a signaling complex associated with the receptor, which further initiates a cascade of intracellular biochemical responses resulting in degranulation and cytokine production.  Recent studies reveal that mast cells share a common signaling pathway with T cells in using the SLP-76 and LAT scaffold proteins to mediate and regulate FceR activation. The current project is to dissect the function of an adaptor protein, GADS, which serves to coordinate SLP-76 and LAT activity in FceR signaling in mast cells. We will analyze the defect of GADS-deficient primary mast cells derived from bone marrow (BMMC). We will use retroviral expression strategy in GADS mast BMMC to examine the structure and function of GADS. Finally, we will examine a novel mechanism involving effector caspases in the apoptotic pathway in regulating FceR function. We observed that mast cells in vitro under specific differentiation conditions exhibit caspase activity, resulting in the cleavage and inactivation of GADS. We will characterize the caspase activity and determine its relevance to FceR function in mast cells. In sum, this project will reveal how mast cell function is regulated by the SLP-76/GADS pathway. The study of the novel role of caspases is likely to lead to new breakthrough in understanding how effector proteins employed by the death pathway plays a role in regulating mast cell function.

David W. Christianson, Ph.D.

Structural and Chemical Biology of Arginase in Asthma

Arginase is a metalloenzyme that catalyzes the hydrolysis of L-arginine to form L-ornithine and urea. We have determined the X-ray crystal structures of human arginases I and II and we have developed tight-binding inhibitors of these human isoforms.  Given our advances with regard to the exploration of arginase function in various human diseases, and given the recent discovery that arginase plays a role in asthma, we propose to focus our study of arginase inhibition on the asthma problem with the support of a SPAR award.   We will explore the structure-based design and development of new inhibitors using our recently determined X-ray crystal structures of human arginases I and II to guide our molecular design rationale.  Specifically, we aim:  (1) to design, synthesize, and assay silanediol and silanetriol amino acids as transition state analogue inhibitors of arginase; (2) to determine X-ray crystal structures of human arginase complexed with silanediol and silanetriol amino acid inhibitors; (3) to evaluate the enhancement of NO-dependent airway smooth muscle relaxation by silanediol, silanetriol, and boronic acid arginase inhibitors in ex vivo organ bath experiments; and (4) to evaluate the potentially beneficial in vivo effects of these arginase inhibitors in suppressing elevated arginase levels detected in animal models of asthma.  The best inhibitors identified in this work will represent potential lead candidates in the development of a new family of arginase-targeted drugs for asthma therapy.

David E. Clapham, M.D., Ph.D.

TRP Ion Channels as New Targets in Asthma

The hypothesis of this proposal is that the airway smooth muscle Transport Receptor Potential (TRP) channels are activated in the inflammatory and mechanically mediated changes that result in asthma.  Based on their tissue distribution and results from genetically targeted mice, TRP channels regulate airway smooth muscle as well as airway epithelia and blood-derived inflammatory cells.  As newly identified Ca²⁺-permeable ion channels, TRPs have not been the target of previous asthmatic therapies.  In the proposed studies we will determine the role of airway smooth muscle TRP channels in airway constriction and remodeling.

 The specific Aims of this proposal are to:  1)   Determine the subtypes of transient receptor potential (TRP) channels present in the human and mouse airway smooth muscle.  2)   Test whether blockade or elimination of TRP channels alters airway smooth muscle contractility and/or cell morphology.  3)  Determine the long-term consequences of the elimination of TRP channels in the airway smooth muscle of genetically targeted mice. 

Marco Conti, M.D.

Role of Phosphodiesterases in Asthma

Cyclic nucleotide signaling plays a crucial modulatory role in the airways and in inflammatory cells. An increase in cAMP is associated with the inhibition of airway smooth muscle contraction, activation of T cell, and migration and/or function of effector cells at sites of allergic inflammation. Here, we will test the hypothesis that genes coding for cyclic AMP-specific phosphodiesterases (PDE4s), components of the cAMP signaling cascade, are major determinants of allergen-induced airway hyperreactivity (AHR), a hallmark of asthma. Of the four PDE4 genes present in humans and other mammals, three (PDE4A, B,and D) are expressed in the airways and in inflammatory cells. We have established in vivo models in which individual PDE4 genes have been inactivated by homologous recombination, and have shown that allergen-induced AHR does not develop in mice deficient in PDE4B or PDE4D. These preliminary findings indicate that these PDEs, and the signaling functions they serve, play an essential role in the pathogenesis of asthma. We propose to use these genetically altered mice to define the role of individual PDEs and cAMP homeostasis in the differentiation and the recruitment of inflammatory cells to the lungs and in the development of specific characteristics of asthma, including AHR. Using these mouse models, we will test the possibility that polymorphisms/mutations in the PDE4 genes in humans contribute to the genetic background predisposing to allergic asthma. These studies will also provide "proof of principle" for the development of a new class of drugs useful for the treatment of this disease.

Michael Croft, Ph.D.

Targeting OX40 in Asthma

Asthma is a disease induced by T cells and Type 2 cytokines.  For T cells to respond, they require costimulatory interactions from cell surface molecules.  We hypothesize that a novel costimulatory pair, OX40/OX40L, may be a major determinant in asthma.  OX40 signals are integral to priming of T cells, and to secretion of Type 2 cytokines.  We predict that OX40 interactions may regulate T cell priming in the bronchial lymph nodes, entry of T cells and other cells into the lungs, and secretion of cytokines within the lungs.

OX40 knockout mice will be used to assess whether asthmatic symptoms are inhibited in the absence of these interactions, and whether blocking CD28 or CD40 has synergistic effects.  Further studies will define whether OX40 is required during the priming phase of asthma, during the effector/challenge phase, or both, using blocking reagents.  Adoptive transfer experiments with OVA-specific CD4 and CD8 T cells from OT-I and OT-II TCR transgenics will determine the contributions of these cells to the asthmatic response and comparison to OTxOX40-/-T cells will determine the role of OX40.  Transfer of Th2/Tc2 effector cells will show the role of OX40 in the lungs.  Transfer of Th1/Tc1 cells will determine whether these subsets inhibit asthma and the involvement of OX40.  Lastly, experiments will determine whether inhibitory or stimulatory OX40 reagents increase the therapeutic efficiency of altered peptide, cytokine, and tolerization therapy.  We predict these studies will provide novel therapeutic strategies for alleviating asthmatic reactions.

David A. Dean, Ph.D.

Targeting Airway Smooth Muscle for Asthma Gene Therapy

At present, there are few methods to selectively transfer genes to non-dividing airway smooth muscle cells. This is a major problem in the development of gene therapy approaches to treat the airway hyperresponsiveness and remodeling associated with asthma. We have identified a DNA sequence that increases nuclear localization and subsequent gene expression uniquely in smooth muscle cells, a critical target in asthma gene therapy. We hypothesize that the cell-selective nuclear import of the smooth muscle gamma actin (SMGA) promoter is mediated by the transcription factors SRF and Nkx3.1/3.2 that are expressed in smooth muscle cells but not other cells of the airway. With our development of a new method for highly efficient gene delivery to the lungs and airways of living animals using electroporation, we are in a unique position to test the effects of this cell-selective nuclear import sequence on smooth muscle transfection in animal models for asthma. We hypothesize that the SMGA DNA nuclear targeting sequence will lead to gene transfer and expression only in airway smooth muscle cells, and not in airway epithelial or other lung cells of living animals. This proposal is designed to test this hypothesis and will led to the creation of new airway gene therapy vectors that are both cell-specific and capable of greater gene transfer efficiencies. Finally, we will use the information gained to test the efficacy of these vectors in an animal model for airway hyperresponsiveness using PKC and MLCK dominant-negative mutants and MLCP gene transfer.

Joseph L. DeRisi, Ph.D.

A Systematic, Genomics-based Investigation of the Role of Viral Infection in Acute Exacerbations of Asthma

There is a general agreement that acute exacerbations of asthma are often precipitated by respiratory viral infection. However, accurate information about the prevalence of infection in asthma and a full definition of the causative agents remains elusive (despite occasional assertions to the contrary). Here we propose a genomics-based investigation of the role of viral infection as a precipitant of asthma. cDNA from respiratory secretions of acute asthmatics will be examined by hybridization to DNA microarrays bearing sequences representative of all known families of human and animal viruses. Arrays will harbor multiple genes from any members of each sequenced family of viruses; this should allow not only detection of infection but (in many cases) identification of viral subtypes as well. Our goal is to produce a comprehensive picture of the role of viral infection in asthma using state of the art technology for pathogen detection.

Colin S. Duckett Ph.D.

IAP Proteins as Novel Molecular Targets for the Treatment of Asthmatic Disease

The Inhibitor of Apoptosis (IAP) proteins were originally described as a family of intracellular proteins with key regulatory roles in the suppression of the apoptotic cell death pathway. Recently, however, studies performed by a number of laboratories, including ours, has revealed numerous cellular functions for the IAPs that appear unrelated to apoptosis, and genetically targeted Iap-deficient mice exhibit very few, if any, differences in terms of apoptotic sensitivity. However, using an established model of asthma induced by administration of cockroach antigen, we have found dramatic alterations in the lungs of mice deficient in one member of the family c-IAP1. Consistent with these structural changes, c-IAP1-null mice exhibit a profound alteration in response to respiratory syncytial virus (RSV) challenge, which is known to greatly potentiate the severity of asthmatic disease. We therefore propose to explore the role of the IAPs using genetically targeted mice, as well as a range of IAP antagonists available to our laboratory. Although these antagonists were initially generated through structure-based synthetic design as targeted anti-cancer reagents, our initial findings raise the exciting possibility that by phenocopying IAP-null mice, they might additionally function as a novel class of anti-asthmatic drugs.

Sean B. Fain, Ph.D.

Non-Invasive Imaging of Airway Closure, Edema and Cellular Activation in an Animal Model of Asthma

 Non-invasive imaging tools have begun to reveal a complex regional patho-physiology of asthma not captured by traditional pulmonary function measures. Moreover, the fusion of functional images from multiple modalities can map the regional physiology during an allergic response. Previous work in our laboratory has combined hyperpolarized gas MRI with proton MRI, and micro-PET to measure regional ventilation, inflammation, and cell metabolism for an allergic inflammation model of asthma in Brown Norway (BN) rats. In preliminary experiments, regional physiology measured after segmental allergen challenge is consistent with the location and cell concentrations observed with histology. Our hypothesis is that imaging can quantify the location and severity of airway response on a regional basis for both single time-point and longitudinal studies in a chronic asthma model. We propose to refine and apply these functional imaging methods to measure the extent and severity of inflammation relative to regions of airway narrowing and closure in chronic asthma. The specific aims of this proposal are: (1) To develop noninvasive imaging tools for functional imaging of airway obstruction, inflammation, and cellular activation associated with asthma in an allergic inflammation model, (2) To validate the measurements derived from the imaging tools developed in Aim 1 with histology, and (3) To measure the severity, extent, and pattern of ventilation changes, gas trapping, and inflammation in a model of chronic asthma. These experiments will improve our understanding of the timing and role of inflammation and cell activation in the onset and location of airway narrowing and closure.

Richard Flavell, Ph.D.

Investigating the Role of IL-9 in the Pathogenesis of Asthma

Allergic asthma is a chronic inflammatory disorder of the airways associated with reversible airway obstruction and bronchial hyperresponsiveness.  The immune response to antigen in the airways involves complex interactions between various inflammatory cells including lymphocytes, eosinophils, and mast cells.

Interleukin-9, a Th2 cell-derived cytokine with pleiotropic functions has been proposed to play a major role in the pathology of asthma.   Transgenic mice overexpressing IL-9 selectively within their lungs show many features of human asthma including eosinophilic and lymphocytic inflammation of the airways, mucus hypersecretion, subepithelial fibrosis mast cell hyperplasis and bronchial hyperresponsiveness.  In this study IL-9 transgenic mice will be used as a unique murine model to further define the role of IL-9 and mast cells in basic mechanisms involved in the pathogenesis of asthma.  This will be achieved by elimination of selected components of the immune response including mast cells, lymphocytes, eosinophils, and cytokines to reveal their impact in phenotypic changes in the lungs of IL-9 transgenic mice.  These studies will include experiments on mast cell degranulation and its effect on lung pathophysiology.  Recently generated, inducible IL-9 transgenic mice will be included in all these studies but especially used to study the effect of lung-specific IL-9 expression in a timely and coordinated manner.  A final specific aim will be the generation of IL-9-deficient mice to clarify the role of IL-9 in Th2 responses and T cell differentiation.  The study of IL-9 and its involvement in the pathogenesis of allergic asthma might reveal new important ways to develop alternative therapeutic strategies for more effective treatments of asthma.

David L. Garbers, Ph.D.

Guanylyl Cyclase Receptors: New Targets for Airway Remodeling Intervention

This proposal focuses on the plasma membrane class of guanylyl cyclase receptors (pGCs) and their ligands as one of, or possibly the most important brake for halting the pathophysiological developments that occur during asthma. Four members of the pGCs (3 with known ligands:1 an orphan), and at least one of the ligands, are expressed locally in airway tissue, but virtually nothing is known about their function. Our work shows a dramatic, rapid, specific adversarial relationship between various mitogens and pGC signaling pathways. We will concentrate on: 1) defining the molecular mechanisms by which mitogen signaling rapidly and specifically shuts down pGCs, 2) defining the mechanisms by which airway cells regulate the local production of cyclase receptor ligands, 3) identifying and defining the mechanisms of regulation of the protein kinases/phosphatases that regulate pGC activity, 4) constructing multiple mouse genetic models to define which of the various cyclase receptors are essential in blocking asthma-like phenotypes, or predispose mice to asthma when eliminated, and 5) discovering and determining the importance of the putative ligand for the orphan pGC expressed in lung/eosinophils. Although little is known about the role of these receptors in the airway, we can infer from our studies in other tissues/cells that these cyclase receptors severely oppose the actions of various mitogens at the level of cell chemotaxis, hyperplasia, hypertrophy, fluid secretion, ciliary beat frequency and extracellular matrix production, and therefore a plethora of positive effects may occur upon intervention to specifically regulate these cyclase receptor signaling pathways.

K. Christopher Garcia, Ph.D.

Targeting Interfaces in the IL-4 Receptor Complex: Structure and Design of Asthma Therapeutics

 Asthma is profoundly influenced by the cytokines IL-4 and IL-13, which interact with the IL-4 receptor (IL-4Ra), and subsequently signal through recruitment of either the common gamma chain (g_c) or IL-13 receptor (IL-13Ra1).  Certain immune cells release IgE antibodies in response to activation of the IL-4/13 receptors, ultimately resulting in the symptoms of allergic asthma.  IL-4Ra is the recycled component of this activation system and is therefore a compelling therapeutic target, which would benefit from structural information to guide the design of drugs.  The structural basis by which IL-4Ra signals through its tri-molecular receptor complexes is not known.  We propose to carry out a comprehensive structural analysis of IL-4Ra complexes, in tandem with combinatorial libraries to probe the newly resolved receptor-cytokine interfaces.

 Our aims are to:

1. Determine the structure of IL-4 in complex with IL-4Ra and g_c.
2. Determine the structure of IL-4 in complex with IL-4Ra and IL-13Ra1.
3. Determine the structure of IL-13 in complex with IL-13Ra1 and IL-4Ra.
4. Evolve IL-4Ra antagonists, using yeast display in vitro evolution.

This set of tri-molecular complexes will reveal new receptor-ligand and receptor-receptor interfaces towards which drugs, both protein and small molecule, can be targeted.  This enabling component of our proposal is a recent technological breakthrough which has resulted in determining the structure of the quaternary signaling complex of human IL-2 with its a, b and g_c receptors.  The methodologies developed for biophysical studies of this system will now be applied to IL-4Ra complexes.

Sankar Ghosh, Ph.D.

Cell-Permeable Peptide Inhibitors of NF-kB as Novel Therapies for Asthma

Inflammation plays a critical role in the pathogenesis of asthma. However, unlike many other widely prevalent inflammatory diseases such as rheumatoid arthritis, inflammation in asthma is primarily a T-cell driven process. In particular T-cell cytokines such as IL-4, IL-5, IL-9 and IL-13 are critical mediators of the overall disease process. Hence the ability to modulate the release of these cytokines from T-cells is likely to be beneficial for treatment of asthma. The inducible transcription factor NF-kB appears to be a particularly attractive target for therapeutic modulation since it not only controls the synthesis of the major pro-inflammatory cytokines IL-1 and TNF-a , but also affects the synthesis of the T-cell cytokines by regulating the development of TH2 cells. Our recent identification and characterization of a cell-permeable, peptide inhibitor of NF-kB activation has opened up the possibility of testing the hypothesis that inhibition of NF-kB in the lung will have a major effect in suppressing airway hyper-reactivity. To more specifically test the hypothesis that inhibition of NF-kB in T-cells will be sufficient, we have identified another peptide inhibitor that blocks the activation of the protein kinasePKCq, and hence specifically suppresses the activation of the NF-kB in T-cells. We believe that further characterization of such peptide inhibitors that can specifically block the activation of NF-kB when administered intranasally is likely to represent a novel strategy for the treatment of asthma.

Gennady Gololobov, Ph.D.

Catalytic Anti-IgE Antibodies

This proposal is devoted to isolating proteolytic antibodies capable of cleaving IgE efficiently and specifically.   Noncatalytic antibodies to human IgE are currently in clinical trials for treatment of allergic asthma.  Catalytic antibodies are predicted to inactivate IgE with superior potency compared to reversibly binding antibodies, because permanent inactivation of IgE will occur as a consequence of the catalytic reaction and because a single catalyst molecule can be reused to cleave multiple IgE molecules.

We proposed to target the CH2-CH3 interdomain junction site in the epsilon chain of IgE by the catalysts.  This peptide determinant is necessary for the binding of IgE to its high affinity Fc receptor (FceRI) found on basophils and mast cells.  Cleavage of IgE at this determinant is hypothesized to render it incapable of binding the inflammatory cells, thus precluding inflammatory mediator release responsible for the allergic reaction.  Moreover, cleavage of IgE expressed on B cells should render the cells incapable of binding the allergen, preventing allergen-driven IgE synthesis.

The source of the catalysts will be pooled lymphocytes from patients with autoimmune disease and asthma, who are known to express autoantibodies capable of binding IgE and autoantibodies with proteolytic activity.   A library of Fv constructs will be prepared from the peripheral blood lymphocytes and expressed on the surface of phage particles.  A chemically reactive analog of the CH2-Ch3 junction peptide containing a phosphonate diester capable of reacting covalently with serine protease type of antibodies will be applied for selecting proteolytic Fv constructs.  The selected Fv constructs will be characterized for the catalytic properties (kinetic parameters, specificity and cleavage sites).  Thereafter, they will be studied for the ability to suppress IgE-induced degranulation of basophils.

Michael J. Grusby, Ph.D.

The Role of IL-13 Receptor Signaling in the Pathogenesis of Asthma

The Th2 cytokine IL-13 has recently been demonstrated to be an important mediator of allergic asthma. Although a number of components of the IL-13 signaling pathway have been identified, their respective roles in contributing to the pathogenesis of this disease remain unclear. The experiments described in this proposal aim to reveal the molecular mechanisms by which IL-13 receptor signaling contributes to the allergic asthma phenotype. Our approach will be to generate mice deficient in the expression of IL-13Ra1 and IL-13Ra2 to further understand the distinct roles that these two receptors play in mediating the biologic effects of IL-13. We will also generate mice harboring a conditional allele of Stat6 so that we may examine its role in the effector function of mature Th2 cells and other cell types in the lung. Finally, we seek to identify new IL-13-regulated and Stat6-dependent genes that may be useful therapeutic targets for the treatment of allergic asthma.

Leonard P. Guarente, Ph.D.

Sirtuins as Novel Regulators of Inflammatory Responses in Asthma

Asthma is a disease involving local and systemic allergic inflammation, which leads to airway obstruction and respiratory insufficiency. A positive correlation has been found between asthma (and certain other inflammatory diseases) and obesity (and the related metabolic syndrome). Calorie restriction (CR) triggers physiological changes opposite to metabolic syndrome, and has been shown to extend life span and mitigate many diseases in rodent models. This proposal will test whether asthma can be mitigated by CR, and whether genetic alterations in the SIRT gene family, encoding mammalian sirtuins, impact this disease. In Aim 1, we will test the effects of CR or high fat diets in a mouse asthma model. We will also determine the effects of reducing or increasing the activity of SIRT1 and other sirtuin genes on this disease. If effects are found, we will pinpoint tissues in which SIRT1 or other sirtuins affect asthma by using tissue specific knockout mice and bone marrow transplantation. In Aim 2, the effects of resveratrol and other activ