2006 Research Grant Awardees

The American Asthma Foundation awards grants to investigators who are pursuing new, breakthrough pathways for treating, preventing, and curing asthma.

Senior Investigator Awards

• John P. Atkinson, M.D.

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

  Washington University School of Medicine

  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.
  Back to Top

• Michael B. Brenner, M.D.

  New Class of Allergens in Asthma

  Harvard Medical School

  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.
  Back to Top

• David W. Christianson, Ph.D.

  Structural and Chemical Biology of Arginase in Asthma

  University of Pennsylvania

  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.
  Back to Top

• V. Michael Holers, M.D.

  Mechanisms of Complement Mediated Inflammation in Asthma

  University of Colorado at Denver and Health Sciences Center

  The complement system has been proposed to play a major role in the pathogenesis of asthma. This hypothesis is based on the presence of increased complement pro-inflammatory activation fragments and their receptors in patients with asthma as well as protection from the development of asthma with complement blockade in mouse models. There are at least three activation pathways and seven unique receptors whereby complement can promote inflammation in target organs such as the lung; however, blocking individual complement receptors has not led to clinical benefit. We have devised a novel method of dampening the entire complement system by the discovery that one pathway, designated the alternative pathway, is required for all seven downstream pro-inflammatory mechanisms to be engaged, no matter how the system is activated. Importantly, preliminary data have shown that blockade of the alternative pathway with a therapeutic with potential application to humans is highly effective in a murine model of asthma. Additional potential limitations of systemic complement inhibition include infectious risk and the necessity to block serum proteins that are present at high concentrations. Using a novel recombinant complement receptor (CR2)-based target inhibition strategy, these two limitations have been overcome and therapeutics can be directed specifically to sites of complement activation. We propose to further develop and test alternative pathway inhibitors for asthma as well as test the hypothesis that systemically administered, CR2-targeted alternative pathway inhibition of complement can ameliorate asthma.
  Back to Top

• Alexandra L. Joyner, Ph.D.

  The Role of Sonic Hedgehog Signaling During Airway Remodeling in Asthma

  Sloan-Kettering Institute/HHMI

  Very little is known about what triggers airway remodeling in asthma, or the molecules that lead to hyperplasia of smooth muscle and fibroblasts. Shh and TGFß are good candidates for being involved in airway remodeling, since they work together to regulate normal lung development and have been implicated in fibrosis. Furthermore, Shh is an ideal target for therapy since effective small molecule antagonists are available, some of which have been shown to be successful in mouse models of diseases. Using a novel fate mapping approach to study cells responding to Shh and conditional mutants recently developed, we will determine whether Shh signaling through Gli proteins is involved in this process, and whether the TGFß and Shh pathways intersect or are largely independent in airway remodeling. Hypothesis and Specific Aims: 1. Determine the fate of Shh-responding cell populations in the mouse during normal lung airway development and airway remodeling in asthma, using Genetic Inducible Fate Mapping (GIFM) of Shh-responding cells. Hypothesis: Shh-responding cells contribute to the expansion of smooth muscle, fibroblast and epithelial cells during normal airway development and airway remodeling caused by chronic asthma. 2. Determine whether Shh signaling through the Gli proteins is necessary for lung airway remodeling in asthma, and whether TGFß acts with Shh to regulate airway remodeling by inducing asthma in Gli1 null mutants and mice carrying conditional mutations in Gli2 and Gli3 in the adult lung mesoderm. Hypothesis: Shhsignaling and TGF are required for airway remodeling caused by asthma.
  Back to Top

• Christopher L. Karp, M.D.

  The Role of Aeroallergen Mimics of TLR Complex Proteins in Asthma Pathogenesis

  Cincinnati Children’s Hospital Medical Center

  These studies aim at a mechanistic understanding of why specific proteins act as aeroallergens in susceptible hosts. Early exposure to high amounts of ambient lipopolysaccharide (LPS) appears to protect against atopy and allergic asthma. On the other hand, LPS can also exacerbate established asthma. Mouse studies have shown that: (a) airway sensitization with an LPS-free model antigen induces tolerance; (b) sensitization with antigen along with low doses of LPS drives Th2 immune responses and allergic asthma; and (c) sensitization with antigen along with high doses of LPS drives Th1 and/or counter-regulatory responses. Mouse models have also provided mechanistic confirmation of the ability of LPS to exacerbate established asthma. TLR4 is the signaling receptor for LPS. Signaling depends on M.D.-2, the LPS-binding member of the receptor complex. The group II major house dust mite allergens (e.g., Der p 2) are members of a novel group of M.D.-2-related lipid-recognition proteins. M.D.-2 and Der p 2 have high structural similarity. Preliminary data suggest that Der p 2 interacts directly with the TLR4 complex, facilitating LPS signaling. These data suggest that: (a) Der p 2 tends to be a target of adaptive immune responses because Der p 2 has intrinsic adjuvant activity; (b) Der p 2 drives aero-allergic responses under conditions of low LPS exposure by shifting the LPS-response curve into the Th2-inducing range; and (c) Der p 2 promotes asthma exacerbation by facilitating TLR4 signaling in airway cells.
  Back to Top

• James L. Manley, Ph.D.

  Alternative Splicing of mRNA Precursors: Links to Asthma

  Columbia University

  Alternative splicing of mRNA precursors is a common mechanism of gene control in human cells. Furthermore, splicing defects are now known to play a role in a large number of human diseases. Although only a few possible examples of association between asthma and defects in splicing have been described, the possibility that this is the case in asthma merits investigation. Here we propose several lines of study to address this issue, and the following Specific Aims are proposed: 1. We will first systematically study the regulation of alternative splicing of glucocorticoid receptor (GR) pre-mRNA. Alterations in GR splicing result in corticosteroid insensitivity and lead to clinically difficult cases of asthma in affected patients. The proposed experiments are aimed at identifying the mechanism of this regulation, and the RNA sequences and protein factors involved, with the ultimate goal of discovering novel targets for future therapy. 2. We propose a series of experiments designed to rigorously test the possible connection between asthma and alternative splicing of the mRNA encoding a molecular pattern recognition receptor, NOD1, and to explore the details of the mechanism that regulates this alternative splicing event. 3. We propose a large-scale microarray-based study aimed at identifying novel asthma-specific changes in alternative splicing. Newly identified genes will be investigated further to gain a broader understanding of the molecular and physiological basis of asthma and as potential therapeutic targets.
  Back to Top

• Ira Mellman, Ph.D.

  Dendritic Cell Biology and Asthma

  Yale University School of Medicine

  Dendritic cells (DCs) are well known to play critical roles in regulating the airway immune response and in the pathogenesis of asthma. However, little is known about how they maintain tolerance to environmental allergens while initiating immunity to invading pathogens, often at the same time. Nor is it clear how DC function is altered in the asthmatic lung, making it difficult to contemplate DC-targeted therapeutic strategies. We propose to apply a range of approaches to characterize the cell biology and function of airway DCs in the normal lung and in murine models of asthma. Using animals bearing genetically-encoded reporters and actuators to probe DC function, three fundamental issues will be addressed. (1) We will examine the distribution and dynamics of myeloid and plasmacytoid DCs in normal and diseased lung using high resolution confocal and multiphoton fluorescence microscopy to determine the distribution and dynamics of distinct DC populations in fixed and living tissue. (2) We will determine how airway DCs interact with allergens and pathogens using imaging and biochemical approaches to reveal mechanisms of capture as well as the abilities of distinct DC populations to induce tolerance or immunity to allergens or microbes. (3) Based on our finding that alterations in cell-cell adhesion (via E-cadherin/ß-catenin) triggers a potentially tolerogenic pathway of DC maturation, we will determine if adhesion molecule-dependent signals contribute to tolerance. If so, we will test a pharmacologic approach aimed at augmenting tolerance in an effort to diminish the inflammatory Th2 response characteristic of asthma.
  Back to Top

• Eric N. Olson, Ph.D.

  Toward Transcriptional Therapies for Asthma

  University of Texas Southwestern Medical Center

  Asthma is a chronic inflammatory disease of the airways characterized by reversible airway obstruction and inflammation. Inappropriate contraction of airways smooth muscle cells in addition to smooth muscle cell hyperplasia and hypertrophy have been implicated in the development of airway obstruction which accompanies asthma. Recently, exciting progress has been made toward understanding gene regulatory changes associated with asthma. In particular, it has been shown that the proinflammatory state associated with severe stages of asthma is dependent on and controlled by alterations in chromatin structure resulting from a change in the balance between histone acetylation and histone deacetylation. Our laboratory has discovered key roles for histone deacetylases (HDACs) and stress-dependent signaling pathways in the control of muscle hypertrophy and pathological remodeling. Given the involvement of abnormal growth and function of airway smooth muscle cells and HDACs in asthma and inflammatory airway disease, we propose to apply our knowledge and unique genetic tools to dissect the mechanisms involved in the pathogenesis of asthma. These studies have the potential to lead to the development of small molecule therapeutics or novel nonsteroidal anti-inflammatory treatments that mimic the actions of corticosteroids on inflammatory gene regulation to prevent or reverse asthma.
  Back to Top

• Ralph Weissleder, M.D., Ph.D.

  In Vivo Imaging of Cellular and Molecular Mediators in Asthma

  Massachusetts General Hospital

  Novel molecular imaging techniques have had a significant impact on the understanding and clinical management of cancer and cardiovascular diseases but have not been used to their full extent to further our understanding of asthma. In addition, immune cells and cellular mediators often behave differently in intact in vivo microenvironments than in in vitro assays. The overall goal of this proposal is to develop and validate novel imaging approaches to identify key molecular and cellular events in experimental airway inflammation and asthma. The discovery effort will utilize a number of agents and approaches pioneered in our laboratory, including amplifiable “smart” enzyme sensing probes, novel cell trackers based on fluorescent nanomaterials, fiber optical detection technology, optical tomography and nuclear imaging techniques. In the first aim we will utilize recently developed protease specific probes to assess pulmonary tryptase, MMP and cathepsin activity during disease progression and its modulation after therapeutic intervention. In the second aim we will use recently developed multimodal (detectable by different techniques), metabolically inert, cell-tagging reagents for serial imaging to assess the mechanisms employed by both anti-allergic Treg cells and CpG motifs to prevent deleterious Th2 responses. We will obtain a signature pattern that has not been perturbed by invasive protocols, and therefore closely represents the unfolding molecular and cellular activities in the effector organ and other sites such as the draining lymph nodes and spleen. The novel imaging techniques will allow us to uncover events in asthma in vivo, ultimately allowing for clinical translation.
  Back to Top

Extension Awards

• Richard A. Bond, Ph.D.

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

  University of Houston

  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.
  Back to Top

• Danuta Radzioch

  Preclinical Studies on the Application of TLR7 as a Therapeutic Agent Against Allergic Asthma

  McGill University

  The Toll-like receptor (TLR) family plays a crucial role in both innate and adaptive immune responses. Asthma pathology is primarily caused by a dysregulated inflammatory response to innocuous allergens. Our results have clearly demonstrated the powerful anti-inflammatory effects of the TLR7 ligand imidazoquinoline S28463 (TLR7L) in both mice and rats. Therefore, we believe that further preclinical investigations are now required so this promising treatment might be introduced into preclinical trials and eventually into clinical practice. Our proposed studies will characterize the kinetics of the protective effect of TLR7L against allergic asthma in Brown Norway rats and A/J and C57BL/6 mice. Next, we will determine the effect of TLR7L against allergen induced asthma in Rhesus monkeys, by monitoring the physiological and molecular consequences resulting from the treatment. We will also perform complete genomic, proteomic and metabolic analysis of TLR7L-treated animals (mice, rats, monkeys), in order to reveal the exact molecular mechanism of the observed protective effect of TLR7L against allergic asthma. These studies exploring the mechanism of TLR ligand-mediated effects on allergic asthma may also explain how environmental factors impact the adaptive immune system, protecting the host against asthma. Based on the preliminary data generated using this promising compound, it is very likely that modulating the interaction between TLRs and their ligands will have significant therapeutic benefits for asthma patients.
  Back to Top

Early Excellence Awards

• Robinna G. Lorenz, M.D., Ph.D.

  Modulation of Allergic Asthma by Gastric Helicobacter Infection

  University of Alabama at Birmingham

  Over the past 50 years, the incidence of asthma has been increasing in developed countries. This increased incidence has been attributed to a decrease in infections acquired early in childhood, which may shape subsequent immune responses. Although there have been multiple clinical associations between gastrointestinal infections and decreased asthma incidence, it is difficult to move beyond a simple correlation when studying human patients. We propose to directly test this association and its subsequent disease modifications through the use of our laboratory’s well-established murine model of gastric helicobacter colonization and inflammation. Our hypothesis states that both the lung and the stomach are components of the common mucosal immune system, and therefore, gastrointestinal colonization and infection can shape the response of the lung to inhaled allergens. Our preliminary data indicates that pulmonary expression of several key initiators of inflammation, such as TSLP and CXCL15, are altered by gastric helicobacter infection. The following specific aims are proposed: 1) Determine the role of helicobacter-induced helper and regulatory T-cells in modulating a murine model of allergic asthma; 2) Investigate the effects of early mucosal exposure to pathogenic bacteria on subsequent development of allergic asthma; 3) Define the importance of changes in TSLP and CXCL15 in helicobacter modulation of allergic asthma. Our expertise in murine models of gastrointestinal inflammation and mucosal immunity will allow us a unique focus on the mechanisms through which pulmonary inflammation develops to inhaled allergens. These results should stimulate further research into novel therapeutic approaches for allergic asthma.
  Back to Top

• Boris Reizis, Ph.D.

  Dendritic Cells in the Pathogenesis and Therapy of Chronic Asthma

  Columbia University

  Asthma is a chronic disease caused by aberrant T cell response to innocuous inhaled antigens. As the primary antigen-presenting cell type in the body, dendritic cells (DC) appear critical for asthma development, and therefore represent potential targets of asthma therapy. However, the precise role of DC in the course of chronic asthma and in the action mode of emerging asthma therapies is poorly understood. We hypothesize that conventional DC and interferon-producing plasmacytoid DC (PDC) play important and specific roles in the normal lung homeostasis and at each step of chronic asthma development. We further propose that immunosuppressive drugs used or developed for asthma therapy, such as cyclosporine and peroxisome proliferator activated receptor gamma (Pparg) agonists, might work at least in part by modulating DC functions. To address these hypotheses, we are developing genetic approaches for constitutive, long-term cell ablation and gene targeting in DC and/or PDC in vivo. We will now use DC-specific gene targeting to dissect the role of DC in chronic asthma and in anti-asthma therapies, including: (i) the function of DC subsets in the maintenance of tolerance in normal lung and at different stages of chronic asthma; (ii) the role of Pparg in the control of lung DC activation and in asthma therapy with Pparg agonists; (iii) the role of calcium/calcineurin signaling in DC in the development of chronic asthma and its treatment with cyclosporine. Altogether, these studies might elucidate important mechanisms of asthma pathogenesis, and facilitate the development of more selective immunosuppressive therapies for the disease.
  Back to Top

• John B. Wallingford, Ph.D.

  Molecular Basis of Mucociliary System Development and Maintenance

  University of Texas at Austin

  Defects in mucus clearance from the airway contribute significantly to mortality in patients with asthma. The mucociliary system, consisting of mucus-secreting goblet cells and ciliated cells, normally generates a constantly overturning layer of protective mucus that lines the airway epithelium. In asthmatic airways, goblet cell hyperplasia leads to an excess of secreted mucus, while a coincident decrease in the number of ciliated cells leads to a deficit in mucus clearance. Moreover, the normally motile cilia atop ciliated airway cells fail to beat effectively in asthmatics, exacerbating defects in tracheobronchial clearance. The molecular mechanisms underlying these defects remain very poorly understood. Here, we propose first to use a very simple model ciliated epithelium to develop specific hypotheses concerning the molecular mechanisms of differentiation in the mucociliary system. We then propose to use mouse models to ask how this knowledge may be used to revert or prevent the mucociliary system defects associated with asthma. Specific Aims include: 1. Determine the role of the Wnt/PCP signal transduction cascade in cilia morphogenesis and function in a model ciliated epithelium. 2. Using data from a pilot screen, determine the molecular mechanisms by which Hedgehog signaling controls ciliated versus goblet cell fate choices in a model ciliated epithelium. 3. Extend findings from Aims 1 and 2 to the mammalian airway epithelium. 4. Test potentially therapeutic manipulations in mouse models for asthma. The data from these experiments will provide a major step forward in understanding and potentially combating deadly defects in the mucociliary systems of asthmatics.
  Back to Top

• Ding Xue, Ph.D.

  Molecular Genetic and Pharmacological Studies of the ADAM33 Regulatory Network in C. elegans and Asthma

  University of Colorado, Boulder

  ADAM33 is the first asthma susceptibility gene cloned by the positional cloning method and has been implicated in regulating airway remodeling and the functions of lung cells. As a metalloprotease with several distinct protein-protein interaction motifs, how ADAM33 acts as a protease to affect the activities of other membrane proteins and how it interacts with other membrane and extracellular proteins to regulate airway cell interaction, adhesion, and migration become a fundamental issue and are critical for the understanding of ADAM33’s involvement in the pathogenesis of asthma and bronchial hyperresponsiveness. We propose to conduct molecular genetic and biochemical studies in C. elegans to identify substrates, regulators, and interaction partners of two C. elegans ADAM33 homologues. In addition, we will apply a newly developed drug screen protocol in C. elegans to search for compounds that can modulate the activity of the ADAM33 regulatory network in combination with using mice asthma models. These systematic genetic, biochemical, and pharmacological studies not only will advance our understanding of the basic biology of ADAM33 but also will reveal the complex network regulated by ADAM33 to promote proper airway remodeling. These studies will lead to identification of new compounds or targets for therapeutic interventions in the treatments of asthma and bronchial hyperresponsiveness.
  Back to Top

2005 Research Grant Awardees

Senior Investigator Awards

• Albert Bendelac, M.D., Ph.D.

  NKT Cell Regulation of Human Asthma

  University of Chicago/HHMI

  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.
  Back to Top

• Dan R. Littman, M.D., Ph.D.

  Dendritic Cell Function in Asthma Pathogenesis

  NYU School of Medicine, Skirball Institute of Biomolecular Medicine/HHMI

  Several recent studies have suggested that dendritic cells have key roles in the pathogenesis of asthma. Dendritic cells contribute to activating allergen-specific T helper 2 cells, but they are also thought to activate regulatory T cells that prevent aberrant inflammatory responses to airway antigens. Recent studies have shown that mice with null mutations for the transcription factors Runx3 and CBFß2, which form heterodimers that regulate numerous target genes, develop airway inflammation characteristic of asthma and display accelerated dendritic cell maturation. We will study how loss of Runx/CBFß contributes to the development of the asthma-like syndrome and will focus on the following aims: (1) We will generate mouse strains that lack expression of Runx1, Runx3, and CBFß in distinct myeloid and lymphoid lineages, and will determine which cells are responsible for airway inflammation. (2) We will eliminate regulatory T cells systemically and in lung by administering diphtheria toxin to mice in which diphtheria toxin receptor is expressed exclusively in regulatory T cells, and we will determine if the animals can be tolerized to airway antigen and if they become more susceptible to spontaneous and allergen-induced asthma. The influence of regulatory T cell depletion in mice with Runx/CBFß deficiencies will be examined. (3) Genetic targets of Runx/CBFß in different subsets of dendritic cells will be characterized by RNA expression analysis with mutant and wild type animals. We anticipate that this approach will provide insight into pathways of asthma pathogenesis and will provide novel targets for therapeutic intervention.
  Back to Top

• Yong-Jun Liu, M.D., Ph.D.

  Function of TSLP-activated Dendritic Cells in the Maintenance of Human TH2 Memory T Cells

  University of Texas M.D. Anderson Cancer Center

  The immunopathological hallmark of allergic diseases is the infiltration and accumulation of polarized CD4 TH2 effector T cells at the sites of inflammation. Recent studies suggested that the memory-like TH2 T cells are the principle cell population responsible for the maintenance of chronic allergic inflammation and the rapid relapse of acute allergic inflammation upon re-exposure to allergens. However the nature of TH2 memory cells and mechanisms regulating their maintenance has remained elusive. Recently a subset of human CD4+ T cells that express a novel G-protein-coupled receptor for prostaglandin D2 (CRTH2) was isolated from human blood, which produced IL-4, IL-5, and IL-13, but not IFN-? immediately upon TCR-triggering. Our microarray analyses show that human CRTH2+ T cells express the gene signatures of TH2 central memory T cells and reveal the new molecular mechanism underling the maintenance of TH2 memory T cells. We also show that DCs activated by a novel cytokine, TSLP, have the capacity to prime TH2 responses, induce more than a 10-fold expansion of the CRTH2+ T cells, and maintain their TH2 functions. Our specific aims are: 1) To determine whether CRTH2+ T cells represent TH2 memory cells; 2) to investigate the molecular mechanisms by which TSLP-activated DCs expand and maintain the CRTH2+ T cells; and 3) to compare CRTH2+ T cells from normal versus asthmatic human subjects. Isolation and characterization of TH2 memory T cells and understanding the molecular mechanisms underlying their generation, maintenance, and responses holds the future promise for developing cures for allergic diseases.
  Back to Top

• Deborah A. Meyers, Ph.D.

  Gene-gene Interaction in Mouse and Man

  Wake Forest University Health Sciences

  There is evidence that multiple genes are important in determining individual susceptibility to the development of asthma. Therefore, it is important to test for gene-gene interaction in performing genetic studies of asthma. In family studies, evidence from genome wide screening and linkage analysis for interactive effects between genes on different chromosomes would facilitate gene mapping and positional cloning. In addition, previous genome wide screen linkage analyses have not shown consistent results for asthma in different family populations. Gene-by-gene interactions may contribute to this lack of reproducibility in these previous studies. Two-locus gene-gene interactions for all possible pairs of loci will be modeled across the genome in several samples of asthma families that have already been genotyped. In addition, similar analyses will be performed on two genome wide screens performed in the mouse for bronchial hyperresponsiveness. Homolog mapping will then be performed using man-mouse homology as a powerful approach to narrow chromosomal regions of interest and identify disease genes. This approach has been very productive in identifying genes for cardiovascular diseases. Evidence for these genes will be then replicated in several case-control populations. These novel approaches, utilizing data and DNA from multiple studies in both man and mouse, are needed because interactions of multiple genes influence individual risk for developing asthma.
  Back to Top

• Alexander Y. Rudensky, Ph.D.

  Genetic Analysis of the Role of Regulatory Foxp3+ T Cells in Asthma

  University of Washington/HHMI

  Chronic Th2 type responses to airborne environmental antigens or microorganisms in the lung and bronchial tissue result in allergic asthma manifested by airway inflammation and hyper-reactivity. Regulatory T cells (TR) expressing transcription factor Foxp3 serve as a critical control mechanism of autoimmunity. Recent studies have provided evidence that CD25+ TR cells likely suppress immune responses associated with the asthma pathogenesis. However, this work has been limited by the fact that CD25 is also expressed on all recently activated T cells and, unlike Foxp3, does not serve as a definitive TR marker. Furthermore, we found that the majority of TR present within the lung tissue lack or exhibit low level of CD25 expression. Therefore, TR role in asthma remains largely unknown. In this proposal we will employ genetically manipulated mice to unambiguously investigate TR dynamics and function in asthma and to dissect TR-mediated effector mechanisms. Specifically, we will investigate a role for Foxp3+ TR cells in allergic asthma using Foxp3gfp knock-in mice and mice with an inducible suicide gene “knocked” into the Foxp3 locus. We will also examine a role for TR-produced major anti-inflammatory cytokines IL-10 and TGF-ß in limiting airway inflammation using mice with the TR-specific ablation of these genes. Understanding the TR mediated control of asthma will provide rationale for assessment of the immunological status of asthma patients by monitoring dynamics of Foxp3+ TR cells. In addition, our studies may facilitate development of novel therapeutic approaches to treatment of asthma by harnessing specific immunosuppressive mechanisms of TR cells.
  Back to Top

• Jenny P-Y Ting, Ph.D.

  Innate Immune Genes and Asthma

  University of North Carolina at Chapel Hill

  Evolutionarily conserved pathways in host defense and immunity have been pivotal in uncovering novel bases of immunologic diseases, and in providing new drug targets. The newly discovered CATERPILLER (CLR) family share structural similarities with the NBS-LRR (nucleotide-binding sequence, leucine-rich repeat) super-family of plant disease resistance (R) proteins,,. In plants, the R proteins mediate an array of host responses to contain the spread of pathogens. Among the CLR genes, Monarch-1 (Pypaf7) is expressed by granulocytes, eosinophils, and monocytes, cells of significant relevance to asthma. Its expression is dramatically reduced by TLR2 and TLR4 agonists. Preliminary data indicate that among patients who have a history of mild asthma, the level of Monarch-1 transcript in sputum samples is reduced relative to normal controls. A reduction of Monarch-1 expression by interference RNA (RNAi) caused a profound enhancement of TLR-induced NF-kB activation, and proinflammatory cytokine/chemokine expression. These all strongly support our working hypothesis that Monarch-1 is a negative regulator of proinflammatory responses and potentially of asthma. Negative regulators of immune responses are necessary to prevent uncontrolled and overzealous responses including allergic inflammation and asthma. The goal of this proposal is to test the hypothesis that Monarch-1 is a negative regulator of allergic inflammation and asthma. The Aims are to: 1. Profile Monarch-1 expression in induced sputum samples obtained from asthmatic individuals. 2. Define the role of Monarch-1 in a mouse model of allergic inflammation. 3. Assess the binding and modulation of Monarch-1 by nucleotides. This is important because nucleotide analogs are candidates for drug therapy.
  Back to Top

Junior Investigator Awards

• K. Christopher Garcia, Ph.D.

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

  Stanford University/HHMI

  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 (?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 gc.
  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, ß and ?c receptors. The methodologies developed for biophysical studies of this system will now be applied to IL-4Ra complexes.
  Back to Top

• Anne-Laure Perraud, Ph.D.

  The Oxidant and ADP-ribose Gated TRPM2 Ion Channel and the Inflammatory Response in Asthma

  National Jewish Medical and Research Center

  Asthma is a disease of the immune system, resulting in a chronic inflam mation of the airways. Activated inflammatory cells recruited to the lungs release chemokines and oxidants, leading to tissue damage, and ultimately persistent airway hy per-responsiveness. Immunocytes recovered from BAL-fluid and blood samples of asthma patients have been shown to generate higher amounts of oxidants than cells from healthy subjects, further contributing to the general redox imbalance observed in the context of the disease. More recently, the concept of oxidant signaling has been intro duced, as it appears that ROS production is a common signaling event in immunology, implying that even subtle changes in the amount of generated Reactive Oxygen Species (ROS) might affect the inflammatory response. Although ROS signaling was mostly thought to be mediated by the direct oxidation of biomolecules, it has been recently shown that mitochondrial ADP-ribose production following oxidant exposure leads to the activation of the ADP-ribose gated TRPM2 ion channel. TRPM2 is the unique fusion of a Ca2+-permeable channel with an ADP-ribose hydrolase, and we have shown that TRPM2 gene expression is differentially regulated in primary mouse leukocytes, further support ing the potential role of TRPM2 and ADPR in modulating the immune response. ADP-ribose generating molecules, such as CD38, a puzzling multifunctional receptor with NAD-glycohydrolase activity, might also play a role in regulating Ca2+-entry via TRPM2. The proposed study aims at analyzing the potential involvement of the TRPM2/ADP-ribose pathway in the inflammatory response following allergen sensitization and chal lenge in a mouse model of asthma.
  Back to Top

• Christine Pham, M.D.

  Role of Cathespins in Murine Models of Asthma

  Washington University, St. Louis

  Although proteases have been implicated in airway diseases, the role of cathepsins is not well studied. Cathepsin C (CtsC) is a ubiquitously expressed lysosomal protease with specific function in many effector cells, including neutrophils, mast cells, cytotoxic T lymphocytes (CTL), and natural killer (NK) cells. In contrast, cathepsin W (CtsW) is unique among the cathepsins because of its restricted pattern of expression, namely CTL and NK cells, and its unusual endoplasmic reticulum localization. Here we showed that cathepsins are important in modulating the innate and adaptive immunity in allergen- and viral-induced airway inflammation. In a model of airway inflammation induced by ovalbumin (OVA) sensitization, we found that CtsW-/- mice developed more airway hyper-reactivity (AHR) and goblet cell hyperplasia. Similarly, CtsW-/- mice harbored significantly higher number of IFNã-producing immune cells in their lungs following infection with the mouse parainfluenza virus type 1 (Sendai virus or SeV). Surprisingly, CtsC-/- mice display a much more attenuated response following SeV infection. These results suggest that a better understanding of the mechanisms by which these proteases control effector functions may potentially lead to novel therapeutic interventions in the treatment of airway diseases. To this end, we propose to 1) define the in vivo mechanisms by which CtsW regulates the development of allergen-induced airway inflammation and hyper-reactivity; 2) define the role of CtsW and CtsC in the development of acute and chronic airway hyper-responsiveness following SeV infection; and 3) determine the mechanisms that confer relative protection against SeV infection in the absence of CtsC.
  Back to Top

2004 Research Grant Awardees

Senior Investigator Awards

• David E. Clapham, M.D., Ph.D.

  TRP Ion Channels as New Targets in Asthma

  Children’s Hospital of Boston/HHMI

  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.
  Back to Top

• Vivek Malhotra, Ph.D.

  Molecular Basis of Mucin Secretion in Airway Goblet Cells

  University of California, San Diego

  Mucus hyper-secretion is a major cause of pathology in asthma and in several respiratory diseases. It leads to plugging of small airways and a dramatic reduction of airflow. Nearly all cases of lethal asthma are associated with airway occlusion by mucus plugs. A pharmacological control of mucus secretion would greatly improve asthmatic symptoms and reduce long-term damages. At present, there is no such treatment available. Mucus is composed mostly of mucins, which are secreted largely by surface epithelial goblet cells. Goblet cells accumulate mucins in secretion granules and release their content upon stimulation. The molecular mechanisms controlling mucin granule formation and release are poorly understood at present. We plan to develop an airway goblet cell line where exogenous horseradish peroxidase enzyme is targeted to mucin granules, allowing for a simple, robust and quantitative assay of secretion. We will use this assay to study the formation and exocytosis of mucin granules. We will also use this system to screen specific gene families by siRNA and perform a high-throughput screen of chemical libraries. These approaches will give us a better understanding of mucin granules biology and reveal new regulatory genes of mucin secretion and new chemicals affecting this process. Furthermore, the chemical library screen could yield entirely new leads for treatment of obstructive pulmonary diseases in general and asthma in particular.
  Back to Top

• Tobias Meyer, Ph.D.

  Mast Cell Signaling Networks

  Stanford University

  Mast cells are key players in acute allergic reactions and have also been strongly implicated in the pathogenesis of asthma. Mast cells express the FceR1 receptors which bind IgE antibodies with high affinity. When multivalent antigens contact IgE bound to FceR1 receptors, the receptors aggregate, triggering a signaling cascade that leads to the extracellular release of histamine, serotonin, proteases, cytokines, and other mediators from the mast cell’s cytoplasmic granules. Many of these secreted mediators have been found to enhance airway inflammation and to cause bronchial and vascular smooth muscle contraction. The secretion response in mast cells occurs rapidly – within minutes – and is regulated by a complex network of potentially tens of second messengers and hundreds of signaling proteins. In this proposal, we will use novel high-throughput single-cell imaging and analysis techniques, together with comprehensive DNA expression and siRNA signaling protein libraries, to carry out large-scale primary and secondary screens of the ~2000 signaling proteins estimated to be in mast cells and to quantitate their effects on mast cell secretion and intermediate signaling. Our strategy will likely lead to two important results. First, we will identify new players in the mast-cell signaling system that can explain how FceRI activation triggers histamine release. Second, the timecourse data obtained simultaneously at multiple points in the signaling network will be useful to identify the overall modular structures and cross-talk in the mast cell signaling system. Both results will provide new insights into weak spots in the mast cell signaling response that can then be exploited as potential drug targets.
  Back to Top

• Sem H. Phan, M.D., Ph.D.

  Bone Marrow Progenitor Cells in Airway Remodeling

  University of Michigan

  Airway remodeling in asthma contributes to bronchial hyperreactivity and reversible airflow obstruction. The overall objective of this project is to elucidate the origin of the fibroblasts/myofibroblasts involved in airway remodeling. Published and preliminary studies suggest that bone marrow derived progenitor cells can repopulate a variety of distal organs, including the lung. For instance, bone marrow derived cells can give rise to lung endothelial cells and fibroblasts, as well as alveolar and bronchial epithelium. Thus the central hypothesis of the project is that certain subpopulations of fibroblasts/myofibroblasts involved in airway remodeling are derived from bone marrow progenitor cells recruited by chemokine signals emitted by activated airway cells. To test this hypothesis four Specific Aims are proposed. First, the extent and kinetics of recruitment of bone marrow derived progenitor cells into remodeling airway wall will be examined in a murine cockroach allergen-induced model of asthma using GFP expressing bone marrow chimera mice. Second, the identity of the bone marrow progenitor cells will be determined. Third, the identity of the chemokines responsible for cell recruitment and their cellular source will be determined. Fourth, the significance of these recruited cells will be examined by evaluating the effects of abrogating their recruitment on airway remodeling. In this manner using a combination of immunological, cell biological and molecular techniques, the role of bone marrow progenitor cells in airway remodeling and the mechanisms of their recruitment can be elucidated. If this is determined to be important, then potentially novel therapeutic approaches could be envisioned for future studies.
  Back to Top

• Daphne Preuss, Ph.D.

  Immune Responses to Pollen Surface Components: Implications for Allergy and Asthma

  University of Chicago/HHMI

  Traditional searches for pollen allergens have identified cytoplasmic proteins encoded by pollen cDNAs, but have overlooked much of the extracellular pollen matrix, a region where allergens likely reside. The pollen extracellular matrix is encoded by cDNAs expressed in developing floral tissue, and its proteins contain hydrophobic domains and are typically absent from commercial pollen extracts. My research has focused on the pollen surface; we have identified Arabidopsis mutations that alter lipid content, examined protein functions and wall composition, identified the entire extracellular proteome, and used genomics to characterize pollen matrix components from several species. Our preliminary studies have shown that Arabidopsis pollen surface proteins elicit an immediate inflammatory response in naïve rabbits and cause non-hypersensitive animals to produce antisera that are ~200-fold more potent than those raised against typical cytosolic plant proteins. This proposal has three specific aims: 1) Extracellular and intracellular pollen fractions will be prepared from several species and probed with sera from individuals living in urban and rural environments; SNP genotypes and pedigrees will be used to examine genetic correlations with IgE binding. 2) Fractions containing allergens will be purified; we will isolate non-protein allergens and clone genes corresponding to novel protein allergens. 3) In collaborations at the University of Chicago, purified extracellular allergens will be tested for their ability to alter pulmonary physiology in mice or tostimulate responses in dendritic cells. This research could enhance allergy and asthma therapy through improved diagnostics or therapeutics that mitigate symptoms or promote immune tolerance.
  Back to Top

• Klaus Rajewsky, M.D.

  Cellular and Molecular Dissection of the Roles of NF-kB Activation Pathways and NKT Cell Autoreactivity in Asthma

  CBR Institute for Biomedical Research

  In asthma, allergens induce a complex interplay between various cell-lineages and tissues, ultimately leading to airway inflammation, obstruction, hyperreactivity and remodeling. These processes are accompanied and sustained by extensive reprogramming of gene expression. NF-?B proteins are a family of transcription factors that activate transcription of multiple genes involved in inflammation. These transcription factors play diverse roles in individual cell-types; therefore it is of great interest to elucidate their cell-type specific role in conditions of allergic lung inflammation. Recently it was discovered that Natural Killer T cells, whose generation depends on two forms of NF-?B activation, are essential for the induction of asthma in various mouse models. To date it is not clear whether activation of these autoreactive T cells, normally kept in check through inhibitory mechanisms, relies on recognition of lipid-antigens by their TCR, or on signaling through engagement of other receptors in the context of the immune response. Conditional gene targeting offers spatial and temporal control of genetic manipulations in the mouse. We propose to utilize the unique features of this technology, to explore aspects of NF-?B activation and NKT cell autoreactivity in the context of asthma as follows: Aim 1: To identify the role of individual NF-?B activation pathways in cell-types and tissues relevant for the induction of airway inflammation, hyperresponsiveness and remodeling. Aim 2: To analyze antigen recognition, differentiation potential and autoreactivity mediated by a canonical NKT cell receptor switched on in conventional T cells, replacing their original TCR.
  Back to Top

• William C. Sessa, Ph.D.

  Role of Nogo Isoforms in Airway Hypersensitivity and the Pathogenesis of Asthma

  Yale University School of Medicine

  Using proteomics as an inductive approach for discovery, our lab has identified the protein Nogo-B highly expressed in pulmonary endothelial cells, airway smooth muscle cells and in pulmonary epithelial cells. The functional role of this Nogo isoform is unknown. In preliminary data, we show that the amino terminus of Nogo-B, unlike Nogo-A, promotes the adhesion of endothelial and smooth muscle cells, and serves as a chemoattractant for endothelial cells while antagonizing PDGF-induced smooth muscle cell migration. Moreover, in a paradigm of vascular injury, the loss of Nogo-A/B results in an exaggerated inflammatory response and neointimal proliferation, and therapeutic gene transfer of Nogo-B rescues these knockout phenotypes. Thus, goal of this proposal is to examine the role of Nogo-B as a newly found endogenous regulator of vascular and epithelial cell homeostasis in the lung. We surmise that the loss of Nogo-function contributes to both the vascular and inflammatory aspects of asthma. In this grant, we plan to generate transgenic models to overexpress soluble Nogo in the lung, examine the pathogenesis of asthma in both Th2 cytokine driven and allergic forms of asthma and to develop therapeutic strategies for delivering Nogo in different models of asthma.
  Back to Top

• Ann-Bin Shyu, Ph.D.

  Cytokine and Chemokine mRNA Turnover and Airway Inflammation

  University of Texas HSC at Houston

  The conservation of an AU-rich RNA-destabilizing element (ARE) in the 3′ non-translated regions of mRNAs coding for ~90% of cytokines and chemokines, including all Th2-type cytokines, suggests that regulation of cytokine and chemokine mRNA turnover via AREs is critical in determining the duration and level of cytokine and chemokine production. It is clear that ARE-containing cytokine mRNAs are differentially stabilized after lymphoid cell activation. Several ARE-binding proteins (ARE-BPs) are known to either accelerate or block ARE-mediated mRNA decay. We hypothesize that rapid decay of cytokine and chemokine mRNAs mediated by the AREs are compromised by changes in ARE-BP functions during allergic asthma, leading to persistently high levels of cytokines and chemokines. The specific aims are: 1) Develop an in vitro model to study the mechanism and regulation of chemokine and Th2-type cytokine mRNA turnover under conditions relevant to allergic responses in asthma; 2) Identify destabilizing and stabilizing ARE-BPs that regulate bronchial epithelial chemokine and Th-2 type cytokine mRNA stability during allergic inflammatory responses; 3) Characterize the dynamic interplay between destabilizing and stabilizing ARE-BPs in regulating chemokine and cytokine mRNA levels; 4) Identify cell type(s) critical to compromised ARE-mediated RNA decay, leading to development of allergic inflammation in an established mouse model. These studies should yield novel and crucial insights into major basic mechanisms by which asthma-related cytokine and chemokine mRNA stability is controlled, an important area of asthma research that is presently ill studied.
  Back to Top

• Yong-Rui Zou, Ph.D.

  The Pathophysiological Role of CXCR4 in the Adult Immune System and in Asthma

  Columbia University

  Asthma is an inflammatory disease characterized by polarized Th2 responses that leads to excessive IgE production and increased recruitment of leukocytes into the airways. An important chemokine involved in the progression of asthmatic inflammation has been shown to be CXCL12, as the treatment of allergen-sensitized mice with specific antagonists to its receptor, CXCR4, significantly reduces eosinophilia and suppresses airway hypersensitivity. We propose to analyze the mechanism underlying CXCR4-mediated pathogenesis in the mouse model of asthma. CXCR4-/- mice die in uterus, thus preventing analysis of the later function of CXCR4 in the immune system under physiological and pathological conditions. We have generated several mouse strains in which the CXCR4 gene has been deleted respectively in granulocytes, macrophages, B-lineage cells or T lymphocytes. First, we will characterize whether the inactivation of CXCR4 affects development, localization and function of these cells. Second, we will examine asthmatic responses in allergen-sensitized mice in which CXCR4 is inactivated in individual leukocyte lineages. Lastly, using additional mouse strains that carry the CXCR4 gene mutated in sequences encoding different conserved signaling domains, we will determine the precise biological effects and pathogenic roles contributed by different signaling pathways downstream of CXCR4. Together, these studies will help to identify the key leukocyte lineage and the important signaling pathway that contribute to the progression of asthmatic responses. Results obtained from our studies could facilitate the development of new therapeutic strategies in immune disorders.
  Back to Top

Junior Investigator Awards

• Sean B. Fain, Ph.D.

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

  University of Texas HSC at Houston

  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.
  Back to Top

• Peter J. Murray, Ph.D.

  Role of Macrophage Arginase in Asthma

  St. Jude Children’s Research Hospital

  Recent progress in asthma research has identified dramatic increases in genes involved in arginine metabolism in inflamed lungs. Arginase I (Arg I) is one of the most highly increased genes and expression of Arg I is most prominent in the lung inflammatory cells, including macrophages. We have been elucidating the mechanisms that lead to Arg I induction in macrophages. The strongest signals to switch on Arg I expression are the Th2 cytokines IL-4 and IL-13 that dominate the immune response in asthmatic lungs. Through the hydrolysis of arginine, Arg I can control tissue repair by promoting collagen biosynthesis and regulate nitric oxide (NO) levels. A gap in understanding Arg I function in asthma is linking the relationship between the increase in expression and the production of downstream metabolites that could influence different elements of asthma such as tissue repair and NO levels. To understand the role of macrophage Arg I in asthma, we have created mice that specifically lack, or overexpress, Arg I in macrophages. The proposed research will use these mice in asthma models to test the possibility that macrophage Arg I controls tissue repair and remodeling by controlling collagen biosynthesis (Aim 1), reducing NO levels in the inflamed lung and thereby reducing inflammatory damage (Aim 2) and potentially regulating the overall asthmatic response (Aim 3). We predict these studies will establish macrophage Arg I as an important element in asthma pathogenesis.
  Back to Top

• Troy D. Randall, Ph.D.

  BALT, the Hygiene Hypothesis and the Development of Asthma

  Trudeau Institute (Now at University of Rochester)

  The incidence of asthma has dramatically increased over the last 20 years and coincides with improved hygiene and a reduced incidence of childhood infections. This has led to the hygiene hypothesis, which states that the immune system does not receive proper stimulation early in development and is subsequently unable to respond appropriately to aeroallergens. Recent observations suggest that neonatal exposure to pathogens or microbial products, such as endotoxin, substantially reduces the risk of developing asthma. However, it is unclear how this effect is achieved. We hypothesize that prior to the development of vaccines to many of the childhood infections, most children were exposed to multiple respiratory infections and that these infections triggered the development of Bronchus Associated Lymphoid Tissue (BALT). Unlike other lymphoid organs, BALT develops postnatally in response to inflammation or infection. However, once developed, BALT persists due the activities of homeostatic chemokines, such as SLC and BLC. Thus, we suspect that BALT is actually a “normal” component of the lymphoid system that is missing or underdeveloped in most people (and mice) due to the lack of the normal triggers that initiate its development. We also hypothesize that once developed, BALT allows the respiratory tract to respond to aeroallergens in a way that does not promote type II inflammation or lead to asthma. Therefore, the experiments in this proposal will determine the microbial triggers that initiate BALT development and test whether the presence of BALT alters immune responses to aeroallergens and reduces the incidence or severity of asthma.
  Back to Top

Extension Awards

• Michael R. Blackburn, Ph.D.

  Adenosine Deaminase Enzyme Therapy and Asthma Exacerbations

  University of Texas HSC at Houston

  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.
  Back to Top

2003 Research Grant Awardees

Senior Investigator Awards

• Gökhan Hotamisligil, M.D., Ph.D.

  Novel Pathways Controlling Inflammatory Responses in Asthma

  Harvard School of Public Health

  Asthma is a chronic inflammatory condition of the airways. While the prevalence of this disease is increasing at alarming rates worldwide, the therapeutic modalities are limited in variety and efficacy and the mechanisms underlying the disease are not clearly understood. Here, we propose a line of research involving the role of fatty acid binding proteins (FABPs) in lipid signaling and inflammatory responses and their contribution to the pathogenesis and treatment of asthma. In asthma, it is generally accepted that many cell types contribute to the overall inflammatory response. These include T cells, particularly the Th2 phenotype, eosinophils and mast cells as primary effectors leading to further activation and recruitment of other inflammatory cells, such as macrophages, and alterations of the bronchial epithelium function. We recently showed an unexpected role for FABPs in regulation of macrophage inflammatory responses. Interestingly, recent experiments have also detected aP2 and mal1 production in the bronchial epithelium during asthma and specifically in response to IL-4 and IL-13. We hypothesize that members of the fatty acid binding protein (FABP) family, aP2 and mal1, play an important role in the generation of inflammatory responses and airway hyperresponsiveness in asthma; blocking the activity of these proteins will result in protection from development of asthma and its chronic complications. This hypothesis will be tested in aP2-, mal1-, and combined aP2-mal1-deficient cells and mice
  Back to Top

• Lee Niswander, Ph.D.

  Lung Development and Disease: Genetic and Experimental Approaches

  Sloan-Kettering Institute/HHMI (Now at University of Colorado Health Sciences Center)

  Embryonic development of the lung requires a coordinated series of cellular and molecular events that results in the elaborate branching pattern observed in the adult lung. Extensive interactions between the lung endoderm and mesenchyme induce the initial lung buds to form and to further elaborate the intricate branching pattern (Fig. 1a). Once the initial pattern is established, additional interactions are necessary to establish the aveoli and the vascular smooth muscle as well as the specific differentiated cell types required to facilitate efficient gas/air exchange. Furthermore, airway remodeling that occurs in asthmatic patients involves communication between different cell types in the lung in a process that is thought to be analogous to that which occurs between the endoderm and the mesenchyme during lung development11, 30. Relatively little is known of the molecular events that are required to make a fully functional lung. Moreover, the genetic basis of lung defects is not well understood. In this proposal we will identify genes involved in critical aspects of embryonic lung morphogenesis by expanding an on-going mutagenesis screen in mice (Aim 1). We will characterize the phenotype associated with mutations in these critical genes to determine the mechanism of protein action (Aim 2). Finally we will test the hypothesis that genes that control embryonic lung function are abnormally expressed in a mouse model of asthma and that mutations in these genes may confer asthma susceptibility (Aim 3). These studies will establish the key genetic, cellular and molecular events that regulate embryonic lung development and generate new animal models of lung development and disease. In addition, they have the potential to uncover new therapeutic targets for asthma treatment and repair of damaged lung tissue.
  Back to Top

• Hidde L. Ploegh, Ph.D.

  Application of a New Cell Biological and Chemical Strategy to the Study of Asthma

  Harvard Medical School

  Class II Antigen presentation is pivotal to asthma pathophysiology. We propose to study presentation of aerosolized antigen (ovalbumin; OVA) in the mouse model of asthma. First, we shall generate mice that harbor antigen-specific B cells capable of isotope switching to the pathophysiologically relevant IgE. Second, the antigen-presenting cell (APC) itself will be investigated. A recently generated mouse, where all class II molecules are EGFP tagged, allows visualization of respiratory APC and even the class II molecules themselves. Finally, we shall develop a novel chemical strategy to visualize the fate of the offending antigen. Together, these approaches will allow unprecedented access to quantitative and qualitative aspects of antigen presentation in asthmatic disease.
  Back to Top

• Danuta Radzioch, Ph.D.

  Genetic Dissection of Susceptibility to Allergic Asthma

  McGill University

  Asthma represents a complex trait with a strong genetic component. Previous studies in our Centre and by others have demonstrated major gene effects underlying the traits of susceptibility or resistance to this disease as well as of several intermediate phenotypes related to certain components of the immune/inflammatory pathways that the host exposed to an allergen. Furthermore, the proposed studies will test the hypothesis that the immunization with M.bovis BCG or/and imidazoquinolines prior to antigenic challenge may slow down or totally inhibit the development of allergic asthma phenotype in asthma-susceptible individuals. Our tool to dissect such complex phenotypes is a unique, newly-developed gene-discovery platform of recombinant congenic strains (RCS) of mice derived from inbred progenitors that are either susceptible to asthma (A/J, abbreviated A) or resistant to asthma (C57BL/6J, abbreviated B). The first objective of the proposed studies is to identify chromosomal loci/genetic factors which control the differences in susceptibility to antigen-induced murine model of asthma. The second objective is to identify discrete mechanistic phenotypes affected by these loci and biological markers of susceptibility to this inflammatory disorder. A particular emphasis will be on the regulation of T helper subset selection, an immunological pathway of pivotal importance in the susceptibility or resistance to pathological inflammation. Finally, we will explore the possible protective effect of M. bovis BCG vaccine/and a potent immunoadjuvant immidazoquinoline against development of allergen induced asthma. The discovery of genetically linked, quantitative biomarkers of asthma will contribute not only to better understanding of the disease pathogenesis but also will facilitate the genetic analysis of this disease in humans.
  Back to Top

• Danuta Radzioch, Ph.D.

  A New Therapeutic Strategy for Asthma: Inhibition of Cortisol Metabolism in the Lung

  University of California, San Francisco

  11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) is the enzyme responsible for conversion of the anti-inflammatory hormone cortisol to cortisone in the lung and kidney, thus inactivating glucocorticoid response in these tissues. We propose to develop the first potent and selective small-molecule inhibitor of 11ß-HSD2 as a potential new therapy for asthma. Our hypothesis is that inhibition of 11ß-HSD2 in the lung will prevent conversion of the anti-inflammatory steroid cortisol to inactive cortisone in the lung epithelium, resulting in reduced bronchial tube swelling associated with asthma attacks. Vastly different levels of 11ß-HSD2 are expressed in the lung compared to other organs, providing an avenue for potent inhibition in the lung without significant inhibition systemically. Interestingly, a steroidal natural product isolated from licorice, glycyrrizic acid, has been used to treat asthma since the 16th century and is a potent inhibitor of 11ß-HSD2. Glycyrrizic acid binds to the steroid binding site of 11ß-HSD2 which is common to several cortisol/cortisone metabolizing enzymes and is thus pleiotropic in its effects. We propose to generate inhibitors of 11ß-HSD2 which bind to the NAD+ binding pocket to achieve highly specific and tight binding inhibitors. In support of this approach, our laboratory has discovered a potent and selective NADPH competitive inhibitor of carbonyl reductase 1, an enzyme in the short-chain dehydrogenase reductase family which includes 11ß-HSD2. Using this inhibitor as a lead structure, we propose synthesize analogs which are specific for 11ß-HSD2 as a potential alternative or adjunct to synthetic corticosteroid treatment for asthma.
  Back to Top

• Jonathan S. Stamler, M.D.

  The Role of S-Nitrosothiols in the Pathogenesis of Asthma

  Duke University/HHMI

  Asthma is characterized by intermittent obstruction, hyperresponsivity and chronic inflammation. Levels of nitric oxide (NO) are increased in asthmatic airways, but it has been difficult to argue for a significant role of NO in the asthmatic response – either as ameliorative or pathogenic – because NO synthase (NOS) inhibitors exert only modest effects in patients with asthma, and mice deficient in NOS2 (inducible NOS) have no significant change in airway responsivity. S-nitrosothiols (SNO’s) are stable endogenous molecules with bioactivity similar to NO. It has recently been shown that S-nitrosoglutathione (GSNO) (NO complexed to glutathione), a major source of NO-derived bronchodilator and antimicrobial activity, is substantially reduced in the airway lining fluid of asthmatics. Thus, NO production is increased in asthmatics, but overall NO bioactivity is decreased. This paradox is best explained by an augmentation of airway denitrosylating activity. We have recently identified an enzyme in mammalian cells, GSNO reductase, which is a primary regulator of cellular GSNO metabolism, and therefore a likely regulator of airway GSNO levels. Moreover, we have produced a GSNO reductase knock-out mouse which is hyporesponsive to asthma. In this grant we will test the hypothesis that dysregulation of SNO metabolism is important in the pathogenesis of bronchial hyperreactivity and airway inflammation in asthma.
  Back to Top

Junior Investigator Awards

• Richard Bond, Ph.D.

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

  University of Houston

  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.
  Back to Top

• Robert Brenner, Ph.D.

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

  University of Texas HSC at San Antonio

  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.
  Back to Top

• William C. Sha, M.D., Ph.D.

  Regulation of Effector Responses in Asthma by B7h-ICOS Costimulatory Molecules

  University of California, Berkeley

  The costimulatory ligand-receptor pair B7h-ICOS has been implicated as a critical regulator in the pathogenesis of asthma. However, in different experimental models of asthma, perturbation of B7h-ICOS signaling interactions resulted in strikingly different outcomes. Systemic blockade of B7h-ICOS interactions during the effector phase of T-cell responses inhibited multiple indices of allergic lung inflammation including cellular infiltrates and IgE production, suggesting that blockade of B7h-ICOS interactions might be an effective strategy in treatment of asthma. In contrast, local blockade of B7h-ICOS interactions in the lung during aerosol tolerization prevented generation of antigen-specific TR cells that control allergic lung inflammation, suggesting that clinical blockade of B7h-ICOS interactions could actually exacerbate asthma. Although B7h is expressed on multiple cell lineages important in asthma, including B cells, dendritic cells, and lung endothelial cells, how distinct effector responses of ICOS+ T cells are regulated is poorly understood with respect to when, where, and with which B7h-expressing cells these T cells interact in vivo. We will examine the pathogenesis of asthma in mouse models where expression of B7h is restricted to distinct cell lineages, using crosses of lineage-specific B7h transgenic mice to B7h-/- mice. Using two distinct antigen-sensitization protocols that induce allergic lung inflammation by either systemic intraperitoneal or local intranasal routes, we will compare the nature and significance of B7h-ICOS interactions during local and systemic sensitization, and determine the contribution of individual lineages of B7h-expressing cells to either induction or inhibition of asthma.
  Back to Top

• Ivaylo Stoilov, M.D.

  Asthma and the Cytochrome p450 System of the Lung

  University of Connecticut Health Center

  Cytochrome P450s are responsible for the metabolic transformation of nearly every known class of endogenous organic molecules as well as various xenobiotics entering the human body. Although the potential role of the P450 super-family in the etiology, pathogenesis and pharmacogenetics of asthma appears to be appreciated from a conceptual viewpoint, a very large number of known P450 genes have not yet been investigated in terms of lung expression, and those that have been studied have not yet been characterized extensively. Comparative expression profiling of the mouse P450 gene family, conducted in the PI’s laboratory, found that as much as 60% of the known P450 genes could be expressed in the adult mouse lung. Such large number of P450s offers numerous metabolic routes to the xenobiotics entering the lung via the airways, some of which may produce toxic and/or asthmogenic compounds. We propose comprehensive study of the lung cytochrome P450 system. Our first objective will be to determine the normal P450 composition in the developing, adult and aging lung. Next we will evaluate the reaction of the lung P450 system in a mouse model of asthma. The data from these studies will be used to select lung P450s for the development of knock-out mouse models. Such models will make it possible to evaluate the role of the P450 system in sustaining lung homeostasis and the etiology and pathogenesis of asthma. Finally, we will establish a library of functionally characterized polymorphisms from human lung P450s for pharmacogenetic studies.
  Back to Top

• Raul Torres, Ph.D.

  Regulation of GTP-binding Proteins in Asthma and Airway Hypersensitivity

  University of Colorado/National Jewish Medical & Research Center

  Inflammation and recruitment of eosinophils, mast cells, and lymphocytes to the lung are critical components in the pathophysiology of asthma. The precise mechanism(s) by which leukocytes are attracted to the lung are ill-defined, although chemokine-mediated cellular recruitment to the lung has been implicated and is consistent with the defined role of chemokines in coordinating leukocyte movement prior to, and during, an immune response. We have generated a mouse mutant whose hematopoietic cells lack an intracellular signaling molecule, lsc, which regulates the activity of both heterotrimeric Gasubunits and RhoA, and thus couples the two families of GTP-binding proteins in a common signal transduction pathway. Hematopoietic cells lacking lsc (lsc-/-) exhibit aberrant G-protein coupled receptor (GPCR) signaling as exemplified by an inability to fully desensitize particular chemokine receptors to chemokine stimulation. A consequence of dysregulated GPCR signaling is that an increased proportion of lsc-/- hematopoietic cells migrate towards particular chemoattractants. Importantly, upon challenge and allergen sensitization, lsc-deficient mice mount an antigen-specific IgE response but do not display typical hallmarks of allergen-induced asthma such as a predominance of Th2 cytokines in bronchoalveolar lavage, airway hyperresponsiveness, or goblet cell hyperplasia. These data together suggest that lsc regulation of GTP-binding proteins is essential for allergen-induced inflammation. In this proposal we wish to define the role of this hematopoietic signaling molecule in a mouse model of asthma.
  Back to Top

2002 Research Grant Awardees

Senior Investigator Awards

• David A. Dean, Ph.D.

  Targeting Airway Smooth Muscle for Asthma Gene Therapy

  Northwestern University

  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.
  Back to Top

• Beverly H. Koller, Ph.D.

  Animal Models for Functional Screening of Polymorphisms Associated with Susceptibility to Asthma

  University of North Carolina at Chapel Hill

  Polymorphisms that are associated with increased risk for asthma have been identified. However, a causative role for these alleles in the pathogenesis of asthma has not been established. The goal of this project is to develop mouse models for testing for causality of human genetic variants in functional screens of allergic airway disease. We suggest that these animals will advance this field of study by providing: 1.) a mechanism for direct in vivo testing of the propensity for alleles present in the human population to promote allergic airway disease and other inflammatory diseases. 2.) mouse models of asthma that more closely resemble the human disease. 3.) a means to identify novel targets for the treatment of asthma. 4.) “humanized” mouse models for direct testing of pharmacological reagents that will be applicable to humans. Two strategies will be used to generate these mouse lines. In the first, amino acids present in the mouse gene are changed in situ, to those associated with asthma in the human population. In the second, the entire mouse gene is deleted and replaced with the corresponding human sequences. While a number of loci conferring susceptibility to asthma and atopy have been identified, some of the most compelling data implicates the genes for IL-4, IL-13, and their respective receptors. We will therefore begin with the humanization of these genes and testing of normal and disease associated alleles in mouse models.
  Back to Top

• Gary A. Koretzky, M.D., Ph.D.

  The Role of Adapter Proteins in Mast Cell Function

  University of Pennsylvania

  Engagement of cell surface receptors results in the activation of numerous biochemical second messenger cascades. These signaling pathways must be integrated as the appropriate cellular response is programmed. Recent evidence from numerous cell types has demonstrated the critical role played by adapters, proteins with modular domains which mediate intermolecular interactions, in the creation of multimolecular complexes essential for coordinating signaling events. Mast cells, critical effectors in the initiation and propagation of airway hyperreactivity, express adapter molecules which are known to play important roles in the regulation of signal transduction cascades. This project will investigate the mechanism by which two of these adapters, SH2 domain containing leukocyte phosphoprotein of 76 kDa (SLP-76) and adhesion and degranulation promoting adapter protein (ADAP), regulate receptor mediated activation of mast cells. We have initiated experiments to assess the structural features of SLP-76 and ADAP important for function in the primary mast cells ex vivo by measuring the role of these proteins on signaling events downstream of the high affinity receptor for IgE and evaluating how these adapters integrate signals important for integrin receptor function. Experiments are also described to generate genetically altered mice with targeted mutations in these proteins to examine the effect of these manipulations on mast cell function in vivo and how this will impact murine models of asthma. Collectively, we anticipate that our studies will provide new insights into the roles these adapter proteins play in mast cell function and will hopefully provide clues to potential avenues for novel asthma therapies.
  Back to Top

• Roy A. Mariuzza, Ph.D.

  Assembly and Structure of LAT-based Signaling Complexes

  University of Maryland Biotechnology Institute

  Mast cells play a crucial role in the allergic airway response of asthma via the high-affinity receptor for IgE, FceRI. The recruitment and activation of mast cells is orchestrated by T cells, which are also involved in pathogenesis. The transmembrane adapter protein LAT is essential for FceRI-mediated signaling in mast cells and for T cell activation following TCR ligation. LAT is believed to direct the assembly of a multiprotein signaling complex comprising the adapter molecules Gads and SLP-76, and the enzyme phospholipase Cg1 (PLCg1). While rapid progress is being made towards the identification of LAT-associated proteins, and on understanding their role in lymphocyte development and function, much less is known about the biochemical properties and three-dimensional structure of LAT-nucleated signaling complexes. We propose to study the assembly and structure of the postulated LAT/Gads/SLP-76/PLCg1 complex using a multidisciplinary approach combining biophysical methods to measure thermodynamic binding parameters with X-ray crystallography to determine three-dimensional structures. First, we will produce full-length and truncated versions of LAT, Gads, SLP-76 and PLCg1 in milligram amounts using bacterial or insect cell expression systems. Second, we will assess the role of cooperative binding interactions in assembly of the LAT/Gads/SLP-76/PLCg1 complex. Third, we will reconstitute binary, and higher-order (ternary and quarternary), LAT-based complexes in vitro for the purpose of visualizing specific protein-protein interfaces by X-ray crystallography. These studies will significantly advance our knowledge of the molecular basis for LAT-mediated signal transduction. Moreover, they will contribute to the development of structure-based therapies for the treatment of asthma based on specific blockade of mast cell or T cell activation using small molecules to disrupt the assembly of LAT-nucleated signaling complexes.
  Back to Top

• Eric G. Pamer, M.D.

  CD4 T Cells in Aspergillus-Induced Asthma

  Memorial Sloan-Kettering Cancer Center

  CD4 T cell responses to fungal antigens can result in allergic asthma. Spores (conidia) from the ubiquitous fungus Aspergillus fumigatus are commonly inhaled and, in a subset of individuals, prime Th2 CD4 T cell responses that lead to increased pulmonary mucus secretion, peribronchial fibrosis, eosinophil recruitment and IgE synthesis. The experiments described in this grant proposal will investigate the early, in vivo events that result in the activation and differentiation of Aspergillus fumigatus specific CD4 T cells following inhalation of conidia. It is our hypothesis that conidia and associated fungal molecules induce inflammatory processes that sway the subsequent CD4 T cell response. Furthermore, we hypothesize that the particulate nature of Aspergillus fumigatus conidia contributes to the priming and differentiation of CD4 T cells, distinguishing our proposed model from existing ones investigating T cell responses to inhaled soluble antigens. Our first aim is to generate and characterize CD4 T cell lines and hybridomas from mice immunized with Aspergillus fumigatus antigens and conidia. Our second aim is to clone T cell receptor (TCR) genes from Aspergillus fumigatus specific T cells clones and generate TCR transgenic mouse strains. In the third aim we describe adoptive transfer studies that characterize the trafficking, activation, proliferation and differentiation of Aspergillus fumigatus specific CD4 T cells during the early stages of allergic asthma pathogenesis. Generation of TCR transgenic mice specific for fungal antigens is essential for the in vivo analysis of the early events leading to inflammation and pulmonary remodeling of asthma. Defining the connections between inflammatory responses to particulate fungal antigens and Th2 CD4 T cell responses to Aspergillus fumigatus conidia may result in novel therapeutic strategies to treat and possibly prevent allergic asthma.
  Back to Top

• Daniele Piomelli, Ph.D.

  Endogenous Cannabinoids as Modulators of Bronchial Responsiveness

  University of California, Irvine

  The active principle in marijuana, D9-tetrahydrocannabinol (D9-THC), dilates bronchial smooth muscle in humans, suggesting that cannabis-like (cannabinoid) compounds may be applied for the treatment of airway diseases. The therapeutic significance of this finding is obscured, however, by our limited understanding of the roles played by cannabinoid receptors and their endogenous ligand, anandamide, in airway physiology. We have recently shown that anandamide is produced in rodent lungs. We have also found that this compound exerts multiple effects on bronchial responsiveness, which are mediated by activation of CB1 cannabinoid receptors in the airways. Based on these findings, we hypothesize that anandamide participates in the pathophysiological control of bronchial responsiveness. Our experiments will address four questions that are pertinent to a test of this hypothesis.

  1. What are the receptor mechanisms responsible for anandamide release in the lungs? We will use a perfused lung model to determine whether neurotransmitters and inflammatory mediators stimulate anandamide release.
  2. What are the mechanisms responsible for anandamide inactivation in the lungs? In the same model, we will also examine the pathways of anandamide clearance.
  3. Does anandamide affect neurotransmitter function in the lungs? Additional in vitro models will be utilized to study the effects of anandamide on (a) neurotransmitter release and (b) contractile responses to neurotransmitters and inflammatory mediators.
  4. Is anandamide involved in allergic asthma? We will address this question by using in vivo models, including CB1 receptor knock-out mice.

  These studies may provide a framework to the development of more selective cannabinoid-based agents for the treatment of respiratory pathologies.
  Back to Top

• Anjana Rao, Ph.D.

  Differential Role of NFAT Family Members in IL-13 Regulation in Mast Cells

  Harvard Medical School

  Allergic asthma is a complex disorder characterized by local and systemic allergic inflammation and reversible airway obstruction. Interleukin-13 is a central mediator of allergic asthma; it is produced by Th2 cells and mast cells, and its levels are increased in airway tissues of human asthmatics and animal models of asthma. In Th2 cells as well as mast cells, IL-13 production is controlled by the calcium-regulated transcription factor NFAT. NFAT consists of four family members, at least three of which (NFAT1 (p, c2), NFAT2 (c, c1), NFAT4 (x, c3)) are expressed in both cell types. Strong but circumstantial evidence indicates that the different NFAT family members exhibit cell-type- and gene-specific differences in their ability to regulate gene transcription in activated immune cells. Of specific relevance to this proposal, we have shown that NFAT1, but not NFAT2, is essential for IL-13 transcription by mast cells. This is very different from the situation in Th2 cells, where both family members contribute to IL-13 expression. Here we propose to investigate the molecular mechanisms underlying the differential regulation of the IL-13 gene by these two NFAT proteins in mast cells, and to determine whether these two NFAT family members have distinct effects on asthma induction and development in an in vivo model of allergen-induced asthma in mice.
  Back to Top

• Michael G. Rosenfeld, M.D.

  Role of p50 Depression in Airway Remodeling in Asthma

  University of California, San Diego/HHMI

  NF-kB is composed of either heterodimeric subunits (e.g. p50-p65 heterodimers) or homodimeric subunits (e.g. p50 homodimers). In this study we are interested to determine the role of the subset of genes controlled by the p50 homodimers, and regulated by a novel derepression mechanism, to the pathogenesis of asthma. p50 homodimers are detected in the nucleus and are able to function as repressors. In this study we propose to focus on increasing our understanding of the genes repressed by p50 homodimers as we hypothesize that derepression of these genes in asthma may play an important role in epithelial repair and airway remodeling. Cytokine stimulation results in derepression of a specific subset of p50 homodimer regulated genes, including the epithelial-expressed gene KAI-1, which encodes a tetraspanin, and may play an important role in airway remodeling as it associates with the Epidermal Growth Factor (EGF) receptor and attenuates EGF receptor signaling. As p50 homodimers induce expression of KAI-1, and KAI-1 attenuates epithelial EGF receptor signaling and function, p50 homodimers could attenuate EGF receptor signaling and reduce epithelial airway repair in asthma. In addition, levels of TGF-beta released by airway epithelial cells are significantly increased if repair is retarded, suggesting that if KAI-1 inhibited EGF receptor signaling increased levels of TGF-b could result and lead to enhanced peribronchial fibrosis. Therefore in this study we propose to investigate the role of p50 homodimers in the repression of genes important to the pathogenesis of asthma, focusing on KAI-1, and identifying “novel” genes regulated by p50 homodimers and determining their role in asthma.
  Back to Top

Junior Investigator Awards

• Marc C. Levesque, M.D., Ph.D.

  Nitric Oxide, LPS and the Pathogenesis of Asthma

  Duke University (now at University of Pittsburgh)

  Asthma is a significant cause of morbidity and mortality for African Americans and asthma is increasing in prevalence. Both environmental and genetic factors contribute to the pathogenesis of asthma. It is our hypothesis that genetic regulation of endogenous nitric oxide (NO) production, in the setting of environmental stimuli such as endotoxin, contributes to the pathogenesis of asthma in humans. Asthma, airway hyperresponsiveness, and atopy are all associated with increased exhaled NO levels. Importantly, NO has several proinflammatory activities that contribute to the pathogenesis of asthma and other diseases associated with chronic inflammation. The production of NO in normal individuals and in individuals with asthma is mediated by bronchial epithelial cells that express the enzyme inducible nitric oxide sythase (NOS2). Lipopolysaccharide (LPS) is a form of endotoxin that is an important inducer of NOS2 expression in human cells. Therefore, we believe that environmental exposure to LPS is associated with production of pathogenic levels of NO in individuals with the appropriate genetic background. To date, we have identified three single nucleotide polymorphisms (SNP) in Africans with malaria that are associated with increases in systemic NO production. Therefore, to test our hypothesis, we will identify functional SNPs in the NOS2 gene that are associated with either the basal or LPS-induced overproduction of exhaled NO in healthy African Americans. These studies will establish NO as a treatment target in asthma, identify the relationship of environmental exposure to LPS on exhaled NO levels and identify genetic markers that may be used to target preventative strategies and predict therapeutic responses for patients with asthma
  Back to Top

• Ruslan Medzhitov, Ph.D.

  Role of Toll-like Receptors in Allergic Inflammation

  Yale University/HHMI

  Asthma is a chronic inflammatory disorder of the airways. The contribution of Th2-mediated adaptive immune responses including the recruitment and activation of mast cells and eosinophils is well established in the pathogenesis of asthma, yet almost nothing is known about the underlying mechanisms that trigger allergic inflammation. Mammalian Toll-like receptors (TLRs) have recently been established as critical signal transducing receptors that initiate innate immune and inflammatory responses. The overall objective of this proposal is to investigate a possible role of TLRs in the pathogenesis of allergic inflammation. The airway epithelium is continuously exposed and challenged by a variety of environmental stimuli, including pathogens and pathogen-derived products known to trigger TLRs. Stimulation of TLRs leads to the activation of the NF-kB signaling pathway that plays a crucial role in allergic and other types of inflammation, in part due to the induction of chemokines and cytokines that mediate Th2 effector responses. We have generated two types of transgenic mice that express constitutively active TLR4 under control of inducible promoters. In one transgenic line, the expression of constitutively active TLR4 is restricted to the airway epithelium, while in the other line, this expression is restricted to dendritic cells. These mice will be used to study the role of TLRs in the initiation of allergic inflammation and to evaluate the contribution of the TLR pathway to the negative regulation of Th2 responses. In addition, we will analyze the induction of allergic inflammation in mice that are genetically deficient for MyD88, a critical downstream component of the TLR signaling pathway.
  Back to Top

2001 Research Grant Awardees

Senior Investigator Awards

• Soman N. Abraham, Ph.D.

  Mast Cell Uptake of Particulate Allergens

  Duke University

  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.
  Back to Top

• Mark R. Boothby, M.D., Ph.D.

  Memory Regulation and Allergic Airway Responses

  Vanderbilt University

  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.
  Back to Top

• Marco Conti, M.D.

  Role of Phosphodiesterases in Asthma

  Stanford University

  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.
  Back to Top

• Richard Flavell, Ph.D.

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

  Yale University/HHMI

  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.
  Back to Top

• David L. Garbers, Ph.D.

  Guanylyl Cyclase Receptors: New Targets for Airway Remodeling Intervention

  UT Southwestern Medical Center, Dallas & HHMI

  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.
  Back to Top

• Sankar Ghosh, Ph.D.

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

  Yale University/HHMI

  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.
  Back to Top

• Michael J. Grusby, Ph.D.

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

  Harvard University

  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-13R&alpah;1 and IL-13R&alpah;2 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.
  Back to Top

• Michael Karin, Ph.D.

  IKK/NF-kB: Role in Airway Inflammation and Remodeling

  University of California, San Diego

  The goal of this proposal is to assess the importance of I?B kinase (IKK) in regulating expression of NF-?B target genes important to the genesis of allergic inflammation and airway remodeling in a mouse model of asthma. The importance of NF-?B to asthma is suggested by mouse models in which mice deficient in either the p50 or c-Rel subunits of NF-?B do not develop eosinophilic airway inflammation or airway responsiveness following allergen challenge. Inhibition of eosinophilic inflammation in p50-deficient mice is not due to defective T cell priming or proliferation, nor deficient expression of endothelial adhesion molecules (ICAM, VCAM), which bind circulating eosinophils. Rather, p50- and C-Rel-deficient mice exhibit a major defect in production of NF-?B regulated chemokines and cytokines may play an important role in eosinophil recruitment and airway hyperreactivity. Chemokine and cytokine genes, as well as NF-?B, are expressed in several airway cell types following allergen challenge. To determine which cell type is the most critical site of NF-?B activation leading to development of full blown allergic inflammation and airway remodeling in a mouse model of acute and chronic asthma. These studies will define the importance of IKK in difference cell types relevant to the pathogenesis of asthma.
  Back to Top

• Michael W. Lieberman, M.D., Ph.D.

  Genetic Models of Cysteinyl Leukotriene Function in Asthma

  Baylor University

  Cysteinyl leukotrienes (CLTs) are powerful mediators of bronchial and vascular smooth muscle contraction, edema and mucus formation, and granulocyte function. They are known to play a key role in asthma, but to date there has been no way to assess the function of individual CLTs. Studies from our laboratory have changed the paradigm of CLT metabolism. Leukotriene C4 is now known to be converted in in vivo to leukotriene D4 by g-glutamyl leukotrienase (GGL), an enzyme newly characterized by us, and by g-glutamyl transpeptidse (GGT) in vitro as the older literature suggests. We have also determined that membrane bound dipeptidase 1 (MBD-1) and MBD-2, which we have recently identified and cloned, are the specific dipeptidases that convert leukotriene D4 to leukotriene E4 . We have developed mice deficient in GGT, GGL, and MBD-1 and plan to make MBD-2 deficient mice. Although it is generally believed that in asthma leukotriene D4 is much more potent than leukotriene C4 and that leukotriene E4 is much less active, our preliminary data using mice deficient in GGT and GGL suggest a more complex story.
  
  
  
  Genetically deficient mice allow isolation of contributions of individual enzymes and CLTs to asthma. We will use mice deficient in GGT, GGL, MBD-1 and MBD-2 to test hypotheses about asthma induced by “complete Aspergillus antigen” or IL-13. We will characterize the asthmatic response, evaluate the ability of CLTs alone to induce an asthma-like response, examine GGL/GGT expression, and investigate potential CLT/cytokine interdependence.
  Back to Top

Junior Investigator Awards

• Jonathan M. Backer, M.D.

  Role of PI 3-Kinases in Mast Cell Degranulation

  Albert Einstein College of Medicine

  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.
  Back to Top

• Alec M. Cheng, Ph.D.

  Regulation of Mast Cell Function by the Signaling Adaptor GADS

  Washington University, St. Louis

  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.
  Back to Top

• Joseph L. DeRisi, Ph.D.

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

  University of California, San Francisco

  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.
  Back to Top

2001 Research Grant Awardees

Senior Investigator Awards

• Soman N. Abraham, Ph.D.

  Mast Cell Uptake of Particulate Allergens

  Duke University

  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.
  Back to Top

• Robert Lefkowitz, M.D.

  Role of GPCR Regulatory Mechanisms in the Pathophysiology of Asthma

  Duke University/HHMI

  Asthma is a complex disease characterized by excessive airway inflammation, reversible airway obstruction and inappropriate airway responsiveness. Since airway responsiveness is primarily mediated by G protein-coupled receptors (GPCRs), we hypothesize that regulation of GPCR signaling may be implicated in the pathophysiology of asthma. G protein-coupled receptors (GPCRs), characterized by seven transmembrane domains, transduce a wide variety of extracellular signals into intracellular events. GPCRs are so named because they couple to guanine nucleotide-binding, or G, proteins. Interaction of the agonist-bound GPCR with the G protein results in modulation of an intracellular effector system which elicits a physiological response. Cellular responses to many hormones and neurotransmitters wane rapidly despite continuous exposure of cells to these stimuli. This phenomenon, termed desensitization, results from uncoupling of the agonist-activated receptor from the G protein. Desensitization of agonist-activated GPCRs is principally mediated by two protein families: G protein-coupled receptor kinases (GRKs) and arrestins. GRK-phosphorylated GPCRs exhibit diminished signaling and increased affinity for arrestin proteins. Binding of arrestin proteins further uncouples agonist-activated GPCRs from their cognate G proteins thus quenching signal transcution. Many in vitro experiments demonstrate the importance of GRKs and arrestins for desensitizing agonist-activated GPCRs. Although, relatively few studies have explored the functional significance of these proteins in vivo, our recent work shows that GRK3 desensitizes muscarinic receptor-mediated bronchoconstriction of murine airways. Using genetically altered mice which lack individual GRK or arrestin proteins, we will determine the role of these GPCR regulatory proteins in regulation of airway smooth muscle under both normal circumstances and in an induced asthmatic state.
  Back to Top

• Jeremy Luban, M.D.

  Cyclophilin A and the Regulation of CD4+ T Cell Function in Asthma

  Columbia University (Now at University of Geneva)

  Cyclophilins, a ubiquitous family of proteins, exhibit peptidyl-prolyl isomerase activity in vitro suggesting that they regulate protein folding in cells. Cyclophilins also bind the immunosuppressive drug cyclosporine but the drug’s activity is not due to cyclophilin inactivation. Rather, immunosuppression results from inhibition of the phosphatase calcineurin by a composite surface created by the cyclophilin-cyclosporine complex. Cyclophilin A (CYPA) was discovered fifteen years ago but its biological function remains unknown. Using targeted gene disruption we have shown that CYPA is not essential for mouse development, fertility, or immune system development. However, cypa -1- animals spontaneously exhibit splenomegaly, blepharitis with tissue infiltration by eosinophils, and elevated serum IgE levels. Consistent with these signs of allergy, cypa -1- CD4+ T cells produce elevated levels of Th2 cytokines in vitro. IL-2 production following stimulation of naive CD4+ T cells is normal but extremely elevated in cells of memory/effector phenotype. Thus, CYPA modulates CD4+ T cell function in the absence of cyclosporine. Animal models have revealed a critical role for Th2 cells in the pathogenesis of asthma. To assess the biological consequences of the cypa -1- T cell cytokine abnormalities in a reproducible and quantitative fashion we will exploit a well-established murine asthma model. Within the context of this disease model and complementary in vitro assays we will test several hypotheses regarding how CYPA regulates CD4+ T cell function. By studying CYPA function we expect to learn about CD4+ memory/effector T cells and thereby contribute to understanding the pathogenesis of asthma.
  Back to Top

• L. Jackson, II Roberts, M.D.

  Oxidant Stress and Oxidized Lipids in Allergic Inflammation

  Vanderbilt University

  Asthma is an inflammatory disease of the airways which is strongly linked to allergic inflammation. Our central hypothesis is that allergic responses in the airways are mediated in part by the production of reactive oxidant species which cause lipid peroxidation of cell membranes, specifically in bronchial epithelial cells, resulting in functional changes in the cell as well as the release of biologically active lipid peroxidation products. We have shown that the measurement of F2-isoprostanes (F2-IsoPs) is a sensitive and reliable measure of oxidant stress. Using mass spectrometry for measurement of F2-IsoPs, we have provided evidence for oxidant stress following allergic stimulation of human and murine airways. We have developed methods for immunohistochemical staining of biopsy tissues for the presence of esterified F2-Isops. Using these tools in association with manipulation of antioxidant defenses, we will test the hypothesis that allergic responses (inflammation and airway responsiveness) are modulated by oxidant stress. Platelet activating factor hydrolase (PAF-AH) is the predominant phosopholipase responsible for hydrolyzing esterified F2-Isops. Using aerosols of PAF-AH and a transgenic mouse hyperexpressing PAF-AH in the airway, we will test the hypothesis that PAF-AH will reduce allergic responses in the airway by hydrolyzing esterified F2-Isops and other oxidized fatty acids from cellular membranes, thus restoring membrane structural integrity. The proposed experiments will yield novel information about the role of oxidant stress and oxidized lipids in allergic airway disorders which in turn could lead to new paradigms regarding the pathophysiology of asthma with the potential for new therapeutic approaches.
  Back to Top

• Julian Solway, M.D.

  Genome-Wide Mutagenesis to Find Genes That Regulate Airway Responsiveness

  University of Chicago

  Airway hyperresponsiveness is characteristic of both atopic and non-atopic asthmatics, but little is known about its genetic basis. The major objective of this proposal is to identify genes that regulate native airway constrictor responsiveness, through phenotypic and genetic analyses of mice expressing gene mutations induced with N-ethyl-N-nitrosourea (ENU). Our rationale for this approach is: 1) it can identify all genes that positively or negatively regulate airway responsiveness; 2) it does not require advance knowledge of the pathways involved; 3) it does not depend upon serendipitous differences in these pathways among existing mouse strains, which now provide the predominant basis for genetics studies in mouse asthma models; and 4) the role of individual genes can be assessed. The key premise underlying our proposal is that identification of genes that control murine airway responsiveness will lead to better understanding of parallel mechanisms that operate in human asthma, and subsequently to development of novel therapies that reduce AHR and asthma symptoms. Our experimental plan is: 1) Identify third generation descendents of ENU-mutagenized male BTBR mice that express heritable mutations responsible for abnormally elevated or reduced non-specific bronchoconstrictor responsiveness. We will use non-invasive whole body plethysmography to identify mice with extremely high or low airway constrictor responses to methacholine, generate homozygous mutant lines from mice whose abnormal phenotype is heritable, and make these mice available to outside investigators. 2) Identify the genetic abnormality in each mutagenized mouse line, through linkage analysis, positional candidate gene sequencing, and where appropriate phenotype rescue through gene complementation in BAC-transgenic animals. 3) Determine the functional and/or structural abnormality in each mutant line. To reveal the pathophysiological consequence of each gene mutation, we will assess lung and airway wall structure, more fully characterize airway and lung function in vivo and in vitro, and evaluate selected biochemical features, expression patterns, and/or signal transduction mechanisms in smooth muscle cells as suggested by initial structure/function studies. Together, these studies should reveal new information about the genetic regulation of native airway responsiveness, which in turn may lead to novel treatment strategies to reduce airway responsiveness in asthma.
  Back to Top

Junior Investigator Awards

• H. Leonardo Aguila, Ph.D.

  NK Cells in Allergic Asthma

  University of Connecticut

  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.
  Back to Top

• Michael R. Blackburn, Ph.D.

  Role of the Adenosine A3 Receptor in Experimental Asthma

  University of Texas – Houston HSC

  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.
  Back to Top

• Michael Croft, Ph.D.

  Targeting OX40 in Asthma

  La Jolla Institute for Allergy and Immunology

  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.
  Back to Top

• Gennady Gololobov, Ph.D.

  Catalytic Anti-IgE Antibodiesa

  University of Texas – Houston HSC

  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.
  Back to Top

• I-Cheng Ho, M.D., Ph.D.

  Transcriptional Regulation of the Murine IL-13 Gene

  Brigham and Women’s Hospital

  CD4+ type 2 helper T (Th2) cells and their secreted cytokines play a pivotal role in the pathogenesis of allergic asthma. Previous data suggest that IL-13 may be the effector Th2 cytokine most responsible for the airway hyperresponsiveness and mucus overproduction seen in allergic asthma. However, it remains unclear how the expression of the IL-13 gene is regulated. While two Th2 cell-specific transcription factors, c-maf and GATA-3, are responsible for the cell type-specific expression of other Th2 cytokine genes, such as IL-4 and IL-5, it appears that other transcription factors, in addition to c-maf and GATA-3, are required for high level expression of IL-13. Recently, we have cloned a region of the murine IL-13 promoter that is sufficient to confer cell type-specificity in vitro. This promoter contains several regions whose sequence is conserved between human and mouse. One of these conserved regions binds at least two Th2 cell-specific protein complexes. The proposal outlined below is based on our preliminary data and is designed to investigate the molecular mechanisms that control the Th2 cell-specific expression of the IL-13 gene.
  Back to Top