Awardees and Abstracts
2001 Awardees
Senior Awards
Marco Conti, M.D.Stanford University (Now at University of California, San Francisco)
Reproductive Biology
Role of Phosphodiesterases in Asthma
David L. Garbers, Ph.D.UT Southwestern Medical Center, Dallas & HHMI
Pharmacology
Guanylyl Cyclase Receptors: New Targets for Airway Remodeling Intervention
Sankar Ghosh, Ph.D.Yale University/HHMI (Now at Columbia University)
Immunobiology
Cell-Permeable Peptide Inhibitors of NF-kB as Novel Therapies for Asthma
Michael J. Grusby, Ph.D.Harvard University
Immunology and Infectious Diseases
The Role of IL-13 Receptor Signaling in the Pathogenesis of Asthma
Michael Karin, Ph.D.University of California, San Diego
Pharmacology
IKK/NF-kB: Role in Airway Inflammation and Remodeling
Junior Awards
Jonathan M. Backer, M.D.Albert Einstein College of Medicine
Molecular Pharmacology
Role of PI 3-Kinases in Mast Cell Degranulation
Alec M. Cheng, Ph.D.Washington University, St. Louis
Developmental Biology
Regulation of Mast Cell Function by the Signaling Adaptor GADS
Joseph L. DeRisi, Ph.D.University of California, San Francisco
Biochemistry
A Systematic, Genomics-based Investigation of the Role of Viral Infection in Acute Exacerbations of Asthma
Soman N. Abraham, Ph.D. — 2001 Senior Award
Duke University
Mast Cell Uptake of Particulate Allergens
The most common form of asthma is allergic asthma, which is developed by inhalation of airborne allergenic particles such as pollen, animal hair, or fur. One of the major effector cells of asthma are mast cells which are positioned in the asthmatic airways so that they immediately interact with inhaled allergens via IgE antibodies bound on their surfaces and respond by the release mediators that initiate the early phase of allergic asthma. Recently, we discovered that pathogenic bacteria can co-opt the endocytic activities of mast cells and gain entry via a route that avoids intracellular degradation (Shin et al Science 289:785-788, 2000). This process involved distinct cellular entities comprising of caveolink cholesterol and glycolipids called caveolae (or rafts). Moreover, infected MC exhibited a remarkably high level of sustained mediator release.
We examined the involvement of caveolae in the uptake of particulate allergens and discovered remarkable similarities between bacterial entry and IgE-mediated entry of allergens into mast cells. We plan to confirm and extend our observations by (i) determining whether caveolae are involved in the uptake of particulate allergens, (ii) identifying the specific role(s) of caveolin in the uptake of allergens and (iii) determining the fate of the internalized allergens in the mast cell and how it affects cellular secretion of inflammatory mediators. These studies could reveal remarkable convergence in the entry of allergens and pathogenic bacteria in mast cells and should help us develop effective strategies to prevent asthma as well as infectious diseases.
Jonathan M. Backer, M.D. — 2001 Junior Award
Albert Einstein College of Medicine
Role of PI 3-Kinases in Mast Cell Degranulation
Mast cells are critical mediators of physiological responses to allergens, and are also strongly implicated in the development of asthma. Mast cells function through the release of both pre-formed mediators, which are stored in secretory granules, as well as the de novo synthesis of bioactive lipids. Mast cell degranulation is mediated primarily by the crosslinking of IgE-bound FceRI receptors on the cell surface. However, coincident stimulation with adenosine leads to an increase in the magnitude of FceRI-mediated degranulation. This synergistic regulation of degranulation by allergens and adenosine is likely to be an important component of the pathophysiology of asthma. This proposal examines the role of Class I and Class III PI 3-kinases in mast cell degranulation. Aims I and II are based on preliminary data showing that the Class III VPS34 PI 3-kinase is required for degranulation mediated by G-protein-coupled receptors (GPCRs), and for adenosine-mediated enhancement of IgE-stimulated degranulation. Aim I studies the subcellular localization of VPS34 in basal and stimulated mast cells, and the effects of overexpression of wild-type and mutant VPS34. Aim II tests whether VPS34 activity is regulated by GPCRs. Finally, Aim III examines the role of PKCb and PKCd as downstream mediators of Class I PI 3 -kinases in IgE-stimulated degranulation, and tests whether constitutively active PKC mutants can rescue degranulation in cells treated with PI 3-kinase inhibitors. These studies should yield new insights into mast cell physiology. Moreover, our focus on distinct isoforms of PI 3-inase may identify new targets for the pharmacological treatment of asthma.
Mark R. Boothby, M.D., Ph.D. — 2001 Senior Award
Vanderbilt University
Memory Regulation and Allergic Airway Responses
The first encounter of an atopic individual with an antigen can lead to TH2-dominated allergic process whereas a non-atopic person would not develop this problem. Microbial exposures are linked with susceptibility to atopic asthma. Similarly, virus infections are a major cause of asthma flares but immunologic mechanisms whereby a virus would reactivate a Th2-dominated allergic response are lacking. Airway obstruction in asthma is intermittent and reversible; patients experience periods of clinical quiescence during which inflammation is attenuated but some allergen-specific T helper (Th)2 cells must become memory cells. However, little is known about contributions of allergen-specific memory T cells to asthma. We have developed a mouse model that permits us to investigate rigorously the potential for memory T cells to influence disease susceptibility. Using this model, we will investigate cellular and molecular mechanisms by which viruses and memory T cells can affect allergic disease. Our hypothesis is that virus infections can influence the development, maintenance, and reactivation of pools of allergen-specific memory Th1 and Th2 cells and thereby influence allergic susceptibility. We will use single- and dual-specificity T cells to establish how virus infections can be programmed to affect allergic airway inflammation. Further, we will investigate whether signals induced during virus infection influence memory Th2 compartments. Together, these studies will yield crucial insights into the role of viruses and memory in asthma.
Alec M. Cheng, Ph.D. — 2001 Junior Award
Washington University, St. Louis
Regulation of Mast Cell Function by the Signaling Adaptor GADS
Mast cells play a critical role in allergic response by virtue of the high affinity FcεR which binds to the Fc portion of the IgE. Anitigen binding to IgE on FcεR 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 FcεR activation. The current project is to dissect the function of an adaptor protein, GADS, which serves to coordinate SLP-76 and LAT activity in FcεR 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 FcεR 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 FcεR 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.
Marco Conti, M.D. — 2001 Senior Award
Stanford University (Now at University of California, San Francisco)
Role of Phosphodiesterases in Asthma
Cyclic nucleotide signaling plays a crucial modulatory role in the airways and in inflammatory cells. An increase in cAMP is associated with the inhibition of airway smooth muscle contraction, activation of T cell, and migration and/or function of effector cells at sites of allergic inflammation. Here, we will test the hypothesis that genes coding for cyclic AMP-specific phosphodiesterases (PDE4s), components of the cAMP signaling cascade, are major determinants of allergen-induced airway hyperreactivity (AHR), a hallmark of asthma. Of the four PDE4 genes present in humans and other mammals, three (PDE4A, B,and D) are expressed in the airways and in inflammatory cells. We have established in vivo models in which individual PDE4 genes have been inactivated by homologous recombination, and have shown that allergen-induced AHR does not develop in mice deficient in PDE4B or PDE4D. These preliminary findings indicate that these PDEs, and the signaling functions they serve, play an essential role in the pathogenesis of asthma. We propose to use these genetically altered mice to define the role of individual PDEs and cAMP homeostasis in the differentiation and the recruitment of inflammatory cells to the lungs and in the development of specific characteristics of asthma, including AHR. Using these mouse models, we will test the possibility that polymorphisms/mutations in the PDE4 genes in humans contribute to the genetic background predisposing to allergic asthma. These studies will also provide "proof of principle" for the development of a new class of drugs useful for the treatment of this disease.
Joseph L. DeRisi, Ph.D. — 2001 Junior Award
University of California, San Francisco
A Systematic, Genomics-based Investigation of the Role of Viral Infection in Acute Exacerbations of Asthma
There is a general agreement that acute exacerbations of asthma are often precipitated by respiratory viral infection. However, accurate information about the prevalence of infection in asthma and a full definition of the causative agents remains elusive (despite occasional assertions to the contrary). Here we propose a genomics-based investigation of the role of viral infection as a precipitant of asthma. cDNA from respiratory secretions of acute asthmatics will be examined by hybridization to DNA microarrays bearing sequences representative of all known families of human and animal viruses. Arrays will harbor multiple genes from any members of each sequenced family of viruses; this should allow not only detection of infection but (in many cases) identification of viral subtypes as well. Our goal is to produce a comprehensive picture of the role of viral infection in asthma using state of the art technology for pathogen detection.
David L. Garbers, Ph.D. — 2001 Senior Award
UT Southwestern Medical Center, Dallas & HHMI
Guanylyl Cyclase Receptors: New Targets for Airway Remodeling Intervention
This proposal focuses on the plasma membrane class of guanylyl cyclase receptors (pGCs) and their ligands as one of, or possibly the most important brake for halting the pathophysiological developments that occur during asthma. Four members of the pGCs (3 with known ligands:1 an orphan), and at least one of the ligands, are expressed locally in airway tissue, but virtually nothing is known about their function. Our work shows a dramatic, rapid, specific adversarial relationship between various mitogens and pGC signaling pathways. We will concentrate on: 1) defining the molecular mechanisms by which mitogen signaling rapidly and specifically shuts down pGCs, 2) defining the mechanisms by which airway cells regulate the local production of cyclase receptor ligands, 3) identifying and defining the mechanisms of regulation of the protein kinases/phosphatases that regulate pGC activity, 4) constructing multiple mouse genetic models to define which of the various cyclase receptors are essential in blocking asthma-like phenotypes, or predispose mice to asthma when eliminated, and 5) discovering and determining the importance of the putative ligand for the orphan pGC expressed in lung/eosinophils. Although little is known about the role of these receptors in the airway, we can infer from our studies in other tissues/cells that these cyclase receptors severely oppose the actions of various mitogens at the level of cell chemotaxis, hyperplasia, hypertrophy, fluid secretion, ciliary beat frequency and extracellular matrix production, and therefore a plethora of positive effects may occur upon intervention to specifically regulate these cyclase receptor signaling pathways.
Sankar Ghosh, Ph.D. — 2001 Senior Award
Yale University/HHMI (Now at Columbia University)
Cell-Permeable Peptide Inhibitors of NF-kB as Novel Therapies for Asthma
Inflammation plays a critical role in the pathogenesis of asthma. However, unlike many other widely prevalent inflammatory diseases such as rheumatoid arthritis, inflammation in asthma is primarily a T-cell driven process. In particular T-cell cytokines such as IL-4, IL-5, IL-9 and IL-13 are critical mediators of the overall disease process. Hence the ability to modulate the release of these cytokines from T-cells is likely to be beneficial for treatment of asthma. The inducible transcription factor NF-κB 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-α , 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-κB activation has opened up the possibility of testing the hypothesis that inhibition of NF-κB in the lung will have a major effect in suppressing airway hyper-reactivity. To more specifically test the hypothesis that inhibition of NF-κB 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-κB in T-cells. We believe that further characterization of such peptide inhibitors that can specifically block the activation of NF-κB when administered intranasally is likely to represent a novel strategy for the treatment of asthma.
Michael J. Grusby, Ph.D. — 2001 Senior Award
Harvard University
The Role of IL-13 Receptor Signaling in the Pathogenesis of Asthma
The Th2 cytokine IL-13 has recently been demonstrated to be an important mediator of allergic asthma. Although a number of components of the IL-13 signaling pathway have been identified, their respective roles in contributing to the pathogenesis of this disease remain unclear. The experiments described in this proposal aim to reveal the molecular mechanisms by which IL-13 receptor signaling contributes to the allergic asthma phenotype. Our approach will be to generate mice deficient in the expression of IL-13Rα-1 and IL-13Rα-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.
Michael Karin, Ph.D. — 2001 Senior Award
University of California, San Diego
IKK/NF-kB: Role in Airway Inflammation and Remodeling
The goal of this proposal is to assess the importance of IκB kinase (IKK) in regulating expression of NF-κB target genes important to the genesis of allergic inflammation and airway remodeling in a mouse model of asthma. The importance of NF-κB to asthma is suggested by mouse models in which mice deficient in either the p50 or c-Rel subunits of NF-κB 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-κB regulated chemokines and cytokines may play an important role in eosinophil recruitment and airway hyperreactivity. Chemokine and cytokine genes, as well as NF-κB, are expressed in several airway cell types following allergen challenge. To determine which cell type is the most critical site of NF-κB 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.
Michael W. Lieberman, M.D., Ph.D. — 2001 Senior Award
Baylor University (Now at Methodist Hospital Research Institute)
Genetic Models of Cysteinyl Leukotriene Function in Asthma
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.