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.

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.

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.

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.

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.

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.

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.

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.

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.

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.