749 Mast Cells Mediate Multiple Features of Asthmatic Respons-es in a Mouse Model of Chronic Asthma

M. Yu, M. Tsai, S. Tam, S. J. Galli; Pathology, Stanford University Schoolof Medicine, Stanford, CA.RATIONALE: The role of mast cells in the development of inflammato-ry, pathophysiological and chronic tissue changes in asthma is controver-sial. We investigated the importance of mast cells in the expression ofcharacteristic features of this disorder by developing and using a mousemodel of chronic asthma.METHODS: Mast cell-deficient WBB6F1- KitW/KitW-v (W/Wv) mice,wild type WBB6F1- +/+ (+/+) mice, and W/Wv mice that had been recon-stituted i.v. with bone marrow-derived cultured mast cells (BMCMCs) of+/+ origin, were sensitized (i.p.) and then repeatedly challenged (i.n.) withovalbumin (OVA) without artificial adjuvant.RESULTS: OVA sensitization/challenge significantly increased mastcell numbers in the lungs of +/+ and +/+BMCMC-reconstituted W/Wv

mice, compared with values in PBS-treated controls. Mast cellsenhanced significantly the expression of antigen-induced acute bron-choconstriction, lung inflammation, airway hyper-responsiveness tomethacholine, mucus gland hyperplasia and numbers of goblet cells inthe airway epithelium, subepithelial and total lung collagen depositionand hyperplasia/hypertrophy of bronchial smooth muscle. cDNAmicroarray analysis revealed an induction of distinct patterns of antigen-induced and mast cell-dependent gene expression in the lung. Theseupregulated genes included some already known to be associated withthe key features of asthma.CONCLUSIONS: We have developed a mouse model of chronic, anti-gen-induced asthma in which mast cells play a critical role in severalimportant acute, late phase and chronic manifestations of the disorder.Funding: NIH

750 Identification of Specific Gene Expression Profile in HumanMast Cells Via Nucleotide-Binding Oligomerization Domain 2(NOD2)

Y. Okayama, S. Okumura, H. Saito; Research Unit for Allergy Tran-scriptome, RIKEN Yokohama Institute, Research Center for Allergy andImmunology, Yokohama, JAPAN.RATIONALE: The purpose of this study was to examine the expressionof nucleotide-binding oligomerization domain 2 (NOD2) by human mastcells (MCs) and its function in MCs.METHODS: Human adult peripheral blood-derived cultured and humantissue lung MCs were used in this study. The expression of NOD2 mRNAwas examined by real-time RT-PCR. The NOD2 protein expression inMCs was confirmed by using confocal laser scanning with anti-NOD2mAbs. High-density oligonucleotide probe arrays were used to comparethe gene expression profiles between the muramyl dipeptide (MDP)-,LPS- and IgE/anti-IgE-specific programs. Secretion of proteins by theMCs via NOD2 was measured by ELISA. Cell surface protein expressionwas examined by FACS.RESULTS: The NOD2 expression and its function were up-regulated byIFN-�. Analysis by high-density oligonucleotide probe arrays revealedthat up-regulation of urinary-type plasminogen activator (uPA) expressionin MCs is specific to NOD2-mediated activation. In contrast, LPS- andIgE/anti-IgE-mediated MC activation induced PA inhibitor-1(PAI-1)expression. Furthermore, compared to the MDP-stimulated gene expres-sion profile of human peripheral blood mononuclear cells, MDP-stimu-lated MCs specifically induced a subset of genes that expressed uPA.MDP induced production of both uPA and tissue-type PA (tPA), with min-imal production of PAI-1.CONCLUSIONS: These results suggest that NOD2 in human MCsmight have different roles from that in monocytes.Funding: RIKEN Yokohama Institute

751 Global Cytokine Profile of Patients With SystemicMastocytosis

J. H. Butterfield; Mayo Clinic & Foundation, Rochester, MN.RATIONALE: Systemic mastocytosis (SM) remains a difficult disorderto understand and to treat effectively. To date there has been scant infor-mation about which cytokines are present in the circulation of thesepatients. We report the relative cytokine levels for SM patients with vary-ing percentages of bone marrow (BM) involvement.METHODS: Serum samples collected from 6 patients with SM (5->20%BM involvement) were analyzed using a human cytokine protein array(RayBiotech Human Cytokine Array V) featuring membrane-bound anti-bodies to 79 cytokines. Membranes were incubated with serum aliquots,washed, and developed as per the manufacturer’s instructions. X-ray filmimages were scanned using Adobe Photoshop 6.0, plotted, and analyzedusing NIH Image 1.63f software. Pixel counts of scanned membraneimages for cytokines were expressed as a fraction of the average pixelcount for positive control images for each membrane.RESULTS: Fifteen or more cytokines were detectable in the serum ofeach SM patient. For the patients with 5-10% BM involvement the serumcytokines with the highest levels of expression were (in order): TIMP-2,Angiogenin, PARC, IGFBP-2, TIMP-1. For those with 10-20% involve-ment: TIMP-2, Angiogenin, PARC, NAP-2, TIMP-1, Leptin. For thosewith >20% involvement: Angiogenin, PARC, TIMP-2, PDGF-BB,IGFBP-2, MIP-1d. (Abbreviations: TIMP: tissue inhibitor of metallopro-teinases; PARC: pulmonary and activation-regulated cytokine; NAP-2neutrophil activating peptide 2; IGFBP-2: insulin-like growth factor bind-ing proteins-2; MIP-1d: macrophage inflammatory protein-1d)CONCLUSIONS: Numerous cytokines are present in the serum of SMpatients. These results suggest that changes in the cytokine profiles maybe helpful in monitoring progression and understanding pathogeneticfindings in SM.Funding: Mayo Foundation

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752 �2-Adrenoceptor Agonist Induced Inhibition of Human MastCells is Dependent on Actin Degradation

S. C. Bischoff, T. Gebhardt, S. Bedoui, M. P. Manns; Medical School ofHannover, Hannover, GERMANY.RATIONALE: To characterize �2-adrenoceptor (�2AR)-mediated mod-ulation of growth and function of human intestinal mast cells (MC).METHODS: MC were isolated from intestinal surgery specimens, purifiedup to 95% using the MACS™ system, and subsequently cultured for 14-21days in the presence of stem cell factor (50ng/ml) with our without additionof adrenaline, noradrenaline, or salbutamol. Mediator release was induced byIgE receptor crosslinking upon incubation with mAb22E7 (100ng/ml) in thepresence or absence of �2AR agonists. Histamine and prostaglandin D2(PGD2) in the supernatant were measured by RIA and EIA, respectively. MCadhesion assays were performed with fibronectin coated plates and humanumbilical vein endothelial cells (HUVEC). Actin polymerization in activat-ed MCs was assessed by flow cytometry using fluorescent phallacidin. RESULTS: �2AR agonists dose-dependently reduced MC growth to23.4% (salbutamol), 25.3% (adrenaline), and 40.5% (noradrenaline) ofcontrol conditions (all at 10-6M, n=5). Activation-induced histaminerelease was inhibited to 4.1% (salbutamol), 21.6% (adrenaline), and 35.8%(noradrenaline, all at 1�M, n=5) of control conditions in a dose-dependentfashion. PGD2 release was nearly abolished in the presence of all �2ARagonists at 1�M (n=3). Moreover, the presence of �2AR agonists strong-ly inhibited MC adhesion to HUVECs as well as activation-induced adhe-sion to fibronectin (n=3). In addition, all �2AR agonists clearly inhibitedactin polymerization in MC induced by SCF (10ng/ml) or mAb22E7. CONCLUSIONS: Our data show that �2AR agonists profoundly regu-late human intestinal MC survival and function. Such findings may be ofimportance for our understanding of intestinal neuroimmune functionsand of therapeutic strategies used in allergic disorders.