1001

Mast cell Modulation of Immune Response During Bacterial Infections

Abraham SN, MacLachlan J, Shelburne C, Pizzo SV, Hart J, Staats HF

Department of Pathology, Molecular Genetics & Microbiology and Immunology, Duke University, Durham, NC, USA

The fact that mast cells have the capacity to secrete a wide range of immunomodulatory mediators together with their well documented role as major effectors in several  inflammatory disorders strongly suggest that these cells have an intrinsic capacity to modulate the immune system. For the past several years we have sought to identify an immunomodulatory role for mast cells during bacterial infection. With the help of wild type and mast cell deficient mice, we showed that mast cells are critical for the initiation of the host’s innate immune responses to infection. We also observed that mast cells at sites of infection regulate the migration of dendritic cells from sites of infection to the draining nodes and the sequestration of T cells from the circulation in these nodes. Mast cell regulation is achieved by “remote control” by the draining of primarily mast cell derived TNF from the sites of infection to the nodes via the lymphatic system. The draining node represents the epicenter where the adaptive immune response is developed and facilitating the interaction of dendritic cells and lymphocytes in this lymphoid organ is critical to the development of the immune response to the infection. Thus, following bacterial infection, mast cells at infected sites mediate simultaneous activation of both arms of the immune system. In view of the importance of mast cells in mobilizing immune defenses against infecting bacteria, we have recently attempted to boost the host’s immune defenses against infection by manipulating mast cells. Data from these studies will also be presented. 

1002

LFA-1: THE MULTI-FACETED REGULATOR OF LEUKOCYTE ADHESION AND MIGRATION

Ronen Alon

Dept. of Immunology, Weizmann Institute of Science, Rehovot, Israel

1003                                            Keynote Lecture

NETWORK MOTIFS: SIMPLE BUILDING BLOCKS OF COMPLEX REGULATION NETWORKS

Uri Alon

Depts. Molecular Cell Biology and Physics of Complex Systems Weizmann Institute of Science, Rehovot, Israel

We will discuss design principles of transcriptional regulation networks that control gene expression in cells. Recent advances in data collection and analysis are generating unprecedented amounts of information about gene regulation networks. To understand these complex wiring diagrams we sought to break them down into basic building blocks. We generalized the notion of motifs, widely used for sequence analysis, to the level of networks. Network motifs are patterns of interconnections that recur in many different parts of a network at frequencies much higher than those found in randomized networks. The known transcription networks across organisms are composed of repeated appearances of a few highly significant motifs. Each network motif has a specific function in determining gene expression, such as generating temporal expression programs and governing the responses to fluctuating external signals. The motif structure also allows an easily interpretable view of the entire known transcriptional network of the organism. This approach may help define the basic computational elements of other biological networks.

1004

DYNAMIC MEMBRANE INTERACTIONS DURING IGE RECEPTOR SIGNALING

Barbara Baird, Ryan Young, Julie Gosse, Alice Wiesner, Daniel Larson, Min Wu,

David Holowka

Dept. of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA

RBL mast cells have proven to be a valuable model for understanding hematopoietic cell signaling and the roles of plasma membrane domains in this process.  Crosslinking of IgE-FceRI, causes their stable association with detergent resistant lipid rafts and their consequent phosphorylation by Lyn kinase. Moreover, Lyn in the ordered lipid environment of these rafts is protected from inactivation caused by certain tyrosine phosphatases. High basal phosphorylation of FceRI by hyperactive Lyn in a CHO cell expression system can be regulated by co-transfection of a transmembrane phosphatase, PTPa, confering crosslink-dependent receptor phosphorylation. Other tyrosine phosphatases, including a lipid-raft preferring version of PTPa fail to do so.  To explore the role of transmembrane segments in immunoreceptor signaling, a series of single chain chimeric receptors for human IgE were stably expressed in RBL cells, and their widely different signaling capacities correlate strongly with crosslink-dependent lipid raft association. Thus, segregation of an ordered lipid-preferring tyrosine kinase from disordered lipid-preferring phosphatases provides a means for initiating IgE-FceRI signaling when crosslinking drives those receptors to coalesce with active Lyn in those ordered lipid domains. A dynamic view of later stages of the membrane interactions is provided by fluorescence correlation spectroscopy, which reveals that antigen crosslinking reduces the diffusion of IgE-FceRI and also Lyn with intermittent interactions occurring between these two components over tens of minutes. In other microscopy studies, spatial definition of crosslinked IgE-FceRI on the micron scale with surfaced-patterned haptens enables visualization of interacting components with spatial and temporal resolution. Phosphorylation by Lyn precedes actin-dependent concentration of Lyn and other inner leaflet raft components that relates to downstream signaling.

1005

IDENTIFICATION AND CHARACTERIZATION OF MAST CELL MEMBRANE AND SECRETED HYDROLASES USING A FUNCTIONAL PROTEOMICS APPROACH

Amos Baruch, Douglas A. Jeffery, Zhengying Pan, Kareem Chehade, James M. Clark

Celera Genomics, South San Francisco, CA, USA

Mediators that are released by mast cells have been implicated in multiple allergic and inflammatory disorders. Among these pro-inflammatory mediators, serine hydrolases/proteases such as tryptase play a prominent role in inflammation and in the regulation of mast cell functions such as migration and degranulation. Several mast cell serine hydrolases have been previously identified, however an unbiased biochemical approach to identify all members of this enzyme family is lacking. We have identified cell surface and secreted serine hydrolases using a unique set of activity-based small-molecule probes directed against that family of enzymes.  Furthermore, we have generated activity-based probes that selectively target the b and g isoforms of human tryptase. Specific inhibition of b-tryptase by selective inhibitors was evaluated both in vitro and in situ by monitoring their ability to compete with probe labeling. By administering the probes intra-nasally to mice, an increase in serine protease activity could be detected in vivo that was associated with airway hyperresponsiveness. In addition, results are presented in which we used activity-based probes for screening inhibitors against Guinea Pig tryptase present in crude lung homogenate. These studies have shown which inhibitors should be used in pre-clinical efficacy studies using Guinea Pig models of asthma.  In summary, functional proteomics approaches can be utilized for the identification and characterization of serine protease drug targets, for monitoring inhibition of those targets within a complex mixture, and for the selection and validation of animal models.  

1006

Sphingosine kinase 1 activation in mast cells – consequences for Lyn activation and LAT phosphorylation

T. Baumruker, A. Billich, E. Bofill-Cardona, N. Urtz

Novartis Institutes for Biomedical Research, Vienna, Austria

Sphingosine kinases have lately been recognized as essential signaling molecules that mediate the intracellular conversion of sphingosine to sphingosine-1-phosphate. In mast cells, induction of sphingosine kinase activity by the immunoglobulin E / antigen trigger was shown to be pivotal to the activation of this cell type by shifting the balance of “inhibitory” concentrations of sphingosine to activating concentrations of sphingosine-1-phosphate (rheostat concept). 

Here we provide evidence that sphingosine kinase 1 interacts directly with the tyrosine kinase Lyn and that this interaction - within minutes after triggering - leads to the recruitment of this lipid kinase to the FceRI. As a consequence of the complex formation between Lyn and sphingosine kinase 1, both show enhanced enzymatic activity.  Furthermore, sphingosine even further stimulates Lyn activity while sphingosine-1-phosphate is inhibitive. This picture at the molecular level is opposite to the sphingolipid rheostat defined at cellular level (here sphingosine is inhibitory). Therefore, additional mechanisms of regulation by sphingosine must apply. As one of these mechanisms, we show the induction of a currently uncharacterized phosphatase, bound to the adaptor molecule LAT. It results in an attenuated phosphorylation of this adaptor after mast cell activation and consequently in an inhibition of the MAP kinase pathway and PLCg. Contrary, all prior phosphorylation / activation steps upon immunoglobulin E / antigen triggering, i.e., b- and g-chain phosphorylation and Lyn and Syk activation, proceed normally or are even enhanced under high intracellular levels of sphingosine. 

1007

INDUCTION OF INHIBITORY REGULATORS OF MAST CELL SIGNALING BY GLUCOCORTICOIDS

Michael A. Beaven, Takaaki Hiragun, Marcus V. Andrade, Ze Peng

Laboratory of Molecular Immunology, NHLBI, National Institutes of Health, Bethesda, MD, USA

Dexamethasone and other glucocorticoids suppress FcRI-mediated release of inflammatory mediators from mast cells. The anti-inflammatory activity of glucocorticoids is generally attributed to suppression of cytokine gene transcription and their metabolic/endocrine effects to activation of gene transcription by processes that are referred to as transrepression and transactivation, respectively. However, suppression of cytokine gene transcription would not account for the inhibition of degranulation and generation of eicosanoids from arachidonic acid in mast cells. We have shown that dexamethasone potently inhibits the Erk1/2 pathway (J. Biol. Chem. 275:7066, 2000) and phosphatidylionositol 3-kinase-dependent pathways (J. Immunol. 172:7254, 2004) and as a consequence the release of arachidonic acid, degranulation, and possibly production of cytokines.  Recent studies now suggest that the glucocorticoids regulate signaling pathways by stimulating transcription of genes that encode inhibitory regulators. These include the inhibitory adaptor protein, downstream of tyrosine kinase (Dok)-1, MAP kinase phosphatase-1 (MKP-1), (Mol. Pharmacol., online ahead of print), and other less well investigated inhibitory regulators (unpublished data). Collectively, the studies suggest that the glucocorticoid-induced suppression of mast cell activation is mediated via glucocorticoid receptor and is dependent on activation as well as repression of transcription of various genes. If so, glucocorticoids selected for selectivity towards transrepression to minimize metabolic side effects via transactivation (the so called dissociated glucocorticoids) may lack an important component of the antiinflammatory activity of these drugs.

1008

ROLE AND MECHANISM OF SIGNALING OF THE KIT RECEPTOR TYROSINE KINASE IN MAST CELL DEVELOPMENT

Peter Besmer

Sloan Kettering Institute for Cancer Research, New York, USA

1009

HUMAN INTESTINAL MAST CELL BIOLOGY: ROLE IN IBD, IBS AND FOOD ALLERGY

Stephan Bischoff

Medical School of Hannover, Germany

1010

NEW EFFECTORS IN THE REGULATION OF MAST CELL EXOCYTOSIS

Ulrich Blank, C. Brochetta, C. Guérin-Marchand, Sophie Martin-Verdeaux, Isabel Pombo

INSERM U699, Bichat Medical School, Paris France

Exocytosis of mast cell granules requires a vesicular- and plasma membrane-associated SNARE fusion machinery. Here, we discuss some key components of this molecular machinery that regulate the final steps of fusion between the granular and plasma membrane, notably the role of regulatory syntaxin-binding Munc 18 proteins.  We did not detect the neuronal isoform Munc18-1, but mast cells express Munc18-2, which interacts with target SNAREs syntaxin 2 or 3, as well as Munc18-3, which interacts with syntaxin 4. Munc18-2 was localized to secretory granules whereas Munc18-3 was found on the plasma membrane. Increased expression of Munc18-2 and derived peptides containing an interfering effector loop inhibited IgE-triggered exocytosis, while increased expression of Munc18-3 showed no effect. Munc18-2 localization on granules is polarized, however, upon stimulation Munc18-2 redistributed into forming lamellipodia and persisted on granules that were aligned along microtubules, but was excluded from F-actin ruffles. Disruption of the microtubule network with nocodazole provoked Munc18-2 redistribution and affected mediator release. These findings suggest a role for Munc18-2 and the microtubule network in the regulation of secretory granule dynamics in mast cells. We further examined the distribution of SNARE membrane fusion and Munc18 accessory proteins in lipid rafts. Cognate SNARE complexes of syntaxin 3 with SNAP-23 and VAMP-8 were enriched in rafts, whereas Munc18-2/syntaxin 3 complexes were excluded. This demonstrates a spatial separation between these two types of complexes and suggests that Munc18-2 acts in a step different from SNARE complex formation and fusion.

1011

DEVELOPMENT OF SA001, AN INHIBITOR OF MAST CELL TRYPTASE

Jennifer A Cairns

Sanofi Aventis, Bridgewater, New Jersey, USA

Tryptase-beta is a mast cell-selective serine protease stored in the granules of mast cells and released following mast cell activation. Once released, tryptase-beta has been shown to act on a variety of cells and to degrade protein substrates, leading to downstream pro-inflammatory effects. All the extracellular actions of tryptase have been shown to require its catalytic activity. This abstract describes the development of SA001, an inhibitor of tryptase. SA001 inhibited tryptase activity with a Ki of 4.1 nm, with good selectivity over other related serine proteases. The compound displayed good pk properties in four species, with oral bioavalaibility of 46-78% and a t1/2 up to 4.3 hr. SA001 inhibited antigen-induced airway hyper-reactivity in a guinea pig model with an ed50 of 0.3-0.45 mg/kg p.o. The duration of action in the guinea pig was out to at least 8 hours. There was a dose-dependent inhibition of cysteinyl leukotriene release into the balf, suggesting an inhibition of mast cell degranulation. Histopathology studies showed a reduction in tissue eosnophilia and mast cells in guinea pigs dosed with sa001. In a mouse model, SA001 was also found to attenuate ovalbumin-induced airway hyper-responsiveness although there was a drop-off in the efficacy compared to that observed in the guinea pig. In a mouse inflammation model, sa001 inhibited the release of cytokines such as IL-4, IL-13, KC, MCP-1, IL-6 and MIP-1alpha. In conclusion, we have shown that SA001, an inhibitor of tryptase, can attenuate inflammation and airway hyper-responsiveness in two animal models supporting the rationale that inhibitors of tryptase can have therapeutic benefit in asthma.

1012

ORIGINS OF Functional diversity iN mammalian mast cell peptidases

George H. Caughey

Cardiovascular Research Institute and Dept. of Medicine, University of California at San Francisco, CA, USA

Serine peptidases of mast cell granules exhibit a range of form and function. The extent, origins and functional consequences of diversity within the tryptases and chymases are beginning to be understood. Recent discoveries by several investigators provide structural and genetic explanations for functional variations in soluble primate alpha/beta/delta and rodent MCP-6/7 tryptases. Our studies of canine mastin, a soluble tryptase-related peptidase, and of gamma-tryptases, which are type I membrane-anchored peptidases, provide fresh insights regarding origins of secreted mast cell tryptic enzymes. These data encourage us to hypothesize that soluble tryptases evolved from Type I transmembrane peptidases by losing their anchors then acquiring an ability to form inhibitor-resistant oligomers, an ancestral characteristic shared by many latter-day tryptase-like enzymes. Mastins have evolved even more rapidly than tryptases, with at least one modern mastin (porcine T30) losing the ability to protect itself from inhibition by oligomerizing, and with others converting to pseudogenes (primate mastin). Diversity and multiplicity among mast cell chymase-like genes is also striking, especially in rodents, which express a variety of chymases, some of which acquired mutations causing loss of chymotryptic and gain of elastolytic activity. Although dogs and primates express only one active chymase (alpha), the canine genome now reveals an apparent pseudogene related to beta-chymase genes, when were previously found only in rodents. We conclude that alpha and beta chymases both originate deep in mammalian or vertebrate evolution and have since diversified in form and function, leading to major differences between mammals in the consequences of mast cell peptidase release.

1013                                               Opening Lecture

SILENCE OF THE GENES

Howard Cedar

Hebrew University Medical School, Jerusalem, Israel

A large fraction of the animal genome is maintained in a repressed state throughout development.  This is probably carried out by utilizing fundamental epigenetic mechanisms such as trans acting repressors, DNA methylation and late replication timing which are all characterized by their unique ability to be preserved autonomously through cell division.  These basic pathways operate either individually or in combination by affecting local patterns of histone tail modification and other aspects of chromatin, thus generating a structural code for regulating repression paradigms.

1014

FcgRIIB AMPLIFY AUTONOMOUS NEGATIVE REGULATION OF FceRI SIGNALING IN MAST CELLS

Renaud Lesourne, Odile Malbec, Karine Roget, Pierre Bruhns, Marc Daëron

Unité d’Allergologie Moléculaire & Cellulaire, Institut Pasteur, Paris, France

Mast cell activation results from the transient displacement of a balance between positive and negative signals which are generated by activating and inhibitory receptors and integrated by transmembrane adapter molecules within intracellular signaling complexes. Thus, FceRI signaling is negatively regulated by low-affinity receptors for IgG (FcgRIIB) that are co-expressed with FceRI on mast cells. Inhibition depends on an Immunoreceptor Tyrosine-based Inhibition Motif (ITIM) located in the intracytoplasmic domain of murine and human FcgRIIB. It occurs when the FcgRIIB ITIM is tyrosyl-phosphorylated upon co-aggregation of the two receptors by immune complexes, and recruits the SH2 domain-containing phopshatidylinositol 5-phosphatase SHIP1. SHIP1 inhibits FceRI-triggered intracellular pathways leading to an increase in intracellular Ca²⁺ and to the activation of MAP kinases. FceRI signaling is also dampened by autonomous negative regulation. The engagement of FceRI indeed generates both positive and negative signals whose integration determine the intensity of mast cell responses. Autonomous regulation of FceRI signaling also depends on SHIP1. Negative signals take over positive signals as FceRI aggregation increases with the concentration of extracellular ligand and as receptor aggregates associate with the F-actin skeleton. We found that the F-actin skeleton also critically contributes to FcgRIIB-dependent negative regulation. Following co-aggregation with FceRI, FcgRIIB associate with the F-actin skeleton. This compartment contains the high-molecular weight-isoform of SHIP1 that is constitutively associated with the actin-binding protein Filamin-1 and is preferentially recruited by FcgRIIB. Increasing amounts of SHIP1 colocalize with FcR patches as these enlarge with time, while Filamin-1 and F-actin become excluded. Based on these results, we propose a dynamic model according to which the F-actin skeleton functions as an inhibitory compartment for both constitutive and FcgRIIB-dependent negative regulation of FceRI signaling. There, Filamin-1 serves as a donor of SHIP1 that FcgRIIB concentrate close to FceRI signaling complexes in which SHIP1 substrates are generated.

1015

ROLES OF THE NON-T CELL ACTIVATION LINKER (NTAL) IN MAST CELL SIGNALING

Petr Dráber

Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic

Early FceRI-mediated activation events include tyrosine phosphorylation of two transmembrane adaptor proteins, the linker for activation of T cells (LAT) and the non-T cell activation linker (NTAL). To clarify the role of NTAL in mast cell (MC) activation, we compared FceRI-mediated signaling events in bone marrow MC (BMMC) from NTAL-deficient and wild-type mice. Although NTAL is structurally similar to LAT, antigen (Ag)-mediated degranulation responses were unexpectedly enhanced in NTAL^-/- mice. The earliest event affected was enhanced tyrosine phosphorylation of LAT. This was accompanied by enhanced tyrosine phosphorylation and enzymatic activity of PLCg1 and PLCg2, resulting in elevated levels of free intracellular Ca²⁺. These data indicate that NTAL has a negative regulatory role in FceRI signaling. Interestingly, BMMC from mice deficient in the two adaptor proteins exhibited lower FceRI-mediated activation than LAT^-/- BMMC, suggesting that NTAL has not only negative but also positive regulatory roles. We also determined activation events in RBL-derived mast cells in which the NTAL levels were either enhanced by transfection of NTAL cDNA (NTAL+ cells) or decreased by RNA interference technique (NTAL- cells). Detailed analyses indicated that both NTAL+ and NTAL- cells exhibited decreased secretory responses after FceRI engagement, supporting the concept that only optimal levels of NTAL allow optimal degranulation. Immunogold electron microscopy on isolated membrane sheets demonstrated that in resting as well as FceRI-activated RBL cells NTAL as well as LAT formed small clusters which did not mix. Thus, both NTAL and LAT are located in distinct nonoverlapping regions of the plasma membrane.

1016

DIVERSE POTENTIAL ROLES OF MAST CELLS IN THE DEVELOPMENT AND EXPRESSION OF ADAPTIVE IMMUNE RESPONSES

Stephen J. Galli

Stanford University School of Medicine, Stanford, CA, USA

1017

INFLAMMATORY RECRUITMENT OF MAST CELLS TO THE LUNGS

Michael F. Gurish

Div. of Rheumatology Immunology and Allergy, Dept of Medicine

Brigham and Women’s Hospital and Harvard Medical School

Boston, MA, USA

Mast cells (MC) are critical innate effector cells involved in the response to certain parasitic and bacterial infections and in allergic responses most often associated with interfaces with the environment. MC are often divided into 2 major subclasses based on phenotypic differences, yet both are derived from a common MC progenitor (MCp) that arises from hematopoietic stem cells. A large pool of MCp is present in the small intestine of normal mice and this pool requires that the MCp express the a4β7 integrin and the chemokine receptor CXCR2. Surprising, antibody blocking experiments demonstrate that this pool is dynamic; the cells demonstrate a half life of less than 1 week in the small intestine. Allergic inflammation and other T helper type 2 (TH2) mediated reactions exhibit increased numbers of MC and we have found an early increase in the number of MCp at the site of TH2-mediated inflammation.  The increase is attenuated in mice lacking certain adhesion molecules, notably beta-7 integrin and CXCR2, or by administration of blocking antibodies to various adhesion molecules. These studies have allowed us to identify the alpha-4 integrins and their endothelial ligands that are important in directing constitutive homing of these cells to the small intestine and to contrast this with the specific requirements of these cells for recruitment to a site of TH2-mediated inflammation.

1018

INHIBITION OF PATHOLOGIC INFLAMMATION BY GP49B1

Joseph S. Zhou,¹ Thomas R. Murphy,¹ Daniel S. Friend,^1,2 K. Frank Austen,¹ Howard R. Katz¹

Departments of Medicine¹ and Pathology, ² Harvard Medical School, and Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Boston, MA, USA

gp49B1 is a member of the immunoglobulin superfamily that is constitutively expressed on the surface of mast cells.  The cytoplasmic domain of gp49B1 has two immunoreceptor tyrosine-based inhibitory motifs, which inhibit FceRI-dependent activation of mast cells in vitro by recruiting the tyrosine phosphatase SHP-1 from the cytoplasm to the plasma membrane.  gp49B1-null (gp49B^-/-) mice exhibit increased FceRI-dependent mast cell activation and ensuing rapid inflammation compared with gp49B1-sufficient (gp49B^+/+) mice.  In addition, dermal mast cells in gp49B^-/- mice are more sensitive to activation induced in vivo by stem cell factor (SCF).  Neutrophils express gp49B1 constitutively, and LPS increases the expression in vivo.  An intradermal injection of LPS in gp49B^-/- mice elicits in 24 h a severe neutrophil-dependent thrombohemorrhagic reaction that does not occur in gp49B^+/+ mice.  In addition to inhibiting acute inflammatory reactions, gp49B1 also suppresses the in situ production of cytokines and chemokines that are essential to the pathogenesis of a neutrophil- and mast cell-dependent model of proliferative synovitis that combines innate and adaptive signals.  Thus, gp49B1 inhibits pathologic inflammation initiated by a functionally diverse group of activating agents involved in adaptive, intrinsic, and innate responses.  

1019

MOLECULAR NETWORKS IN PROGRAMMED CELL DEATH

Adi Kimchi

Weizmann Institute of Science, Rehovot, Israel

1020                                               Keynote Lecture

IGE-MEDIATED PATHWAYS IN MAST CELLS: A GLIMPSE AT THERAPEUTIC STRATEGIES

Jean-Pierre Kinet

Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, MA, USA

1021

ENZYMES AND ADAPTERS REGULATE MAST CELL ACTIVATION

Michael Silverman, Jennifer Wu, Andrew Singer, Martha Jordan, Benjamin Olenchock, Xiao-Ping Zhong, Gary Koretzky

Abramson Family Cancer Research Institute and the Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA 

Stimulation of the high affinity receptor for IgE on mast cells leads to activation of numerous signal transduction cascades whose integration is critical for determining the cellular response.  Our laboratory is interested in identifying these signaling pathways and investigating how the different second messenger cascades are coordinated. We anticipate that such knowledge will provide clues to how aberrant mast cell function may be modulated.  One of the key signaling intermediates critical for mast cell activation is diacylglycerol (DAG), a lipid second messenger generated by hydrolysis of phosphatidylinositol-4,5-bisphosphate.  Once produced, DAG activates members of the protein kinase C family and stimulates GTP for GDP exchange on Ras, both essential steps in mast cell cytokine production and degranulation.  DAG signaling can be terminated by DAG phosphorylation by DAG kinases (DGKs) into phosphatidic acid (PA)  We have focused recently on one member of the DGK family, DGKz, by generating mice deficient in this enzyme through homologous recombination.  As expected, DGKz-deficient mast cells demonstrate decreased FceRI-mediated PA production as well as enhanced Ras activation and cytokine production.  Surprisingly, however, mice deficient in DGKz exhibit reduced rather than augmented passive cutaneous anaphylaxis.  This finding correlates with decreased antigen-stimulated degranulation ex vivo.  We are currently investigating the biochemical basis for this complex mast cell phenotype.

Upstream of DAG production, IgE receptor engagement leads to stimulation of several families of protein tyrosine kinases (PTKs).  PTK signaling is translated into production of key second messengers by the formation of multimolecular complexes nucleated by adapter proteins.  One such adapter, SH2 domain containing leukocyte phosphoprotein of 76 kDa (SLP-76), is critical for mast cell function.  We have shown previously that the IgE receptor on SLP-76-deficient mast cells is uncoupled from most but not all second messenger cascades.  More recently, we have been working to engineer a dominant negative version of SLP-76 that is predicted to interfere with mast cell function.  To date we have found that this dominant negative form blocks subcellular localization of SLP-76 in the RBL model and interferes with cytokine production and degranulation in primary murine mast cells.  Experiments are underway to determine if this reagent can be used in in vivo models of mast cell function.

1022

THE CORONARY MAST CELL – AN EFFECTOR CELL WITH MANY FUNCTIONS IN ATHEROGENESIS

P.T. Kovanen

Wihuri Research Institute, Helsinki, Finland

Immunohistochemical observations on human atherosclerotic lesions have revealed that the lesions contain mast cells. Studies with rat serosal mast cells in vitro and studies with rats in vivo have shown that the heparin proteoglycans of exocytosed mast cell granules bind low density lipoproteins (ldl), carry the bound ldl into macrophages, and so induce their conversion into foam cells. Granule chymase and tryptase also proteolyze hdl and reduces the hdl-dependent efflux of cholesterol from the foam cells. Functions for intimal mast cells other than those related to lipid metabolism are emerging; infiltrates of activated mast cells were detected at the site of human coronary atheromatous erosion or rupture in myocardial infarction. Both chymase and tryptase may degrade extracellular matrix of the fibrous cap of the atheromas, either directly, or indirectly by activating metalloproteinases secreted by other cells in the vulnerable regions. Mast cell chymase also degrades pericellular matrices and so disrupts the outside-in survival signaling. This way mast cells induce apoptosis of cultured smooth muscle cells and endothelial cells. Coronary mast cells, as sources of potent proinflammatory cytokines such as tnf-a, may also lead to production of certain metalloproteinases by macrophages in the lesions, and so further weaken the rupture-prone areas of atheromas. Finally, the products released from mast cells may also regulate thrombus formation; heparin by inhibiting platelet-collagen interaction, and chymase by degrading and inactivating thrombin. The results point to multiple functions of mast cells in human atherogenesis.

1023

THE TETRASPANIN CD9 IS ESSENTIAL FOR THE IL-16-MEDIATED CHEMOTAXIS AND ACTIVATION OF HUMAN MAST CELLS

Steven A. Krilis

Immunology, Allergy & Infectious Diseases, St. George Hospital, Kogarah, Australia

IL-16 plays a key role in numerous immune responses due to its ability to induce the chemotaxis and activation of T cells, monocytes, dendritic cells, eosinophils, and mast cells. The ability of IL-16 to inhibit HIV -1 infection also has implicated a protective role for this proinflammatory cytokine in AIDS patients. While CD4 appears to be the primary IL-16 receptor on the surface of the T cell, other IL-16-receptors must exist because monocytes and dendritic cells isolated from CD4-null mice are IL-16 responsive. We now show that the IL-16-reponsive mast cell line HMC-1 lacks CD4 protein on its surface, and that the IL-16-mediated chemotactic and Ca2+ mobilization responses of this cell line can be blocked effectively by anti-CD9 mAbs but not by anti-CD4 mAbs or mAbs directed against other tetraspanins. Similar findings were obtained with non-transformed cord blood-derived human mast cells. The chemotactic response of HMC-l cells to IL-16, as well as the binding of the cytokine to the cell's plasma membrane, were inhibited by CD9-specific antisense oligonucleotides. The tetraspanin therefore is essential for the IL-16-mediated chemotaxis and activation of human mast cells. In support of this conclusion, IL-16 bound to CD9-expressing CHO cell transfectants and activated a CD9-dependent, phosphatidylinositol 3-kinase/phospholipase Cγ/ inositol trisphosphate-dependent signalling pathway. This is the first report of a tetraspanin that plays an essential role in a cytokine-mediated chemotactic response of the mast cell.

1024

IgE ACTIVATION OF MAST CELLS: THE BEGINNING OF THE END OR THE END OF THE BEGINNING?

Francesca Levi-Schaffer, Ido Bachelet, Beata Berent, Ariel Munitz,

Department of Pharmacology, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Israel

An allergic response is typically initiated by the allergen cross-linking of IgE antibodies bound to FceRI receptors on the mast cells. Consequently, much information exists on IgE-triggering of mast cells and its consequences. In contrast, the possible network of inhibitory and pro-apoptotic signaling has not been extensively investigated as yet.

We have recently demonstrated that human cord blood derived mast cells (CBMC) express the inhibitory receptor IRp60, a non-MHC specific Ig-superfamily inhibitory receptor expressed and the death receptor for TRAIL (TNF-related apoptosis inducing ligand). Our aim was to evaluate the function of these two receptors on CBMC and their interaction with IgE-dependent activation. Cross-linking of IRp60 on CBMC inhibited IgE but not compound 48/80 mediated b-hexosaminidase and IL-4 release. IRp60, whose expression was found to be down-modulated by eosinophil MBP, acts via tyrosine phosphorylation and recruitment of SHP-1 and SHIP-1 but not of SHP-2. 

Activation of TRAIL, in the presence of an optimal concentration of SCF induced caspase-3 dependent apoptotic death of the CBMC after 12-24 hrs of incubation. CBMC activated via IgE and further incubated with TRAIL ligand for 12-24 hrs displayed a significantly increased apoptotic death.

Taken together these two results indicate that it is possible to down-modulate the allergic response at the mast cell level both by activation of the inhibitory receptor IRp60 and/or by ligation of the TRAIL receptor. It is still to be investigated whether these mechanisms are active in vivo during an allergic inflammatory reaction and, if not, how we can intervene to up-regulate them in this pathologic setting.

1025

APOPTOSIS-INDUCING CHIMERIC PROTEINS FOR TARGETED ALLERGY TREATMENT

R. Belostotsky¹, N. Wald¹, H. Lorberboum-Galski,^1,2

¹Department of Cellular Biochemistry and Human Genetics, Hebrew University, Hadassah Medical School, Jerusalem, ² Target-In Ltd.

The allergic response is initiated through the high-affinity receptor to IgE (FcεRI), which is found almost exclusively on the surface of mast cells and basophils. Since these cells play a central role in the allergic response, the targeted elimination of mast cells and basophils may be a promising approach for allergy treatment. In the last few years a new approach for targeted therapy of human diseases was developed using a class of molecules termed chimeric proteins. These molecules comprise both a cell targeting and a cell killing moiety, fused at the cDNA level.

We designed novel chimeric protein composed of the receptor-binding domain of the human IgE as the targeting moiety and pro-apoptotic proteins such as Bak, and Bax, as the killing components (hFcε-Bak/Bax/). Treatment of the target mast cells with the novel chimeric proteins had a dramatic effect on cell survival. Using Western blot, FACS analyses and confocal microscopy we demonstrated that the hFcε-chimeric protein binds to the surface of target cells, enters inside the cells and leads to their death via apoptosis. The effect was highly specific to cells expressing the FceRI. Moreover, interaction of the chimeric proteins with the mast cells did not cause degranulation.

Fce-Bak/Bax/ are new chimeric proteins of human origin, and, as such, are expected to be both less immunogenic and less toxic. Targeted elimination of mast cells and basophils via apoptosis is a novel concept for anti-allergy treatment and these promising reagents are now being developed for the targeted therapy of allergic diseases.

1026

SIGNAL TRANSDUCTION IN HUMAN BASOPHILS INDUCED BY HISTAMINE RELEASING FACTOR

Susan MacDonald

Johns Hopkins Asthma and Allergy Center, Baltimore, MD, USA

1027

REGULATION OF SECRETION IN HUMAN BASOPHILS

Donald MacGlashan

Johns Hopkins Asthma and Allergy Center, Baltimore, MD, USA

1028

ACTIVATION OF HUMAN BASOPHILS AND MAST CELLS BY SUPERALLERGENS

Gianni Marone, Francesca W. Rossi, Valentina Maglione, Giuseppe Spadaro, Virginia Forte, Massimo Triggiani

Department of Clinical Immunology and Allergy, University of Naples Federico II, Naples, Italy

Superantigens have the unique ability to interact specifically with most lymphocytes expressing antigen receptors from a particular variable region gene family. Classical superantigens are T-cell superantigens. However, some naturally occurring proteins have the properties of superantigens for B-lymphocytes. These proteins are endowed with unconventional immunoglobulin-binding capacities. Human basophils and mast cells are the only cells expressing the high affinity receptor for IgE and they play a prime role in the pathophysiology of allergic disorders through the elaboration and release of numerous proinflammatory and immunoregulatory molecules. We conducted experiments to determine whether immunoglobulin superantigens activate human basophils and mast cells to release pro-inflammatory mediators and cytokines. Protein Fv is released in the biological fluids of patients affected by viral hepatitis. Protein Fv preparations from viral hepatitis patients stimulated histamine and cytokine release from purified basophils. IL-4 and IL-13 mRNA, constitutively present in basophils, were increased after stimulation by protein Fv. Basophils from which IgE have been removed no longer released IL-4 in response to protein Fv and anti-IgE. Human monoclonal IgM V_H3⁺, but not V_H6⁺, concentration-dependently inhibited protein Fv-induced secretion of IL-4 and histamine from basophils and lung mast cells. HIV-1 gp120 is a superantigen, which might explain the activation of B-lymphocytes in patients with AIDS. We found that recombinant gp120 from divergent HIV-1 isolates increased IL-4 and IL-13 mRNA expression parallel to histamine secretion from basophils and lung mast cells. Gp120 activates FceRI⁺ cells through interaction with IgE, since removal of IgE completely blocked the glycoprotein’s effects on secretion. Preincubation of gp120 with monoclonal IgM V_H3⁺ also inhibited its effect on secretion from FceRI⁺ cells. Peptostreptococcus magnus is a bacterium expressing a cell wall protein L that binds human immunoglobulin through high-affinity interaction with immunoglobulin light chains. Increasing concentrations of Peptostreptococcus magnus and protein L induced histamine release from basophils and mast cells. Protein L and a recombinantly expressed fragment covering the immunoglobulin-binding domains B1-B4 also induced IL-4 and IL-13 release from basophils. Preincubation of protein L with IgE from myelomas expressing l chains did not affect the activating property of protein L, whereas IgE from myeloma with k chains completely blocked the activity of protein L. These results indicate that protein L interacts with the k light chains of IgE on human FceRI⁺ cells to induce the release of mediators. Thus, a novel mechanism may be envisaged by which endogenous, viral and bacterial proteins specifically activate human FceRI⁺ cells thereby acting as immunoglobulin superantigens. The in vivo implications of IgE-mediated activation of human basophils and mast cells by these immunoglobulin superantigens are yet to be defined.

1029

Anopheles mosquito bites activate cutaneous mast cells leading to lymph node hyperplasia and suppression of locally induced immune response

C. E. Demeure^*, K. Brahimi^†, F. Hacini^*, F. Marchand^*, R. Péronet^*, M. Huerre^‡ P. St-Mezard^§, J-F. Nicolas^§, P. Brey^†, G. Delespesse^¶, S. Mécheri^*

^*RPPI, ^†BBMI, ^‡Histotechnology and Pathology Units, Institut Pasteur, Paris, France; ^§INSERM, Lyon, France, ^¶ CHUM, Montreal, Québec, Canada

While Anopheles mosquitoes probe the skin for blood feeding, they inject saliva in dermal tissue. Mosquito saliva is known to exert various biological activities, but its modulatory effect on the immune system and its role in parasite transmission remain poorly understood. Here we report on the cellular changes occurring in the mouse skin and draining lymph nodes after Anopheles stephensi mosquito bite. We show that mosquito bites induce dermal mast cell degranulation leading to fluid extravasation  and neutrophil influx. This inflammatory response does not occur in mast cell-deficient W/W^v mice, unless these are reconstituted specifically with mast cells. Mast cell activation caused by mosquito bites is followed by hyperplasia of the draining lymph node due to the accumulation of CD3⁺, B220⁺, CD11b⁺, and CD11c⁺ cells. The T cell enrichment of the draining lymph nodes results from their sequestration from the circulation rather than local proliferation. These data demonstrate the role of mosquito saliva in triggering rapid innate immune responses and emphasize the critical contribution of peripheral mast cells in inducing T cell and dendritic cell recruitment within draining lymph nodes, a pre-requisite for the elicitation of the adaptive immune response.

1030

MAST CELL - T CELL INTERACTION:  AN OVERVIEW

Yoseph A. Mekori

Sackler Faculty of Medicine, Tel Aviv University, Israel

1031

MAST CELL DEVELOPMENT IN HEALTH AND DISEASE

Dean Metcalfe

NIAID, NIH, Bethesda, MD, USA

1032

ROLE OF CHEMOKINES IN MAST CELL ACTIVATION AND RECRUITMENT

Santa J. Ono

University College London, UK

1033

BASOPHIL PRODUCTION OF IL-4 AS VISUALIZED WITH CELLS FROM A GFP-INDICATOR MOUSE

Booki Min, Jinfang Zhu, William E. Paul

Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD, USA

Using mice in which the eGfp gene replaced the first exon of the Il4 gene (G4 mice), we examined production of IL-4 during infection by the intestinal nematode Nippostrongylus brasiliensis (Nb). Nb infection induced green fluorescent protein (GFP)^pos cells that were FcRI^pos, CD49b^bright, c-kit^neg, and Gr1^neg. These cells had lobulated nuclei and granules characteristic of basophils. They were found mainly in the liver and lung, to a lesser degree in the spleen, but not in the lymph nodes. Although some liver basophils from naive mice express GFP, Nb infection enhanced GFP expression and strikingly increased the number of tissue basophils. Similar basophil GFP expression was found in infected Stat6^–/– mice.  Basophils did not increase in number in infected Rag2^–/– mice nor did they in mice in which GATA3 was conditionally deleted in CD4 T cells.  Rag2^–/– mice reconstituted with CD4 T cells displayed significant basophil accumulation, indicating that GATA3-expressing CD4 T cells can direct both tissue migration of basophils and enhanced IL-4 production. IL-4 production was immunoglobulin independent and only partially dependent on IL-3. Thus, infection with a parasite that induces a "Th2-type response" resulted in accumulation of tissue basophils, and these cells, stimulated by a non-FcR cross-linking mechanism, are a principal source of in vivo IL-4 production.

1034

CROSS-TALK AMONG MEMBRANE RECEPTORS: REGULATION OF MAST CELLS' SECRETORY RESPONSE.

J. Abramson, A.E. Barbu and I. Pecht

Dept. of Immunology, The Weizmann Institute of Science, Rehovot, 76100, Israel.

Current understanding of the stimulus-response coupling networks triggered by the multi-chain immuno-recognition receptors (MIRRs) has markedly advanced while knowledge of its regulation is only emerging. Control of the secretory response of mast cells to the type I Fce receptor (FceRI) stimulus is a major topic of our interest. Several mast cell membranal receptors capable of inhibiting both immediate and late responses have so far been identified. However, their mode(s) of operation are only partly resolved. Moreover, control of mast cells response to the FceRI by desensitization, a wide spread process of control of many receptors, is still hardly understood.

We have previously shown that the FceRI-mediated degranulation is efficiently suppressed upon clustering the inhibitory receptor–Mast cell function-associated antigen (MAFA), previously discovered and characterized in our laboratory. MAFA clustering is also suppressing the FceRI -induced secretion of de novo synthesized cytokines and leukotriens and we have now shown that it interferes with activation of Erk-1/2 and p38 MAP kinase resulting in a selective suppression of cytokine gene transcription and leukotriene synthesis. Dok-1 and Dok-2 were found to undergo tyrosine phosphorylation upon MAFA clustering with the concomitant increase in Dok-1 binding to RasGAP. This is apparently essential for MAFA-mediated down-regulation of RasGTP levels, suppression of Erk activity and the subsequent reduced cytokine and leukotriene de novo synthesis. Both Dok molecules also undergo tyrosine phosphorylation upon FceRI clustering. Further, RBL-2H3 cells overexpressing Dok-1 exhibit significantly lower Ras and Erk-1/2 activation, and a concomitantly reduced level de-novo synthesis and secretion of TNF-a and LTC4. These findings suggest that Dok-1 functions as a built-in autoregulatory element, keeping in check the FceRI induced de-novo synthesis of pro inflammatory mediators. Most recently, we found that MAFA, so far considered only as an inhibitory receptor, may also produce activating signals as its clustering alone (without an activating stimulus of the FceRI) induces rapid activation of MAP kinases (Erk-1/2, p38, JNK), which subsequently enhances transcription of several genes (e.g. MCP-1).

1035

HOW ION CHANNELS ORCHESTRATE CALCIUM OSCILLATIONS

Rheinhold Penner

University of Hawaii, Honolulu, HI, USA

1036

THE CROSS TALK BETWEEN THE SIGNALING AND CYTOSKELETAL SYSTEMS THAT LEADS TO DIRECTED CELL MOTILITY

Liron Even-Faitelson, Daniel Ronen, Shoshana Ravid

Department of Biochemistry, Institute of Medical Sciences, Hadassah Medical School, The Hebrew University, Jerusalem, Israel

Many signaling pathways regulate the function of the cellular cytoskeleton, however we know very little about the identity of the proteins involved in the cross talk between the signaling and cytoskeletal systems.  PAK and Rock are the effectors of Rac and Rho small GT Pases proteins that play a key role in the regulation of cytoskeletal organization.  Non-muscle myosin II is an important component of the cytoskeleton, it has been shown to play a major part in cell motility and chemotaxis.  We investigated the role of PAK and Rock in the regulation of myosin II in prostate cancer cells in response to EGF stimulation.  We found that both PAK and Rock affect EGF-dependent myosin II phosphorylation, localization and chemotaxis, in an opposite manner.  We found that myosin II resides in a complex with PAK and aPKCz and that the interaction between these proteins is EGF-dependent.  We showed that aPKCz phosphorylates myosin II directly and specifically. In addition we found that myosin II undergoes dephosphorylation in response to EGF stimulation via Rho kinase (Rock) pathway.  The signaling pathway involving PAK1, aPKCz Rock, and myosin II that provides the link between the signaling and the cytoskeletal systems leading to chemotaxis will be discussed.

1037

THE INTERPLAY BETWEEN MITF, PIAS3 AND STAT3 IN CYTOKINE ACTIVATED MAST CELLS.

Carmit Levy, Amir Sonnenblick, Shelley Hachohen and Ehud Razin

Hebrew University Medical School, Jerusalem, Israel

The primary cell types affected in microphthalmia transcription factor (MITF)-deficient mice are melanocytes, osteoclasts and mast cells. A search for MITF associated proteins, using a mast cell library that was screened with a construct that encodes the bHLH-Zip domain of MITF, resulted in the isolation of the STAT3 inhibitor, PIAS3.  PIAS3 functions in vivo as a key molecule in suppressing the transcriptional activity of MITF.

Here we report that the zip domain is the region of MITF that is involved in the direct interaction between MITF and PIAS3. Additionally, we investigated the effect of phosphorylation of MITF on its interaction with PIAS3.  We found that phosphorylation of MITF on ser409, significantly reduced the inhibitory affect of PIAS3 on MITF and also modulated MITF’s transcriptional activity.  Thus, phosphorylation of MITF could be considered as a fine and alternative tuning of its transcriptional machiner

The PIAS family of proteins contains several conserved domains: the SAP domain, the Miz-Zn finger/RING domain and the PINIT motif, which is required for nuclear retention of PIAS3. PIAS3 functions in vivo as a key molecule in suppressing the transcriptional activity of both MITF and STAT3. In the present work we showed that PIAS3 directly and specifically interacts with MITF and STAT3 via 50 amino acids, which is located at the N terminus of its “PINIT” domain. We also showed that the interaction of MITF and STAT3 with the 50aa domain of PIAS3 affects their transcriptional activity in melanocyte and mast cells.

Moreover we explored how the MITF-PIAS3-STAT3 network of interactions occur, how this interactions regulate gene expression, and how cytokines activation mediated are involved in the in vivo interplay between these three proteins.

Since STAT3 is known to be activated by IL-6 in immune hematopoietic cells, we determined in mast cells whether IL-6 could regulate the associations in vivo between MITF, PIAS3 and STAT3. We found that IL6 trigger caused a transfer of PIAS3 from MITF to STAT3. Thus activation of mast cells by SCF or IL6, can lead to the mobility of PIAS3 between the two transcription factors induced changes in the association of MITF, STAT3 and PIAS3.

1038

FREE IMMUNOGLOBULIN LIGHT CHAINS MEDIATE MAST CELL ACTIVATION IN IMMEDIATE AND DELAYED HYPERSENSITIVITY RESPONSES

Frank Redegeld

Department of Pharmacology and Pathophysiology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands

Mast cells play an important role in allergic and non-allergic immune reactions. Mast cells can be activated non-specifically and via antigen-specific routes e.g. by crosslinking of receptors for IgE or IgG₁. We have recently identified a novel pathway for antigen-specific activation of mast cells via free immunoglobulin light chains (FLC).

Passive immunization of mice with FLC followed by crosslinking with cognate antigen resulted in rapid mast cell activation and edema formation in ear tissue.

Mast cells were shown to be crucial in the hypersensitivity response elicited by FLC: no response in genetically mast cell-deficient animals could be initiated. Moreover, mast cells in ear tissue showed morphological signs of degranulation. Current research focuses on the nature of the FLC receptor on mast cells. We have excluded an involvement of common gamma-chain associated receptors. Mast cell-bound FLC’s seem to colocalize with membrane lipid rafts after crosslinking.

Contact sensitization with low molecular weight compounds such as dinitrofluorobenzene, picryl chloride and oxazolone results in rapid production of FLC’s specific for the hapten used. Moreover, in mouse models for contact sensitivity and non-atopic asthma, we were able to inhibit development of clinical signs of disease by treating the animals with F991, an FLC antagonist. F991 is a peptide compound homologous to the Ig light chain-binding moiety of Tamm-Horsfall protein.

In conclusion, we propose that antigen-specific mast cell activation through FLC may be an alternative pathway to induce hypersensitivity responses. Our studies in preclinical models for contact sensitivity and asthma indicate that FLC or its receptor may be interesting therapeutic targets.

1039

MAST CELLS AND ANGIOGENESIS IN HEMATOLOGICAL MALIGNANCIES

Domenico Ribatti

Department of Human Anatomy and Histology, University of Bari Medical School, Bari, Italy

The importance of angiogenesis for growth and viability of solid and hematological tumors has been established. Tumor microvessel density predicts a risk of progression in multiple myeloma, in b-cell non-hodgkin’s lymphomas and matches in step with the growth of acute and chronic leukemias and myelodysplastic syndromes.

Tumor cells are surrounded by an infiltrate of inflammatory cells, namely lymphocytes, neutrophils, macrophages and mast cells (MC), which communicate via a complex network of intercellular signaling pathways mediated by surface adhesion molecules, cytokines and their receptors. MC density is highly correlated with the extent of both normal and pathological angiogenesis, such as that in chronic inflammatory diseases and tumors. The mechanism of MC-mediated neovascularization has been mainly ascribed to the effects of various products released by MC, including vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2) and tryptase.

We have demonstrated in multiple myeloma, B-cell non-Hodgkin’s lymphoma, myelodysplastic syndromes and B-cell chronic lymphocytic leukemia that there is a striking association between MC and microvessel counts and that both increase in function of tumor progression, as defined by its increasing malignancies grades. Moreover, angiogenesis is highly correlated with the total and MC tryptase-positive counts. Finally, a common features of our studies is the presence of MC granules with a piecemeal partial degranulation morphological aspect, expression of a progressive release of angiogenic factors in response to a chronic stimulation of MC degranulation.

1040                                          Keynote Lecture

Molecular regulation of mast cell activation and the allergic response

Stephen Brooks, Kazuyasu Chihara, Yasuko Furumoto, Gregorio Gomez, Martina Kovarova, Sandra Odom, Ana Olivera, Mikako Takasugi, Juan Rivera
Molecular Inflammation Section, NIAMS, NIH

Molecular signals are required for homeostatic control of mast cells in resting and activated states. Dysequilibrium of these molecular controls can cause detrimental outcomes.  Thus, regulatory events preceding and following engagement of the high affinity IgE receptor (FceRI) are important in determining the responsiveness of a mast cell.  This governing of a mast cells’ response may be key in controlling the inflammatory response, by these cells, that can cause allergic inflammation.  Among the many regulators, the “receptor phosphorylation initiating” SrcPTK family member, Lyn, plays an important role in negatively controlling the extent of a mast cells response.  The activity of another FceRI-proximal Src PTK, Fyn, was demonstrated to drive and enhance mast cell degranulation. The cellular role of these kinases as negative and positive regulators of a mast cells response is recapitulated in vivo in allergy models.  Additional studies show that the loss of Lyn interaction with FceRI has a modest inhibitory effect on mast cell degranulation while enhancing lymphokine production, even when many biochemical signals (including FceRI phosphorylation) are significantly impaired.  Lipids also control cell signaling  as modulators of both phosphatidylinositol and sphingomyelin metabolism cause altered mast cell responses.  The collective impact of these molecular regulators is exquisite control of mast cell responses.  Herein, we attempt to integrate these regulatory steps in the context of IgE-dependent mast cell activation and the allergic response.   

1041

NEURONAL CALCIUM SENSOR-1 (NCS-1), A NOVEL REGULATOR OF FCeRI-INDUCED SIGNALING IN MAST CELLS

Yaara Kapp-Barnea ¹, Koret Hirschberg² , Andreas Jeromin³, Ronit Sagi-Eisenberg¹

¹Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Israel, ²Department of Pathology, Sackler School of Medicine, Tel Aviv University, Israel, ³Division of Neuroscience, Baylor College of Medicine, Houston, TX, USA

Neuronal Calcium Sensor-1 (NCS-1), a member of the superfamily of EF-hand calcium-binding proteins, regulates synaptic transmission and plasticity. Previously, we have shown that NCS-1 is expressed in mast cells and by stimulating the activity of PI4Kb and increasing PI(4)P and PI(4,5)P₂ production NCS-1 potentiates FceRI-triggered generation of lipid-derived second messengers and exocytosis¹. Here we show that NCS-1 significantly stimulates recycling from the perinuclear endocytic recycling compartment (ERC) in the mast cell line rat basophilic leukemia (RBL-2H3). We further demonstrate that recycling through the ERC is an essential for FceRI-induced activation of the MAP kinases ERK1/2, whereby a close correlation exists between the amount of ERC localized non-active ERK1/2 and the amount of active ERK1/2 in the nucleus. Consistent with this finding, NCS-1 potentiates and extends receptor-triggered activation and nuclear translocation of ERK1/2. Conversely, knock-down of synaptotagmin IX, a crucial factor for ERC recycling, abrogates FceRI-induced ERK1/2 activation. Finally, we show that secretion of Arachidonic-Acid (AA)/metabolites, a downstream process to ERK1/2 activation, is enhanced in NCS-1 over-expressing RBL cells. That NCS-1 action is mediated by activation of PI4Kb is indicated by our findings that expression of a kinase dead mutant of PI4Kb or PH PI(4)P binding domain of FAPP, result in inhibition of recycling, ERK1/2 activation and AA/metabolies release. Taken together, our implicate NCs-1 as a novel role of FceRI signaling and inflammatory reactions in mast cells.

1     Kapp-Barnea, Y., Melnikov, S., Shefler, I., Jeromin, A. and Sagi-Eisenberg, R. “Neuronal Calcium Sensor-1 (NCS-1) and Phosphatidylinositol 4-Kinase beta Regulate IgE Receptor Triggered Exocytosis in Cultured Mast Cells”. J. Immunol. 171, 5320-5327 (2003).

1042

MAST CELLS IN THE BRAIN

Rae Silver

Columbia University, New York, USA

1043

FUNCTIONAL DISSECTION OF THE ROLE OF SYK IN SIGNALING IN MAST CELLS

Reuben Siraganian

NIDCR, NIH, Bethesda, MD, USA

1044

REGULATION OF MAST CELLS AND OTHER HEMATOPOIETIC CELLS BY THE RASGRP FAMILY OF SIGNALING PROTEINS

Richard L. Stevens

Harvard Medical School, Brigham and Women's Hospital, Boston, MA, USA

The nucleotide sequences of the mouse, rat, and human cDNAs and genes that encode the fourth member of the Ras Guanine Nucleotide Releasing Protein (RasGRP) family of signaling proteins have been deduced.  RasGRP4 is a mast cell (MC) restricted, cation-dependent, guanine nucleotide exchange factor. It also is a diacylglycerol/phorbol ester receptor that plays a prominent role in dictating which protease and eicosanoid mediators are expressed in rat and human MC lines.  RasGRP4 appears to act downstream of the surface receptor Kit/CD117 and upstream of the transcription factor MITF.  Allelic variants of RasGRP4 have been identified, as well as functionally different isoforms that are the result of variable splicing of its gene.  Earlier gene-linkage studies revealed a site on chromosome 7A3-B1 that controls intrinsic airway reactivity to methacholine in backcrossed C3H/HeJ and A/J mice.  The 18-exon mRasGRP4 gene resides on chromosome 7A3-B1, and recent studies revealed that the MCs developed from the hyporesponsive C3H/HeJ mouse strain preferentially produce a defective isoform of mRasGRP4.  These and other data suggest that RasGRP4 is of critical importance in MC development and that the expression of abnormal isoforms of the protein can lead to MC dysfunction.  Others have shown that RasGRP1, RasGRP2, and RasGRP3 regulate the final stages of development of T cells, platelets, and B cells, respectively.  Our recent discovery of a RasGRP-like gene in C. elegans indicates that foundering member of this family of signaling proteins appeared quite early in evolution presumably because of its importance in immunity. 

1045

CONFORMATIONAL DYNAMISM DICTATE ANTIBODY SPECIFICITY AND CROSS-REACTIVITY

Leo C. James¹, Dan S. Tawfik²

¹ MRC Laboratory of Molecular Biology, Cambridge, United Kingdom,

² Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel

Our knowledge regarding transient protein conformations and binding intermediates is rather partial. We combined solution pre-steady-state kinetics and crystal structures to determine the entire sequence of events that lead to antibody complex formation. As paradoxical as it may seem, i will show how, the conformational diversity of an antibody (ige spe7) is the source of its cross-reactivity as well as its fine specificity. Spe7 equilibrates between different pre-existing isomers: one isomer with a wide, shallow binding site (ab¹), binds a promiscuous protein antigen; the other isomer (ab²), possesses a deep and narrow cavity that binds aromatic ligands. Including the immunizing hapten. Our most recent results show that ab² is a highly promiscuous antibody conformer that binds both specific, and non-specific ligands, to form the same encounter, low-affinity complex. Yet only specific ligand complexes undergo a subsequent conformational rearrangement to yield the final, high-affinity complex ab³. We determined the structure of ab² in complex with a non-specific ligand. Its comparison with ab³ reveals rearrangements involving backbone and side-chain movements of up to 14å. This conformational switch, combined with a pre-equilibrium isomerisation between ab² and ab¹, provide spe7 with a ‘kinetic discrimination’ mechanism that enables it to distinguish, by factor of up to 10⁴, between highly-related ligands that initially bind ab² with the same affinity.

References

James, L.C., Roversi, P. & Tawfik, D.S. (2003) Antibody multi-specificity mediated by conformational diversity. Science 299, 1362-1367.

James, L.C & Tawfik, D.S. (2003) The specificity of cross-reactivity: Promiscuous antibody binding involves specific hydrogen-bonds rather than non-specific hydrophobic stickiness. Protein Sci. 12, 2183-2193.

James, L.C & Tawfik, D.S. (2003) Conformational diversity and protein evolution – a 60 year old hypothesis revisited. Trends Biochem. Sci. (TiBS) 28, 361-8.

James, L.C & Tawfik, D.S. (2005) Structure and kinetics of a transient antibody binding intermediate along a kinetic discrimination mechanism. Submitted.

1046

CRH AND IL-1 TRIGGER SELECTIVE SECRETION OF EITHER VEGF OR IL-6 FROM HUMAN MAST CELLS

Theoharis C. Theoharides^1,2,3, Jing Cao², Nikoletta G. Papadopoulou¹, Jill Donelan¹, Lauren Oleson¹, Duraisamy Kempuraj¹, Kristiana Kandere-Grzybowska^1,*

Dept. of ¹Pharmacology, ²Biochemistry, ³Internal Medicine, Tufts University School of Medicine and Tufts-New England Medical Center, Boston, MA, USA

A number of internal and external stressors can affect most tissues of the body, while many inflammatory diseases worsen by stress.  Corticotropin-releasing hormone (CRH) is secreted under stress and regulates the hypothalamic-pituitary-adrenal (HPA) axis, but is also secreted outside the brain where it exerts proinflammatory effects, possibly through mast cell activation.  We report that both CRHR-1 and CRHR-2 are expressed on human umbilical cord blood-derived cultured mast cells (hCBMCs) by RT-PCR, Western blot analysis and immunocytochemistry. Stimulation by CRH induces selective synthesis and release of vascular endothelial growth factor (VEGF) without histamine, tryptase, IL-6, IL-8 or TNF-α, a process blocked by the selective CRHR-1 antagonist Antalarmin. In contrast, stimulation by the selective CRHR-2 agonist urocortin III induces selective release of IL-6 without tryptase. Pretreatment of hCBMCs with IL-1 upregulates CRHR-2 only and IL-1, itself, stimulates selective release of IL-6 without tryptase and without degranulation.  Neither CRH nor IL-1 can augment secretion triggered by FcεRI activation.  However, intradermal administration of CRH leads to skin mast cell degranulation and increased vascular permeability, which a mediated by neurotensin (NT), as pretreatment with a NT-receptor antagonist blocks the CRH effect. This is the first evidence that activation of different CRHR types leads to selective release of inflammatory mediators from normal human mast cells without degranulation and that IL-1 can also trigger selective IL-6 release. These results implicate CRH and mast cells in initiating or potentiating inflammatory diseases, especially those affected by stress.