Irit Shefler

Cognitive-Performance Recovery of Alzheimer's Disease Model Mice by Modulation of Early Soluble Amyloidal Assemblies†

A rationally designed oligomerization inhibitor interacts with early intermediate assemblies of amyloid-beta polypeptide (Abeta) through the aromatic elements and inhibits their assembly into the toxic oligomers that cause Alzheimers disease by a unique C(alpha)-methylation beta-breakage strategy. The electrostatic potential of the low-energy conformation of the dipeptide inhibitor bound to Abeta is shown.

T Cell-Induced Mast Cell Activation: A Role for Microparticles Released from Activated T Cells

Close physical proximity between mast cells and T cells has been demonstrated in several T cell-mediated inflammatory processes. However, the way by which mast cells are activated in these T cell-mediated immune responses has not been fully elucidated. We previously identified and characterized a novel mast cell activation pathway initiated by physical contact with activated T cells and showed that this pathway is associated with degranulation and cytokine release. In this study, we provide evidence that mast cells may also be activated by microparticles released from activated T cells that are considered miniature versions of a cell. Microparticles were isolated from supernatants of activated T cells by Centricon filtration or by high-speed centrifugation and identified by electron microscopy, flow cytometry (Annexin stain), and expression of the integrin LFA-1. Stimulated T cells were found to generate microparticles that induce degranulation and cytokine (IL-8 and oncostatin M) release from human mast cells. Mast cell activation by T cell microparticles involved the MAPK signaling pathway. The results were similar when mast cells were stimulated by activated fixed T cells or by whole membranes of the latter. This suggests that microparticles carry mast cell-activating factors similar to cells from which they originate. By releasing microparticles, T cells might convey surface molecules similar to those involved in the activation of mast cells by cellular contact. By extension, microparticles might affect the activity of mast cells, which are usually not in direct contact with T cells at the inflammatory site.

Gi-Mediated Activation of the Syk Kinase by the Receptor Mimetic Basic Secretagogues of Mast Cells: Role in Mediating Arachidonic Acid/Metabolites Release

Syk kinase is essential for FcεRI-mediated signaling and release of inflammatory mediators from mast cells. We now show that activation of rat peritoneal mast cells by the nonimmunological, Gi-mediated pathway also results in the activation of Syk. We show that compound 48/80 (c48/80), a receptor analogue that activates directly G proteins, activates Syk in a pertussis toxin-sensitive fashion. We further show that Syk activation by c48/80 is blocked by the protein kinase C inhibitor GF109203X, by the phosphatidylinositol 3-kinase inhibitors, wortmannin and LY294002, by EGTA, and by the selective src-like kinase inhibitor PP1. These results suggest that in the nonimmunological, Gi-mediated pathway, Syk is located downstream from phospholipase C and phosphatidylinositol 3-kinase. However, in common with the FcεRI-mediated pathway, activation of Syk by c48/80 is dependent on a src-like protein tyrosine kinase. Finally, we show that in the nonimmunological pathway, Syk plays a central role in the release of arachidonic acid/eicosanoid metabolites, but not in the release of prestored mediators such as histamine.

Neuronal Calcium Sensor-1 and Phosphatidylinositol 4-Kinase β Regulate IgE Receptor-Triggered Exocytosis in Cultured Mast Cells

We examined the possible occurrence and function of neuronal Ca2+ sensor 1 (NCS-1/frequenin) in the mast cell line rat basophilic leukemia, RBL-2H3. This protein has been implicated in the control of neurosecretion from dense core granules in neuronal cells as well as in the control of constitutive secretory pathways in both yeast and mammalian cells. We show that RBL-2H3 cells, secretory cells of the immune system, endogenously express the 22-kDa NCS-1 protein as well as an immune-related 50-kDa protein. Both proteins associate in vivo with phosphatidylinositol 4-kinase β (PI4Kβ) and colocalize with the enzyme in the Golgi region. We show further that overexpression of NCS-1 in RBL-2H3 cells stimulates the catalytic activity of PI4Kβ, increases IgE receptor (FcεRI)-triggered hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), and stimulates FcεRI-triggered, but not Ca2+ ionophore-triggered, exocytosis. Conversely, expression of a kinase-dead mutant of PI4Kβ reduces PI4Kβ activity, decreases FcεRI-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis, and blocks FcεRI-triggered, but not Ca2+ ionophore-triggered, exocytosis. Our results indicate that PI(4)P, produced by the Golgi-localized PI4Kβ, is the rate-limiting factor in the synthesis of the pool of PI(4,5)P2 that serves as substrate for the generation of lipid-derived second messengers in FcεRI-triggered cells. We conclude that NCS-1 is involved in the control of regulated exocytosis in nonneural cells, where it contributes to stimulus-secretion coupling by interacting with PI4Kβ and positive regulation of its activity.

Basic secretagogues activate protein tyrosine phosphorylation and release of arachidonic acid in mast cells via a novel protein kinase C and phosphatidylinositol 3‐kinase‐dependent mechanism

Mast cells play a central role in inflammatory and immediate‐type allergic reactions. These granulated cells release by a process of regulated exocytosis a variety of biologically active substances which are either preformed (e.  g. histamine), or synthesized de novo following activation [e.  g. metabolites of arachidonic acid (AA) and multifunctional cytokines]. Exocytosis in mast cells is activated either in response to aggregation of the receptors for immunoglobulin E (FcϵRI) or by the direct activation of pertussis toxin‐sensitive G‐proteins by a class of receptor mimetic agents, collectively known as basic secretagogues of mast cells. In the present study we show that compound 48/80 (c48/80), a synthetic member of the class of basic secretagogues, stimulates protein tyrosine phosphorylation of a number of as yet unidentified cellular substrates.These phosphorylations were inhibited by the tyrphostin AG‐18, by the phosphatidylinositol 3‐kinase inhibitor wortmannin and by the protein kinase C inhibitors K252a and GF109203X. These inhibitors also inhibited the release of AA induced by c48/80 but had no effect on exocytosis. Taken together, our findings suggest that basic secretagogues induce protein tyrosine phosphorylation as part of their parallel multiple signaling pathways which are presumably mediated by more than one G‐protein. Both protein kinase C and phosphatidylinositol 3‐kinase serve as intermediates in this signaling pathway. The protein tyrosine kinase signaling pathway, which mediates the activation of AA release, does not contribute to secretion of the preformed mediators such as histamine, but it might largely contribute to the de novo production of inflammatory mediators like leukotrienes and prostaglandins.

Mast cells as sources and targets of membrane vesicles.

In addition to being major effector cells in the elicitation of allergic responses, mast cells have been found to play a significant role in the establishment of innate and adaptive immune responses. This occurs, in part, by regulating the phenotype and function of immune cells such as T cells, B cells and dendritic cells, and by acting as antigen presenting cells. Indeed, mast cells have been found to be activated in various T cell-mediated inflammatory processes and to reside in close physical proximity to T cells. Such observations have led investigators to propose a functional relationship between these two cell populations. Mast cells can interact with other cells including T cells in several ways such as cell-cell interaction via membrane associated receptors, release of cytokines and chemokines or by heterotypic adhesion to activated T cells. In this review, we focus on a novel communication pathway between mast cells and other inflammatory cells that occurs by the release of or response to membrane vesicles. Membrane vesicles are circular fragments, released from the endosomal compartment as exosomes or shed from the cell plasma membrane as microparticles. Because their membrane orientation is the same as that of the donor cell, they can be considered to be miniature versions of a cell. Growing evidence indicates that microparticles play a pivotal role in cell to cell communication. The functional consequences of such membrane transfers include the induction, amplification and/or modulation of immune responses, as well as the acquisition of new functional properties by recipient cells.

Inhibition of Basic Secretagogue-Induced Signaling in Mast Cells by Cell Permeable Gαi-Derived Peptides

Background: Basic secretagogues of connective tissue mast cells act as receptor mimetic agents that trigger mast cells by activating G proteins. This leads to simultaneous propagation of two signaling pathways: one that culminates in exocytosis, while the other involves protein tyrosine phosphorylation and leads to release of arachidonic acid metabolites. We have previously shown that introduction of a peptide that comprises the C-terminal end of Gαi3 into permeabilized mast cells inhibits basic secretagogue-induced exocytosis [Aridor et al., Science 1993;262:1569–1572]. We investigated whether cell-permeable peptides, composed of the C-terminus of Gαi3 fused with importation sequences, affect mast cell function. Methods: Following preincubation with the fused peptides, rat peritoneal mast cells were activated by compound 48/80 and analyzed for histamine and prostaglandin D2 release and protein tyrosine phosphorylations. Results: We demonstrate that out of three importation sequences tested only Gαi3 peptide fused with the Kaposi fibroblast growth factor importation sequence (ALL1) inhibited release of histamine. ALL1 as well as a cell-permeable peptide that corresponds to Gαi2 also blocked compound 48/80-stimulated protein tyrosine phosphorylation, though the latter did not block histamine release. ALL1 effect was G protein-specific, as it was incapable of blocking protein tyrosine phosphorylation stimulated by pervanadate. Conclusion: ALL1, a transducible Gαi3-corresponding peptide, blocks the two signaling pathways in mast cells: histamine release and protein tyrosine phosphorylation. Cell permeable peptides that block these two signaling cascades may constitute a novel approach for preventing the onset of the allergic reaction.

Characterization of ERK Activation in Human Mast Cells Stimulated by Contact with T Cells

Close physical proximity between mast cells and T cells has been demonstrated in several human conditions. We have identified and characterized a novel mast cell activation pathway initiated by contact with T cells, and showed that this pathway is associated with cytokine release. It has been shown recently that Ras is activated in this pathway. Thus, in the present study we further explore the downstream events associated with Ras activation and cytokine release in human mast cells stimulated by contact with T cells. ERK activation in human mast cells stimulated by either contact with T cells or by crosslinking the FC epsilon receptor was studied. Photobleaching experiments were used to study ERK localization. Enzyme linked immunosorbent assay was used to study the cytokine release by human mast cells. We show that stimulation of human mast cells by contact with activated T cells results is sustained ERK activation. Furthermore, sustained ERK activation in these cells is associated with increased dwell time at the nucleus and with IL-8 release. Interestingly, when mast cells were stimulated by crosslinking the FC epsilon receptor I, ERK activation was transient. ERK activation was associated with a shorter dwell time at the nucleus and with TNF-α release. Thus, retaining ERK in the nucleus might be a mechanism utilized by human mast cells to generate different cytokines from a single signaling cascade.

Immunotherapy with commercial venoms is efficacious for anaphylactic reactions to Vespa orientalis stings.

Background:Vespa orientalis (VO) stings occasionally induce anaphylaxis. In the absence of commercial VO venom, allergists use commercial venoms for immunotherapy, despite having no indication regarding efficacy. We attempted to examine the effectiveness of immunotherapy with commercial venoms in patients with VO allergy and to identify the venom accountable for this effect. Methods: Patients who unequivocally identified VO as the culprit insect were administered venom immunotherapy with the commercial venoms available in Israel to which they had positive skin tests. Patients were also skin tested with VO venom sac extracts and, after reaching the maintenance dose, were sting challenged by a live insect. The allergenic components in the venom were determined by immunoblotting. Results: Twelve patients were recruited and, based on their skin test results, all were treated with yellow jacket (YJ) venom, either alone or combined with other venoms. All 8 patients who were sting challenged by VO demonstrated positive skin test responses to VO venom. Six of the stung patients tolerated the sting challenge uneventfully. Two patients developed minimal transient symptoms that resolved spontaneously. SDS-PAGE with patient sera suggested cross-reactivity between VO and YJ venoms at molecular weights of 39–42 kDa. Using phospholipases, antigen 5 and hyaluronidase derived from several Vespa, Dolichovespula and Vespula species, hyaluronidase is possibly accountable for inducing the allergic reaction. Conclusion: In the absence of commercial VO venom the practice of treating patients allergic to this insect with available commercial venoms seems to be efficacious and YJ venom is probably responsible for this effect.

Gi-mediated activation of the p42/p44 mitogen-activated protein kinases by receptor mimetic basic secretagogues is abrogated by inhibitors of endocytosis

Exocytosis in mast cells, effector cells of allergic and inflammatory reactions, can be activated, in a receptor-independent manner, by a family of polycationic molecules (e.g. the Basic Secretagogues of mast cells) that activate directly heterotrimeric G-proteins that control exocytosis. We have recently shown that pertussis toxin (Ptx)-sensitive Gi-protein(s), activated directly by Basic Secretagogues, also stimulate protein tyrosine phosphorylation and activation of the p42/p44 MAP kinases, via a mechanism that involves protein kinase C (PKC), phosphatidylinositol-3-kinase and Ca2+ as intermediates (J. Pharmacol. Exp. Ther. 289 (1999) 1654). In this paper, we have investigated the role of endocytosis in this receptor-independent, G-protein-mediated signaling. Using mechanistically distinct inhibitors of clathrin-mediated endocytosis, we demonstrate that protein tyrosine phosphorylation and activation of p42/p44 MAP kinases are endocytosis-dependent. In contrast, Gi-stimulated exocytosis is unaffected. We show further that Gi activation results in recruitment of clathrin from the cytosol to the plasma membrane. Taken together, our results indicate that signal transduction between G-proteins and the components of the MAP kinase activation cascade is dependent on clathrin-mediated endocytosis and can occur independently of a 7 TM cell surface receptor.