R P Siraganian

National Institutes of Health, 베서스다, Maryland, United States

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Publications (182)834.89 Total impact

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    ABSTRACT: Monomeric IgE molecules, when bound to the high-affinity receptor, exhibit a vast heterogeneity in their ability to induce survival promotion and cytokine production in mast cells. At one end of this spectrum, highly cytokinergic (HC) IgEs can induce potent survival promotion, degranulation, cytokine production, migration, etc., whereas at the other end, poorly cytokinergic (PC) IgEs can do so inefficiently. In this study, we investigated whether IgEs recognize autoantigens and whether IgEs' binding of autoantigens correlates with difference s in HC versus PC properties. Enzyme-linked immunosorbent assays were performed to test whether IgEs bind antigens. Histamine-releasing factor in human sera was quantified by western blotting. Cultured mast cells derived from human cord blood were used to test the effects of human sera on cytokine production. Most (7/8) of mouse monoclonal HC IgEs exhibited polyreactivity to double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), β-galactosidase, thyroglobulin and/or histamine-releasing factor. By contrast, mouse PC IgEs failed to react with these antigens. A human monoclonal HC IgE also showed polyreactivity to histamine-releasing factor, dsDNA and ssDNA. Interestingly, sera from atopic dermatitis patients showed increased reactivity to ssDNA and β-galactosidase and increased levels of histamine-releasing factor. Some atopic dermatitis patients, but not healthy individuals, had substantial serum levels of HRF-reactive IgE. Sera from atopic dermatitis patients with high titers of DNA-reactive IgE could induce several fold more IL-8 secretion in human mast cells than sera from healthy individuals. The results show that most HC, but not PC, IgEs exhibit polyreactivity to autoantigens, supporting the autoimmune mechanism in the pathogenesis of atopic dermatitis.
    Allergy, asthma & immunology research 11/2012; 4(6):332-40. DOI:10.4168/aair.2012.4.6.332 · 2.43 Impact Factor
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    ABSTRACT: Mast cell activation results in the release of stored and newly synthesized inflammatory mediators. We found that Zeb2 (also named Sip1, Zfhx1b), a zinc finger transcription factor, regulates both early and late mast cell responses. Transfection with small interfering RNA (siRNA) reduced Zeb2 expression and resulted in decreased FcεRI-mediated degranulation, with a parallel reduction in receptor-induced activation of NFAT and NF-κB transcription factors, but an enhanced response to the LPS-mediated activation of NF-κB. There was variable and less of a decrease in the Ag-mediated release of the cytokines TNF-α, IL-13, and CCL-4. This suggests that low Zeb2 expression differentially regulates signaling pathways in mast cells. Multiple phosphorylation events were impaired that affected molecules both at early and late events in the signaling pathway. The Zeb2 siRNA-treated mast cells had altered cell cycle progression, as well as decreased expression of several molecules including cell surface FcεRI and its β subunit, Gab2, phospholipase-Cγ1, and phospholipase-Cγ2, all of which are required for receptor-induced signal transduction. The results indicate that the transcription factor Zeb2 controls the expression of molecules thereby regulating signaling in mast cells.
    The Journal of Immunology 05/2012; 188(12):6278-86. DOI:10.4049/jimmunol.1102660 · 4.92 Impact Factor
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    ABSTRACT: Activation of the high affinity IgE-binding receptor (FcεRI) results in the tyrosine phosphorylation of two conserved tyrosines located close to the COOH terminus of the protein-tyrosine kinase Syk. Synthetic peptides representing the last 10 amino acids of the tail of Syk with these two tyrosines either nonphosphorylated or phosphorylated were used to precipitate proteins from mast cell lysates. Proteins specifically precipitated by the phosphorylated peptide were identified by mass spectrometry. These included the adaptor proteins SLP-76, Nck-1, Grb2, and Grb2-related adaptor downstream of Shc (GADS) and the protein phosphatases SHIP-1 and TULA-2 (also known as UBASH3B or STS-1). The presence of these in the precipitates was further confirmed by immunoblotting. Using the peptides as probes in far Western blots showed direct binding of the phosphorylated peptide to Nck-1 and SHIP-1. Immunoprecipitations suggested that there were complexes of these proteins associated with Syk especially after receptor activation; in these complexes are Nck, SHIP-1, SLP-76, Grb2, and TULA-2 (UBASH3B or STS-1). The decreased expression of TULA-2 by treatment of mast cells with siRNA increased the FcεRI-induced tyrosine phosphorylation of the activation loop tyrosines of Syk and the phosphorylation of phospholipase C-γ2. There was parallel enhancement of the receptor-induced degranulation and activation of nuclear factor for T cells or nuclear factor κB, indicating that TULA-2, like SHIP-1, functions as a negative regulator of FcεRI signaling in mast cells. Therefore, once phosphorylated, the terminal tyrosines of Syk bind complexes of proteins that are positive and negative regulators of signaling in mast cells.
    Journal of Biological Chemistry 01/2012; 287(11):8194-204. DOI:10.1074/jbc.M111.326850 · 4.57 Impact Factor
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    ABSTRACT: IgE-mediated activation of mast cells and basophils underlies allergic diseases such as asthma. Histamine-releasing factor (HRF; also known as translationally controlled tumor protein [TCTP] and fortilin) has been implicated in late-phase allergic reactions (LPRs) and chronic allergic inflammation, but its functions during asthma are not well understood. Here, we identified a subset of IgE and IgG antibodies as HRF-interacting molecules in vitro. HRF was able to dimerize and bind to Igs via interactions of its N-terminal and internal regions with the Fab region of Igs. Therefore, HRF together with HRF-reactive IgE was able to activate mast cells in vitro. In mouse models of asthma and allergy, Ig-interacting HRF peptides that were shown to block HRF/Ig interactions in vitro inhibited IgE/HRF-induced mast cell activation and in vivo cutaneous anaphylaxis and airway inflammation. Intranasally administered HRF recruited inflammatory immune cells to the lung in naive mice in a mast cell- and Fc receptor-dependent manner. These results indicate that HRF has a proinflammatory role in asthma and skin immediate hypersensitivity, leading us to suggest HRF as a potential therapeutic target.
    The Journal of clinical investigation 12/2011; 122(1):218-28. DOI:10.1172/JCI59072 · 13.22 Impact Factor
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    Reuben P Siraganian · Rodrigo O de Castro · Emilia A Barbu · Juan Zhang ·
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    ABSTRACT: The aggregation by antigen of the IgE bound to its high affinity receptor on mast cells initiates a complex series of biochemical events that result in the release of inflammatory mediators. The essential role of the protein tyrosine kinase Syk has been appreciated for some time, and newer results have defined the mechanism of its activation. The use of siRNA has defined the relative contribution of Syk, Fyn and Gab2 to signaling and has made possible a screening study to identify previously unrecognized molecules that are involved in these pathways.
    FEBS letters 12/2010; 584(24):4933-40. DOI:10.1016/j.febslet.2010.08.006 · 3.17 Impact Factor
  • Rodrigo O de Castro · Juan Zhang · Maria C Jamur · Constance Oliver · Reuben P Siraganian ·
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    ABSTRACT: The Syk tyrosine kinase family plays an essential role in immunoreceptor tyrosine-based activation motif (ITAM) signaling. The binding of Syk to tyrosine-phosphorylated ITAM subunits of immunoreceptors, such as FcepsilonRI on mast cells, results in a conformational change, with an increase of enzymatic activity of Syk. This conformational change exposes the COOH-terminal tail of Syk, which has three conserved Tyr residues (Tyr-623, Tyr-624, and Tyr-625 of rat Syk). To understand the role of these residues in signaling, wild-type and mutant Syk with these three Tyr mutated to Phe was expressed in Syk-deficient mast cells. There was decreased FcepsilonRI-induced degranulation, nuclear factor for T cell activation and NFkappaB activation with the mutated Syk together with reduced phosphorylation of MAP kinases p38 and p42/44 ERK. In non-stimulated cells, the mutated Syk was more tyrosine phosphorylated predominantly as a result of autophosphorylation. In vitro, there was reduced binding of mutated Syk to phosphorylated ITAM due to this increased phosphorylation. This mutated Syk from non-stimulated cells had significantly reduced kinase activity toward an exogenous substrate, whereas its autophosphorylation capacity was not affected. However, the kinase activity and the autophosphorylation capacity of this mutated Syk were dramatically decreased when the protein was dephosphorylated before the in vitro kinase reaction. Furthermore, mutation of these tyrosines in the COOH-terminal region of Syk transforms it to an enzyme, similar to its homolog ZAP-70, which depends on other tyrosine kinases for optimal activation. In testing Syk mutated singly at each one of the tyrosines, Tyr-624 but especially Tyr-625 had the major role in these reactions. Therefore, these results indicate that these tyrosines in the tail region play a critical role in regulating the kinase activity and function of Syk.
    Journal of Biological Chemistry 08/2010; 285(34):26674-84. DOI:10.1074/jbc.M110.134262 · 4.57 Impact Factor
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    Juan Zhang · Mary Mendoza · Michel F Guiraldelli · Emilia Alina Barbu · Reuben P Siraganian ·
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    ABSTRACT: Mast cells play pivotal roles in the initiation of the allergic response. To gain an understanding of the functions played by phosphatases in IgE-mediated mast cell activation, a small interfering RNA (siRNA) library that targets all mouse phosphatase genes was screened in a mouse mast cell line, MMC-1. Of 198 targets, 10 enhanced and 7 inhibited FcepsilonRI-induced degranulation. For seven of the strongest hits, four different siRNAs per target were tested, and at least two out of the four single siRNA per target had similar effects as the pool suggesting that these were true hits. Bone marrow-derived mast cells from normal mice further validated these results for six definite positive targets. The mechanism of the reduced mast cell degranulation due to calcineurin B deficiency was investigated. Calcineurin B deficiency reduced the phosphorylation of MAPKs and the phosphorylation of protein kinase D/protein kinase Cmu and protein kinase Cdelta, which are involved in FcepsilonRI signaling. The screen, therefore, has identified several new molecules that are critical for FcepsilonRI-induced degranulation. Regulating the function of these proteins may be potential targets for the treatment of allergic inflammation. The result also indicates that the system used is efficient for searching molecules implicated in complex receptor-induced signaling.
    The Journal of Immunology 06/2010; 184(12):7178-85. DOI:10.4049/jimmunol.0904169 · 4.92 Impact Factor
  • Emilia Alina Barbu · Juan Zhang · Reuben P Siraganian ·
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    ABSTRACT: Several studies with mast cells from knock-out mice have suggested that the tyrosine kinase Fyn and its downstream substrate Gab2 may play a role in high affinity IgE receptor (FcepsilonRI)-mediated mast cell activation. To better understand the role of these two molecules and of Syk, we transiently transfected mast cells with small interference RNA (siRNA) targeted to Fyn, Gab2, or Syk to specifically decrease their expression. The siRNA suppression of Gab2 but not Fyn reduced activation of the phosphoinositide-3-kinase (PI3K) pathway as demonstrated by the change in phosphorylation of Akt; this indicates that Gab2 but not Fyn regulates this pathway. The decreased expression of Gab2 and Fyn had minor effects on degranulation. There were also some minor changes in activation of the NFAT or NFkappaB transcription factors in cells with reduced expression of Fyn or Gab2. Decreased Gab2 but not Fyn reduced the FcepsilonRI-induced activation of the Erk, Jnk, and p38 MAP kinases and the release of TNF-alpha. In contrast, decreased expression of Syk dramatically reduced FcepsilonRI-induced degranulation, activation of NFAT and NFkappaB. Therefore, the reduction in expression of these proteins in mast cells indicates that Syk is the major regulator of FcepsilonRI-mediated reactions, whereas Fyn has minor if any effects and Gab2 regulates primarily late events including MAP kinase activation and release of cytokines.
    Journal of Biological Chemistry 03/2010; 285(21):15761-8. DOI:10.1074/jbc.M110.109413 · 4.57 Impact Factor
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    Ana Cristina G Grodzki · Kyungduk D Moon · Elsa H Berenstein · Reuben P Siraganian ·
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    ABSTRACT: High affinity IgE receptor (FcvarepsilonRI)-induced activation of mast cells results in degranulation and generation of leukotrienes and cytokines. FcvarepsilonRI-induced mast cell activation was analyzed at a single cell basis using a rat basophilic leukemia (RBL-2H3) cell line transfected with a reporter plasmid containing three tandem NFAT (nuclear factor of activated T cells) binding sites fused to enhanced green fluorescent protein (GFP). Surprisingly, with this sensitive detection system, there is activation of IgE sensitized cells at concentrations of antigen as low as 10pg/ml, which was 10-fold lower than was detected by degranulation. There were differences in signaling pathways leading to degranulation compared to NFAT-mediated gene activation. Both signaling to NFAT activation and degranulation required Syk and calcineurin. However inhibitors of the phosphatidylinositol 3-kinase pathway blocked degranulation but did not NFAT activation. The results also indicate that NFAT was activated at lower intracellular signals compared to degranulation. Therefore, FcvarepsilonRI activation can result in nuclear signals in the absence of the release of mediators.
    Molecular Immunology 07/2009; 46(13):2539-47. DOI:10.1016/j.molimm.2009.05.013 · 2.97 Impact Factor
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    ABSTRACT: We show in this study that the ability of five different monomeric IgEs to enhance murine bone marrow-derived mast cell (BMMC) survival correlates with their ability to stimulate extracellular calcium (Ca(2+)) entry. However, whereas IgE+Ag more potently stimulates Ca(2+) entry, it does not enhance survival under our conditions. Exploring this further, we found that whereas all five monomeric IgEs stimulate a less robust Ca(2+) entry than IgE+Ag initially, they all trigger a more prolonged Ca(2+) influx, generation of reactive oxygen species (ROS), and ERK phosphorylation. These prolonged signaling events correlate with their survival-enhancing ability and positively feedback on each other to generate the prosurvival cytokine, IL-3. Interestingly, the prolonged ERK phosphorylation induced by IgE appears to be regulated by a MAPK phosphatase rather than MEK. IgE-induced ROS generation, unlike that triggered by IgE+Ag, is not mediated by 5-lipoxygenase. Moreover, ROS inhibitors, which block both IgE-induced ROS production and Ca(2+) influx, convert the prolonged ERK phosphorylation induced by IgE into the abbreviated phosphorylation pattern observed with IgE+Ag and prevent IL-3 generation. In support of the essential role that IgE-induced ROS plays in IgE-enhanced BMMC survival, we found the addition of H(2)O(2) to IgE+Ag-stimulated BMMCs leads to IL-3 secretion.
    The Journal of Immunology 10/2008; 181(6):3850-60. DOI:10.4049/jimmunol.181.6.3850 · 4.92 Impact Factor
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    ABSTRACT: The mast cell specific monoclonal antibody, mAb BGD6, is a mast cell lineage marker [Jamur, M.C., Grodzki, A.C., Berenstein, E.H., Hamawy, M.M., Siraganian, R.P., Oliver, C., 2005. Identification and characterization of undifferentiated mast cells in mouse bone marrow. Blood 105, 4282-4289]. In rat basophilic leukemia (RBL-2H3) cells, mAb BGD6 precipitates cell-surface proteins of approximately 110 and 40-60 kDa. An expression cloning strategy was used to identify proteins that interact with mAb BGD6. A RBL-2H3 cDNA library in plasmids was transfected into PEAK cells, which do not bind mAb BGD6, and positive cells were selected with mAb BGD6. The plasmids recovered from the positive cells were amplified; retransfected into PEAK cells and after several screening cycles a positive clone was identified. This clone showed almost complete identity to Fc gamma RIIB (CD32), the low affinity IgG receptor. However, in contrast to the sequence in GenBank, this clone had an insert of 141 bp which codes for a longer isoform of this molecule with an extra 47 aa in its cytoplasmic domain. In RBL-2H3 cells both isoforms were expressed, with higher expression of the shorter form. The mechanism of binding of mAB BGD6 on both RBL-2H3 and CD32 transfected PEAK cells was then examined. Intact mAb BGD6 bound to both RBL-2H3 and CD32 expressing PEAK cells, but F(ab')(2) fragments bound only to RBL-2H3 cells demonstrating that mAb BGD6 binds to Fc gamma RIIB only through its Fc portion. On RBL-2H3 cells, the Fab of an anti-CD32 mAb partially inhibited the binding of intact mAb BGD6. The binding pattern of mAb BGD6 inhibited with anti-CD32 resembled that of the F(ab')(2) fragment of the antibody suggesting that the Fc portion of mAb BGD6 contributes to its binding on cells that have Fc gamma RIIB. These results are consistent with a model where mAb BGD6 binds through its Fab portion to a approximately 110 kDa protein and the Fc tail interacts with Fc gamma RIIB (CD32).
    Molecular Immunology 05/2008; 45(8):2411-8. DOI:10.1016/j.molimm.2007.07.041 · 2.97 Impact Factor
  • Juan Zhang · Katsuhiro Suzuki · Tomohiro Hitomi · Reuben P Siraganian ·
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    ABSTRACT: Protein-tyrosine kinase Lyn and Syk are critical for antigen-receptor-induced signal transduction in mast cells. To identify novel Lyn/Syk substrates, we screened an RBL-2H3 bacterial expression library for proteins that were tyrosine phosphorylated with baculoviral expressed Lyn or Syk. Five clones as potential Lyn substrates and eight clones as Syk substrates were identified including known substrates such as SLP-76, LAT, and alpha-tubulin. A potential substrate of Lyn identified was the molecule TOM1L1, which has several domains thought to be important for membrane trafficking and protein-protein interactions. Because the function of TOM1L1 is unclear, the rat TOM1L1 full-length cDNA was isolated and used to express the protein in COS-1 and RBL-2H3 mast cells. In COS-1 cells, the co-transfection of TOM1L1 and Lyn, but not Syk, resulted in the tyrosine phosphorylation of TOM1L1. In RBL-2H3 mast cells, the overexpressed TOM1L1 was strongly tyrosine phosphorylated in non-stimulated cells, and this phosphorylation was enhanced by FcepsilonRI aggregation. By subcellular fractionation, wild-type TOM1L1 was mainly in the cytoplasm with a small fraction constitutively associated with the membrane; this association was markedly reduced in deletion mutants lacking several of the protein interaction domains. The overexpression of TOM1L1 enhanced antigen-induced tumor necrosis factor (TNF) alpha generation and release. Both protein interaction domains (VHS and the coiled-coil domains) were required for the increased TNFalpha release, but not the increased TNFalpha generation. These results suggest that TOM1L1 is a novel protein involved in the FcepsilonRI signal transduction for the generation of cytokines.
    Journal of Biological Chemistry 01/2008; 282(52):37669-77. DOI:10.1074/jbc.M705168200 · 4.57 Impact Factor
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    ABSTRACT: The high affinity immunoglobulin E receptor (FcepsilonRI) complex is dedicated to immunoglobulin E-mediated allergic responses. Expression of the FcepsilonRI receptor is thought to be relatively stable and limited to mast cells, basophils, eosinophils, monocytes, Langerhans cells, platelets, and neutrophils. We now report that the FcepsilonRIalpha and FcepsilonRIgamma polypeptides are expressed in the pinealocyte, the melatonin-secreting cell of the pineal gland. Moreover, Fcer1a mRNA levels increased approximately 100-fold at night to levels that were higher than in other tissues examined. Pineal FcepsilonRIalpha protein also increased markedly at night from nearly undetectable daytime levels. Our studies indicate that pineal Fcer1a mRNA levels are controlled by a well described neural pathway that controls pineal function. This pathway includes the master circadian oscillator in the suprachiasmatic nucleus and passes through central and peripheral structures. The circadian expression of FcepsilonRIalpha in the pineal gland is driven by this neural circuit via an adrenergic/cyclic AMP mechanism. Pineal FcepsilonRIalpha and FcepsilonRIgamma may represent a previously unrealized molecular link between the neuroendocrine and immune systems.
    Journal of Biological Chemistry 12/2007; 282(45):32758-64. DOI:10.1074/jbc.M705950200 · 4.57 Impact Factor
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    ABSTRACT: The high affinity immunoglobulin E receptor (FcϵRI) complex is dedicated to immunoglobulin E-mediated allergic responses. Expression of the FcϵRI receptor is thought to be relatively stable and limited to mast cells, basophils, eosinophils, monocytes, Langerhans cells, platelets, and neutrophils. We now report that the FcϵRIα and FcϵRIγ polypeptides are expressed in the pinealocyte, the melatonin-secreting cell of the pineal gland. Moreover, Fcer1a mRNA levels increased ∼100-fold at night to levels that were higher than in other tissues examined. Pineal FcϵRIα protein also increased markedly at night from nearly undetectable daytime levels. Our studies indicate that pineal Fcer1a mRNA levels are controlled by a well described neural pathway that controls pineal function. This pathway includes the master circadian oscillator in the suprachiasmatic nucleus and passes through central and peripheral structures. The circadian expression of FcϵRIα in the pineal gland is driven by this neural circuit via an adrenergic/cyclic AMP mechanism. Pineal FcϵRIα and FcϵRIγ may represent a previously unrealized molecular link between the neuroendocrine and immune systems.
    Journal of Biological Chemistry 11/2007; 282(45):32758-32764. · 4.57 Impact Factor
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    ABSTRACT: Recent studies have shown that, in mast cells, membrane microdomains rich in cholesterol and glycosphingolipids called lipid rafts play an important role in FcepsilonRI signaling. The present study demonstrates that, in RBL-2H3 cells following stimulation, the mast cell-specific gangliosides associated with FcepsilonRI are internalized from lipid rafts along with the receptor. When the cells are labeled with iodinated antibodies against the gangliosides or against FcepsilonRI and the cell components are then fractionated on Percoll density gradients, in stimulated cells the gangliosides are internalized with the same kinetics as FcepsilonRI and at 3 hr are present in the dense lysosome fraction. Using transmission electron microscopy, with antibody against the gangliosides conjugated to horseradish peroxidase and antibody against FcepsilonRI conjugated to colloidal gold, it was possible to demonstrate that the gangliosides and FcepsilonRI are internalized in the same coated vesicles. At 5 min, the gangliosides and FcepsilonRI can be identified in early endosomes and at 3 hr are found together in acid phosphatase-positive lysosomes. This study demonstrates that the mast cell-specific gangliosides are internalized from lipid rafts in the same vesicles and traffic intracellularly with the same kinetics as FcepsilonRI. This study contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.
    Journal of Histochemistry and Cytochemistry 05/2007; 55(4):315-25. DOI:10.1369/jhc.6A7037.2006 · 1.96 Impact Factor
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    Haifeng Song · Juan Zhang · Y Jeffrey Chiang · Reuben P Siraganian · Richard J Hodes ·
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    ABSTRACT: Deficiency in the adaptor protein B cell linker protein (BLNK) results in a substantial but incomplete block in B cell development, suggesting that alternative pathways exist for B lineage differentiation. Another adaptor protein, c-Cbl, plays a negative regulatory role in several BCR-signaling pathways. We therefore investigated the role of c-Cbl during B cell development and addressed the possibility that redundancies in pathways for B cell differentiation could be further revealed by eliminating negative effects mediated by c-Cbl. Strikingly, c-Cbl inactivation reversed a number of the critical defects in early B cell differentiation that are seen in BLNK-deficient mice. c-Cbl(-/-)BLNK(-/-) mice exhibited normalized down-regulation of pre-BCR and CD43, up-regulation of MHC class II, and augmented L chain rearrangement, resulting in a successful transition from pre-B cells to immature B cells. c-Cbl inactivation also reversed the potentially tumor-predisposing hyperproliferative response of BLNK(-/-) pre-B cells to IL-7. Pre-BCR cross-linking induced enhanced and prolonged tyrosine phosphorylation in c-Cbl(-/-)BLNK(-/-) pre-BCR(+) pre-B cells compared with c-Cbl(+/-)BLNK(-/-) cells, including elevated phosphorylation of Lyn, Syk, Btk, and phospholipase C-gamma2. Our studies suggest that some, but not all, pre-BCR-triggered developmental events can be mediated by BLNK-independent pathways that are negatively regulated by c-Cbl, and further suggest that different events during early B cell development require different strength or duration of pre-BCR signaling.
    The Journal of Immunology 02/2007; 178(2):926-35. DOI:10.4049/jimmunol.178.2.926 · 4.92 Impact Factor
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    Alessandra Mazzoni · Reuben P Siraganian · Cynthia A Leifer · David M Segal ·
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    ABSTRACT: The cytokines secreted by pathogen-activated human dendritic cells (DC) are strongly regulated in vitro by histamine, a major component of mast cell granules, ultimately modulating the capacity of the DC to polarize naive T cells. Because DC and mast cells are located in close proximity in peripheral compartments, we hypothesized that mast cell products would influence the maturation of DC and hence the Th balance of an immune response in vivo. In this study, we show that specific mast cell degranulation stimuli, given s.c. in mice with Ag and adjuvant, produce effector T cells that proliferate to Ag but secrete dramatically reduced levels of IFN-gamma and increased amounts of IL-4 compared with control T cells primed in the absence of a mast cell stimulus. Immunization with Ag and adjuvant in the presence of a degranulation stimulus also resulted in the accumulation of DC in the draining lymph nodes that had reduced capacity to induce Ag-specific Th1 cells, in comparison with DC from mice lacking a degranulation stimulus. Therefore, by acting upon DC at sites of inflammation, mast cells play a critical role in determining the polarity of Ag-specific T cell responses in vivo.
    The Journal of Immunology 10/2006; 177(6):3577-81. DOI:10.4049/jimmunol.177.6.3577 · 4.92 Impact Factor
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    ABSTRACT: We have previously demonstrated that adrenomedullin (AM) plays a critical role as an autocrine/paracrine tumor cell survival factor. We now present evidence that AM is an important regulator of mast cell (MC) function and that this modulation is potentially involved in tumor promotion. AM induced histamine or beta-hexosaminidase release from rat and human MCs through a receptor-independent pathway. AM was chemotactic for human MCs and stimulated mRNA expression of vascular endothelial growth factor, monocyte chemoattractant protein-1, and basic fibroblast growth factor in this cell type. Differentiated but not undifferentiated human MCs responded to hypoxic insult with elevated AM mRNA/protein expression. Using confocal microscopy, we identified AM-producing MCs in tumor infiltrates of human breast and lung cancer patients. In mixed culture assays the AM-producing human MC line HMC-1 augmented both anchorage-dependent and -independent growth of human lung cancer A549 cells, an effect that was suppressed by MC-targeted siRNA AM knockdown. Finally, HMC-1 cells induced in vivo angiogenesis as assessed by directed in vivo angiogenesis assay analysis; neutralizing anti-AM monoclonal antibody blocked this ability. Our collective data suggest a new role for AM as a cross-talk molecule that integrates tumor and MC communication, underlying a unique promotion mechanism of human cancers.
    American Journal Of Pathology 02/2006; 168(1):280-91. DOI:10.2353/ajpath.2006.050291 · 4.59 Impact Factor
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    ABSTRACT: Sequential immunomagnetic isolation with 2 monoclonal antibodies was used to purify and characterize an undifferentiated mast cell in adult mouse bone marrow that had not been previously recognized. This cell represents 0.02% of the cells in the bone marrow, is CD34(+), CD13(+), and c-kit(+), and does not express FcepsilonRI. However, by polymerase chain reaction (PCR) the cell contains message for the alpha and beta subunits of FcepsilonRI, mast cell-specific proteases, and carboxypeptidase A. Morphologically, this cell has a large nucleus, little cytoplasm, few cytoplasmic organelles, and no cytoplasmic granules. In vitro, in the presence of interleukin-3 (IL-3) and stem cell factor (SCF) these cells differentiate only into a granulated mast cell that now expresses CD13, c-kit, mast cell-specific gangliosides, FcepsilonRI, and binds immunoglobulin E (IgE). When injected into lethally irradiated mice, these cells are able to reconstitute the mast cell population in the spleen.
    Blood 07/2005; 105(11):4282-9. DOI:10.1182/blood-2004-02-0756 · 10.45 Impact Factor
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    ABSTRACT: To investigate the role of phospholipase D (PLD) in FcepsilonRI signaling, the wild-type or the catalytically inactive forms of PLD1 or PLD2 were stably overexpressed in RBL-2H3 mast cells. FcepsilonRI stimulation resulted in the activation of both PLD1 and PLD2. However, PLD1 was the source of most of the receptor-induced PLD activity. There was enhanced FcepsilonRI-induced degranulation only in cells that overexpressed the catalytically inactive PLD1. This dominant-negative PLD1 enhanced FcepsilonRI-induced tyrosine phosphorylations of early signaling molecules such as the receptor subunits, Syk and phospholipase C-gamma which resulted in faster release of Ca(2+) from intracellular sources. Therefore, PLD1 negatively regulates signals upstream of the Ca(2+) response. However, FcepsilonRI-induced PLD activation required Syk and was downstream of the Ca(2+)response, suggesting that basal PLD1 activity rather than that activated by cell stimulation controlled these early signaling events. Dominant-negative PLD1 reduced the basal phosphatidic acid formation in unstimulated cells, which was accompanied by an increase in FcepsilonRI within the lipid rafts. These results indicate that constitutive basal PLD1 activity by regulating phosphatidic acid formation controls the early signals initiated by FcepsilonRI aggregation that lead to mast cell degranulation.
    Blood 01/2005; 104(13):4122-8. DOI:10.1182/blood-2004-06-2091 · 10.45 Impact Factor

Publication Stats

7k Citations
834.89 Total Impact Points


  • 1975-2012
    • National Institutes of Health
      • • Branch of Oral Infection and Immunity
      • • Section on Molecular Signal Transduction
      • • Laboratory of Immunology
      • • Section of Inflammation Immunobiology
      • • Laboratory of Tumor Immunology and Biology
      베서스다, Maryland, United States
  • 2011
    • La Jolla Institute for Allergy & Immunology
      • Division of Cell Biology
      La Jolla, CA, United States
  • 2005
    • University of São Paulo
      • Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos (Ribeirão Preto)
      São Paulo, Estado de Sao Paulo, Brazil
  • 2002
    • Sapienza University of Rome
      • Laboratory of Experimental Medicine and Pathology Environmental
      Roma, Latium, Italy
  • 2001
    • Terry Fox Laboratory
      Vancouver, British Columbia, Canada
  • 1994
    • National Institute of Allergy and Infectious Diseases
      베서스다, Maryland, United States
  • 1991
    • Salk Institute
      لا هویا, California, United States
    • United States Naval Research Laboratory
      Washington, Washington, D.C., United States
  • 1988
    • Howard Hughes Medical Institute
      Ашбърн, Virginia, United States
  • 1979
    • National Cancer Institute (USA)
      Maryland, United States