Fc epsilon RI gamma-ITAM is differentially required for mast cell function in vivo

The cross-linking of IgE-bound FcepsilonRI by Ags triggers mast cell activation leading to allergic reactions. The in vivo contribution of FcepsilonRIgamma signaling to IgE/FcepsilonRI-mediated mast cell responses has not yet been elucidated. In this study FcepsilonRIgamma(-/-) mast cells were reconstituted with either wild-type or mutant FcepsilonRIgamma in transgenic mice and transfected mast cells in vitro. We demonstrate that FcepsilonRIgamma-immunoreceptor tyrosine-based activation motif is essential for degranulation, cytokine production, and PG synthesis as well as for passive systemic anaphylaxis. Recent reports have suggested that cell surface FcepsilonRI expression and mast cell survival are regulated by IgE in the absence of Ag, although the molecular mechanism is largely unknown. We also found that the promotion of mast cell survival by IgE without Ags is mediated by signals through the FcepsilonRIgamma-immunoreceptor tyrosine-based activation motif. In contrast, the IgE-mediated up-regulation of FcepsilonRI is independent of FcepsilonRIgamma signaling. These results indicate that FcepsilonRIgamma-mediated signals differentially regulate the receptor expression, activation, and survival of mast cells and systemic anaphylaxis.     

Src homology 2 domain-containing protein-tyrosine phosphatases, SHP-1 and SHP-2, are required for platelet endothelial cell adhesion molecule-1/CD31-mediated inhibitory signaling

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a newly assigned member of the Ig immunoreceptor tyrosine-based inhibitory motif superfamily, and its functional role is suggested to be an inhibitory receptor that modulates immunoreceptor tyrosine-based activation motif-dependent signaling cascades. To test whether PECAM-1 is capable of delivering inhibitory signals in B cells and the functional requirement of protein-tyrosine phosphatases (PTPs) for this inhibitory signaling, we generated chimeric Fc gamma RIIB1-PECAM-1 receptors containing the extracellular and transmembrane portions of murine Fc gamma RIIB1 and the cytoplasmic domain of human PECAM-1. These chimeric receptors were stably expressed in chicken DT40 B cells either as wild-type or mutant cells deficient in SHP-1(-/-), SHP-2(-/-), SHIP(-/-), or SHP-1/2(-/-) and then assessed for their ability to inhibit B cell Ag receptor (BCR) signaling. Coligation of wild-type Fc gamma RIIB1-PECAM-1 with BCR resulted in inhibition of intracellular calcium release, suggesting that the cytoplasmic domain of PECAM-1 is capable of delivering an inhibitory signal that blocks BCR-mediated activation. This PECAM-1-mediated inhibitory signaling correlated with tyrosine phosphorylation of the Fc gamma RIIB1-PECAM-1 chimera, recruitment of SHP-1 and SHP-2 PTPs by the phosphorylated chimera, and attenuation of calcium mobilization responses. Mutational analysis of the two tyrosine residues, 663 and 686, constituting the immunoreceptor tyrosine-based inhibitory motifs in PECAM-1 revealed that both tyrosine residues play a crucial role in the inhibitory signal. Functional analysis of various PTP-deficient DT40 B cell lines stably expressing wild-type chimeric Fc gamma RIIB1-PECAM-1 receptor indicated that cytoplasmic Src homology 2-domain-containing phosphatases, SHP-1 and SHP-2, were both necessary and sufficient to deliver inhibitory negative regulation upon coligation of BCR complex with inhibitory receptor.     

125I-fibrin deposition in IgE-dependent immediate hypersensitivity reactions in mouse skin. Demonstration of the role of mast cells using genetically mast cell-deficient mice locally reconstituted with cultured mast cells

We investigated the clotting associated with IgE-dependent immediate hypersensitivity reactions in the mouse by injecting monoclonal mouse anti-dintrophenyl IgE antibodies i.d. and, the next day, administering 125I-guinea pig fibrinogen i.v. 10 to 30 min before i.v. antigen (2,4-dinitrophenylated human serum albumin) challenge. In normal mice, 2-hr passive cutaneous anaphylaxis (PCA) reactions were associated with substantial leakage of 125I-fibrinogen and deposition of 125I-fibrin. Thus, ears injected with IgE contained up to six times the total cpm of 125I and up to 30 times the cross-linked 125I-fibrin-associated cpm of 125I than did control ears. Several lines of evidence indicated that the 125I-fibrin deposition associated with the PCA reactions was dependent on the activity of mast cells: 1) Mast cell degranulation occurred at sites of PCA reactions. 2) Antigen-induced influx of 125I-fibrinogen and deposition of 125I-fibrin were virtually abolished by heating the IgE (56 degrees C, 1 hr) before i.d. injection. 3) Little or no IgE-dependent 125I-fibrinogen influx or 125I-fibrin deposition occurred in mast cell-deficient WBB6F1-W/Wv or WCB6F1-S1/S1d mice X 4) Adoptive transfer of cutaneous mast cell populations into WBB6F1-W/Wv mice (by each of three approaches: i.v. transplantation of normal bone marrow cells or local i.d. injection of cultured, growth factor-dependent mast cells 2 days or 9 to 10 wk before antigen challenge) conferred on the recipients the ability to express the 125I-fibrinogen influx and 125I-fibrin deposition associated with PCA reactions. These data demonstrate that 125I-fibrinogen influx and 125I-fibrin deposition occurs in association with PCA reactions in the mouse, and that the reaction is largely or entirely dependent on the function of cutaneous mast cells. The experiments also demonstrate the utility of a novel model system for the analysis of mast cell function in vivo: WBB6F1-W/Wv mice locally reconstituted with mast cells by the injection of mast cell populations generated in vitro.     

Integrin activation by regulated dimerization and oligomerization of platelet endothelial cell adhesion molecule (PECAM)-1 from within the cell

Platelet endothelial cell adhesion molecule (PECAM)-1 is a 130-kD transmembrane glycoprotein having six Ig homology domains within its extracellular domain and an immunoreceptor tyrosine-based inhibitory motif within its cytoplasmic domain. Previous studies have shown that addition of bivalent anti-PECAM-1 mAbs to the surface of T cells, natural killer cells, neutrophils, or platelets result in increased cell adhesion to immobilized integrin ligands. However, the mechanism by which this occurs is not clear, and it is possible that anti-PECAM-1 mAbs elicit this effect by simply sequestering PECAM-1, via antibody-induced patching and capping, away from stimulatory receptors that it normally regulates. To determine whether dimerization or oligomerization of PECAM-1 directly initiates signal transduction pathways that affect integrin function in an antibody-independent manner, stable human embryonic kidney-293 cell lines were produced that expressed chimeric PECAM-1 cDNAs containing one or two FK506-binding protein (FKBP) domains at their COOH terminus. Controlled dimerization initiated by addition of the bivalent, membrane-permeable FKBP dimerizer, AP1510, nearly doubled homophilic binding capacity, whereas AP1510-induced oligomers favored cis PECAM-1/PECAM-1 associations within the plane of the plasma membrane at the expense of trans homophilic adhesion. Importantly, AP1510-induced oligomerization resulted in a marked increase in both adherence and spreading of PECAM/FKBP-2-transfected cells on immobilized fibronectin, a reaction that was mediated by the integrin alpha(5)beta(1). These data demonstrate that signals required for integrin activation can be elicited by clustering of PECAM-1 from inside the cell, and suggest that a dynamic equilibrium between PECAM-1 monomers, dimers, and oligomers may control cellular activation signals that influence the adhesive properties of vascular cells that express this novel member of the immunoreceptor tyrosine-based inhibitory motif family of regulatory receptors.     

The effect of IgE-mediated mast cell degranulation on the expression of experimental contact sensitivity in the mouse

The effects of mast cell activation/degranulation on the elicitation of contact sensitivity (CS) to oxazolone and dinitrofluorobenzene were investigated. Mice were actively sensitized to oxazolone by epicutaneous painting followed by ear challenge. Passive sensitization to DNFB was induced by intradermal injections of dinitrophenol (DNP)-specific cloned T cells in the ears. Mast cells in the challenged ears were activated in various time periods by inducing a passive cutaneous anaphylaxis reaction where passive sensitization with monoclonal IgE anti-DNP antibodies was followed by iv injection of DNP-BSA. This combination of immediate and delayed-type hypersensitivity reactions resulted in a significant increase of ear swelling without any noticeable effect on cellular infiltration when the contact response was evaluated a short time (3-4 hr) after mast cell activation. The very same results were obtained in naive (unsensitized) mice, indicating that this reaction was nonspecific. However, when the CS reaction was evaluated at its peak, i.e., 24 hr post challenge, mast cell activation that had been induced 0.5-11 hr after ear challenge did not have any significant effect on both swelling and cellular infiltration when the latter was evaluated by a radiometric assay. We conclude that in these systems mast cell activation/degranulation makes little or no contribution to the modulation of T-cell activity.     

Antiallergic/antiasthmatic effect of novel antiallergic hexapeptide-95/220 in various experimental models

The effects of newly synthesized antiallergic hexapeptide 95/220 was investigated on various allergic and asthmatic test models. This newly developed peptide was found to be more potent than clinically used drug disodium cromoglycate (DSCG). Hexapeptide 95/220 inhibited immediate hypersensitivity reactions such as passive cutaneous anaphylaxis (PCA) and mast cell degranulation in rats, antigen-induced bronchoconstriction in actively sensitized guinea pigs in dose dependent manner like DSCG. Antigen-induced contraction of guinea pig ileum was also markedly inhibited by this newly developed hexapeptide in the same fashion as ketotifen and DSCG did but at comparatively lower dose. Egg albumin-induced histamine release was also blocked by this hexapeptide from chopped lung tissues of sensitized guinea pigs. These results suggest that hexapeptide' 95/220 has potent inhibitory effect on immediate hypersensitivity reactions thereby inhibiting mediator release from mast cell. Moreover, this newly synthesized peptide is orally active and effective at lower doses as compared to standard drugs.     

A new role for platelet-endothelial cell adhesion molecule-1 (CD31): inhibition of TCR-mediated signal transduction.

Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kDa transmembrane glycoprotein expressed by endothelial cells, platelets, monocytes, neutrophils, and certain T cell subsets. The PECAM-1 extracellular domain has six Ig-homology domains that share sequence similarity with cellular adhesion molecules. The PECAM-1 cytoplasmic domain contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) that, when appropriately engaged, becomes phosphorylated on tyrosine residues, creating docking sites for nontransmembrane, Src homology 2 domain-bearing protein tyrosine phosphatase (SHP)-1 and SHP-2. The purpose of the present study was to determine whether PECAM-1 inhibits protein tyrosine kinase (PTK)-dependent signal transduction mediated by the immunoreceptor tyrosine-based activation motif-containing TCR. Jurkat cells, which coexpress PECAM-1 and the TCR/CD3 complex, were INDO-1AM-labeled and then incubated with anti-CD3epsilon mAbs, anti-PECAM-1 mAbs, or both, and goat anti-mouse IgG was used to cross-link surface-bound mAbs. Calcium mobilization induced by CD3 cross-linking was found to be attenuated by coligation of PECAM-1 in a dose-dependent manner. PECAM-1-mediated inhibition of TCR signaling was attributable, at least in part, to inhibition of release of calcium from intracellular stores. These data provide evidence that PECAM-1 can dampen signals transduced by ITAM-containing receptors and support inclusion of PECAM-1 within the family of ITIM-containing inhibitors of PTK-dependent signal transduction.     

Blunted IgE-mediated activation of mast cells in mice lacking the Ca2+-activated K+ channel KCa3.1

Mast cell stimulation by Ag is followed by the opening of Ca(2+)-activated K(+) channels, which participate in the orchestration of mast cell degranulation. The present study has been performed to explore the involvement of the Ca(2+)-activated K(+) channel K(Ca)3.1 in mast cell function. To this end mast cells have been isolated and cultured from the bone marrow (bone marrow-derived mast cells (BMMCs)) of K(Ca)3.1 knockout mice (K(Ca)3.1(-/-)) and their wild-type littermates (K(Ca)3.1(+/+)). Mast cell number as well as in vitro BMMC growth and CD117, CD34, and FcepsilonRI expression were similar in both genotypes, but regulatory cell volume decrease was impaired in K(Ca)3.1(-/-) BMMCs. Treatment of the cells with Ag, endothelin-1, or the Ca(2+) ionophore ionomycin was followed by stimulation of Ca(2+)-activated K(+) channels and cell membrane hyperpolarization in K(Ca)3.1(+/+), but not in K(Ca)3.1(-/-) BMMCs. Upon Ag stimulation, Ca(2+) entry but not Ca(2+) release from intracellular stores was markedly impaired in K(Ca)3.1(-/-) BMMCs. Similarly, Ca(2+) entry upon endothelin-1 stimulation was significantly reduced in K(Ca)3.1(-/-) cells. Ag-induced release of beta-hexosaminidase, an indicator of mast cell degranulation, was significantly smaller in K(Ca)3.1(-/-) BMMCs compared with K(Ca)3.1(+/+) BMMCs. Moreover, histamine release upon stimulation of BMMCs with endothelin-1 was reduced in K(Ca)3.1(-/-) cells. The in vivo Ag-induced decline in body temperature revealed that IgE-dependent anaphylaxis was again significantly (by approximately 50%) blunted in K(Ca)3.1(-/-) mice. In conclusion, K(Ca)3.1 is required for Ca(2+)-activated K(+) channel activity and Ca(2+)-dependent processes such as endothelin-1- or Ag-induced degranulation of mast cells, and may thus play a critical role in anaphylactic reactions.     

A novel role for PECAM-1 (CD31) in regulating haematopoietic progenitor cell compartmentalization between the peripheral blood and bone marrow

Although the expression of PECAM-1 (CD31) on vascular and haematopoietic cells within the bone marrow microenvironment has been recognized for some time, its physiological role within this niche remains unexplored. In this study we show that PECAM-1 influences steady state hematopoietic stem cell (HSC) progenitor numbers in the peripheral blood but not the bone marrow compartment. PECAM-1(-/-) mice have higher levels of HSC progenitors in the blood compared to their littermate controls. We show that PECAM-1 is required on both progenitors and bone marrow vascular cells in order for efficient transition between the blood and bone marrow to occur. We have identified key roles for PECAM-1 in both the regulation of HSC migration to the chemokine CXCL12, as well as maintaining levels of the matrix degrading enzyme MMP-9 in the bone marrow vascular niche. Using intravital microscopy and adoptive transfer of either wild type (WT) or PECAM-1(-/-) bone marrow precursors, we demonstrate that the increase in HSC progenitors in the blood is due in part to a reduced ability to migrate from blood to the bone marrow vascular niche. These findings suggest a novel role for PECAM-1 as a regulator of resting homeostatic progenitor cell numbers in the blood.     

Key role for mast cells in nonatopic asthma

The mechanisms involved in nonatopic asthma are poorly defined. In particular, the importance of mast cells in the development of nonatopic asthma is not clear. In the mouse, pulmonary hypersensitivity reactions induced by skin sensitization with the low-m.w. compound dinitrofluorobenzene (DNFB) followed by an intra-airway application of the hapten have been featured as a model for nonatopic asthma. In present study, we used this model to examine the role of mast cells in the pathogenesis of nonatopic asthma. First, the effect of DNFB sensitization and intra-airway challenge with dinitrobenzene sulfonic acid (DNS) on mast cell activation was monitored during the early phase of the response in BALB/c mice. Second, mast cell-deficient W/W(v) and Sl/Sl(d) mice and their respective normal (+/+) littermate mice and mast cell-reconstituted W/W(v) mice (bone marrow-derived mast cells-->W/W(v)) were used. Early phase mast cell activation was found, which was maximal 30 min after DNS challenge in DNFB-sensitized BALB/c, +/+ mice but not in mast cell-deficient mice. An acute bronchoconstriction and increase in vascular permeability accompanied the early phase mast cell activation. BALB/c, +/+ and bone marrow-derived mast cell-->W/W(v) mice sensitized with DNFB and DNS-challenged exhibited tracheal hyperreactivity 24 and 48 h after the challenge when compared with vehicle-treated mice. Mucosal exudation and infiltration of neutrophils in bronchoalveolar lavage fluid associated the late phase response. Both mast cell-deficient strains failed to show any features of this hypersensitivity response. Our findings show that mast cells play a key role in the regulation of pulmonary hypersensitivity responses in this murine model for nonatopic asthma.     

SHP-1 Regulation of Mast Cell Function in Allergic Inflammation and Anaphylaxis

Allergic inflammation and severe allergic reactions (anaphylaxis) are important in allergen induced diseases. Bacterial products such as lipopolysaccharide (LPS) are ubiquitous and can facilitate allergen induced Th2 immune responses. Phosphatase SHP-1 is critical in regulating immunological homeostasis and in allergen induced Th2 immune responses in the lung. However, the mechanisms underlying the initiation of allergic inflammation and allergen induced anaphylaxis are still not completely elucidated and it is unclear whether SHP-1 plays any role in LPS-induced airway inflammation and in allergen-induced anaphylaxis. In this study we tested the hypothesis that phosphatase SHP-1 plays an important role in allergic inflammation and anaphylaxis and determined whether its effects are through regulation of mast cell functions. SHP-1 deficient (mev/+ and mev/mev) and mast cell deficient (Kit^W-sh) mice were examined in their responses to LPS airway stimulation and to ovalbumin (OVA) allergen induced systemic anaphylaxis. Compared to wild type mice, mev/+ mice had significantly enhanced LPS induced airway inflammation and OVA induced anaphylactic responses, including hypothermia and clinical symptoms. These changes were mast cell dependent as Kit^W-sh mice had reduced responses whereas adoptive transfer of mast cells restored the responses. However, T and B cells were not involved and macrophages did not play a significant role in LPS induced airway inflammation. Interestingly, basophil differentiation from SHP-1 deficient bone marrow cells was significantly reduced. These findings provided evidence that through regulation of mast cell functions SHP-1 plays a critical role as a negative regulator in allergic inflammation and in allergen induced anaphylaxis. In addition, SHP-1 seems to be required for normal basophil development.     

Immune Response to Snake Envenoming and Treatment with Antivenom; Complement Activation,Cytokine Production and Mast Cell Degranulation

Background

Snake bite is one of the most neglected public health issues in poor rural communities worldwide. In addition to the clinical effects of envenoming, treatment with antivenom frequently causes serious adverse reactions, including hypersensitivity reactions (including anaphylaxis) and pyrogenic reactions. We aimed to investigate the immune responses to Sri Lankan snake envenoming (predominantly by Russell''s viper) and antivenom treatment.

Methodology/Principal Findings

Plasma concentrations of Interleukin (IL)-6, IL-10, tumor necrosis factor α (TNFα), soluble TNF receptor I (sTNFRI), anaphylatoxins (C3a, C4a, C5a; markers of complement activation), mast cell tryptase (MCT), and histamine were measured in 120 Sri Lankan snakebite victims, both before and after treatment with antivenom. Immune mediator concentrations were correlated with envenoming features and the severity of antivenom-induced reactions including anaphylaxis. Envenoming was associated with complement activation and increased cytokine concentrations prior to antivenom administration, which correlated with non-specific systemic symptoms of envenoming but not with coagulopathy or neurotoxicity. Typical hypersensitivity reactions to antivenom occurred in 77/120 patients (64%), satisfying criteria for a diagnosis of anaphylaxis in 57/120 (48%). Pyrogenic reactions were observed in 32/120 patients (27%). All patients had further elevations in cytokine concentrations, but not complement activation, after the administration of antivenom, whether a reaction was noted to occur or not. Patients with anaphylaxis had significantly elevated concentrations of MCT and histamine.

Conclusions/Significance

We have demonstrated that Sri Lankan snake envenoming is characterized by significant complement activation and release of inflammatory mediators. Antivenom treatment further enhances the release of inflammatory mediators in all patients, with anaphylactic reactions characterised by high levels of mast cell degranulation but not further complement activation. Anaphylaxis is probably triggered by non allergen-specific activation of mast cells and may be related to the quality of available antivenom preparations, as well as a priming effect from the immune response to the venom itself.     

Hybrid resistance to parental mast cell precursors in the skin of (WB X C57BL/6)F1-W/Wv mice

When bone marrow cells of (WB X C57BL/6)F1-+/+ (WBB6F1-+/+) and WB-+/+ (WB) mice were directly injected into the skin of genetically mast cell-deficient WBB6F1-W/Wv mice, mast cell clusters appeared at the injection sites. However, the number of WB bone marrow cells necessary for appearance of mast cell clusters was significantly larger than when bone marrow cells of WBB6F1-+/+ mice were used. When WB bone marrow cells were mixed either with WB thymus cells or with silica particles, the proportion of injection sites at which mast cell clusters appeared increased to the level that was observed after the injection of the same number of WBB6F1-+/+ bone marrow cells. When suckling WBB6F1-W/Wv mice of less than or equal to 18 days of age were used as recipients, bone marrow cells of WBB6F1-+/+ and WB mice produced mast cell clusters with a comparable efficiency. Both syngeneic thymus cells and silica particles are known to abrogate the hybrid resistance that is observed in the spleen against parental hematopoietic stem cells. The hybrid resistance in the spleen is not detectable in suckling mice, either. Thus, the poor growth of mast cell precursors in the skin and the poor growth of hematopoietic stem cells in the spleen seem to be regulated by the same mechanism.     

Points of control exerted along the macrophage-endothelial cell-polymorphonuclear neutrophil axis by PECAM-1 in the innate immune response of acute colonic inflammation

PECAM-1 is expressed on endothelial cells and leukocytes. Its extracellular domain has been implicated in leukocyte diapedesis. In this study, we used PECAM-1(-/-) mice and relevant cells derived from them to assess the role of PECAM-1 in an experimental model of acute colonic inflammation with a predominant innate immune response, i.e., 2,4,6-trinitrobenzine sulfonic acid (TNBS). Using chimeric approaches, we addressed the points of control exerted by PECAM-1 along the macrophage-endothelial cell-polymorphonuclear neutrophil (PMN) axis. In vivo, TNBS-induced colitis was ameliorated in PECAM-1(-/-) mice, an event attributed to PECAM-1 on hematopoietic cells rather than to PECAM-1 on endothelial cells. The in vivo innate immune response was mimicked in vitro by using a construct of the vascular-interstitial interface, i.e., PMN transendothelial migration was induced by colonic lavage fluid (CLF) from TNBS mice or macrophages (MPhi) challenged with CLF. Using the construct, we confirmed that endothelial cell PECAM-1 does not play a role in PMN transendothelial migration. Although MPhi activation (NF-kappaB nuclear binding) and function (keratinocyte-derived chemokine production) induced by CLF was diminished in PECAM-1(-/-) MPhi, this did not affect their ability to promote PMN transendothelial migration. By contrast, PECAM-1(-/-) PMN did not adhere to or migrate across endothelial cell monolayers in response to CLF. Further, as compared with PECAM-1(+/+) PMN, PECAM-1(-/-) PMN were less effective in orientating their CXCR2 receptors (polarization) in the direction of a chemotactic gradient. Collectively, our findings indicate that PECAM-1 modulation of PMN function (at a step before diapedesis) most likely contributes to the inflammation in a colitis model with a strong innate immune component.     

Biology of marrow stromal cell lines derived from long-term bone marrow cultures of Trp53-deficient mice.

To investigate the effect of Trp53 (formerly known as p53) on stromal cells of the hematopoietic microenvironment, long-term bone marrow cultures were established from mice in which the Trp53 gene had been inactivated by homologous recombination (Trp53(-/-)) or their wild-type littermates (Trp53(+/+)). Long-term bone marrow cultures from Trp53(-/-) mice continued to produce nonadherent cells for 22 weeks, while Trp53(+/+) cultures ceased production after 15 weeks. There was a significant increase in the number of nonadherent cells produced in Trp53(-/-) long-term bone marrow cultures beginning at week 9 and continuing to week 22 (P < 0.02). The Trp53(-/-) cultures also showed significantly increased cobblestone island formation indicative of early hematopoietic stem cell-containing colonies beginning at week 10 (P < 0.01). Cobblestone islands persisted until weeks 15 and 22 in Trp53(+/+) and Trp53(-/-) cultures, respectively. Co-cultivation experiments in which Trp53(+/+) Sca1(+)lin- enriched hematopoietic stem cells were plated on Trp53(-/-) stromal cells showed increased cobblestone island formation compared to Trp53(-/-) Scal+lin- cells plated on Trp53(+/+) or Trp53(-/-) stromal cells. Radiation survival curves for clonal bone marrow stromal cells revealed a similar D0 for the Trp53(+/+) and Trp53(-/-) cell lines (1.62 +/- 0.16 and 1.49 +/- 0. 08 Gy, respectively; P = 0.408), and similar n (8.60 +/- 3.23 and 10.71 +/- 0.78, respectively) (P = 0.491). Cell cycle analysis demonstrated a G2/M-phase arrest that occurred 6 h after irradiation for both Trp53(+/+) and Trp53(-/-) stromal cell lines. After 10 Gy irradiation, there was no significant increase in the frequency of apoptosis detected in Trp53(+/+) compared to Trp53(-/-) marrow stromal cell lines. In the stromal cell lines, ICAM-1 was constitutively expressed on Trp53(+/+) but not Trp53(-/-) cells; however, a 24-h exposure to TNF-alpha induced detectable ICAM-1 on Trp53(-/-) cells and increased expression on Trp53(+/+) cells. To test the effect of Trp53 on the radiation biology of hematopoietic progenitor cells, the 32D cl 3 cell line was compared with a subclone in which expression of an E6 inserted transgene accelerates ubiquitin-dependent degradation of Trp53, thus preventing accumulation of Trp53 after genotoxic stress. The radiation survival curves were similar with no significant difference in the D0 or n, or in the percentage of cells undergoing apoptosis after 10 Gy irradiation between the two cell lines. Cells of the 32D-E6 cell line displayed a G2/M-phase arrest 6 h after 10 Gy, while cells of the parent line exhibited both a G2/M-phase arrest and a G1-phase arrest at 24 and 48 h. The results suggest a complex mechanism of action of Trp53 on the interactions between stromal and hematopoietic cells in long-term bone marrow cultures.     

Effects of Prunella vulgaris on mast cell-mediated allergic reaction and inflammatory cytokine production

In this study, we investigated the effect of aqueous extract of Prunella vulgaris (Labiatae; PVAE) on the mast cell-mediated allergy model. We found that PVAE (0.001-0.1 g/kg) dose dependently inhibited compound 48/80-induced systemic anaphylaxis and serum histamine release in mice. PVAE decreased the IgE-mediated local allergic reaction, passive cutaneous anaphylaxis. In addition, PVAE attenuated phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187-stimulated TNF-alpha, IL-6, and IL-8 secretion in human mast cells. The inhibitory effect of PVAE on proinflammatory cytokines was nuclear factor-kappaB (NF-kappaB) dependent. PVAE suppressed PMA and A23187-induced NF-kappaB/DNA binding activity and NF-kappaB-dependent gene reporter assay. Our findings provide evidence that PVAE inhibits mast cell-derived immediate-type allergic reactions and involvement of proinflammatory cytokines and NF-kappaB in these effects.     

Phosphatidylinositol-3-kinase C2β and TRIM27 function to positively and negatively regulate IgE receptor activation of mast cells

Cross-linking of the IgE receptor (FcεRI) on mast cells plays a critical role in IgE-dependent allergy, including allergic rhinitis, asthma, anaphylaxis, and immediate-type hypersensitivity reactions. Previous studies have demonstrated that the K(+) channel, KCa3.1, plays a critical role in IgE-stimulated Ca(2+) entry and degranulation in both human and mouse mast cells. We now have shown that the class II phosphatidylinositol-3-kinase C2β (PI3KC2β) is necessary for FcεRI-stimulated activation of KCa3.1, Ca(2+) influx, cytokine production, and degranulation of bone marrow-derived mast cells (BMMC). In addition, we found that the E3 ubiquitin ligase, tripartite motif containing protein 27 (TRIM27), negatively regulates FcεRI activation of KCa3.1 and downstream signaling by ubiquitinating and inhibiting PI3KC2β. TRIM27(-/-) mice are also more susceptible in vivo to acute anaphylaxis. These findings identify TRIM27 as an important negative regulator of mast cells in vivo and suggest that PI3KC2β is a potential new pharmacologic target to treat IgE-mediated disease.     

Role of immunoreceptor tyrosine-based inhibitory motifs of PECAM-1 in PECAM-1-dependent cell migration

Platelet endothelial cell adhesion molecule (PECAM-1), a transmembrane glycoprotein, has been implicated in angiogenesis, with recent evidence indicating the involvement of PECAM-1 in endothelial cell motility. The cytoplasmic domain of PECAM-1 contains two tyrosine residues, Y663 and Y686, that each fall within an immunoreceptor tyrosine-based inhibitory motif (ITIM). When phosphorylated, these residues together mediate the binding of the protein tyrosine phosphatase SHP-2. Because SHP-2 has been shown to be involved in the turnover of focal adhesions, a phenomenon required for efficient cell motility, the association of this phosphatase with PECAM-1 via its ITIMs may represent a mechanism by which PECAM-1 might facilitate cell migration. Studies were therefore done with cell transfectants expressing wild-type PECAM or mutant PECAM-1 in which residues Y663 and Y686 were mutated. These mutations eliminated PECAM-1 tyrosine phosphorylation and the association of PECAM-1 with SHP-2 but did not impair the ability of the molecule to localize at intercellular junctions or to bind homophilically. However, in vitro cell motility and tube formation stimulated by the expression of wild-type PECAM-1 were abrogated by the mutation of these tyrosine residues. Importantly, during wound-induced migration, the number of focal adhesions as well as the level of tyrosine phosphorylated paxillin detected in cells expressing wild-type PECAM-1 were markedly reduced compared with control cells or transfectants with mutant PECAM-1. These data suggest that, in vivo, the binding of SHP-2 to PECAM-1, via PECAM-1’s ITIM domains, promotes the turnover of focal adhesions and, hence, endothelial cell motility. platelet endothelial cell adhesion molecule-1; endothelial cells; angiogenesis     

Syk protein tyrosine kinase involves PECAM-1 signaling through tandem immunotyrosine inhibitory motifs in human THP-1 macrophages

Although recent evidence supports a functional relationship between platelet endothelial cell adhesion molecule (PECAM-1) and Syk tyrosine kinase, little is known about the interaction of Syk with PECAM-1. We report that down-regulation of Syk inhibits the spreading of human THP-1 macrophage cells. Moreover, our data indicate that Syk binds PECAM-1 through its immune tyrosine-based inhibitory motif (ITIM), and dual phosphorylation of the ITIM domain of PECAM-1 leads to activation of Syk. Our results indicate that the distance between the phosphotyrosines could be up to 22 amino acids in length, depending on the conformational flexibility, and that the dual ITIM tyrosine motifs of PECAM-1 facilitate immunoreceptor tyrosine-based activation motif-like signaling. The preferential binding of PECAM-1 to Src homology region 2 domain-containing phosphatase-2 or Syk may depend on their relative affinities, and could provide a mechanism by which signal transduction from PECAM-1 is internally regulated by both positive and negative signaling enzymes.