Mast cells have been implicated in the pathogenesis of both atopic and non-atopic asthma. Ras guanine nucleotide-releasing protein 4 (RasGRP4) is a mast cell-restricted guanine nucleotide exchange factor and diacylglycerol (DAG)/ phorbol ester receptor whose function has not been deduced. RT-PCR analysis of 40 asthmatic patients and 40 non-asthmatic controls demonstrated a higher hRasGRP4 mRNA expression in a subgroup of the asthmatics. A RasGRP4-defective variant of the human mast cell line HMC-1 was used to create stable clones expressing green fluorescent protein-labeled human RasGRP4 for monitoring the movement of this signaling protein inside mast cells before and after exposure to phorbol-12-myristate 13-acetate (PMA) and for evaluating the protein???s ability to control the development, phenotype, and function of mast cells. Transcript-profiling approaches revealed hRasGRP4 constitutively regulates the expression of numerous genes in the HMC-1 cell line. For example, expression of hRasGRP4 in HMC-1 cells substantially decreased GATA-1 levels without altering GATA-2 levels, suggesting that hRasGRP4 regulates mast cell commitment of multipotential progenitors in part by controlling the intracellular levels of at least one lineage-dependent transcription factor for hematopoietic cells. hRasGRP4 resided primarily in the cytosol before HMC-1 cells were stimulated with PMA. After exposure to PMA, hRasGRP4 translocated to the inner leaflet of the cell???s plasma membrane and then to perinuclear and Golgi compartments. Extracellular signal-regulated kinases 1 and 2 were activated during this translocation process, and the PMA-treated cells transiently increased their expression of the transcripts encoding the interleukin 13 receptor IL-13R??2 and numerous other proteins. The accumulated data in our mast cell model suggest hRasGRP4 translocates to various intracellular compartments via its DAG/PMA-binding domain to regulate those signaling pathways that allow mast cells to respond quickly to changes in their tissue microenvironments.

Nitric oxide (NO) is a short-lived free radical that plays a critical role in the regulation of cellular signalling. Mast cell (MC) derived NO and exogenous NO regulate MC activities including the inhibition of MC degranulation. At a molecular level the intermediate metabolites of NO modify protein structure and function through several mechanisms including protein tyrosine nitration. To begin to elucidate the molecular mechanisms underlying the effects of NO in MC, we investigated protein tyrosine nitration in human mast cell lines HMC-1 and LAD2 treated with the NO donor S-nitrosoglutathione (SNOG). Using two dimensional gel western blot analysis with an anti-nitrotyrosine antibody together with mass spectroscopy we identified aldolase A, an enzyme of the glycolytic pathway, as a target for tyrosine nitration in MC. S-nitrosoglutathione treatment also reduced the Vmax of aldolase in HMC-1 and LAD2. Nuclear magnetic resonance (NMR) analysis showed that despite these changes in activity of a critical enzyme in glycolysis, there was no significant change in total cellular ATP content, although the AMP/ATP ratio was altered. Elevated levels of lactate and pyruvate suggested that SNOG treatment enhanced glycolysis. Reduced aldolase activity was associated with increased intracellular levels of its substrate, fructose-1,6-bisphosphate (FBP). Interestingly, FBP inhibited IgE-mediated MC degranulation and intracellular Ca2+ levels in LAD2 cells. In addition to aldolase, 15-hydroxy prostaglandin dehydrogenase (PGDH), a critical enzyme in the metabolism of PGE2, was identified as a prominent target for tyrosine nitration in LAD2 cells. Thus for the first time we report evidence of protein tyrosine nitration in human MC lines and identify aldolase A as a prominent target in HMC-1 and LAD2; and PGDH in LAD2 cells. The post translational nitration of aldolase A and PGDH may be important pathways that regulate MC phenotype and function. / Experimental Medicine

Mast cells (MCs), normally classified into connective tissue MCs and mucosal MCs, are highly granulated cells found in the interface between the interior and the exterior environment of our body, e.g. skin, airways and gastro-intestinal tract. They react to bacteria, parasites, viruses, and allergens by degranulation and release of premade and newly synthesized inflammatory mediators. The MC-proteases (tryptases, chymases and carboxypeptidase A), histamine and serglycin (SG) proteoglycans are premade mediators. Among these, SG is also expressed in a variety of other immune and non-immune cells. Heparin and chondroitin sulphate glycosaminoglycan chains confer highly negative charge to SG, by which MC-proteases are retained in secretory granules. Deletion of SG cause impaired packing and storage of most MC-proteases. During challenge with Toxoplasma gondii the SG-deficient mice showed significant lower inflammatory cytokine levels in comparison to wild-type mice. Results were consistently similar in vitro, bringing forward the importance of SG in inflammatory cytokine and innate immune responses towards T. gondii. Infection with Trichinella spiralis in SG-/- mice caused increased intestinal enteropathy, a tendency of delayed worm expulsion and increased larval burden in the muscle tissue as compared to wild-type animals. An altered TH2 cytokine response was also observed, and all these effects were not repaired by wild-type MC reconstitution of the SG-/- mice. Altogether, our results suggest that SG is important for tissue homeostasis, and that SG expressing cells seem capable of switching from a SG-dependent storage mode to a SG-independent secretory mode upon infection. The chymase (MCPT4) expressed by connective tissue MC has been implicated to have a protective role during infection and in limiting inflammation. We explored a protective role by inducing T. gondii infection in the Mcpt4-null mice, and found MCPT4-mediated recruitment of neutrophils and eosinophils via control of cytokine signaling. Endogenous proteins “alarmins” released by dead cells can trigger tissue and cell damage. We conclusively show that chymase efficiently degrades Hsp70 both in vitro and in vivo and that the degradation of other alarmins, e.g. HMGB1, biglycan and IL-33 may also depend on chymase.

Inflammatory response in the airway may lead to asthma. Asthma may develop during the childhood in some asthmatic patients. Both environmental and genetic factors may influence the onset and progress of asthma. It is well-known that there may be complex neural innervation and reflex mechanisms between trachea and esophagus. Intraesophgeal infusion of 1N HCl could lead to tracheal inflammation by activating neural reflex pathway and cause tachykinin-like substance to release. In this study, we first sensitized rats with 1ml of OVA-Al［OH］3 mixture containing 200£gg OVA via intraperitoneal injection on days 1, 2, 3 and 11, then perform intraesophageal infusion of ovalbumin to see whether stimulation of esophagus in sensitized rat model could involve inflammatory response in the lower airways. Animals were perfused with saline and fixative at various time points and the esophagus and airway tissues were processed for the subsequent analysis. We observed the extent of plasma leakage and migration of leukocytes in the lower airway. India ink was used to label the leaky blood vessels.The magnitude of plasma leakage was expressed by the area density of India ink-labeled blood vessels. The results showed that the intraesophageal infusion of ovalbumin 75 mg/kg caused an increase in plasma leakage in the lower airways. The plasma leakage peaked at 30 min, the area density of plasma leakage in trachea was 22.43 ¡Ó 3.34¢H; and 20.57 ¡Ó 4.91¢H in right bronchus; 18.47 ¡Ó 5.03¢H in left bronchus and 27.85 ¡Ó 2.71¢H in epiglottis. The extent of leakage gradually diminished 3 hours after ovalbumin infusion. However, a second increased plasma leakage peaked at about 4 hours of ovalbumin infusion. Tissue sections clearly showed degranulation of mast cells in OVA infusion group. Experimental data showed that pretreatment with either bilateral vagotomy, or mepyramine, the histamine H1 receptor antagonist, significantly inhibited the inflammatory response in the lower airways induced by intraesophageal infusion of OVA. In conclusion, there were clearly two phases, early and late phase responses, in inflammatory response in OVA-sensitized rats receiving intra-esophageal OVA challenge. The underlying mechanisms may involve vagal C-fibers and histamine H1 receptors.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成25年3月25日 水野紘樹氏の博士論文として提出された

Mast cells are granulocytes that are a key component of the innate and adaptive immune system, and contribute to allergic disorders. Mast cell activation following clustering of the high affinity IgE receptor (FcepsilonRI) by multivalent antigens requires reversible tyrosine phosphorylation of myriad signaling proteins. Activated mast cells rapidly release granule contents (eg. histamine and serine proteases) that cause vascular permeability, and in a more delayed manner they also synthesize and secrete eicosanoids and numerous cytokines (eg. IL-6 and TNFalpha) that recruit activated leukocytes. FcepsilonRI signaling is initiated by Lyn, a Src Family Kinase (SFK), that phosphorylates immunoreceptor tyrosine-based activation motifs (ITAMs) found on the FcepsilonRI beta and gamma chains. This allows recruitment of Fyn SFK and Syk kinase that bind ITAMs and phosphorylate numerous downstream targets. Src Homology 2 domain-containing Phosphatase 2 (SHP2, encoded by ptpn11/shp2) is known to be recruited to several phosphorylated proteins following FcepsilonRI aggregation in mast cells, however attempts to define the role of SHP2 have been hampered by its essential role during embryonic development and hematopoiesis in mice. Recently, conditional SHP2 knock-out mice (shp2fl/fl) have been created allowing for shp2 inactivation in a tissue-specific manner by Cre recombinase. Here we describe the use of transgenic mice expressing a modified estrogen receptor-Cre Recombinase (TgCreER*) on a shp2fl/fl genetic background, that allows for maturation of bone marrow-derived mast cells (BMMCs) prior to shp2-inactivation using 4-hydroxytamoxifen (4OH-TM). SHP2-depleted BMMCs display reduced phosphorylation of the FcepsilonRI beta chain, but exhibit extended phosphorylation of Syk kinase. Additionally, SHP2-deficient cells display a defect in the activation of both Erk mitogen-activated protein kinase and Akt, which correlates with an observed defect in the production of TNFalpha and Leukotriene C4. Finally, we show that SHP2-deficient BMMCs display elevated FcepsilonRI-evoked phosphorylation of Csk-Binding Protein (Cbp or PAG) on residue Y317, which recruits C-terminal Src kinase (Csk) that phosphorylates SFKs on an inhibitory tyrosine. This hyperphosphorylation of Cbp correlates with elevated phosphorylation of the C-terminal inhibitory tyrosine on Fyn kinase. This study provides new insights into the role of SHP2 as a positive effector of FcepsilonRI signaling and cytokine production in mast cells. / Thesis (Master, Biochemistry) -- Queen's University, 2008-08-20 13:51:18.751

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No description available.

Mast cells have been implicated in the pathogenesis of both atopic and non-atopic asthma. Ras guanine nucleotide-releasing protein 4 (RasGRP4) is a mast cell-restricted guanine nucleotide exchange factor and diacylglycerol (DAG)/ phorbol ester receptor whose function has not been deduced. RT-PCR analysis of 40 asthmatic patients and 40 non-asthmatic controls demonstrated a higher hRasGRP4 mRNA expression in a subgroup of the asthmatics. A RasGRP4-defective variant of the human mast cell line HMC-1 was used to create stable clones expressing green fluorescent protein-labeled human RasGRP4 for monitoring the movement of this signaling protein inside mast cells before and after exposure to phorbol-12-myristate 13-acetate (PMA) and for evaluating the protein???s ability to control the development, phenotype, and function of mast cells. Transcript-profiling approaches revealed hRasGRP4 constitutively regulates the expression of numerous genes in the HMC-1 cell line. For example, expression of hRasGRP4 in HMC-1 cells substantially decreased GATA-1 levels without altering GATA-2 levels, suggesting that hRasGRP4 regulates mast cell commitment of multipotential progenitors in part by controlling the intracellular levels of at least one lineage-dependent transcription factor for hematopoietic cells. hRasGRP4 resided primarily in the cytosol before HMC-1 cells were stimulated with PMA. After exposure to PMA, hRasGRP4 translocated to the inner leaflet of the cell???s plasma membrane and then to perinuclear and Golgi compartments. Extracellular signal-regulated kinases 1 and 2 were activated during this translocation process, and the PMA-treated cells transiently increased their expression of the transcripts encoding the interleukin 13 receptor IL-13R??2 and numerous other proteins. The accumulated data in our mast cell model suggest hRasGRP4 translocates to various intracellular compartments via its DAG/PMA-binding domain to regulate those signaling pathways that allow mast cells to respond quickly to changes in their tissue microenvironments.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 4 uppsatser.