Molecular Biology Of Mast Cell Growth And Differentiation

肥大细胞生长和分化的分子生物学

• 批准号: 7732464
• 负责人: Dean D Metcalfe
• 金额: $ 84.8万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7732464
• 关键词: Allergic; Animal Model; Bacteria; Behavior; Bone Marrow; Bone Marrow Cells; C-KIT Gene; Calcium; Carboxypeptidase; Cell Line; Cells; Characteristics; Chymase; Clinical Sensitivity; Column Chromatography; Comparative Study; Condition; Cytoplasmic Granules; Data; Development; Differentiation and Growth; Disease; Endopeptidases; Endotoxins; Environment; Evaluation; Exposure to; Gastrointestinal tract structure; Goals; Growth; Growth and Development function; Human; Hypersensitivity skin testing; IL8 gene; Immune response; In Vitro; Individual; Inflammation; Inflammatory; Interleukin-1; Interleukin-12; Interleukin-6; Knockout Mice; Lead; Leukotriene C4; Lung; Mediator of activation protein; Messenger RNA; Molecular Biology; Mus; Numbers; Peptide Hydrolases; Peptidoglycan; Pertussis Toxin; Play; Production; Prostaglandin D2; Reaction; Reagent; Recombinants; Role; Shrimp; Site; Stem cells; Surface; TLR4 gene; Tissues; Toll-Like Receptor 1; Tropomyosin; Tryptase; cell growth; cytokine; food allergen; in vivo; inhibitor/antagonist; mast cell; mast cell protease 4; mucosal site; prevent; research study; size

Human mast cells originate from pluripotential progenitor cells and migrate as immature cells from the bone marrow to tissue sites including the lung and gastrointestinal tract. There these precursors mature and participate in both innate and acquired immune responses with production of cytokines and other inflammatory mediators. Mast cell growth and development thus may occur in a tissue that interfaces with the external environment, potentially exposing mast cells during their development to bacterial products which could have an impact on their subsequent behavior. Consistent with this idea is the observation that mast cells are known to express Toll-like receptors (TLR) 1-7, and 9 both in vitro and in vivo; and exposure to such bacterial products as endotoxin or peptidoglycan leads to expression and release of cytokines. However, and in a related question, we explored whether bacteria-derived products alter the growth and development of HuMC. We thus performed long and short-term cultures to which we added LPS or PGN. We followed specific mast cell characteristics including growth; surface FcepsilonRI and CD117 expression; degranulation, LTC4 and PGD2 release; protease expression and composition, and cytokine release. Over 6 wks of culture, LPS had minimal effect on HuMC numbers but increased CD117, tryptase and chymase expression. PGN inhibited HuMC development. For mature mast cells, LPS in the presence of rhSCF (10 ng/ml) increased CD117, tryptase, chymase and carboxypeptidase expression, primarily in CD117low HuMC. LPS decreased FcepsilonRI expression and granule release; but had no effect on LTC4 and PGD2 production. PGN reduced HuMC numbers; and CD117 and tryptase expression. IL-1 and IL-6 (in addition to IL-8 and IL-12) were detected in short-term culture supernatants of LPS treated cells, and reproduced the increases in CD117, tryptase, chymase, and carboxypeptidase expression observed in the presence of LPS. Comparative studies with mouse bone marrow-derived mast cells from wild type, but not TLR4 knockout mice, showed increases in mRNA of mouse mast cell chymases MMCP-1, MMCP-2 and MMCP-4. We thus concluded PGN inhibits human mst cell growth, while LPS exerts its primary effects on mature mast cells by altering cytokine production and protease composition, particularly at low concentrations of SCF. These data thus demonstrate the ability of bacterial products to alter human mast cell mediator production, granular content, and number which may be particularly relevant at mucosal sites where mast cells are exposed to these products. In a question relevant to the evaluation of allergic sensitization, we are examining the ability of a component of shrimp, a major food allergen, to directly activate human mast cells. It is known that shrimp extracts, used as skin testing reagents, cannot be used at higher concentrations, as this leads to non-specific reactions; that is, these reactions occur in all individuals and thus do not correlate with clinical sensitivity. To identify the component of shrimp that directly activates mast cells, we fractionated shrimp extracts by size using column chromatography (S-300) and screened fractions for direct mast cell degranulating activity using the LAD2 human mast cell line. Active fractions were sequenced. Tropomyosin (Pen a 1) was identified as the active material. Recombinant Pen a 1 (rPen a 1) was used in degranulation experiments and did directly degranulate mast cells. In vitro degranulation experiments were then undertaken. Pertussis toxin and inhibitors of PI3K, PLCgamma, PKC and Src did not prevent degranulation. rPen a 1 did increase cytosolic calcium concentrations by promoting liberation from intracellular stores and calcium entry. These observations appear to explain how shrimp extracts at higher concentrations lead to non-specific skin test reactivity. In vivo experiments in an animal model are underway.

人肥大细胞来源于多能祖细胞，并作为未成熟细胞从骨髓迁移到包括肺和胃肠道在内的组织部位。在那里，这些前体成熟并参与先天性和获得性免疫应答，产生细胞因子和其他炎症介质。因此，肥大细胞的生长和发育可能发生在与外部环境接触的组织中，在肥大细胞发育期间可能将肥大细胞暴露于细菌产物，这可能对其随后的行为产生影响。与该想法一致的是，已知肥大细胞在体外和体内表达Toll样受体（TLR）1-7和9;并且暴露于诸如内毒素或肽聚糖的细菌产物导致细胞因子的表达和释放。然而，在一个相关的问题中，我们探讨了细菌衍生的产物是否会改变HuMC的生长和发育。 因此，我们进行了长期和短期培养，我们加入LPS或PGN。 我们遵循特定的肥大细胞特征，包括生长;表面Fc受体RI和CD 117表达;脱粒，LTC 4和PGD 2释放;蛋白酶表达和组成，以及细胞因子释放。 在6周的培养中，LPS对HuMC数量的影响最小，但增加了CD 117、类胰蛋白酶和糜蛋白酶的表达。PGN抑制HuMC的发育。对于成熟肥大细胞，LPS在rhSCF（10 ng/ml）存在下增加CD 117、类胰蛋白酶、糜蛋白酶和羧肽酶表达，主要在CD 117低HuMC中。LPS可降低FcepsilonRI的表达和颗粒释放，但对LTC 4和PGD 2的产生无影响。PGN减少HuMC数量;和CD 117和类胰蛋白酶表达。在LPS处理的细胞的短期培养上清液中检测到IL-1和IL-6（除了IL-8和IL-12之外），并且再现了在LPS存在下观察到的CD 117、类胰蛋白酶、糜蛋白酶和羧肽酶表达的增加。与来自野生型而非TLR 4敲除小鼠的小鼠骨髓源性肥大细胞的比较研究显示小鼠肥大细胞糜酶MMCP-1、MMCP-2和MMCP-4的mRNA增加。 因此，我们得出结论，PGN抑制人肥大细胞的生长，而LPS发挥其主要影响成熟肥大细胞通过改变细胞因子的产生和蛋白酶的组成，特别是在低浓度的SCF。因此，这些数据证明了细菌产物改变人肥大细胞介质产生、颗粒含量和数量的能力，这在肥大细胞暴露于这些产物的粘膜部位可能特别相关。 在一个与过敏性致敏性评价相关的问题中，我们正在研究虾的一种成分（一种主要的食物过敏原）直接激活人类肥大细胞的能力。已知用作皮肤测试试剂的虾提取物不能以较高浓度使用，因为这会导致非特异性反应;也就是说，这些反应发生在所有个体中，因此与临床敏感性无关。为了鉴定直接激活肥大细胞的虾的组分，我们使用柱色谱法（S-300）通过大小分级分离虾提取物，并使用LAD 2人肥大细胞系筛选用于直接肥大细胞脱粒活性的级分。对活性组分进行测序。 原肌球蛋白（Pen a 1）被确定为活性物质。 重组Pen a 1（rPen a 1）用于脱颗粒实验并直接脱颗粒肥大细胞。 然后进行体外脱粒实验。百日咳毒素和PI 3 K、PLC γ、PKC和Src的抑制剂不能防止脱粒。 rPen a 1通过促进胞内钙库的释放和钙内流而增加胞浆钙浓度。 这些观察似乎解释了较高浓度的虾提取物如何导致非特异性皮肤试验反应性。 动物模型的体内实验正在进行中。

项目成果

Mast cells express connexins on their cytoplasmic membrane.

肥大细胞在其细胞质膜上表达连接蛋白。

• DOI: 10.1016/s0091-6749(99)70239-3
• 发表时间: 1999
• 期刊: The Journal of allergy and clinical immunology
• 作者: Vliagoftis,H;Hutson,AM;Mahmudi-Azer,S;Kim,H;Rumsaeng,V;Oh,CK;Moqbel,R;Metcalfe,DD
• 通讯作者: Metcalfe,DD

Kit signal transduction.

试剂盒信号转导。

• DOI: 10.1016/s0889-8588(05)70294-x
• 发表时间: 2000
• 期刊: Hematology/oncology clinics of North America
• 作者: Taylor,ML;Metcalfe,DD
• 通讯作者: Metcalfe,DD

Receptor-mediated modulation of murine mast cell function by alpha-melanocyte stimulating hormone.

α-黑素细胞刺激激素对小鼠肥大细胞功能的受体介导调节。

• 发表时间: 1999
• 期刊: Journal of immunology (Baltimore, Md. : 1950)
• 作者: Adachi,S;Nakano,T;Vliagoftis,H;Metcalfe,DD
• 通讯作者: Metcalfe,DD