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.

人的肥大细胞起源于多潜能的祖细胞，以未成熟细胞的形式从骨髓迁移到组织部位，包括肺和胃肠道。在那里，这些前体成熟并参与先天和后天免疫反应，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，可能会在肥大细胞发育期间暴露在细菌产物中，这可能会对它们随后的行为产生影响。与这一观点一致的是观察到肥大细胞在体外和体内都表达Toll样受体(TLR)1-7和9；接触内毒素或肽聚糖等细菌产物会导致细胞因子的表达和释放。然而，在一个相关的问题中，我们探索了细菌衍生产品是否会改变HUMC的生长和发育。 因此，我们进行了长期和短期培养，并在其中添加了内毒素或PGN。我们观察了肥大细胞的特征，包括生长，表面FcepsilonRI和CD117的表达，脱颗粒，LTC4和PGD2的释放，蛋白酶的表达和组成，以及细胞因子的释放。 培养6wk以上，内毒素对HUMC数影响不大，但CD117、类胰蛋白酶和糜乳酶表达增加。PGN可抑制HUMC的发育。在成熟肥大细胞中，加入重组人干细胞因子(10 ng/ml)后，CD117、类胰蛋白酶、凝乳酶和羧基肽酶的表达增加，主要是在CD117低的HUMC中。脂多糖降低FcepsilonRI的表达和颗粒释放，但不影响LTC4和PGD2的产生。PGN使HUMC数减少，CD117和类胰蛋白酶表达减少。在短期培养上清液中检测到IL-1和IL-6(除IL-8和IL-12外)，并复制了在内毒素存在下CD117、类胰蛋白酶、凝乳酶和羧基肽酶的表达增加。与野生型小鼠骨髓来源的肥大细胞的比较研究表明，小鼠肥大细胞糜酶MMCP-1、MMCP-2和MMCP-4的mRNA表达增加。 因此，我们得出结论：PGN抑制人MST细胞的生长，而内毒素通过改变细胞因子的产生和蛋白水解酶的组成，尤其是在低浓度的SCF时，对成熟的肥大细胞起主要作用。因此，这些数据表明细菌产品能够改变人类肥大细胞介质的产生、颗粒含量和数量，这可能与肥大细胞接触这些产品的粘膜部位特别相关。 在一个与过敏反应评估相关的问题中，我们正在检测虾的一种成分直接激活人类肥大细胞的能力，虾是一种主要的食物过敏原。众所周知，用作皮肤测试试剂的虾提取物不能在较高浓度下使用，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人身上，因此与临床敏感性无关。为了确定虾中直接激活肥大细胞的成分，我们使用柱层析(S-300)对虾提取物按大小进行分级，并使用LAD2人肥大细胞系筛选出直接肥大细胞脱颗粒活性的组分。对活性组份进行了测序。原肌球蛋白(Pen A 1)为活性物质。重组PEN A 1(RPEN A 1)用于脱颗粒实验，直接脱颗粒肥大细胞。 然后进行体外脱颗粒实验。百日咳毒素和PI3K、PLCGamma、PKC和Src的抑制剂不能阻止脱颗粒。RPEN A 1通过促进细胞内钙离子的释放和钙内流而增加细胞内钙离子浓度。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。动物模型的活体实验正在进行中。

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