REGULATION OF CELL DEATH IN EOSINOPHILOPOIESIS

研究提出，以检查细胞凋亡的作用， 嗜酸性粒细胞生成和细胞外基质类肝素的作用 硫酸盐（HS），通过一个潜在的受体，CD 31调节生存和 嗜酸性粒细胞的发展，嗜酸性粒细胞是哮喘发病机制的核心细胞。 这一建议的核心是受体介导的 前体细胞与HS的相互作用导致 前体细胞中的存活机制有助于增加 在嗜酸性粒细胞的产生中。 为了验证这一假设，我们提出了三个 具体目的：（1）确定HS和CD 31连接对 嗜酸性粒细胞（CDE）体外产生 祖细胞 硫酸乙酰肝素增强 将检查体外CDE生产。 实验将：a）评估 IL-3和IL-5指导的从分离的脐带中产生成熟CDE 在组织培养板中的血液祖细胞（CD 34+）， 在确定的剂量范围内的HS或不相关的基质分子; B） 确定CDE对乙酰肝素反应的受体特异性 通过使用表位特异性mAb测定硫酸盐; c）评估CD 31 刺激性单克隆抗体在嗜酸性粒细胞产生方面， 与HS刺激相比。 (2)确定敏感性 发展CDE以通过“被动”细胞因子诱导细胞凋亡 撤回和'主动' Fas连接和机制的作用， Bcl-2家族和ICE家族在这些过程中的作用。 我们将测量相对于精氨酸诱导的祖细胞， 细胞对细胞因子撤出和Fas连接的易感性;B） 检测Bcl-2相关基因和ICE mRNA及蛋白的表达 在不同成熟阶段的嗜酸性粒细胞中，使用RT-PCR， 通过北方、西方和流式细胞术检测， 细胞凋亡调节的细胞内机制; c）使用特异性 Bcl-2和ICE抑制易感和耐药嗜酸性粒细胞 前体，评估这些监管机构的相对作用， 发展 (3)确定硫酸乙酰肝素和/或CD 31的作用 结扎抑制发育中的嗜酸性粒细胞的凋亡， 分子机制包括抑制这些凋亡的细胞， 事件 使用脐带血来源的祖细胞和CDE，我们将 a）确定培养物对硫酸乙酰肝素或刺激的影响， 抗CD 31对细胞因子撤药和Fas诱导的凋亡反应的影响 在不同成熟阶段的嗜酸性粒细胞中进行连接; B）评估 特异性Bcl-2和ICE家族成员对CD 31/HS的贡献 调节对凋亡刺激的反应。 我们预计， 研究将建立一种新的策略，检查嗜酸性粒细胞生成 并将阐明基质介导的调节机制， 造血

Studies are proposed to examine the role of apoptosis during eosinophilopoiesis and the role of the extracellular matrix heparans sulfate (HS), via a potential recepto, CD31 on modulating survival and development of eosinophils, cells central to pathogenesis of asthma. The central gypothesis of this proposal is tht ceptor-mediated interaction of precursor cells with HS causes the upregulation of survival mechansims in the precursor cells contributing to an increase in eosinophil production. To test this hypothesis, we propose three specific aims: (1) Determine the effects of HS and CD31 ligation on the in Vitro production of eosinophils (CDE) developing from isolated progenitor cells. The mechanism by which heparan sulfate enhances CDE production in vitro will be examined. Experiments will: a) assess Il-3 and IL-5-directed production of maturing CDE from isolated cord blood progenitor (CD34+) cells in tissue-culture plates pretreated with HS or irrelevant matrix molecules over a determined dose range; b) determine receptor specificity of the response of CDE to heparan sulfate by using epitope-specific mAb; c) assess effects of CD31 stimulatory monoclonal antibody in terms of eosinophil production and compare to HS stimulation. (2) Determine the susceptibility of developing CDE to induction of apoptosis through 'passive' cytokine withdrawal and 'active' Fas ligation and the mechanistic roles of the Bcl-2 family and the ICE family in these processes.a) Using 'naive' or cytokine-induced progenitor cells, we will measure the relative susceptibility of the cells to cytokine withdrawl and to Fasligation;b) measure the expression of Bcl-2-related and ICE mRNA and proteins in eosinophils at different maturational stages, using RT-PCR, Northern, Western and flow cytometric assays to identify specific intracellular mechanisms of regulation of apoptosis; c) using specific Bcl-2 and ICE inhibition of susceptible and resistant eosinophil precursors, assess the relative roles of these regulators during development. (3) Determine the role of Heparan sulfate and/or CD31 ligation in the suppression of apoptosis of developing eosinophils and the molecular mechanisms involved suppression of these apoptotic events. Using cord blood-derived progenitor cells and CDE, we will a) detemine the effect of culture on heparan sulfate or stimulation by anti-CD31 on the apoptotic response to cytokine withdrawal and Fas ligation in different maturational stages of eosinophils; b) assess the contributions of specific Bcl-2 and ICE family members to CD31/HS modulated response to apoptotic stimuli. We anticipate that these studies will establish a novel strategy of examining eosinophilopoiesis and will elucidate mechanisms of matrix-mediated regulation of hematopoiesis.