A New Aspect of Neutrophil Function Dependent on Their Protein Synthesis.

中性粒细胞功能依赖于其蛋白质合成的新方面。

• 批准号: 01480166
• 负责人: YOSHINAGA Masaru
• 金额: $ 4.29万
• 依托单位: Kumamoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-01480166/
• 关键词: Inflammation; Neutrophil; Polymorphonuclear leukocyte; Cytokine; Protein synthesis; RNA synthesis; サイトカイン; タンパク合成能; RNA合成能; IL1; 差し引きライブラリ-

Generally, mature polymorophonuclear leukocytes (PMN) are thought to be terminally differentiated end cells and have only limited protein synthetic capability, if any. This belief seems to be consistent with the relative scarcity of ribosomes and endoplasmic reticulum and with the ability of PMN in achieving their functions of phagocytosis, metabolic burst and lysosomal discharge when RNA and/or protein synthesis are blocked.While studying the production of an immune-potentiation factor at the site of inflammation, we noticed this immune-potentiation factor was produced by infiltrating PMN. Finally, we concluded that this immune-Potentiation factor was interleukin 1beta (IL-1beta) by cloning and sequencing of its cDNA. Although, IL-1beta is generally believe to be synthesized by macrophages and not by PMN, we definitely proved that PMN were the major producer of IL-1beta during the caseininduced acute inflammation in rabbits, with respect to a single cell level by using immunostaining. … More Furthermore, this production of IL-1beta by PMN was blocked by inhibitors of protein synthesis. In addition, poly A^+RNA fraction from purified PMN of an early stage of the inflammation was proved by Northern analysis to include the specific mRNA for rabbit IL-1beta.Next, we chose IL-1 inhibitor as another target molecule of PMN-synthesizing protein, because the circulating leukocytes did not have the inhibitor, while PMN of inflammatory site became to have the factor. After final purification and cloning of cDNA for this factor, we concluded this inflammatory IL-1 inhibitor was a rabbit homologue of human IL-1 receptor antagonist (IL-1ra). The rabbit IL-1ra production was observed during 5 and 96 hr of inflammation. PMN were thought to be major producer of this inhibitor at least during a relatively earlier stage (5-24 hr). In a later stage, the producer of the IL-1ra was switched to change to macrophages according to the progression of the inflammation.In order to perform a systemic study of the protein synthesis-dependent function by PMN, we constructed two cDNA libraries of PMN preparations from a representative early stage (5 hr-old lesion) and a later stage (24 hr-old lesion) of the inflammation. We isolated the genes expressed specific in PMN at early inflammatory stage by subtraction between 5 hr-PMN and 24 hr-PMN and that expressed at late inflammatory stage by subtraction between 24 hr-PMN and 5 hr-PMN. Further, 100 candidate clones of each libraries were screened by differential hybridization Finally, 9 clones were found to be preferentially expressed in 5 hr-PMN and one clone was in 24-hr PMN. These clones were divided into 4 groups : group A include clones that only expressed in 5 hr-PMN ; group B, clones dominantly expressed in 5 hr-PMN but also weakly expressed in late leukocytes ; group C, clones dominantly in 5 hr-PMN and also strongly in late leukocytes, and group D, clone dominantly in 24-hr PMN but not in 5 hr-PMN. This indicate that both the inflammatory exuded PMN in early and late stages had synthesized at least 12 independent substances according to the progression of inflammation. Less

通常，成熟的多形核白细胞（PMN）被认为是终末分化的终末细胞，如果有的话，只有有限的蛋白质合成能力。这与中性粒细胞缺乏核糖体和内质网，以及当RNA和/或蛋白质合成受阻时，中性粒细胞能完成吞噬、代谢爆发和溶酶体释放等功能是一致的。在研究炎症部位免疫增强因子的产生时，我们注意到这种免疫增强因子是由浸润的中性粒细胞产生的。最后，通过克隆和测序，我们得出结论，这种免疫增强因子是白细胞介素1 β（IL-1 β）。虽然IL-1 β通常被认为是由巨噬细胞而不是由PMN合成的，但我们明确地证明了在酪蛋白诱导的兔急性炎症中，PMN是单个细胞水平上IL-1 β的主要产生者。 ...更多信息 此外，白细胞介素-1 β的生产由中性粒细胞被阻断蛋白质合成的抑制剂。另外，北方分析证实，从炎症早期PMN中提取的poly A^+RNA中含有兔IL-1 β的特异性mRNA。经过最终的纯化和克隆的cDNA，我们得出结论，这种炎性IL-1抑制剂是一个兔同源的人IL-1受体拮抗剂（IL-1 ra）。在炎症的5和96小时期间观察兔IL-1 ra的产生。PMN被认为是这种抑制剂的主要生产者，至少在相对较早的阶段（5-24小时）。为了对PMN的蛋白合成依赖性功能进行系统的研究，我们构建了两个PMN制备物的cDNA文库，这两个cDNA文库分别来自炎症的代表性早期阶段（5 h-老病变）和晚期阶段（24 h-老病变）。我们用5 hr-PMN和24 hr-PMN相减法分离了炎症早期PMN特异性表达的基因，用24 hr-PMN和5 hr-PMN相减法分离了炎症晚期PMN特异性表达的基因。进一步，每个文库的100个候选克隆通过差异杂交筛选，最终发现9个克隆在5小时PMN中优先表达，1个克隆在24小时PMN中优先表达。将这些克隆分为4组：A组包括仅在5小时PMN中表达的克隆; B组，主要在5小时PMN中表达但在晚期白细胞中也有弱表达的克隆; C组，主要在5小时PMN中表达但在晚期白细胞中也有强表达的克隆;这表明，无论是早期还是晚期的炎性渗出的PMN，随着炎症的进展，至少合成了12种独立的物质。少

项目成果

Ohkawara S.,Goto F.,and Yoshinaga M.: "Interleukin 1 as an inflammatory hormone" Acta Pathologica Japonica. 39. 85-100 (1989)

Ohkawara S.、Goto F. 和 Yoshinaga M.：“白细胞介素 1 作为炎症激素”Acta Pathologica Japonica。

後藤文正,後義久美子,森俊輔,松川昭博,吉永秀: "炎症メディエ-タ-としてのサイトカイン" 炎症. 10. 17-23 (1990)

Fumimasa Goto、Kumiko Gogi、Shunsuke Mori、Akihiro Matsukawa、Hide Yoshinaga：“细胞因子作为炎症介质”10. 17-23 (1990)。

Mori S, Goto F, Goto K, Ohkawara S, Maeda S, Shimada K and Yoshinaga M: "Cloning and sequence analysis of a cDNA for lymphocyte proliferation potentiating factor of rabbit polymorphonuclear leukocytes : Identification as rabbit interleukin 1beta." Biochem

Mori S、Goto F、Goto K、Ohkawara S、Maeda S、Shimada K 和 Yoshinaga M：“兔多形核白细胞淋巴细胞增殖增强因子 cDNA 的克隆和序列分析：鉴定为兔白细胞介素 1β。”

Ohkawara S, Goto K, Mori S, Goto F, Saita N, Sagara T and Yoshinaga M: "Interleukin 1 production by polymorphonuclear leukocytes during the course of acute inflammation in rabbits." Arch. Dermatol.179(Supple 1). 84-90 (1989)

Ohkawara S、Goto K、Mori S、Goto F、Saita N、Sagara T 和 Yoshinaga M：“兔子急性炎症过程中多形核白细胞产生白细胞介素 1。”

Ohkawara S,Goto K,Mori S,Goto F,Saita N,Sagara T and <Yoshinaga M>___ー: "Interleukin production by polymorphonuclear leukocytes during the course of acute inflammation in rabbits." Archives of Dermatology. 179(Supple 1). 84-90 (1989)

Ohkawara S、Goto K、Mori S、Goto F、Saita N、Sagara T 和 <Yoshinaga M>___ー：“兔子急性炎症过程中多形核白细胞产生白细胞介素。”（皮肤病学档案 1） ）84-90（1989）。