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

一般来说，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。这一观点似乎与核糖体和内质网的相对稀缺性以及当RNA和/或蛋白质合成受阻时PMN实现吞噬、代谢爆发和溶酶体排出功能的能力一致。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通过渗透PMN而产生的。通过克隆和测序，确定该免疫增强因子为白介素1β(IL-1β)。虽然一般认为IL-1β是由巨噬细胞合成的，而不是由PMN合成的，但我们通过免疫染色从单细胞水平证实了在酪蛋白诱导的兔急性炎症过程中，PMN是产生IL-1β的主要来源。…此外，这种由PMN产生的IL-1β被蛋白质合成的抑制剂所阻断。此外，从炎症早期纯化的PMN中提取的Poly A^+RNA组分经Northern分析证实含有兔IL-1β的特异性mRNA。接下来，我们选择IL-1抑制物作为PMN合成蛋白的另一个靶分子，因为循环中的白细胞不含该抑制物，而炎症部位的PMN变得具有该因子。经过最终纯化和克隆该因子的cDNA，我们认为该炎性IL-1抑制物是人IL-1受体拮抗剂(IL-1ra)的兔同系物。在炎症反应的5小时和96小时内观察兔IL-1ra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)是这样。为了系统研究PMN的蛋白质合成依赖功能，我们构建了具有代表性的早期炎症阶段(5小时病变)和晚期炎症阶段(24小时病变)的两个PMN制剂的cDNA文库。我们通过5hr-PMN和24hr-PMN之间的差减分离了炎症早期PMN中特异表达的基因，并通过24hr-PMN和5hr-PMN之间的差减分离了炎症晚期表达的基因。进一步对每个文库的100个候选克隆进行差异杂交筛选，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。这些克隆分为4组：A组为仅在5hr-PMN中表达的克隆，B组为在5hr-PMN中主要表达但在晚期白细胞中也有弱表达的克隆，C组为在5hr-PMN中主要表达但在晚期白细胞中也有强表达的克隆，D组为在24hr-PMN中主要表达但在5hr-PMN中不表达的克隆。这表明炎症早期和晚期的炎性渗出的PMN根据炎症的进展至少合成了12种独立的物质。较少

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