| Budget Amount *help |
¥6,700,000 (Direct Cost: ¥6,700,000)
Fiscal Year 1990: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1989: ¥4,100,000 (Direct Cost: ¥4,100,000)
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| Research Abstract |
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.
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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
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