|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1992 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1991 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Neutrophils have long been considered to be terminally differentiated end cells and to possess little RNA and protein synthesis capability. Using a casein-induced rabbit peritonitis, we found that neutrophils synthesize interleukin 1beta(IL-1beta)de novo only at early stages of inflammation. This observation suggested that protein synthesis of neutrophils is involved with inflammatory reaction. With the focus on RNA/protein synthesis capability by neutrophils and its dynamic change during the inflammatory process, we carried out gene library subtraction studies. We performed gene subtraction with the ss cDNA library hybridization method and the resulted clones of 5 hr-minus 24 hr-neutrophils were further confirmed by differential hybridization. The cDNA from candidate clones were converted to sense- and anti-sense-RNA and using these RNA probes, we performed Northern blot and cytoplasmic slot blot analyses.
Immune activation gene 2(Act-2), MIF related protein-8(MRP-8), MRP-14, fMLP rece
ptor (fMLPR) and gamma-actin homologues were isolated as genes representing neutrophils at an early inflammatory stage. Ferritin light(L)chain cDNA was cloned as the gene representing neutrophils at a late inflammatory stage. In addition, mRNA expression of rabbit neutrophil attractant/activation protein-1/interleukin 8(NAP-1/IL-8), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor alpha(TNFalpha)and ferritin heavy(H)chain mRNASs expression during inflammation were examined by cytoplasmic dot blot analysis. The patterns of mRNAs of Act-2, NAP-1/IL-8, MRP-14 and gamma-actin homologues were induced immediately after the onset of inflammation. This pattern is similar to that of IL-1beta mRNA expression. Pattern2 : mRNA expression of the ferritin H chain in neutrophils reached a peak at intermediate periods (12hr)of inflammation, with a monophagic pattern. Pattern3 : the level of ferritin L chain mRNA increased with progression of the inflammatory process. The mRNA expressions of TNFalpha and MCP-1 was not significant. These data suggest that neutrophils contribute to the defense reactions by synthesizing various proteins at the site of inflammation. Even in a later stage of inflammation, neutrophils were viable and retain protein synthetic capability, although they are generally believed to have a very short life-span and may die at a different site of inflammation. Less