| Research Abstract |
(1) Separation of novel genes highly expressed in macrophages : 1). Cloning of the genes : We have cloned a variety of cDNA which are highly expressed in myelomonocytic cells. These includes CD14, osteopontin CD156, ferritin heavy subunit, SAA3, unique murine retrotransposon. In this project, we newly cloned genes for BS9, BS10, AD56, AD65, AD89, AD104, SY-2 and 40 as novel proteins. 2).Sequencing, separation of recombinant proteins and genomes : Of these clones, sequencing of several important clones including BS9, BS10, AD56, SY-2 and 40 were performed. (2) Analysis of CD14 : 1) Cloning of rat CD14 (rCD14) cDNA and analysis of its expression : Rat CD14 cDNA clones were isolated. The levels of rat CD14 mRNA expression in resident peritoneal macrophages (PM), alveolar macrophages (AM) and peripheral blood monocytes (BM) were constitutively high, whereas that in Kupffer cells (KC) was low. Upon intravenous injection with LPS,the expression of rat CD14 mRNA in KC increased markedly, wher
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eas the increases in PM,AM and BM were mild. Similar features of expression of rat CD14 in these cells were observed after stimulation with LPS in vitro. The levels of cytokine expression in KC after stimulation with LPS in vivo was relatively low compared with that in PM,AM and BM.2) Analysis of transgenic mice : Two different metallothionein promoter (MT)-mouse CD14 (mCD14) fusion genes were constructed. Fusion genes of the membrane form of mCD14 fusion gene, designated M14M and the soluble form fusion gene, designated M14S,was used. Sera from M14S mice demonstrated significantly. l'ower levels of TNF-alpha than those from nontransgenic mice (TM). Levels of IL-1beta were comparable between TM and non-TM whereas those of IL-6 in M14S mice were significantly lower than in M14M and nontransgenic mice. Survival rates in the lethal Schwartzman reaction induced by the priming and challenge injections of LPS (S.enteritidis) were significantly higher in M14M and M14S mice than in nontransgenic mice. 3) Analysis of knock out mice : The role of CD14 in bacterial-induced and LPS-induced shock was tested in CD14-deficient mice produced by gene targetting in embryonic stem cells. CD14-deficient mice were found to be highly resistant to shock induced by either live Gram-negative bacteria of LPS ; however, at very high concentrations of LPS or bacteria, responses through non-CD14 receptors could be detected. CD14-deficient mice also showed dramatically reduced levels of bacteria, suggesting an unexpected role for CD14 in the dissemination of Gramnegative bacteria. 4). Expression and use of truncated mouse CD14, (3) Analysis of CD156 (MS2, ADAM 8) : 1) Cloning of CD156 : cDNA for human CD156 was isolated from cDNA libraries from the human macrophage cell line THP-1 and from human granulocytes. The CD156 cDNA detected mRNA from human macrophage cell lines, granulocytes, monocytes, and B cell but not T cell lines. The nucleotide sequence of the CD156 cDNA showed 65.6% homology with Less
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