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SummaryWestern blot analysis showed that a monoclonal antibody against recombinant mouse CD14 (mCD14), designated rmC5-3, specifically reacted with mouse macrophage cell line J774, but not myeloma cell line NS1. Fluorographic and immunocytochemical analysis demonstrated specific binding of rmC5-3 with mouse resident macrophages, inflammatory monocytes and neutrophils, and macrophage cell lines. Immunohistochemical staining using rmC5-3 showed that CD14-positive Kupffer cells (KC) were small in number in the liver in nonstimulated mice. The number of stained KC, which were rich in the midzonal and periportal regions, gradually increased with time after intraperitoneal injection of lipopolysaccharide (LPS), peaked 6 h after injection, and returned to normal by 20 h after injection. Staining intensity over time was proportional to the number ofKC. A slight increase in mCD14 expression was observed in peritoneal macrophages 2 h after LPS administration in vivo using flow cytometric analysis, mCD14 mRNA became detectable at 1 h after the intraperitoneal injection of LPS (20 gg/mice), and the level dramatically increased with time, peaking at 3 h, and sharply dropped at 6 h. The resident peritoneal macrophages demonstrated a constitutively high mCD14 mRNA expression, which slightly increased 2 h after LPS (100 ng/ml) stimulation in vitro. The level of mCD14 expression in macrophages did not increase after intraperitoneal injection of LPS (20 gg/mice).

We isolated the human osteopontin (hOP) gene and the 5' upstream region, and analysed its exon-intron structure and potential regulatory sequences of the promoter region in comparison with those of the mouse and porcine gene. The coding sequence is split into 7 exons which are similar to those of the mouse gene, although the hOP gene is longer than the mouse gene. The difference in length is mainly due to variations in intron 3, which is approximately 2.7-fold longer than that of the mouse OP gene. The 5' upstream region of the hOP, which is highly conserved up to nucleotide -250, contains a number of potential cis regulatory consensus sequences. A series of sequentially 5'-deleted chimeric clones was tested for the ability to stimulate chloramphenicol acetyltransferase (CAT). Initial CAT analysis demonstrated that nucleotides at positions -474 to -270, -124 to -80, and -55 to -39 contained cis-acting enhancing sequences in a human monocyte cell line, SCC-3, although the -124 to -80 region was much more active than other regions. Deletion of the sequences between -474 and -270 localized this cis region to the sequence at positions -439 to -410, whereas the deletion between -124 to -80 localized the regions to -124 to -115, and -94 to -80. Gel-shift analysis using as probes synthesized double-stranded DNA corresponding to the 10 and 15 bp region at positions -124 to -115 and -94 to -80 respectively revealed that each probe formed a major band complexed with nuclear proteins prepared from SCC-3 cells.

cDNA clones which strongly hybridized with a 3.1 kb mRNA from mouse macrophages and macrophage cell lines and weakly with mRNA from P815 but not from a variety of other cell lines and tissues were isolated from cDNA libraries constructed using mRNA from murine macrophage cell lines and peritoneal macrophages. Treatment of a macrophage cell line with macrophage stimulators significantly enhanced transcription of the mRNA. Sequencing analysis of these clones demonstrated that the cDNA consisted of 3036 bp insert containing a 2478 bp open reading frame followed by a 538 bp 3' untranslated region. The amino acid sequence, deduced from the nucleotide sequence of the cDNA, predicted a protein containing a signal peptide, an extracellular region, a transmembrane domain, and a cytoplasmic tail. The extracellular region had five putative N-glycosylation sites and a cysteine-rich domain, whereas the cytoplasmic region consisted of a proline-rich amino acid sequence significantly similar to CD2. SDS-PAGE and NEPHGE SDS-PAGE analysis of the immunoprecipitated membrane of the macrophage cell lines prepared by using rabbit anti-MS2 peptide antibody raised against a synthetic peptide preparation relative to a hydrophilic region of the MS2 amino acid sequence confirmed that MS2 protein is a cross-linked protein having approximate molecular sizes of 89 kd and pl 6.5-7.0. These results show that MS2 protein is a novel cell surface antigen expressed mainly in monocytic lineages.

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