Yasunori Higuchi

ADAM family proteins in the immune system

CD156 is a member of a family proteins characterized by a disintegrin and a metalloprotease domain (ADAM). These molecules are phylogenically conserved but have individual roles in a variety of cells. Here, Shunsuke Yamamoto and colleagues discuss data suggesting that ADAM family proteins have important roles in the immune system.

Mouse and human CD14 (myeloid cell-specific leucine-rich glycoprotein) primary structure deduced from cDNA clones

cDNA clones complementary to MS7-4 (Setoguchi et al. (1988) Somat. Cell Mol. Genet. 14, 427-438) from a mouse macrophage cDNA library were separated. Sequence analysis of these clones demonstrated that the longest cDNA clone, MS7X, had a 1366 bp insert and high homology with that of the human CD14 gene (Ferrero and Goyert (1988) Nucleic Acids Res. 16, 4173). Using the MS7X cDNA probe, cDNA clones were separated from cDNA libraries constructed from a human macrophage cell line and macrophages. The total cDNA sequence was 1364 bp in length, with an open reading frame of 1125 nucleotides matching that of the human CD14 gene except for one nucleotide difference. The amino-acid sequence (mouse CD14), deduced from the nucleotide sequence of the MS7X insert consisted of 351 amino-acid residues with a high leucine content (17.66%) and five putative N-glycosylation sites, and in vitro translation predicted a protein of molecular mass of 37.5 kDa. Human CD14 had 356 amino-acid residues, with high leucine content (15.5%), and contained four putative N-glycosylation sites. Mouse CD14 showed 13 building blocks, of which internal nine blocks have a conserved leucine motif and significant homology with human leucine-rich alpha 2-glycoprotein.

Primary structure of rat CD14 and characteristics of rat CD14, cytokine, and NO synthase mRNA expression in mononuclear phagocyte system cells in response to LPS.

Rat CD14 cDNA clones were isolated. The predicted protein sequence exhibits 82,61.6, and 64% identity with the mouse, rabbit, and human CD14, respectively. 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. On intravenous injection with lipopolysaccharide (LPS), the expression of rat CD14 mRNA in KC increased markedly, whereas 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 level of tumor necrosis factor α (TNF‐α) mRNA expression in KC after stimulation with LPS in vivo was comparable to that in PM, AM, and BM, whereas that of TNF‐α mRNA expression in KC and PM after stimulation with LPS in vitro was lower than that in AM and BM. Interleukin (IL)‐1β and iNOS mRNA expressions in KC after stimulation with LPS in vivo and in vitro were low, whereas those in PM, AM, and BM were high. Little or no expression of IL‐6 was observed in KC after stimulation with LPS in vivo and in vitro, whereas higher expression was observed in PM, AM, and BM than in KC. J. Leukoc. Biol. 61: 736–744; 1997.

Structure of the Murine CD156 Gene, Characterization of Its Promoter, and Chromosomal Location

The murine cell surface antigen mCD156 is a glycoprotein that is expressed in monocytic cell lines and consists of a metalloprotease domain, a disintegrin domain, a cysteine-rich domain, and an epidermal growth factor-like domain in the extracellular region. The mCD156 gene is composed of 24 exons and 23 introns and spans approximately 14 kilobases. The first exon encodes most of the signal peptide sequence, and the transmembrane region is encoded by a single exon (19). In contrast, the other regions are composed of multiple exons. Of these, exons 7–12 and 12–15 encode a metalloprotease domain and a disintegrin domain, respectively. Sequence analysis of the 5′-flanking DNA revealed many potential regulatory motifs. Chloramphenicol acetyltransferase analysis demonstrated that nucleotides at positions −183, −334, and −623 containedcis-acting enhancing elements in a mouse monocytic cell line, aHINS-B3. Nucleotides at positions −183 and −390 contained elements responsible for lipopolysaccharide (LPS) inducibility, although several other 5′-flanking regions were also involved in LPS responsiveness. Regions −202, −507, and −659 play a role in interferon-γ inducibility. Some of the potential regulatory motifs and other unknown cis elements may be involved in the constitutive expression, and LPS and interferon-γ inducibilities. The mCD156 gene was mapped to chromosome 7, region F3-F4.

Expression of Osteopontin in a Macrophage Cell Line and in Transgenic Mice with Pulmonary Fibrosis Resulting from the Lung Expression of a Tumor Necrosis Factor‐α Transgene

The expression level of osteopontin (OPN) mRNA was found to be increased in a macrophage cell line in the presence of recombinant tumor necrosis factor-alpha (TNF-alpha). OPN mRNA level was also explored in the lungs of transgenic mice which were expressing TNF-alpha in type II pneumocytes, a condition leading to pulmonary alveolitis and progressive fibrosis. OPN mRNA was significantly increased in the lungs of these transgenic mice. In situ hybridization showed that it was localized mostly in alveolar macrophages. Since OPN can be induced in macrophages by TNF-alpha stimulation and since on the other hand osteopontin appears to decrease the level of nitric oxide synthase, and thus the production of nitric oxide, osteopontin might also indirectly have an antifibrotic effect. The role played by osteopontin in fibrotic lesions resulting from the release of TNF-alpha deserves further study, since it may be involved in the balance of opposite effects eventually leading to local tissue damage ending in fibrosis.

An autopsy case of human T-lymphotropic virus type I—Associated myelopathy

This report describes the first autopsy case of human T-lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM). The disease mainly affected the spinal cord, particularly the lateral and anterior columns, where loss of myelin and axon was observed. The changes were bilateral and occurred mainly along the tract. Perivascular and parenchymal infiltration with lymphocytes and macrophages, as well as astrocytosis, were observed in the white and grey matters of the spinal cord. Blood vessels in the spinal cord and in the subarachnoid space of the spinal cord showed hyalinoid thickening of media and adventitia associated with infiltration of lymphocytes. These findings are similar to those of tropical spastic paraparesis (TSP).

Expression of Splice Variants of the Human ADAM15 Gene and Strong Interaction between the Cytoplasmic Domain of One Variant and Src Family Proteins Lck and Hck

Objective: The aim of the present study was to show variant species of ADAM15 and unique Src homology 3 (SH3)-binding motifs, which strongly bound Src family proteins compared with ADAM15. Methods and Results: RT-PCR using primers for the cytoplasmic domain revealed the presence of different species, designated ADAM15v1 and ADAM15v2, which had characteristic SH3-binding class I and class II motifs. The mRNA of ADAM15v1 and ADAM15v2 was mainly found in peripheral blood mononuclear cells, T lymphocytes and monocytic cell lines. ADAM15v2 protein interacted more strongly with the Src family proteins Lck and Hck than did ADAM15 protein, as examined by pull-down analysis and immunoprecipitation followed by immunoblot analysis. The binding with Lck and Hck was enhanced by the phosphorylation of ADAM15v2 protein. Conclusions: These results suggest that the cytoplasmic domain of ADAM15v2 strongly interacts with Lck and Hck and regulates leukocyte function.

Enhancement of murine serum amyloid A3 mRNA expression by glucocorticoids and its regulation by cytokines.

Macrophages are regulated by hormones, cytokines, and other substances. We examined the effects of glucocorticoids (GC) on serum amyloid A (SAA) mRNA expression using an antisense SAA3 probe, and found that dexamethasone (DEX) and triamcinolone (TC) increased SAA3 mRNA expression by macrophage cell lines in a time‐ and concentration‐dependent manner. Both an RNA polymerase II antagonist, α‐amanitin, and a specific GC antagonist, RU38486, inhibited the enhancement of SAA3 mRNA expression by DEX. These results show that GC lead to enhanced SAA3 mRNA transcription. Nuclear run‐on experiments supported these results. Enhanced expression of SAA3 mRNA by DEX was accompanied by production of SAA3 protein. Interferon‐γ (IFN‐γ) alone showed any effect on SAA3 mRNA expression. Enhanced SAA3 expression by DEX was inhibited by treatment with IFN‐γ in a dose‐dependent manner. Either transforming growth factor (TGF)‐β1 or interleukin (IL)‐4 alone showed no effect on SAA3 mRNA expression, but suppressed DEX‐induced SAA3 expression. The timing of the effects of IFN‐γ and TGF‐βl on DEX‐ induced SAA3 expression differed: IFN‐γ showed its effect between 30 h before and 18 h after DEX administration, whereas TGF‐β1 showed an effect when administered concomitantly with DEX and in the late stages after DEX administration. IL‐4 mildly suppressed DEX‐induced SAA3 expression when given 12 h before and after DEX administration. J. Leukoc. Biol. 56: 797–806; 1994.

Time course study of H-2 and other antigen expression by hybrids of a myeloma cell line with inflammatory macrophages

The hybrids (the CANS lines) between inflammatory macrophages from C57BL/6N (B6) mice (H-2b) and BALB/c mouse (H-2d)-derived myeloma cell line NS1 in the early period after cell fusion showed no macrophage functions. However, most of the hybrids expressed these functions after prolonged cultivation accompanied with chromosome loss. In contrast, the hybrids initially displaying myeloma functions (κ light chain production) lost this function when they exhibited macrophage functions. We studied the expression of cell-surface antigens in these hybrids and found that hybrids in the early period after cell fusion codominantly expressed both parental cell H-2 antigens (H-2Kb, H-2Kd, and H-2Dd) but not the H-2Db antigen. On the other hand, aged hybrids strongly expressed the H-2 d antigen but lacked the H-2Kb antigen. Alternatively, these aged hybrids with macrophage functions expressed antigen(s) as detected with antiaged CANS-196 cell sera and asialo GM1 antigen, both of which were thought to be found exclusively on macrophages. Thus, the expression of cell-surface antigens in these hybrids was greatly altered after cell fusion.

Close Localization of Mouse CD14 and CD32/16 in the Cell Surface of Monocytic Cell Lines

The binding of a rat anti-mouse CD14 monoclonal antibody (mAb) (rmC5-3) was inhibited by pretreatment of a mouse monocytic cell line WEHI-3 cells with anti-mouse CD32/16 mAb (2.4G2), whereas that of 2.4G2 was not inhibited by pretreatment of WEHI-3 cells with rmC5-3. An enzyme-linked immunosorbent assay showed that rmC5-3 detected peptide 9 corresponding to amino acid position 308–322 of CD14 but 2.4G2 did not. A Western blot analysis of sera revealed that rmC5-3 and 2.4G2 detected the bands thought to be soluble CD14 and CD32/16, respectively. rmC5-3 reacted with mouse CD14-transfected CHO cells, CD14-CHO-K1 cells, but 2.4G2 did not. Lipopolysaccharide-induced tumor necrosis factor (TNF)-α release was enhanced when a monocyte cell line (J774) was pretreated with rmC5-3. The enhancement was abolished by pretreatment with 2.4G2. The release of TNF-α was observed following treatment of J774 cells with 2.4G2 followed by anti-rat IgG F(ab′)2.