Biomedical subjects

T Marunouchi

Publications and source records attributed to T Marunouchi.

At least 19 recordsLinked to original sources

PubMed1992

Down regulation of CD4 expression in cultured microglia by immunosuppressants and lipopolysaccharide.

Microglia, brain macrophages, are thought to be the primary target of HIV-1 infection in the brain, because they exclusively express the CD4 antigen which is effectively used for viral entry. The expression of CD4 mRNA in cultured microglia could be detected by the reverse-PCR method. Using this and immunohistochemical staining, we found that the immunosuppressants cyclosporin A and FK506 decreased CD4 expression in cultured murine microglia without causing any significant decrease in cell viability. FK506 was more potent than cyclosporin A. Lipopolysaccharide also decreased CD4 mRNA expression in microglia. The effects of immunosuppressants and lipopolysaccharide seemed to be specific for microglia since these chemicals did not alter the CD4 expression in lymphocytes or peritoneal macrophages. These agents, if modified to pass through the blood-brain barrier, may prevent viral spread of HIV-1 infection in the central nervous system and the AIDS-dementia complex.

Animals↗

PubMed1992

Induction of cytokines in glial cells by trans activator of human T-cell lymphotropic virus type I.

Human T-cell lymphotropic virus type I (HTLV-I), the cause of adult T-cell leukemia, is also associated with the neurological disease, TSP/HAM (tropical spastic paraparesis/HTLV-I associated myelopathy). The HTLV-I genome encodes a protein, Tax, that trans activates viral and cellular gene transcription. To understand the mechanisms for the production of cytokines by HTLV-I in nervous tissue, we examined their expression in glial cells which carried the Tax-expressing vector. We demonstrated that Tax expression enhanced the production by glial cells of interleukin (IL)-1, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor (TGF) beta. We suggest that the excessive production of cytokines in nervous tissue may play a role in pathogenesis of TSP/HAM. Glial cells that carry the tax gene may provide a model useful for in vitro study of the mechanism of production of cytokines in the nervous system.

Animals↗

PubMed1992

TNF alpha induces IL-6 production by astrocytes but not by microglia.

Astrocytes and microglia, both produced interleukin-6 (IL-6) in culture by lipopolysaccharide (LPS) stimulation. IL-6 activity was detected 3-5 h after LPS stimulation and reached a maximum at 10 h. Microglia responded faster than astrocytes. Tumor necrosis factor alpha and interleukin 1 also induced IL-6 mRNA and biological activity in astrocytes, but not in microglia. Among these stimuli, LPS was the most potent inducer of IL-6 production by astrocytes. Our results suggest that different regulatory mechanisms for cytokine production exist in glial cells. The possible roles of astrocytes and microglia in CNS immune responses are also discussed.

Animals↗

PubMed1992

Interleukin-1 up-regulates transcription of its own receptor in a human fibroblast cell line TIG-1: role of endogenous PGE2 and cAMP.

The regulation of interleukin-1 receptor (IL-1R) mRNA expression by IL-1 in a human lung fibroblast cell line (TIG-1) was investigated. After 2 h of stimulation with human recombinant IL-1 alpha or IL-1 beta, the levels of T cell/fibroblast-type IL-1R mRNA increased, and the elevation was sustained for at least 72 h. IL-1 also stimulated synthesis of prostaglandin E2 (PGE2) and secondary cAMP accumulation. Exogenously added PGE2 increased the levels of both IL-1R mRNA and intracellular cAMP. Forskolin, cholera toxin and 8-Bromo adenosine (8-Br-cAMP) all increased IL-1R mRNA levels. Indomethacin blocked IL-1 stimulation of IL-1R mRNA expression, PGE2 production and cAMP. 125I-labeled IL-1 alpha-binding studies showed that this cell line expresses 2.6 x 10(4) IL-1R per cell with a Kd of 5.1 x 10(-10) M. After treatment of the cells with IL-1, the level of IL-1R increased over that of control cells. PGE2 also increased IL-1R without alteration in its affinity. Cross-linking experiments indicate that this cell line expresses the 80-kDa receptor molecule before and after treatment with PGE2; the molecular mass corresponds to the T cell/fibroblast type I IL-1R. These results indicate that IL-1 does not directly stimulate expression of IL-1R mRNA or cell surface IL-1R, but only indirectly by stimulation of endogenous PGE2.

Cell Line↗

PubMed1992

Specific distribution of an epididymal 50 kDa protein revealed by an anti-Mos protein monoclonal antibody.

An anti-Mos protein monoclonal antibody, 4A6, was used to investigate the distribution of the antigen in the epididymis, in which the c-mos gene is reportedly expressed. The 4A6-reactive antigen was found on the basement membrane and luminal surface of the epithelial cells in the caput epididymis of BALB/c male mice as well as in the proximal corpus epididymis, the cauda epididymis, and the vas deferens. The 4A6 antigen was also found on the luminal surface of the epithelial cells in the epididymis of male germ cell-deficient C57BL/6J-Wv/Wv mice. This confirmed that the 4A6 antigen does not derive entirely from the testicular c-Mos protein but is synthesized in the epididymis. Western blot analysis revealed that the molecular weight of the epididymal 4A6 antigen was 50 kDa, which is unusually high for the c-Mos protein. With its specific distribution in the epididymis, the protein should play a specific role in functions of the epididymis.

Animals↗

PubMed1992

Differentiation and dedifferentiation of the human monocytic leukemia cell line, U937.

U937 cells were differentiated into macrophages after being treated with 12-o-tetradecanoyl-phorbol-13-acetate (TPA) for the first two days and dedifferentiated with daily medium renewal for 10 days. Cell proliferation slowed down and the number of cells reached the maximum level on day 2. By day 4, all of the cells had spread and attached firmly to the culture dish, and more than 90% of the cells expressed the Fc-receptor and produced superoxide anion. From there on, the number of adherent, living cells decreased gradually to about half the initial count. Most of the cells eliminated from the culture by cell death were in the S phase at the time of TPA treatment. After day 8, the number of cells expressing macrophage-specific phenotypes gradually decreased, cell adhesion was weakened, and at the same time, DNA synthesis was initiated anew. The cells became round and began to proliferate as floating cells on days 9 to 10, and thereafter they became sensitive to the second round of TPA treatment. On the basis of all the results taken together, it is suggested that fully differentiated U937 cells were dedifferentiated after being cultured with frequent medium renewal.

Autoradiography↗

PubMed1992

Establishment of mouse oligodendrocyte/type-2 astrocyte lineage cell line by transfection with origin-defective simian virus 40 DNA.

A permanent glial cell line has been established from the neonatal mouse primary mixed glial cell cultures by transfection with replication origin-defective simian virus 40 DNA. This cell line, designated OS3, has morphological similarity to type-2 astrocyte and expresses an astrocyte-specific marker, glial fibrillary acidic protein (GFAP), when cultured in the presence of 10% calf serum (CS). OS3 cells do not express the O4 antigen, galactocerebroside (GalC) and A2B5 under this culture condition. When cultured in a medium containing 2% CS or a chemically defined medium, these cells undergo morphological transformation. Some of these cells express O4 antigen and/or GalC, and the percentage of GFAP positive cells decreases under these conditions. Thus depending on the culture conditions, the OS3 cells display either type-2 astrocyte properties or immature oligodendrocyte characteristics. Furthermore, the OS3 cells show similar responses to the various growth factors as do oligodendrocyte/type-2 astrocyte (O-2A) progenitors. Therefore, the OS3 cell line is an unique mouse bipotential permanent O-2A lineage cell line which may be useful to analyze the developmental properties of these glial cells.

Animals↗

PubMed1991

Morphological transformation of microglia in vitro.

Isolated microglia underwent in vitro morphological transformation to ameboid, rod-shaped and ramified forms in response to several cytokines, factors from astrocytes, or factors which affect macrophage function. These morphological transformations were reversible. Examination of the enzymatic activity, superoxide anion formation and proliferative activity of these transformed microglia indicated that the ameboid forms were activated, rod-shaped microglia were proliferating, and the ramified forms were resting microglia.

Animals↗

PubMed1991

Production and characterization of a monoclonal antibody to the v-mos oncogene protein.

Valuable information about proto-oncogenes and their physiological functions has been obtained by studying their expression in normal cells. However, the protein product of the c-mos gene, the cellular homologue of the transforming gene (v-mos) of Moloney murine sarcoma virus, has not been detected in normal mouse cells or tissues. Here, we have constructed a v-mos expression vector, pRI-delta mos, which directs the synthesis of a truncated v-mos gene product, a protein A fusion protein. Using the truncated v-mos oncoprotein produced in Escherichia coli as immunogen, we prepared anti-v-mos monoclonal antibodies (MAbs). In immunoblotting assays, the MAb was reactive with v-mos oncoprotein and detected bands at 43 KDa or 39 kDa in the tissue extract of mouse testes or ovaries, respectively, in which the c-mos protooncogene mRNA is expressed. These results demonstrate that the v-mos MAb obtained is suitable for elucidating the physiological functions of v-mos gene product and may also be utilized to detect c-mos gene product at the cellular level.

Animals↗

PubMed1991

Strain difference in expression of the adult-type polycystic kidney disease gene, pcy, in the mouse.

DBA/2FG-pcy and C57BL/6FG-pcy congenic strains were established by transferring the polycystic kidney disease gene, pcy, to DBA/2 and C57BL/6 mice. We carried out pathological and hematological examinations of these strains at 4, 8, 16 and 30 weeks of age. In DBA/2FG-pcy mice more than 8 weeks of age, macroscopic renal cysts were observed on the surface of both kidneys. Their kidneys weight was significantly greater than in DBA/2 mice at all ages examined. Microscopic renal cysts were evenly distributed at 4, 8 and 16 weeks of age. At 30 weeks of age, the kidneys were filled with numerous polycysts. In C57BL/6FG-pcy mice, no macroscopic renal cysts were found until the animals were 30 weeks old, and the weight of their kidneys was greater than in B6 mice of the same age. From 8 weeks of age on, a limited number of microscopic renal cysts was observed, and many renal cysts were found adjacent to the enlarged Bowman's capsules. With age, the red blood cell count and hematocrit level decreased while the platelet count increased in both strains, with greater changes occurring in DBA/2FG-pcy mice than in C57 BL/6FG-pcy mice. These findings demonstrate that polycystic kidney disease exhibits strain differences in animals with a DBA/2 and C57BL/6 background. Our results suggest that phenotypic expression of the pcy gene in the mouse depends on genetic background, and that variations in the severity of human polycystic kidney disease may be explained, at least in part, by individual differences in genetic background.

Animals↗

PubMed1991

[Functional and immunohistochemical studies of cultured rat microglia].

We studied functional and immunohistochemical characteristics of cultured rat microglia. Unstimulated microglia did not proliferate. Microglia stimulated with LCM (L929 conditioned medium: colony stimulating factor-1) had proliferative activity and increased acid phosphatase activity. LPS (lipopolysaccharide) and IFN gamma (interferon-gamma) but did not affect proliferative activity. Immunohistochemically, RCA-1 lectin and GS-1 lectin, which react to beta-D-galactose and alpha-D-galactose respectively, strongly reacted to the cytoplasm and membrane of unstimulated microglia. After stimulation with LCM, microglia elongated processes and decreased response to these lectins. On the other hand, microglia stimulated with LCM showed increased reactivity to monoclonal antibody of vimentin. Microglia stimulated with LPS had round shape and had response to these lectins and vimentin. Microglia stimulated with IFN gamma had adhesive activity and weakly stained with these lectins but not with vimentin. ED-1 (monoclonal antibody of rat monocytes/macrophages) reacted to unstimulated and stimulated microglia. In flow cytometry, unstimulated microglia expressed OX-18 (MHC class I) and W3/25 (CD4) antigen. After stimulation with IFN gamma, microglia were induced to express these antigens. CD4 antigen is a marker of helper/inducer T cells and thought to be a receptor of HIV. The results that microglia had CD4 antigen which was further induced with IFN gamma are important to investigate infection of the CNS with HIV. OX-6 (Ia) antigen was induced with IFN gamma. This indicates that the microglia plays a central role in the CNS immune reaction. These characteristics of cultured rat microglia provide useful informations to investigate the pathogenesis of the CNS disorders.

Animals↗

PubMed1990

Production of granulocyte/macrophage colony-stimulating factor by cultured astrocytes.

We investigated the production of interleukin-3 (IL-3)-like factor by murine astrocytes. Supernatants from lipopolysaccharide (LPS)-stimulated astrocytes induced proliferation of IC-2, an IL-3- and granulocyte/macrophage colony-stimulating factor (GM-CSF)-dependent cell line. This activity was completely neutralized by the antibody against GM-CSF but not by the anti-IL-3 monoclonal antibody. Northern blot analysis revealed the expression of GM-CSF mRNA, but not of IL-3 mRNA, in cultured astrocytes. These results indicate that with proper stimuli murine astrocytes produce GM-CSF.

Animals↗

PubMed1990

Activation and proliferation of the isolated microglia by colony stimulating factor-1 and possible involvement of protein kinase C.

Microglia were isolated from primary mixed brain cell culture of normal newborn mice and then cultivated. They were able to be maintained in vitro for 1-2 months, but incorporated little [3H]thymidine under normal culture conditions. When treated with the conditioned medium of L929 mouse fibroblast cells as a crude CSF-1 (mouse macrophage-colony stimulating factor) or purified CSF-1, microglia showed morphological changes and increased in both cell number and [3H]thymidine uptake. In addition, crude CSF-1 increased lysosomal enzyme activity and superoxide anion formation of microglia up to 2 and 3.8 fold as control value, respectively. These effects of CSF-1 were not observed in the purified astrocyte culture. Purified microglia had CSF-1 receptors which were recognized by the anti-CSF-1 receptor antibody that arose from a peptide of a product of proto-oncogene, c-fms. 12-O-Tetradecanoylphorbol-13-acetate (TPA) also increased microglia cell number and their biochemical activities, suggesting the possible involvement of protein kinase C activation. Protein kinase inhibitors, such as staurosporine or H-7, inhibited the effects of both CSF-1 and TPA. These results indicate that microglia may be regulated in its biochemical and proliferation activities by CSF-1 and that this may occur via activation of protein kinase C.

Animals↗

PubMed1990

Effects of colony stimulating factors on isolated microglia in vitro.

Effects of colony stimulating factors (CSF), known regulators for cells in monocytic lineage, on isolated microglia were examined. Interleukin-3 (IL-3) induced only morphological changes in rod-shaped microglia, while granulocyte-macrophage CSF (GM-CSF) and CSF-1 induced both morphological changes and proliferation of microglia. CSF-1 also activated the enzyme activity of microglia. These observations indicated that, in terms of regulation by cytokines, microglia are similar to mature cells in monocytic lineage. Although astrocytes reportedly produce IL-3 and GM-CSF, the effects of astrocyte-conditioned medium (Ast-Sup) were different from those of either IL-3 or GM-CSF. Ast-Sup caused ameboid microglia to become ramified, and did not induce proliferation of microglia. Factors from astrocytes may have a role in the transformation of microglia which occurs in either normal developing brain or inflammation in the brain.

Animals↗

PubMed1990

Expression of protein kinase C isozyme in epidermal Langerhans cells of the mouse.

Protein kinase C (PKC) is encoded by a complex of a gene family, and its multiple isozymes are expressed in various mammalian tissues. We examined whether PKC is expressed in epidermal Langerhans cells (LC) of the mouse by using monoclonal antibodies specific to PKC I, PKC II, and PKC III isozymes (respective products of PKC genes gamma, beta, and alpha). Immunohistochemical and immunoblotting studies revealed that LC of adult C57BL/6 mice express PKC II, while PKC I and PKC III were not detected. In keratinocytes, none of the isozymes were detected. These results suggest that PKC II is a novel marker of LC and that it possibly plays a regulatory role in epidermal LC of the mouse in vivo.

Animals↗

PubMed1990

Isolation and culture of panning method-enriched Langerhans cells from dispase-dissociated epidermal cells of the mouse.

Langerhans cells (LCs) are bone marrow-derived, Ia-positive antigen-presenting cells in the epidermis which constitute 2-4% of the total epidermal cells. We examined the usefulness of a combination of dispase treatment and the panning method for enriching and culturing mouse LCs. Trunk skin was treated with partially purified dispase (Godo Shusei, type II) to separate epidermal sheets and to dissociate epidermal cells. Suspended cells were treated with ascites or culture supernatant containing anti-Ia monoclonal antibody, and LCs were enriched by the Ia-mediated panning method. Per mouse, 3-4 X 10(5) LCs were recovered with greater than 95% purity and greater than 90% viability. Enriched LCs potently stimulated the allogeneic mixed-leukocyte reaction. Ultrastructural observations revealed that enriched LCs contained many vesicles but almost no Birbeck granules. A laminal structure, which was apparently adhesive to the surface of LCs, was observed when ascites were employed as the anti-Ia antibody. These results indicate that a combination of dispase treatment and the Ia-mediated panning method is very useful for isolating high yields of functionally mature murine Langerhans cells with high purity and viability.

Animals↗

PubMed1990

Human T-cell leukemia virus type I trans activator induces class I major histocompatibility complex antigen expression in glial cells.

Transfection of the tax gene encoding the trans activator of human T-cell leukemia virus type I into glial line cells induced class I major histocompatibility complex (MHC) antigens on these cells. This occurred through the interaction of tax protein with the gene encoding class I MHC antigens but not through any soluble factors, such as interferons, or factors from glial cells. Since neural cells do not usually express MHC antigens, this novel mechanism may be an intermediate event between viral infection and subsequent immune-mediated pathology in the central nervous system.

Animals↗

PubMed1990

Expression of protein kinase C isozyme in human Langerhans' cells.

Protein kinase C is a key molecule controlling signal transduction into the cell. We recently reported that protein kinase C II isoenzyme, but neither I nor III isozyme, was expressed in epidermal Langerhans' cells of the adult mouse, and that none of these isozymes was detected in keratinocytes. In this study, we examined the expression of protein kinase C isozymes in human Langerhans' cells in vivo to see whether the expression of protein kinase C II isozyme in Langerhans' cells is a mouse-specific trait. Immunohistochemical studies revealed that protein kinase C II isozyme, but neither I nor III isozyme, was expressed in epidermal Langerhans' cells. None of these isozymes was detected in keratinocytes. These results suggest that the expression of protein kinase C II isozyme in epidermal Langerhans' cells in vivo is not a mouse-specific trait and that protein kinase C II isozyme is a novel phenotypic marker for epidermal Langerhans' cells in human as well as mouse skin.

Adolescent↗