Transfer Agent of Immunity

An immune ribonucleic acid (RNA) preparation was extracted with phenol from the spleens of guinea pigs immunized with diphtheria toxoid. Antibody-carrying cells were detected by immunocyte adhesion as rosette-forming cells. When germ-free rats, conventional guinea pigs or mice were injected intraperitoneally with this preparation, the rosette-formers were detected in either peritoneal exudate cells or spleen cells, whereas serum antibodies were unable to be detected thus far in such animals. Two injections with this preparation did not cause any remarkable increase in the number of rosette-formers, and serum antibody was also not detectable. By contrast, a high titer of serum antibody was demonstrated and the number of rosette-formers increased shortly after an injection of a small amount of diphtheria toxoid into guinea pigs which had previously received an injection with immune RNA. This reaction indicates a secondary response of antibody formation. However, secondary responses were not induced by injections of immune RNA preparations in guinea pigs primed with either diphtheria toxoid or immune RNA preparation. These facts suggest that immune RNA preparations did not contain antigens or fragments thereof and the immune response induced by RNA preparation is not the same as that induced by stimulation by the antigen itself. These results moreover can be accounted for by the notion that the immune RNA preparation is able to induce “memory” cells capable of responding to a secondary stimulus with an antigen and producing a high titer of serum antibody.     

Purine metabolic enzymes in lymphocytes. II. Adenosine deaminase and purine nucleoside phosphorylase activities in the immune response

ICR mice were immunized with sheep red blood cells (sRBC). Both adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in spleen lymphocytes increased faster than the serum antibody titer and reached a peak one week after the immunization. ADA activity increased significantly in T lymphocytes but not in B lymphocytes collected from the spleens of the immunized mice. A statistically significant increase in PNP activity was found in both T and B lymphocytes from the spleens of the immunized mice. Spleen lymphocytes collected from ICR mice which had been immunized with mitomycin C-treated sarcoma 180 (S180) cells one week earlier showed cytotoxic activity against viable S180 cells. Both ADA and PNP activities in spleen lymphocytes of S180-immunized mice increased significantly, and both activities increased in T lymphocytes prepared from spleen of immunized mice. In contrast, an increase was found in PNP activity but not in ADA activity in B lymphocytes. These results suggest that an increase in both ADA and PNP activities may by necessary for the T-cell response in both humoral and cellular immune responses, and that an increase in PNP activity may be necessary for the B-cell response.     

Inhibitory effect of interferon-gamma-dependent induction of major histocompatibility complex class II antigen by expressing exogenous poly(ADP-ribose) synthetase gene

Ia antigen, one of class II major histocompatibility complexes, was induced by treatment of mouse macrophage P388D1 cells with interferon-gamma. During the process, expression of poly(ADP-ribose) synthetase gene was depressed. We examined whether or not down-regulation of the synthetase was required for the interferon-gamma-dependent induction of Ia antigen. We constructed expression plasmid harboring metallothionein promoter-regulated synthetase gene. The enzyme activity and mRNA level of the synthetase were increased in exogenous synthetase gene-transfected clones. Induction of Ia antigen gene was strongly inhibited by expressing the exogenous synthetase gene upon treatment of the transfected clones with interferon-gamma. The result suggests that interferon-gamma-dependent down-regulation of the synthetase may be involved in a step of the signal transduction to induce Ia antigen.     

Fluctuation of gene expression for poly(ADP-ribose) synthetase during hemin-induced erythroid differentiation of human leukemia K562 cells and its reversion process

We have studied the regulation of gene expression for poly(ADP-ribose) synthetase during erythroid differentiation and its reversion process. When human leukemia K562 cells were incubated in the presence of 80 microM hemin, benzidine-positive cells appeared at day 2 and 90% of the cells became positive at day 6. However, RNA blot analysis reveals that mRNA for gamma-globin was already abundant in untreated K562 cells and the level of the message was slightly increased by hemin-treatment. Spectroscopic analysis and polyacrylamide gel electrophoresis of the induced cell extracts indicate that hemoglobin molecules were not detected in untreated cells, and increased successively up to day 6. The hemin-induced cells were thoroughly washed, and then recultured in the absence of hemin. The benzidine-positive cells mostly disappeared 3 days after the elimination of the inducer. During the hemin-induced erythroid differentiation, the activity and mRNA for poly(ADP-ribose) synthetase decreased to 50% and 20% of the initial level at day 3 and a low level of the gene expression was maintained afterwards, whereas the activity and mRNA returned to the initial value 1 day after hemin elimination. The results indicate that the hemin-induced erythroid differentiation of K562 cells is a reversible process and depression of the synthetase may be involved in the progress of differentiation.     

A chemiluminescent probe with a Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one, specific and sensitive for O2- production in phagocytizing macrophages

When a Cypridina luciferin analog (the title compound) was added to a macrophage suspension in Hank's balanced salt solution (control), the system emitted a weak, but detectable light, which was not altered in the presence of superoxide dismutase. The same system, however, emitted a much stronger light, just after the addition of a trigger, opsonized zymosan. The luminescence was suppressed to the control level in the presence of superoxide dismutase, while it was only slightly influenced, if at all, by NaN3, a scavenger of singlet oxygen and an inhibitor of myeloperoxidase. Some other results obtained also indicate the participation of O2- in the luciferin analog-dependent luminescence in macrophages during phagocytosis.     

Experimental Salmonellosis XI. Induction of Cellular Immunity and Formation of Antibody by Transfer Agent of Mouse Mononuclear Phagocytes

When mice were injected intraperitoneally with a ribonucleic acid (RNA) preparation extracted from the peritoneal mononuclear phagocytes (termed monocytes) of immunized mice, these macrophages developed cellular immunity and cellular antibodies. The peritoneal monocytes were obtained from normal mice and maintained in tissue culture bottles in a homogeneous cell population. When they were treated in vitro with an immune RNA preparation, they acquired cellular immunity, and cellular antibodies were detectable in such monocytes. These results suggest that the mononuclear phagocytic cell line constitutes a cell line responsible for antibody formation.     

Secoisolariciresinol and isotaxiresinol inhibit tumor necrosis factor-alpha-dependent hepatic apoptosis in mice

The effects of secoisolariciresinol (1) and isotaxiresinol (2), two major lignans isolated from the wood of Taxus yunnanensis, on tumor necrosis factor-alpha (TNF-alpha)-dependent hepatic apoptosis induced by D-galactosamine (d-GalN)/lipopolysaccharide (LPS) were investigated in mice. Co-administration of d-GalN (700 mg/kg) and LPS (10 microg/kg) resulted in a typical hepatic apoptosis characterized by DNA fragmentation and the formation of apoptotic bodies. Serum glutamic pyruvic transaminase (sGPT) and glutamic oxaloacetic transaminase (sGOT) levels were also raised at 8 h after d-GalN/LPS intoxication due to a severe necrosis of hepatocytes. Pre-administration of 1 or 2 (50, 10 mg/kg, i.p.) 12 and 1 h before d-GalN/LPS significantly reduced DNA fragmentation and prevented chromatin condensation, apoptotic body formation and hepatitis. Pro-inflammatory cytokines such as TNF-alpha and interferon-gamma (IFN-gamma) secreted from LPS-activated macrophages are important mediators of hepatocyte apoptosis in this model. Pre-treatment with 1 or 2 significantly inhibited the elevation of serum TNF-alpha and IFN-gamma levels. In a separate experiment, both lignans had a significant dose-dependent protective effect on d-GalN/TNF-alpha-induced cell death in primary cultured mouse hepatocytes and TNF-alpha-mediated cell death in murine L929 fibrosarcoma cells. These results indicated that 1 and 2 prevent d-GalN/LPS-induced hepatic injury by inhibiting hepatocyte apoptosis through the blocking of TNF-alpha and IFN-gamma production by activated macrophages and direct inhibition of the apoptosis induced by TNF-alpha.     

Immunological Properties of TtT/M-87 Cell Line Established from Murine Pituitary Tumor-Associated Macrophages

TtT/M-87 cell is a macrophage cell line established from thyrotropic pituitary tumor tissues in mouse. In this paper, we report the immunological properties of M-87 cells as a model of tumor-associated macrophage. Contrasting with resident peritoneal macrophages, M-87 cells constitutively secreted small but significant amounts of TNF-α and IL-1α, which were detectable in both biological assays (cytotoxic activity for L929 and co-mitogenic activity for Con A-induced T cell proliferation, respectively) and ELISA, and produced larger amounts of these cytokines upon stimulation with LPS. They expressed MHC class II molecules on their cell surface without stimulation by IFN-γ. The accessory or antigen-presenting cell activity in antibody-producing response of spleen lymphocytes to sheep red blood cells was shown to be much higher in M-87 cells than normal peritoneal macrophages. In addition, when normal spleen lymphocytes were cultured with allogeneic tumor cells, such as EL-4 and S-180, in the presence of M-87 cells, lymphocytes reactive to stimulator cells were activated to manifest inhibitory effect on the tumor cell growth and also to manifest specific cytotoxic effect on the allogeneic tumor cells. These results show that M-87 cells derived from tumor-associated tissue are activated macrophages and that they are inhibitory to tumor cell growth and augmentative in the induction of T-cell-mediated immune responses.