Functional activation of immune lymphocytes by antigenic stimulation in cell-mediated immunity. III. A soluble factor released from LPS-stimulated peritoneal adherent cells effective in antigenic activation of sensitized lymphocytes.

Antigen-induced production of migration inhibitory factor (MIF) by sensitized lymphocytes requires macrophages to effectively stimulate lymphocytes with soluble antigen in vitro. The present study showed that macrophage-depleted lymphocytes of sensitized guinea pigs could be activated with antigens when the culture supernatant of peritoneal adherent cells pulse-stimulated with a macromolecular fraction of bacterial lipopolysaccharide (LPS) was added to the lymphocyte culture. The apparent macrophage-replacing activity was found in the fraction which emerged slightly ahead of serum albumin upon gel filtration of the culture supernatant, and the activity was shown to be destroyed by heating at 65 °C for 30 min or by trypsin digestion. These results appeared to show that the activity was due to a protein component, most probably released from macrophages. Two-step culture experiments revealed that the soluble factor should be present in the early stage of the culture to activate the macrophage-depleted immune lymphocytes with antigen, as well as in the later stage when the presence of antigen in the medium is no longer required. Furthermore, the factor was shown to act in the activation of a T-cell-enriched fraction of immune lymphocytes. The factor appeared to be playing some essential role in making an antigenic stimulus effective for the activation of immune lymphocytes.     

Dissection of the role of macrophages in triggering T lymphocytes for interleukin 2 production by monoclonal antibody OKT3

Unfractionated human peripheral blood mononuclear cells produce a small amount of interleukin 2 (IL 2) by stimulation with a monoclonal anti-T3 antibody (OKT3) in vitro. The IL 2 production could be greatly augmented by the addition of a phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). In the presence of TPA, the T cell enriched fraction deprived of macrophages did not produce IL 2, but the T cells pulse-incubated with OKT3 and reconstituted with macrophages efficiently produced IL 2 in subsequent culture in the presence of TPA as did T cells reconstituted with OKT3-pulse-incubated macrophages. The stimulating effect of OKT3 in the presence of macrophages was inhibited dose-dependently by the addition of immunoglobulins, particularly by mouse IgG2a which is the same isotype as that of the OKT3 antibody, showing that it inhibits by blocking the binding of OKT3 to Fc receptors on macrophages. The same extent of IL 2 production was induced in T cells when paraformaldehyde-fixed macrophages were substituted for intact macrophages. Remarkable IL 2 production was also induced by OKT3 when latex beads coated with rabbit anti-mouse IgG2a antibody and TPA were added to the culture. It was confirmed that the production induced by these stimulations was due to an increase of IL 2 mRNA. These results show that effective signals for IL 2 production are generated by efficient crosslinking of T3 molecules which results from multi-interaction of T3 molecules on the T cell membrane and anti-T3 antibody molecules on macrophage membrane or on the surface of the latex particle.     

Photosynthetic Characteristics of Photoautotrophically Cultured Cells of Tobacco

The photosynthetic characteristics of photoautotrophically culturedcells of tobacco (Nicotiana tabacum cv. Samsun NN) as well asthose of photomixotrophically cultured cells and green leaveswere investigated. Analyses revealed that on a fresh weightbasis cultured tobacco cells had lower chlorophyll contentsthan cells of green leaves. The chlorophyll content per chloro-plast,however, was almost the same in both types of cell, and thechloroplast number per cell accounted for only small differencesin the cellular chlorophyll content. This indicates that thelarger cell volume of cultured cells is the main factor in thedifference in the chlorophyll content of these cells. Photosynthetic activity measurements also showed differencesin the chloroplasts of cultured and leaf cells. The maximumactivities of photosystem I and the Hill reaction for the culturedcells were about half those for leaf cells on a per unit chlorophyllbasis. Moreover, photo-autotrophic cells had relatively constantphotosystem I and Hill reaction activities during growth; whereas,on a fresh weight basis these activities in leaf cells reflecteddevelopmental changes in the chlorophyll content. Lithium dodecyl sulfate-polyacrylamide gel electrophoresis showedqualitatively similar thylakoid polypeptide compositions forcultured and leaf cells at all stages of growth even thoughthere were quantitative decreases in the contents of severalpolypeptides in the cultured green cells (especially in photomixotrophiccells) in comparison to the polypeptide contents of tobaccoleaves. We speculate that the lower photosynthetic activityof the cultured cells may be caused by this reduction in thecontents of certain thylakoid polypeptides. (Received November 14, 1988; Accepted June 19, 1989)     

Studies of Sl/Sld in equilibrium with +/+ mouse aggregation chimaeras. II. Effect of the steel locus on spermatogenesis

Mutant mice of Sl/Sld genotype are deficient in melanocytes, erythrocytes, mast cells and germ cells. Deficiency of melanocytes, erythrocytes and mast cells is not attributable to an intrinsic defect in their precursor cells but to a defect in the tissue environment that is necessary for migration, proliferation and/or differentiation. We investigated the mechanism of germ cell deficiency in male Sl/Sld mice by producing aggregation chimaeras from Sl/Sld and +/+ embryos. Chimaeric mice with apparent white stripes were obtained. Two of four such chimaeras were fertile and the phenotypes of resulting progenies showed that some Sl/Sld germ cells had differentiated into functioning sperms in the testis of the chimaeras. In cross sections of the testes of chimaeras, both differentiated and nondifferentiated tubules were observed. However, the proportions of type A spermatogonia to Sertoli cells in both types of tubules were comparable to the values observed in differentiated tubules of normal +/+ mice. We reconstructed the whole length of four tubules from serial sections. Differentiated and nondifferentiated segments alternated in a single tubule. The shortest differentiated segment contained about 180 Sertoli cells and the shortest nondifferentiated segment about 150 Sertoli cells. These results suggest that Sertoli cells of either Sl/Sld or +/+ genotype make discrete patches and that differentiation of type A spermatogonia does not occur in patches of Sl/Sld Sertoli cells.     

Contra-IL 2; a suppressor lymphokine that inhibits IL 2 activity

Suppressive activity of culture supernatant of AS-9 (AS-9 CS), a T cell hybridoma line that was derived from fusion of BW5147 thymoma and splenic T cells of anti-lymphocyte serum-treated C3H mice, was analyzed. AS-9 CS inhibited allogeneic cytotoxic T lymphocyte (CTL) generation as well as T cell proliferation to alloantigens and mitogens, but failed to inhibit B cell response to lipopolysaccharide or growth of tumor and fibroblast cells. Although addition of AS-9 CS to the allogeneic sensitization culture as late as on day 2 of incubation resulted in maximal inhibiton of CTL generation, removal of AS-9 CS on day 3 of incubation abolished its inhibitory effect. Addition of purified IL 2 together with AS-9 CS to the allogeneic sensitization cultures only partially abrogated the suppression. Experiments with IL 2-dependent cytotoxic T cell line (CTLL) showed that AS-9 CS suppressed the IL 2-induced proliferation of CTLL. Preincubation of AS-9 CS with CTLL removed its inhibitory effect on CTL generation. These results indicate that AS-9 CS interferes with the mechanism of T cell activation by IL 2. On this basis, AS-9 CS was named contra-IL 2.     

Antitumor activities and tumor necrosis factor producibility of traditional Chinese medicines and crude drugs

Summary The antitumor activities and capacity for tumor necrosis factor (TNF) production of traditional Chinese herbal preparations (Zhu-ling-tang, Xiao-chai-hu-tang), crude drugs (Polyporus, Hoelen, Bupleuri radix, Angelica radix, Cnidii rhizoma, Cinnamomum cortex), and Krestin (PSK) were investigated. These drugs were given to DDY mice in the drinking water before and after transplantation of Ehrlich tumors, and the development of the intradermally transplanted Ehrlich tumors and survival rate were observed. A good survival rate and sometimes a complete cure were found in the groups administered Bupleuri radix, Xiao-chai-hu-tang, Angelica radix, or Cinnamomum cortex, while the group given Hoelen showed poor results. To examine the capacity for TNF production these drugs were given to DDY mice PO as initial stimulating agents, to stimulate the reticuloendothelial system (RES) prior to lipopolysaccharide injection. The TNF activity was tested from the cytotoxicity against L cells. Significant differences in capacity for TNF production were observed among the drugs. Relatively high levels of TNF activity were noted in the groups given Angelica radix, Bupleuri radix, Cnidii rhizoma, or Cinnamomum cortex, very low activities in the groups given Xiao-chai-hu-tang, Zhu-ling-tang, or Krestin, and no TNF activities in the groups given Polyporus or Hoelen. The TNF capacity for production broadly paralleled the survival rate of the mice transplanted to Ehrlich tumors. Our findings suggest that one mechanism underlying the antitumor activities of these drugs is based on stimulation of the RES and is closely related of TNF production.This work was supported in part by a grant-in-aid from the Ministry of Education, Japan     

Structural studies of Fc receptors. V. Effect of phospholipase C treatment on the binding activities of the Fc receptor of macrophage or its isolated plasma membrane

The effect of phospholipase C treatment on the binding activity of the Fc receptor of guinea pig macrophage was studied to analyze the interaction of the Fc receptor with membrane phospholipids necessary for the activity. It was confirmed by subcellular fractionation that the receptor is localized on the plasma membrane. Treatment of the whole cell or isolated plasma membrane with phospholipase C of Clostridium perfringens diminished the binding of soluble IgG2-immune complex to Fc receptors on the cell or membrane. On the other hand, phospholipase C of Bacillus cereus did not affect the activity when it acted on the whole cell but it did diminish the activity when it acted on the isolated plasma membrane. Analysis of the phospholipids of untreated and treated macrophages or plasma membrane showed that phosphatidylcholine molecules, particularly those located in the membrane (not accessible to attack from the cell surface by phospholipase C of B. cereus), appear to be crucial for efficient interaction of macrophage Fc receptors with immune complex. Ligand-binding experiments with macrophages showed that the diminished binding activity was due to a decrease of the avidity for immune complex, but did not seem to be due to a decrease in the number or affinity of Fc receptors for monomeric IgG2. Taken together with the previous results which demonstrated that Fc receptors which had apparently lost the activity due to delipidation could be reconstituted with phosphatidylcholine but not with most other phospholipids, the results seem to indicate that the diminution of the binding activity to the immune complex of macrophage or its plasma membrane caused by phospholipase C treatment is due to the impairment of multivalent interaction between Fc receptor molecules on the membrane and IgG2 molecules in the immune complex, probably as a result of the loss of interaction of the head groups of phospholipids with Fc receptor molecules and the change in membrane properties resulting from the increase of diglycerides.     

Phorbol ester increases the level of interleukin 2 mRNA in mitogen-stimulated human lymphocytes

TPA alone did not induce the production of IL 2 in human tonsillar lymphocytes but enhanced the PHA-induced IL 2 production by seven-fold. That the effect of TPA was due to an increase in IL 2 mRNA was demonstrated by examining the amount of IL 2 mRNA translatable in Xenopus laevis oocytes, and by Northern blotting analysis using IL 2 cDNA as a probe. In these ways, it was shown that TPA alone did not induce any significant IL 2 mRNA synthesis, but when added together with PHA it increased the level of IL 2 mRNA by at least 10-fold, as compared with that induced by PHA alone.