Ethanol inhibition of the chemiluminescent response of stimulated macrophages.

The effect of ethanol on the luminol-dependent chemiluminescence and cAMP level in mice peritoneal macrophages was investigated. Ethanol was shown to inhibit the chemiluminescence of macrophages by acting both as a "trap" for active radicals and as a suppressor of the cellular functional activity. A short preincubation of macrophages with ethanol results in a dose-dependent decrease of the chemiluminescent response to the stimulatory agent (opsonized zymosan). Ethanol was also shown to induce a peakwise rise of the intracellular cAMP after a 2-min incubation. The observed effects are correlated both in time and concentration, which allows the presumption that inhibition of the functional activity of macrophages is mediated by the increase of the intracellular cAMP level.     

Differentiation of murine macrophages to express nonspecific cytotoxicity for tumor cells results in L-arginine-dependent inhibition of mitochondrial iron-sulfur enzymes in the macrophage effector cells

Previous studies show that cytotoxic activated macrophages cause a reproducible pattern of metabolic inhibition in viable tumor target cells. This includes inhibition of DNA synthesis, two oxidoreductases of the mitochondrial electron transport chain (NADH: ubiquinone oxidoreductase and succinate: ubiquinone oxidoreductase), and the citric acid cycle enzyme aconitase. This pattern of metabolic inhibition is induced by a cytotoxic activated macrophage associated biochemical pathway with L-arginine deimination activity that synthesizes L-citrulline from L-arginine and oxygenated nitrogen derivatives from the imino nitrogen removed from the guanido group of L-arginine. Here we report that macrophages activated in vivo by infection with bacillus Calmette-Guérin or in vitro by murine rIFN-gamma or murine IFN-alpha/beta (in the presence of the second signal LPS in all cases) develop inhibition of aconitase and the same two oxidoreductases of the mitochondrial electron transport chain as was documented earlier in target cells of cytotoxic activated macrophages. In addition, this pattern of metabolic inhibition which develops in cytotoxic activated macrophages is caused by the L-arginine-dependent effector mechanism. Inhibition of mitochondrial respiration by effectors of the L-arginine-dependent cytotoxicity system results in a compensatory increase in activity of the glycolytic pathway. We speculate that the pattern of metabolic inhibition induced in cytotoxic activated macrophages by the L-arginine-dependent effector system causes changes in the macrophage intracellular environment that increases resistance to certain facultative and obligate intracellular pathogens.     

Tumoricidal activity of syngeneic macrophages from melanoma-bearing mice: Changes with progressive tumor growth

Summary Changes in the cytostatic and cytotoxic activity of macrophages from tumor-bearing (TBM) and control mice were studied in a murine model of malignant melanoma. Syngeneic macrophages from TBM were initially noncytotoxic, but became cytotoxic and achieved their maximum destructive ability after 14 days of tumor growth. With continued tumor growth these macrophages either lost or had reduced cytotoxic activity. In contrast, macrophages from the same melanoma-bearing animals were significantly cytostatic at an earlier stage of tumor growth, but with continued melanoma growth these macrophages were no more cytostatic than controls. Moreover, melanomas grew slowly during the time when macrophages were observed to be cytostatic but grew rapidly at those stages when macrophages had a reduced ability to inhibit melanoma DNA synthesis. When these effector cells became cytotoxic melanomas were growing rapidly and changes in cytotoxicity had little effect on tumor mass. Thus, macrophages do not completely suppress melanoma proliferation and, although exhibiting cytotoxicity they were relatively ineffective in controlling a large mass of tumor cells.     

Effect of modifiers of natural biological response on the functional activity of macrophages (oncological aspect)

Induction of cytotoxicity by biological response modifiers (BRMs) is only one aspect of macrophage activation. After the use of BRMs there were other changes in the functional activity of cells and in particular their increased production or secretion of a number of growth factors. Thus, activation of macrophage antitumor activity induced by BCG vaccine was transitory while activation of growth factor production was more stable in time which finally led to increased proliferation of tumor cells. Combined use of cyclophosphamide and BCG vaccine significantly increased not only the toxicity induced by BCG vaccine but also their liberation of the growth factors. Such macrophages lost their ability to control the growth of a small number of the tumor cells cultivated in their presence. Development of ways for directed activation of macrophages aimed at elimination of the tumor cells which survived the chemotherapy should include evaluation of the combined effect of various BRMs and chemotherapeutics on both antitumor and protumor activity i. e. ability to produce the factors stimulating the tumor growth.     

Production of arachidonic acid metabolites by operationally defined macrophage subsets

The antitumor activity and arachidonic acid metabolism of operationally defined macrophage populations was examined. Macrophages from mice injected with Mycobacterium bovis (strain BCG) or with pyran-copolymer were cytotoxic for tumor cells. The major arachidonic acid metabolite of these cells was PGE2. Neither resident nor elicited macrophages were cytotoxic. However, elicited macrophages as well as macrophages from BCG injected mice inhibited tumor cell growth. The production of arachidonic acid metabolites by elicited cells, while low initially, was followed by a rapid increase in PGE2. The major metabolites of resident cells were PGE2 and prostacyclin. The cAMP:cGMP ratio correlated with the metabolic activity of the cells.     

The role of host lymphocytes and host macrophages in antitumor reactions after injection of sensitized lymphocytes and tumor target cells into naive mice

Summary DBA/2 mice were immunized i.p. against syngeneic SL2 lymphosarcoma cells. At various days after the last immunization peritoneal and spleen lymphocytes were collected. The lymphocyte suspensions were enriched for T-cells by nylon wool filtration.The peritoneal T-cells from immunized mice (a) expressed direct specific antitumor cytotoxicity in vitro, (b) induced macrophage cytotoxicity in vitro, and (c) exerted tumor neutralization measured in a Winn-type assay. Spleen T-cells from these immunized mice (a) expressed no direct specific antitumor cytotoxicity in vitro, (b) only induced moderate macrophage cytotoxicity in vitro, but (c) exerted tumor neutralization in a Winn assay.For effective tumor neutralization in vivo effector target cell ratios of 1000:1 were required. When the effector/target ratio of 1000:1 was maintained but the absolute numbers of effector and target cells were lowered from 10⁶ to 10⁵ lymphocytes and 10³ to 10² target cells respectively, no tumor neutralization was obtained.The major effect of the sensitized-transferred T-lymphocytes seemed to be the induction of cytotoxic macrophages in the (naive) recipient mice, as the peritoneal macrophages collected from the recipient mice 7 days after i.p. injection of a mixture of sensitized T-cells and tumor cells were cytotoxic. Purified peritoneal T-lymphocytes collected from these recipient mice were able to induce macrophage cytotoxicity in vitro but expressed no cytotoxic T-cell activity.In conclusion, our results show that in the tumor system used, tumor neutralization after transfer of sensitized lymphocytes is not dependent on the presence of cytotoxic T-lymphocytes. Lymphocytes with the strongest potency to render macrophages cytotoxic (in vitro and in vivo) also induce the best tumor neutralization in vivo, suggesting an important role for host macrophages as antitumor effector cells.     

Production of arachidonic acid metabolites by operationally defined macrophage subsets

The antitumor activity and arachidonic acid metabolism of operationally defined macrophage populations was examined. Macrophages from mice injected with (strain BCG) or with pyran-copolymer were cytotoxic for tumor cells. The major arachidonic acid metabolite of these cells was PGE₂. Neither resident nor elicited macrophages were cytotoxic. However, elicited macrophages as well as macrophages from BCG injected mice inhibited tumor cell growth. The production of arachidonic acid metabolites by elicited cells, while low initially, was followed by a rapid increase in PGE₂. The major metabolites of resident cells were PGE₂ and prostacyclin. The cAMP:cGMP ratio correlated with the metabolic activity of the cells.     

The cytolytic function of mononuclear phagocytes. I. The dependence of the mechanisms regulating and realizing the cytolytic function of macrophages on their origin and the mode of activation

It has been shown that the induction of cytolytic activity in macrophages cause remarkable metabolic and morpho-structural shifts in effector cells, such as: 1) an expression of trypsin-sensitive receptors responsible for macrophage binding to malignant cells; 2) some differences in macrophage sensitivity to the cAMP system modifiers dependent on the activation inducer nature; 3) an enhanced production of oxygen metabolites directly involved in oxygen-dependent cytotoxicity of macrophages; 4) augmentation of lysosomal contents and activation of lysosomal enzymes. The inhibitor analysis enabled us to identify the main lytic components acting in the process of cytolytic functioning of macrophages of different origin and activation.     

Release of tumor necrosis factor-alpha from macrophages. Enhancement and suppression are dose-dependently regulated by prostaglandin E2 and cyclic nucleotides

PGE2 has previously been shown to suppress various leukocyte functions. In this study, we examined whether PGE2 would affect release of TNF-alpha from rat resident peritoneal macrophages. Two different, dose-dependent effects were observed: low PGE2 concentrations (0.1 to 10 ng/ml) stimulated, whereas higher concentrations (greater than 10 ng/ml) suppressed TNF-alpha release. PGE2-stimulated TNF-alpha production was dependent on de novo protein synthesis and was associated with an intracellular rise of cGMP. The importance of cGMP as an intracellular messenger for PGE2 was confirmed by the following evidence: (1) low PGE2 concentrations preferentially increased cGMP and not cAMP and (2) cGMP, either exogenously added or endogenously generated by sodium nitroprusside, were efficient stimulators of TNF-alpha production. In contrast, agents increasing intracellular cAMP concentrations such as PGE1, higher PGE2 doses, isoproterenol, and theophylline, all suppressed TNF-alpha synthesis. Only resident, but not casein-elicited or Corynebacterium parvum-activated macrophages, were stimulated by low PGE2 concentrations to increase TNF-alpha production. In tumor cytotoxicity assays, PGE2-activated macrophages were active only against TNF-alpha-sensitive target cells. These findings demonstrate that TNF-alpha synthesis in macrophages is up-regulated by cGMP and down-regulated by cAMP, which indicates that cyclic nucleotides act as intracellular messengers for extracellular signals of macrophage activation.     

Activities of cAMP-dependent protein kinase and enzymes of 2′,5′-oligoadenylate metabolism in NIH 3T3 cells deepening into the resting state

The activity of cAMP-dependent protein kinase was found to increase continuously in the NIH 3T3 cells, deepening into the resting state. The increase correlated with intracellular level of heat-stable protein inhibitor of the protein kinase rather than with the cAMP content. The elevation of 2',5'-oligo(A) synthetase activity and the decrease in 2'-phosphodiesterase activity were also observed in the cells sinking into the resting state. The variations in enzyme activities were similar to those caused by the increase in the intracellular cAMP content described elsewhere. These results agree with the idea that the cAMP-dependent protein kinase is involved in the regulation of the enzymes of 2',5'-oligo(A) metabolism.     

Thrombin inhibits proliferation of the human megakaryoblastic MEG-01 cell line: a possible involvement of a cyclic-AMP dependent mechanism.

Thrombin, a potent platelet activating agent, has previously been found to increase intracellular calcium levels and/or thromboxane A2 synthesis in leukemic cell lines exhibiting specific markers of the megakaryocyte/platelet lineage. However, its functional role on these cells has not been defined. As thrombin is implicated in the regulation of cellular proliferation or differentiation in various other cell types, we investigated the functional effects of thrombin on the megakaryoblastic MEG-01 cell line, and further explored its receptor coupling mechanisms on these cells. We observed that thrombin caused in 1% serum containing culture medium, a reduction in the proliferation of MEG-01 cells, without affecting their differentiation stage as determined by the expression of platelet glycoproteins GPIIb/IIIa and GPIb, FVIII-related-antigen and cell-size measurement, which are specific markers for megakaryocyte maturation. In addition, incubation of MEG-01 cells with thrombin resulted in dose-dependent increases in cAMP levels, and in inositol-trisphosphate formation and intracellular Ca2+ levels. All these responses required thrombin proteolytic activity. The lipoxygenase inhibitor, nordihydroguaiaretic acid, blunted thrombin-induced calcium increase without affecting thrombin-induced increase in cAMP levels, suggesting different thrombin coupling mechanisms with these two second messenger pathways. In addition, the inhibitory effect of thrombin on MEG-01 cell growth was mimicked by cAMP level enhancing agents such as forskolin, prostaglandin E1 and Bt2cAMP. These results suggest the involvement of a cAMP-dependent mechanism in the thrombin-induced reduction in MEG-01 cell growth.     

Mechanism and specificity of macrophage-mediated cytotoxity.

The cytotoxic effect of macrophages derived from alloimmunized mice (immune macrophages) was found to be immunologically specific. The immune macrophages killed only target macrophages carrying the alloantigens used for immunization in mixed macrophage cultures (MMC) under optimal conditions of contact between effector and target cells. T-sensitized lymphocytes, but not B cells, were capable of arming nonimmune macrophages and conferring upon them cytotoxic activity; the arming factor, which seemed to be a T mediator or T-cell receptor (membrane component) was removable by trypsin. Frequent rinsing or addition of hydrocortisone significantly decreased the cytotoxicity of the MMC. Pretreatment of peritoneal cells with anti-θ antisera and complement markedly decreased immune macrophage cytotoxic activity. It is suggested that the presence of a very small number of T-sensitized lymphocytes is required for strong cytotoxic activity to be manifested by the macrophages.     

Cytotoxic T cells in the peritoneal cavity of mice infected with ectromelia virus.

Peritoneal exudate cells from mice infected with ectromelia virus were cytotoxic for virus-infected target cells as measured in a 51Cr release assay. Cytotoxic activity seemed to be T cell-dependent as it was largely abolished by treatment with anti-theta serum and complement but was not impaired by macrophage depletion. The kinetics of development of cytotoxicity in the peritoneal cavity lagged behind spleen cytotoxicity by 1-2 days. Peak activity in peritoneal cells was present about 6 days after intravenous infection with virus. These studies suggest that macrophages present in the free peritoneal cell populations of ectromelia-infected mice are not cytotoxic for virus-infected target cells. The effect of macrophages in virus clearance is therefore likely to be due to phagocytic rather than cytotoxic effects.     

Studies of the mechanisms for the induction of in vivo tumor immunity. VI. Induction of specific and nonspecific cell-mediated immunity in tumor-bearing hosts and its correlation with transplantation tumor immunity

Studies were performed to determine the development of cell-mediated cytotoxic response at tumor site in C57BL/6 mice bearing progressively growing FBL-3 ascites leukemia. The effectors isolated from tumor ascites are found to be highly cytotoxic for leukemic target cells. The levels of cytotoxicity obtained with effectors isolated from tumor site are generally higher than those obtained with immune mice. This cytotoxicity is both specific and nonspecific. The specific cytotoxicity against tumor-associated antigen is mainly mediated by T cells and the nonspecific cytotoxicity against unrelated tumor cells is mediated largely by macrophages. The T-cell-enriched preparation did not give significant natural killer activity. When testing the ability of these effectors to produce in vivo immunity against the challenge of FBL-3, it was found that only T cells could confer the transplantation-type immunity, but the immunity was transient. The macrophage-enriched preparation isolated from tumor ascites failed to give in vivo protection. These findings indicate that in FBL-3 system, mice with progressively growing tumors are able to develop immune response against tumor cells. However, this immunity is probably interfered with by a suppressor factor(s) or suppressor cells which restrict their activity to eliminate the tumor cells effectively.     

Effect of levamisole on cytotoxic T-cell-mediated immune resistance to L1210 murine leukemia in hyperimmune mice

Summary The effect of levamisole (LMS) on T-cell-mediated antitumor immunity was examined in adult and aged mice hyperimmune to L1210 leukemia. The immune resistance of aged mice was depressed compared with that of adult mice, which almost completely rejected 5×10⁷ L1210 cells inoculated IP. A significant level of tumor-specific cytotoxicity was detected in the spleen cells of adult hyperimmune mice by the ⁵¹Cr-release assay after in vitro sensitization with mitomycin C-treated L1210 cells. This was mediated by cytotoxic T cells, since in vivo administration of antithymocyte serum or in vitro treatment of the spleen cells with anti-Thy 1.2 antibody and complement abrogated the cytotoxicity completely. In aged mice, however, cytotoxic T-cell activity was lower although the animals were immune to L1210.Administration of LMS (0.38 mg/kg) restored the depressed cytotoxicity of aged mice to the level seen in adult mice. Furthermore, in adult hyperimmune mice LMS augmented T-cell-mediated cytotoxic activity and restored the reduced cytotoxicity caused by in vivo administration of antithymocyte serum. These results indicate that LMS was effective in augmenting T-cell-mediated tumor immunity in immunologically competent or deficient hosts.     

Effect of insulin and isoproterenol on macrophage phagocytic activity

To reveal the influence of cyclic adenosine monophosphate (cAMP) on the completion of the phagocytosis of salmonellae, the influence of insulin and isoproterenol on the phagocytic activity of peritoneal macrophages obtained from mice infected with S. typhimurium strains differing in virulence was studied in vitro. The study showed that isoproterenol, while increasing the intracellular content of cAMP, suppressed the bactericidal properties of macrophages with respect to salmonellae, whereas insulin decreased the level of cAMP in the cells and thus facilitated more rapid and complete digestion of ingested bacteria irrespective of their virulence.     

Suppression of natural killer cell cytotoxicity by splenocytes from Corynebacterium parvum-injected, bone marrow-tolerant, and infant mice.

Natural killer (NK) cell cytotoxicity to YAC-1 lymphoma was investigated in mice tolerant to bone marrow grafts (BM-tolerant), Corynebacterium parvum- (C. parvum) treated mice, and infant mice. Also the comparison was made between the NK cell and the hemopoietic-resistance effector (HR-E) cells. It was found that the BM-tolerant mice and C. parvum-treated mice showed either no or markedly decreased NK cell cytotoxicity. These mice were also nonresponders to bone marrow grafts in vivo. The lack of or decreased reactivity was apparently caused by the regulatory cell activities of the suppressor cell since the splenocytes from C. parvum-treated and BM-tolerant mice suppressed significantly the cytotoxic activities of otherwise fully functional NK cells. Similar suppressive effect on NK cells was mounted by splenocytes from infant mice, indicating again the suppressor cell regulation of NK cell cytotoxicity.     

Effects of intracellular cyclic AMP and cyclic GMP levels on DNA synthesis of young-adult rat hepatocytes in primary culture.

Possible roles of dibutyryladenosine 3',5'-cyclic monophosphate (cAMP) and dibutyryl-guanosine 3',5'-cyclic monophosphate (cGMP) in regulation of hepatocyte DNA synthesis were examined using primary cultures of young-adult rat hepatocytes maintained in arginine-free medium. Throughout the experimental period, nonparenchymal cells were hardly observed in the selective medium. When epidermal growth factor (EGF) was added to the cultures, a transient increase in the intracellular cAMP level preceded the elevation of hepatocyte DNA synthesis. EGF-stimulated hepatocyte DNA synthesis was remarkably enhanced by the elevation of the intracellular cAMP level induced by treatment with cAMP alone or a combination of cAMP and theophylline, an inhibitor of cyclic nucleotide phosphodiesterase. Furthermore, the early elevation of intracellular cAMP alone, which was induced by treatment with the combination of cAMP and theophylline, caused a remarkable increase in hepatocyte DNA synthesis. On the other hand, addition of EGF to the cultures caused a rapid decrease in the intracellular cGMP level followed by an increase in hepatocyte DNA synthesis. EGF-stimulated hepatocyte DNA synthesis was severely suppressed or completely inhibited by the elevation of the intracellular cGMP level induced by treatment with cGMP alone or a combination of cGMP and dipyridamole, a specific inhibitor of cGMP phosphodiesterase. These findings indicate that cAMP and cGMP act oppositely on the regulation of DNA synthesis of young-adult rat hepatocytes in primary culture: cAMP plays a positive role, whereas cGMP plays a negative role. Also it is strongly suggested that an early elevation of the intracellular cAMP level is essential for the onset of DNA synthesis in hepatocyte primary cultures.     

Human monocyte activation for cytotoxicity against intracellular Leishmania donovani amastigotes: induction of microbicidal activity by interferon-gamma

Macrophages are pivotal cells in interactions of man and leishmania. Leishmanial disease results from intracellular infection of macrophages: parasitized cells are seen in smears or biopsy specimens of lesions; macrophages cultured in vitro support replication of parasites. Paradoxically, parasite destruction is also mediated by macrophages, which become highly cytotoxic after exposure to immune lymphocytes or their lymphokine (LK) products. The precise molecular mechanisms by which lymphocytes or LK induce macrophage activation for leishmanicidal activity, however, are not yet known. We analyzed interactions of leishmania amastigotes with human monocytes cultured in vitro as a nonadherent cell pellet. Leishmania donovani and L. major replicated in freshly isolated monocytes. Monocytes treated with greater than 200 IU/ml of the LK, human Interferon-gamma (IFN-gamma), destroyed tumor cells and L. donovani, but not L. major. Phorbol myristate acetate, endotoxic bacterial lipopolysaccharide, and recombinant human IFN-alpha and IFN-beta did not induce cytotoxicity. The time course for induction of cytotoxicity contrasted sharply with that of previously described monocyte antileishmanial activity: IFN-gamma induced cytotoxicity even when added after infection with L. donovani; induction of cytotoxicity did not require that IFN-gamma be present throughout the period of culture after infection: a 30-min preinfection pulse of IFN-gamma was sufficient to induce 70% of maximal activity; and freshly isolated monocytes and cells cultured for up to 4 days in vitro prior to infection and IFN-gamma treatment were equally responsive to IFN-gamma. These studies provide convincing evidence for intracellular cytotoxicity for L. donovani by freshly isolated human monocytes. This system provides an important base for further analysis of induction and expression of cytotoxic mechanisms against leishmania and other intracellular organisms that cause human disease.     

Regulation of cyclic AMP levels in Arthrobacter crystallopoietes and a morphogenetic mutant.

The extracellular levels of cyclic AMP (cAMP), cAMP phosphodiesterase activity, and adenylate cyclase activity were measured at various intervals during growth and morphogenesis of Arthrobacter crystallopoietes. There was a significant rise in the extracellular cAMP level at the onset of stationary phase, and this rise coincided with a decrease in intracellular cAMP. The phosphodiesterase activity measured in vitro increased in the early exponential phase of growth as intracellular cAMP decreased, and, conversely, prior to the onset of stationary phase the phosphodiesterase activity decreased as the intracellular cAMP levels increased. Adenylate cyclase activity was greater in cell extracts prepared from cells grown in a medium where morphogenesis was observed. Pyruvate stimulated adenylate cyclase activity in vitro. A morphogenetic mutant, able to grow only as spheres in all media tested, was shown to have altered adenylated cyclase activity, whereas no significant difference compared to the parent strain was detectable in either the phosphodiesterase activity or the levels of extracellular cAMP. The roles of the two enzymes, adenylate cyclase and phosphodiesterase, and excretion of cAMP are discussed with regard to regulation of intracellular cAMP levels and morphogenesis.