Effect of an acidic heteropolysaccharide (ARAGAL) from the gum of Anadenanthera colubrina (Angico branco) on peritoneal macrophage functions

Brazilian flora are a source of interesting polysaccharides which, either in their native state or when submitted to structural modifications, might have potential applications as biological response modifiers (BRM). A complex acidic heteropolysaccharide, containing mainly galactose and arabinose (ARAGAL), isolated from the gum of the native leguminous tree Anadenanthera colubrina (Angico branco), was studied for its immunological properties on peritoneal exudate cells, namely their superoxide anion production, phagocytic activity, morphological alterations and percentage content of activated macrophages. Activation of macrophages showing increased cytoplasm, bright and large nuclei, various cytoplasmatic projections and spreading ability, was detected following in vitro cell exposure to ARAGAL or in cells obtained from treated animals. In vitro exposure to ARAGAL increased the occurrence of activated macrophages in a time- and a dose-dependent pattern, since approximately 82% of the cells were activated in the presence of 300 microg/ml of ARAGAL after 24 h of incubation and approximately 91% after 48 h. The occurrence of activated macrophages was also evident in cell preparations from ARAGAL-treated mice, their percentage showing a dose-dependent pattern. There were approximately 60, 75 and 75% following treatment with 100, 250 and 500 mg/kg of ARAGAL, respectively. A phagocytic assay showed that 25 microg/ml ARAGAL was sufficient to impose a maximum phagocytic ability, although this effect was dose-dependent. O(2)(-) production by macrophages from ARAGAL-treated mice was 70% higher than that of cells from untreated mice. Moreover, cells from treated mice responded to PMA, the effect being 25% higher than that of the control using untreated mice. These results thus suggest a possible role of ARAGAL from A. colubrina as a BRM.     

Induction of tumoricidal activity of human and murine peritoneal macrophages by antitumor chemotherapy

Institute of Chemical Physics, Academy of Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR S. M. Navashin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 108, No. 9, pp. 330–332, September, 1989.     

A novel rat monoclonal antibody reactive with murine tumoricidal Kupffer cells and activated peritoneal macrophages from BCG-infected mice

A rat monoclonal antibody, termed 1A2.10D and raised against mouse BCG-activated peritoneal-macrophages, was used to detect antigenic determinant(s) on mouse tumoricidal activated peritoneal and liver macrophages from BCG-infected mice by indirect immunofluorescence flow cytometry and immunoblot analysis. This antibody was of the rat IgG2b subclass and not cytolytic to BCG-activated macrophages in the presence of rabbit complement. The antigen(s) was expressed on tumoricidal activated peritoneal and liver macrophages from BCG-infected mice and some macrophage cell lines. Its expression could not be detected on resident peritoneal cells, peritoneal exudate cells containing non- or low tumoricidal macrophages, thymocytes, resting bone marrow cells, normal spleen cells, various established mouse tumour cells or one macrophage cell line. Immunoblot analysis showed the antibody to bind to one major protein of 56,000 MW, and to be expressed on the peritoneal and liver macrophages from BCG-infected mice. Using this antibody and a fluorescence-activated cell sorter, selection was made of antibody-positive or -negative macrophages from BCG-infected mice to examine their tumour killing activity. The antibody-positive macrophages clearly showed higher tumour killing activity than the negative macrophages.     

Immunoregulation by macrophages II. Separation of mouse peritoneal macrophages having tumoricidal and bactericidal activities and those secreting PGE and interleukin I

Macrophage subpopulations having bactericidal or tumoricidal activities and secreting interleukin I (IL1) or prostaglandin E (PGE) were identified through primary or secondary infection with Salmonella enteritidis and separated by sedimentation velocity. Bactericidal activity was measured by [3H]-thymidine release from Listeria monocytogenes and tumoricidal activity by 51Cr-release from C-4 fibrosarcoma or P815 mastocytoma cells. Macrophages with bactericidal activity were distinguished from those with tumoricidal activity a) during secondary infection when cytolytic activity occurred only at days 1-4 post injection and bactericidal activity remained high throughout and b) after sedimentation velocity separation. Cytolysis was consistently greatest among adherent cells of low sedimentation velocity, whereas cells with bactericidal activity increased in size during the infection. Tumour cytostasis (inhibition and promotion of [3H]-thymidine uptake) differed from cytolysis in that the former was more prolonged during infection and was also detected among large cells. Secretion of immunoregulatory molecules PGE and IL1 occurred maximally among different macrophage subpopulations separated by sedimentation velocity and depending on the type of stimulus used in vitro. There was an inverse correlation between IL1 production and PGE production after stimulation with C3-zymosan or lipopolysaccharide (LPS). The development of immunity during infection may therefore be dependent upon the relative proportions of effector and regulatory macrophage subpopulations and the selective effects of environmental stimuli on these functions.     

Induction of the respiratory burst in turtle peritoneal macrophages by Salmonella muenchen

Peritoneal macrophages were collected from juvenile turtles 72h after intraperitoneal inoculation with a 3% Sephadex suspension. The macrophages were assayed for their chemiluminescent (CL) properties, reflecting their respiratory burst activity, after stimulation with Zymosan A, phorbol 12-myristate 13-acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and calcium ionophore A23187. Except for fMLP, all triggering agents induced a marked CL response. Luminol was used as the chemiluminescent probe. When comparing CL responses in temperatures ranging from 15 to 35 degrees C, lower assay temperatures induced lower and slower CL responses. Stimulation with viable Salmonella muenchen resulted in a distinct response. Bacteria, inactivated by means of heat or acetone, induced a faster and stronger oxidative burst. Opsonization of either viable or heat-inactivated S. muenchen with non-inactivated anti-S. muenchen serum, prepared in turtles, induced faster and higher CL responses. On the other hand, opsonization of acetone-inactivated S. muenchen caused CL responses to be slower and weaker. S. muenchen, opsonized with heat-inactivated turtle anti S. muenchen serum, induced higher responses than non-opsonized bacteria, but slower and weaker responses than bacteria opsonized with native turtle antiserum. No response was recorded after stimulation with LPS and the supernatant of heat-inactivated bacteria.     

Facsimile epithelioid cells obtained from stimulated peritoneal macrophages and their secretory activity in vitro

Mouse peritoneal macrophages collected after exposure to new born calf serum in vivo were found to have some of the morphological and ultrastructural features attributed to epithelioid cells. These features were accentuated by short term culture in vivo or in vitro. Three accepted varieties of epithelioid cell were reproduced, i.e., those with predominant rough ER, those with predominant smooth vesicles and the plasmacytoid variety. Cells resembling fibroblasts were not seen, nor were lymphoid cells. The facsimile epithelioid cells had considerable secretory activity for a range of macrophage enzymes. They retained the phagocytic capacity and the surface receptors of macrophages but to a reduced extent. It is suggested that epithelioid cells are a form of stimulated macrophage, especially effective in enzyme secretion and liable to appear after excitation of the cell membrane by pinocytosis rather than phagocytosis.     

Induction of chemotaxis in mouse peritoneal macrophages by activators of protein kinase C

Two activators of calcium and phospholipid dependent protein kinase (protein kinase C), the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and the synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), were compared as chemotactic agents for mouse peritoneal macrophages. Both of these compounds were found to induce chemotaxis in the macrophages to a similar extent in a time and dose dependent manner. Induction of chemotaxis was observed in the concentration range of 10-100 nM for TPA and 25-250 microM for OAG. Two structurally related synthetic sn 1,2-diacylglycerols, 1,2-dioctanoylglycerol (diC8) and 1,2-didecanoylglycerol (diC10), were also found to be chemotactic for macrophages, while monoacylglycerol (2-monoolein) was inactive. Of the diacylglycerols, OAG was found to be the most active followed by diC8 and diC10. In contrast to TPA, the synthetic diacylglycerols had no effect on superoxide anion release by the cells, suggesting that the mechanism of superoxide anion release by TPA in macrophages is distinct from chemotaxis. Phorbol-12,13-diacetate, a biologically inactive phorbol ester analog that inhibits the binding of TPA to its cellular receptors, inhibited macrophage chemotaxis induced by TPA, the synthetic diacylglycerols, and the complement fragment, C5a. Taken together, our results suggest that chemotaxis in macrophages may be mediated by activation of protein kinase C.     

In vitro killing of Ehrlichia risticii by activated and immune mouse peritoneal macrophages.

Normal resident murine peritoneal macrophages inoculated in vitro with Ehrlichia risticii readily phagocytized the organism but were unable to suppress ehrlichial replication as determined by indirect fluorescent-antibody staining of the inoculated cells. In contrast, macrophages from Corynebacterium parvum-inoculated and E. risticii-recovered mice rapidly eliminated the ehrlichiae. Macrophages from E. risticii-recovered mice were as effective as the C. parvum-activated cells in phagocytizing and eliminating the organism. Opsonization of E. risticii with homologous antiserum prior to inoculation of macrophage cultures resulted in enhancement of phagocytosis and greater suppression of E. risticii replication in all macrophage groups. These findings indicate that the pathogenesis of E. risticii infection centers on the ability of the organism to enter and replicate within the macrophage with avoidance of macrophage antimicrobial effects. An immune response results in macrophage activation with enhancement of the macrophage's ability to eliminate E. risticii. Opsonization of E. risticii with anti-E. risticii serum renders E. risticii more susceptible to macrophage destruction.     

Phagocytosis, peritoneal influx, and enzyme activities in peritoneal macrophages from germfree, conventional, and ex-germfree mice.

Peritoneal macrophages from germfree mice showed a higher basic activity of lysosomal enzymes than did macrophages from conventional mice, whereas oil-induced peritoneal influx, induction of lysosomal enzymes, and phagocytosis via the C3b receptor after endotoxin stimulation were reduced or absent. After germfree mice had been housed with conventional mice for 1 week, peritoneal influx and C3b receptor-mediated phagocytosis reached normal levels; after 4 weeks, enzyme activities also reached normal levels.     

Induction of DNA synthesis in rat peritoneal macrophages in culture by a pleural inflammatory exudate

Peritoneal macrophages in culture are blocked in the G₀ phase of the cell cycle, but retain many of their functional characteristics such as phagocytic ability. Peritoneal macrophages have been thought to be a terminal cell type. It has been investigated whether such properties could be modified by a substance released in acute inflammatory exudates. For this purpose a pleural exudate obtained from rats injected with dextran (40,000) 4 hours before, was centrifuged to eliminate cells, sterilized by filtration on Millipore filter 0.22 m and diluted 50% with 199 medium culture. This medium was used to treat normal and activated peritoneal macrophages in culture. The effects were observed 24, 48, 72, 96 hours after the beginning of treatment. An enhancement of spreading and capacity of phagocytosis was observed 24 hours after the beginning of treatment. After 48 hours, the number of cells incorporating tritiated thymidine increased and became highest 4 days later. These phenomena were also obtained with pleural exudate of inbred rats (Lewis, Wag) treating macrophages of the same strain and with rat pleural exudate treating mouse macrophages. No effects were observed with dextran alone.The chemical nature of the stimulatory factor remains to be elucidated.     

Effects of adrenergic agents on rat peritoneal macrophages activated in vitro by acetylated low-density lipoprotein

Macrophages interact with modified lipoproteins and alter their functional status. In this study, the effects of the adrenergic agents adrenaline, isoproterenol, and dobutamine on macrophages activated with acetylated low-density lipoprotein were tested. The aim of this investigation was to determine whether adrenergic agents influence biologically significant functions of these cells in an in vitro model of macrophage-lipoprotein acute interaction. Rat peritoneal macrophages were incubated with acetylated low-density lipoprotein for 16 h, with or without adrenergic agents. Hydrogen peroxide and nitric oxide production and acid phosphatase activities in the supernatant and cell lysate were assayed. Adrenaline and isoproterenol inhibited the production of hydrogen peroxide, stimulated the production of nitric oxide, and increased the extracellular activity of acid phosphatase in the lipoprotein-activated cells. Dobutamine increased the extracellular, but decreased the intracellular acid phosphatase activity. Adrenaline and dobutamine also produced changes in the cell size and nuclear morphology of the macrophages. Macrophages are activated in vitro by acetylated low-density lipoprotein, and their functions and morphology are modified under the influence of adrenergic agents. Certain effects could be attributed to changes in cyclic AMP levels.     

In vitro activation of murine peritoneal exudate cells (PEC) and peritoneal macrophages by ST 789.

ST 789 is a new synthetic compound characterized by an amino acidic group joined to the N9 position of the hypoxanthine ring, which has been shown recently to have immunomodulating properties and minimal toxicity. The drug has been reported to protect immunosuppressed mice from microbial infections and tumour growth, and to restore the mitogen-induced proliferation of splenocytes from immunosuppressed young mice. In this study, we show that in vitro addition of ST 789 is able to markedly augment the sheep red blood cells (SRBC) phagocytosis by PEC, and to potentiate the cytotoxic activity of peritoneal exudate (PE) macrophages (M phi) vs the L-M tumour cell line. We also found that ST 789 enhanced the rIFN-gamma-induced NO2- release from cultured PE M phi. Similarly, in vitro addition of ST 789 to the latter cultures significantly increased the production of interleukin 1 (IL-1) and tumour necrosis factor (TNF) induced by lipopolysaccharide (LPS). These studies demonstrate that ST 789 is a potent phagocyte activator for the induction of cytokine release, phagocytosis and cytotoxic activity against tumour cells in vitro.     

Induction of serine and threonine protein phosphorylation by endotoxin-associated protein in murine resident peritoneal macrophages.

Endotoxin-associated protein (EP) from Salmonella typhi activated murine resident peritoneal macrophages to produce prostaglandin E2 (PGE2). Cells from both endotoxin nonresponder (C3H/HeJ) and the endotoxin responder (C3H/OuJ) mouse strains were activated by EP. This EP-induced prostaglandin E2 production was blocked by the protein kinase C (PKC) inhibitor H-7 as well as the tyrosine kinase inhibitor genistein, suggesting the involvement of both serine and threonine phosphorylation and tyrosine phosphorylation pathways in the activation of resident peritoneal macrophages by EP. Immunoblot analysis using antiphosphoserine and antiphosphothreonine antibodies showed that EP induced the serine and threonine phosphorylation of a 14-kDa protein (p14). This phosphorylation was not induced by phorbol myristic acid or by lipopolysaccharide endotoxin. Inhibitors of PKC, PKA, and PKG did not block the phosphorylation of p14. However, the tyrosine kinase inhibitor piceatannol blocked p14 serine and threonine phosphorylation, suggesting that this phosphorylation is dependent upon and preceded by a tyrosine phosphorylation step.     

Production of reactive nitrogen intermediates (RNI) by peritoneal macrophages from rats with experimental autoimmune prostatitis (EAP)

Peritoneal macrophages from experimental autoimmune prostatitis (EAP) rats were examined for their capacity to secrete reactive nitrogen intermediates (RNI), measured by the release of nitrite (NO ₂ ^– ). Under basal conditions, there was a significant increase of NO ₂ ^– secretion by cells from autoimmune rats in relation to resident cells. After stimulation in vitro with lipopolysaccharide (LPS), the NO ₂ ^– production was higher in cells from autoimmune rats compared to treated and non-treated controls. The NO ₂ ^– production was dependent upon the presence of L-arginine in the culture medium. The addition of L-N^G-monomethyl arginine, an inhibitor of nitric oxide synthesis, to the medium reduced the amount of measurable NO ₂ ^– . Kinetic studies in cells from EAP rats showed that in basal conditions there was an significant release of NO ₂ ^– at day 7 of immunization that was maintained during the whole period studied. After LPS stimulation, there was a similar behavior and maximum values were reached at day 28 of immunization. These results, together with the lesion observed in the prostate gland, suggest that RNI may be of pathogenic importance in the development of early tissue inflammation and autoimmune disease of the prostate.     

Role of interleukins and nitric oxide secretion by peritoneal macrophages in differential tumoricidal effect to transplantable melanomas as regarding their biological properties

The secretion of interleukins (IL-18, IL-12) and Nitric Oxide (NO) by peritoneal macrophages from hamsters bearing two lines of transplantable melanoma was estimated. Macrophages of animals with melanoma lines secreted less IL-18 but more IL-12 and NO in comparison with the control macrophages. The distinctly higher cytotoxic activity of macrophages from animals with amelanotic line in comparison with the melanotic line was not accompanied by significant differences in the IL-18 and IL-12 secretion between studied groups of macrophages. Thus, it seems that IL-18 play the role in innate immunity but not in adaptive cellular immunity, whereas IL-12 and NO take part in macrophages tumoricidal activity.     

Induction of interferon synthesis and cytotoxicity by murine peritoneal macrophages exposed to glycoprotein ligands.

Thioglycollate-induced murine C57BL/6 and C3H/HeN peritoneal macrophages synthesized interferon-beta (IFN-beta) in response to exposure to glycoproteins such as horseradish peroxidase (HRP) or mannosyl or fucosyl bovine serum albumin (BSAman of BSAfuc, respectively), but not glucosylated or galactosylated BSA (BSAglu or BSAgal, respectively). These results suggest participation of the mannosyl-fucosyl receptor (MFR) in this response. IFN synthesis was augmented by culturing macrophages in L cell-conditioned medium prior to exposure to these substances. Macrophages obtained from lipopolysaccharide (LPS)-resistant C3H/HeJ mice did not produce IFN in response to HRP. Furthermore, IFN-induction by HRP was blocked by polymyxin B. In addition, exposure of macrophages to HRP or BSAman induced cytotoxicity against NIH 3T12 cells. Cytotoxicity was not inhibited by the presence of anti-IFN-alpha/beta. In contrast to IFN induction, however, macrophages activation was LPS-independent, since this activity was demonstrated in macrophages from C3H/Hej mice. The carbohydrate specificity of these responses suggests that the MFR or an another scavenger receptor may be involved in the responses to these substances, and that cytotoxicity and IFN-induction by glycoproteins follow unique pathways.     

Weight loss associated with an endotoxin-induced mediator from peritoneal macrophages: the role of cachectin (tumor necrosis factor)

Endotoxin-induced cells of the reticuloendothelial system were shown to produce mediator(s) that evoke a state of cachexia in recipient animals. The factor(s) responsible were assayed in endotoxin-resistant (C3H/HeJ) mice, which were injected with dialyzed conditioned medium obtained from lipopolysaccharide-induced peritoneal macrophages. The mice exhibited weight loss and anorexia, and they died if sufficient quantities of medium were administered. The syndrome was reversible if injections were discontinued. Endotoxin alone did not produce this effect, and no gross pathologic lesions were discernable in the treated animals. In this model system, cachexia appears to result from the action of soluble macromolecules produced by activated macrophages in vitro. Cachectin (murine tumor necrosis factor) is thought to play a central role in this phenomenon.     

Killing of Plasmodium falciparum by cytokine activated effector cells (neutrophils and macrophages)

Macrophages display natural antibody independent killing of asexual blood stages of Plasmodium falciparum in vitro. In contrast, the neutrophil killing of P. falciparum requires the presence of antibodies. Cytokines such as TNF alpha have very little effect on the macrophage-induced antiplasmodial activity, but significantly increase the damage of parasites by neutrophils. Cytokines, TNF alpha, IFN-gamma and TNF beta at very high concentrations were not toxic to P. falciparum in culture. It is postulated that the basis for cytokine modulated antiplasmodial activity of leukocytes is increased expression of Fc and complement receptors, which leads to a more efficient interaction between the parasite and neutrophils. It is also postulated that the parasite evades natural macrophage killing mechanisms by inducing factors which suppress this macrophage activity. Cytokine inhibitors may be induced during the course of a malarial infection. These could be involved in attempts to attain a balance between the host and the parasite, by protecting the parasite from the damaging effect of the immune system and protecting the host from the deleterious effects of cytokines.