Activated Mouse Peritoneal Macrophages Research Articles

In vivo enhancement of cytotoxic activity of mouse peritoneal macrophages by Met-enkephalin

In vivo enhancement of cytotoxic activity of mouse peritoneal macrophages by Met-enkephalin

In vivo enhancement of cytotoxic activity of mouse peritoneal macrophages by Met-enkephalin

In vivo enhancement of cytotoxic activity of mouse peritoneal macrophages by Met-enkephalin

Interleukin-12 synthesis is a required step in trehalose dimycolate-induced activation of mouse peritoneal macrophages.

Trehalose dimycolate (TDM), a glycolipid present in the cell wall of Mycobacterium spp., is a powerful immunostimulant. TDM primes murine macrophages (Mphi) to produce nitric oxide (NO) and to develop antitumoral activity upon activation with low doses of lipopolysaccharide (LPS). In this study, we investigated the ability of TDM to induce interleukin 12 (IL-12) and the role of this cytokine in TDM-induced activation of murine Mphi. RNA isolated from peritoneal exudate cells (PEC) collected at different times after TDM injection was used to determine IL-12 (p35 and p40 subunits) and gamma interferon (IFN-gamma) mRNA levels by semiquantitative reverse transcriptase-PCR. Constitutive expression of IL-12p35 was observed in PEC from untreated as well as from TDM-injected mice. In contrast, expression of the IL-12p40 subunit was almost undetectable in control PEC but was dramatically upregulated in PEC from TDM-injected mice. IL-12p40 expression peaked at 8 h and subsided to baseline levels at 39 h postinjection. TDM was also able to induce IFN-gamma expression; however, kinetics of induction of IFN-gamma was different from that of IL-12p40. Maximal levels of IFN-gamma mRNA were reached by 24 h and did not return to baseline by 4 days. In addition, pretreatment of mice with neutralizing monoclonal antibodies directed against IL-12 (C15.6.7 and C15.1.2) blocked IFN-gamma mRNA induction in PEC from TDM-treated mice. We further determined if the induction of IL-12 and/or IFN-gamma contributes to the in vivo priming effect of TDM on peritoneal Mphi. TDM-injected mice were treated in vivo with anti-IL-12 or anti-IFN-gamma (XMG.1.6) monoclonal antibodies. TDM-primed Mphi were then activated in vitro with LPS and tested for their ability to produce NO and to develop cytostatic activity toward cocultivated L1210 tumor cells. Priming of Mphi by TDM was completely blocked by in vivo neutralization of either IL-12 or IFN-gamma as demonstrated by an absence of tumoricidal activity and NO production by TDM-elicited Mphi in the presence of LPS. Taken together our results show that TDM, a defined molecule from M. tuberculosis, induces in vivo production of IL-12. Moreover, synthesis of IL-12 mediates TDM priming of mouse peritoneal Mphi through IFN-gamma induction.

Differential Effects of Granulocyte/Macrophage Colony-Stimulating Factor (GM-CSF) in Enhancing Macrophage Resistance toLegionella pneumophilavsCandida albicans

It has been reported that granulocyte/macrophage colony-stimulating factor (GM-CSF), one of the hemopoietic growth factors which regulates the function of phagocytic cells, is a potent activator of cultured macrophages and induces antimicrobial activities as well as differentiation of precursor cells. In this study, we examined the ability of recombinant murine GM-CSF to activate mouse peritoneal macrophages to restrict the growth of two different microorganisms,Candida albicansandLegionella pneumophila,both of which are important opportunistic pathogens in an immunocompromised host. Treatment of thioglycollate-elicited BDF1 mouse macrophages with GM-CSF for 24 hr enhanced the anti-C. albicansactivity of the macrophages in terms of inhibiting growth of the fungi. Reactive oxygen (H2O2) and IL-1 production by the macrophages were also enhanced by treatment with GM-CSF. However, no enhancement of anti-L. pneumophilaactivity of macrophages obtained from either susceptible A/J or resistant BDF1 mice toL. pneumophilainfection after treatment with up to 1000 units/ml GM-CSF was observed under the same conditions. When the treatment time was extended to 72 hr, GM-CSF was still unable to induce anti-L. pneumophilaactivity. As a control study, treatment with recombinant IFN-γ enhanced both anti-Candidaand anti-Legionellaactivity in cultured macrophages under the same conditions used in the GM-CSF study. Measurement of cellular iron content revealed the low iron content in IFN-γ-treated macrophages, but no decrease of iron in GM-CSF-treated macrophages compared with the control group, indicating a possible involvement of iron as a key factor in anti-L. pneumophilaactivity. Thus, the results of the study show that GM-CSF activation of elicited peritoneal macrophages is selective with regard to the type of antimicrobial activity induced.

Cystatins Up-regulate Nitric Oxide Release from Interferon-γ- activated Mouse Peritoneal Macrophages

Up-regulation of nitric oxide (NO) production by activated murine macrophages was observed during infection by Trypanosoma cruzi, the etiological agent of Chagas' disease. Cell infection by T. cruzi depends at least in part on cruzipain, a membrane-associated papain-related proteinase which is sensitive to inhibition by synthetic inhibitors of cysteine proteinases. Using the natural cysteine proteinase inhibitor chicken cystatin, a representative member of cystatin family 2, to investigate the effect of cruzipain on macrophage infection and NO release, we found that the inhibitor alone up-regulated NO release from interferon-gamma-activated macrophages. A 12-fold increase in NO production was observed in the presence of 1 microM chicken cystatin. This overproduction was concentration-dependent and could be detected at concentrations as low as 10 nM and remained in the presence of polymyxin B. Representative members of the other cystatin families, i.e. stefin B (family 1), T-kininogen, and its inhibitory domains (family 3), were also able to enhance NO production from interferon-gamma-activated macrophages. Neither E64, an irreversible inhibitor of cysteine proteinases, nor inhibitors of aspartyl and serine proteinases (aprotinin, pepstatin, and soybean trypsin inhibitor) enhanced NO production. Upon complexation with saturating amounts of reduced-alkylated papain, cystatins still remained active in increasing NO production, suggesting that the cystatin inhibitory site was not involved in the mechanism. The results demonstrate that members of all 3 cystatin families share another common property unrelated to their function of cysteine proteinase inhibitors, i.e. up-regulation of NO production, which biological significance remains to be elucidated.

Regulation of interleukin-10 production by beta-adrenergic agonists.

Catecholamines have been shown to inhibit some aspects of macrophage activation through a beta receptor-dependent mechanism. This study was undertaken to analyze the effects of isoproterenol, a specific beta-adrenergic agonist, on the synthesis of interleukin-10 (IL-10), a major macrophage-deactivating factor. Isoproterenol increased IL-10 release from lipopolysaccharide-(LPS)-activated mouse peritoneal macrophages in a dose-dependent manner. A significant effect was already observed with 1 microM isoproterenol, while a 4.5-fold increase was achieved with 10 microM. This increase was observed only if macrophages were exposed to isoproterenol for at least 2 h before LPS challenge. It was apparent within 0.5 h and persisted through 24 h at all the LPS concentrations used. A similar increase was observed at the IL-10 mRNA level, as judged by enzyme-linked immunosorbent assay-polymerase chain reaction. The macrophage response to isoproterenol that led to cyclic AMP accumulation was markedly inhibited by H-89, a potent inhibitor of protein kinase A. These data suggest the involvement of cyclic AMP in the regulation of IL-10 synthesis by isoproterenol. IL-10 was in turn partly responsible for a reduction in tumor necrosis factor-alpha synthesis. In vivo, the administration of oxprenolol, a beta-receptor antagonist, significantly reduced serum IL-10 levels 90 min after LPS challenge. Thus, the present study provides the first evidence that endogenous catecholamines are of critical importance in determining the magnitude of the IL-10 response in experimental endotoxemia.

Concomitant transcriptional activation of nitric oxide synthase and heme oxygenase genes during nitric oxide-mediated macrophage cytostasis.

During in vitro activation of mouse peritoneal macrophages with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS), their synthesis of peroxynitrite and their cytostatic activity against mouse lymphocytic leukemia (L1210) cells were examined. The activation of the genes for nitric oxide synthase (iNOS) and heme oxygenase (HO-1) was also determined during the activation of the macrophages. Results showed that activation of peroxynitrite synthesis in macrophages was accompanied by the transcriptional activation of iNOS and HO-1 genes. Both genes seem to be activated simultaneously upon activation of the macrophages. Simultaneous activation of iNOS and HO-1 genes may be important because degradation of heme by HO-1 is one of the most important reaction that produces CO in higher organisms, and nitric oxide (NO) and carbon monoxide (CO) can react with heme-containing guanylate cyclase.

Induction of cystine transport activity in mouse peritoneal macrophages by bacterial lipopolysaccharide

The transport of cystine has been investigated in mouse peritoneal macrophages cultured in vitro. The transport activity for cystine was very low in freshly isolated macrophages but was potently induced during culture in the presence of bacterial lipopolysaccharide (LPS) at concentrations as low as 0.1 ng/ml. The transport activity for cystine was enhanced when the cells were incubated with tumour necrosis factor-alpha (TNF-alpha), but not with interferon-gamma (IFN-gamma) or interleukin-1. IFN-gamma was rather repressive in the induction of the activity by LPS or TNF-alpha. The transport activity for cystine induced by LPS has been characterized. Cystine was transported mainly by Na(+)-independent system and the uptake of cystine was inhibited by extracellular glutamate and homocysteate, but not by aspartate, indicating that the transport of cystine in macrophages treated with LPS is mediated by System xc-. Glutathione content of the macrophages increased when they were exposed to LPS, and this increase was, at least in part, attributable to the induced activity of the cystine transport.

Phosphatidylinositolmannoside-Based Liposomes Induce No Synthase in Primed Mouse Peritoneal-Macrophages

Liposomes prepared from phosphatidylinositolmannosides (extracted from BCG) and cholesterol are efficiently endocytosed by macrophages. Phagocytosis of particles or microbes modifies macrophage metabolism and in some cases, delivers potent stimulating signals to macrophages. We examined the effect of phosphatidylinositolmannoside-based liposomes on three macrophage functions especially important for host defenses : nitric oxide production, oxidative burst and TNF-α secretion. Phosphatidylinositolmannoside-based liposomes, added as empty vesicles, induced a strong NO synthase activity in mouse peritoneal macrophages primed either by interferon-γ or by trehalose dimycolate. They also induced a moderate production of TNF-α. Phosphatidylinositolmannosides conferred activating properties to pH-sensitive liposomes. In contrast, liposomes composed of phosphatidylcholine and phosphatidylserine were unable to activate primed macrophages.

Spin trapping isotopically-labelled nitric oxide produced from [15N]L-arginine and [17O]dioxygen by activated macrophages using a water soluble Fe(++)-dithiocarbamate spin trap.

The unique capabilities of EPR spin trapping of nitric oxide based on a ferrous-dithiocarbamate spin trap have been demonstrated in a study verifying the source of the nitrogen and oxygen atoms in nitric oxide produced from activated macrophages. Spin trapping experiments were performed during nitric oxide generation from activated mouse peritoneal macrophages using the ferrous complex of N-methyl D-glucamine dithiocarbamate as a spin trap. When 15N-substituted arginine was given to the activated macrophages in the presence of the spin trap, a characteristic EPR spectrum of the nitric oxide spin adduct was obtained, which indicates the presence of the 15N atom in the nitric oxide molecule. The hyperfine splitting (hfs) constant of the 15N nucleus was 17.6 gauss. When 17O-containing dioxygen (55%) was supplied to the medium, an EPR spectrum consistent with the 17O-substituted nitric oxide spin adduct was observed in the composite spectrum. The hfs of 17O was estimated to be 2.5 gauss. The 14NO spin adduct observed after prolonged incubation in the medium which contains [15N]L-arginine as the only extracellular source of arginine demonstrates that arginine is recycled through its metabolite in activated macrophages.

Suppression of syntheses of high molecular weight nonmuscle tropomyosins in macrophages

In mouse fibroblasts, at least five TM isoforms are identified and they can be grouped into the high (TM1, TM2, and TM3) and low molecular weight TM isoforms (TM4 and TM5). Suppression of one of the high molecular weight tropomyosin (TM) isoforms in nonmuscle cells is implicated to be one of the causes for disorganization of actin microfilament bundles and subsequent changes in cell motility and cell shape. In this study, we studied the expression of tropomyosin isoforms in macrophages that exhibit high motility and ability to change cell shape. Two-dimensional gel electrophoresis followed by Western blot analysis using polyclonal anti-TM antiserum revealed that the high molecular weight TM isoforms were lacking in both resident and activated mouse peritoneal macrophages. Analyses of newly synthesized TM isoforms, Northern blot analyses using isoform-specific cDNA probes, and immunostaining with monoclonal anti-TM antibody that recognizes only the high molecular weight TM isoforms also demonstrated that the syntheses of the high molecular weight TM isoforms (TM1, TM2, and TM3) were completely suppressed, whereas the low molecular weight TM isoforms (TM4 and TM5) were expressed in macrophages. These results indicate that macrophages intrinsically lack the high molecular weight TM isoforms. In order to obtain information about cellular localization of the low molecular weight TM isoforms in macrophages, they were immunostained with polyclonal anti-TM antiserum that recognizes both the high and low molecular weight TM isoforms. The results showed that the low molecular weight TM isoforms were co-localized with F-actin in punctate and short fibrous structures. In addition, we performed in situ hybridization analysis to examine localizations of the TM mRNAs in fibroblasts and macrophages. The results showed that TM mRNAs were localized throughout the cytoplasm.

Optimizing the detection of cell surface antigens on elicited or activated mouse peritoneal macrophages.

Blocking conditions that are optimal for the detection of surface antigens on resident peritoneal macrophages (PM phi) by flow cytometry are not ideal for elicited or activated PM phi. A blocking step of 10% goat serum can be used routinely to detect the F4/80 and Mac-1 antigens on resident PM phi. In contrast, high concentrations (33-50% each) of combined goat and mouse sera were required to reduce nonspecific binding and to improve the detection of the F4/80 antigen on PM phi elicited by thioglycollate broth (TG) or activated by maleic anhydride divinyl ether copolymer (MVE-2). However, even low concentrations of goat serum masked the expression of the Mac-2 antigen on TG and MVE-2 PM phi. Thus, within a given elicited or activated PM phi population, different blocking conditions may be necessary to detect different surface antigens optimally. In addition to blocking, the use of isotypic controls that match the monoclonal antibody isotypes was found to be necessary for the optimal detection of antigen expression on TG and MVE-2 PM phi.

Role of Calcium in the Activation of Mouse Peritoneal Macrophages: Induction of NO Synthase by Calcium Ionophores and Thapsigargin

Bacterial lipopolysaccharide (LPS) has been recognized as one of the most potent activating signals for mouse peritoneal macrophages. In macrophages primed by interferon-γ (IFN-γ) or trehalose dimycolate (TDM), LPS induces NO synthase and the events associated with a high nitric oxide output: antitumor and antiparasitic activities. In the present report, it is shown that drugs (calcium ionophores or thapsigargin) which elevate the concentration of cytosolic calcium, [Ca 2+] i, induce NO synthase and antitumor activities in primed macrophages, mimicking LPS action. Calcium ionophores and thapsigargin trigger NO synthase activity in macrophages primed in vivo by TDM, in thioglycollate-elicited macrophages primed in vitro by IFN-γ, and in IFN-γ-treated EMT6 adenocarcinoma cells. However, activation of TDM-primed macrophages by LPS does not seem to involve calcium fluxes: (i) no change in [Ca 2+] i was detectable in TDM-primed macrophages loaded with Fura-2 and exposed to LPS, and (ii) activation of TDM-primed macrophages by LPS can be obtained in the presence of 4 m M EGTA. NO synthase expression is thus controlled in primed macrophages by two different pathways; calcium ionophores can replace LPS but do not act through the same intracellular cascade.

Role of Acetylsalicylic Acid in Cytokine Stimulation of Macrophages in Antibody-Dependent Cellular Cytotoxicity (ADCC)

In addition to the spectrum of biological action already known to be exhibited by acetylsalicylic acid (ASA) as an analgesic, anti-inflammatory and platelet aggregation inhibitor, there is growing evidence of a stimulatory effect on the immune system. ASA has been found to increase the production ofcytokines and to increase the activity of various leukocytes. The action of ASA on the activity of mouse peritoneal macrophages was therefore investigated in the present study. Therapeutically effective concentrations of ASA, which are known to decrease levels of prostaglandins, had neither a stimulating nor an inhibiting influence on antibody-dependent cellular cytotoxicity (ADCC) or on the binding capacity of macrophages with regard to SW 948 tumour cells. Likewise ASA had little or no adverse effect on the capacity of the macrophages for stimulation by interferon-gamma (IFN-gamma) and interleukin-4 (IL-4). Taken together, the immunostimulant effect of ASA shown in the literature as an increased production of interleukin-2 (IL-2) and IFN, could not be confirmed on the basis of the macrophage cytotoxiclty.

Activation of Fc receptor-mediated phagocytosis by mouse peritoneal macrophages following the intraperitoneal administration of liposomes.

The effects of liposomes on the phagocytic activity of mouse peritoneal macrophages were investigated using IgG-opsonized sheep red blood cells (SRBC). The highest ingestion index of opsonized SRBC via Fc receptors of macrophages from BALB/c mice was observed for macrophages harvested on day 4 following the intraperitoneal injection of liposomes (2.27 mumol lipid/mouse). An increase in the ingestion index was observed irrespective of liposomal charge. Binding parameters of Fc receptors of macrophages from liposome- or saline-injected mice were determined using horseradish peroxidase-conjugated IgG, and an increase in the number of binding sites with the same binding constant was observed in macrophages from liposome-injected mice. The activation mechanism of mouse peritoneal macrophages by liposomes differed from that by lipopolysaccharides. Liposomes thus appear to contribute to the activation of immune response.

Mechanisms of suppression of macrophage nitric oxide release by transforming growth factor beta.

Activated mouse peritoneal macrophages produce nitric oxide (NO) via a nitric oxide synthase that is inducible by interferon gamma (IFN-gamma): iNOS. We have studied the mechanisms by which transforming growth factor beta 1 (TGF-beta) suppresses IFN-gamma-stimulated NO production. TGF-beta treatment reduced iNOS specific activity and iNOS protein in both cytosolic and particulate fractions as assessed by Western blot with monospecific anti-iNOS immunoglobulin G. TGF-beta reduced iNOS mRNA without affecting the transcription of iNOS by decreasing iNOS mRNA stability. Even after iNOS was already expressed, TGF-beta reduced the amount of iNOS protein. This was due to reduction of iNOS mRNA translation and increased degradation of iNOS protein. The potency of TGF-beta as a deactivator of NO production (50% inhibitory concentration, 5.6 +/- 2 pM) may reflect its ability to suppress iNOS expression by three distinct mechanisms: decreased stability and translation of iNOS mRNA, and increased degradation of iNOS protein. This is the first evidence that iNOS is subject to other than transcriptional regulation.

Adenosine and a related carbocyclic nucleoside analogue selectively inhibit tumor necrosis factor-alpha production and protect mice against endotoxin challenge.

Adenosine (ADO) and its structurally related analogues are known to regulate the activities of immune and inflammatory cells, including a number of key functions of mononuclear phagocytes. In this study ADO and the synthetic ADO analogue MDL201112 inhibited TNF-alpha, but not IL-1, production by activated mouse peritoneal macrophages and the macrophage-like cell lines J774 and RAW-264. Northern blot analysis indicated that MDL201112 selectively inhibited the expression of steady-state TNF-alpha RNA in LPS+IFN-gamma-activated J774 and RAW-264 cells. This effect could not be attributed to changes in TNF-alpha RNA stability. In contrast, ADO had no effect on RNA levels for TNF-alpha and IL-1, suggesting that ADO acts at a post-transcriptional biosynthetic step. To determine whether either compound inhibited TNF-alpha-mediated inflammatory responses, mice were treated with ADO or MDL201112 and challenged with a lethal dose of endotoxic LPS and D-galactosamine, an hepatotoxin that sensitizes mice to lethal LPS challenge. A single i.p. injection of MDL201112 (100 mg/kg) protected over 90% of the mice, whether injected 1 h before or at the time of LPS challenge. MDL201112 also inhibited the appearance of TNF-alpha in the serum of LPS-challenged animals. The compound did not block D-galactosamine sensitization nor did it prevent lethality caused by the injection of rTNF-alpha. ADO failed to protect animals against endotoxin lethality, most likely due to the rapid metabolism of the nucleoside in vivo. These results establish ADO and MDL201112 as potent inhibitors of TNF-alpha biosynthesis and suggest that MDL201112 or similar analogues warrant further study as potential agents for the treatment of endotoxin shock and other diseases in which TNF-alpha plays an important pathogenic role.

Identification of phospholipase D (PLD) activity in mouse peritoneal macrophages.

It is now believed that PLD may contribute to the sustained generation of diacylglycerol (DAG) within activated cells. DAG can be formed from phosphatidylcholine by the sequential actions of PLD and phosphatidic acid phosphohydrolase. Phorbal myristate acetate (PMA, 1 microM), A23187 (10 microM) or platelet-activating factor (PAF, 100 nM) caused significant enhancement of intracellular 14C-phosphatidic acid levels 2-5 min after the addition of stimulus, in cultures of peritoneal macrophages pre-labelled with 14C-palmitate. Bacterial lipopolysaccharide (LPS) (5 micrograms/ml) or zymosan (375 micrograms/ml) also stimulated the production of 14C-phosphatidic acid, but over a longer time course (15-60 min). In the presence of 1% ethanol each stimulus caused significant production of 4C-phosphatidylethanol at the expense of 14C-phosphatidic acid, thus confirming a contribution of PLD in these reactions. This is the first report of PLD activity in this cell type.

Tumoricidal activity of mouse peritoneal macrophages treated with Viscum album extract.

Macrophages from mice treated with Viscum album extract were shown to be active in inhibiting the proliferation of tumour cells in culture. These activated macrophages have now been shown to protect mice from dying of progressive tumours when injected intraperitoneally into the animals. Prophylactic as well as multiple treatments with macrophages activated with Viscum album extract seemed more effective than a single treatment. The present finding suggests that in addition to a direct cytotoxic effect of Viscum album extract, the activation of macrophages may contribute to the overall antitumor activity of the drug.

Studies on thermophile products. VI. Activation of mouse peritoneal macrophages by bis(2-hydroxyethyl) trisulfide.

The biological effects of a cytotoxic substance (BS-1), isolated from Bacillus stearothermophilus UK563 and identified as bis(2-hydroxyethyl) trisulfide, on elicited mouse peritoneal macrophages induced by a casein injection, were investigated in vitro. BALB/c mouse macrophages treated or pretreated with BS-1 (1-10 micrograms/ml) showed cytotoxicity against syngeneic DBA/2 mouse P815 mastocytoma. BS-1 also showed weak cytotoxicity directly against P815 in the absence of macrophages. BS-1 significantly increased the glucose consumption of macrophages without producing cytotoxicity. This trisulfide compound increased nitric oxide formation, interleukin-1 production and prostaglandin E2 release in macrophages. It did not, however, increase the production of active oxygen species in macrophages, but it reduced cytochrome c in the presence of phagocytes. These results indicate that BS-1 activates macrophages to the cytolytic stage.