Abilities of activated macrophages to manifest tumoricidal activity and to generate reactive nitrogen intermediates: a comparative study in vitro and ex vivo

The abilities of lymphokines and heat-killed bacteria to induce and to maintain tumoricidal activity and/or the secretion of reactive nitrogen intermediates (RNI) were comparatively assessed in bone marrow-derived mononuclear phagocytes (BMM phi) in vitro and in adherent peritoneal cells (APC) ex vivo. In showing that the kinetics of tumoricidal activity and of secretion of RNI induced by macrophage-activating agents in BMM phi and/or in peritoneal cells do largely parallel each other, the present findings provide evidence for a role of RNI in tumor cell killing by activated macrophages both in vitro and in vivo.     

The interaction of macrophages and bacteria: Escherichia coli species, bacterial lipopolysaccharide, and lipid A differ in their ability to induce tumoricidal activity and the secretion of reactive nitrogen intermediates in macrophages.

The ability of nine Escherichia coli strains, and of bacterial lipopolysaccharide (LPS)3 and lipid A preparations, to elicit in a pure population of bone marrow-derived mononuclear phagocytes (BMM phi) tumoricidal activity and/or the generation of reactive nitrogen intermediates (RNI) was compared. Generally, low concentrations of E. coli organisms were able to trigger the generation of RNI: however, for induction of tumoricidal activity, higher concentrations were required. Nonisogenic E. coli species exhibited different ability; isogenic E. coli organisms that differed only in the expression of K antigen exhibited similar ability to elicit the macrophage activities. LPS proved to be highly efficient in triggering the secretion of reactive nitrogen intermediates; lipid A was clearly less potent, but evidence is presented to suggest that this was due to the diminished solubility of these reagents. On the other hand, all LPS and lipid A samples were very poor inducers of tumoricidal activity. Although RNI secretion and expression of tumoricidal activity are both strongly dependent on L-arginine, various evidence suggests that the two functions are not closely correlated and are induced by different bacterial structures.     

Tumor necrosis factor-alpha-dependent production of reactive nitrogen intermediates mediates IFN-gamma plus IL-2-induced murine macrophage tumoricidal activity.

We have previously established that IFN-gamma plus IL-2 induces murine macrophage tumoricidal activity. The purpose of this study was to identify the effector molecules that account for the IFN-gamma plus IL-2-induced macrophage cytotoxicity against P815 mastocytoma cells. ANA-1 macrophages and normal thioglycollate-elicited mouse peritoneal macrophages produced little or no detectable nitrite (NO2-) after incubation with IFN-gamma alone or IL-2 alone; however, IL-2 synergized with IFN-gamma for the production of NO2-. IFN-gamma plus IL-2 did not induce NO2- production or tumoricidal activity in ANA-1 macrophages that were cultured in medium devoid of L-arginine or in ANA-1 macrophages that were incubated with NG-monomethyl-L-arginine. As observed previously with ANA-1 macrophage tumoricidal activity, IL-4 inhibited IFN-gamma plus IL-2-induced, but not IFN-gamma plus LPS-induced, NO2- production. IL-4 also selectively decreased the ability of IFN-gamma and/or IL-2 to augment TNF-alpha mRNA expression in ANA-1 macrophages. Lastly, incubation of ANA-1 macrophages with anti-TNF mAb selectively inhibited the ability of IFN-gamma plus IL-2 to induce NO2- production and tumoricidal activity. These results indicate that IFN-gamma plus IL-2-induced tumoricidal activity is dependent upon the metabolism of L-arginine to reactive nitrogen intermediates, and they establish a role for TNF-alpha as a required intermediate for IL-2-dependent NO2- production and tumoricidal activity.     

Interferon-gamma-treated murine macrophages inhibit growth of tubercle bacilli via the generation of reactive nitrogen intermediates

Murine peritoneal macrophages were isolated and their ability to restrict growth of a virulent Mycobacterium tuberculosis in response to IFN-gamma was assessed in various conditions. Doses of IFN-gamma ranging from 10 to 100 U stimulated high levels of antimycobacterial activity, as seen by inhibition of growth. Addition of catalase, superoxide dismutase, and other scavengers of reactive oxygen species before infection failed to abrogate this restriction of growth, suggestive of a lack of involvement of reactive oxygen species in this phenomenon. Addition of arginase before infection inhibited the bacteriostatic ability of IFN-gamma-pulsed macrophages as did addition of NG-monomethyl L-arginine, an inhibitor of the synthesis of inorganic nitrogen oxide. In both cases, this inhibition was reversed by adding excess L-arginine in the medium. Moreover, nitrite production in macrophages was correlated with their ability to restrict tubercle bacilli growth. These results imply that nitric oxide or another inorganic nitrogen oxide is an important effector molecule in restricting growth of M. tuberculosis in IFN-gamma-pulsed murine macrophages.     

The macrophage response to bacteria: flow of L-arginine through the nitric oxide and urea pathways and induction of tumoricidal activity

The consequences of the interaction of heat-killed bacteria and lipopolysaccharide (LPS) with a pure population of bone marrow-derived mononuclear phagocytes (BMM?) were investigated, utilizing changes in the flow of L-arginine and expression of tumoricidal activity as parameters of macrophage (m?) function. Gram negative bacteria and LPS proved potent in inducing the flow of L-arginine through the nitric oxide and the urea pathways but were mostly poor in eliciting tumoricidal activity. Gram positive bacteria affected the metabolism of L-arginine only little but were often efficient in triggering tumoricidal activity. The findings show that the m? response to bacteria, which may determine the outcome of their interaction with the host, may differ considerably depending on the type of bacteria.     

Localized reactive oxygen and nitrogen intermediates inhibit escape of Listeria monocytogenes from vacuoles in activated macrophages

Listeria monocytogenes (Lm) evades being killed after phagocytosis by macrophages by escaping from vacuoles into cytoplasm. Activated macrophages are listericidal, in part because they can retain Lm in vacuoles. This study examined the contribution of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) to the inhibition of Lm escape from vacuoles. Lm escaped from vacuoles of nonactivated macrophages within 30 min of infection. Macrophages activated with IFN-gamma, LPS, IL-6, and a neutralizing Ab against IL-10 retained Lm within the vacuoles, and inhibitors of ROI and RNI blocked inhibition of vacuolar escape to varying degrees. Measurements of Lm escape in macrophages from gp91(phox-/-) and NO synthase 2(-/-) mice showed that vacuolar retention required ROI and was augmented by RNI. Live cell imaging with the fluorogenic probe dihydro-2',4,5,6,7,7'-hexafluorofluorescein coupled to BSA (DHFF-BSA) indicated that oxidative chemistries were generated rapidly and were localized to Lm vacuoles. Chemistries that oxidized DHFF-BSA were similar to those that retained Lm in phagosomes. Fluorescent conversion of DHFF-BSA occurred more efficiently in smaller vacuoles, indicating that higher concentrations of ROI or RNI were generated in more confining volumes. Thus, activated macrophages retained Lm within phagosomes by the localization of ROI and RNI to vacuoles, and by their combined actions in a small space     

Separation of soluble amoebicidal and tumoricidal activity of activated macrophages.

Macrophage-conditioned medium (M phi CM) prepared from mouse peritoneal macrophages activated in vivo with bacillus Calmette-Guérin (BCG) or Propionibacterium acnes and triggered with lipopolysaccharide in vitro contained tumoricidal and amoebicidal activity. The murine fibroblast cell line L929 was used as the indicator of tumoricidal activity and Naegleria fowleri amoeba was used to detect amoebicidal activity in M phi CM. The protease inhibitor, soybean trypsin inhibitor, decreased tumoricidal activity but had little effect on amoebicidal activity in M phi CM. Anti-TNF alpha antiserum inhibited tumoricidal activity in M phi CM. The antiserum reduced amoebicidal activity in BCG-activated M phi CM but had no effect on amoebicidal activity in P. acnes-activated M phi CM. Recombinant TNF alpha, rIL-1 alpha, or rIL-1 beta independently did not affect cytolysis of amoebae. Also, rTNF alpha had no effect on the growth of amoebae. Preparative flat-bed electrofocusing of BCG-activated M phi CM yielded fractions that exhibited different amoebicidal and tumoricidal activity profiles. Three domains of activity were analyzed (acidic, neutral, and basic). Anti-TNF alpha antiserum eliminated tumoricidal activity, but not amoebicidal activity, in fractions from the acidic domain. A combination of anti-TNF alpha and anti-IL-1 alpha antisera failed to eliminate amoebicidal activity in fractions from the basic domain. These results indicate that different factors are responsible for macrophage amoebicidal and tumoricidal activity. The amoebicidal factors in M phi CM affected cytolysis of several species of amoebae.     

Disappearance and reappearance of resident macrophages: importance in C. parvum-induced tumoricidal activity

We have investigated the role of resident macrophages in the early tumoricidal response to C. parvum. The bacteria were labeled with FITC and resident cells were labeled in situ with blue fluorescent covaspheres to enable subsequent monitoring of cellular changes by flow cytometry. Macrophages disappeared within 5 hr of administration of bacteria. At 24 hr, fibrinous adhesions containing double labeled macrophages were observed at numerous sites on the peritoneum. Macrophages associated with large numbers of bacteria, levels of beads similar to control animals, and elevated plasminogen activator-like activity did not reappear in washings in significant numbers until 72 hr. Thus, the large bacteria-containing cells that account for the majority of the early tumoricidal activity are likely to be derived from resident macrophages.     

Response of Mycobacterium tuberculosis to reactive oxygen and nitrogen intermediates.

BACKGROUND: Mycobacterium tuberculosis is a significant human pathogen capable of replicating in mononuclear phagocytic cells. Exposure to reactive oxygen and nitrogen intermediates is likely to represent an important aspect of the life cycle of this organism. The response of M. tuberculosis to these agents may be of significance for its survival in the host. MATERIALS AND METHODS: Patterns of de novo proteins synthesized in M. tuberculosis H37Rv exposed to compounds that generate reactive oxygen and nitrogen intermediates were studied by metabolic labeling and two-dimensional electrophoresis. RESULTS: Menadione, a redox cycling compound which increases intracellular superoxide levels, caused enhanced synthesis of seven polypeptides, six of which appeared to be heat shock proteins. Chemical release of nitric oxide induced eight polypeptides of which only one could be identified as a heat shock protein. Nitric oxide also exhibited a mild inhibitory action on general protein synthesis in the concentration range tested. Hydrogen peroxide did not cause differential gene expression and exerted a generalized inhibition in a dose-dependent manner. Cumene hydroperoxide caused mostly inhibition but induction of two heat shock proteins was detectable. CONCLUSIONS: The presented findings indicate major differences between M. tuberculosis and the paradigms of oxidative stress response in enteric bacteria, and are consistent with the multiple lesions found in oxyR of this organism. The effect of hydrogen peroxide, which in Escherichia coli induces eight polypeptides known to be controlled by the central regulator oxyR, appears to be absent in M. tuberculosis. Superoxide and nitric oxide responses, which in E. coli overlap and are controlled by the same regulatory system soxRS, represent discrete and independent phenomena in M. tuberculosis.     

Roles of reactive nitrogen intermediates and transforming growth factor-beta produced by immunosuppressive macrophages in the expression of suppressor activity against T cell proliferation induced by TCR stimulation

The suppressor activity of splenic macrophages induced by Mycobacterium intracellulare infection (MI-M phi s) against T cell concanavalin A (Con A) mitogenesis is mediated by MI-M phi's mediators, such as reactive nitrogen intermediates (RNIs), phosphatidylserine, free fatty acids, prostaglandin E(2) and to a minor extent TGF-beta. Here, we have compared the roles of RNIs and TGF-beta in the expression of MI-M phi's suppressor activity against Con A mitogenesis and anti-CD3 monoclonal antibody (mAb)- and anti-CD28 mAb-induced mitogenesis (TCR signal-induced mitogenesis) of the target T cells, and have found the following. First, N(G)-monomethyl-L-arginine (NMMA) inhibited MI-M phi's suppressor activity against TCR signal-induced mitogenesis as well as Con A mitogenesis. Second, anti-TGF-beta mAb weakly restored the MI-M phi-mediated suppression only in the case of Con A mitogenesis, under limited conditions, such as very low cell densities of MI-M phi s. Third, the blocking effects of NMMA plus anti-TGF-beta mAb were somewhat more prominent in the case of Con A mitogenesis than in the case of TCR signal-induced mitogenesis. Fourth, Con A- or TCR signal-stimulated MI-M phi s secreted significant amounts of the latent TGF-beta but not the active one. These findings indicate that RNIs, but not TGF-beta, play important roles in the MI-M phi-mediated suppression of TCR signal-induced mitogenesis, as well as Con A mitogenesis, of the target T cells.     

Phagocytosis of bacteria by macrophages: changing the carbohydrate of lipopolysaccharide alters interaction with complement and macrophages

Salmonella transductants and recombinants differing the O-antigenic side chain of their lipopolysaccharide are taken up at different rates by the murine macrophage-like cell line J774. Bacteria containing abequose, mannose, rhamnose, and galactose in O-antigenic side chain were taken up at the slowest rate; the one containing tyvelose instead of abequose was taken up at an intermediate rate; and the one containing mannose, N-acetylglucosamine, and glucose, instead of the above sequence, was taken up at the highest rate. These rates correlate well with the known virulence of these strains; the most virulent is the one taken up slowest, the one taken up at an intermediate rate is less virulent, and the one taken up fastest is the least virulent. The differences in ingestion rates reflect differences in affinity of the bacteria for the macrophages and not in the rate of ingestion once interaction has occurred, suggesting a receptor-mediated process. The majority of uptake is probably dependent on complement, as shown by the requirement for a serum component(s) destroyed by heating at 56 degrees C or by incubation with zymosan. Specific antibody is not required. We therefore postulate that relative virulence in vivo may reflect the relative ability of the polysaccharide of bacterial lipopolysaccharide to activate complement, thus determining the susceptibility of the bacteria to ingestion via the complement receptor of phagocytic cells.     

NADH-dependent microsomal interaction with ferric complexes and production of reactive oxygen intermediates

The interaction of NADPH with ferric complexes to catalyze microsomal generation of reactive oxygen intermediates has been well studied. Experiments were carried out to characterize the ability of NADH to interact with various ferric chelates to promote microsomal lipid peroxidation and generation of .OH-like species. In the presence of NADH and iron, microsomes produced .OH as assessed by the oxidation of a variety of .OH scavenging agents. Rates of NADH-dependent .OH production were 50 to 80% those of the NADPH-catalyzed reaction. The oxidation of dimethyl sulfoxide or t-butyl alcohol was inhibited by catalase and competitive .OH scavengers but not by superoxide dismutase or carbon monoxide. NADH-dependent .OH production was effectively catalyzed by ferric-EDTA and ferric-diethylenetriaminepentaacetic acid (DTPA), whereas ferric-ATP and ferric-citrate were poor catalysts. All these ferric chelates were reduced by microsomes in the presence of NADH (and NADPH). H2O2 was produced in the presence of NADH in a reaction stimulated by the addition of ferric-EDTA, consistent with the increase in .OH production. The latter appeared to be limited by the rate of H2O2 generation rather than the rate of reduction of the ferric chelate. NADH-dependent lipid peroxidation was much lower than the NADPH-catalyzed reaction and showed an opposite response to catalysis by ferric complexes compared to .OH generation as production of thiobarbituric acid-reactive material was increased with ferric-ATP and -citrate, but not with ferric-EDTA or- DTPA, and was not affected by catalase, SOD, or .OH scavengers. These results indicate that NADH can support microsomal reduction of ferric chelates, with the subsequent production of .OH-like species and peroxidation of lipids. The pattern of response of the NADH-dependent reactions with respect to catalytic effectiveness of ferric chelates and sensitivity to radical scavengers is similar to that found with NADPH. Many of the metabolic actions of ethanol have been ascribed to production of NADH as a consequence of oxidation by alcohol dehydrogenase. Since the cytosol normally maintains a highly oxidized NAD+/NADH redox ratio, it is interesting to speculate that increased availability of NADH from the oxidation of ethanol may support microsomal reduction of iron complexes, with the subsequent generation of reactive oxygen intermediates.     

The role of reduced pterins in resistance to reactive oxygen and nitrogen intermediates in the protozoan parasite Leishmania

During its life cycle, the protozoan parasite Leishmania experiences oxidative stress when interacting with macrophages. Reduced pterins are known scavengers of reactive oxygen and nitrogen intermediates. Leishmania has a pteridine reductase, PTR1, whose main function is to provide reduced pterins. We investigated the role of PTR1 in resistance to oxidative and nitrosative stress in Leishmania tarentolae, Leishmania infantum, and Leishmania major PTR1^?/? mutants. The PTR1^?/? cells of the three species were more sensitive to H₂O₂- and NO-induced stress. Using a fluorescent probe allowing ROI quantification, we demonstrated an increase in intracellular oxidant molecules in the PTR1^?/? mutants. The disruption of PTR1 increased metacyclogenesis in L. infantum and L. major. We purified metacyclic parasites from PTR1^?/? mutants and control cells and tested their intracellular survival in the J774 mouse cell line and in human monocyte-derived macrophages. Our results showed that PTR1^?/? null mutants survived less in both macrophage models compared to control cells and this decrease was more pronounced in macrophages activated for oxidant production. This study demonstrates that one physiological role of reduced pterins in Leishmania is to deal with oxidative and nitrosative species, and a decreased ability to provide reduced pterins leads to decreased intracellular survival.     

Up-regulation of NK cytotoxic activity via IL-15 induction by different viruses: a comparative study.

IL-15 is a recently identified cytokine that belongs to the four alpha-helix bundle cytokine family and possesses biological activities similar to those of IL-2. Its ability to induce effectors of NK activity suggests its involvement in innate immunity. In this study, we analyzed the effect of different viruses (HSV, EBV, respiratory syncitial virus, vesicular stomatitis virus, influenza virus, reovirus, and Sendai virus) on the up-regulation of NK activity in vitro. Exposure of human PBMC to the these viruses resulted in an immediate up-regulation of NK activity of PBMC via IL-15 induction; this effect was abrogated in the presence of mAbs to IL-15. Results of experiments conducted in parallel using mAbs to IL-15, as well as to other cytokines (IL-2, IL-12, IFN-gamma, and TNF-alpha), clearly indicated that IL-15 was specifically responsible for the observed effect. Furthermore, supernatants of virus-infected PBMC cultures significantly enhanced NK activity of uninfected PBMC in vitro. An increase of IL-15 protein levels 20 h postinfection was also confirmed in a bioassay using the IL-2-dependent cell line CTLL. Kinetic analysis of IL-15 mRNA expression using a semiquantitative RT-PCR revealed that the level of IL-15 messages peaked at different time points (up to 12 h) postinfection, depending on the nature of the virus. Taken together, these results suggest that the IL-15 response of the host to viral infection and the subsequent NK cell activation represent an important effector mechanism of the innate immune surveillance of the host against viral infections.     

Growth inhibition of Francisella tularensis live vaccine strain by IFN-gamma-activated macrophages is mediated by reactive nitrogen intermediates derived from L-arginine metabolism.

We have examined the abilities of the recombinant murine lymphokines IFN-gamma, granulocyte-macrophage (GM)-CSF, and IL-4 to stimulate the in vitro antimicrobial activity of macrophages against the live vaccine strain (LVS) of Francisella tularensis. Resident peritoneal macrophages from C57BL/6 strain mice were cultured overnight with IFN-gamma, GM-CSF, or IL-4, and then infected with LVS. In macrophages treated with IFN-gamma, the growth of LVS was suppressed by a factor of 100- to 1000-fold in comparison with untreated cells. This effect was dose-dependent and was enhanced by the addition of LPS. In contrast, macrophages treated with either GM-CSF or IL-4 exhibited no such enhanced antitularemic activity, even in the presence of LPS. Because reactive nitrogen intermediates derived from L-arginine metabolism have been implicated in the killing of various infectious organisms, we evaluated the possibility that such a mechanism might contribute to the antitularemic activity of IFN-gamma-stimulated macrophages. Macrophages were treated with NG-monomethyl-L-arginine (NMMA), an inhibitor of L-arginine metabolism in mammalian cells, during the activation procedure and throughout the course of infection. NMMA had no effect on the growth of LVS in unstimulated macrophages. In macrophages activated with IFN-gamma, however, NMMA suppressed their capacity to inhibit LVS growth. This effect was proportional to the dose of NMMA added and reversible by supplementing the medium with additional L-arginine, and there was a direct correlation between the production of nitrite by activated macrophages and their ability to inhibit LVS growth. Furthermore, the growth of LVS was inhibited by nitrogen metabolites in a cellfree system. The results of this study indicate that the mechanism of action of IFN-gamma on the resistance of macrophages to LVS growth is related, at least in part, to the production of reactive nitrogen metabolites.     

Binding of activated macrophages to tumor cells through a macrophage lectin and its role in macrophage tumoricidal activity

A 45-60 kDa Gal/GalNAc-specific macrophage lectin was found to participate in the interaction between tumor cells and tumoricidal macrophages activated by an antitumor streptococcal preparation, OK-432, and in the tumoricidal activity of the activated macrophages. The binding between OK-432-elicited activated macrophages and murine mastocytoma P-815 cells was inhibited on preincubation of the macrophages with a neoglycoprotein (Gal-BSA) or a complex-type glycopeptide (unit B) which was a specific inhibitor of the macrophage lectin. This binding of the macrophages to P-815 cells was also inhibited on the addition of anti-macrophage lectin antiserum. Contrary to the case of OK-432-elicited macrophages, the binding of thioglycolate-elicited (responsive) macrophages to P-815 cells was inhibited only a little by Gal-BSA and unit B, and not inhibited by the antiserum. Furthermore, the tumoricidal activity of the activated macrophages was inhibited by the addition of the anti-macrophage lectin antiserum. These results suggest that the binding of activated macrophages to tumor cells through the Gal/GalNAc-specific macrophage lectin is an important part of the tumor cell killing mechanism.     

Induction of the tumoricidal activity of human and murine peritoneal macrophages under the action of antitumor chemical preparations

Platidiam, cyclophosphamide and adriamycin induced tumoricidal activity of peritoneal macrophages from patients with disseminated ovarian carcinoma when applied in the autologous tumor cells in vitro. This effect was not observed with 10 micrograms/ml concentration of 5-fluorouracil. The mice peritoneal macrophages after incubation in vitro with 0.01-1.0 micrograms/ml of aclarubicin showed cytostatic action on syngeneic and semisyngeneic P388 cells. The peritoneal macrophages from mice treated with 2.5 mu/kg of aclarubicin intraperitoneally 1-4 days before were cytotoxic for tumor cells too.     

Discovery of a novel Toxoplasma gondii conoid-associated protein important for parasite resistance to reactive nitrogen intermediates

Toxoplasma gondii modifies its host cell to suppress its ability to become activated in response to IFN-γ and TNF-α and to develop intracellular antimicrobial effectors, including NO. Mechanisms used by T. gondii to modulate activation of its infected host cell likely underlie its ability to hijack monocytes and dendritic cells during infection to disseminate to the brain and CNS where it converts to bradyzoites contained in tissue cysts to establish persistent infection. To identify T. gondii genes important for resistance to the effects of host cell activation, we developed an in vitro murine macrophage infection and activation model to identify parasite insertional mutants that have a fitness defect in infected macrophages following activation but normal invasion and replication in naive macrophages. We identified 14 independent T. gondii insertional mutants out of >8000 screened that share a defect in their ability to survive macrophage activation due to macrophage production of reactive nitrogen intermediates (RNIs). These mutants have been designated counter-immune mutants. We successfully used one of these mutants to identify a T. gondii cytoplasmic and conoid-associated protein important for parasite resistance to macrophage RNIs. Deletion of the entire gene or just the region encoding the protein in wild-type parasites recapitulated the RNI-resistance defect in the counter-immune mutant, confirming the role of the protein in resistance to macrophage RNIs.