Killing of Bacillus spores is mediated by nitric oxide and nitric oxide synthase during glycoconjugate–enhanced phagocytosis

Nitric oxide (NO) is a signaling and defense molecule of major importance. NO endows macrophages with bactericidal, cytostatic as well as cytotoxic activity against various pathogens. Bacillus spores can produce serious diseases, which might be attenuated if macrophages were able to kill the spores on contact. Present research was carried out to study whether glycoconjugates stimulated NO and nitric oxide synthase (NOS2) production during phagocytosis killing of Bacillus spores. Murine macrophages exposed to glycoconjugate-treated spores induced NOS2 and NO production that was correlated with high viability of macrophages and killing rate of bacterial spores. Increased levels of inducible NOS2 and NO production by macrophages in presence of glycoconjugates suggested that the latter provide an activation signal directed to macrophages. Glycoconjugates were shown to exert a protective influence, sparing macrophages from spore-induced cell death. In presence of glycoconjugates, macrophages efficiently kill the organisms. Without glycoconjugate activation, murine macrophages were ineffective at killing Bacillus spores. These results suggest that glycoconjugates promote killing of Bacillus spores by blocking spore-induced macrophage cell death, while increasing their activation level and NO and NOS2 production. Glycoconjugates suggest novel antimicrobial approaches to prevention and treatment of infection caused by bacterial spores.     

Phagocytosis of Leishmania enhances macrophage activation by IFN-gamma and lipopolysaccharide

This investigation describes the ability of Leishmania promastigotes to enhance activation of bone marrow-derived murine macrophages in vitro if added together with rIFN-gamma in the presence or absence of LPS. Activation was defined as the capacity for arginine-derived NO2- production and the killing of intracellular Leishmania. Enhanced NO2- production was observed for either CBA or C3H/HeJ macrophages undergoing phagocytosis at the time of activation. Other phagocytic stimuli including inert polystyrene latex beads were as effective as Leishmania. No correlation could be demonstrated between the enhanced NO2- release and secretion of products of the respiratory burst or PGE2. However, TNF-alpha secretion was elevated in cultures undergoing phagocytosis and a relationship between hexosemonophosphate shunt activity and NO2- levels was evident. These studies confirm and extend previous reports that phagocytosis plays an important role in the regulation of macrophage physiology.     

Macrophage cytotoxicity against Entamoeba histolytica trophozoites is mediated by nitric oxide from L-arginine.

The killing of Entamoeba histolytica trophozoites by phagocytes involves oxidative and nonoxidative mediators. In this study, we determine whether L-arginine-derived nitric oxide (NO) is involved in the killing of E. histolytica trophozoites by activated murine macrophages in vitro. Elicited peritoneal and bone marrow-derived macrophages activated with IFN-gamma alone or with IFN-gamma and LPS killed 62 to 73% of amebae, concomitant with increased levels of nitrate (NO2). Depletion of L-arginine by addition of arginase to culture medium abrogated macrophage amebicidal activity. NG-monomethyl L-arginine, an L-arginine analog, competitively inhibited NO2 release and amebicidal activity in a dose-dependent fashion, without affecting H2O2 production; however, the addition of excess L-arginine competitively restored macrophage amebicidal effects. In culture, sodium nitrite and sodium nitroprusside were cytotoxic to E. histolytica and this was reversed by the addition of myoglobin. Exogenously added FeSO4 prevented macrophage cytotoxicity. Addition of superoxide dismutase, a scavenger of O2-, partially inhibited amebicidal activity, without influencing NO2 production. Untreated and LPS-exposed macrophages produced high levels of H2O2 independent from NO2 production and amebicidal effects. However, the addition of catalase, a scavenger of H2O2, inhibited both amebicidal activity and NO2 production by activated macrophages. Our results demonstrate that NO is the major cytotoxic molecule released by activated macrophages for the in vitro cytotoxicity of E. histolytica and that O2- and H2O2 may be cofactors for the NO effector molecule.     

Nitric oxide production by Leishmania-infected macrophages and modulation by cytokines and prostaglandins

Nitric oxide (NO) produced by an inducible nitric oxide synthase (iNOS or NOS2) plays a major microbicidal role in murine macrophages and its importance is now emerging also in the dog and human models. In dogs we demonstrated that macrophages in vitro infected with Leishmania infantum produced NO, after stimulation with cytokine-enriched peripheral blood mononuclear cell supernatants. In addition, parasite killing was reduced by the NOS inhibitor L-NG monomethylarginine. On the contrary, canine blood monocytes before macrophage differentiation did not release NO, and their leishmanicidal activity was instead correlated with superoxide anion and interferon (IFN)-gamma production. In human macrophage cultures, after infection with Leishmania infantum, we showed both iNOS expression by immunofluorescence and western blotting and NO release by the Griess reaction for nitrites. Various cytokines and prostaglandins can differently modulate NO synthesis. In our experiments, stimulation by recombinant human IFN-gamma and bacterial lipopolysaccharide greatly enhanced iNOS expression and NO production in human macrophages. In addition, the prostaglandin E2 increased NO release in activated, Leishmania-infected human macrophages. These results are interesting in the light of a possible immunological or pharmacological regulation of NO synthesis and microbicidal functions of macrophages.     

PECAM-1 ligation negatively regulates TLR4 signaling in macrophages

Uncontrolled TLR4 signaling may induce excessive production of proinflammatory cytokines and lead to harmful inflammation; therefore, negative regulation of TLR4 signaling attracts much attention now. PECAM-1, a member of Ig-ITIM family, can mediate inhibitory signals in T cells and B cells. However, the role and the mechanisms of PECAM-1 in the regulation of TLR4-mediated LPS response in macrophages remain unclear. In this study, we demonstrate that PECAM-1 ligation with CD38-Fc fusion protein negatively regulates LPS-induced proinflammatory cytokine TNF-alpha, IL-6, and IFN-beta production by inhibiting JNK, NF-kappaB, and IFN regulatory factor 3 activation in macrophages. In addition, PECAM-1 ligation-recruited Src homology region 2 domain-containing phosphatase 1 (SHP-1) and Src homology region 2 domain-containing phosphatase 2 (SHP-2) may be involved in the inhibitory effect of PECAM-1 on TLR4 signaling. Consistently, silencing of PECAM-1 enhances the macrophage response to LPS stimulation. Taken together with the data that PECAM-1 is constitutively expressed in macrophages and its expression is up-regulated by LPS stimulation, PECAM-1 might function as a feedback negative regulator of LPS inflammatory response in macrophages. This study may provide a potential target for intervention of inflammatory diseases.     

ATP-mediated killing of intracellular mycobacteria by macrophages is a P2X(7)-dependent process inducing bacterial death by phagosome-lysosome fusion.

Mycobacterium tuberculosis survives within host macrophages by actively inhibiting phagosome fusion with lysosomes. Treatment of infected macrophages with ATP induces both cell apoptosis and rapid killing of intracellular mycobacteria. The following studies were undertaken to characterize the effector pathway(s) involved. Macrophages were obtained from p47(phox) and inducible NO synthase gene-disrupted mice (which are unable to produce reactive oxygen and nitrogen radicals, respectively) and P2X(7) gene-disrupted mice. RAW murine macrophages transfected with either the natural resistance-associated macrophage protein gene 1 (Nramp1)-resistant or Nramp1-susceptible gene were also used. The cells were infected with bacille Calmette-Guérin (BCG), and intracellular mycobacterial trafficking was analyzed using confocal and electron microscopy. P2X(7) receptor activation was essential for effective ATP-induced mycobacterial killing, as its bactericidal activity was radically diminished in P2X(7)(-/-) macrophages. ATP-mediated killing of BCG within p47(phox-/-), inducible NO synthase(-/-), and Nramp(s) cells was unaffected, demonstrating that none of these mechanisms have a role in the ATP/P2X(7) effector pathway. Following ATP stimulation, BCG-containing phagosomes rapidly coalesce and fuse with lysosomes. Blocking of macrophage phospholipase D activity with butan-1-ol blocked BCG killing, but not macrophage death. ATP stimulates phagosome-lysosome fusion with concomitant mycobacterial death via P2X(7) receptor activation. Macrophage death and mycobacterial killing induced by the ATP/P2X(7) signaling pathway can be uncoupled, and diverge proximal to phospholipase D activation.     

The timing of TNF and IFN-gamma signaling affects macrophage activation strategies during Mycobacterium tuberculosis infection

During most infections, the population of immune cells known as macrophages are key to taking up and killing bacteria as an integral part of the immune response. However, during infection with Mycobacterium tuberculosis (Mtb), host macrophages serve as the preferred environment for mycobacterial growth. Further, killing of Mtb by macrophages is impaired unless they become activated. Activation is induced by stimulation from bacterial antigens and inflammatory cytokines derived from helper T cells. The key macrophage-activating cytokines in Mtb infection are tumor necrosis factor-α (TNF) and interferon (IFN)-γ. Due to differences in cellular sources and secretion pathways for TNF and IFN-γ, the possibility of heterogeneous cytokine distributions exists, suggesting that the timing of macrophage activation from these signals may affect activation kinetics and thus impact the outcome of Mtb infection. Here we use a mathematical model to show that negative feedback from production of nitric oxide (the key mediator of mycobacterial killing) that typically optimizes macrophage responses to activating stimuli may reduce effective killing of Mtb. Statistical sensitivity analysis predicts that if TNF and IFN-γ signals precede infection, the level of negative feedback may have a strong effect on how effectively macrophages kill Mtb. However, this effect is relaxed when IFN-γ or TNF+IFN-γ signals are received coincident with infection. Under these conditions, the model suggests that negative feedback induces fast responses and an initial overshoot of nitric oxide production for given doses of TNF and IFN-γ, favoring killing of Mtb. Together, our results suggest that direct entry of macrophages into a granuloma site (and not distal to it) from lung vascular sources represents a preferred host strategy for mycobacterial control. We examine implications of these results in establishment of latent Mtb infection.     

Cutting edge: purinergic signaling regulates radical-mediated bacterial killing mechanisms in macrophages through a P2X7-independent mechanism.

Signaling by extracellular nucleotides through P2 purinergic receptors affects diverse macrophage functions; however, its role in regulating antimicrobial radicals during bacterial infection has not been investigated. Mycobacterium tuberculosis-infected macrophages released ATP in a dose-dependent manner, which correlated with nitrite accumulation. P2 receptor inhibitors, including oxidized ATP, blocked NO synthase (NOSII) up-regulation and NO production induced by infection with M. tuberculosis or bacille Calmette-Guérin, or treatment with LPS or TNF-alpha. Oxidized ATP also inhibited oxygen radical production and activation of NF-kappaB and AP-1 in response to infection and inhibited NO-dependent killing of bacille Calmette-Guérin by macrophages. Experiments using macrophages derived from P2X7 gene-disrupted mice ruled out an essential role for P2X7 in NOSII regulation. These data demonstrate that P2 receptors regulate macrophage activation in response to bacteria and proinflammatory stimuli, and suggest that extracellular nucleotides released from infected macrophages may enhance production of oxygen radicals and NO at sites of infection.     

Identification and characterization of monoclonal antibodies specific for macrophages at intermediate stages in the tumoricidal activation pathway

Macrophage activation for tumor cell killing is a multistep pathway in which responsive macrophages interact sequentially with priming and triggering stimuli in the acquisition of full tumoricidal activity. A number of mediators have been identified which have activating capability, including in particular IFN-gamma and bacterial LPS. Although the synergistic functional response of normal macrophages to sequential incubation with these activation signals has been well-established, characterization of the intermediate stages in the activation pathway has been difficult. We have developed a model system for examination of various aspects of macrophage activation, through the use of the murine macrophage tumor cell line, RAW 264.7. These cells, like normal macrophages, exhibit a strict requirement for interaction with both IFN-gamma and LPS in the development of tumor cytolytic activity. In addition, these cells can be stably primed by the administration of gamma-radiation. In the studies reported here, we have used RAW 264.7 cells treated with IFN-gamma alone or with IFN-gamma plus LPS to stimulate the production of rat mAb probes recognizing cell surface changes occurring during the activation process. In this way we have identified three Ag associated with intermediate stages of the activation process. One Ag, TM-1, is expressed on RAW 264.7 cells primed by IFN-gamma or gamma-radiation. This surface Ag thus identifies cells at the primed cell intermediate stage of the tumoricidal activation pathway regardless of the mechanism of activation. A second Ag, TM-2, is expressed on IFN-treated RAW 264.7 cells but not on RAW 264.7 cells primed with gamma-radiation alone. Expression of this Ag can be induced by treatment of irradiated cells with IFN-gamma, but is not induced by IFN-gamma treatment of a noncytolytic cell line, WEHI-3. This Ag thus appears to be an IFN-inducible cell surface protein associated specifically with macrophage activation for tumoricidal activity. Finally, Ag TM-3 is detectable on RAW 264.7 cells primed by either IFN-gamma or gamma-radiation, after subsequent triggering of the primed cells with LPS. The addition of the mAb recognizing this antigen to the function assay of tumor cell killing can inhibit they lytic activity of both triggered cells. Thus, this Ag may play a role in the antitumor effector functions of activated macrophages. Overall, the results suggest that these mAb can serve as useful tools for identification of molecules associated with the process of macrophage activation for tumor cell killing.     

EPR detection of heme and nonheme iron-containing protein nitrosylation by nitric oxide during rejection of rat heart allograft.

The paramagnetic molecule nitric oxide (NO), produced from L-arginine by a specific enzyme (NO synthase), has been shown to be involved in a surprising variety of mammalian cellular responses, including the regulation of T cell immunity to alloantigens in vitro. In cytotoxic activated macrophages, NO production results in a characteristic pattern of alteration of iron-containing enzyme function that is mimicked by exposure to NO. Electron paramagnetic resonance (EPR) studies have shown the formation of iron-nitrosyl species during macrophage activation and also during sepsis, indicating that alteration of iron-containing protein function may be the result of the well-documented tendency of NO to bind to metal ions. We have recently shown that the NO synthesis induced during alloantigenic activation of rat splenocytes inhibits lymphocyte proliferation and cytotoxic T-lymphocyte generation. This report demonstrates that iron-nitrosyl EPR signals similar to those observed in macrophages and during sepsis are present in the blood and in the grafted tissue of rats during the rejection of allogeneic (but not syngeneic) heart grafts. These signals are found in the blood and at the site of allograft rejection, but are not found in other tissues (such as spleen and lung), and are obliterated by administration of the immunosuppressant FK506. These results directly demonstrate the formation of iron-nitrosyl complexes during vascularized allograft rejection and suggest that consequent destruction of iron-containing protein function plays an important role in the rejection response.     

MyD88-dependent but Toll-like receptor 2-independent innate immunity to Listeria: no role for either in macrophage listericidal activity

We have assessed the requirements for Toll-like receptor (TLR) signaling in vivo during early infection with Listeria monocytogenes. Mice deficient for TLR2, a receptor required for the recognition of Gram-positive peptidoglycan, showed equivalent Listeria resistance to wild-type mice. However, mice deficient for MyD88, an adaptor molecule used by all TLRs, showed profound susceptibility with 3-4 logs greater Listeria burden and severe spleen and liver pathology at day 3 postinfection. Listeria-infected MyD88-deficient mice also showed markedly diminished IFN-gamma, TNF-alpha, and NO responses, despite evidence of macrophage activation and up-regulation of MHC class II molecules. We demonstrate that although minor MyD88-independent responses to live Listeria do occur, these are insufficient for normal host defense. Lastly, we performed experiments in vitro in which macrophages deficient in TLR2 or MyD88 were directly infected with Listeria: Although TLR signaling was required for macrophage NO and cytokine production in response to Listeria, handling and direct killing of Listeria by activated macrophages occurred by TLR2- and MyD88-independent mechanisms.     

Both tumor necrosis factor and nitric oxide participate in lysis of simian virus 40-transformed cells by activated macrophages.

SV40 transformation of rodent fibroblasts generally produces cells that are highly sensitive to killing by activated macrophages. The cell line SV-COL-E8 (E8) is typical of SV40-transformed mouse fibroblasts in that it is readily lysed when exposed to activated macrophages. This killing is not due solely to TNF, because soluble TNF alone is incapable of lysing these cells. TNF is, however, necessary for lysis since antibodies to TNF will prevent macrophage-mediated lysis. Similarly, E8 is not sensitive to nitric oxide (NO); however, NO is also necessary for lysis since inhibition of NO generation (by coincubation with the arginine analogue NG-monomethyl-1-arginine) with Fe(II)) blocks lysis of E8 by activated macrophages. Cytolysis by macrophages is contact dependent, suggesting that the cell-associated TNF precursor may be involved in mediating cytolysis. However, transfected cell lines bearing cell-associated TNF precursor do not mediate killing of E8. Thus, killing of E8 either involves both TNF and NO in addition to a third, as yet unidentified, lytic mechanism, or killing requires the contact-dependent delivery of TNF and NO from the macrophage to its target.     

A selective role for the TNF p55 receptor in autocrine signaling following IFN-gamma stimulation in experimental autoimmune uveoretinitis

IFN-gamma stimulates macrophage activation and NO production, which leads to destruction of the retina in experimental autoimmune uveoretinitis. In this study, we investigate the mechanism of disease resistance in TNF p55 receptor-deficient animals. We show that although T cell priming is relatively unaffected, macrophages lacking the TNF p55 receptor fail to produce NO following IFN-gamma stimulation because of a requirement for autocrine TNF-alpha signaling through the TNF p55 receptor. In contrast to the impaired activation of NO synthesis, MHC class II up-regulation was indistinguishable in wild-type and TNFRp55-/- mice stimulated with IFN-gamma. These defects could be overcome by stimulating macrophages with LPS. Together, these results show that selected aspects of IFN-gamma activation are controlled by autocrine secretion of TNF-alpha, but that this control is lost in the presence of signals generated by pathogen-associated molecular patterns recognizing receptors.     

Increased mitogen-activated protein kinase activity and TNF-alpha production associated with Mycobacterium smegmatis- but not Mycobacterium avium-infected macrophages requires prolonged stimulation of the calmodulin/calmodulin kinase and cyclic AMP/protein kinase A pathways

Previous studies have shown the mitogen-activated protein kinases (MAPKs) to be activated in macrophages upon infection with Mycobacterium, and that expression of TNF-alpha and inducible NO synthase by infected macrophages was dependent on MAPK activation. Additional analysis demonstrated a diminished activation of p38 and extracellular signal-regulated kinase (ERK)1/2 in macrophages infected with pathogenic strains of Mycobacterium avium compared with infections with the fast-growing, nonpathogenic Mycobacterium smegmatis and Mycobacterium phlei. However, the upstream signals required for MAPK activation and the mechanisms behind the differential activation of the MAPKs have not been defined. In this study, using bone marrow-derived macrophages from BALB/c mice, we determined that ERK1/2 activation was dependent on the calcium/calmodulin/calmodulin kinase II pathway in both M. smegmatis- and M. avium-infected macrophages. However, in macrophages infected with M. smegmatis but not M. avium, we observed a marked increase in cAMP production that remained elevated for 8 h postinfection. This M. smegmatis-induced cAMP production was also dependent on the calmodulin/calmodulin kinase pathway. Furthermore, stimulation of the cAMP/protein kinase A pathway in M. smegmatis-infected cells was required for the prolonged ERK1/2 activation and the increased TNF-alpha production observed in these infected macrophages. Our studies are the first to demonstrate an important role for the calmodulin/calmodulin kinase and cAMP/protein kinase A pathways in macrophage signaling upon mycobacterial infection and to show how cAMP production can facilitate macrophage activation and subsequent cytokine production.     

Macrophage-colony-stimulating factor-induced proliferation and lipopolysaccharide-dependent activation of macrophages requires Raf-1 phosphorylation to induce mitogen kinase phosphatase-1 expression

Macrophages are key regulators of immune responses. In the absence of an activating signal, murine bone marrow-derived macrophages undergo proliferation in response to their specific growth factor, namely M-CSF. The addition of bacterial LPS results in macrophage growth arrest and their engagement in a proinflammatory response. Although participation of ERKs is required for both macrophage proliferation and activation, ERK phosphorylation follows a more delayed pattern in response to activating agents. In primary macrophages, mitogen kinase phosphatase-1 (MKP-1) is a key regulator of the time course of MAPK activity. Here we showed that MKP-1 expression is dependent on Raf-1 activation. The time course of Raf-1 activation correlated with that of ERK-1/2. However, whereas ERK phosphorylation in response to M-CSF is Raf-1 dependent, in response to LPS, an alternative pathway directs the activation of these kinases. Inhibition of Raf-1 activity increased the expression of cyclin-dependent kinase inhibitors and growth arrest. In contrast, no effect was observed in the expression of proinflammatory cytokines and inducible NO synthase following LPS stimulation. The data reported here reveal new insights into how signaling determines opposing macrophage functions.     

Modulation of macrophage function by gamma-irradiation. Acquisition of the primed cell intermediate stage of the macrophage tumoricidal activation pathway

Macrophage activation for tumor cell killing is a multistep pathway in which responsive macrophages interact sequentially with priming and triggering stimuli in the acquisition of full tumoricidal activity. Although this synergistic response of normal macrophages to sequential incubation with activation signals has been well established, characterization of the intermediate stages in this pathway has been difficult, due in large measure to the instability of the intermediate cell phenotypes. We have developed a model system for examination of macrophage-mediated tumor cell lysis, with the use of the murine macrophage tumor cell line RAW 264.7. These cells, like normal macrophages, exhibit a strict requirement for interaction with both interferon-gamma (IFN-gamma, the priming signal) and bacterial lipopolysaccharide (LPS, the triggering signal) in the development of tumor cytolytic activity. In this system, the priming effects of IFN-gamma decay rapidly after withdrawal of this mediator and the cells become unresponsive to LPS triggering. We have recently observed that gamma-irradiation of the RAW 264.7 cells also results in development of a primed activation state for tumor cell killing. The effects of gamma-radiation on the RAW 264.7 cell line are strikingly similar to those resulting from incubation with IFN-gamma, with the exception that the irradiation-induced primed cell intermediate is stable and responsive to LPS triggering for at least 24 hr. Treatment with gamma-radiation also results in increased cell surface expression of major histocompatibility complex-encoded class I antigens; however, class II antigen expression is not induced. Irradiation-induced development of the primed phenotype is not solely the result of cytostatic effects as treatment of the cells with a radiomimetic drug, mitomycin C, results in decreases in [3H]thymidine incorporation that are similar to those observed after irradiation, without concomitant development of cytolytic potential. In addition, priming by gamma-radiation does not appear to be mediated by the release of soluble autoregulatory factors. This alternate pathway for induction of the primed macrophage activation state should serve as a useful tool for identification of molecules important to the functional potential of primed cells, and for elucidation of the biochemical mechanisms of the priming event in tumoricidal activation.