Endogenous tumor necrosis factor alpha is required for enhanced antimicrobial activity against Toxoplasma gondii and Listeria monocytogenes in recombinant gamma interferon-treated mice.

In vitro studies have shown that macrophages stimulated with recombinant gamma interferon (rIFN-gamma) produce tumor necrosis factor alpha (TNF-alpha), which in an autocrine fashion activates these cells. The aim of the present study was to determine whether endogenously formed TNF-alpha also is required for rIFN-gamma-induced macrophage activation and enhanced antimicrobial activity in vivo. After an intraperitoneal injection of rIFN-gamma into CBA/J mice, their peritoneal macrophages released enhanced amounts of NO2- and inhibited the intracellular proliferation of Toxoplasma gondii. Injection of neutralizing antibodies against TNF-alpha simultaneously with the rIFN-gamma completely inhibited both the release of NO2- by macrophages and their toxoplasmastatic activity. Similar results were observed after intraperitoneal injection of a competitive inhibitor of L-arginine, NG-monomethyl-L-arginine, together with rIFN-gamma, demonstrating that in vivo L-arginine-derived reactive nitrogen intermediates are essential for the induction of toxoplasmastatic activity. Intravenous injection of rIFN-gamma inhibited the growth of Listeria monocytogenes in the livers and spleens of mice; this effect was abrogated by antibodies against TNF-alpha. Intravenous injection of a large dose of rTNF-alpha resulted in a decrease in the number of bacteria in the liver and spleen, but an injection of rIFN-gamma and rTNF-alpha did not result in enhanced inhibition of the proliferation of L. monocytogenes. Together, the results of the present study are the first to demonstrate that endogenous TNF-alpha is required in vivo for the expression of macrophage activation with respect to the release of reactive nitrogen intermediates and toxoplasmastatic activity and for enhanced listericidal activity in the livers and spleens of mice stimulated with rIFN-gamma.     

Mycobacterial 65-kilodalton heat shock protein induces tumor necrosis factor alpha and interleukin 6, reactive nitrogen intermediates, and toxoplasmastatic activity in murine peritoneal macrophages.

The 65-kDa heat shock protein (Hsp65) is supposed to play a role in host defense against infections with various microbial pathogens and in autoimmune inflammatory disorders. These effects are thought to result mainly from an Hsp65-specific T-lymphocyte-mediated immune response that recognizes conserved epitopes. The aim of the present study was to assess whether mycobacterial Hsp65 has a direct effect on resident murine peritoneal macrophages, independent of Hsp65-sensitized T lymphocytes. Exposure of peritoneal macrophages from naive C57BL/6 mice to the mycobacterial Hsp65 in vitro induced an enhanced release of tumor necrosis factor alpha (TNF-alpha) and interleukin 6. These cells also produced large amounts of reactive nitrogen intermediates (RNI) and inhibited the intracellular proliferation of Toxoplasma gondii. Small amounts of gamma interferon acted synergistically with Hsp65. Thus, exposure of murine macrophages to Hsp65 results in activation of these cells. The acquisition of these characteristics by peritoneal macrophages occurred in the absence of sensitized T lymphocytes. Addition of anti-TNF-alpha antiserum resulted in an attenuation of the Hsp65-induced release of RNI and toxoplasmastatic activity, indicating that endogenous TNF-alpha is involved in the Hsp65-induced macrophage activation. The conclusion of this study is that in vitro exposure of peritoneal macrophages to the mycobacterial Hsp65 induces the release of proinflammatory cytokines and RNI and results in inhibition of the intracellular proliferation of T. gondii. These effects on murine macrophages occur independently of Hsp65-specific T lymphocytes. The proinflammatory effect of Hsp65 demonstrated in this study suggests that this heat shock protein may play a role in the initiation of inflammation that adds to a non-species-specific resistance in the early stages of infections.     

Bone marrow progenitors cultured in the presence of granulocyte-macrophage colony-stimulating factor versus macrophage colony-stimulating factor differentiate into macrophages with distinct tumoricidal capacities

Bone marrow-derived cells from C3H/HeJ mice were cultured in the presence of recombinant murine granulocyte-macrophage colony-stimulating factor (rGM-CSF) or highly purified murine macrophage colony-stimulating factor (CSF-1) for 7 days. Following this 7-day culture period, mature macrophages were harvested and replated at precise densities in the absence of exogenous rGM-CSF or CSF-1, and assayed in a two-signal tumoricidal assay. Cultures were stimulated with medium only or with combinations of recombinant interferon-gamma (rIFN-gamma) as the "priming" signal, and/or butanol-extracted lipopolysaccharide (But-LPS) as the "triggering" signal for 24 hr. At this time, 51Cr-labeled, P815 tumor target cells were added, and the percent tumor cell cytotoxicity was determined after 16 hr. Macrophages derived under the influence of rGM-CSF exhibited significant tumoricidal capacity with medium alone (16 +/- 5%). The addition of "priming" signal only (i.e., rIFN-gamma, 10.0 U/ml) significantly increased tumoricidal capacity to 31 +/- 9%. Treatment with But-LPS alone did not alter the basal tumoricidal activity of rGM-CSF-derived macrophages. Combinations of rIFN-gamma (10.0 U/ml) and But-LPS (0.5-5.0 micrograms/ml) generated highly tumoricidal macrophages (50-60% tumor cell cytotoxicity). In contrast, medium-treated CSF-1-derived macrophages exhibited a significantly lower basal level of tumor cytotoxicity (6 +/- 3%). Unlike rGM-CSF-derived macrophages, treatment of CSF-1-derived macrophages with high concentrations of rIFN-gamma alone did not increase significantly the level of cytotoxicity above that of medium-treated cultures. However, CSF-1-derived macrophages responded to the highest concentrations of But-LPS (5.0 micrograms/ml) to increase tumoricidal activity from 6 +/- 3% to 17 +/- 5%. Optimal tumoricidal activity (44 +/- 17%) was observed when CSF-1-derived macrophages were treated simultaneously with high concentrations of both rIFN-gamma and But-LPS. Thus, macrophages derived from bone marrow progenitors in either rGM-CSF or CSF-1 exhibited tumoricidal capacities that differed in basal activity as well as in their requirements for and sensitivities to "priming" and "triggering" signals.     

Interferon-gamma and tumor necrosis factor induce the L-arginine-dependent cytotoxic effector mechanism in murine macrophages

We tested several monokines and muramyl dipeptide (MDP) to determine whether they induce the L-arginine-dependent effector mechanism in cultured murine macrophages. Recombinant interferon-gamma (rIFN-gamma) and recombinant tumor necrosis factor (rTNF) synergize to induce nitrite (NO2-) and nitrate (NO3-) synthesis from L-arginine as well as to cause inhibition of the iron-dependent enzyme aconitase in macrophages. Unlike rTNF, recombinant interleukin 1 (rIL 1) and rIL 6/B cell stimulatory factor 2 (rIL 6/BSF-2) did not act as cofactors when added to macrophages in the presence of rIFN-gamma. rIFN-gamma plus MDP induced the L-arginine-dependent effector mechanism in murine macrophages. However, induction by rIFN-gamma plus MDP was inhibited by anti-rTNF antibodies which suppressed both NO2-/NO3- synthesis and aconitase inhibition. This result indicates that endogenously produced TNF is involved in the induction of the L-arginine-dependent effector mechanism when MDP is the co-stimulant with rIFN-gamma. In contrast, anti-rTNF antibodies did not fully suppress the effect of combining rIFN-gamma and lipopolysaccharide, suggesting that, in this case, activation of the L-arginine-dependent effector pathway may involve more than induction of TNF synthesis by the macrophages. These results provide information, at a biochemical level, on a mechanism through which combination of IFN-gamma and TNF can modulate macrophage functions involved in the control of cell proliferation.     

Nitric Oxide Mediates the Toxoplasmastatic Activity of Murine Microglial Cells in vitro

Toxoplasma gondii (T. gondii), an opportunistic protozoan, is an important cause of central nervous system (CNS) infections in immunosuppressed patients. The present study focused on the interaction between T. gondii and microglial cells from the brain of neonatal Balb/c mice.

Preincubation of the murine microglial cells with recombinant interferon-gamma (rIFN-gM) and lipopolysaccharide (LPS) induced significant inhibition of T. gondii replication in a dose dependent manner. This antiparasitic effect in microglial cells was correlated with the induction of the L-arginine-dependent generation of reactive nitrogen intermediates (RNIs). Tumor necrosis factor-alpha (TNF-aL) was also involved in the toxoplasmastatic activity. Microglial cells incubated with recombinant TNF-aL in combination with a non-activating concentration of rIFN-gM released substantial amount of RNIs. Neutralizing antibodies against mouse TNF-aL inhibited the release of RNI by rIFN-gM activated macrophages.

In summary, the present results show that activation of microglial cells by rIFN-gM and LPS induce the production of nitric oxide (NO) by these cells via an L-arginine dependent pathway. NO appears to be the effector molecule mediating the toxoplasmastatic effects in these cells.     

Effect of IFN-gamma on the proliferation of Toxoplasma gondii in monocytes and monocyte-derived macrophages from AIDS patients.

This study was undertaken to determine whether the activity of monocytes and monocyte-derived macrophages (MDM) from acquired immune deficiency syndrome (AIDS) patients against Toxoplasma gondii is altered and whether this activity can be modulated by recombinant interferon-gamma (rIFN-gamma). Untreated and rIFN-gamma-treated monocytes or MDM from AIDS patients and from healthy controls were infected with T. gondii and the proliferation of these protozoa was determined. The H2O2 release by monocytes from AIDS patients and healthy controls was measured upon stimulation with phorbol myristate acetate (PMA) and formyl methionyl leucyl phenylalanine (FMLP). Monocytes from AIDS patients exhibited significantly lower toxoplasmastic activity compared to monocytes from healthy controls. The H2O2 release by monocytes from AIDS patients was also diminished. Incubation of monocytes from AIDS patients with rIFN-gamma for 2 days, but not 1 day, restored their toxoplasmastatic activity. The rate of proliferation of T. gondii was higher in MDM from AIDS patients than in MDM from healthy controls. Treatment of MDM from AIDS patients with rIFN-gamma for 1, 2 or 3 days resulted in partial inhibition of the proliferation of T. gondii. Collectively, these results demonstrate that the reduced toxoplasmastatic activity of monocytes and MDM from AIDS patients can be enhanced by in vitro treatment with rIFN-gamma, which supports the clinical use of rIFN-gamma for the treatment of opportunistic infections in these patients.     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) reduces toxoplasmastatic activity of human monocytes via induction of prostaglandin E2 (PGE2).

In this study we investigated the effect of human GM-CSF on the toxoplasmastatic activity and release of H2O2 and PGE2 by human monocytes. Incubation of monocytes from healthy controls with GM-CSF resulted in a dose-dependent reduction of toxoplasmastatic activity and a decrease in H2O2 production. Furthermore GM-CSF-treated monocytes released more PGE2 than untreated cells. To investigate the role of PGE2 in the reduced toxoplasmastatic activity of GM-CSF-treated monocytes, these cells were incubated with indomethacin. This resulted in a reduction of PGE2 release and restoration of toxoplasmastatic activity of monocytes treated with GM-CSF. GM-CSF reduces the toxoplasmastatic activity of monocytes via production of PGE2.     

Nitric Oxide Mediates the Toxoplasmastatic Activity of Murine Microglial Cells in vitro

Toxoplasma gondii (T. gondii), an opportunistic protozoan, is an important cause of central nervous system (CNS) infections in immunosuppressed patients. The present study focused on the interaction between T. gondii and microglial cells from the brain of neonatal Balb/c mice.

Preincubation of the murine microglial cells with recombinant interferon-gamma (rIFN-gM) and lipopolysaccharide (LPS) induced significant inhibition of T. gondii replication in a dose dependent manner. This antiparasitic effect in microglial cells was correlated with the induction of the L-arginine-dependent generation of reactive nitrogen intermediates (RNIs). Tumor necrosis factor-alpha (TNF-aL) was also involved in the toxoplasmastatic activity. Microglial cells incubated with recombinant TNF-aL in combination with a non-activating concentration of rIFN-gM released substantial amount of RNIs. Neutralizing antibodies against mouse TNF-aL inhibited the release of RNI by rIFN-gM activated macrophages.

In summary, the present results show that activation of microglial cells by rIFN-gM and LPS induce the production of nitric oxide (NO) by these cells via an L-arginine dependent pathway. NO appears to be the effector molecule mediating the toxoplasmastatic effects in these cells.     

Production of reactive nitrogen intermediates by bone marrow-derived macrophages on treatment with cisplatin in vitro.

L929 culture medium (a source of macrophage colony stimulating factor (M-CSF) or recombinant granulocyte-macrophage colony stimulating factor (rGM-CSF)-derived bone marrow macrophages treated with cisplatin or lipopolysaccharide (LPS) (10 micrograms/ml) were effective in the production of L-arginine-dependent reactive nitrogen intermediates (RNI) and generation of tumouricidal activity. The abilities of RNI secretion and related tumouricidal activity against P815 mastocytoma cells were compared. These parameters were found to be closely correlated in various experiments. RNI secretion and generation of bone marrow macrophage-mediated tumouricidal activity were significantly inhibited by L-N-monomethyl arginine (L-NMMA), a specific inhibitor of the L-arginine pathway, but L-NMMA did not inhibit macrophage-mediated killing of tumour necrosis factor (TNF)-sensitive Wehi cells, suggesting that activated macrophages exhibit at least two cytolytic mechanisms, one by L-arginine-dependent nitric oxide pathway and another by TNF-mediated killing. The present findings suggest that the mechanism of tumour cell killing by activated macrophages may differ, depending on the tumour cell type, and reactive nitrogen intermediates play a major role in cisplatin-mediated activation of bone marrow-derived macrophages.     

Malaria antigen and cytokine-induced production of reactive nitrogen intermediates by murine macrophages: no relevance to the development of experimental cerebral malaria.

The in vitro production of reactive nitrogen intermediates (RNI) by murine macrophages was evaluated in response to heat-stable malaria antigen and cytokines. Malaria antigen, interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF) induced RNI production in macrophages in a dose-dependent way. RNI production steadily increased over a 2-day period and was enhanced when the malaria antigen was co-incubated with IFN-gamma and/or TNF. RNI production induced by either IFN-gamma or malaria antigen or a combination of the two was suppressed by pentoxifylline in a dose-dependent manner. Pentoxifylline did not significantly influence TNF-induced RNI production. L-N-monomethyl arginine reduced malaria antigen, IFN-gamma and TNF-induced RNI production when these reagents were used in combination or alone. An anti-TNF monoclonal antibody (mAb) reduced IFN-gamma-induced RNI production, but did not significantly alter the malaria antigen-induced RNI synthesis by macrophages. The influence of inhibitors of nitric oxide synthase, L-N-monomethyl arginine and N omega-nitro-L-arginine, was studied in experimental cerebral malaria. They did not exert any significant effect on the development of cerebral malaria in Plasmodium berghei ANKA-infected CBA/J mice.     

Macrophage function in response to PGE2, L-arginine deprivation, and activation by colony-stimulating factors is dependent on hematopoietic stimulus.

Different macrophage preparations were compared for functional capacity in conditions of high prostaglandin E2 (PGE2) or low L-arginine concentrations. Macrophages derived in vitro from bone marrow progenitor cells (bone marrow-derived macrophages, BMDMs) using colony-stimulating factor 1 (CSF-1) as the myelopoietic stimulus displayed a greater sensitivity to PGE2-induced suppression of tumor necrosis factor alpha (TNF-alpha) secretion than did macrophages derived using granulocyte-macrophage colony-stimulating factor (GM-CSF). Neither BMDM population was inhibited by PGE2 for the direct cytolysis of L929 cells (TNF-alpha sensitive), and only GM-CSF-derived macrophages showed decreased killing of TNF-alpha-resistant K562 targets. Exogenous cAMP inhibited TNF-alpha secretion, but not nitrite secretion, by both BMDM populations. GM-CSF-derived macrophages accumulated less cAMP following PGE2 treatment than did CSF-1-derived macrophages. Removing L-arginine from the medium did not inhibit cytotoxicity or PGE2 secretion, but the listeriacidal activity specific to interferon-gamma plus lipopolysaccharide (LPS)-activated GM-CSF-derived macrophages was blocked by removal of L-arginine. Treatment with CSF-1 or GM-CSF alone did not activate the macrophages, but GM-CSF efficiently primed both BMDM populations for augmented TNF-alpha secretion in response to secondary stimulation using LPS. However, GM-CSF augmented the LPS-induced production of nitrite and PGE2 by CSF-1-derived macrophages only. These results demonstrate the potential for differential macrophage function within inflammatory sites based on the hematopoietic stimulus under which the macrophage is derived and the specific conditions present in the lesion.     

Phagocytosis enhances murine macrophage activation by interferon-gamma and tumor necrosis factor-alpha

Previously, we reported that exposure of bone marrow-derived macrophages (M phi) to a phagocytic stimulus in the simultaneous presence of interferon-gamma (IFN-gamma) induced these cells to generate nitrite (NO2-). This effect was achieved using both living (i.e. promastigotes of the protozoan parasite Leishmania enriettii) and inert (latex beads) particles. When the phagocytic stimulus was Leishmania, enhanced intracellular killing accompanied elevated NO2- secretion. As shown here, the capacity of phagocytosis to elicit NO2- production by IFN-gamma-treated M phi was inhibited by antibody to murine recombinant tumor necrosis factor-alpha (rTNF-alpha), suggesting that phagocytosis enabled IFN-gamma to activate M phi via the induction of TNF-alpha as an autocrine second signal. M phi NO2- production in response to rIFN-gamma and either exogenous TNF-alpha or Leishmania was strongly enhanced by prostaglandin E2, consistent with such a mechanism. However, addition of either Leishmania promastigotes or latex beads to M phi cultures simultaneously exposed to both IFN-gamma and exogenous murine or human rTNF-alpha further potentiated activation as measured by NO2- release. Furthermore, anti-TNF antibody failed to inhibit M phi responses to rIFN-gamma and bacterial lipopolysaccharide (LPS) in the presence or absence of Leishmania; also exogenous rTNF-alpha did not significantly affect NO2- production by IFN-gamma/LPS cultures despite a strong enhancement by Leishmania. These results suggest that phagocytosis enhances M phi responses by a process more complex than the sole induction of TNF-alpha. Phagocytosis also increased M phi NO2- production elicited by IFN-gamma plus TNF-alpha in L-arginine-deficient media. These results indicate that phagocytosis may be an important mechanism of up-regulating M phi microbicidal activity, and could be particularly relevant upon arginine depletion which occurs during an inflammatory response.     

Regulation of interferon production by human monocytes: requirements for priming for lipopolysaccharide-induced production.

Macrophages are uniquely responsive to bacterial lipopolysaccharide (LPS) for activation of a number of host defense functions and production of bioactive mediators. One potentially important mediator produced by LPS-stimulated macrophages is interferon (IFN-alpha/beta). In contrast to murine observations, we have observed that freshly isolated human monocytes, purified by counter-current centrifugal elutriation, do not produce interferon in response to LPS. This is not due to a lack of response to LPS, as assessed by the induction of other monokines, or to an incapacity for IFN production, since IFN was inducible by poly-I,C treatment of monocytes in the absence of any other exogenous stimulus. However, human monocytes can be primed for the production of IFN in response to LPS if they are cultured in the presence of either granulocyte-macrophage colony stimulating factor (GM-CSF) or interferon-gamma (IFN-gamma). The IFN secreted is of the alpha subtype. Monocytes primed with GM-CSF or IFN-gamma also maintained LPS responses for production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1). M-CSF did not prime monocytes for LPS-induced IFN production, although it did enhance production of TNF-alpha and promoted monocyte survival. Northern analysis indicated that the induction of IFN-alpha by LPS was regulated primarily at the mRNA level. The highly regulated production of IFN-alpha by monocytes/macrophages has important implications for autocrine action of interferons in the activation and differentiation of these cells.     

Enhancing effect of oxygen radical scavengers on murine macrophage anticryptococcal activity through production of nitric oxide

We examined the roles of reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI) in interferon-gamma (IFN-γ)-induced cryptococcostatic activity of murine peritoneal macrophages using N^G-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of RNI synthesis, and superoxide dismutase (SOD) and catalase, oxygen radical scavengers. IFN-γ-activated macrophages produced nitric oxide (NO) in a dose-dependent manner, as measured by increased nitrite concentration in the culture supernatant. IFN-γ also enhanced the suppressive effect on cryptococcal growth in a similar dose-dependent manner. The induction of killing activity and NO production by an optimal dose of IFN-γ (100 U/ml) was virtually suppressed by 500 μM L-NMMA. These results confirmed the importance of the RNI-mediated effector mechanism in anticryptococcal activity of macrophages. SOD and catalase significantly enhanced the cryptococcostatic activity of macrophages induced by a suboptimal dose of IFN-γ (20 U/ml). The augmenting effect of these reagents was mediated by NO, since they potentiated the production of NO by macrophages and their effects were totally blocked by L-NMMA. Our results indicate that the IFN-γ-induced anticryptococcal activity of macrophages is dependent mostly on RNI, and suggest that the ROI system down-regulates the effector mechanism for cryptococcostasis by suppressing the RNI system.     

Tumor necrosis factor and granulocyte macrophage-colony stimulating factor stimulate human macrophages to restrict growth of virulent Mycobacterium avium and to kill avirulent M. avium: killing effector mechanism depends on the generation of reactive nitrogen intermediates

An avirulent and a virulent strain of Mycobacterium avium were selected on the basis of their growth patterns in human monocyte-derived macrophages. The virulent 7497 M. avium grew progressively in untreated macrophages, whereas the avirulent LR/149 M. avium was killed to a moderate extent by untreated human macrophages (50% of the original infectious inoculum killed 7 days after infection). We set out to investigate the possibility of modulating these growth patterns by cytokine treatment. Application of tumor necrosis factor (TNF) (100 U/ml) led to macrophages restricting significantly the growth of virulent M. avium 7497 (tenfold decrease at 7 days). TNF was also effective at modulating positively the interaction between avirulent LR/149 M. avium and macrophages inasmuch as TNF-treated cells killed 99% of infecting mycobacteria at 7 days. Granulocyte macrophage-colony stimulating factor (GM-CSF) (100-10,000 U/ml) treatment led to macrophages being as mycobacteriostatic for virulent 7497 M. avium as TNF-alpha-treated cells (i.e., tenfold reduction in growth). Treatment of macrophages with both GM-CSF and TNF-alpha was shown to have additive effects on bacteriostatic activity on M. avium. The mechanism of killing of avirulent M. avium by TNF-alpha was shown to be dependent on the generation of reactive nitrogen intermediates, as seen by inhibition of effector mechanisms by NG-monomethyl-arginine and arginase. Moreover, there was a correlation between NO2- generation and mycobactericidal activity of macrophages. Addition of superoxide dismutase reversed the killing of avirulent M. avium by untreated or TNF-treated macrophages. This abrogation was also apparent in chronic granulomatous disease (CGD) macrophages, which were inefficient at generating reactive oxygen intermediates. Moreover, macrophages from CGD patients killed avirulent M. avium as efficiently as cells from normal individuals. We conclude from these results that 1) GM-CSF and TNF-alpha, alone or in combination, increase effector functions of macrophages against virulent and avirulent strains of M. avium; 2) reactive nitrogen intermediates seem to be involved in this effector mechanism; and 3) superoxide dismutase protected M. avium against macrophage effector function, seemingly by protecting the bacteria against endogenous superoxide anion. The implications of these findings for host resistance to atypical mycobacteria are discussed.     

Differential potentiation of anti-mycobacterial activity and reactive nitrogen intermediate-producing ability of murine peritoneal macrophages activated by interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α)

The anti-mycobacterial activities of IFN-γ and TNF-α-treated murine peritoneal macrophages were determined. Resident macrophages pretreated with IFN-γ or TNF-α for 2 days were infected with test organisms and subsequently cultured for up to 7 days. First, the early-phase growth of Mycobacterium tuberculosis (days 0–3) was strongly suppressed in IFN-γ-treated macrophages, and progressive bacterial elimination was subsequently observed. Although TNF-α treatment of macrophages did not affect the early phase growth of organisms, bacterial killing was observed in the later phase of cultivation. Second, although IFN-γ-treated macrophages killed M. avium during the first 3 days of culture, regrowth of the intracellular organisms was subsequently observed. TNF-α treatment of macrophages did not influence the mode of intracellular growth of M. avium. Third, IFN-γ but not TNF-α enhanced production of reactive nitrogen intermediates (RNI) by macrophages infected with M. tuberculosis or M. avium, whereas both cytokines increased macrophage release of reactive oxygen intermediates (ROI). The present findings therefore show that IFN-γ and TNF-α potentiated the anti-mycobacterial activity of murine peritoneal macrophages in different fashions. They also suggest that RNI played more important roles than did ROI in the expression of macrophage anti-mycobacterial, particularly anti-M. avium, activity.     

Tumor necrosis factor alpha augments nitric oxide-dependent macrophage cytotoxicity against Entamoeba histolytica by enhanced expression of the nitric oxide synthase gene.

Nitric oxide (NO measured as nitrite, NO2-) is the major effector molecule produced by activated macrophages for in vitro cytotoxicity against Entamoeba histolytica trophozoites. In this study, we determine whether tumor necrosis factor alpha (TNF-alpha) produced by activated bone marrow-derived macrophages (BMM) is involved in the induction of the inducible NO synthase gene (mac-NOS) for NO-dependent amebicidal activity. TNF-alpha alone did not directly induce macrophage NO2- production to kill amebae; however, in combination with increasing concentrations of TNF-alpha and gamma interferon (IFN-gamma), BMM amebicidal activity and NO2- production progressively increased and showed a significant linear correlation. Antiserum to TNF-alpha and the NO synthase inhibitor NG-monomethyl L-arginine (L-NMMA) inhibited the synergistic effects of TNF-alpha and IFN-gamma. BMM activated with increasing concentrations of lipopolysaccharide (LPS) and IFN-gamma showed a significant linear correlation between TNF-alpha release and NO2- production. Antiserum to TNF-alpha suppressed TNF-alpha release, NO2- production, and amebicidal activity by 93, 53, and 86%, respectively. L-NMMA diminished NO2- production by 74% and macrophage amebicidal activity by 83% but had no effect on TNF-alpha release. Quantification by Northern (RNA) blot analyses demonstrated that IFN-gamma in combination with TNF-alpha or LPS increased markedly the accumulation of mac-NOS and TNF-alpha mRNAs in a time-dependent manner with a concomitant increase in NO and TNF-alpha production. Peak induction of mac-NOS occurred after 24 h, whereas TNF-alpha mRNA was rapidly expressed after 4 h and remained stable for 48 h. Taken together, these data argue that TNF-alpha augments NO-dependent macrophage cytotoxicity against E. histolytica via elevated levels of mac-NOS mRNA expression which may be associated with the accumulation of TNF-alpha mRNA.     

Interleukin 1 and/or tumor necrosis factor-alpha synergize with granulocyte-macrophage colony-stimulating factor to enhance histamine synthesis in hematopoietic cells: role of prostaglandin E2

Previously, we have shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates histamine synthesis by normal murine hematopoietic cells. Addition of either interleukin (IL) 1 (alpha or beta) or murine recombinant tumor necrosis factor (TNF)-alpha to murine recombinant GM-CSF (at optimal or suboptimal concentrations) enhances its activity on bone marrow histamine synthesis up to 70%. Evidence is provided that these synergies between GM-CSF and IL 1 or TNF-alpha are mediated by prostaglandin E2 (PGE2) production since (a) GM-CSF together with either IL 1 or TNF-alpha stimulates PGE2 synthesis by bone marrow cells, while none of these factors does it alone; (b) exogenous PGE2 (ranging from 10(-6) M to 10(-10) M) potentiates GM-CSF-induced histamine synthesis in a dose-dependent manner; and (c) indomethacin, a cyclooxygenase inhibitor, completely abrogates the synergistic action of IL 1 and TNF-alpha on GM-CSF-induced histamine generation. Conversely, histamine synthesis promoted by IL 3, the unique cytokine sharing this property with GM-CSF, cannot be modulated by IL 1, TNF-alpha or PGE2, suggesting two distinct mechanisms for the induction of this biological activity in hematopoietic progenitor cells.     

Induction of tumor necrosis factor alpha by spherules of Coccidioides immitis.

The cytokine tumor necrosis factor alpha (TNF-alpha) functions as an immunomodulatory protein and as a mediator of cachexia. We report that viable or Formalin-killed spherules of Coccidioides immitis induced the secretion of TNF-alpha by peritoneal-exudate cells from BALB/c mice. The identification of the cytokine as TNF-alpha was based on its lytic activity against the TNF-alpha-sensitive LS murine fibrosarcoma cell line but not the TNF-alpha-resistant LR cell line, its neutralization by rabbit anti-TNF-alpha, and its secretion by peritoneal cells having characteristics of macrophages. The induction of TNF-alpha was to spherules and not to contaminating lipopolysaccharide (endotoxin), as evidenced by the finding that polymyxin B, a reagent that blocks the TNF-alpha-inducing component of lipopolysaccharide, did not negate the production of TNF-alpha in response to spherules, whereas pretreatment of spherules with hyperimmune goat antiserum to spherulin neutralized the induction of TNF-alpha by these cells. The demonstration that C. immitis activates macrophages to secrete TNF-alpha in vitro is a new finding and warrants studies to determine whether this cytokine is produced during active coccidioidomycosis.