Effect of PGE2 and of agents that raise cAMP levels on macrophage activation induced by IFN-gamma and TNF-alpha.

The effect of prostaglandin (PG) E2 on macrophage activation by interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) was evaluated. Murine macrophages infected with Leishmania enriettii or Leishmania major were activated by exposure to IFN-gamma (10-50 U/ml) and TNF-alpha (30-3000 U/ml), leading to intracellular parasite destruction within 24-48 h. Leishmanicidal activity was markedly increased when activation was performed in the presence of PGE2 (10(-9)-10(-7) M) or arachidonate (10(-5) M, a PG precursor), concomitant with enhanced nitrite release and glucose oxidation through the hexose monophosphate shunt pathway. Conversely, activation was reduced by indomethacin and hydrocortisone, two inhibitors of PG synthesis. Parasite killing and nitrite production were fully restored by exogenous PGE2, indicating that inhibition by these drugs was related to their ability to block PG production. PG can stimulate adenylate cyclase, thus raising intracellular cAMP levels. Accordingly, dibutyryl-cAMP, theophylline (which prevents cAMP breakdown), and forskolin (an activator of adenylate cyclase) all stimulated macrophage activation. Finally, PGE2 and cAMP enhanced expression of inducible nitric oxide synthase mRNA in response to IFN-gamma and TNF-alpha, and this effect was inhibited by the cAMP antagonist 2'-O-methyl adenosine. These findings are consistent with the hypothesis that PGE2 acts as a positive agonist in macrophage activation by IFN-gamma and TNF-alpha via its capacity to modulate intracellular cAMP levels.

Role of glutathione in macrophage activation: effect of cellular glutathione depletion on nitrite production and leishmanicidal activity.

We have examined the effects of two agents depleting the intracellular pool of glutathione (GSH) on macrophage activation induced by IFN-gamma + LPS, as measured by nitrite production and leishmanicidal activity. Diethylmaleate (DEM), which depletes intracellular GSH by conjugation via a reaction catalyzed by the GSH-S-transferase, strongly inhibited nitrite secretion and leishmanicidal activity when added before or at the time of addition of IFN-gamma + LPS; this inhibition was progressively lost when addition of DEM was delayed up to 10 hr. A close correlation was observed between levels of intracellular soluble GSH during activation and nitrite secretion. Inhibition was partially reversed by the addition of glutathione ethyl ester (GSH-Et). Buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, also inhibited macrophage activation, although to a lesser extent than DEM despite a more pronounced soluble GSH depletion. This inhibition was completely reversed by the addition of GSH-Et. DEM and BSO did not alter cell viability or PMA-triggered O2- production by activated macrophages, suggesting that the inhibitory effects observed on nitrite secretion and leishmanicidal activity were not related to a general impairment of macrophage function. DEM and BSO treatment reduced iNOS specific activity and iNOS protein in cytosolic extracts. DEM also decreased iNOS mRNA expression while BSO had no effect. Although commonly used as a GSH-depleting agent, DEM may have additional effects because it can also act as a sulhydryl reagent; BSO, on the other hand, which depletes GSH by enzymatic inhibition, has no effect on protein-bound GSH. Our results suggest that both soluble and protein-bound GSH may be important for the induction of NO synthase in IFN-gamma + LPS-activated macrophages.

Effect of increasing intravesicular pH on nitrite production and leishmanicidal activity of activated macrophages.

We examined the effect of bafilomycin A1 (BAF), an inhibitor of vacuolar-type H(+)-ATPases, on macrophages activation (measured as increased nitrite production and leishmanicidal activity) induced by interferon gamma alone or together with lipopolysaccharide or tumour necrosis factor alpha. BAF increased intravesicular pH and enhanced nitrite release by activated macrophages; however, the NO concentration necessary to kill parasites was higher in BAF-exposed than control macrophages, suggesting that microbicidal nitrogen derivatives were less active at alkaline pH. Antibody to tumour necrosis factor alpha inhibited BAF-induced nitrite production in interferon-activated cultures. To determine if enhanced NO synthesis was related to vesicular alkalinization, macrophages were incubated with the lysosomotropic bases NH4Cl and methylamine. These agents also increased intravesicular pH and nitrite production. Nitrite production was correlated with enhanced NO synthase activity in cytosolic extracts of the activated cells.

Transforming growth factor beta 1 regulation of macrophage activation depends on the triggering stimulus.

We have examined the effects of transforming growth factor beta 1 (TGF-beta 1) on the regulation of murine bone marrow-derived macrophage function. TGF-beta, added simultaneously with or up to 4 h before interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS), inhibited macrophage leishmanicidal activity, nitrite (NO2-) production, and secretion of prostaglandin E2. In contrast, no effect of TGF-beta could be demonstrated on macrophages stimulated with IFN-gamma plus tumor necrosis factor-alpha (TNF-alpha) under the same conditions. These results suggested that TGF-beta inhibited LPS-induced triggering of macrophage activation, which was confirmed by studies with IFN-gamma-primed cells. Interestingly, when macrophages were pretreated with TGF-beta for 24 h, NO2- production in response to IFN-gamma plus TNF-alpha was also inhibited. Although control and IFN-gamma/LPS-stimulated macrophages were found to secrete latent TGF-beta, only the IFN-gamma/LPS cultures produced biologically active TGF-beta. Significantly, active TGF-beta was present at concentrations shown earlier to inhibit macrophage function.

Induction of macrophage nitric oxide production by interferon-gamma and tumor necrosis factor-alpha is enhanced by interleukin-10.

Interleukin-10 (IL-10) has been reported to inhibit nitric oxide (NO) synthesis and microbicidal activity of interferon-gamma (IFN-gamma)-stimulated macrophages (M phi) by preventing the secretion of tumor necrosis factor-alpha (TNF-alpha) which serves as an autocrine activating signal. We have examined the effects of recombinant IL-10 on the capacity of IFN-gamma together with exogenous TNF-alpha to induce NO synthesis by bone marrow-derived M phi. Under these conditions and in contrast to its reported deactivating potential, IL-10 strongly enhanced NO synthesis measured as nitrite (NO2-) release (half maximal stimulation at approximately 10 U/ml). IL-10 further increased NO2- production by M phi stimulated in the presence of optimal concentrations of prostaglandin E2, a positive modulator of M phi activation by IFN-gamma/TNF-alpha. Increased steady state levels of NO synthase mRNA were observed in 4-h IFN-gamma/TNF-alpha cultures and enhanced NO2(-)-release was evident 24 h but not 48 h after stimulation. These results suggest that the effects of IL-10 on M phi function are more complex than previously recognized.

Macrophage activation for intracellular killing as induced by a Ca2+ ionophore. Dependence on L-arginine-derived nitrogen oxidation products.

Mouse macrophages activated by interferon-gamma kill intracellular Leishmania by a process that depends on the generation of L-arginine-derived nitrogen oxidation products. Interferon-induced intracellular killing can be mimicked by exposure of macrophages to the Ca2+ ionophore A23187 in the presence of lipopolysaccharide. The mechanisms of this effect were therefore investigated. Destruction of the parasite was accompanied by accumulation of nitrite in the macrophage culture fluids. Leishmanicidal activity and nitrite production in cultures stimulated with ionophore A23187 and lipopolysaccharide were abrogated when cells were activated in medium containing arginase or the L-arginine analogues L-canavanine, guanidine or NG-monomethyl-L-arginine. L-Arginine was required during the lipopolysaccharide-induced triggering phase only. Indeed, macrophage priming with ionophore A23187 in L-arginine-depleted medium led to full microbicidal activity and nitrite generation provided that L-arginine was present during subsequent triggering by lipopolysaccharide. Addition of NG-monomethyl-L-arginine to ionophore-activated macrophages increased O2- production on phorbol myristate stimulation, while inhibiting glucose oxidation through the hexose monophosphate shunt pathway. Leishmanicidal activity and nitrite production were also inhibited when ionophore-treated cultures were incubated with excess iron, implying a role for iron as a defence mechanism against the toxicity of nitrogen derivatives. These results indicate that the ionophore-induced leishmanicidal activity occurs through a process similar to that evoked by interferon-gamma, i.e. the production of L-arginine-derived nitrogen oxidation products.

3-amino-1,2,4-triazole inhibits macrophage NO synthase.

Murine macrophages activated by interferon-gamma and lipopolysaccharide become leishmanicidal through a process involving L-arginine-derived nitrogen oxidation products. Both nitrite secretion and parasite killing by activated macrophages were inhibited by 3-amino-1,2,4-triazole as well as the related compound, 3-amino-1,2,4-triazine. Moreover, NO synthase activity in cytosolic extracts of activated cells was inhibited by both compounds. 4-amino-1,2,4-triazole, an isomer of 3-amino-1,2,4-triazole, was without effect. Our results suggest that besides its known inhibitory effect on catalases and peroxidases, 3-amino-1,2,4-triazole is an inhibitor of NO synthase. The resemblance between the tautomeric form of 3-amino-1,2,4-triazole and the guanidino group of L-arginine, the natural substrate for NO synthase, might be responsible for the observed inhibition.

Differential effects of prostaglandins on macrophage activation induced by calcium ionophore A23187 or IFN-gamma.

Calcium ionophore A23187 can mimic IFN-gamma-induced macrophage activation for intracellular Leishmania killing and secretion of L-arginine-derived nitrite. Because the effects of ionophore are not restricted to calcium mobilization but also involve alterations of phospholipid metabolism, we have examined the role of PGE2 in the activation process. Macrophages exposed to A23187 or IFN-gamma in the presence of LPS and FCS secreted significant amounts of PGE2 independently of the presence of L-arginine in the incubation medium. The addition of the cyclooxygenase inhibitor indomethacin or omission of FCS abrogated PGE2 secretion but had little effect on nitrite production or intracellular killing. The addition of exogenous PGE2, of agents increasing PGE2 production such as arachidonic acid and colchicine, or of an analogue of cAMP, dibutyryl cAMP inhibited A23187 + LPS-induced activation whereas that mediated by IFN-gamma + LPS remained unimpaired. Our results indicate that PGE2 can modulate activation induced by A23187 but not by IFN-gamma, probably by a process involving cAMP. Conceivably, ionophore can mimic IFN-gamma for the induction of activation but lacks the capacity to help maintain the activated state because of its inability to desensitize macrophages to negative regulation by PGE2, as suggested previously for IFN-gamma-dependent activation.

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.

Killing of Leishmania parasites in activated murine macrophages is based on an L-arginine-dependent process that produces nitrogen derivatives.

The experiments described in this report were aimed at determining whether L-arginine (L-arg)-derived nitrogen oxidation products (nitric oxide, nitrous acid, nitrites) are involved in the intracellular killing of Leishmania parasites by activated murine macrophages in vitro. Peritoneal or bone marrow-derived macrophages were infected with L. enriettii or L. major, then activated by exposure to recombinant murine interferon-gamma or to macrophage activating factor (MAF)-rich media in the presence of lipopolysaccharide. Activation of macrophages in regular (i.e., arginine-containing) culture medium led to complete destruction of the microorganisms within 24 h (L. enriettii) or 48 h (L. major), concomitant with accumulation of nitrites (NO2-) in the culture fluids. When macrophage activation was carried out in L-arg-free medium, however, neither parasite killing nor NO2- production was obtained. A similar inhibition of macrophage leishmanicidal activity and of NO2- release was observed using media treated with arginase (which converts L-arg to urea and ornithine), or supplemented with NG-monomethyl-L-arg or guanidine (which inhibit the conversion of L-arg to nitrogen oxidation products). In all these situations, an excellent correlation between the levels of NO2- production by macrophages and intracellular killing of Leishmania was observed, whereas no strict correlation was detectable between leishmanicidal activity and superoxide production. Intracellular parasite killing by activated macrophages could be prevented by addition of iron salts to the incubation fluids. Incubation of free parasites with NaNO2 at acid pH (which permits the production of nitrous acid) led to immobilisation, multiplication arrest, and morphological degeneration of the microorganisms. Similarly, exposure of infected cells to NaNO2 led to killing of the intracellular parasite without affecting macrophage viability. These experiments strongly suggest that the leishmanicidal effect of activated murine macrophages involves the agency of L-arg-derived nitrogen oxidation products.

Macrophage activation for intracellular killing as induced by calcium ionophore. Correlation with biologic and biochemical events.

Changes in the concentration of cytosolic Ca2+ are known to affect various macrophage functions; in particular, exposure in vitro to the Ca2+ ionophore A23187 primes macrophages for tumor cell killing. In the present report, it is shown that treatment with this ionophore similarly mimics IFN-gamma as a priming signal for induction of microbicidal activity. Incubation of mouse bone marrow-derived macrophages with 10(-7) to 10(-6) M A23187 (in the presence of Ca2+) led to intracellular killing of the protozoan parasite Leishmania enriettii within 24 h, provided LPS (1 ng/ml) was also present; no microbicidal activity was observed using either compound alone. A 4-h exposure to the ionophore in the presence of Ca2+ (priming phase) was sufficient to induce leishmanicidal activity upon reincubation with LPS, here acting as a necessary second signal. Addition of EGTA during the priming phase blocked intracellular killing upon subsequent LPS treatment; microbicidal activity could be restored by excess Ca2+, but not Mg2+, suggesting that changes in the concentration of cytosolic Ca2+ are sufficient to mediate the molecular events that lead to acquisition of microbicidal potential. Ionophore-induced leishmanicidal activity was paralleled by a stimulation of the hexosemonophosphate shunt pathway and production of nitrites, which are biochemical correlates of the activated state. In addition, sequential exposure to A23187 and LPS markedly stimulated macrophages to release TNF and PGE2, two agents thought to act as modulators of macrophage activation.

Lead inhibits oxidative metabolism of macrophages exposed to macrophage-activating factor.

The present experiments were designed to evaluate the effect of lead on the capacity of macrophages to respond to activating signals by increased respiratory-burst activity. When mouse peritoneal macrophages were exposed for 24 h to macrophage-activating factor (MAF) and/or bacterial lipopolysaccharide in the presence of lead acetate, a marked inhibition of their oxidative metabolism was observed. The hexosemonophosphate-shunt (HMPS) activity and the release of oxygen derivatives upon triggering by phorbol myristate acetate (PMA) were impaired. Treatment with the metal for 1 h led, however, to stimulation rather than inhibition of the PMA-triggered superoxide production, suggesting that the metal interfered with neither the triggering steps nor the activity of the NADPH oxidase. Moreover, the lead-induced inhibition of macrophage oxidative metabolism did not result from blockade of enzymes of the HMPS pathway. Glucose-6-phosphate dehydrogenase in macrophage extracts, as well as CO2 production from glucose, remained unaffected by the presence of lead, and extracts of lead-treated macrophages were as active as extracts from control cells in those two assays. Lead appeared to interfere with an early event in the MAF-induced activation process. In addition, lead decreased the uptake of 2-deoxyglucose by macrophages, suggesting that the metal might inhibit trans-membrane glucose-transport systems, a phenomenon that might explain in part the metabolic inhibition observed in lead-treated cells.

Lead inhibits intracellular killing of Leishmania parasites and extracellular cytolysis of target cells by macrophages exposed to macrophage activating factor.

Activation of Leishmania enriettii-infected mouse macrophages in vitro by treatment with macrophage activating factor (MAF)-rich media supplemented with lipopolysaccharide (LPS) leads to rapid killing of the microorganism. When exposed to MAF + LPS in the presence of 30-100 microM lead acetate, however, macrophages failed to destroy the parasites. This effect was not due to lead toxicity for macrophages. Decreased microbicidal activity correlated with depressed respiratory burst as determined by measurements of glucose oxidation through the hexose monophosphate shunt (HMPS). Lead had little effect on intracellular parasite killing induced by exposure of macrophages to the electron carrier methylene blue; HMPS in such cells was similarly little affected, indicating that chemical triggering of this pathway bypassed the lead-imposed blockade. Lead also abolished macrophage activation measured by the lysis of tumor target cells in vitro. The metal failed, however, to interfere with target-cell lysis by macrophages activated in lead-free medium, suggesting that lead inhibited the acquisition of the activated state rather than the functional expression of such state. Lead did not prevent the binding of radiolabelled interferon-gamma to macrophages; it did, however, slow down receptor turnover and degradation of bound interferon. Lead also inhibited the LPS-triggered cytotoxicity in macrophages previously exposed to interferon-gamma in lead-free medium, suggesting that depressed intracellular killing might result from an effect on both the priming (interferon or MAF-dependent) and the triggering (LPS-dependent) steps of activation.

Impairment of the oxidative metabolism of mouse peritoneal macrophages by intracellular Leishmania spp.

When stimulated in vitro with macrophage-activating factor or lipopolysaccharide, mouse peritoneal macrophages acquire the capacity to develop a strong respiratory burst when they are triggered by membrane-active agents. The presence of intracellular parasites of the genus Leishmania (L. enriettii, L. major) significantly inhibited such activity, as measured by chemiluminescence, reduction of cytochrome c and Nitro Blue Tetrazolium, and hexose monophosphate shunt levels. On the contrary, inert intracellular particles such as latex beads strongly increased the macrophage respiratory burst, suggesting that the Leishmania-linked inhibition resulted from a specific parasite effect. Impairment of macrophage oxidative metabolism by intracellular Leishmania spp. was a function of the number of infecting microorganisms and was more pronounced in macrophages infected with living than with dead parasites. Moreover, the metabolic inhibition was less apparent in L. enriettii-infected macrophages that were exposed to both macrophage-activating factor and lipopolysaccharide, i.e., conditions leading to complete parasite destruction. The mechanisms of respiratory burst inhibition by intracellular Leishmania spp. are unclear, but these observations suggest that such effects may contribute significantly to intracellular survival of the microorganisms.

Effect of lipopolysaccharide on intracellular killing of Leishmania enriettii and correlation with macrophage oxidative metabolism.

The effect of lipopolysaccharide (LPS) on the lymphokine (LK)-dependent activation of murine peritoneal macrophages for intracellular killing of Leishmania enriettii parasites was investigated. Exposure to LPS alone did not induce macrophages to kill the parasite. In the presence of LK or recombinant interferon-gamma, however, which by themselves rendered the macrophages only weakly cytotoxic, considerable stimulation of intracellular parasite killing was achieved already at a LPS concentration of 1 ng/ml. The response to LPS was of the same magnitude in macrophages tested for intracellular killing as in parallel assays of extracellular cytolysis of target cells. Acquisition of leishmanicidal activity by macrophages exposed to LK and LPS correlated with stimulation of the respiratory burst, as shown by increased hexose monophosphate shunt levels, and priming for elevated chemiluminescence and O2- and H2O2 production. Polymyxin B blocked both this LPS-dependent metabolic activity and intracellular parasite destruction. Intracellular killing was, however, not solely dependent on oxidative metabolism of macrophages since in the absence of LK, LPS stimulated respiratory burst activity, yet no intracellular killing was observed, and triggering of the respiratory burst by phorbol myristate acetate or zymosan did not affect intracellular parasite survival. These results suggest that, in this experimental model, efficient intracellular parasite killing depends both on increased production of oxygen metabolites and on the availability of so far unidentified factor(s), the synthesis of which requires exposure of macrophages to both LK and LPS.

Correlation between enhanced oxidative metabolism and leishmanicidal activity in activated macrophages from healer and nonhealer mouse strains.

A test system that allows a precise monitoring of intracellular killing of Leishmania parasites was used to measure the leishmanicidal capacity of activated macrophages from different strains of mice. Activation was obtained by exposure to dilutions of lymphokine (LK)-rich medium in the presence of ng/ml amounts of E. coli lipopolysaccharide (LPS). Highest leishmanicidal activity was displayed by macrophages from the healer mouse strain CBA/T6, whereas cells from the nonhealer strains DBA/2 and BALB/c were less effective. C3H/HeJ macrophages from a healer but LPS-unresponsive mouse strain failed to destroy leishmanias under these conditions. Experiments were then performed to determine the level of respiratory burst activity in the various macrophage populations. Hexose monophosphate shunt stimulation was higher in CBA/T6 than in BALB/c or DBA/2 macrophages, and only marginal in C3H/HeJ cells, correlating with differing leishmanicidal activities of such macrophages under the present experimental conditions. Measurements of O2- and H2O2 secretion and of chemiluminescence led to similar findings, i.e., CBA/T6 macrophages released higher amounts of oxygen metabolites than BALB/c cells activated under the same conditions, whereas C3H/HeJ cells were the least active. The results obtained by the four assays of oxidative metabolism were consistent in that endotoxin itself (in the 10 to 30 ng/ml range) stimulated the oxidative response of macrophages to a level close to that achieved by using LPS and LK together, however, only in the latter situation were parasites destroyed.