Macrophage activation to kill Leishmania tropica: defective intracellular killing of amastigotes by macrophages elicited with sterile inflammatory agents

Resident peritoneal macrophages and macrophages elicited by injection of C3H/HeN mice with sterile inflammatory agents were exposed to amastigotes of Leishmania tropica in vitro and treated with lymphokines. Resident macrophages developed the capacity to kill intracellular parasites; microbicidal activity of activated resident cells ranged between 60 and 80%. In contrast, inflammatory macrophages responded poorly to lymphokines for intracellular killing of amastigotes; microbicidal activity of cells elicited with chronic inflammatory agents ranged between 0 and 45%. Defective intracellular killing of L. tropica by inflammatory macrophages was independent of the agent used to elicit the cells, but was clearly associated with the number of immature macrophages in the population. That intracellular killing capacity may reflect the presence of a killing mechanism in tissue-derived cells that is not yet developed in undifferentiated macrophages is supported by studies with peripheral blood monocytes: these cells were also incapable of eliminating intracellular amastigotes in the presence of potent activating factors. These observations on inflammatory macrophage interactions with amastigotes may provide important insights into the chronic nature of leishmanial disease.     

Macrophage activation to kill Leishmania tropica: characterization of P/J mouse macrophage defects for lymphokine-induced antimicrobial activities against Leishmania tropica amastigotes

Macrophages from P/J mice demonstrated both quantitative and qualitative defects in lymphokine (LK)-induced activated macrophage antileishmanial effector reactions: a) these cells recognized the same LK signals that generated resistance to infection in responsive C3H/HeN macrophages, but more signal was required to observe maximal activity; b) LK-induced intracellular destruction of Leishmania tropica by P/J macrophages was minimal (less than 20%), and was induced by only one of three LK signals that regulate antimicrobial activities in C3H/HeN macrophages. The defective microbicidal activity of P/J macrophages observed with LK activation in vitro could also be demonstrated in vivo. Macrophages from P/J mice exposed to the macrophage-activating agent Mycobacterium bovis strain BCG in vivo were capable of restricting the intracellular replication of L. tropica but could not eliminate intracellular parasites, even with further incubation with LK during the 72-hr culture period. The defect of P/J macrophages for intracellular destruction of L. tropica, then, occurred in the activation sequence before the triggering stage that characterizes the macrophage defect of C3H/HeJ mice. Genetic regulation of the P/J macrophage defect appears to be by a single autosomal gene, with defective microbicidal activity as a recessive trait in these animals.     

Macrophage activation to kill Leishmania tropica: characterization of a T cell-derived factor that suppresses lymphokine-induced intracellular destruction of amastigotes

Factors obtained from phorbol myristate acetate (PMA)-stimulated EL-4 thymoma cells, a continuous T cell line, suppressed lymphokine-induced macrophage activation to kill intracellular Leishmania tropica amastigotes. Suppression of this macrophage effector activity was dependent upon concentration of EL-4 fluids admixed with lymphokines in infected macrophage cultures, and was not due to residual PMA or factors released from unstimulated EL-4 cells. Fluids from PMA-stimulated EL-4 cells did not affect the expression of microbicidal activity by macrophages activated in vivo as a consequence of infections with Mycobacterium bovis strain BCG, nor did they abrogate intracellular killing activities by C3H/HeJ macrophages primed by BCG infection and triggered by lymphokines in vitro. That the action of this EL-4 suppressor activity was at the priming stage of macrophage activation was confirmed by kinetic studies: EL-4 fluids added to lymphokine-treated cells in the first 4 hr of treatment completely suppressed intracellular killing of L. tropica; fluids added after 4 hr were not effective. The effects of these EL-4 factors appeared to be selective: of three effector activities of activated macrophages tested, induction of resistance to infection, tumor cytotoxicity, and intracellular destruction of L. tropica, only intracellular killing by lymphokine-treated macrophages was significantly suppressed. These T cell-derived soluble suppressor factor(s) may provide insight into mechanisms of immunosuppression during leishmanial disease and perhaps other intracellular parasitic infections.     

Antileishmanial activities of macrophages from C3H/HeN and C3H/HeJ mice treated with Mycobacterium bovis strain BCG

C3H/HeN and C3H/HeJ mice were infected ip with viable BCG, a macrophage-activating agent, and their peritoneal exudate macrophages exposed to Leishmania tropica amastigotes. Macrophages from BCG-infected C3H/HeN mice had both leishmanicidal activities described for lymphokine activation of C3H/HeN macrophages in vitro: increased resistance to L. tropica infection, followed by intracellular killing of the parasite. Macrophages from BCG-infected C3H/HeN mice were also activated to kill tumor cells in vitro. In contrast, macrophages from BCG-treated C3H/HeJ mice were not resistant to L. tropica infection, did not kill intracellular amastigotes over 72 hr in culture, and were not cytotoxic to tumor cells.     

Resistance to macrophage-mediated killing as a factor influencing the pathogenesis of chronic cutaneous leishmaniasis

Cutaneous leishmaniasis can be either a spontaneously healing or chronic disease, depending upon the strain of parasite and the immunological status of the host. We have investigated parasite factors responsible for the variable pathogenesis observed in leishmanial infections by testing the sensitivity of several leishmanial strains to intracellular killing in lymphokine (LK) activated mouse macrophages. Significant microbicidal activity against Leishmania tropica, a strain which heals in C57BL/6 (B6) mice, was found. In contrast, a strain (Maria) which has previously been shown to induce chronic nonhealing cutaneous lesions in B6 mice was resistant to killing in activated macrophages. This resistance to killing was observed in macrophages activated by LK obtained from either Bacille Calmette-Guérin-, L. tropica, or the Maria strain infected mice. The inability of LK activated macrophages to kill the Maria strain was shown not to be due to parasite induced inhibition of killing mechanisms, since Maria strain infected, LK treated macrophages exhibited tumoricidial activity similar to uninfected macrophages. Furthermore, LK activated macrophages simultaneously infected with the Maria strain and another intracellular pathogen, Toxoplasma gondii, killed Toxoplasma, but not the Maria strain. Temperature was also found to significantly influence the multiplication and killing of Leishmania parasites. As would be expected from their cutaneous nature, L. tropica and Maria strain parasites multiplied better at 35 degrees C than at 37 degrees C. Also consistent with the failure of cutaneous strains to visceralize in immunocompetent mice was the observation that the killing of leishmanial parasites was enhanced at the higher temperature. Thus, the temperature dependent growth capacity and sensitivity to killing of a given leishmanial strain in macrophages may be important factors influencing the pathogenesis of cutaneous leishmaniasis.     

Susceptibility of inbred mice to Leishmania tropica infection: correlation of susceptibility with in vitro defective macrophage microbicidal activities

Eleven mouse strains were inoculated in footpads with amastigotes of Leishmania tropica and observed for 12 weeks. Liver and spleen impression smears from infected mice were examined for the presence of intracellular parasites. Four strains (BALB/cJ, C57L/J, NZW/N, and P/J) failed to heal the subcutaneous lesion and showed evidence of systemic infection; the remaining seven strains (A/J, C3H/HeJ, C3H/HeN, C3HeB/FeJ, C57BL/6J, C57BL/10J, and C57BL/10ScN) were each resistant to infection and resolved their lesions by Week 10. Macrophages from the four susceptible strains could not be activated to kill L. tropica amastigotes by treatment with soluble lymphocyte products in vitro. In contrast, macrophages from all seven resistant strains responded to lymphokine treatment and eliminated 80-90% of intracellular parasites. These results suggest that in vitro macrophage microbicidal activities predict the course of systemic leishmanial disease.     

Regulation of activated macrophage antimicrobial activities. Cooperation of lymphokines for induction of resistance to infection

Macrophages treated with the soluble products of Ag-stimulated spleen cells from bacillus Calmette-Guérin-infected C3H/HeN mice (lymphokines) (LK] before infection developed the capacity to resist infection with obligately intracellular amastigotes of the protozoan parasite, Leishmania major: 40 to 60% fewer cells in LK-treated cultures were infected 2 h after exposure to parasites than cells in medium-treated controls. Macrophages treated with LK depleted of IFN-gamma failed to acquire this activated macrophage effector activity. Paradoxically, IFN-gamma by itself was also not effective. Activity of the ineffective, IFN-gamma-depleted LK was restored, however, by addition of 10 to 100 U/ml IFN-gamma, itself inactive. The induction of this antimicrobial activity was the result of the interaction of macrophages and several molecularly distinct LK, and IFN-gamma was a necessary but insufficient activation signal. The activation of macrophage resistance to infection by LK was 1) not signal sequence dependent, 2) absent in cells treated with the second signal at lower (4 degrees C) temperatures and in the presence of protein synthesis inhibitors, and 3) induced by the cooperation of IFN-gamma and LK of m.w. 45,000 and 33,000. These factors in LK constituted more than 85% total LK activity for induction of resistance to infection. A minor activity in LK, of m.w. 20,000, could apparently induce this effector activity in the absence of IFN-gamma: this activity was less than 15% of total LK activity.     

Macrophage activation for microbicidal activity against Leishmania major: inhibition of lymphokine activation by phosphatidylcholine-phosphatidylserine liposomes

Resident peritoneal macrophages from untreated mice develop microbicidal activity against amastigotes of the protozoan parasite Leishmania tropica (current nomenclature = Leishmania major) after in vitro exposure to LK from antigen-stimulated leukocyte culture fluids. This LK-induced macrophage microbicidal activity was completely abrogated by addition of 7:3 phosphatidylcholine: phosphatidylserine liposomes. Liposome inhibition was not due to direct toxic effects against the parasite or macrophage effector cell; factors in LK that induce macrophage microbicidal activity were not adsorbed or destroyed by liposome treatment. Other phagocytic particles, such as latex beads, had no effect on microbicidal activity. Moreover, liposome inhibition of activated macrophage effector function was relatively selective: LK-induced macrophage tumoricidal activity was not affected by liposome treatment. Liposome inhibition was dependent upon liposome dose (5 nmoles/culture) and time of addition of leishmania-infected, LK-treated macrophage cultures. Addition of liposomes through the initial 8 hr of culture completely inhibited LK-induced macrophage microbicidal activity; liposomes added after 16 hr had no effect. Similarly, microbicidal activity by macrophages activated in vivo by BCG or Corynebacterium parvum was not affected by liposome treatment. Liposome treatment also did not affect the increased resistance to infection induced in macrophages by LK. These data suggest that liposomes interfere with one or more early events in the induction of activated macrophages (macrophage-LK interaction) and not with the cytotoxic mechanism itself (parasite-macrophage interaction). These studies add to the growing body of data that implicate cell lipid in regulatory events controlling macrophage effector function.     

Colony-stimulating factors and regulation of macrophage tumoricidal and microbicidal activities

Conditioned medium from antigen- or mitogen-stimulated spleen cells, lymphokines, contained factors that induced formation of granulocyte and macrophage colonies in cultures of bone marrow cells (CSF). Lymphokines also contained factors that induced macrophage non-specific tumoricidal activity against fibrosarcoma 1023, antibody-dependent tumoricidal activity against lymphoma 18-8, and antimicrobial activities against amastigotes of the protozoan parasite, Leishmania tropica. The factors that regulated macrophage effector functions, however, were different from those that induced colony formation, and could be distinguished from CSF by Sephadex gel chromatography or heat sensitivity. To further analyze a role for CSF in induction of macrophage effector activities, conditioned medium from several nonlymphoid cell sources (L-929, WEHI-3, and endotoxin-treated lung cells) were assayed for CSF activities and capacity to induce tumoricidal and microbicidal activities. Conditioned medium that contained either macrophages CSF (CSF-1) or the factor that induced formation of both macrophage and granulocyte colonies failed to activate macrophages for effector activities against fibrosarcoma 1023, lymphoma 18-8, and L. tropica amastigotes (either resistance to infection or intracellular destruction). These data suggest that CSF has no direct role in activation of macrophages for tumoricidal and microbicidal activities against these targets.     

A spectrum in the susceptibility of leishmanial strains to intracellular killing by murine macrophages

The susceptibility of 26 strains and clones of Leishmania to in vitro killing by lymphokine (LK)-activated macrophages was determined. A spectrum in the susceptibility of Leishmania to macrophage killing was observed. Some leishmanias were completely resistant to killing, including some but not all of the L. mexicana strains studied. This resistance was expressed in amastigotes and stationary growth-phase promastigotes, but not in logarithmic promastigotes. In contrast, some L. braziliensis parasites failed to survive within either activated or nonactivated macrophages. Between these two extremes were strains that survived within nonactivated macrophages, but were readily killed within activated macrophages. These included L. donovani, L. major, and some L. mexicana strains. Finally, one L. mexicana strain (WR357) was found to be susceptible to killing at high LK concentrations, but was relatively resistant at lower LK concentrations or at cutaneous temperatures. The observed differences in susceptibility to macrophage-mediated microbicidal activity may explain, in part, the variable pathogenesis of leishmanial infections.     

Regulation of activated macrophage antimicrobial activities. Identification of lymphokines that cooperate with IFN-gamma for induction of resistance to infection

Macrophages exposed to lymphokines (LK) before exposure to parasites develop the capacity to resist infection with amastigotes of Leishmania major. Activity of LK for induction of this activated macrophage effector function is abrogated by depleting the LK of IFN-gamma, yet IFN-gamma is incapable of inducing the activity by itself. To identify the factors in LK that serve as second signals for induction of resistance to infection, we exposed macrophages to the following cytokines available as recombinant or highly purified reagents: CSF-1, granulocyte/macrophage colony-stimulating factor (GM-CSF), IL-1, -2, -3, -4, and -5, and IFN-alpha/beta. None of these factors induced resistance to infection by themselves or in combination with each other; in the presence of 50 U/ml IFN-gamma, three cytokines were active: GM-CSF, IL-2, and IL-4. IFN-gamma was an essential component of the activation cascade but was insufficient by itself to induce the effector reaction. Cytokines that act as cofactors with IFN-gamma worked directly on macrophages and not through another cell in the peritoneal cell (PC) cultures. Activation of PC depleted of Thy-1.2+ cells (85 +/- 5% macrophages) and bone marrow-derived macrophages (100% macrophages) showed that 50% maximal doses of GM-CSF, IL-2, and IL-4 for these macrophage-enriched populations were not different than for untreated PC. Unlike other effector reactions of activated macrophages, bacterial LPS did not synergistically enhance the activity of any of the cytokines, alone or in combination with IFN-gamma. Antibody depletion of the active cytokines from LK, singly or in combination, failed to alter the dose response of the active factors in whole LK for induction of resistance to infection. Thus, multiple factors can provide the second signal for IFN-gamma in the induction of resistance to infection, namely, GM-CSF, IL-2, IL-4, and at least two additional undefined factors in whole LK. Resistance to infection may be the first example of an activated macrophage effector reaction that has an absolute requirement for more than one endogenous signal for its induction.     

Differential susceptibility of activated macrophage cytotoxic effector reactions to the suppressive effects of transforming growth factor-beta 1

We examined the effects of TGF-beta 1 on induction of several activated macrophage antimicrobial activities against the protozoan parasite Leishmania, and on induction of tumoricidal activity against the fibrosarcoma tumor target 1023. TGF-beta by itself did not affect the viability of either the intracellular or extracellular target in concentrations up to 200 ng/ml. As little as 1 ng/ml TGF-beta, however, suppressed more than 70% of the intracellular killing activity of macrophages treated with lymphokines. In contrast, more than 100 ng/ml TGF-beta was required to suppress intracellular killing by cells activated with an equivalent amount of recombinant IFN-gamma. Addition of TGF-beta for up to 30 min after exposure to activation factors significantly reduced macrophage killing of intracellular parasites. Pretreatment of macrophages with TGF-beta was even more effective: treatment of cells with TGF-beta for 4 h before addition of activation factors abolished all macrophage intracellular killing activity. Regardless of treatment sequence, however, TGF-beta had absolutely no effect, at any concentration tested, on activated macrophage resistance to infection induced by lymphokines or by the cooperative interaction of IFN-gamma and IL-4. Effects of TGF-beta on tumoricidal activity of activated macrophages was intermediate to that of its effects on intracellular killing or resistance to infection. Lymphokine-induced tumor cytotoxicity was marginally (25%) affected by TGF-beta; 200 ng/ml was able to suppress IFN-gamma-induced tumoricidal activity by 40%. Thus, TGF-beta dramatically suppressed certain activated macrophage cytotoxic effector reactions, but was only partially or not at all effective against others, even when the same activation agent (IFN-gamma) was used. The biochemical target for TGF-beta suppressive activity in these reactions may be the pathway for nitric oxide production from L-arginine, because TGF-beta also inhibited the generation of nitric oxide by cytokine-activated macrophages.     

Human monocyte activation for cytotoxicity against intracellular Leishmania donovani amastigotes: induction of microbicidal activity by interferon-gamma

Macrophages are pivotal cells in interactions of man and leishmania. Leishmanial disease results from intracellular infection of macrophages: parasitized cells are seen in smears or biopsy specimens of lesions; macrophages cultured in vitro support replication of parasites. Paradoxically, parasite destruction is also mediated by macrophages, which become highly cytotoxic after exposure to immune lymphocytes or their lymphokine (LK) products. The precise molecular mechanisms by which lymphocytes or LK induce macrophage activation for leishmanicidal activity, however, are not yet known. We analyzed interactions of leishmania amastigotes with human monocytes cultured in vitro as a nonadherent cell pellet. Leishmania donovani and L. major replicated in freshly isolated monocytes. Monocytes treated with greater than 200 IU/ml of the LK, human Interferon-gamma (IFN-gamma), destroyed tumor cells and L. donovani, but not L. major. Phorbol myristate acetate, endotoxic bacterial lipopolysaccharide, and recombinant human IFN-alpha and IFN-beta did not induce cytotoxicity. The time course for induction of cytotoxicity contrasted sharply with that of previously described monocyte antileishmanial activity: IFN-gamma induced cytotoxicity even when added after infection with L. donovani; induction of cytotoxicity did not require that IFN-gamma be present throughout the period of culture after infection: a 30-min preinfection pulse of IFN-gamma was sufficient to induce 70% of maximal activity; and freshly isolated monocytes and cells cultured for up to 4 days in vitro prior to infection and IFN-gamma treatment were equally responsive to IFN-gamma. These studies provide convincing evidence for intracellular cytotoxicity for L. donovani by freshly isolated human monocytes. This system provides an important base for further analysis of induction and expression of cytotoxic mechanisms against leishmania and other intracellular organisms that cause human disease.     

Stimulation by granulocyte-macrophage colony-stimulating factor of Leishmania tropica killing by macrophages.

The intracellular protozoan parasite Leishmania tropica was found to survive unharmed and to multiply for several days in normal mouse peritoneal macrophages. In contrast, when infected monolayers were treated with GM-CSF, there was a continuous decrease in the percentage of infected cells, reaching less than 10% on day 4 in culture, compared to about 30% in normal controls. Microscopic observations showed an increased number of dead parasites in GM-CSF treated infected cells. Within 5 hr of incubation with GM-CSF, almost 40% of intracellular parasites showed morphologic damage, compared to less than 10% in untreated cells. Pretreatment of macrophage monolayers with pure GM-CSF before infection led to an increased level of phagocytosis of L. tropica parasites as reflected by the percentage of infected cells and the increased number of parasites in each infected cell. GM-CSF treated cultures showed 73% infected cells containing a mean of five parasites per cell, as compared to controls in which only about 50% of macrophages were infected with only two parasites per cell. The number of dead parasites per cell was 5-fold higher in the GM-CSF treated cultures at 2 hr. After 24 hr the percentage of infected GM-CSF treated cells was less than one-third that in the control cultures.     

Leishmania major amastigotes initiate the L-arginine-dependent killing mechanism in IFN-gamma-stimulated macrophages by induction of tumor necrosis factor-alpha

Macrophages exposed to IFN-gamma and infected with amastigotes of Leishmania major develop the capacity to eliminate the intracellular pathogen. This antimicrobial activity of activated macrophages correlates with the initiation of nitrogen oxidation of L-arginine, yet other reports suggest that two signals are required for induction of this biochemical pathway for effector activity. In the present studies, macrophages treated with up to 100 U/ml IFN-gamma, or 100 ng LPS, or 10(7) amastigotes produced minimal quantities (less than 9 microM) of NO2- and failed to develop cytotoxic effector activities. In contrast, the combination of IFN-gamma and either LPS (greater than 0.1 ng) or amastigotes (10(6) induced high concentrations (much greater than 30 microM) of NO2- and macrophage cytotoxicity against intra- and extracellular targets. The induction of nitrogen oxidation by amastigotes could be dissociated from LPS-induced events by 1) performing the assays in the presence of polymyxin B (which blocked LPS effects, but not amastigote effects), 2) determining the threshold of IFN-gamma required to prime cells for subsequent trigger (1 U/ml for LPS trigger effects; 10-fold higher for amastigotes), and 3) determining the heat sensitivity of the two trigger agents (amastigote effects abolished at 100 degrees C; LPS effects unaffected at this temperature). Further, culture fluids from amastigote-infected macrophages did not contain detectable LPS (less than 6 pg/ml). Possible parasite and cell-associated factors that could contribute to the induction of nitrogen oxidation and cytotoxic activity of IFN-gamma treated macrophages were examined: only certain intact microorganisms, LPS from a variety of bacteria, and the cytokine TNF alpha were effective. Both NO2- production and intracellular killing were abolished by the addition of anti-TNF-alpha mAb in the assay. TNF-alpha was produced by amastigote-infected macrophages and IFN-gamma dramatically enhanced secretion of this cytokine; IFN-gamma alone had no effect. Endogenous TNF-alpha produced during infection of macrophages with L. major acted in an autocrine fashion to trigger the production of L-arginine-derived toxic nitrogen intermediates that killed the intracellular parasites.     

Leishmania major and Leishmania tropica: I the in vitro effects of an immunomodulator, S2-Complex

This study was undertaken to try to determine the possible anti-leishmanial activity of S2-Complex, an organic complex of copper chloride, ascorbic acid, and nicotinamide. The promastigotes, axenic amastigotes, and intracellular amastigotes of both Leishmania major and Leishmania tropica were incubated with different concentrations of S2-Complex. The EC50 for each form was calculated. Results show that all forms of the parasites were dose dependently inhibited by S2-Complex. The promastigotes of both parasites were the most resistant with highest EC50 followed by axenic amastigotes. While intracellular amastigotes were the most sensitive with the lowest EC50.These results indicate that S2-Complex has a direct anti-leishmanial effect. When mice were treated with S2-Complex or BCG for four days before harvesting the macrophages, and the macrophages infected with both L. major and L. tropica, they showed increased phagocytosis and increased parasite killing. The results of S2-Complex were not statistically different from the immunomodulating agent BCG. These results indicate that S2-Complex has an immunomodulating effect in addition to the direct anti-leishmanial effect.     

Suppression of macrophage antimicrobial activity by a tumor cell product

Medium conditioned by tumor cells (TCM) and certain nonmalignant cells contains a trypsin-sensitive factor that suppresses macrophage oxidative metabolism. Because the killing of intracellular pathogens such as Toxoplasma gondii and Leishmania donovani by macrophages is largely oxygen-dependent, we tested the effect of TCM on the antiprotozoal activity of mouse peritoneal macrophages. After 24 hr of cultivation with TCM, in vivo and in vitro activated macrophages could no longer kill toxoplasmas or inhibit their replication. In vivo administration of TCM resulted in similar impairment. The leishmanicidal activity of resident and activated macrophages, when measured 6 hr after infection, was markedly suppressed by in vitro exposure to TCM. The addition of exogenous H2O2 in the form of glucose-glucose oxidase reconstituted the capacity of TCM-exposed macrophages to kill L. donovani promastigotes as quickly as control cells. Thus, TCM appears to deactivate macrophages by the functional criteria of suppressed antitoxoplasmal and antileishmanial activity, as well as by the biochemical criterion of suppressed oxidative metabolism.     

Sabotage and exploitation in macrophages parasitized by intracellular protozoans

Macrophages are crucial in immunity to infection. They possess potent antimicrobial function, and efficiently process and present peptide antigens for T-cell activation. Despite this, the intracellular protozoan parasites Toxoplasma gondii, Trypanosoma cruzi and Leishmania spp. target macrophages for infection. Each has adopted unique strategies to subvert macrophage antimicrobial functions. The parasites sabotage killing activities through sophisticated manipulation of intracellular macrophage signaling pathways. These subversive activities are probably dictated by the need to evade microbicidal effector function, as well as to avoid proinflammatory pathology that can destabilize the host-parasite interaction. The molecular details of how intracellular protozoans manipulate macrophage signal transduction pathways for their own ends are beginning to emerge.     

Kinetics and requirements for activation of macrophages for fungicidal activity: effect of protein synthesis inhibitors and immunosuppressants on activation and fungicidal mechanism.

Peritoneal-and pulmonary macrophages can be activated in vitro with lymphokines (LK) or IFN-gamma, without exogenous lipopolysaccharide, for fungicidal activity against several pathogenic fungi. However, neither the biochemical nor metabolic events of the activation process or of the effector phase have been defined. In the present work we sought to elucidate these events with time-course studies using inhibitors of protein synthesis as well as immunosuppressive agents. We found that protein synthesis inhibitors abrogated the activation process, because cycloheximide (CHX) (1-2 micrograms/ml) prevented activation of macrophages for fungicidal activity against Candida albicans, Blastomyces dermatitidis, and Paracoccidioides brasiliensis. Blocking of the activation process by CHX was not due to macrophage cytotoxicity, and CHX did not impair the ability of nonactivated macrophages to kill Candida parapsilosis. In kinetic studies we showed that activation of macrophages was induced in 4 hr of LK treatment and that CHX had no effect if added after this time. In contrast to CHX, therapeutic concentrations of hydrocortisone (HC), such as less than or equal to 5 micrograms/ml, or cyclosporin A (CsA), 5 micrograms/ml, did not significantly inhibit LK activation of macrophages for killing of fungi. In the effector phase, the fungicidal capacity of activated macrophages in short-term (less than or equal to 4 hr) killing assays could not be abrogated by CHX (5 micrograms/ml), HC (100 micrograms/ml), or CsA (10 micrograms/ml). These results demonstrate that the activation but not the effector mechanism of macrophages for fungicidal activity is blocked by inhibition of protein synthesis. In contrast, therapeutic concentrations of HC or CsA may not interfere with activation of macrophages or their killing mechanisms, thus providing a rationale for antifungal immunotherapy in certain clinical situations (e.g., infection in the immunosuppressed patient).     

Macrophage activation to kill Leishmania major: activation of macrophages for intracellular destruction of amastigotes can be induced by both recombinant interferon-gamma and non-interferon lymphokines

Macrophages treated with lymphokine (LK)-rich culture fluids from antigen- or mitogen-stimulated spleen cells or the hybridoma T cell 24/G1, or murine recombinant interferon-gamma (IFN-gamma) from either transfected monkey kidney cells (cos rIFN-gamma) or bacterial (E. coli) DNA (rIFN-gamma) developed the capacity to kill intracellular amastigotes of Leishmania major. Removal of IFN activity from LK by neutralizing fluid phase monoclonal anti-rIFN-gamma antibody, or by solid phase immunoadsorption, left residual macrophage activation factors that induced approximately 50% of the macrophage anti-leishmanial activity of untreated LK. In contrast, rIFN-gamma subjected to the same antibody treatments lost all capacity to induce this macrophage effector function. These results suggest that the intracellular destruction of amastigotes is regulated by several different factors. One of these factors is clearly IFN-gamma, which is pleiotropic in its effects on macrophage functions. The other non-IFN LK factors are immunochemically unrelated to IFN-gamma, and may regulate macrophage microbicidal activities in a more selective manner.