Interferon-gamma inhibits the intrahepatocytic development of malaria parasites in vitro

In this study, we examined the activity of recombinant interferon (IFN)-gamma against Plasmodium berghei exoerythrocytic forms (EEF) grown in vitro within the highly differentiated human hepatoma cell line HEPG2. We assayed the effect of IFN-gamma on parasite growth by DNA hybridization using a P. berghei specific DNA probe. The specific activity of IFN-gamma against EEF is very high, and depends upon the time of lymphokine addition. When IFN-gamma is added to HEPG2 cells containing intracellular EEF, 6 hr after sporozoite invasion, parasite DNA replication is inhibited by approximately 75% at 10(3) U/ml and 50% at 1 U/ml. This treatment can either abolish or greatly reduce the infectivity of EEF for mice. When added earlier, 3 hr after completion of sporozoite invasion, IFN-gamma inhibits parasite replication to an even greater degree. The highest levels of inhibition were obtained when IFN-gamma was added 6 hr prior to sporozoite invasion (100% inhibition at 10(2) U/ml, approximately 55% inhibition at 0.1 U/ml, and 17% inhibition at 0.001 U/ml). We found that HEPG2 cells express approximately 44,000 surface receptors for IFN-gamma. These data are consistent with the view that IFN-gamma exerts its antimalarial activity by binding to surface receptors on hepatocytes and inducing intracellular changes unfavorable for parasite development. Tryptophan starvation does not appear to be involved in this process. These findings also support the idea that IFN-gamma, released from immune T cells upon encountering sporozoite antigen, may be an important effector mechanism in sterile immunity to sporozoite challenge.     

Use of a DNA probe to measure the neutralization of Plasmodium berghei sporozoites by a monoclonal antibody

A specific DNA probe has been used to quantify the neutralizing effects of monoclonal antibodies (3D11) against the circumsporozoite protein of Plasmodium berghei sporozoites. The amount of parasite DNA was measured in the livers of Norway Brown rats at the peak of proliferation of the exoerythrocytic forms (EEF). In vitro treatment of 1.5 X 10(5) sporozoites with 0.36 microgram/0.5 ml of whole 3D11 IgG neutralized about 90% of the sporozoite infectivity. When the dose was 3.6 micrograms no signal was detected, indicating that less than ten sporozoites developed into EEF in the liver. In contrast, 3.6 micrograms of Fab obtained from 3D11 neutralized sporozoite infectivity by only 60%. Although the neutralizing effect of 3D11 was very marked, the infected rats developed parasitemias after a prolonged delay in patency, suggesting that a small proportion of sporozoites was resistant to the effects of 3D11. The sporozoites were subjected to four cycles of 3D11-mediated selection, each one involving treatment of sporozoites with the antibodies, injection of the mixture into rats, infection of hamsters with blood stage parasites obtained from the rats, feeding of Anopheles stephensi on these hamsters, and obtaining sporozoites from the salivary glands of the infected mosquitoes. After four cycles of selection, the susceptibility of the resulting sporozoites to different concentrations of 3D11 was compared with that of nonselected sporozoites. No differences were detected, indicating that the capacity of a few sporozoites to escape the neutralizing effect of 3D11 antibodies is not inherited.     

In vivo administration of recombinant IFN-gamma induces macrophage activation, and prevents acute disease, immune suppression, and death in experimental Trypanosoma cruzi infections

Recombinant murine IFN-gamma (rMu-IFN-gamma) was demonstrated to be a potent in vivo activator of mouse peritoneal macrophages to kill Trypanosoma cruzi in vitro and to be capable of conferring protection against death from acute T. cruzi infection. Following i.p. injections of rMu-IFN-gamma, resident peritoneal macrophages were cultured and infected with T. cruzi in vitro. Numbers of intracellular parasites were determined at different times thereafter. Ten or 100 micrograms (1 microgram = 6.5 X 10(5) U) of Mu-IFN-gamma, injected both 24 and 4 h before macrophage harvest, induced up to 99% inhibition of T. cruzi. One microgram of rMu-IFN-gamma was not effective under these conditions. In vitro inhibition of T. cruzi by peritoneal macrophages occurred by 24 h after infection and continued until at least 120 h after infection. There were no significant differences in initial parasite uptake by macrophages from IFN-gamma-treated or control mice, indicating that the rMu-IFN-gamma induced parasite killing. One i.p. dose of 10 micrograms was as effective as two doses if the single injection was given 24 h before macrophage harvest. In subsequent experiments, mice were given multiple injections of 10 micrograms rMu-IFN-gamma beginning 24 h before or 2 h after infection with virulent T. cruzi. Mice treated with rMu-IFN-gamma had significantly lower parasitemias and decreased morbidity compared with control mice. Proliferative responses to Con A and antibody responses to SRBC were not significantly lowered in IFN-gamma-treated mice, in contrast to untreated infected controls. All of the IFN-gamma-treated mice survived acute T. cruzi infection, whereas 100% of saline-treated infected mice died. It was demonstrated in this study that rMu-IFN-gamma activated mouse macrophages in vivo to kill T. cruzi and that rMu-IFN-gamma significantly reduced morbidity and immune suppression, and eliminated mortality resulting from acute infection with this parasite.     

DNA from pre-erythrocytic stage malaria parasites is detectable by PCR in the faeces and blood of hosts

Following the bite of an infective mosquito, malaria parasites first invade the liver where they develop and replicate for a number of days before being released into the bloodstream where they invade red blood cells and cause disease. The biology of the liver stages of malaria parasites is relatively poorly understood due to the inaccessibility of the parasites to sampling during this phase of their life cycle. Here we report the detection in blood and faecal samples of malaria parasite DNA throughout their development in the livers of mice and before the parasites begin their growth in the blood circulation. It is shown that parasite DNA derived from pre-erythrocytic stage parasites reaches the faeces via the bile. We then show that different primate malaria species can be detected by PCR in blood and faecal samples from naturally infected captive macaque monkeys. These results demonstrate that pre-erythrocytic parasites can be detected and quantified in experimentally infected animals. Furthermore, these results have important implications for both molecular epidemiology and phylogenetics of malaria parasites. In the former case, individuals who are malaria parasite negative by microscopy, but PCR positive for parasite DNA in their blood, are considered to be “sub-microscopic” blood stage parasite carriers. We now propose that PCR positivity is not necessarily an indicator of the presence of blood stage parasites, as the DNA could derive from pre-erythrocytic parasites. Similarly, in the case of molecular phylogenetics based on DNA sequences alone, we argue that DNA amplified from blood or faeces does not necessarily come from a parasite species that infects the red blood cells of that particular host.     

Antigen presentation by interferon-gamma-treated endothelial cells and fibroblasts: differential ability to function as antigen-presenting cells despite comparable Ia expression

The effect of interferon-gamma (IFN-gamma) on endothelial cell (EC) and fibroblast (FB) class II major histocompatibility complex (MHC) gene product expression and antigen presenting ability was examined. Control FB did not express class II MHC gene products, whereas a small (less than 1%) population of passaged EC expressed class II gene products. IFN-gamma induced a comparable density of HLA-DR expression on nearly all EC and FB. IFN-gamma-treated EC and FB also expressed HLA-DP but at a lower density, whereas HLA-DQ expression was barely detectable on either cell type. Control FB were not able to stimulate allogeneic T4 cell DNA synthesis or function as antigen-presenting cells (APC). Control EC were also unable to stimulate allogeneic T4 cell DNA synthesis unless large numbers of stimulator cells were used. Small numbers of IFN-gamma-treated EC were able to stimulate allogeneic T4 cell DNA synthesis, whereas larger numbers were markedly more effective than control EC. In contrast, IFN-gamma-treated FB were ineffective stimulators of allogeneic T4 cell DNA synthesis. IFN-gamma-treated FB were able to present the exogenous antigen SKSD to autologous but not allogeneic T4 cells, but they were extremely inefficient APC. The inability of IFN-gamma-treated FB to function as APC could not be explained by FB-mediated immunosuppression, Ia density, or HLA-DQ expression. This limited capacity of IFN-gamma-treated FB to participate in Ia-restricted functional interactions with T4 cells correlated with a similar diminished capacity to support nonspecific mitogen-induced proliferation of T4 cells before IFN-gamma-induced Ia expression. This accessory cell function was not enhanced by IFN-gamma treatment. Monocytes syngeneic to the responding T4 cells but not interleukin 1 (IL 1) permitted IFN-gamma-treated FB but not control FB to stimulate allogeneic T4 cell DNA synthesis, but they remained markedly less effective stimulators than monocytes. Moreover, IFN-gamma-treated FB were effective stimulators of alloprimed T4 cells, in contrast to their inability to stimulate fresh T4 cells. Furthermore, monocytes and IFN-gamma-treated FB were comparably effective stimulators of alloreactive T cell lines. These data suggest that accessory cells perform functions unrelated to Ia and IL 1 that are necessary for mitogen-, alloantigen-, and antigen-induced proliferation of freshly isolated T cells. Monocytes and EC effectively perform this function, but FB do not. This accessory cell function does not seem to be as important for the activation of primed T cells.     

Differential effects of interferon-gamma and tumor necrosis factor-alpha on Toxoplasma gondii proliferation in organotypic rat brain slice cultures

Organotypic slice culture explants of rat cortical tissue infected with Toxoplasma gondii tachyzoites were applied as an in vitro model to investigate host-pathogen interactions in cerebral toxoplasmosis. The kinetics of parasite proliferation and the effects of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in infected organotypic cultures were monitored by light microscopy, transmission electron microscopy (TEM), and quantitative polymerase chain reaction (PCR) assay. As assessed by the loss of the structural integrity of the glial fibrillary acidic protein-intermediate filament network, tachyzoites infected and proliferated mainly within astrocytes, whereas neurons and microglia remained largely unaffected. Toxoplasma gondii proliferation was severely inhibited by IFN-y. However, this inhibition was not linked to tachyzoite-to-bradyzoite stage conversion. In contrast, TNF-alpha treatment resulted in a dramatically enhanced proliferation rate of the parasite. The cellular integrity in IFN-gamma-treated organotypic slice cultures was severely impaired compared with untreated and TNF-alpha-treated cultures. Thus, on infection of organotypic neuronal cultures, IFN-gamma and TNF-alpha exhibit largely detrimental effects, which could contribute to either inhibition or acceleration of parasite proliferation during cerebral toxoplasmosis.     

Th2-like CD8 T cells: their role in protection against infectious diseases

The expression of cytolytic activity and production of interferon gamma (IFN-gamma) by CD8(+) T cells is thought to play a fundamental role in protection against infection by viruses and intracellular parasites. Fran?ois Erard and Graham Le Gros have recently shown that CD8(+) T cells activated in the presence of interleukin 4 (IL-4) can switch development to a CD8(-)CD4(-)Th2-like phenotype that is not cytolytic and that does not produce IFN-gamma. Here they speculate on whether this IL-4-induced switch is used by the host to make a more-effective response against parasite invasion, or i f it is a host mechanism used by the parasite to evade protective CD8(+) T-cell responses.     

Asexual blood stages of Plasmodium falciparum exhibit signs of secondary necrosis, but not classical apoptosis after exposure to febrile temperature (40 C)

It has been shown by others that after cultures of Plasmodium falciparum were exposed to a febrile temperature of 40 C, parasitemia was reduced in the subsequent generation, suggesting a temperature-induced inhibition of trophozoites and schizonts. In the current study, influences unique to cultivation were ruled out, demonstrating that 40 C impacted the parasites directly. Metabolic profiling of DNA synthesis, protein synthesis, and glucose utilization clearly indicated that febrile temperatures had a direct effect on parasite development, beginning 20-24 hr after erythrocyte invasion. The mechanism of parasite death was investigated for evidence of temperature-induced apoptosis. Lack of typical physiological hallmarks, namely, caspase activation, characteristic mitochondrial membrane potential changes, and DNA degradation as indicated by DNA laddering, eliminated 'classical' apoptosis as a mechanism of parasite death. Parasites dying under the influence of heat, staurosporine, and chloroquine initially appeared pyknotic by light and electron microscopy (as in apoptosis), but eventual swelling and lysis of the food vacuole membrane led to secondary necrosis. Chloroquine did induce DNA laddering, but it was later attributed to occult white blood cell contaminants. While not apoptosis, the results do not rule out other forms of temperature-induced programmed cell death.     

Mechanism of interferon action: inhibition of vesicular stomatitis virus replication in human amnion U cells by cloned human gamma-interferon. I. Effect on early and late stages of the viral multiplication cycle

The molecular basis of the inhibition of vesicular stomatitis virus (VSV) replication by pure recombinant gamma-interferon (IFN-gamma) in human amnion U cells was examined. A saturating concentration of IFN-gamma induced, at maximum, about a two log10 reduction in infectious VSV yield. The kinetics of induction of the antiviral activity by IFN-gamma were first order over the period of about 6-18 h, following a lag of about 3 h, after treatment with a saturating concentration of IFN-gamma. The relationship of the inhibition in VSV infectivity to the early and late events of the VSV multiplication cycle was investigated. IFN-gamma treatment had no detectable effect on the adsorption and penetration of VSV virions or on their uncoating to yield viral nucleocapsids. The polypeptides of adsorbed or uncoated VSV particles were neither preferentially degraded nor detectably altered in IFN-gamma-treated U cells, as compared to untreated U cells. Progeny virions isolated from IFN-gamma-treated U cells, although greatly reduced in number, were found to be equally as infectious as those isolated from untreated U cells. Progeny virions from IFN-gamma-treated cells also possessed the same composition of viral proteins as was observed for virions from untreated cells. These results suggest that conditions of IFN-gamma treatment sufficient to reduce the yield of infectious VSV progeny 100-fold do not detectably affect either the early or the late stages of the VSV multiplication cycle.     

Plasmodium falciparum: studies on mature exoerythrocytic forms in the liver of the chimpanzee, Pan troglodytes

Mature exoerythrocytic forms (EEF) of Plasmodium falciparum from the chimpanzee were examined by light- and transmission electron microscopy from a liver biopsy taken on Day 6 after sporozoite inoculation. Infectivity of the sporozoites obtained from whole mosquitoes which were membrane fed on cultured gametocytes was about 4-6%. In comparison, salivary gland sporozoites added to human hepatocytes in vitro had only a developmental percentage of 0.02 to 0.05% at Day 5. The EEF found in the liver biopsy were not all at the same stage of development. Immature compact parasites were seen simultaneously with stages with fully formed merozoites, indicating a rapid final maturation or asynchrony. At Day 7.5, large numbers of rings were already seen in the peripheral blood, indicating a duration of the liver development of P. falciparum in the chimpanzee of about 5.5-6 days. The process of merogony at the fine structural level was comparable to that described for rodent and other primate parasites in vivo. Compared to the fine structure of EEF in vitro in cultured human hepatocytes, the parasites described here were much more advanced in development. There appeared to be some cell infiltration with collagen deposition around the intracellular parasite; however, no marked degeneration of EEF was observed.     

IL-10 inhibits parasite killing and nitrogen oxide production by IFN-gamma-activated macrophages.

IL-10, a cytokine produced by CD4+ T lymphocytes belonging to the Th-2 subset, has previously been shown to inhibit the synthesis of IFN-gamma by both T cells and NK cells. We now demonstrate that IL-10 can also down-regulate IFN-gamma-dependent immunity by blocking the ability of that lymphokine to activate macrophages. Thus, IL-10, in a dose-dependent manner, inhibits the microbicidal activity of IFN-gamma-treated inflammatory macrophages against intracellular Toxoplasma gondii as well as the extracellular killing of schistosomula of Schistosoma mansoni. This suppression correlates with the inhibition by IL-10 of IFN-gamma-induced production of toxic nitrogen oxide metabolites, an effector mechanism previously implicated in the killing by macrophages of both parasite targets. IL-10 inhibition of nitric oxide production was shown to occur when the cytokine is given before or together with the IFN-gamma-activating stimulus, but not after its removal from the cultures and to require 12 h of contact for maximal suppressive effect on macrophage function. These results, taken together with previous findings on the down-regulation of Th1 lymphokine production by IL-10, indicate that the induction of IL-10 may be an important strategy by which parasites evade IFN-gamma-dependent, cell-mediated immune destruction.     

IFN-gamma induces apoptosis in mouse embryonic stem cells, a putative mechanism of its embryotoxicity

It has been reported that interferon (IFN)-gamma should inhibit in vitro mouse embryo growth by direct cell toxicity. However, the mechanism involved has not been clearly established. In the present study, this question was addressed using the embryonic stem (ES) cell model. It was found that IFN-gamma, induces a dose-dependent apoptosis in ES cells, as assessed by trypan-blue staining, by Annexin-V labeling and DNA analysis, Moreover, IFN-gamma treatment cooperates with Fas-mediated apoptosis, a phenomenon that has been recently reported. As Bcl-2 oncoprotein functions as a death repressor molecule in an evolutionarily conserved cell death pathway, its expression was analyzed by flow cytometry. It was demonstrated that Bcl-2 is expressed in ES cells. When compared to untreated ES cells, IFN-gamma-treated, apoptotic cells expressed a lower Bcl-2 level and a normal level of Fas, whereas surviving cells expressed a normal level of Bcl-2 but a lower Fas expression. Altogether, these data suggest that IFN-gamma may influence early mouse embryo development by promoting apoptosis, which may constitute a novel mechanism of IFN-gamma embryotoxicity.     

IL-10 synergizes with IL-4 and transforming growth factor-beta to inhibit macrophage cytotoxic activity.

After activation with IFN-gamma, thioglycollate-elicited murine peritoneal macrophages kill schistosomula of Schistosoma mansoni in vitro by an L-arginine-dependent mechanism which involves the production of reactive nitrogen oxides (NO). In the present study we demonstrate that the regulatory cytokines IL-10, IL-4, and transforming growth factor-beta (TGF-beta) are potent inhibitors of this extracellular killing function of activated macrophages. Each cytokine was found to suppress killing of schistosomula in a dose-dependent fashion. The activity of IL-10 was not permanent, because subsequent treatment with additional IFN-gamma 2 to 6 h later reversed the inhibition of macrophage larval killing. More importantly, the combination of suboptimal levels of any two of these three cytokines was found to give a potent synergistic suppression of schistosomulum killing by IFN-gamma-treated macrophages. Similarly, IL-10, IL-4, or TGF-beta alone blocked the production of NO, and when used in combination these cytokines exhibited an enhanced inhibitory effect on nitrite production. Macrophage-mediated killing of schistosomula through the generation of NO has been shown previously to be a major effector mechanism of schistosome immunity. The results presented here suggest that the suppression of this mechanism by induction of the regulatory cytokines IL-10, IL-4, and TGF-beta, which are known to be produced during schistosome infection, may be an important strategy used by the parasite to evade macrophage-mediated immune destruction.     

Stage- and time-dependent effects of crisis form factor on Plasmodium falciparum in vitro

To determine the stage- and time-dependent effects of crisis form factor (CFF) on Plasmodium falciparum metabolism in vitro, the parasite erythrocytic cycle was divided into sequential 8 hr time intervals, and highly synchronous parasites were exposed to CFF for various lengths of time. Hypoxanthine and phenylalanine incorporation into parasite nucleic acids and proteins, respectively, and glucose consumption by the parasites were compared in cultures grown in CFF-containing or nonimmune sera. The most profound derangement of metabolism occurred in parasites 0-8 hr post-invasion. Inhibition correspondingly decreased in tests started with progressively older parasites. Cultivation in CFF serum for 8 hr caused maximal inhibition of purine and amino acid incorporation; longer periods of exposure did not increase inhibition. In contrast, CFF's effect on glucose consumption varied inversely to the duration of exposure to CFF. As the parasites matured in the presence of CFF, inhibition of the rate of glucose utilization decreased, with little or no reduction in consumption observed as parasites entered schizogony. Of the 3 metabolic parameters studied, hypoxanthine was the most sensitive indicator of metabolic inhibition throughout the cycle.     

Calcium signal regulates temperature-dependent transformation of sporozoites in malaria parasite development

The infection by the malaria parasite of its mammalian host is initiated by the asexual reproduction of the parasite within the host hepatocyte. Before the reproduction, the elongated sporozoites undergo a depolarizing morphogenesis to the spherical exo-erythrocytic form (EEF). This change can be induced in vitro by shifting the environmental conditions, in the absence of host hepatocytes. Using rodent malaria parasites expressing a FRET-based calcium sensor, YC3.60, we observed that the intracellular calcium increased at the center of the bulbous structure during sporozoite transformation. Modulators of intracellular calcium signaling (A23187 and W-7) accelerated the sporozoite-rounding process. These data suggest that calcium signaling regulates the morphological development of the malaria parasite sporozoite to the EEF, and support a fundamental role for calcium as a universal transducer of external stimuli in the parasitic life cycle.     

Selective augmentation by recombinant interferon-gamma of the intracellular content of S-adenosylmethionine in murine macrophages

Treatment of mouse peritoneal macrophages with IFN-gamma augmented the intracellular content of S-adenosylmethionine, as measured by quantitative high-performance liquid chromatography. Accumulation of S-adenosylhomocysteine, a competitive product of S-adenosylmethionine, was not detectable, either by direct measurement of absorbance or by radioisotopic techniques in IFN-gamma-treated macrophages. However, accumulation of S-adenosylhomocysteine was observed after treatment of macrophages with known inhibitors of S-adenosylhomocysteine catabolism. No inhibition of phospholipid methylation was observed upon IFN-gamma treatment, indicating that no reduction of the S-adenosylmethionine to S-adenosylhomocysteine ratio is induced by IFN-gamma in murine macrophages. The increased content of S-adenosylmethionine was associated with the acquisition of tumoricidal activity by macrophages upon IFN-gamma treatment. LPS also augmented the cellular content of S-adenosylmethionine and activated macrophages to become cytotoxic, suggesting a common mechanism of action for IFN-gamma and LPS in macrophage activation. Treatment of macrophages with cycloleucine, an agent that induces depletion of cellular S-adenosylmethionine, made the macrophages refractory to induction of cytolytic activity by IFN-gamma, suggesting a critical role for S-adenosylmethionine in macrophage activation.     

Induction of suppressor of cytokine signaling-1 by Toxoplasma gondii contributes to immune evasion in macrophages by blocking IFN-gamma signaling

Toxoplasma gondii is an intracellular parasite that survives and multiplies in professional phagocytes such as macrophages. Therefore, T. gondii has to cope with the panel of antimicrobial host immune mechanisms, among which IFN-gamma plays a crucial role. We report in this study that in vitro infection of murine macrophages with viable, but not with inactivated, parasites results in inhibition of IFN-gamma signaling within the infected cells. Thus, infection of RAW264.7 macrophages with tachyzoites inhibited IFN-gamma-induced STAT-1 tyrosine phosphorylation, mRNA expression of target genes, and secretion of NO. These effects were dependent on direct contact of the host cells with living parasites and were not due to secreted intermediates. In parallel, we report the induction of suppressor of cytokine signaling-1 (SOCS-1), which is a known feedback inhibitor of IFN-gamma receptor signaling. SOCS-1 was induced directly by viable parasites. SOCS overexpression in macrophages did not affect tachyzoite proliferation per se, yet abolished the inhibitory effects of IFN-gamma on parasite replication. The inhibitory effects of T. gondii on IFN-gamma were diminished in macrophages from SOCS-1-/- mice. The results suggest that induction of SOCS proteins within phagocytes due to infection with T. gondii contributes to the parasite's immune evasion strategies.     

Protective T cell immunity against malaria liver stage after vaccination with live sporozoites under chloroquine treatment

In this study we present the first systematic analysis of the immunity induced by normal Plasmodium yoelii sporozoites in mice. Immunization with sporozoites, which was conducted under chloroquine treatment to minimize the influence of blood stage parasites, induced a strong protection against a subsequent sporozoite and, to a lesser extent, against infected RBC challenges. The protection induced by this immunization protocol proved to be very effective. Induction of this protective immunity depended on the presence of liver stage parasites, as primaquine treatment concurrent with sporozoite immunization abrogated protection. Protection was not found to be mediated by the Abs elicited against pre-erythrocytic and blood stage parasites, as demonstrated by inhibition assays of sporozoite penetration or development in vitro and in vivo assays of sporozoite infectivity or blood stage parasite development. CD4(+) and CD8(+) T cells were, however, responsible for the protection through the induction of IFN-gamma and NO.     

Trypanosoma cruzi: cytokine effects on macrophage trypanocidal activity

Mouse macrophages infected with Trypanosoma cruzi in vitro may be activated to reduce parasite infection by interferon gamma (IFN-gamma). The addition of up to 10,000 units of IFN-gamma however, does not result in a 100% reduction of intracellular parasites. We, therefore, investigated the possibility that macrophages require an additional signal or signals to completely clear T. cruzi infection. Because the combination of IFN-gamma with lipopolysaccharide greatly enhanced macrophages ability to decrease the number of intracellular parasites, the interaction of IFN-gamma with tumor necrosis factor (TNF) was examined. TNF alone and the combination of TNF with IFN-gamma did not have a significant effect on reducing parasite numbers below that obtained with IFN-gamma alone. This was also true for lymphotoxin, a lymphokine similar to TNF in structure and function. The effect of IFN-gamma in combination with a cytokine-rich supernatant containing IL-2, IL-3, IL-4, IL-5, and IFN-gamma on macrophage clearance of the parasite was also examined. The cytokine-rich supernatant alone had no effect on reducing parasite infection of the macrophages; indeed, in some experiments the addition of the supernatant resulted in an increase in the level of parasite infection. However, 1000 units of IFN-gamma combined with the complex cytokine mixture caused a decrease in parasite infection of nearly 100% compared to that of control cultures treated with media alone. To determine which cytokine or cytokines in the supernatant were responsible for this synergistic activity, anti-cytokine antibodies were added to the supernatant prior to its addition with IFN-gamma to the cultures. Anti-IL-4 was the only antibody found to inhibit the synergism of IFN-gamma with the cytokine-rich supernatant. IL-4, however, did not significantly enhance the ability of IFN-gamma to induce macrophage clearance of the parasite, and IL-4 alone caused a slight increase in parasite infection in vitro. These results further define the role that cytokines play in T. cruzi infection of macrophages in vitro and suggest that the interaction of cytokine networks within this system is complex.