Expression of a 120,000 dalton protein during tumoricidal activation in murine peritoneal macrophages

Modulation of protein expression during interferon-gamma (IFN-gamma)-lipopolysaccharide (LPS)-mediated macrophage tumoricidal activation has been examined by metabolic radiolabeling of various murine peritoneal macrophage populations with [35S]methionine followed by SDS-PAGE analysis. Although both IFN-gamma and LPS are capable of stimulating the expression of several proteins when used independently, combined treatment induced the enhanced or de novo expression of a 120,000 dalton polypeptide. The expression of this protein was synergistically regulated by both IFN-gamma and LPS in a manner strongly reminiscent of the functional synergism that these two agents exhibit with respect to induction of tumoricidal activity. p120 expression could be seen first at approximately 3 hr after the addition of both agents, reached optimal expression by 6 hr, and maintained elevated synthesis for up to 24 hr. This time course corresponds closely to that seen for the acquisition of tumoricidal competence. Macrophages elicited in the primed state of activity in vivo with methyl vinyl ether co-polymer II (MVE-II) did not express p120, but could be induced to do so when treated with low doses of LPS. Under similar conditions, MVE-II-elicited cells also acquire tumoricidal activity. Macrophages obtained from mice chronically infected with bacillus Calmette-Guerin constitutively expressed both p120 and cytolytic activity. If such macrophages were cultured for 24 hr, the expression of both events decayed and was lost, but could be restored by treatment with low doses of LPS. Thus the data support a strong correlation between the expression by macrophages of a novel 120,000 dalton protein and the expression of tumor cytotoxicity.     

Alteration of in vitro murine peritoneal macrophage function by dietary enrichment with eicosapentaenoic and docosahexaenoic acids in menhaden fish oil

The effects of diets containing menhaden fish oil (MFO), compared with those of diets containing safflower oil (SAF) or an essential fatty acid deficient hydrogenated coconut oil (HCO), on in vitro activation of tumoricidal capacity by murine macrophages were assessed. Mice fed the experimental diets for 4 weeks were injected intraperitoneally with sterile thioglycollate broth 3 days before use. There was no difference between any of the groups with respect to total peritoneal exudate cells or the percentage of macrophages, although the fatty acid profile of purified adherent macrophages closely paralleled that of the diets. Macrophages from mice fed MFO killed fewer P815 mastocytoma cells upon activation with recombinant interferon gamma (IFN gamma) and lipopolysaccharide. Macrophages from all diets were equally competent for tumoricidal capacity when activated pharmacologically with calcium ionophore, phorbol 12-myristate 13-acetate, and lipopolysaccharide (LPS), suggesting that MFO diet macrophages were hyporesponsive to IFN gamma. Priming with higher concentrations of IFN gamma restored the partial defect in activation of MFO macrophages. When activated for 24 hr with high levels of LPS, macrophages from mice fed SAF displayed little cytolytic capacity; addition of indomethacin. (1 microM) resulted in enhanced levels of P815 kill. In contrast, MFO and HCO diet macrophages were highly cytolytic with similar LPS treatment with or without indomethacin. Macrophages from mice fed SAF produced threefold more prostaglandin E in response to LPS than did MFO and HCO diet macrophages. These results suggest that dietary manipulation of fatty acids can alter activation of tumoricidal capacity of macrophages, possibly both dependent and independent of changes in eicosanoid synthesis.     

The correlation between specific protein synthesis and tumoricidal function in murine peritoneal macrophages

Macrophages from the lipopolysaccharide (LPS)-responsive C3H/HeN mouse strain and the closely related LPS-nonresponsive C3H/HeJ strain were compared for tumoricidal activation and protein synthetic changes following in vivo and in vitro stimulation, utilizing two-dimensional polyacrylamide gel electrophoresis of [35S]methionine-labeled proteins. Peritoneal macrophages elicited from C3H/HeN mice with heat-killed Propionibacterium acnes exhibited tumoricidal activity in a 16-hr cytolytic assay and expressed cytoplasmic levels of a 23.5-kDa protein during 48 hr of culture. The inability to detect persistent expression of p23.5 in P. acnes-stimulated C3H/HeJ macrophages correlated with the cytolytic impotence of those cells in the 16-hr chromium release assay. C3H/HeN macrophage populations lacking tumoricidal capacity could be rendered lytic, as could P. acnes-elicited C3H/HeJ macrophages, following in vitro stimulation with bacterial lipopolysaccharide. Concomitant with the LPS-induced expression of new functional activity was the appearance of augmented levels of several macrophage-specific proteins, including p23.5. This effect was dependent upon the lipid A moiety of LPS as the effects of LPS could be blocked by inclusion of polymyxin B sulfate in the culture medium. However, neither tumoricidal function nor protein modulation could be readily induced in C3H/HeJ proteose peptone-elicited or resident macrophages. These results identify biochemical responses to stimuli which may be requisite to acquisition or execution of cytolytic activity.     

Maleyl-BSA and fucoidan induce expression of a set of early proteins in murine mononuclear phagocytes

We examined the effect of maleyl-BSA on specific protein expression in murine peritoneal macrophages by radiolabeling treated macrophages with [35S]methionine followed by SDS-polyacrylamide gel electrophoresis. Such treatment induces the expression of a set of at least seven proteins (38, 42, 57, 65, 75, 80, and 85 kD). A similar set of proteins is also induced by treatment of macrophages with the algal polysaccharide fucoidan. The proteins resemble those induced in response to treatment of this same cell population with bacterial lipopolysaccharide (LPS), as judged by co-migration in both one- and two-dimensional electrophoresis. Two proteins induced by either LPS or maleyl-BSA (e.g., p57 and p85) show similar primary structure, as assessed by partial proteolytic peptide mapping confirming their identity. The induction of these proteins by maleyl-BSA is a transient phenomenon, being expressed as early as 1 hr after treatment and declining after 8 hr even in the continuous presence of the stimulus. The dose of maleyl-BSA required to induce the response varies to some extent with the protein in question, but agrees with the Kd for ligand-receptor binding. Chloroquine, which blocks the degradation of ligand, does not inhibit the induction of early protein synthesis. Whereas the induction of these proteins is blocked by inhibition of RNA synthesis with actinomycin D, the reversible inhibition of protein synthesis with cycloheximide during the induction phase does not prevent their expression. LPS, maleyl-BSA, and fucoidan previously have been shown to stimulate protease secretion and tumoricidal function in appropriately primed macrophages. The present findings now demonstrate that all three agents can also mediate the expression of early genes which may participate in the acquisition of functional competence.     

Effect of cisplatin, lipopolysaccharide, muramyl dipeptide and recombinant interferon-gamma on murine macrophages in vitro. I. Macrophage-mediated tumor cell lysis

Murine macrophage monolayers treated with cisplatin, lipopolysaccharide (LPS), muramyl dipeptide (MDP) or recombinant interferon-gamma (rIFN gamma) were observed to have significantly increased tumoricidal activity. rIFN gamma had synergistic effects with cisplatin, LPS or MDP in activating macrophages. However, MDP showed much more pronounced synergism with cisplatin and LPS than with rIFN gamma. Supernatants collected from these activated macrophage monolayers also showed increased tumoricidal activity. Tumor cell lysis mediated by cisplatin-treated macrophages did not require priming with rIFN gamma though it may be necessary as a first signal for the increased macrophage activation with LPS and MDP.     

Characterization of protein kinase C activity in interferon gamma treated murine peritoneal macrophages

Macrophage activation for tumoricidal and microbicidal functions can be achieved in part by treatment with recombinant interferon gamma (IFN gamma) in vitro. We have previously demonstrated that IFN gamma treatment of murine peritoneal macrophages results in a two- to five-fold increase in the activity of Ca++, phospholipid dependent protein kinase C (Hamilton et al., J. Biol. Chem., 260:1378, 1985). We now report that this effect was not dependent upon continuing protein synthesis since treatment with cycloheximide under conditions where normal protein synthesis was inhibited by greater than 95% had no effect upon the development of increased enzyme activity. Examination of Ca++ and phospholipid requirements revealed no differences between enzyme isolated from control or IFN gamma treated cells. Similarly, protein kinase C from control and IFN gamma-treated cells could not be distinguished in terms of the diacylglycerol (DG) or phorbol diester (PMA) concentration required for stimulation of activity. Kinetic analysis of the ATP (as substrate) concentration dependence revealed that both control and treated enzyme preparations (either basal or stimulated) had comparable Km values. Maximum velocity (Vmax) was increased both by IFN gamma treatment and also by stimulation with DG or PMA. The major difference which could be discerned between protein kinase C derived from control versus IFN gamma-treated macrophages was the magnitude of the response to DG or PMA; IFN gamma treatment increased the stimulation index (i.e., ratio of basal to stimulated activity) by a factor of two to four fold. These results suggest that IFN gamma treatment leads to reversible modulation of existing protein kinase C resulting in increased catalytic efficiency when exposed to an appropriate stimulant.     

Inhibition of macrophage activation by calcium channel blockers and calmodulin antagonists

The biochemical mechanisms by which macrophages become activated to the tumoricidal state are poorly understood. To investigate the role of calcium in this process, the effect of calcium channel blockers and calmodulin antagonists on the acquisition of tumoricidal properties by macrophages activated by a number of different agents was examined. Activation of thioglycollate-stimulated C57BL/6 mouse peritoneal macrophages by macrophage activation factor (MAF) plus LPS, IFN-gamma plus LPS or the calcium ionophore, A23187, was inhibited in a dose-dependent fashion by the calcium channel blockers nifedipine and verapamil. These agents blocked the influx of 45Ca into macrophages activated by MAF plus LPS. Macrophage activation was also inhibited by chlorpromazine, W-7, and calmidazolium at concentrations known to perturb calmodulin function. The data suggest that activation of macrophages to the tumoricidal state is a calcium-dependent process involving the participation of calcium-regulated biochemical reactions whose activities can be modulated by pharmacological agents that frustrate transmembrane calcium fluxes and/or inhibit calmodulin function.     

Production of tumor necrosis factor by rIFN-gamma-primed C3H/HeJ (Lpsd) macrophages requires the presence of lipid A-associated proteins

Previous studies have shown that the activation of murine macrophages to a fully tumoricidal state requires that specific environmental signals be delivered to the macrophage in a step-wise manner: a "priming" signal first renders the macrophage stimulated, but not cytolytic. The addition of a second or "trigger" signal to the primed macrophage results in tumoricidal activity. One potent priming signal has been identified as IFN-gamma and one often used trigger signal for endotoxin-responsive (Lpsn) macrophages is LPS. In contrast to LPS-responsive macrophage, rIFN-gamma-primed C3H/HeJ (Lpsd) macrophages fail to become cytolytic in response to protein-free, phenol-water-extracted LPS preparations, but become tumoricidal when exposed in vitro to protein-rich butanol-extracted LPS or purified lipid A-associated proteins. Further characterization of the activation requirements of the C3H/HeJ macrophages revealed that for optimal elaboration of TNF in vitro, two signals were also required: rIFN-gamma and a second signal that contained LAP. C3H/HeJ macrophages macrophages primed with rIFN-gamma failed to produce TNF in response to any concentration of protein-free phenol-water extracted LPS, even when supernatants were concentrated before assaying for functional activity in a standard TNF L929 fibroblast assay. Although exposure of rIFN-gamma-primed C3H/HeJ macrophages to LAP resulted in a fully tumoricidal state equivalent to that exhibited by C3H/OuJ macrophages, the levels of TNF produced remained discrepant. Under identical conditions, C3H/OuJ macrophages produced approximately fivefold more TNF (11,776 U/ml) than C3H/HeJ macrophages (2,399 U/ml). This suggests that although C3H/HeJ macrophages can respond functionally in a "normal" manner given the correct signals, they remain quantitatively deficient in the production of certain proteins. In this system, the elaboration of TNF and macrophage-mediated tumor cell lysis were shown to be dissociable events. The tumor target used in these studies (P815) was shown to be resistant to as much as 40,000 U/ml of purified rTNF. In addition, C3H/OuJ macrophage cultures exposed to LPS only (which resulted in the production of high levels of TNF), failed to lyse these targets. Lastly, anti-mouse TNF antibody added to macrophage cultures had no effect on the induction of tumor cell lysis.     

IFN-gamma/lipopolysaccharide activation of macrophages is associated with protein kinase C-dependent down-modulation of the colony-stimulating factor-1 receptor.

IFN gamma/LPS treatment increases macrophage tumoricidal and microbicidal activity and inhibits CSF-1-induced macrophage proliferation. The mechanism underlying the latter effect was investigated in the CSF-1-dependent mouse macrophage cell line, BAC-1.2F5. IFN-gamma and LPS together dramatically reduced the total number of CSF-1 receptors (CSF-1R) via selective degradation of the cell surface form. Processing and transport of intracellular CSF-1R to the cell surface were unaffected. IFN-gamma alone had no effect but significantly enhanced LPS-induced CSF-1R down-regulation. The reduction in CSF-1R number was protein kinase C-dependent and involved changes in serine phosphorylation of the receptor at different sites. CSF-1R down-modulation by this mechanism may be important in switching off the energy-consuming processes of CSF-1R-mediated proliferation and chemotaxis in activated macrophages.     

Expression of the transferrin receptor on murine peritoneal macrophages is modulated by in vitro treatment with interferon gamma

The expression of transferrin receptors on murine peritoneal macrophages has been shown to be down regulated during functional activation in vivo. This observation suggested that the level of transferrin receptor expression varies in response to discrete extracellular signals known to induce macrophage activation. We have tested this concept directly and have shown that decreased transferrin receptor expression can be reproduced in vitro by treatment of inflammatory macrophages with preparations of interferon gamma derived from a T cell hybridoma supernatant. The ability of this agent to down regulate the expression of the transferrin receptor exhibited dose and time dependencies similar to those required for development of other macrophage functions in vitro. The addition of LPS produced no further decrease in receptor expression. Furthermore, murine gamma interferon, produced by recombinant DNA technology also caused a downshift in transferrin receptor expression at doses similar to those which have been shown previously to induce activation. The changes in receptor activity were the result of altered numbers of binding sites and the receptor:ligand affinity remained unaffected. These results indicate that altered expression of the transferrin receptor is one element of the pleiotypic change which macrophages undergo in response to IFN gamma. This system may, therefore, provide a useful model in which to study the biochemical basis of IFN gamma action in mononuclear phagocytes.     

Expression of macrophage p120 depends on early protein synthesis

We have previously identified a group of early proteins preceding the expression of a 120-kDa protein (p120) which coincides with tumoricidal activation in peritoneal macrophages. In the present report, we have asked whether the in vitro induction of new or enhanced expression of p120 depends on early protein synthesis and RNA synthesis during the treatment period. Expression of p120 was sensitive to pretreatment of the macrophages with either actinomycin D or cycloheximide, indicating that both active protein synthesis and RNA synthesis were required. When poly-adenylated RNA isolated from various macrophage populations was translated in a rabbit reticulocyte in vitro translation system, only mRNA isolated from cells which express p120 was able to direct synthesis of a 120-kDa polypeptide. This product showed identical mobility to p120 induced in intact activated macrophages radiolabeled with [35S]methionine. The presence of translatable p120 mRNA was dependent upon treatment of thioglycollate-elicited macrophages with both IFN-gamma plus LPS at low doses, as is expression of p120 in intact cells. Accumulation of translatable p120 mRNA was blocked by treatment with cycloheximide, indicating that active protein synthesis was required during the induction period. These results suggest that the presence of specific translatable mRNA encoding the p120 polypeptide is dependent upon the expression of early macrophage gene products.     

Induction of nitrite/nitrate synthesis in murine macrophages by BCG infection, lymphokines, or interferon-gamma

Macrophage synthesis of nitrite and nitrate after activation by BCG infection or by treatment in vitro with both T cell-derived (lymphokines (LK) or recombinant murine interferon-gamma (IFN-gamma] and bacterial (lipopolysaccharide (LPS) and heat-killed bacillus Calmette-Guerin (hk BCG] agents was studied by using macrophages from C3H/He and C3H/HeJ mice. Spleen and peritoneal macrophages isolated from BCG-infected donors that were producing nitrate continued to synthesize nitrite and nitrate in culture. LPS treatment in vitro (25 or 50 micrograms/ml) additionally increased this nitrite/nitrate synthesis. Thioglycolate-elicited macrophages from non-infected C3H/HeJ mice treated with LK also produced nitrite/nitrate, and concurrent LPS (0.1 to 50 micrograms/ml) treatment resulted in enhanced synthesis. Recombinant IFN-gamma also stimulated nitrite/nitrate synthesis by C3H/He and CeH/HeJ macrophages as did LPS (C3H/He only) and hk BCG. When given concurrently with either LPS or hk BCG, IFN-gamma enhanced C3H/He and C3H/HeJ macrophage nitrite/nitrate synthesis over that produced by macrophages treated with either LPS or hk BCG alone. Macrophages activated in vitro exhibited a 4 to 12 hr lag time before engaging in nitrite/nitrate synthesis, which then proceeded for 36 to 42 hr at linear rates. Daily medium renewal did not alter the synthesis kinetics but increased the total amount of nitrite/nitrate produced. Nitrate and nitrite were stable under the conditions of culture and when added did not influence additional macrophage synthesis. Taken together, these results indicate that T cell lymphokines and IFN-gamma are powerful modulators of macrophage nitrite/nitrate synthesis during BCG infection and in vitro, and nitrite/nitrate synthesis appears to be common property of both primed and fully activated macrophage populations.     

Treatment of murine peritoneal macrophages with bacterial lipopolysaccharide alters expression of c-fos and c-myc oncogenes

Expression of the c-fos, c-myc, and c-fms proto-oncogenes has been studied in thioglycollate-elicited murine peritoneal macrophages after exposure to lipopolysaccharide (LPS). After incubation with LPS (20 ng/ml), a transient and rapid induction of the expression of c-fos and c-myc oncogenes could be observed, whereas the RNA levels for c-fms were not affected. Treatment with lipid A, the active moiety of the LPS molecule, increased the c-fos and c-myc expression to a comparable degree. Similar induction of c-fos and c-myc was observed after treatment with phorbol myristate acetate, suggesting that this effect of LPS on murine macrophages might be mediated through stimulation of protein kinase C. Under similar experimental conditions, LPS treatment of macrophages did not trigger DNA synthesis. Treatment with LPS blocked DNA synthesis in macrophages treated with L cell-conditioned medium containing colony-stimulating factor. Thus changes in c-fos and c-myc expression may be elements in the complex series of biochemical events that contribute to macrophage activation and are not necessarily related to induction or priming for cellular proliferation.     

Production of C3 as a marker of lymphokine-mediated macrophage activation

C3 production was assayed using an enzyme-linked immunosorbent assay (ELISA) in cell-free supernatants harvested from thioglycollate-elicited macrophages exposed to a variety of macrophage stimulating and activating agents. Macrophage monolayers treated with the stimulating agents starch, glycogen, and zymosan secreted three- to four-fold less C3 (mean 12 ng/10(5) cells/12 hr) than macrophages exposed to lymphokines containing macrophage-activating factor (MAF) (mean C3 production 44 ng/10(5) cells/12 hr). The increased production of C3 in macrophages exposed to MAF parallels the ability of these macrophages to acquire tumoricidal capacity as monitored in an in vitro 72 hr tumor cell cytotoxicity assay using B16 melanoma cells. Macrophages previously rendered tumoricidal by exposure to MAF and which are refractory to further challenge by MAF following decay of their tumoricidal properties, do not produce C3 on rechallenge with MAF. Exposure of refractory macrophages to liposome-encapsulated MAF overcomes the refractory state and induces re-expression of the tumoricidal phenotype and C3 production. We conclude that quantitative detection of macrophage-generated C3 antigen provides a useful biochemical marker for monitoring the acquisition of tumoricidal properties in macrophages exposed to MAF and offers a sensitive assay for screening novel agents that activate macrophages via mechanisms similar to MAF.     

Triggering of interferon gamma-primed macrophages by various known complement activators for nonspecific tumor cytotoxicity

Five known complement activators were evaluated for their capacity to directly activate murine macrophages and to trigger activation of lymphokine primed macrophages for nonspecific tumor cytotoxicity. Bacterial lipopolysaccharide (LPS), Lipid A, polyinosinic-polycytidylic acid, cobra venom factor (CVF), and zymosan directly activated macrophages in a dose-dependent fashion at high concentrations. Subactivating concentrations of each of these agents were found to effectively trigger macrophages which were preprimed either by macrophage-activating factor or by murine recombinant interferon gamma for enhanced tumoricidal activity. An Fc receptor blockade with opsonized sheep erythrocytes abrogated LPS-mediated direct activation and triggering of interferon gamma-primed macrophages, but had no inhibitory effect on direct activation or triggering by CVF for nonspecific tumor cytotoxicity. This study characterizes the capacity of a diverse group of known complement activators to serve as second signal triggers for culmination of the activation process of interferon-primed macrophages for nonspecific tumoricidal activity. These findings suggest that complement activators may directly activate macrophages by stimulation of interferon beta production by macrophages for self-priming and, as we have shown, act as self-triggers. The putative role of macrophage-associated complement components in the activation process is discussed.     

Stimulatory effects of purified macrophage colony-stimulating factor on murine resident peritoneal macrophages

A purified preparation of macrophage colony-stimulating factor (M-CSF) free of interferon and endotoxin activity was studied for its effects on resident murine peritoneal macrophages. M-CSF was found to induce profound morphologic alterations in resident macrophages. These changes included a marked increase in cell size, membrane ruffling, and cytoplasmic vacuolization. Further, after 72 hr of incubation with 1000 U/ml of M-CSF, there were significant increases in macrophage DNA synthesis as measured by autoradiography (P less than 0.001), and in macrophage monolayer protein content (P less than 0.01). None of these changes was seen in control macrophages or those exposed to recombinant interferon-gamma (IFN). Low activity levels of the ectoenzymes 5'-nucleotidase (5'NTD) and alkaline phosphodiesterase I (APD) have been associated with certain macrophage functions, particularly the expression of tumor cytotoxicity. Macrophage monolayers exposed to M-CSF demonstrated an unaltered level of 5'NTD activity from controls and a significantly increased level of APD activity (P less than 0.01) and did not demonstrate an increased ability to kill tumor cells, as measured by the 51Cr-release assay. On the other hand, IFN caused significant decreases in both 5'NTD (P less than 0.05) and APD (P less than 0.01) and also induced marked tumoricidal activity in macrophage monolayers. These results indicate that purified M-CSF induces highly specific alterations in the functional activity and morphologic appearance of resident macrophages and these changes are distinct from those induced by IFN.     

Induction by immunomodulatory agents of a macrophage antigen recognized by monoclonal antibody 158.2 and correlation with macrophage function

MA158.2, a rat monoclonal antibody with binding specificity for cells of the monocyte-macrophage lineage, reacts with an antigen (158.2) whose expression is enhanced on mononuclear cells activated to the tumoricidal phenotype by treatment with lymphokine supernatant containing macrophage activating factor (MAF). The functional relevance of enhanced expression of this antigen has been examined in mouse peritoneal macrophages treated with a variety of immunomodulatory agents and assayed for augmented macrophage-mediated defense reactions, including O-2 production, microbicidal, and tumoricidal activity. An interferon-gamma (IFN-gamma) preparation produced by recombinant DNA technology induced a dose-dependent increase in expression of the 158.2 antigen in inflammatory macrophages which was accompanied by acquisition of microbicidal activity against Listeria monocytogenes. However, these cells did not express tumoricidal activity and induction of this property required concomitant exposure to lipopolysaccharide (LPS). Similar results were obtained using macrophages elicited with pyran copolymer. Exposure to LPS alone induced enhanced expression of antigen 158.2 but did not elicit microbicidal activity. Macrophages challenged with IFN-alpha, IFN-beta, MDP, and bestatin did not exhibit increased 158.2 and also failed to acquire tumoricidal activity when treated concomitantly with LPS. Collectively, these data indicate that the MA 158.2 antibody recognizes an antigen expressed by macrophage populations displaying the so-called primed phenotype in which microbicidal activity is expressed but in which induction of tumoricidal activity requires the addition of a second signal such as LPS.     

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.