Induction of tumor cell resistance to macrophage-mediated lysis by preexposure to non-activated macrophages

Thioglycollate-elicited peritoneal exudate (non-activated) macrophages do not lyse tumor cells and in contrast to activated macrophages bind less target cells. However, a non-lethal encounter of tumor cells with non-activated macrophages resulted in a pronounced effect on the subsequent tumor cell binding to and lysis by activated macrophages. Our results have shown that binding of tumor cells by non-activated macrophages was Ca2+ and temperature dependent; had a requirement for a Pronase-sensitive structure on macrophage surface membranes; was saturable; and was 2-3X less than that observed for activated macrophages. Experiments were conducted in which syngeneic tumor cells were incubated with a monolayer of non-activated macrophages and then assayed for selective binding and sensitivity to lysis. The important observations were that as a result of a 3-hr incubation with non-activated macrophages at an EC: TC ratio of 5:1 there was an increase in the number of tumor cells that bound to both activated and non-activated macrophages; a loss of selective binding in which the ratio of tumor cells bound to activated/non-activated macrophages (normally greater than 2) was lowered to 1.0; and a concomitant decrease in the susceptibility of tumor cells to macrophage-mediated cytolysis. The induction of tumor cell resistance to macrophage kill required an exposure to an excess number of non-activated macrophages, was reversible upon culturing with or without macrophages for 24 hr and required cell-cell contact. Our results reinforce the importance of selective binding between tumor cells and activated macrophages as an initial phase in tumor cell killing and also illustrates an active role for non-activated macrophages in vivo in allowing tumor cells to escape the immune surveillance by activated macrophages.     

Decrease of the major surface glycoprotein gp160 in activated macrophages

The quantity of surface-radioiodinated gp160, the previously described trypsin- and plasmin-sensitive surface glycoprotein of guinea pig macrophages was reduced 70% in macrophages activated in vivo in comparison to elicited macrophages. The reduction of gp160 was also detected by Coomassie blue staining, demonstrating that the absolute number of gp160 molecules is lower in activated macrophages. Gp160 was present on activated macrophages as single-chain intact molecules; no proteolytically cleaved gp160 was detected. Biosynthesis of gp160 was compared for activated and elicited macrophages by culturing with [³⁵S]methionine. Whereas biosynthetically labeled gp160 was readily detected in elicited macrophages, negligible [³⁵S]methionine-labeled gp160 was detected in parallel cultures of activated macrophages, suggesting that a major cause of decreased surface gp160 on activated macrophages lies at the level of synthesis.     

PAF metabolism in resident and activated alveolar macrophages: role of protein kinase C

Platelet-activating factor (PAF) metabolism was studied in resident and activated alveolar macrophages. Macrophages were obtained from normal Sprague-Dawley rats and from rats previously injected with complete Freund's adjuvant. Macrophages were attached and stimulated for 90 min. Then, cell PAF was extracted and quantitated by thin-layer chromatography. We found that in both resident and activated macrophages, calcium ionophore A23187 was a potent stimulus for PAF production while phorbol myristate acetate (PMA) was not. PMA and ionophore acted synergistically to increase PAF content in resident macrophages. This synergism was not observed in activated macrophages. To examine if this difference between resident and activated macrophages was due to a difference in PAF degradation, we assayed acetylhydrolase, the PAF-degrading enzyme. We found that ionophore stimulated acetylhydrolase activity in activated macrophages, but not in resident macrophages. Furthermore, PMA potentiated the ionophore effect in activated macrophages. This synergism was less obvious in resident cells. We conclude that PAF metabolism is different in activated and resident alveolar macrophages. Protein kinase C may play an important role in acetylhydrolase regulation in these cells.     

Binding of activated macrophages to tumor cells through a macrophage lectin and its role in macrophage tumoricidal activity

A 45-60 kDa Gal/GalNAc-specific macrophage lectin was found to participate in the interaction between tumor cells and tumoricidal macrophages activated by an antitumor streptococcal preparation, OK-432, and in the tumoricidal activity of the activated macrophages. The binding between OK-432-elicited activated macrophages and murine mastocytoma P-815 cells was inhibited on preincubation of the macrophages with a neoglycoprotein (Gal-BSA) or a complex-type glycopeptide (unit B) which was a specific inhibitor of the macrophage lectin. This binding of the macrophages to P-815 cells was also inhibited on the addition of anti-macrophage lectin antiserum. Contrary to the case of OK-432-elicited macrophages, the binding of thioglycolate-elicited (responsive) macrophages to P-815 cells was inhibited only a little by Gal-BSA and unit B, and not inhibited by the antiserum. Furthermore, the tumoricidal activity of the activated macrophages was inhibited by the addition of the anti-macrophage lectin antiserum. These results suggest that the binding of activated macrophages to tumor cells through the Gal/GalNAc-specific macrophage lectin is an important part of the tumor cell killing mechanism.     

Alternatively activated macrophages during parasite infections

Depending on the cytokine environment, macrophages can differentiate into distinct subsets that perform specific immunological roles. In this regard, the functions of macrophages activated by interferon gamma, referred to as classically activated macrophages, have been extensively documented, particularly during immune responses to infection. Recently, it was recognized that macrophages exposed to cytokines generated by T helper cell type 2 (Th2) cells exert an alternative activation program. However, the nature and functions of alternatively activated macrophages are ill defined. Evidence for the presence of alternatively activated macrophages and their possible influence in the outcome of several parasite diseases are discussed here.     

Studies on the regulation of lymphocyte reactivity by normal and activated macrophages.

To determine the potential role of macrophages as regulators of the immune response, the effect of mouse peritoneal macrophages on transforming mouse spleen lymphocytes was investigated. Mitogen and antigen stimulated lymphocyte transformation, as measured by DNA synthesis, was enhanced by all concentrations of normal macrophages tested, but only by low concentrations of activated macrophages. High concentrations of activated macrophages markedly inhibited lymphocyte transformation. This inhibition occurred whether lymphocyte DNA synthesis was measured by incorporation of [³H]TdR or of ³²P. Activated macrophages cultured with lymphocytes within 4 hr of being removed from the peritoneal cavity inhibited lymphocyte transformation. When activated macrophages were cultured alone for 24 or more hours before addition of lymphocytes, enhancement of transformation was noted. Once lymphocytes were exposed to activated macrophages, they could not be induced to undergo transformation in the presence of Con A. Whereas heat-killed activated macrophages, which appeared intact morphologically, lost their capacity to inhibit lymphocyte transformation, macrophages treated with mitomycin C to inhibit DNA synthesis retained this capacity. Syngeneic and allogeneic macrophages had similar inhibitory ability. Supernatants from cultures of many cell types (including normal or activated macrophages, lymphocytes, lymphocytes plus macrophages, and L cells) inhibited [³H]TdR incorporation by both mitogen stimulated lymphocytes and tumor cells. These studies demonstrate the capacity of macrophages to regulate lymphocyte transformation in vitro and suggest a role for these cells as regulators of cell-mediated immunity in vivo.     

Peroxisome proliferator-activated receptor gamma activation promotes infiltration of alternatively activated macrophages into adipose tissue

Obesity is associated with infiltration of macrophages into adipose tissue. Adipose macrophages may contribute to an elevated inflammatory status by secreting a variety of proinflammatory mediators, including tumor necrosis factor alpha and interleukin-6 (IL-6). Recent data suggest that during diet-induced obesity the phenotype of adipose-resident macrophages changes from alternatively activated macrophages toward a more classical and pro-inflammatory phenotype. Here, we explore the effect of peroxisome proliferator-activated receptor gamma activation on obesity-induced inflammation in 129SV mice fed a high fat diet for 20 weeks. High fat feeding increased bodyweight gain, adipose tissue mass, and liver triglycerides. Rosiglitazone treatment further increased adipose mass, reduced liver triglycerides, and changed adipose tissue morphology toward smaller adipocytes. Surprisingly, rosiglitazone markedly increased the number of macrophages in adipose tissue, as shown by immunohistochemical analysis and quantification of macrophage marker genes CD68 and F4/80+. In adipose tissue, markers for classically activated macrophages including IL-18 were down-regulated, whereas markers characteristic for alternatively activated macrophages (arginase 1, IL-10) were up-regulated by rosiglitazone. Importantly, conditioned media from rosiglitazone-treated alternatively activated macrophages neutralized the inhibitory effect of macrophages on 3T3-L1 adipocyte differentiation, suggesting that alternatively activated macrophages may be involved in mediating the effects of rosiglitazone on adipose tissue morphology and mass. Our results suggest that short term rosiglitazone treatment increases infiltration of alternatively activated macrophages in adipose tissue. The alternatively activated macrophages might play a role in peroxisome proliferator-activated receptor gamma-dependent expansion and remodeling of adipose tissue.     

The interaction of Naegleria fowleri amoebae with murine macrophage cell lines

The present study was undertaken to determine whether murine macrophage cell lines exhibited in vitro amoebicidal activity comparable to that elicited by activated murine peritoneal macrophages. Peritoneal macrophages activated in vivo by bacillus Calmette-Guérin or Propionibacterium acnes demonstrated significant cytolysis of Naegleria fowleri amoebae. The macrophage cell line RAW264.7 also effected cytolysis of amoebae, but to a lesser extent than that elicited by activated peritoneal macrophages. However, the macrophage cell lines, J774A.1 and P388D1, did not exhibit amoebicidal activity. Macrophage conditioned medium prepared from RAW264.7 macrophages mediated cytolysis of L929 tumor cells but had no effect on N. fowleri amoebae. In addition, neither recombinant tumor necrosis factor nor recombinant interleukin-1 exhibited amoebicidal activity. Scanning electron microscopy of co-cultures revealed that N. fowleri bound to activated peritoneal macrophages and RAW264.7 macrophages. These results suggest that RAW264.7 macrophages treated in vitro with lipopolysaccharide are similar to macrophages activated in vivo in that they effect contact-dependent cytolysis of Naegleria fowleri amoebae. The RAW264.7 macrophages are unlike primary macrophage cultures in that they either do not release soluble amoebicidal factors into the conditioned medium or they release insufficient quantities.     

Protein changes associated with stages of activation of mouse macrophages for tumor cell killing

Bone marrow-derived mouse macrophages become activated for tumor cell killing by traversing a series of stages. The stages studied here were as follows: unstimulated (exposed to nothing but medium), primed (prepared to become cytolytic), fully activated (primed macrophages exposed to a triggering agent), and postcytolytic (previously activated macrophages that had gradually lost cytolytic activity after the removal of stimuli). Macrophages were labeled with [35S]methionine, lysed, and subjected to 2-D gel electrophoresis and fluorography. The priming agent used was recombinant mouse IFN-gamma, 10 to 20 U/ml. Bacterial lipopolysaccharide (LPS), 0.4 to 1 ng/ml, was used as the triggering agent. A total of 40 major changes was identified in macrophages treated with both agents. Twenty-six of these were seen in macrophages treated with IFN-gamma, and 35 were found in LPS-treated macrophages. Twenty-two of the 40 changes were found in both IFN-gamma- and LPS-treated macrophages. The major reason for this overlap was the autocrine action of IFN-alpha/beta secreted from LPS-treated macrophages. Changes in expression of specific proteins, designated p47b and p71/73, were found to correlate closely with the development and loss of the activated state. With the use of these proteins as markers, phenotypes could be constructed that distinguished unstimulated, LPS-treated, primed, and fully activated macrophages. Postcytolytic macrophages had a phenotype similar to unstimulated macrophages and could be reactivated by reexposure to inducing agents. They also reexpressed the protein markers that were characteristic of fully activated macrophages.     

Cytolytic mechanisms of activated macrophages. Tumor necrosis factor and L-arginine-dependent mechanisms act synergistically as the major cytolytic mechanisms of activated macrophages

We examined the cytolytic mechanisms of activated macrophages by using proteose peptone- or thioglycollate broth-induced mouse peritoneal macrophages or mouse macrophage hybridomas as effector cells, L.P3 cells, a clone of L929 cells, and P815 cells as target cells, and IFN-gamma and LPS as activators. It was determined that TNF is the main cytolytic molecule against L.P3 cells from the following results: 1) activated macrophages can produce TNF; 2) TNF shows cytotoxic activity against L.P3 cells; 3) the addition of anti-TNF antibody inhibited most of the cytolytic activity of activated macrophages against L.P3 cells. On the other hand, it was concluded that the main cytolytic mechanism against P815 cells is the production of NO2-/NO3- from L-arginine, from the following results: 1) activated macrophages can produce NO2-; 2) NaNO2 shows high cytotoxic activity against P815 cells; 3) the depletion of L-arginine from the medium inhibited most of the cytolytic activity of activated macrophages against P815 cells and NO2- production by activated macrophages. In this study, however, cytostatic effects of L-arginine-dependent effector mechanism were not studied. Thus, these results show that activated macrophages can express at least two cytolytic mechanisms independently, namely, the one that appears to be mediated by the L-arginine-dependent effector mechanism and the second that appears to be mediated directly by TNF. Furthermore, it was demonstrated that TNF and L-arginine-dependent NO2- production act synergistically as killing mechanisms of activated macrophages. These mechanisms can explain the cytolytic activity of activated macrophages against a variety of target cells.     

CD14 is a component of multiple recognition systems used by macrophages to phagocytose apoptotic lymphocytes.

Expression of the aminophospholipid phosphatidylserine (PS) on the surface of apoptotic lymphocytes and lipid-symmetric erythrocytes triggers their phagocytosis by macrophages. Phagocytosis by both activated and unactivated macrophages, which utilize different recognition systems, can be blocked by certain monoclonal antibodies directed against the LPS receptor, CD14. Here we investigate the requirement for CD14 in the phagocytosis of both apoptotic thymocytes and lipid-symmetric erythrocytes by both activated and unactivated macrophages. We show that phagocytosis of lipid-symmetric erythrocytes by both activated and unactivated macrophages is completely abolished when CD14 is removed from macrophages by cleaving its glycosylphosphatidylinositol tether with phospholipase C. This treatment also substantially reduces phagocytosis of apoptotic lymphocytes by both types of macrophages. Unactivated LR-9 mouse macrophages which are deficient in CD14 expression are completely unable to phagocytose either apoptotic thymocytes or lipid-symmetric erythrocytes. These results argue that CD14 is an absolute requirement for the phagocytosis of lipid-symmetric erythrocytes by both activated and unactivated macrophages, despite their different recognition systems, that CD14 contributes at least substantially to the phagocytosis of apoptotic lymphocytes by both activated and unactivated macrophages, and that activated macrophages may also possess an alternate, CD14-independent mechanism for phagocytosis of apoptotic lymphocytes.     

The effects of activated macrophages on tumor target cells: Escape from cytostasis

In contrast to normal mouse peritoneal macrophages, activated macrophages almost totally inhibit [³H]TdR uptake by tumor target cells 24 hr after challenge. However, when the period of observation was extended to 48 or 72 hr, renewed [³H]TdR uptake by target cells was often, but not always, observed in the presence of activated macrophages. This apparent escape of target cells from the cytostatic effects of activated macrophages was not due to a subpopulation of resistant target cells, and autoradiographic studies revealed that target cells, inhibited from incorporating [³H]TdR by activated macrophages at 24 hr, were subsequently able to renew DNA synthesis and multiply. These results suggest that in the presence of activated macrophages, the almost total cytostasis of target cells does not necessarily mean that these cells are irreversibly damaged or killed.Escape from or maintenance of cytostasis was not peculiar to any of the target cells (L cells, EMT-6, Bladder 4934) or mouse strains (SW, C57BL, BALB/c) employed nor was it consistent with any of the forms of stimulation used for obtaining activated macrophages (Toxoplasma or Besnoitia infection; C. parvum treatment). However, the results suggest that when escape of target cells from the cytostatic effects of activated macrophages occurred, it may have been due to a qualitative or quantitative inadequacy of the population of macrophages employed.     

Generation of EPR-detectable nitrosyl-iron complexes in tumor target cells cocultured with activated macrophages.

After immunostimulation, murine macrophages oxidize L-arginine into nitric oxide (NO) which acts as an effector molecule. In this study, we attempted to establish whether activated macrophage-derived NO forms paramagnetic complexes in tumor target cells which do not express by themselves the L-arginine:NO pathway. Accordingly, murine L1210 leukemia cells were cocultivated with activated peritoneal macrophages from Bacillus-Calmette-Guérin-infected mice, or activated in vitro with interferon-gamma. In control experiments, macrophages were prevented from producing nitrogen oxides by incubation with NG-monomethyl-L-arginine, a specific inhibitor of the L-arginine:NO pathway. After coculture, L1210 cells were removed from adherent macrophage monolayers and analyzed by electron paramagnetic resonance at 77 K. In the L1210 cells cultured with activated macrophages, we detected a signal typical of nitrosyl-iron-sulfur complexes, with g values of 2.041 and 2.015. This signal was not present when L1210 cells were either cultured alone or cocultured with activated macrophages in the presence of NG-monomethyl-L-arginine. Mitochondria from activated macrophage-injured L1210 cells also exhibited the signal with g values of 2.041 and 2.015. These results show that when tumor target cells undergo cell-to-cell contact with activated macrophages during culture, the macrophages promote target cell nitrosylation in compartments like mitochondria.     

Effector mechanisms of cytolytically activated macrophages. I. Secretion of neutral proteases and effect of protease inhibitors

Peritoneal macrophages from C56BL/6J mice, when activated by bacillus Calmette-Guérin, lysed syngeneic MCA-I sarcoma targets but not syngeneic embryo fibroblasts. Inflammatory macrophages elicited by concanavalin A (Con A) did not appreciably lyse either target. The activated macrophages secreted more neutral proteases into the extracellular compartment, both absolutely and relative to intracellular content, than did the Con A inflammatory macrophages. Bovine pancreatic trypsin inhibitor (BPTI) (750 KIU/ml) and diisopropylfluorophosphate (2 x 10(-3) M) inhibited cytolysis of neoplastic targets by the activated macrophages. The BPTI had to be present during the 48-hr macrophage-tumor cell interaction to reduce cytolysis; pretreatment of either the macrophages or the targets by the BPTI did not reduce cytolysis. The inhibitors, at the concentrations found to inhibit cytolysis, were not toxic to the macrophages as judged by morphology, by the ability of the macrophages to incorporate leucine into protein, and by the potential for cytolytic activation of the macrophages in vitro. It is suggested that neutral serine protease(s) secreted by activated macrophages participate in the cytolytic destruction of neoplastic cells.     

Failure of Trypanosoma cruzi to trigger the respiratory burst of activated macrophages. Mechanism for immune evasion and importance of oxygen-independent killing

Activated macrophages are thought to kill Trypanosoma cruzi, which lack catalase, by the generation of hydrogen peroxide. We investigated triggering of the respiratory burst of activated macrophages induced by phagocytosis of virulent T. cruzi, bloodform trypomastigotes, amastigotes obtained from spleens, and tissue culture organisms; and of relatively nonvirulent epimastigotes. All stages of T. cruzi prompted the release of less than 10% of hydrogen peroxide released by activated macrophages when stimulated with PMA or Candida. Superoxide anion production was not stimulated by PMA or Candida in activated macrophages nor was there a significant qualitative reduction of nitroblue tetrazolium induced by ingestion of virulent T. cruzi. Opsonization of T. cruzi with specific antibody did not promote the release of hydrogen peroxide or the reduction of nitroblue tetrazolium. Similar results were observed with activated spleen macrophages. Incubation of activated macrophages with catalase, catalase and superoxide dismutase, sodium benzoate with or without catalase, and respiratory burst-exhausting PMA failed to inhibit the killing of T. cruzi in vitro. These results indicate that 1) virulent opsonized or unopsonized T. cruzi do not trigger a respiratory burst by activated macrophages and 2) oxygen-independent killing of T. cruzi is of prime importance.     

Fas-Fas ligand interactions are essential for the binding to and killing of activated macrophages by gamma delta T cells

Gammadelta T cells have a direct role in resolving the host immune response to infection by eliminating populations of activated macrophages. Macrophage reactivity resides within the Vgamma1/Vdelta6.3 subset of gammadelta T cells, which have the ability to kill activated macrophages following infection with Listeria monocytogenes (Lm). However, it is not known how gammadelta T cell macrophage cytocidal activity is regulated, or what effector mechanisms gammadelta T cells use to kill activated macrophages. Using a macrophage-T cell coculture system in which peritoneal macrophages from naive or Lm-infected TCRdelta-/- mice were incubated with splenocytes from wild-type and Fas ligand (FasL)-deficient mice (gld), the ability of Vgamma1 T cells to bind macrophages was shown to be dependent upon Fas-FasL interactions. Combinations of anti-TCR and FasL Abs completely abolished binding to and killing of activated macrophages by Vgamma1 T cells. In addition, confocal microscopy showed that Fas and the TCR colocalized on Vgamma1 T cells at points of contact with macrophages. Collectively, these studies identify an accessory or coreceptor-like function for Fas-FasL that is essential for the interaction of Vgamma1 T cells with activated macrophages and their elimination during the resolution stage of pathogen-induced immune responses.     

Effect of Fonsecaea monophora on the Polarization of THP-1 Cells to Macrophages

Background

Chromoblastomycosis is a chronic, progressive fungal disease of the skin and subcutaneous tissue caused by a unique group of dematiaceous fungi. Fonsecaea monophora, a new species distinct from Fonsecaea pedrosoi strains, is the main pathogen responsible for chromoblastomycosis in south China. Macrophages can be polarized into two categories: classically activated and alternatively activated.

Objectives

Little is known about the relationship between F. monophora and macrophage polarization. This study aimed to study the effect of F. monophora on the polarization of THP-1 cells to macrophages.

Methods

We established coculture systems of F. monophora and THP-1-derived macrophages in different activation states.

Results

F. monophora enhanced the phagocytosis by macrophages in the initially activated state and weakened the phagocytosis by classically activated macrophages without affecting that by alternatively activated macrophages. Classically activated macrophages had the strongest killing effect on F. monophora, while the initially activated macrophages had the weakest. The pathogen could not be rapidly cleared by any type of macrophage. F. monophora promoted the expression of proinflammatory cytokines and inhibited that of anti-inflammatory cytokines.

Conclusions

F. monophora promoted the polarization of THP-1 cells to classically activated macrophages and inhibited that of THP-1 cells to alternatively activated macrophages.

     

Antibody-dependent cytolysis (ADCC) of tumor cells by activated murine macrophages is a two-step process: Quantification of target binding and subsequent target lysis

To analyze the antibody-dependent cell-mediated cytotoxicity (ADCC) reaction between tumor cells and activated murine macrophages in detail, it must be first determined if physical binding occurred between the two cell types. Over 15–20 min in vitro, antibody-coated HSB neoplastic targets became so firmly attached to the activated macrophages that they resisted removal with 4 vigorous washes. When a quantitative assay of binding was employed, attachment of tumor cells to activated macrophages was found to depend on the concentration of antibody and on the density of the macrophages. These two variables also determined the subsequent extent of cytolysis. Binding of antibody-coated targets by macrophages elicited with thioglycollate broth or activated by bacillus Calmette-Guerin (BCG) was comparable. Lysis by the activated macrophages, however, was far greater. Binding occurred at 4, 22, or 37 °C, while the subsequent lytic reaction occurred only at 37 °C. Thioglycollate broth effectively inhibited lysis but had no effect on binding. A porous filter placed between activated macrophages and targets resulted in abrogation of binding and lysis, even when antibody-coated targets were placed beneath the filters. When labeled, uncoated targets were added to cultures of macrophages in the presence of unlabeled antibody-coated targets, no lysis of the bystander (i.e., uncoated) targets was seen. The data suggest that ADCC is a multistep reaction, that vigorous physical binding of antibody-coated targets by activated macrophages is an initial and necessary step in ADCC, that such binding is not sufficient for ADCC, that such binding controls the selectivity of lysis in ADCC, and that the second step in ADCC results in target lysis.     

The Interaction of Naegleria fowleri Amoebae with Murine Macrophage Cell Lines

The present study was undertaken to determine whether murine macrophage cell lines exhibited in vitro amoebicidal activity comparable to that elicited by activated murine peritoneal macrophages. Peritoneal macrophages activated in vivo by bacillus Calmette-Guérin or Propionibacterium acnes demonstrated significant cytolysis of Naegleria fowleri amoebae. The macrophage cell line RAW264.7 also effected cytolysis of amoebae, but to a lesser extent than that elicited by activated peritoneal macrophages. However, the macrophage cell lines, J774A.1 and P388D₁, did not exhibit amoebicidal activity. Macrophage conditioned medium prepared from RAW264.7 macrophages mediated cytolysis of L929 tumor cells but had no effect on N. fowleri amoebae. In addition, neither recombinant tumor necrosis factor nor recombinant interleukin-1 exhibited amoebicidal activity. Scanning electron microscopy of co-cultures revealed that N. fowler bound to activated peritoneal macrophages and RAW264.7 macrophages. These results suggest that RAW264.7 macrophages treated in vitro with lipopolysaccharide are similar to macrophages activated in vivo in that they effect contact-dependent cytolysis of Naegleria fowleri amoebae. The RAW264.7 macrophages are unlike primary macrophage cultures in that they either do not release soluble amoebicidal factors into the conditioned medium or they release insufficient quantities.