CB2 receptors in the brain: role in central immune function.

Recently, it has been recognized that the cannabinoid receptor CB2 may play a functionally relevant role in the central nervous system (CNS). This role is mediated primarily through microglia, a resident population of cells in the CNS that is morphologically, phenotypically, and functionally related to macrophages. These cells also express the cannabinoid receptor CB1. The CB1 receptor (CB1R) is constitutively expressed at low levels while the CB2 receptor (CB2R) is expressed at higher levels and is modulated in relation to cell activation state. The relatively high levels of the CB2R correspond with microglia being in 'responsive' and 'primed' states, suggesting the existence of a 'window' of functional relevance during which activation of the CB2R modulates microglial activities. Signature activities of 'responsive' and 'primed' microglia are chemotaxis and antigen processing, respectively. The endocannabinoid 2-arachidonylglycerol has been reported to stimulate a chemotactic response from these cells through the CB2R. In contrast, we have shown in vivo and in vitro that the exogenous cannabinoids delta-9-tetrahydrocannabinol and CP55940 inhibit the chemotactic response of microglia to Acanthamoeba culbertsoni, an opportunistic pathogen that is the causative agent of Granulomatous Amoebic Encephalitis, through activation of the CB2R. It is postulated that these exogenous cannabinoids superimpose an inhibitory effect on pro-chemotactic endocannabinoids that are elicited in response to Acanthamoeba. Furthermore, the collective results suggest that the CB2R plays a critical immune functional role in the CNS.

Cannabinoid receptors in microglia of the central nervous system: immune functional relevance.

Microglia, resident macrophages of the brain, function as immune effector and accessory cells. Paradoxically, they not only play a role in host defense and tissue repair but also have been implicated in a variety of neuropathological processes. Microglia, in addition to exhibiting phenotypic markers for macrophages, express CB1 and CB2 cannabinoid receptors. Recent studies suggest the existence of a third, yet-to-be cloned, non-CB1, non-CB2 cannabinoid receptor. These receptors appear to be functionally relevant within defined windows of microglial activation state and have been implicated as linked to cannabinoid modulation of chemokine and cytokine expression. The recognition that microglia express cannabinoid receptors and that their activation results in modulation of select cellular activities suggests that they may be amenable to therapeutic manipulation for ablating untoward inflammatory responses in the central nervous system.

Naegleria fowleri amoebae express a membrane-associated calcium-independent phospholipase A(2).

Naegleria fowleri, a free-living amoeba, is the causative agent of primary amoebic meningoencephalitis. Previous reports have demonstrated that N. fowleri expresses one or more forms of phospholipase A(2) (PLA(2)) and that a secreted form of this enzyme is involved in pathogenesis. However, the molecular nature of these phospholipases remains largely unknown. This study was initiated to determine whether N. fowleri expresses analogs of the well-characterized PLA(2)s that are expressed by mammalian macrophages. Amoeba cell homogenates contain a PLA(2) activity that hydrolyzes the substrate that is preferred by the 85 kDa calcium-dependent cytosolic PLA(2), cPLA(2). However, unlike the cPLA(2) enzyme in macrophages, this activity is largely calcium-independent, is constitutively associated with membranes and shows only a modest preference for phospholipids that contain arachidonate. The amoeba PLA(2) activity is sensitive to inhibitors that block the activities of cPLA(2)-alpha and the 80 kDa calcium-independent PLA(2), iPLA(2), that are expressed by mammalian cells. One of these compounds, methylarachidonyl fluorophosphonate, partially inhibits the constitutive release of [(3)H]arachidonic acid from pre-labeled amoebae. Together, these data suggest that N. fowleri expresses a constitutively active calcium-independent PLA(2) that may play a role in the basal phospholipid metabolism of these cells.

Immunotoxic effects of diethylstilbestrol on host resistance: comparison with cyclophosphamide.

To evaluate the usefulness of host resistance assays for measurement of immunotoxicologic effects of chemicals, the immunosuppressive effects of exposure to diethylstilbestrol (DES) were compared with the effects of treatment with the known immunosuppressive drug cyclophosphamide (CPS). A panel of six host resistance models was evaluated, including infection with the bacterium Listeria monocytogenes, herpes simplex virus type 2 (HSV-2), and encephalomyocarditis virus (EMC), the yeast Cryptococcus neoformans, the parasite Naegleria fowleri, and transplantation of the B16F10 melanoma tumor. The results demonstrate a general correlation between the effects of CPS and DES on host resistance. Acute treatment with CPS (200 mg/kg) markedly depressed resistance to the microbial infections with L. monocytogenes and HSV, and exposure to DES usually also decreased resistance in a dose dependent manner. Moreover, CPS had no marked effect on resistance to N. fowleri and EMC virus, and exposure to DES also had a neglible or slight effect. There were, however, two model systems in which the effects of CPS and DES diverged. Whereas treatment with DES produced no significant effect on resistance to C. neoformans, acute treatment with CPS prior to the fungal infection produced a marked increase in resistance. Also, while treatment with CPS markedly increased B16F10 lung metastases, treatment with DES significantly decreased the incidence and number of lung metastases. The data support the general validity of host resistance assays, particularly with models of short disease course, for measuring immunosuppression. However, the results also emphasize the complexity of interpreting effects of environmental chemicals on host resistance, because of the interplay of such factors as relative times of exposure to the chemical in relation to pathogenesis of infection, the length of the disease course, the nature of the operative host defense mechanisms, and the compensatory recovery of these mechanisms.

Calcium-dependent protection from complement lysis in Naegleria fowleri amebae.

Pathogenic Naegleria fowleri amebae are resistant to the lytic effects of serum complement. The presence of surface glycoproteins or removal of the membrane attack complex (MAC) of complement from the cell surface by vesiculation serve to protect the amebae from complement lysis. The specific mediators important in stimulating complement resistance are not defined. These studies were undertaken to examine the effect of Ca(2+) ions in initiating complement resistance of N. fowleri in contrast to non-pathogenic complement-sensitive N. gruberi. Chelation of extracellular calcium with ethylene glycol tetraacetic acid (EGTA) or chelation of intracellular calcium with 1,2-bis-(O-Aminophenoxy) ethane-N,N,N,N tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM) increased complement lysis of N. fowleri. Chelation of calcium ions did not affect complement sensitivity of N. gruberi. Increased lysis of ionomycin-treated N. fowleri was detected after exposure to serum complement, suggesting that a threshold level of Ca(2+) mediates complement resistance before survival mechanisms are overwhelmed and lysis occurs. A differential influx of Ca(2+) ions occurred in fura-2 labeled N. fowleri after deposition of complement component C9 to form the MAC complex on the cell surface in comparison to N. gruberi. These studies suggest that Ca(2+) ions influence complement resistance in N. fowleri but do not play a role in altering the sensitivity of N. gruberi to complement.

Differential expression of the CB2 cannabinoid receptor by rodent macrophages and macrophage-like cells in relation to cell activation.

An in vitro model of multi-step activation, in which cells of macrophage lineage are driven sequentially through inflammatory, primed, and fully activated states, was employed to assess for cannabinoid receptor expression. Murine and rat peritoneal macrophages, murine RAW264.7 and P388D, macrophage-like cells, and neonatal rat brain cortex microglia expressed the cannabinoid receptor type 2 (CB2) differentially in relation to cell activation. The CB2 was undetectable in resident peritoneal macrophages, present at high levels in thioglycolate-elicited inflammatory and interferon gamma (IFNgamma)-primed peritoneal macrophages, and detected at significantly diminished levels in bacterial lipopolysaccharide (LPS)-activated peritoneal macrophages. A comparable pattern of differential expression of the CB2 was noted for murine macrophage-like cells and neonatal rat brain cortex microglia. The cannabinoid receptor type 1 (CB1) was not detected in peritoneal macrophages or murine macrophage-like cells regardless of cell activation state but was present in neonatal rat microglia at low levels. These results indicate that levels of the CB2 in cells of macrophage lineage undergo major modulatory changes in relation to cell activation. Furthermore, since inflammatory and primed macrophages express the highest levels of CB2, the functional activities of macrophages when in these respective states of activation may be the most sensitive to the action of cannabinoids.

Anandamide inhibits macrophage-mediated killing of tumor necrosis factor-sensitive cells.

Anandamide (arachidonoylethanolamide) was shown to inhibit macrophage-mediated killing of tumor necrosis factor-sensitive murine L929 fibroblasts. Scanning electron microscopy (SEM) demonstrated that L929 cells, co-cultured with Propionibacterium acnes (P. acnes)-activated peritoneal macrophages from mice treated with vehicle, were either disrupted or had surface abnormalities and numerous punctate lesions. In contrast, L929 cells co-cultured with macrophages from mice receiving P. acnes in concert with Anandamide (20 mg/kg-80 mg/kg) or the exogenous cannabinoid delta-9-tetrahydrocannabinol (THC; 80 mg/kg) did not exhibit ultrastructural abnormalities. Cytotoxicity assays were performed in parallel with SEM in order to determine whether ultrastructural observations correlated with target cell killing as measured by release of radiolabel from L929 target cells. P. acnes-activated macrophages from vehicle-treated mice elicited 41% specific release of radiolabel from [51Cr]-labeled L929 cells. In contrast, macrophages from animals treated with P. acnes and with 20, 40, or 80 mg/kg Anandamide exhibited 38%, 25%, or 28% specific release of radiolabel, respectively. Similarly, macrophages from animals treated with P. acnes and with 80 mg/kg THC exhibited 21% specific release of radiolabel. In vitro cytotoxicity studies using radiolabeled L929 target cells and conditioned medium from RAW264.7 murine macrophage-like cells allowed for determination of the time interval over which Anandamide exerted its inhibitory effect. Maximal inhibition of target cell killing occurred when conditioned medium was obtained from macrophages exposed to Anandamide for 1 hr prior to activation. In contrast, conditioned medium from THC-treated macrophages exerted its maximal inhibition of target cell killing when obtained from RAW264.7 cells pretreated for 24hr-48hr prior to activation. These results indicate that Anandamide and THC exert a similar inhibition of killing of TNF-sensitive target cells. However, the time interval over which these two substances elicit their suppressive effect differs.

Resistance of Acanthamoeba species to complement lysis.

Acanthamoeba species were evaluated for susceptibility to complement lysis as determined by release of radiolabeled uridine. The 3 Acanthamoeba species tested, A. culbertsoni (ATCC 30171), A. castellanii (ATCC 30010), and A. polyphaga (ATCC 30461), depleted hemolytic complement activity from normal human serum (NHS), yet were resistant to its lytic effects. Examination of microtiter plates containing amoebae incubated in NHS demonstrated formation of a pellet in the wells. Pellet formation was not observed when amoebae were incubated in human cord serum, heat-inactivated serum, or C1q-deficient serum. Ultrastructural examination of serum-treated amoebae revealed the presence of a finely granular substance that surrounded the amoebae. Treatment of amoebae with enzymes or metabolic inhibitors prior to incubation in NHS was performed to investigate the mechanism of complement resistance. Cycloheximide or cytochalasin D pretreatment increased the susceptibility of A. culbertsoni and A. castellanii to complement lysis. Cytochalasin D treatment also increased the susceptibility of A. polyphaga to complement lysis. Inhibition of serine protease activity by phenylmethylsulfonylfluoride increased complement susceptibility of all 3 species of Acanthamoeba. Enzymatic removal of surface components from A. polyphaga or A. castellanii, with trypsin, neuraminidase, or phosphatidylinositol-specific phospholipase C (PIPLC), did not affect serum resistance. In contrast, PIPLC treatment of A. culbertsoni significantly increased lysis by complement. The ability of Acanthamoeba species to activate the alternative complement pathway yet resist complement-mediated cellular lysis can be attributed to both the release of a transport-dependent extracellular matrix as well as the presence of complement inhibitory surface proteins.

In vitro destruction of nerve cell cultures by Acanthamoeba spp.: a transmission and scanning electron microscopy study.

Trophozoites of 4 species of Acanthamoeba were cytopathic for cultured rat B103 neuroblastoma cells. Cytopathogenicity was evaluated by a chromium release assay and by transmission and scanning electron microscopy. Acanthamoeba culbertsoni, Acanthamoeba castellanii, and Acanthamoeba polyphaga destroyed B103 target cells at 37 C as evidenced by the release of radiolabel. Acanthamoeba astronyxis did not produce cytopathology at 37 C but destroyed nerve cells at 25 C. Transmission and scanning electron microscopy of cocultures maintained at different time periods revealed that all species of Acanthamoeba exhibited long cylindrical structures, termed digipodia, which made contact with target cells. Following this effector cell-target cell contact, membrane blebbing on the nerve cells was observed. These events were followed either by lysis of target nerve cells or ingestion of the target cells via food-cups and their subsequent channeling into intracytoplasmic food vacuoles. Use of the TUNEL (TdT-mediated dUTP nick end labeling) technique indicated that approximately 40% of B103 cells incubated with A. culbertsoni, 20% of B103 cells cocultured with A. castellanii or with A. polyphaga, and less than 1% of B103 cells incubated with A. astronyxis at 37 C were apoptotic after 24 hr of coculture. Studies using electron microscopy indicated that Acanthamoeba trophozoites destroyed nerve cells both by cytolysis and by ingestion of whole nerve cells via food-cups.

Dependence of growth, metabolic expression, and pathogenicity of Naegleria fowleri on exogenous porphyrins.

The pathogenicity of the free-living amoeba Naegleria fowleri is modulated by the composition of the medium used for cultivation. The constituents that determine the level of pathogenicity of N. fowleri, however, have not been definitively established. The present study examined the effects of selected porphyrins on N. fowleri amoebae. The iron-containing porphyrins, hemin or hematin, or the iron-free porphyrin, protoporphyrin IX, were effective in supporting growth of N. fowleri in Cline medium lacking serum. Iron-binding proteins, including hemoglobin, could not satisfy the growth requirement of the amoebae for exogenous porphyrin. Expression of biological functions including azocaseinase activity, agglutination, mobility, complement susceptibility, and virulence were altered by the composition of the growth medium. Amoebae grown in Cline medium supplemented with either hemin or protoporphyrin IX displayed greater mobility and were more resistant to lysis by complement than those grown in Nelson medium. Similarly, amoebae grown in Cline medium supplemented with either hemin or protoporphyrin IX were more pathogenic for B6C3F1 mice than those grown in Nelson medium. The addition of protoporphyrin IX to Nelson medium resulted in a modest increase in mobility, resistance to complement lysis and virulence when compared to N. fowleri amoebae grown in Nelson medium without added porphyrin.

Cytopathogenicity of Naegleria fowleri and Naegleria gruberi for established mammalian cell cultures.

Amebae of Naegleria fowleri and Naegleria gruberi were cytopathic for nine established mammalian cell cultures, including mouse and human fibroblasts, rabbit and monkey kidney cells, rat and mouse neuroblastoma cells, baby hamster kidney cells, and human epithelioma and carcinoma cells. Nine strains of N. fowleri were equally cytopathic for rodent neuroblastoma cells. As few as one ameba per million neuroblastoma cells destroyed the mammalian target cells after 9 days. The N. fowleri grew and destroyed rat neuroblastoma cells at 30 to 37 C whereas N. gruberi grew and destroyed the target cells at 25 to 30 C. Both N. fowleri and N. gruberi attached efficiently to the target cells at 30 to 37 C; N. gruberi but not N. fowleri attached efficiently at 25 C. Electron microscopic observations of mixed cultures of N. fowleri and neuroblastoma cells established that the amebae, after 12 hr, had ingested portions of the neuroblastoma target cells without causing cell lysis. Conversely, N. gruberi amebae, after attaching to target cells, disrupted the plasma membrane and cytoplasm of the target cells although the target cell nucleus remained intact. The amebae then ingested the target cell debris.

The interaction between the amoeba Balamuthia mandrillaris and extracellular matrix glycoproteins in vitro.

Balamuthia mandrillaris, a soil amoeba, is the causative agent of Balamuthia granulomatous amoebic encephalitis, a life-threatening brain infection. This amoeba is acquired from contaminated soil and may enter the host through cutaneous lesions or through nasal passages, migrating to the lungs or brain. During invasion, B. mandrillaris has access to components of the extracellular matrix (ECM) of the host. Therefore, we investigated the interaction of B. mandrillaris with 3 ECM glycoproteins (collagen-I, fibronectin and laminin-1) that are encountered in host connective tissues and at the basal lamina. Using optical microscopy, amoeba association on ECM-coated surfaces was examined. Binding of amoebae on laminin was greater than that on collagen or fibronectin. Laminin-adhered B. mandrillaris exhibited elongated and spread forms, distinctive from those observed for amoebae on a plastic surface. Collagen and fibronectin-adhered B. mandrillaris presented elongated shapes with cellular expansions. Binding to collagen, fibronectin, or laminin was inhibited when amoebae were pre-treated with sialic acid. Treatment with galactose resulted in diminished binding of amoebae on laminin, while mannose increased binding in all coating conditions tested. Dependence of divalent cations on amoeba binding was demonstrated for laminin-amoeba interaction. Collectively, the results indicate that B. mandrillaris recognizes specific glycoproteins of the mammalian extracellular matrix.

A method for assessing the migratory response of Naegleria fowleri utilizing [3H]uridine-labeled amoebae.

A new procedure is described to assay the migratory response of Naegleria fowleri (ATCC 30894) amoebae to potential chemoattractants. The method utilizes a blind-well Boyden chemotaxis chamber, two micropore filters of different construction, and amoebae-labeled with [3H]uridine. The technique was standardized by determining the influence of incubation time, filter construction, filter pore size and geometry, amoebae to filter pore ratio, and chemoattractant concentration. Radiolabeled amoebae were placed in Boyden chambers that contained the combination of an upper polycarbonate filter with distinct pores with a diameter of 8 microns and a lower filter of nitrocellulose with a 150-micron depth to separate the wells. A ratio of two amoebae to one filter pore and a 2-h incubation period were chosen to obtain optimal migration conditions. Nerve cell extract was used as the chemoattractant. The migratory responses of both highly pathogenic and weakly pathogenic strains of N. fowleri to nerve cell extract were compared using either the radiolabel procedure or the conventional single filter, leading-front method. Using either method, a highly pathogenic cloned strain of N. fowleri amoebae moved in a directional manner (chemotactically) in vitro to B103 rat neuroblastoma cell extract. In contrast, a weakly pathogenic strain of amoebae responded in a nondirectional manner (chemokinetically) to nerve cell extract. While both the leading-front assay and the radiolabel assay give accurate results, the measurement of radiolabeled cells allows one to test a greater number of attractants in one assay and the procedure eliminates observer bias.

Susceptibility of pathogenic and nonpathogenic Naegleria spp. to complement-mediated lysis.

The susceptibility of four species of Naegleria amoebae to complement-mediated lysis was determined. The amoebicidal activity of normal human serum (NHS) and normal guinea pig serum (NGPS) for Naegleria amoebae was measured by an in vitro cytotoxicity assay. Release of radioactivity from amoebae labeled with [3H]uridine and visual observation with a compound microscope were used as indices of lysis. Highly pathogenic mouse-passaged N. fowleri was less susceptible to the lytic effects of NHS and NGPS than the weakly pathogenic, axenically grown N. fowleri or N. australiensis and the nonpathogenic amoebae N. gruberi and N. lovaniensis. However, both pathogenic and nonpathogenic Naegleria spp. depleted complement as assessed by total hemolytic activity. Treatment of serum with EDTA, heat (56 degrees C, 30 min), cobra venom factor, or antibody to C3 or C9 complement components decreased the amoebicidal activity of NHS. The presence of specific agglutinating antibody to N. fowleri enhanced the amoebicidal activity of NGPS for N. fowleri.

Modulation of biological functions of Naegleria fowleri amoebae by growth medium.

Two strains of Naegleria fowleri amoebae were studied when the amoebae were maintained in the same growth medium or in two different media. A weakly pathogenic strain of N. fowleri, LEE, and a highly pathogenic strain, LEEmpC1, were compared for growth properties, the presence or absence of surface structures termed food cups, cytopathogenicity, cellular locomotion, susceptibility to complement-mediated lysis and immunological relatedness by western immunoblot analysis when grown in Nelson medium or in Cline medium. The two different strains of N. fowleri, LEE and LEEmpC1, were more similar in protein profiles and functional activity when both strains were grown in the same nutritional medium. Differences in growth, proteins synthesized, cytopathogenicity, susceptibility to complement lysis and rate of locomotion were noted when the same strain was grown in different media. Naegleria fowleri grown in Cline medium demonstrated an increased rate of growth, an increase in its rate of locomotion, an increased resistance to complement lysis, and destroyed target nerve cells by contact-dependent lysis. In contrast, the same strain of amoeba grown in Nelson medium showed slower growth, destroyed target cells by trogocytosis, and was less resistant to complement-mediated lysis.

Modulation of complement resistance and virulence of Naegleria fowleri amoebae by alterations in growth media.

Highly-pathogenic, mouse-passaged Naegleria fowleri amoebae are complement resistant. The present study evaluates the effect of complement on N. fowleri and the virulence of the amoebae after animal passage and growth in two different axenic media. Pathogenic N. fowleri maintained in "enriched" Cline medium are virulent for mice and resistant to complement lysis. A rapid decline in resistance to complement and virulence for mice is observed when highly-pathogenic N. fowleri are grown in Nelson medium lacking hemin. N. fowleri maintained in Nelson medium can be rendered complement-resistant by shifting the amoebae to growth in Cline medium for 2 h prior to the addition of complement. Cycloheximide treatment of N. fowleri maintained in Nelson medium blocks the transition to a complement-resistant phenotype following a shift in growth medium. Proteins were radiolabeled with [35S] during a shift from Nelson to Cline medium to identify specific polypeptides which may be associated with the functional activities related to virulence and resistance to complement.

Chemotaxis by Naegleria fowleri for bacteria.

Naegleria fowleri amebae demonstrated a chemotactic and chemokinetic response toward live cells and extracts of Escherichia coli and other bacterial species when experiments were performed using a blind-well chemotaxis chamber. The peptide N-formyl-methionyl-leucyl-phenylalanine acted as a chemokinetic rather than a chemotactic factor for N. fowleri amebae. Competition experiments in which nerve cell extracts or bacteria were placed on either side of the filter in chemotaxis chambers resulted in increased movement towards bacteria. A scanning electron microscopy study of the interaction of N. fowleri with different bacterial species confirmed that when the amebae were near ingestible bacteria they moved toward the bacteria by pseudopod formation. Naegleria fowleri appeared to respond to bacteria by three interrelated but distinct processes: chemokinesis, chemotaxis, and formation of food cups.

The arginine-dependent cytolytic mechanism plays a role in destruction of Naegleria fowleri amoebae by activated macrophages.

Mouse peritoneal macrophages activated by different immunomodulators (Mycobacterium bovis bacillus Calmette-Guérin or Propionibacterium acnes) destroy Naegleria fowleri amoebae by a contact-dependent process and by soluble cytolytic molecules secreted by macrophages in response to lipopolysaccharide. The goal of this study was to determine whether the arginine-dependent cytolytic mechanism which results in the production of nitric oxide from arginine by activated macrophages destroys the amoebae. Amoebicidal activity of activated macrophages was determined by coculturing macrophages with N. fowleri amoebae radiolabeled with 3H-uridine. The percent specific release of radiolabel was used as an index of cytolysis of the amoebae. The inhibitors NG-monomethyl-L-arginine and arginase were used to determine whether the arginine pathway was a major effector mechanism responsible for amoebicidal activity of activated macrophages. Both the arginine analog NG-monomethyl-L-arginine and arginase, which breaks down arginine, decreased macrophage amoebicidal activity. Addition of arginine to arginine-free medium restores amoebicidal activity to activated macrophage cultures. These results demonstrate that the arginine pathway is an important mechanism for the destruction of susceptible N. fowleri amoebae.

Cytopathogenicity of Naegleria fowleri for rat neuroblastoma cell cultures: scanning electron microscopy study.

Neuroblastoma cells were inoculated with Naegleria fowleri Lee and examined for cytopathology at various periods post-inoculation by scanning electron microscopy. By 18 h post-inoculation, approximately 50% of neuroblastoma cells were nonviable, as evidenced by trypan blue exclusion and light microscopic examination. This cytopathology resulted from piecemeal consumption of target cells mediated by a sucker apparatus extending from the surface of N. fowleri.

Soluble amoebicidal factors mediate cytolysis of Naegleria fowleri by activated macrophages.

Murine peritoneal macrophages activated in vivo with Corynebacterium parvum or bacille Calmette-Guérin, in contrast to resident macrophages, demonstrated significant cytolysis of the amoeba, Naegleria fowleri. Catalase and superoxide dismutase, both alone and in combination, failed to inhibit cytolysis of amoebae. N. fowleri amoebae demonstrated significant resistance to exogenously added hydrogen peroxide. The hydroxyl radical scavengers mannitol, thiourea, and dimethyl sulfoxide, as well as anaerobic conditions, failed to inhibit the amoebicidal activity of activated macrophages. Actinomycin D, cycloheximide, and puromycin blocked macrophage amoebicidal activity. Conditioned medium (CM) from lipopolysaccharide-stimulated, but not unstimulated, cultures of activated macrophages was capable of mediating cytolysis of N. fowleri amoebae. Cytolytic activity was recovered by ammonium sulfate precipitation of CM. Heat treatment of the CM inactivated cytolytic activity. Results indicate soluble proteins of activated macrophage origin to be responsible for the amoebicidal activity.