Activated macrophages demonstrate direct cytotoxicity, antibody-dependent cellular cytotoxicity, and enhanced binding of Naegleria fowleri amoebae

CELLULAR

IMMUNOLOGY

98, 125- 136 ( 1986)

Activated Macrophages Demonstrate Direct Cytotoxicity, Antibody-Dependent Cellular Cytotoxicity, and Enhanced Binding of Naegleria fowled Amoebae S. F. CLEARY Department

AND F. MARCIANO-CABRAL

of Microbiology and Immunology, Virginia Commonwealth Medical College of Virginia, Richmond, Virginia 23298 Received

July 22, 1985; accepted

October

University,

15, 1985

Macrophages activated in vivo by injection of Corynebacterium parvum or bacillus CalmetteG&in caused direct cytolysis of the pathogenic free-living amoeba, Naegteria fowleri, in vitro. Amoebic&l activity was time and cell density-dependent but was not dependent on the presence of specific antibody. Antibodydependent cellular cytoxicity for amoebae was also expressed by activated macrophages. Resident and thioglycolate-elicited macrophages demonstrated low cytolytic activity under all conditions tested. From scanning electron microscopy it appears that the degree. of target cell binding is directly related to the degree of cytolysis expressed by the macrophage populations. Cell-cell contact was required for cytolysis of amoebae by activated macrophages since cytolysis did not occur when contact was blocked by a porous filter. For each macrophage population, the levels of amoebicidal activity and tumoricidal activity were comparable. o 1986 Academic

Press, Inc.

INTRODUCTION Macrophages are important effector cells in host defense to microbial invaders and malignancies. Mechanisms by which macrophages exert their effector functions include both intracellular and extracellular cytocidal activities. Investigations into the repertoire of macrophage extracellular cytolytic activities have shown that macrophages mediate cytolysis of tumor cells (l-3), multicellular parasites (4), fungi (5, 6), and certain protozoans (7, 8). Macrophages are capable of obtaining an activated state as a result of in vivo stimulation by certain microorganisms or their products. This activated state is associated with morphological changes, altered ectoenzyme levels, and enhanced cytolytic capabilities for both mammalian and nonmammalian cells in vitro (9-l 1). Activated macrophages can be differentiated from resident macrophages or those elicited by sterile irritants on the basis of these characteristics. Cytolysis of target cells by activated macrophages has been shown to occur in the absence of lymphocytes, to be a predominantly nonphagocytic event, and in the case of tumor cell hilling, to require target cell binding by macrophages (8, 12-17). The purpose of this study was to investigate the ability of murine peritoneal macrophages to induce cytolysis of the extracellular amoeba, Naegkriafiwleri. IV. fowled, an opportunistic pathogen, is the causative agent of primary amoebic meningoenceph125 0008-8749186 $3.00 Copyright @ 1986 by Academic FWs, Inc. All lights of reproduction in any form reserved.

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alitis (PAME). We describe an in vitro method for the study of the interactions between murine peritoneal macrophages and the extracellular amoeba N. fiwleri. Macrophages activated by in vivo injections of Corynebacterium parvum (recently designated Propionibacterium acnes) and bacillus Calmette-G&in (BCG) were compared in amoebicidal activity to those elicited by the injection of thioglycolate broth as well as untreated resident macrophages. The ability of the different populations of macrophages to induce direct cytolysis at a variety of time periods and effector-to-target-cell ratios was tested. Cytolysis of the amoebae was also tested in the presence of specific antibody raised to the N. fowled organisms. Scanning electron microscopy (SEM) was performed on coverslip cultures of macrophages and amoebae to investigate binding of amoebae by macrophages. Results of our investigations indicate the existence of both qualitative and quantitative differences between activated and nonactivated macrophages in their abilities to bind and kill an extracellular protozoa. MATERIALS AND METHODS Animals. Eight- to ten-week-old B6C3Fl (C57BL/6 X C3H) female mice (The Charles River Breeding Laboratories, Wilmington, Mass.) were used as the source of peritoneal cells. Peritoneal cells. Mice were sacrificed by cervical dislocation. Peritoneal exudate cells (PECs) were obtained by lavaging the peritoneal cavities with 8 ml of ice-cold Hanks’ balanced salt solution (HBSS) (GIBCO, Grand Island, N.Y.) with penicillin (200 U/ml), streptomycin (200 &ml), and 10 U/ml heparin. Cells were treated with T&buffered NH&l to deplete RBCs, washed three times with HBSS, and suspended in RPM1 1640 medium (GIBCO) supplemented with penicillin (100 U/ml), streptomycin ( 100 pg/ml), 1% glutamine, 25 rmJ4 Hepes (C(2-Hydroxyethyl)- 1-piperazineethanesulfonic acid) buffer, and 10% heat-inactivated fetal calf serum (Flow Laboratories, McLean, Va.). Total cell counts were performed on a hemacytometer. Viability was not less than 95% as determined by trypan blue dye exclusion. Differential cell counts were done on Wright-Giemsa-stained cell smears prepared by cy-tocentrifugation (Shandon Southern Instruments, Sewickly, Pa.). Cells were diluted in supplemented RPM1 1640 to yield 1 X lo6 macrophages/ml and plated in 0.2-ml aliquots in 96-well U-bottom tissue culture plates (Costar, Cambridge, Mass.). Plates were centrifuged at 5OOg for 2 min and incubated at 37°C in the presence of 5% CO1 for 2 hr to allow macrophages to adhere. Wells were then washed three times with warm HBSS to remove nonadherent cells. Resultant cultures consisted of highly purified (>98%) macrophage monolayers as determined by morphology when stained with hematoxylin-eosin and by staining for nonspecific esterase. Resident peritoneal macrophages were obtained from untreated mice. Inflammatory macrophages were elicited by the intraperitoneal (ip) injection of 0.2 ml of thioglycolate broth 3 days prior to harvesting PECs. Activated macrophages were obtained according to two protocols. C. parvum-activated macrophages were obtained from mice injected ip with 0.2 ml (70 mg/kg) of a Formalin-fixed preparation of C. parvum (Lot No. 776/ 1, Burroughs-Wellcome, Research Triangle Park, N.C.) 7 days prior to harvesting PECs. BCG-activated macrophages were obtained from mice immunized 4 to 6 weeks prior to harvest with a subcutaneous injection of 5 X lo6 viable bacillus CalmetteG&in organisms followed by - lo- and -3-day ip injections of 5 X lo6 BCG organisms (Phipps Strain 1012 from Trudeau Institute, Saranac Lake, N.Y.).

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Untreated mice yielded approximately 5 X lo5 PECs per mouse of which 25-30% were macrophages. Mice injected with thioglycolate and C. parvum yielded 2-3 X lo6 and 1 X lo6 PECs per mouse, respectively, 50-60% of which were macrophages. Mice treated with BCG yielded 2-3 X lo6 PECs per mouse of which approximately 35% were macrophages. Amoebae and amoeba labeling. N. fowleri LEE (ATCC-30894) originally isolated from the brain of a patient with PAME was maintained axenically in Nelson medium at 37°C (18). N. fowleri amoebae (5 X lo5 cells) were seeded in 2.0 ml of Nelson medium in 25-cm2 tissue culture flasks 2 days prior to assay. Amoebae were labeled with 10 &i/ml of [3H]uridine (New England Nuclear, Boston, Mass.) 24 hr before coincubation with the macrophages. Radiolabeling of amoebae in this manner yields 0.5 to 1.O count per minute (cpm) per amoeba of TCA (trichloroacetic acid) precipitable label. The release of label correlates with death of the amoebae as determined by viability studies (data not shown). Labeled amoebae were washed twice with Nelson medium and three times with HBSS before being suspended in cell culture medium (CCM) comprised of Neumann-Tytell serumless medium (GIBCO) supplemented with antibiotics, 1% glutamine, 0.1% lactalbumin hydrolysate, and 25 mM Hepes buffer. Preparation of antiserum. N. fowleri maintained in Nelson medium were washed in HBSS and fixed with 2% paraformaldehyde. Fixed amoebae were washed three times in HBSS and mixed with an equal volume of complete Freund’s adjuvant (GIBCO). Adult male New Zealand white rabbits were immunized with three l-ml injections of 1 X lo7 fixed amoebae at weekly intervals. The first injection was given in the footpad, followed by two injections in the rear flanks. One week after the third injection rabbits were bled via cardiac puncture and the sera obtained was heat-inactivated at 56°C for 30 min, filter sterilized through a 0.2-pm filter (Gelman Sciences, Inc., Ann Arbor, Mich.), and stored at -80°C. The same lot of antiserum from a single rabbit was used for all assays. The agglutination titer was determined prior to each assay according to the method described by Reilly et al. (19). Tumor cell targets.Rat neuroblastoma (B- 103) cells, a nerve cell line obtained from D. Schubert (Salk Institute, San Diego, Calif.) were maintained in Eagle minimal essential medium with Hank’s salts (HMEM; GIBCO) and supplemented with 10 m&f Hepes, nonessential amino acids, vitamins, L-glutamine, and 10% fetal calf serum (GIBCO). The B-103 cells were labeled with 10 &i/ml of [3H]thymidine (New England Nuclear, Boston, Mass.) 24 hr prior to assay. Immediately prior to addition to macrophages the tumor cells were washed three times with warm HBSS and suspended in CCM. Cytotoxicity assays.Assays were performed in sterile, U-bottom 96-well microtiter plates (Costar, Cambridge, Mass.). Labeled amoebae, 1, 2, or 4 X 1O4 cells, in 0.1 ml of CCM were added to monolayers of adherent macrophages to obtain effector to target cell ratios of 20: 1, 10: 1, and 5: 1, respectively. Wells .were brought up to 0.2 ml with 0.1 ml of CCM with or witout antibody. Plates were centrifuged at 5OOg for 2 min and incubated at 37°C in the presence of 5% CO2 for 12, 24, or 36 hr. Labeled tumor targets, 2 X lo4 cells in 0.2 ml of CCM, were added to adherent macrophages to obtain an effector to target ratio of 10: 1 and incubated at 37°C in the presence of 5% CO2 for 24 hr. After incubation, plates were centrifuged at 1OOOgfor 2 min, the supernatant fluid was withdrawn, and radioactivity present was determined by liquid

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scintillation spectrometry. The percentage specific release correlating to cytolysis was calculated from the mean specific release of 4 wells according to the formula % specific release =

experimental - spontaneous release x 100. maximum - spontaneous release

Spontaneous release of radiolabel from amoebae was determined in CCM with or without antibody in the absence of macrophages. Spontaneous release was 1.O- 1S%/hr. Spontaneous release of label from tumor cells was determined in CCM in the absence of macrophages. Maximum release of label was determined by incubating amoebae or tumor cells with 0.1 ml of 1% sodium dodecyl sulfate (SDS) to obtain the total label present. The data presented in each figure are representative of at least three experiments. Statistical analysis was performed on the data using the Student t test. Scanning electron microscopy. Samples for SEM were prepared in a manner described previously (20). Briefly, to 2 X 10’ macrophages plated on coverslips were added N. fowleri amoebae suspended in CCM. Coverslip cultures were incubated for 24 hr, fixed for 30 min with warm 2% glutaraldehyde in 0.1 M sodium-cacodylate-hydrochloride buffer (pH 7.2), and stored at 4°C. Cultures were postfixed in 1% OS04 containing 0.15 M cacodylate buffer. After a rinse in 0.15 M cacodylate buffer, cultures were dehydrated in a graded series of ethanol, subjected to critical point drying, and sputter-coated with gold (2 1). Coverslips were examined with a Hitachi HS-500 scanning electron microscope operating at an accelerating voltage of 20 kV. Porous membranejilters. Filters were employed in the manner described by Marino and Adams (22). Polycarbonate membrane filters (Nuclepore, Pleasanton, Calif.) were cut to 16-mm diameter and placed over macrophage monolayers established in 24-well plates (Costar, Cambridge, Mass.). Membranes of l-pm pore size were employed to abrogate contact between macrophages and amoebae. Manually perforated 12qm pore filters allowed passage of amoebae and contact with macrophages (personal observation). Filters were held in place by a 16-mm-diameter plastic ring and amoebae suspended in CCM were place directly on the membrane filters. Control wells contained macrophages and amoebae without filters and spontaneous release wells contained amoebae and filter membranes alone. RESULTS Kinetics of Macrophage-Mediated Macrophage Populations

Cytolysis of Naegleria

fowleri

by the Various

Initial experiments were conducted to define the parameters under which macrophages mediate direct cytolysis of amoebae in the absence of antibody. Effector to target cell (ET) ratios of 5: 1, 10: 1, and 20: 1 were employed at incubation periods of 12,24, and 36 hr. Assays were performed in serumless medium because the cytolytic response in this study was found to be diminished in the presence of 10% fetal calf serum. Resident macrophages and those elicited by thioglycolate were able to induce a minimal release of radiolabel from amoebae (Fig. 1). Increasing the period of coincubation and altering E:T ratios failed to substantially increase this response. At almost all ET ratios and incubation periods tested, thioglycolate macrophages demonstrated a slightly higher cytolytic response than resident macrophages. The highest cytolytic

AMOEBICIDAL

ACTIVITY

GO

OF ACTIVATED

MACROPHAGES

1

129

36h

GO 70 60 50

40 30 20 IO 0 RESIDENT

TG

MACROPHAGE

c PPNWM

ECG

POPULATION

FIG. 1. Amoebicidal activity of the four macrophage populations. Resident, thioglycolate-elicited (TG), C. parvm-activated, and BCG-activated (BCG) macrophages were tested at varying E:T ratios and coincubation periods. Labeled amoebae, 1,2, or 4 X lo4 were added to 2 X lo5 macrophages to obtain E:T ratios of 5:l (I@, 10: I (Cl), and 20~1 (I@, respectively. Cells were coincubated for 12, 24, or 36 hr. Values represent the mean + SE percentage specific release of label from four wells in a representative experiment.

activity produced by thioglycolate macrophages was 10.2% at an E:T ratio of 20: 1 and 36 hr of coincubation. C. parvum and BCG-activated macrophages demonstrated a time and cell densitydependent cytolytic response. Amoebicidal activity increased with increasing time and decreasing target cell density for both populations. C. parvum macrophage-induced release of label ranged from 11.1 to 63.7%. Cytolytic activity of BCG macrophages ranged from 12.4% to a maximum of 80.6% at 36 hr and an E:T ratio of 20: 1. BCG

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macrophages exhibited a consistently higher cytolytic response than C. pawum macrophages at all but the 5:l E:T ratio. C. pawurn macrophages induced a 40.7% and BCG macrophages a 60.7% specific release of radiolabel over 24 hr at an E:T ratio of 10: 1. At this time period and E:T ratio, a substantial and reproducible cy-tolytic response was produced by both populations of activated macrophages. Therefore, further studies were conducted under these conditions.

Eflect of Antibody on Amoebicidal Activity A role for antibody-dependent cellular cytoxicity (ADCC) was investigated and results of a representative experiment can be seen in Fig. 2. The N. fowleri specific antibody employed had an agglutination titer of 1:256 and the subsequent two dilutions were found to be immobilizing for amoebae. Normal rabbit serum was included as a control and produced similar efkcts to medium alone. Addition of specific antibody failed to enhance significantly the response of either resident or thioglycolate macrophages. Antibody added to amoebae in the absence of macrophages had no effect on the spontaneous release of label. ADCC was expressed by both populations of activated macrophages. Addition of an agglutinating titer of antibody (1:250) to C. pawum macrophages induced a slight (13%) but significant enhancement of cytolysis above that of control serum or medium alone. Immobilizing titers of 1500 and 1: 1000 induced levels of cytolysis above that of the agglutinating titer, exhibiting 3 1 and 28% enhancement, respectively, over control serum levels. The addition of each dilution of antibody to the BCG macrophages elevated release of label to maximum release levels indicating complete cytolysis of amoebae. Addition of specific antibody to BCG macrophages led to a 64% increase in cytolysis over that found with control serum.

FmKmT

TG

c. FaWM

6CG

FIG. 2. Effect of addition of N. fowleri specific antiserum to macrophage-amoebae cultures. Antiserum diluted to 1:250(O), 1:500 (RI), or 1:lOOO ( was added to resident, thioglycolateelicited (TG), C. parvumactivated, and BCG-activated (BCG) macrophage cultures. The addition of medium alone (B@ or normal rabbit serum (B!) served as controls. Values represent the mean percentage specific release of label f SE from four wells in a representative experiment. The mean maximum releasable label was 11080 + 56 cpm.

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Scanning Electron Microscopy of Target Cell Binding by Macrophages Scanning electron micrographs of the various populations of macrophages can be seen in Fig. 3. Resident and thioglycolate macrophages exhibited minimal binding of Naegleria in vitro. This was limited to peripheral binding of one or two amoebae per macrophage. In the presence of both resident and thioglycolate macrophages Naegleria remained amoeboid and exhibited a characteristically smooth surface (Figs. 3A, B). C. parvum and BCG macrophages, on the other hand, demonstrated enhanced binding of the amoebae. Binding of several amoebae per macrophage was typical. Greatest binding of amoebae was found with BCG macrophages. BCG macrophages were observed which had 4 to 15 amoebae bound to their surface. Amoebae in the presence

FIG. 3. Scanning electron microscopy of macrophage-amoebae cultures. N. fowl& amoebae (N) were cultured with resident (A), thioglycolate-elicited (B), C. purvum-activated (C), and BCG-activated (D) macrophages for 24 hr at 37’C. Arrows show cellular debris from amoebae. Bars represent 5 pm.

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of activated macrophages are rounded in contrast to their normal amoeboid morphology. Cellular debris from injured amoebae was apparent in C. parvum-activated macrophage cultures (Fig. 3C). Holes could be seen in the outer cell membrane of amoebae bound to BCG-activated macrophages (Fig. 3D). Requirement for E$ector-Target

Cell Contact for Cytolysis

When contact between N. fowleri amoebae and activated macrophages was prevented by the interposition of a l-pm pore filter, cytolysis was almost completely abrogated (Table 1). Cytolysis of amoebae was still seen at appreciable levels in the presence of perforated filters which allowed contact between macrophages and amoebae. Normal levels of cytolysis were seen in the absence of filters. Comparison

of Amoebicidal

and Tumoricidal

Activity

When N. fowleri amoebae and B-103 tumor cells were assayed in parallel with the same populations of macrophages, cytolytic activity for each target cell was similar (Table 2). Resident and thioglycolate macrophages demonstrated low cytolytic activity for both targets. C. parvum and BCG-activated macrophages exhibited significant and very similar levels of cytolysis for both types of target cells. DISCUSSION Investigations into macrophage extracellular mechanisms, especially recognition and cytolysis of individual cells, are of prime importance to the characterization of macrophage-mediated immunity to protozoa and macrophage tumoricidal activities. The ability of macrophages to recognize and specifically bind nonmammalian cells, or to discriminate between neoplastic and nonneoplastic cells, is critical to their selectivity in cytocidal activity (23-27). We have developed an in vitro assay, similar to that used in studying macrophage tumoricidal activity, in order to investigate the interaction which occurs between resident, elicited, and activated macrophages and N. fowleri, an extracellular protozoan. We employed this assay in order to demonstrate

TABLE 1 Requirement for Effector-Target Cell Contact for Macrophage-Mediated

Cytolysis of N. fowleri Amoebae

Macrophage population

Membrane filter”

% Cytolysis f SEb

C. pawum

None Perforated l-pm pore

58.67 f. 1.69 37.88 f 3.37 12.97 t- 7.5 1

BCG

None Perforated I-rrn pore

72.64 f 5.38 46.20 f 3.91 18.95 + 8.36

’ Porous polycarbonate membrane filters were placed directly on top of macrophage monolayers established in 16-mm wells and amoebae added on top of filters at an E:T ratio of 10: 1. ’ Represents the percentage specific release of radiolabel from amoebae correlating to cytolysis.

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MACROPHAGES

TABLE 2 Comparison of Macrophage Cytocidal Activity for Rat Neuroblastoma Cell Line B- 103 and N. fowleri Amoebae Macrophage population Resident Thioglycolate c. parvum

BCG

Target

cell ’

N. fowleri B-103 N. fowleri B-103 N. fowleri B-103 N. fowleri B-103

% Cytolysis + SE b 0.0 12.46 2 0.0 1.14 + 36.84 + 40.01 k 55.60 -t 52.22 +

0.49 0.95 3.03 2.31 4.62 3.12

’ Both tumor cells and amoebae were suspended in CCM and added to monolayer’s of macrophages at an effector to target ratio of 10: 1. b Represents the percentage specific release of radiolabel correlating to cytolysis of amoebae or tumor cells. Results presented for individual macrophage populations are from the same experiment employing the two target cell types in parallel.

a possible role for macrophages in protecting against extracellular protozoa and to extend the range of demonstrable macrophage extracellular cytocidal activities. Resident, thioglycolate-elicited, C. parvum-activated, and BCG-activated murine peritoneal macrophages were tested for amoebicidal activity. Of these four populations of macrophages, only those activated in vivu by the injection of live bacteria (i.e., BCG) or bacterial products (i.e., C. parvum) demonstrated significant levels of amoebicidal activity. The inability of resident and thioglycolate-elicited peritoneal macrophages to mount a cytolytic response against amoebae is in accordance with previous reports which indicate defective tumoricidal and anti-protozoal activity by these populations in the absence of in vitro stimulation by LPS or lymphokines (28-32). The direct cytolysis of N. fowleri amoebae induced by C. parvum and BCG-activated macrophages was not dependent on the presence of antibody and increased with increasing E:T ratios and increasing times of coincubation. In contrast, the low cytolytic activity of resident and thioglycolate macrophages was not substantially affected by altered E:T ratios or incubation periods. The time- and density-dependent amoebicidal activity exerted by the activated macrophages suggests that this cell killing is dependent on cellular interactions between macrophages and amoebae. In the assay employed in this study, cell-cell contact was assured by gently centrifuging the microtiter plates to bring amoebae into contact with the macrophages. The requirement for prolonged coincubation to produce lolling may indicate that a prerequisite for cytolysis is the establishment of tight target cell binding as has been reported in macrophage-tumor cell interactions (33-35). Required synthesis of cytolytic substances by the macrophages may also be involved. In addition to exhibiting direct amoebicidal activity, activated macrophages demonstrated ADCC toward N. fowleri amoebae. BCG macrophages showed the greatest capacity for enhancement by antibody with total release of label by the amoebae occuring in the presence of all titers of antibody tested. A significant, yet less dramatic

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enhancement was seen for C. pawum macrophages and was dependent on the titer employed. The least enhancement was seen with the agglutinating titer of antibody and may have been due to the inability of the C. parvum macrophages or their cytolytic factors to gain access to amoebae secluded within an agglutinated mass of cells. The immobilizing titer, on the other hand, may have enabled increased binding of amoebae by the macrophages which could have been avoided had the amoebae been able to escape. ADCC was not expressed by resident and elicited macrophages. Neither of these populations demonstrated significant enhancement of amoebicidal activity in the presence of either agglutinating or immobilizing titers of antibody. Addition of normal rabbit serum to resident and thioglycolate-elicited macrophages produced similar effects to medium alone; thus nonspecific serum inhibitory factors, which may have masked an enhancement by specific antibody, were apparently absent. The selective binding of tumor cells by macrophages has been shown to exhibit quantitative and qualitative differences among activated, elicited, and resident populations of macrophages (35, 36). Such differences are not evident among the populations in nonspecific binding of red blood cells or nonneoplastic lymphoid cells (35-38). That specific binding, which may entail a foreign recognition step, is necessary for tumor cell cytolysis and has been demonstrated in a number of systems (35, 36, 39). Cytolysis of syngeneic cells does not occur when they are placed in the presence of, or are artificially bound to activated macrophages (35, 37, 39, 40). From SEM studies it is apparent that, between the different macrophage populations, the ability to bind amoebae is directly related to the ability to mediate cytolysis. Resident and thioglycolate-elicited macrophages demonstrated low levels of both target cell binding and cytolysis. C. parvum macrophages, on the other hand, demonstrated a significant capacity for both the binding and cytolysis of amoebae. BCG-activated macrophages demonstrated an even higher level of binding along with a proportionately increased degree of cytolysis compared to the C. parvum macrophages. When target cell binding by activated macrophages was prevented by the interposition of a porous filter between the amoebae and macrophages, low levels of cytolysis were seen. In contrast, significant levels of cytolysis were seen in the absence of a filter or in the presence of a perforated filter which allowed effector-target cell contact. The requirement for effector-target celI contact may be explained by the need for concentration of the macrophage secretory products in the microenvironment between cells in contact in order to effect cytolysis. Target cell binding and foreign cell recognition may also be required for subsequent elaboration of toxic species by activated macrophages. Selective binding of neoplastic over nonneoplastic cells has been demonstrated to be an initial and necessary step in tumoricidal activity by activated macrophages (34-36). Whether recognition and binding of N. fowleri amoebae and tumor cells is mediated by the same macrophage surface structures remains to be determined. Qualitative differences in the binding of amoebae between populations of macrophages were similar to those reported in tumor cell binding (36). Activated macrophages demonstrated extensive binding of amoebae while elicited and resident macrophages exhibited minimal binding of amoebae, predominantly at the periphery of the macrophages. Amoebae bound to the surface of the activated macrophages were rounded rather than amoeboid in morphology, The normally smooth surface of these amoebae was pitted and holes through the outer cell membrane were evident in the presence of BCG macrophages. This suggests that the lytic factor(s) involved may be acting on

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membrane surface structures and mediating cytolysis by disruption of the amoebae cell membrane. In addition to similar qualitative binding characteristics, levels of cytolysis by the different macrophage populations were comparable for both N. fowled amoebae and B- 103 tumor cell targets. Acquisition of tumoricidal activity with activation appeared to be coincident with acquisition of amoebicidal activity. Nonactivated macrophages exhibited low levels of cytolysis for both targets. C. pawurn-activated macrophages killed N. fowleri and B- 103 tumor targets to the same degree. BCG macrophages also demonstrated cytolysis of amoebae and tumor cells at similar levels. Similarities in the interactions between activated macrophages and N. fiwleri amoebae or tumor cells suggests that a similar or common mechanism of recognition and cytolysis may be involved. Elucidation of these events is of considerable interest in the determination of underlying mechanisms of macrophage cellular recognition and cytolytic specificity. ACKNOWLEDGMENTS This research was supported in part by grants from the Virginia Electric and Power Company, Richmond, Virginia, the Thomas F. JelBess and Kate Miller JelTressMemorial Trust, Richmond, Virginia, and a training grant CA092 10 from the National Cancer Institute.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 1 I. 12. 13. 14. 15. 16. 17. 18.

Alexander, P., Annu. Rev. Med. 27,207, 1976. Hibbs, J. B., Chapman, H. A., and Weinberg, J. B., J. Reticuloendothel. Sot. 24, 549, 1978. Adams, D. O., and Snyderman, R. J., Natl Cancer Inst. 62, 134 1, 1979. James, S. L., Leonard, E. J., and Meltzer, M. S., Cell. Immunol. 74, 86, 1982. Diamond, R. D., Huber, E., and Haudenschild, C. C., J. Infect. Dis. 147,474, 1983. Brummer, E., Morozumi, P. A., and Stevens, D. A., J. Reticuloendothel. Sot. 28, 507, 1980. Landolfo, S., Martinotti, G., and Martin&to, P., In “NK Cells and Other Natural Effector Cells” (R. B. Herberman, Ed.), pp. 1099-l 104. Academic Press, New York. Smith, P. D., Keister, D. B., Wahl, S. M., and Meltzer, M. S., Cell Immunol. 85,244, 1984. Chapes, S. K., and Haskill, S., Cell Immunol. 70, 65, 1982. Olivotto, M., and Bomford, R., Int. J. Cancer 13,478, 1974. Alexander, P., and Evans, R., Nature (London) 232, 76, 1971. Keller, R., Immunology 27, 285, 1974. Krahenbuhl, J. L., and Remington, J. S., J. Immunol. 113, 507, 1974. Keller, R., Bregnard, A., Gehring, W. J., and Schroeder, H. E., Exp. Cell. Biol. 44, 108, 1976. Mehzer, M. S., Tucker, R. W., and Breuer, A. C., Cell. Immunol. 17, 30, 1975. Snodgrass, M. J., Harris, T. M., Geeraets, R., and Kaplan, A. M., J. Reticuloendothd Sot. 22, 149, 1977. Melsom, H., Ofiebro, R., and Seljelid, R., Exp. Cell. Res. 80, 388, 1973. Duma, R. J., Rosenblum, W. T., McGhee, R. F., Jones, M. M., and Nelson, E. C., Ann. Intern. Med. 74,923,

1981.

19. Reilly, M. F., Marciano-Cabral, F., Bradley, D. W., and Bradley, S. G., J. Clin. Microbial. 17, 576, 1983. 20. Marciano-Cabral, F., and John, D. T., In&t. Immun. 40, 1214, 1983. 21. Brunk, U., Collins, V. P., and Arro, E., J. Microsc. 123, 121, 1981. 22. Marino, P. A., and Adams, D. O., Cell Immunol. 54, 26, 1980. 23. Meltzer, M. S., Tucker, S. W., Sanford, K. K., and Leonard, E. J., J. Natl. Cancer Inst. 54, 1177, 1975. 24. Adams, D. O., J. Immunol. 124,286, 1980. 25. Hibbs, J. B., Science (Washington, D.C.) 180,868, 1973. 26. Piessons, W. F., Churchill, W. H., and David, J. R., J. Immunol. 114, 273, 1975.

136 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.

CLEARY

AND MARCIANO-CABRAL

Kaplan, A. M., Morahan, P. S., and Regelson, W. J., J. Natl. Cancer Inst. 52, 1919, 1974. Chapman, H. A., and Hibbs, J. B., Science (Washington, D.C.) 197,279, 1977. Ruco, L. P., and Meltzer, M. S., J. Immunol. 121,2035, 1978. Cohn, Z. A., J. Immunoi. 121, 813, 1978. Karnovsky, M. L., and Lazolins, J. K., J. Zmmunol. 121, 809, 1978. Murray, H. W., and Cohn, Z. A., J. Exp. Med. 152, 1596, 1980. Hibbs, J. B., Jr.; In “The Macrophage in Neoplasia” (M. A. Fink, Ed.), p. 83. Academic Press, New York, 1976. Piessens, W. F., Cell. Immunol. 35, 303, 1978. Marino, P. A., and Adams, D. O., Cell. Immunol. 54, 11, 1980. Somers, S. D., Mastin, J. P., and Adams, D. O., J. Immunol. 131,2086, 1983. Adams, D. O., and Marino, P. A., J. Zmmunol. 126,981, 1981. Toge, T., Nakanishi, K., Yamada, Y., Yanagawa, E., and Hattori, T., Gunn 72,305, 1981. Johnson, W. J., Whisnant, C. C., and Adams, D. O., J. Immunol. 127, 1787, 1981. Cabilly, S., and Gallily, R., Immunology 42, 149, 198 1.