Nonspecific Tumoricidal Activity Research Articles

Differences in the Induction of Macrophage Cytotoxicity by the Specific T Lymphocyte Factor, Specific Macrophage Arming Factor (SMAF), and the Lymphokine, Macrophage Arming Factor (SMAF), and the Lymphokine, Macrophage Activating Factor (MAF)

Specific T cell factors, such as specific macrophage arming factor (SMAF), are involved inthe initiation of the immune response. Induction of SMAF-producing T lymphocytes in vivo and of SMAF production by T lymphocytes in vitro is dependent on the presence of intact tumor cells, and is independent of antigen presentation by macrophages. SMAF renders peritoneal macrophages cytotoxic for tumor cells. The armed peritoneal macrophages expressed a specific cytotoxicity. However, antigen-presenting cells can trigger lymphokine-producing T lymphocytes. TheseT lymphocytes produce lymphokines (e.g. macrophage activating factor (MAF)) that activate macrophages. The MAF-activated macrophages express a non-specific tumoricidal activity. In the present study, we investigated the difference in the induction of macrophagecytotoxicity by SMAF and MAF. The following differences were found: 1) SMAF renders peritoneal resident macrophages cytotoxic, whereas MAF could only render peritoneal exudate macrophages cytotoxic. 2) SMAF requires only a 4-h incubation with macrophages, whereas MAF activates macrophages optimally after 12 h. 3) SMAF-armed macrophages recognize only the specific target cell(s), and thus, the cytotoxicity is specific in its expression. MAF activated macrophages were non-specifically cytotoxic. 4) Lipopolysaccharide (LPS) in the culture medium did not enhance the cytotoxicity of SMAF-armed macrophages. In contrast, MAF induced tumoricidal activity was enhanced by adding LPS to the culture medium. 5) After adsorption chromatography with anti-murine interferon-gamma (IFN-γ), the arming capacity of SMAF supernatant was not reduced, whereas the activating capacity of the MAF supernatant was significantly reduced or abrogated. After immunization of mice with allogeneic tumor cells, SMAF-producing lymphocyteswere detected in the draining lymph nodes already 4 days after immunization and up to 12 days. Lymphocytes with the capacity to produce MAF were present in the draining lymph nodes 14-24 days after immunization. Our data indicate that the T cell factors SMAF and MAF can both render macrophages cytotoxic, but act in a different way and during different stages of the cellular immune response against allogeneic tumor cells.

Internalization and degradation of receptor-bound interferon-gamma by murine macrophages. Demonstration of receptor recycling.

Although the interferon-gamma (IFN-gamma) receptor on murine and human mononuclear phagocytes has been defined and partially characterized, very little data exists which describes the ultimate fate of receptor-bound ligand. The current studies were specifically designed to define the metabolic processes which act on murine recombinant IFN-gamma following its interaction with murine macrophages at physiologic temperatures. Ligand internalization was demonstrated by comparing binding of [125I]IFN-gamma to macrophages at 4 degrees C and 37 degrees C. When binding was carried out at 4 degrees C, 96% of the cell-associated [125I]IFN-gamma remained accessible at the plasma membrane and could be stripped from the cell by exposure to pronase. In contrast, at 37 degrees C, only 35% of the cell-associated radioactivity was pronase strippable. Macrophages degraded [125I]IFN-gamma into trichloroacetic acid-soluble material at 37 degrees C at a constant rate of 7000 molecules/cell/hr over a 12-hr time period. The amount of IFN-gamma degraded correlated with the amount of IFN-gamma bound to the cell surface. The receptor was neither up- nor down-regulated by ligand or by other agents known to regulate macrophage functional activity such as IFN-alpha, IFN-beta, lipopolysaccharide, or phorbol myristate acetate. The constant uptake of IFN-gamma by macrophages was due to the presence of an intracellular receptor pool (62% of the total receptor number) and to a mechanism of receptor recycling. Evidence for the latter was obtained using lysosomotropic agents which blocked degradation but not binding and internalization of ligand and caused the intracellular accumulation of receptor. By comparing the relationship between receptor occupancy and biologic response induction, two activation mechanisms became apparent. Induction of certain functions, such as H2O2 secretion, appeared to require only a single round of receptor occupancy. However, induction of more complex functions such as nonspecific tumoricidal activity appeared to require three to four rounds of receptor occupancy. These results thus support the concept that IFN-gamma internalization and receptor recycling are essential in the induction of nonspecific tumoricidal activity by macrophages.

Activation of Mouse Macrophages for Migration Inhibition and for Tumor Cytotoxicity Is Mediated by Interferon-γ Priming and Triggering by Various Agents

The requirements for activation of C3HeB/FeJ mouse peritoneal macrophages to mediate migration inhibition from capillary tubes was compared with those conditions prerequisite for nonspecific tumor cytotoxicity. Both in vitro assays for macrophage activation were found to require a two-stage process that involved priming by murine recombinant interferon-gamma (IFN-gamma) and triggering by subactivating concentrations of bacterial lipopolysaccharide (LPS), Lipid A, Polyinosinic-polycytidylic acid (poly I:C), or cobra venom factor (CVF). A dose-related increase in both migration inhibition and tumor cytotoxicity was shown with increasing concentrations of IFN-gamma (3.0-50.0 U/ml) in synergistic combination with an LPS trigger. IFN-gamma alone produced low levels of migration inhibition or tumor cytotoxicity, only at higher concentrations, that was not attributable to LPS contamination. The concentrations of the various agents required for direct activation or triggering of IFN-gamma-primed macrophages were approximately 2- to 10-fold greater for migration inhibition than for tumor cytotoxicity. Our results indicate that the two-signal process of priming and triggering for mediating mouse macrophage nonspecific tumoricidal activity is also operative in migration inhibition from capillary tubes. Thus, under defined conditions with purified lymphokines, the migration inhibition assay appears to be a reliable alternate in vitro correlate of macrophage activation by IFN-gamma.

Triggering of interferon γ-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 γ for enhanced tumoricidal activity. An Fc receptor blockade with opsonized sheep erythrocytes abrogated LPS-mediated direct activation and triggering of interferon γ-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 β 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.

Role of protein kinase C and intracellular calcium mobilization in the induction of macrophage tumoricidal activity by interferon-gamma.

These studies were designed to test the hypothesis that changes in intracellular Ca2+ levels and activation of the calcium ion- and phospholipid-dependent protein kinase C were required for the induction of macrophage tumoricidal activity by interferon-gamma (IFN-gamma). Phenothiazines and R24571, known antagonists of calcium-binding proteins and therefore nonspecific inhibitors of protein kinase C, blocked in a dose-dependent manner the induction of macrophage cytocidal activity by either natural or recombinant IFN-gamma. Macrophages depleted of intracellular Ca2+ by chelation with Quin 2, were also unresponsive to IFN-gamma. These treatments effected neither the binding of IFN-gamma to its cell surface receptor nor the normal intracellular processing of IFN-gamma. Activators of protein kinase C (such as phorbol esters) and Ca2+ ionophores when added alone did not effect the activation state of the macrophage population. However, macrophages exposed to both drugs in combination were elevated into the primed activation state such that in the presence of a second signal (lipopolysaccharide or heat killed Listeria monocytogenes), the cells were triggered to express full levels of tumoricidal activity. The capacity of phorbol esters to induce cellular activation correlated with their ability to bind and to activate protein kinase C. No synergistic effect was observed between IFN-gamma and protein kinase C activators and/or Ca2+ ionophores, indicating that the drugs could only prime and could not trigger macrophages for tumor cell killing. These results thus support the concept that protein kinase C activation and mobilization of intracellular Ca2+ are essential steps in the pathway of IFN-gamma-dependent induction of non-specific tumoricidal activity in macrophages.

Relationship between Murine Macrophage Fc Receptor-Mediated Phagocytic Function and Competency for Activation for Non-Specific Tumor Cytotoxicity

The relationship between Fc receptor (FcR) function and activation of murine macrophage populations for non-specific tumor cytotoxicity was studied. Oil-elicited inflammatory peritoneal macrophages (PMФ) from C3HeB/FeJ mice had higher FcR function upon harvest than resident PMФ from the same strain or elicited PMФ from genetically deficient C3H/HeJ mice. C3HeB/FeJ inflammatory PMФ were uniformly responsive to activation by MAF and the complement activators: LPS, Poly I:C, cobra venom factor (CVF) and zymosan for tumoricidal activity. Resident cells from the same strain and C3H/HeJ-elicited PMФ were uniformly unresponsive to the same activators. In vitro culture of C3HeB/FeJ resident PMФ with fetal bovine serum for 24–48 h produced unregulation of FcR function which coincided with a conversion from an unresponsive to a responsive state for tumoricidal activity. Reconstitution of the FcR function of C3H/HeJ-elicited PM In vitro during 24–48 h culture with lymphokine or Poly I:C also coincided with the restoration of responsiveness to activation by LPS, CVF, and zymosan for tumor cytotoxicity. Thus, the consistent temporal relationship between upregulated FcR function and the capacity of macrophages to respond to activation for non-specific tumoricidal activity may be more than coincidental. Preincubation of responsive C3HeB/FeJ-elicited PMФ with insoluble immune complex or heat-aggregated IgG was shown to blockade FcR-mediated phagocytosis and to abrogate LPS-mediated tumoricidal activity. Interestingly, FcR blockade by IgG-opsonized sheep erythrocyte conjugates selectively inhibited activation by MAF, LPS, and Poly I:C, but had no inhibitory effect on activation by CVF or zymosan. Similar blockade of C3b receptors produced an identical pattern of selective inhibition of activation. This selective inhibition of non-specific tumoricidal activity by FcR/C3bR blockade suggests the existence of two pathways for antibodyindependent activation of macrophages.

Rapid killing of actinomycin D-treated tumor cells by mononuclear phagocytes: characterization of effector cells in mice.

Pretreatment with Actinomycin D (Act D, 1 microgram/ml for 3 hr) rendered WEHI 164 tumor cells susceptible to killing by mouse resident or peptone-induced peritoneal exudate cells (PEC) in a 6-hr 51Cr release assay. Cytotoxicity was attributed to cells of the monocyte macrophage lineage on the basis of tissue distribution, separation by adherence on plastic and carbonyl iron, membrane antigens, and expression in mice with defective T cell- or NK cell-mediated immunity. Macrophages from four strains of mice (C3H/HeJ, A/J, P/J, C57B1/10 ScCR) previously shown to have defective "classical" nonspecific tumoricidal activity were examined for killing of Act-D-treated WEHI 164 cells. C3H/HeJ peritoneal macrophages had little or no DDCC, whereas cells from A/J, P/J, and C57B1/10 ScCR mice had normal levels of this reactivity. Tumor cells exposed to ActD were heterogenous in their susceptibility to killing by PEC, with five lines showing significant, though variable, lysis, whereas 12 tumor lines, normal fibroblasts, and lymphoblasts were not appreciably killed under the same conditions. Macrophage-mediated DDCC was also detectable in a colony assay. DDCC could explain how macrophages contribute to the antitumor activity of selected chemotherapeutic agents in murine tumor models.

Requirement of endogenous interferon-gamma production for resolution of Listeria monocytogenes infection.

Peritoneal exudate cells and splenic cells of mice infected with Listeria monocytogenes show increased production of interferon-gamma (IFN-gamma) after antigen or mitogen stimulation. When an IFN-gamma-specific enzyme-linked immunosorbent assay was used, increased production was first observed 2 days after infection in peritoneal cells and 4 to 6 days after infection in splenic cells. The increased production of IFN-gamma correlated with the clearance of Listeria from the peritoneal cavity and spleen. Macrophages derived from mice at these times were activated as evidenced by expression of nonspecific tumoricidal activity against 111In-labeled P815 mastocytoma cells. Injection of neutralizing monoclonal anti-IFN-gamma into 1-day-infected mice completely inhibited the generation of activated macrophages. Normal hamster IgG had no effect. In vivo, the monoclonal antibody also abrogated clearance of bacteria from the spleen and peritoneal cavity. Six days after injection of a sublethal dose of Listeria, the peritoneal cavity of control mice treated with normal hamster IgG was devoid of bacteria and the spleen contained less than 10(3) colony-forming units. However, mice treated with anti-IFN-gamma carried more than 8 X 10(6) colony-forming units in either anatomical site at day 6 and exhibited a higher mortality rate. These results indicate that IFN-gamma production is required for the in vivo generation of activated macrophages and the clearance of bacteria during Listeria infection.

Monoclonal antibodies to murine gamma-interferon which differentially modulate macrophage activation and antiviral activity.

Four monoclonal IgG antibodies to purified, recombinant murine gamma-interferon (rIFN-gamma) have been produced by fusion of immune hamster splenocytes with HAT-sensitive murine myeloma cells. Specificity was confirmed either with an enzyme-linked immunosorbent assay (ELISA) that used immobilized rIFN-gamma or with a radioimmunoassay that employed soluble 125I-rIFN-gamma and heat-killed, fixed Staphylococcus aureus-bearing Protein A. Competition binding experiments suggested that the monoclonal antibodies (MoAb) displayed two distinct epitope specificities: one displayed by H1 and H2, and the other displayed by H21 and H22. By using murine-human recombinant IFN-gamma hybrid molecules, the H1/H2 epitope was shown to depend on the amino-terminus of IFN-gamma, whereas the H21/H22 epitope was formed by the carboxy-terminal amino acid sequence. The MoAb also reacted with natural IFN-gamma. When bound to a surface, all four MoAb, but not normal hamster IgG, removed 100% of the antiviral and MAF activities present in supernatants of cultures of the murine 24/G1 T cell hybridoma. In free solution, all four antibodies inhibited IFN-gamma dependent antiviral activity, but with different efficiencies. Soluble H21/H22 also blocked all of the 24/G1-derived activity that induces nonspecific tumoricidal activity in macrophages (MAF) while H1/H2 enhanced MAF activity. The differential inhibitory or enhancing activities of H21 or H1 reflected their ability to inhibit or enhance binding of 125I-rIFN-gamma to macrophages, respectively. Soluble H21/H22 and solid-phase H1/H2 inhibited 100% of the MAF, microbicidal, and Ia-inducing activities from lymphokine preparations produced by mitogen stimulation of normal murine splenic cells. These results help to establish definitive structure-function relationships for the IFN-gamma molecule, and indicate that IFN-gamma is the primary lymphokine responsible for inducing nonspecific tumoricidal activity and Ia antigen expression, and for enhancing microbicidal activity in macrophages.

Evidence for a gamma-interferon receptor that regulates macrophage tumoricidal activity.

Gamma-interferon (IFN-gamma) is the macrophage-activating factor (MAF) produced by normal murine splenic cells and the murine T cell hybridoma 24/G1 that induces nonspecific tumoricidal activity in macrophages. Incubation of 24/G1 supernatants diluted to 8.3 IRU IFN-gamma/ml with 6 X 10(6) elicited peritoneal macrophages or bone marrow-derived macrophages for 4 h at 37 degrees C, resulted in removal of 80% of the MAF activity from the lymphokine preparation. Loss of activity appeared to result from absorption and not consumption because (a) 40% of the activity was removed after exposure to macrophage for 30 min at 4 degrees C, (b) no reduction of MAF activity was detected when the 24/G1 supernatant was incubated with macrophage culture supernatants, and (c) macrophage-treated supernatants showed a selective loss of MAF activity but not interleukin 2 (IL-2) activity. Absorption was dependent on the input of either IFN-gamma or macrophages and was time dependent at 37 degrees C but not at 4 degrees C. With four rodent species tested, absorption of murine IFN-gamma displayed species specificity. However, cultured human peripheral blood monocytes and the human histiocytic lymphoma cell line U937 were able to absorb the murine lymphokine. Although the majority of murine cell lines tested absorbed 24/G1 MAF activity, two murine macrophage cell lines, P388D1 and J774, were identified which absorbed significantly reduced amounts of natural IFN-gamma. Purified murine recombinant IFN-gamma was absorbed by elicited macrophages but not by P388D1. Normal macrophages but not P388D1 bound fluoresceinated microspheres coated with recombinant IFN-gamma and binding was inhibited by pretreatment of the normal cells with 24/G1 supernatants. Scatchard plot analysis showed that 12,000 molecules of soluble 125I-recombinant IFN-gamma bound per bone marrow macrophage with a Ka of 0.9 X 10(8) M-1. Binding was quantitatively inhibitable by natural IFN-gamma but not by murine IFN alpha. IFN-beta competed only weakly. Monoclonal antibodies against IFN-gamma either inhibited or enhanced MAF activity by blocking or increasing IFN-gamma binding to macrophages, respectively. These results indicate that IFN-gamma reacts with a receptor on macrophage in a specific and saturable manner and this interaction initiates macrophage activation.

Identification of gamma-interferon as a murine macrophage-activating factor for tumor cytotoxicity.

Over the past decade, work from a number of laboratories has indicated that, under the proper conditions, the macrophage can express nonspecific effector cell function toward a variety of neoplastic cells (reviewed in Den Otter, 1981; Fidler and Raz, 1981; Keller, 1981; Lohmann-Matthes et al., 1981; Meltzer, 1981b; Piessens et al., 1981). While this type of macrophage tumoricidal activity does not require antibody or antibodylike specificity, it is nevertheless able to discriminate between normal cells and tumor cells (Hibbs, 1974; Piessens et al., 1975; Fidler et al.,1976). The currently accepted concept is that two signals are required to activate macrophages for tumor cell killing (Russell et al., 1977; Ruco and Meltzer, 1978a,b; Weinberg et al., 1978; Weinberg and Hibbs, 1979; Meltzer, 1981a,b; Pace and Russell, 1981). The first signal is a lymphokine that primes the macrophage and makes it receptive to a diverse group of substances that can act as second signals to trigger the development of full cytocidal activity. The lymphokine, denoted macrophage-activating factor (MAF), has been reported to alter a number of functional and biochemical properties in macrophage populations (reviewed in David and Remold, 1979; Rocklin et al., 1980). These alterations include increases in endocytic, biosynthetic, secretory, and effector cell functions as well as changes in membrane physiology and composition. However, since it is unclear whether all these alterations are effected by the same molecular species, it has become critical to define MAF in the context of the activity it induces. This chapter defines MAF exclusively as the factor that primes macrophages for nonspecific tumoricidal activity.

Inhibition of macrophage tumoricidal activity by immune complexes and altered erythrocytes.

Engagement of the macrophage membrane by biologic particles including insoluble immune complexes inhibited the development of lymphokine-mediated nonspecific tumoricidal activity by murine macrophages. The degree of inhibition was dependent on the dose of particles and the lymphokine concentration. Inhibition was not due to macrophage cell death or to diminution of cell adherence after ingestion of the immune complexes. Soluble immune complexes were not inhibitory, although approximately 10% of the complexes became cell-associated. Monomeric or heat-aggregated IgG was also not inhibitory. IgG-opsonized erythrocytes (EA) were inhibitory and inhibition was dependent on the degree of opsonization. In contrast, nonopsonized erythrocytes (E), which did not bind to macrophages, were not inhibitory. Phagocytosis of glutaraldehyde-treated E or E carrying IgM antibody and complement (EAC) also led to a reduction of tumorilytic activity. Insoluble immune complexes were inhibitory when added either before or after lymphokine. Phagocytosis was neither sufficient nor necessary to cause inhibition because 1) ingestion of polystyrene latex beads did not diminish tumoricidal activity, and 2) macrophages plated on IgG-coated surfaces were inhibited with respect to the tumoricidal function. Inhibition was not affected when indomethacin (10(-6) M) was included in the assay, which indicated that prostaglandins were not involved in the process. Thus, macrophage tumoricidal responsiveness may be compromised by interaction of biologic substances with macrophage plasma membranes. This process may thereby inactivate an important host defense mechanism against neoplastic cells.

Macrophage-activating factor produced by a T cell hybridoma: physiochemical and biosynthetic resemblance to gamma-interferon.

Biochemical and biosynthetic evidence has been obtained which indicates that the macrophage activating factor (MAF) produced by the murine T cell hybridoma clone 24/G1, which primes macrophages for nonspecific tumoricidal activity, is a form of gamma-interferon (IFN gamma). MAF and antiviral activities were generated by the clone in proportional amounts under a variety of culture conditions. Production of both activities showed identical dependence on cell density even when production precipitously ceased at cell concentrations greater than 1.2 X 10(6) cells/ml. MAF and antiviral activities displayed identical sensitivities to pH and temperature and were indistinguishable on the basis of binding to insolubilized polynucleotides. Dye-ligand chromatography of a stimulated hybridoma supernatant on a column of Matrex Gel Red A resulted in the 1500-fold purification and 100% recovery of the MAF activity. A qualitatively identical elution profile was also obtained for the antiviral activity; however, only 32% of the original activity was recovered. When subjected to gel filtration on a high performance liquid chromatography system (HPLC), the MAF and antiviral activities present in the Matrex Red pool displayed identical elution profiles and exhibited an apparent m.w. of 50,000. This technique resulted in another 10-fold purification and 50% recovery of the two activities. On HPLC chromatofocusing the MAF activity in the Matrex Red pool could be resolved into seven chromatographically distinct species yet could not be resolved from the antiviral activity on either a qualitative or quantitative basis. These results thus provide quantitative molecular evidence to support the concept that IFN gamma can act as a MAF.

Suppression of ACTH-induced steroidogenesis by supernatants from LPS-treated peritoneal exudate macrophages.

The results from a number of clinical and experimental studies have suggested that during endotoxemia, suppression of adrenocortical steroidogenesis may occur. We have examined the possibility that macrophages are the source of a factor that suppresses adrenocortical steroidogenesis. Resident and peptone-elicited peritoneal exudate macrophages (PEM) from C3HeB/FeJ mice were incubated for 4 hr at 37 degrees C in the presence or absence of T cell hybridoma-derived lymphokine (LK) that contained high concentrations of MAF activity (assessed by induction of nonspecific tumoricidal activity in PEM). The LK was removed by rinsing, and fresh medium was added, followed by Salmonella minnesota R595 LPS (final concentration 10 micrograms/ml). After 18 hr at 37 degrees C the PEM supernatants and control medium from flasks without cells were harvested and stored at -20 degrees C. Explanted rabbit adrenocortical cells in 96-well plates were exposed to 30 microliters of PEM supernatant or control medium and ACTH (10 or 100 mU/ml) in a final volume of 120 microliters for 3 consecutive days. The adrenocortical cell supernatants were harvested each day, followed by replenishment of medium, PEM supernatant, and ACTH. Fluorogenic steroid production in wells that received control medium or supernatants from PEM not treated with LPS was normal (0.22 microgram +/- 0.010 (SD) per 5 X 10(4) cells). However, as much as 75 to 95% suppression of steroidogenesis was observed in wells that received supernatants from PEM treated with LK and LPS, compared to 40% suppression in wells that received supernatant from PEM treated with LPS alone. Continued exposure (over 3 days) of adrenocortical cells to supernatants from LPS-treated PEM resulted in progressively decreasing response to ACTH. Comparable suppressive activity was observed in supernatants from LPS-treated bone marrow-derived macrophages. In further experiments, suppression was observed in wells that were pretreated (22 hr) with the appropriate PEM supernatant, and evidence was obtained that the suppressive activity was not due to carry-over LPS. Finally, results from control experiments demonstrated that suppressive PEM supernatants neither inactivate ACTH nor interfere with the assay of fluorogenic steroids. Thus, these results suggest that during endotoxemia, products from LPS-stimulated macrophages may suppress adrenocortical function.

Colony-stimulating factors and regulation of macrophage tumoricidal and microbicidal activities

Conditioned medium from antigen- or mitogen-stimulated spleen cells, lymphokines, contained factors that induced formation of granulocyte and macrophage colonies in cultures of bone marrow cells (CSF). Lymphokines also contained factors that induced macrophage nonspecific tumoricidal activity against fibrosarcoma 1023, antibody-dependent tumoricidal activity against lymphoma 18-8, and antimicrobial activities against amastigotes of the protozoan parasite, Leishmania tropica. The factors that regulated macrophage effector functions, however, were different from those that induced colony formation, and could be distinguished from CSF by Sephadex gel chromatography or heat sensitivity. To further analyze a role for CSF in induction of macrophage effector activities, conditioned medium from several nonlymphoid cell sources (L-929, WEHI-3, and endotoxin-treated lung cells) were assayed for CSF activities and capacity to induce tumoricidal and microbicidal activities. Conditioned medium that contained either macrophages CSF (CSF-1) or the factor that induced formation of both macrophage and granulocyte colonies failed to activate macrophages for effector activities against fibrosarcoma 1023, lymphoma 18-8, and L. tropica amastigotes (either resistance to infection or intracellular destruction). These data suggest that CSF has no direct role in activation of macrophages for tumoricidal and microbicidal activities against these targets.

Macrophage activation for tumor cytotoxicity: induction of macrophage tumoricidal activity by lymphokines from EL-4, a continuous T cell line.

Supernatant culture fluids from a phorbol myristate acetate (PMA) stimulated variant of the murine EL-4 thymoma cell line activated inflammatory macrophages for nonspecific tumoricidal activity in vitro; active supernatants fluids were not directly toxic to tumor target cells in the absence of macrophages. Macrophages treated with culture fluids of unstimulated and concanavalin A-stimulated EL-4 or PMA-stimulated cells derived from any of ten other continuous T cell lines were not cytotoxic. The titer of activity in lymphokines from PMA-stimulated EL-4 was similar to that from antigen-stimulated spleen cell cultures. Spleen cell and EL-4 derived activities were both retained within a dialysis membrane and destroyed by heating at 100 degrees C for 15 min. However, unlike the activity in spleen cell-derived LK, activity in EL-4 culture fluids was stable to dialysis against pH 2 buffer. Further physicochemical characterization of the EL-4 activity showed two different factors that activated macrophages for cytotoxicity: one was indistinguishable from gamma interferon (IFN) (antiviral activity, 50,000 m.w., pI 4.2 to 6.0, pH 2 labile and neutralized by anti-gamma IFN serum); the other was clearly distinct (no antiviral activity, 23,000 m.w., pH 2 stable and unaffected by anti-gamma IFN serum). These results suggest that the EL-4 cell line may produce one or more of the macrophage activation factors present in spleen cell-derived lymphokines.

Induction of antibody-dependent and nonspecific tumor killing in human monocytic leukemia cells by nonlymphocyte factors and phorbol ester

Induction of human cell lines to express properties characteristic of differentiated mononuclear phagocytes was investigated. Phagocytosis and receptors for immunoglobulin Fc and complement were induced or increased to variable extents in histiocytic lymphoma line U937, myeloblast line KG-1, and myelomonocytic line RC-2A. The active agents used were tumor promoter phorbol myristic acetate (PMA), supernatants of human bladder carcinoma line 5637, and lymphokine preparations. The strong induction of these properties concomitant with growth inhibition of the U937 line was suggestive of a terminal differentiation process. Incubation of U937 with these agents for several days induced antibody-dependent cellular cytotoxicity (ADCC) to tumor targets in the monocyte-related line. Factors inducing U937 maturation appear to be distinct from myeloid colony-stimulating activity (CSA) since the former is present in 2-day LK, whereas more than 2 days is required for the production of most CSA. In addition to ADCC, PMA also induced nonspecific tumoricidal activity in U937. These experiments show that (i) the same monocyte cell type can mediate both nonspecific and antibody-dependent lysis of tumor targets; (ii) exogenous agents and endogenous factors other than lymphokine preparations can induce ADCC; (iii) the U937 line can be used as a model for detecting nonlymphoid as well as lymphoid factors stimulating differentiation or maturation of human monocytic cells.

Macrophages in resistance to rickettsial infection: strains of mice susceptible to the lethal effects of Rickettsia akari show defective macrophage Rickettsicidal activity in vitro.

Activation of macrophages was assessed in strains of mice inoculated intraperitoneally with 1,000 times the 50% lethal dose of Rickettsia akari. Macrophages from mice resistant to R. akari infection (C3H/HeN, C57BL/10J, and BALB/cN) were nonspecifically tumoricidal 2 to 4 days after rickettsial inoculation. Moreover, these macrophages were microbial for R. akari in vitro; cells were resistant to infection with the bacterium and were capable of killing intracellular rickettsiae. In contrast, macrophages from strains of mice susceptible to R. akari (C3H/HeJ, C57BL/10SnCR, and A/J) failed to develop nonspecific tumoricidal activity over the course of lethal disease and became infected with R. akari in vivo within 2 days of rickettsial inoculation. Macrophages from uninfected mice of strains susceptible to R. akari also could not be activated for rickettsicidal or tumoricidal activities by treatment with macrophage-activating agents (Mycobacterium bovis BCG) in vivo or by treatment with lymphokines in vitro.

Macrophages as effector cells of protective immunity in murine schistosomiasis. II. Killing of newly transformed schistosomula in vitro by macrophages activated as a consequence of Schistosoma mansoni infection.

Plastic adherent peritoneal cells from Schistosoma mansoni-infected mice have previously been shown to exhibit nonspecific tumoricidal activity in vitro. In this report we show that these same cell populations kill significant numbers of skin-stage schistosomula in vitro in the absence of added antibody. Larval killing by these activated cells could be enhanced by the use of suspension rather than monolayer cultures and by addition of heat-inactivated immune mouse serum to the cultures. Adherence of cells to schistosomula was also enhanced under the same conditions, suggesting that cell binding to the larvae might be critical in the development or expression of microbicidal activity. In support of this hypothesis, the same level of enhancement of cell binding and larval damage was observed upon substitution of concanavalin A for immune mouse serum. Killing of schistosomula appeared to be mediated solely by activated macrophages in the peritoneal cell suspensions from S. mansoni-infected mice, because partially purified preparations of eosinophils were virtually inactive in these assays. Likewise, inflammatory macrophages from uninfected mice were unable to kill schistosomula under the same conditions, emphasizing the importance of activation in the development of killing capability. The finding that macrophages activated as a consequence of S. mansoni infection are able to kill larval schistosomes in vitro suggests that these cells may play a role in concomitant immunity to schistosomiasis in vivo.

Macrophages as effector cells of protective immunity in murine schistosomiasis: I. Activation of peritoneal macrophages during natural infection

Mice infected with Schistosoma mansoni develop a dramatic (five- to eightfold) increase in numbers of peritoneal leukocytes, and approximately 65% of these cells are macrophages. By several biochemical and cytochemical criteria, these cells were comparable to resident peritoneal macrophages of normal mice. However, macrophages from schistosome-infected mice exhibited significant nonspecific tumoricidal activity in vitro, a function associated with immunologically activated cells. The time course for development of activated macrophages in the peritoneal cavity was dependent upon the route of infection. Cytotoxic cells were present in the peritoneal cavity by 3 weeks after intraperitoneal infection, but were not evident until several weeks later in animals infected percutaneously, subcutaneously, or intravenously. However, by 3 weeks after subcutaneous infection, tumoricidal macrophages appeared in the peritoneal cavity after intraperitoneal challenge with soluble schistosome antigens. Macrophage activation was independent of the development of egg granulomas, since tumoricidal cells could be found prior to the onset of egg production and were also present in mice infected with only male worms. Development of activated macrophages in these instances is thus consistent with previous observations on induction of T lymphocyte reactivity toward schistosomula. Since other manipulations known to activate macrophages have been shown to induce partial resistance to schistosome infection, the finding that macrophage activation results from primary S. mansoni infection itself suggests that these cells may play a major role in acquired immunity to this parasite.