Enhancement of selective tumor cell binding by activated murine macrophages in response to phorbol myristate acetate

The fundamental biology of how stable cell-cell bonds develop between activated macrophages and tumor cells, although essential to lysis of the neoplastic targets, remains poorly understood. To investigate whether this phenomenon could be pharmacologically manipulated, we analyzed the effect of phorbol diesters on tumor cell binding by macrophages. Activated murine peritoneal macrophages, treated in vitro with as little as 1 ng/ml of phorbol myristate acetate (PMA), bound significantly more tumor cells than did untreated macrophages. The effect was induced rapidly by PMA (i.e., maximum enhancement was seen within 15 min) and resulted in an average approximately twofold increase in the number of targets bound. The interaction between PMA-treated activated macrophages and tumor cells was completed much more rapidly than by untreated macrophages. The enhanced binding was seen only in macrophages treated with biologically active phorbol esters. Only the selective interaction between activated macrophages and tumor cells was affected (i.e., PMA treatment had no effect on nonselective interactions between activated macrophages and non-neoplastic targets or between nonactivated macrophages and any type of target). Pretreatment of activated macrophages with PMA apparently altered the requirements for microfilaments and microtubules in establishing binding, because cytochalasin B and colchicine, which inhibited control binding, as well as phagocytosis, had no effect on PMA-enhanced binding. PMA treatment did not alter energy requirements for binding, however, because low temperature (4 degrees C) or inhibitors of glycolysis and oxidative phosphorylation blocked both control and PMA-enhanced binding. The enhancement of binding apparently was not due to large quantities of secreted oxygen metabolites but did correlate closely with increased spreading and surface area of the macrophages. PMA treatment resulted in enhanced expression of trypsin-sensitive tumor-cell binding sites on the macrophage surface. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of macrophage membrane proteins labeled with 125I by the lactoperoxidase method revealed at least four trypsin-sensitive cell surface proteins that were re-expressed after PMA treatment. The data suggest that rearrangement and/or induced expression of surface binding sites may be an important step in the binding of tumor cells and indicate that PMA is a useful pharmacologic probe in dissecting the establishment of such binding into discrete steps.     

Quantification of the strength of cell-cell adhesion: the capture of tumor cells by activated murine macrophages proceeds through two distinct stages

The binding of tumor cells by macrophages activated with Bacillus Calmette-Guerin is a necessary step toward destruction of those cells. Although several characteristics of the interaction have been defined, little is known of how the actual binding process develops. We used a technique to quantify the forces required to disrupt cell-cell interactions. Over a range of applied relative centrifugal forces, the majority of targets that bound to the activated macrophages fell on two distinct plateaus. Approximately 90% of added targets were bound to the monolayers of macrophages over the range of 1 to 100 X G; 25 to 30% remained bound from 1200 X G to 1500 X G. Two strengths of binding, termed weak and strong binding, respectively, were thus defined on the basis of these curves. Strong binding developed only between activated macrophages and tumor cells. By contrast, weak interactions occurred between either activated or nonactivated macrophages and neoplastic or non-neoplastic target cells. The strong binding required time (60 to 90 min), metabolic activity by the macrophages, and trypsin-sensitive surface structures on the macrophages for development, whereas the weak interaction occurred rapidly and required none of these. Additional evidence indicated the weak binding developed into strong when activated macrophages bound neoplastic cells. This stabilization increased the strength of force to separate tumor cells from the macrophages at least approximately 15 fold (i.e., from approximately 16 mu dynes/cell to approximately 240 mu dynes/cell). Of note, the development of strong binding of antibody-coated targets had distinct requirements for establishment. Taken together, the data suggest the stabilization of binding (i.e., the development of weak into strong binding) leading to effective cell-cell interaction is a complex and dynamic process that may vary depending upon the recognition system involved.     

Increased binding of tumor cells by macrophages activated in vitro with lymphocyte mediators.

Following incubation in vitro with lymphocyte mediators, activated macrophages become capable of binding more tumor cells than nonactivated macrophages. Increased binding occurs rapidly (within 1 hr), does not require the presence of serum in the medium, and is inhibited by treatment with trypsin. The increased binding by activated macrophages is quantitatively selective for tumor cells. Incubation with lymphocyte mediators of cell types other than macrophages does not increase the binding of tumor cells to such monolayers. These results indicate that the binding of tumor cells by activated macrophages results from the stimulation of a specific macrophage function during the process of macrophage activation.     

Macrophage binding of cells resistant and sensitive to contact-dependent cytotoxicity

We compared macrophage binding and killing of F5b cells to the binding and killing of P815 mastocytoma cells and to several other nontransformed and transformed cell lines. Formalin fixation of elicited or activated macrophages did not affect binding of F5b or 3T3 cells but did abrogate binding of P815 cells. However, formalin fixation abrogated resident macrophage binding of F5b and 3T3 cells. Therefore, depending on the type of macrophage or target cell, formalin fixation may affect binding. Only the binding of P815 cells was dependent upon activation; macrophage binding of target cells F5b and 3T3 was not. Even though macrophages bound F5b and 3T3 cells, macrophages only mediated contact-dependent cytotoxicity against F5b cells. Macrophages did not kill 3T3 cells. Experiments also compared macrophage binding and killing of the uv-light-induced tumor cell lines 1422, 2237, and 2237a46. Only the cell line 2237a46 was susceptible to contact-dependent killing. Both 1422 and 2237 cells were resistant. In contrast, cell lines 2237a46 and 1422 were bound by activated macrophages while 2237 cells were bound poorly.     

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

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

The unique killing of embryo-derived teratocarcinoma cells by nonactivated murine macrophages is not due to a lack of H-2 antigen expression.

It is well documented that activated macrophages, but not nonactivated ones, kill tumor cells in vitro without damaging normal cells. We, however, have previously shown that embryo-derived teratocarcinoma cells (F9, P19, PCC4) are efficiently killed by nonactivated macrophages as well as by activated ones. Whereas other tumor cells are killed extracellularly by macrophages, we found that F9 teratocarcinoma cells are phagocytosed alive by macrophages and subsequently killed intracellularly by a process dependent on intact lysosomal function. Neither the H-2 antigens nor the mRNAs for the alpha-chain and beta 2-microglobulin are detectable in embryo-derived teratocarcinoma cells. An obvious explanation for this unique killing is that the nonactivated macrophages recognize and kill these cells due to their lack of class I MHC antigen expression, assuming that class I MHC gene products on the target cells switch off the cytolytic machinery of nonactivated macrophages. Our present findings demonstrate that there is no correlation between H-2 antigen expression on tumor cells and their susceptibility to killing by macrophages. Retinoic acid-differentiated F9 cells and P19 cells expressing H-2 antigen after exposure to MAF (IFN-gamma) were sensitive to the killing by nonactivated macrophages. Hybrids that arose from fusion of P19 teratocarcinoma cells with embryonal normal fibroblasts (C57BL/6), which displayed the morphology of embryonal carcinoma stem cells and expressed H-2 antigens, were also sensitive to the killing by nonactivated macrophages. On the other hand, the H-2-negative testicular 402AX teratocarcinoma cells and K1735P melanoma cells were both resistant to the killing by nonactivated macrophages. We concluded that the unique killing of embryo-derived teratocarcinoma cells by nonactivated murine macrophages is not related to a lack of H-2 antigen expression.     

Mechanisms of tumor cell capture by activated macrophages: evidence for involvement of lymphocyte function-associated (LFA)-1 antigen

The lymphocyte function-associated (LFA)-1 molecule is expressed on certain populations of macrophages that have an augmented capacity to capture tumor cells. Accordingly, we analyzed the role of LFA-1 in the establishment of such cell-cell interactions. F(ab')2 fragments of the M17/4, anti-LFA-1 monoclonal antibody (MAb) inhibited the interaction between activated macrophages and tumor cells by up to 80% in a dose-dependent manner. The anti-LFA-1 MAb reduced (between 55 to 79%) the number of P815, LSTRA, or EL-4 tumor cells bound to trypsin-sensitive structures on bacillus Calmette Guerin activated macrophages. The inhibition appeared selective, because a F(ab')2 fragment of anti-Mac-1 did not inhibit such binding. Inhibition of tumor cell capture could be observed as soon as 15 min after the onset of the cell-cell interaction between activated macrophages and tumor cells. Optimal inhibition occurred when both tumor targets and macrophages were precoated with the MAb. Although P815, LSTRA, EL-4, and BW5147 tumor cells all expressed LFA-1, only the first three but not BW5147 cells were bound by activated macrophages. Furthermore, endotoxin-pulsed macrophages elicited by thioglycollate broth expressed the LFA-1 antigen but did not exhibit selective tumor cell capture. Finally, anti-LFA-1 inhibited the development of weak into strong binding. Taken together, the results suggest that LFA-1 molecules can participate in the interaction between activated macrophages and neoplastic cells.     

The cytolytic function of mononuclear phagocytes. II. Factors that determine the binding and lysis of tumor cells by activated macrophages

The effects of various modifiers upon the interaction of LPS- and BCG-activated macrophages with cells of mastocytoma P815 have been investigated. The efficiency of binding and lysis of the tumor cells is to a great extent determined by activation of the effector-cells, expression of the trypsin-sensitive receptors on the surface of macrophages, and by the type of target-cells. Introduction into the analytical system (effector-target) of unlabeled tumor cells or membrane preparations obtained from them inhibits substantially both binding and lytic activity of cytotoxic macrophages. If nontransformed cells or their membranes are applied, no significant changes in the investigated processes can be detected. Trypsinization of tumor cells as well as of activated but not resident macrophages modifies considerably the interaction of effectors with targets. The quantity of tumor cells bound with macrophages does not depend on the fact, which of the partners is subject to trypsinization, but it is much less than that of target-cells bound in the control. The incubation of activated macrophages with actinomycin D results in a substantial suppression of their lytic activity, whereas treatment of tumor cells with this inhibitor of protein synthesis leads to a considerable decrease in stability of the targets against lytic activity of the factor activated by effectors. The obtained data reveal the ways of selective binding and effective lysis of transformed targets by activated macrophages.     

Activated macrophages inhibit human cytotrophoblast invasiveness in vitro

Pre-eclampsia is associated with inadequate cytotrophoblast invasion and remodeling of the uterine spiral arterioles, as well as by an aberrant maternal immune response. This study determined the effect of activated macrophages and one of its products, tumor necrosis factor (TNF)-alpha, on cytotrophoblast invasiveness. Coculture with human lipopolysaccharide-activated macrophages decreased the ability of immortalized HTR-8/ SVneo human trophoblast cells to invade through reconstituted extracellular matrix (P < 0.05). This effect of activated macrophages on trophoblast invasiveness was paralleled by abrogation of a 55-kDa caseinolytic activity corresponding to prourokinase plasminogen activator (pro-uPA) and an increased secretion of plasminogen activator inhibitor 1 (PAI1), as determined by gel zymography and ELISA, respectively. Coculture with nonactivated macrophages did not significantly affect trophoblast invasiveness or pro-uPA and PAI1 secretion. Activated macrophages secreted detectable levels of TNF, and administration of exogenous TNF significantly decreased trophoblast invasiveness (P < 0.05), increased the secretion of PAI1 (P < 0.01), and completely inhibited the pro-uPA-associated caseinolytic activity by binding to the TNF receptor 1. Moreover, addition of up to 10 ng/ml of TNF did not increase the rate of apoptosis in HTR-8/SVneo cells. Finally, the increased secretion of PAI1 by trophoblast cells cocultured with activated macrophages was significantly inhibited when a neutralizing anti-TNF antibody was added to the cocultures. These results suggest that the aberrant presence of activated macrophages around uterine vessels may contribute to inadequate trophoblast invasion and remodeling of the uterine spiral arterioles. Thus, the presence of activated macrophages may be important in the etiology of pre-eclampsia.     

The CD44 Receptor of Lymphoma Cells: Structure-Function Relationships and Mechanism of Activation

Migration of some tumor cells, and their lodgment in target organs, is dependent on the activation of cell surface CD44 receptor, usually detected by its ability to bind hyaluronic acid (HA) or other ligands. In an attempt to reveal the mechanism of tumor cell CD44 activation, we compared the physical and chemical properties of CD44 in nonactivated LB cell lymphoma with those in phorbol 12-myristate 13-acetate (PMA)-activated LB cells and of an LB cell subline (designated HA9) expressing constitutively-active CD44. In contrast to nonactivated LB cells, PMA-activated LB cells and HA9 cells displayed a CD44-dependent ability to bind HA. The ability of activated cell CD44 to bind HA was not dependent on microfilament or microtubule integrity or on changes in CD44 mobility on the membrane plane, indicating that the CD44 activation status is not associated with cytoskeleton function. Aside from the increased expression of CD44 on the surface of PMA-activated LB cells and HA9 cells, qualitative differences between the CD44 of nonactivated and activated LB cells were also detected: the CD44 of the activated lymphoma was (i) larger in molecular size, (ii) displayed a broader CD44 isoform repertoire, including a CD44 variant that binds HA, and (iii) its glycoprotein contained less sialic acid. Indeed, after removal of sialic acid from their cell surface by neuraminidase, LB cells acquired the ability to bind HA. However, a reduced dose of neuraminidase did not confer HA binding on LB cells, unless they were also activated by a low concentration of PMA, which by itself was ineffective. Similarly, under suboptimal conditions, a synergistic effect was obtained with tunicamycin and PMA: each one alone was ineffective but in combination they induced the acquisition of HA binding by the lymphoma cells, while their CD44 expression was not enhanced. Unveiling of the activation mechanism of CD44, by exposing the cells to PMA stimulation or to deglycosylation, is not only academically important, but it also has practical implications, as activated CD44 may be involved in the support of tumor progression.     

The CD44 receptor of lymphoma cells: structure-function relationships and mechanism of activation

Migration of some tumor cells, and their lodgment in target organs, is dependent on the activation of cell surface CD44 receptor, usually detected by its ability to bind hyaluronic acid (HA) or other ligands. In an attempt to reveal the mechanism of tumor cell CD44 activation, we compared the physical and chemical properties of CD44 in nonactivated LB cell lymphoma with those in phorbol 12-myristate 13-acetate (PMA)-activated LB cells and of an LB cell subline (designated HA9) expressing constitutively-active CD44. In contrast to nonactivated LB cells, PMA-activated LB cells and HA9 cells displayed a CD44-dependent ability to bind HA. The ability of activated cell CD44 to bind HA was not dependent on microfilament or microtubule integrity or on changes in CD44 mobility on the membrane plane, indicating that the CD44 activation status is not associated with cytoskeleton function. Aside from the increased expression of CD44 on the surface of PMA-activated LB cells and HA9 cells, qualitative differences between the CD44 of nonactivated and activated LB cells were also detected: the CD44 of the activated lymphoma was (i) larger in molecular size, (ii) displayed a broader CD44 isoform repertoire, including a CD44 variant that binds HA, and (iii) its glycoprotein contained less sialic acid. Indeed, after removal of sialic acid from their cell surface by neuraminidase, LB cells acquired the ability to bind HA. However, a reduced dose of neuraminidase did not confer HA binding on LB cells, unless they were also activated by a low concentration of PMA, which by itself was ineffective. Similarly, under suboptimal conditions, a synergistic effect was obtained with tunicamycin and PMA: each one alone was ineffective but in combination they induced the acquisition of HA binding by the lymphoma cells, while their CD44 expression was not enhanced. Unveiling of the activation mechanism of CD44, by exposing the cells to PMA stimulation or to deglycosylation, is not only academically important, but it also has practical implications, as activated CD44 may be involved in the support of tumor progression.     

Sensitivity to macrophage-mediated cytostasis is cell cycle dependent

We have examined the sensitivity of proliferating lymphoid cells in different phases of the cell cycle to macrophage-mediated cytostatic activity. These studies evaluated the ability of target cells enriched in individual cell cycle phases to pass into the next phase during brief (2–6 hr) periods of coculture with activated or nonactivated peritoneal macrophages. Both normal (concanavalin A-stimulated spleen cells) and neoplastic (Gross virus-induced thymic lymphoma) cells were analyzed. Spleen cells or lymphoma cells were first separated by centrifugal elutriation into populations highly enriched for G₁, S, or G₂/M phases of the cell cycle and cultured in the presence of nonactivated or activated macrophages for periods of 2, 4, or 6 hr. The cellular DNA content of recovered nonadherent target cells was then analyzed by flow cytometry after staining with propidium iodide. Macrophage contamination of target cell populations was insignificant under these conditions. Nonactivated macrophages did not affect target cell cycle traverse when compared with target cells cultured alone. Activated macrophage mediated cytostatic activity resulted in complete block of the transition of cells in G₁ phase into S phase and of the further accumulation of DNA by cells in early S phase. Cells already in mid to late S phase were able to continue DNA replication at rates nearly equivalent to control cells. There was no inhibition of the passage of cells through G₂ or mitosis. These effects were seen by as early as 2 hr of macrophage-target cell coculture and both normal and neoplastic cells exhibited identical patterns of cell cycle phase sensitivity.     

Induction of macrophage-mediated cytolysis of neoplastic cells by mycoplasmas

Unexpected cytolysis was encountered when nonactivated murine peritoneal macrophages were cultured with [³H]TdR-prelabeled syngeneic or allogeneic tumor cells at a 10:1 ratio. The level of specific cytolysis reached 70% within 48 hr of cocultivation. Similar killing was observed whether the macrophages were derived from untreated, thioglycollate-treated, or germ-free mice. Cytolytic activity was also demonstrated when bone marrow-derived or peritoneal macrophages from 9- and 5-day in vitro cultures, respectively, were employed rather than freshly harvested peritoneal macrophages. Thus, the macrophage-mediated killing was neither the result of in vivo preactivation nor a consequence of the presence of lymphocytes in the assay. Moreover, macrophages derived from different strains caused similar effects. Our study revealed that the neoplastic target cell cultures susceptible to cytolysis by nonactivated macrophages were contaminated with mycoplasma. A mycoplasma was isolated from the supernatant of a culture of the A9HT fibrosarcoma line, identified as Mycoplasma orale, and cultivated. Addition of viable mycoplasma from that isolate to mixed cultures of thioglycollate-elicited macrophages and [³H]TdR-prelabeled mycoplasma-free target cells resulted in specific cytolysis of transformed A9 cells, but not of normal mouse fibroblasts. The level of macrophage-dependent cytolysis correlated with the number of viable mycoplasma cells added and was higher than that attained by activation with LPS at optimal concentration. Similar specific cytolysis was observed with heat-killed mycoplasmas. Our results demonstrate that mycoplasmas may cause selective macrophage-mediated cytolysis of neoplastic but not of normal target cells, perhaps via activation of the macrophages. It is suggested that undetected infection of experimental systems by mycoplasmas may account for some reports on lysis of neoplastic cells by nonactivated macrophages.     

Tumor cell killing by macrophages activated in vitro with lymphocyte mediators. II. Inhibition by inhibitors of protein synthesis.

The effect of inhibitors of protein synthesis on the killing of tumor cells by in vitro activated macrophages was determined. Cytotoxicity was inhibited by concentrations of puromycin, pactamycin, and actinomycin D that almost completely inhibited protein synthesis by guinea pig macrophages, but not by concentrations of drug that inhibited protein synthesis by only ± 50%. Cytotoxicity was inhibited when the effector macrophages were pretreated with the metabolic inhibitors, but not when the drugs were added 30 to 60 min after the initiation of the reaction. Pretreatment with puromycin or pactamycin also markedly inhibited the binding of tumor cells by mediator activated macrophages. These results are consistent with the hypothesis that one possible mechanism by which inhibitors of protein synthesis inhibit macrophage mediated cytotoxicity is by inhibiting close contact between effector and target cells. The finding that pretreatment of activated macrophages with trypsin also inhibits tumor cell killing suggests that protein synthesis may be necessary to maintain an adequate number of “recognition structures” on the macrophage membrane, structures that mediate the initial contact between the activated macrophage and the target tumor.     

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

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

Monoclonal antibodies directed against mouse macrophages in different stages of activation for tumor cytotoxicity

Three rat monoclonal antibodies against mouse peritoneal macrophages in different stages of activation were produced and characterized. One of these (AcM.1) bound to activated macrophages induced by pyran and Corynebacterium parvum, but not to resident and thioglycollate medium- (TGC) or proteose peptone- (PP) elicited macrophages. On the contrary, the antigen identified by MM9 monoclonal antibody was expressed only on resident and TGC- or PP-elicited macrophages. WE15 monoclonal antibody, on the other hand, reacted with all of the macrophages described above. In the assay for function, AcM.1 and WE15 monoclonal antibodies in the presence of complement (C) abolished the capacity of activated macrophages induced by pyran or C. parvum but not the capacity of killer T cells and natural killer (NK) cells to kill tumor target cells. On the other hand, MM9 and anti-Thy-1.2 monoclonal antibodies in the presence of C, as expected, did not affect the cytotoxicity of activated macrophages. However, none of the four monoclonal antibodies in the absence of C had any blocking effect on macrophage-mediated cytotoxicity. AcM.1 antibody reacted with two polypeptides with m.w. of 70,000 and 45,000 on pyran-activated macrophages; however, the antigens recognized by WE15 and MM9 have not been determined yet. These results indicate that the three rat monoclonal antibodies define different antigens present on macrophages at different stages of activation for tumor cytotoxicity, and that these antibodies should prove to be useful probes for analyzing the mechanism of activation of macrophages for tumor cytotoxicity.     

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

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

Differential HIV-1 Endocytosis and Susceptibility to Virus Infection in Human Macrophages Correlate with Cell Activation Status

HIV-1 is an enveloped virus that enters target cells by fusion either directly at the plasma membrane or at the endosomal membrane. The latter mechanism follows a rapid engulfment of HIV-1 after its receptor engagement at the cell surface, and its scale depends on cellular endocytosis/degradation rates and virus fusion kinetics. HIV-1 has recently been shown to exploit a novel Pak1-dependent macropinocytosis mechanism as a way to productively infect macrophages. However, macrophages are highly heterogeneous cells that can adapt functionally to their changing environment, and their endosomal/lysosomal pathway is highly regulated upon cell activation. These changes might impact the ability of HIV-1 to exploit endocytosis as a way to productively infect macrophages. In this study, we compared HIV-1 endocytosis/degradation rates in nonactivated, M1-activated, and M2a-activated monocyte-derived macrophages (MDMs). We found that the rate of HIV-1 endocytosis was increased in M1-activated but decreased in M2a-activated MDMs. However, both M1 and M2a activations of MDMs led specifically to a greater clathrin-mediated endocytosis of HIV-1, which was independent of CD4 and CCR5 binding. Furthermore, clathrin-mediated endocytosis is unlikely to result in productive HIV-1 infection, given that it leads to increased viral degradation. Therefore, we suggest that viral fusion following endocytosis is restricted in activated macrophages.     

Macrophage cytotoxicity against schistosomula of Schistosoma mansoni involves arginine-dependent production of reactive nitrogen intermediates

Lymphokine (LK)-activated macrophages are cytotoxic for multicellular larvae of the helminth parasite Schistosoma mansoni. Macrophage-mediated larval killing was found to be arginine dependent, as indicated by inhibition in the presence of exogenous arginase or the competitive inhibitor NG-monomethyl-L-arginine. Culture supernatant fluids from the larvicidal LK-activated macrophages contained nitrite, a product of activated macrophages derived by oxidation of arginine and implicated in the antitumor and antimicrobial effector function of these cells. Nitrite was not detectable in supernatant fluids obtained from nonactivated macrophages or from macrophages stimulated with LK in the presence of arginase or NG-monomethyl-L-arginine. Addition of excess iron or the reductant sodium dithionite to LK-activated macrophage cultures also inhibited larval killing in vitro, under conditions that have been shown by others to stabilize the activity of iron-containing enzymes involved in respiration. Nitrite production was not decreased under these conditions. These observations are consistent with the hypothesis that macrophage-mediated schistosomulum killing is caused, at least in part, by a mechanism proposed for tumor cytotoxicity, whereby production of reactive nitrogen intermediates triggers iron loss from critical target cell enzymes leading to lethal metabolic inhibition. In accordance, schistosomula were shown to be killed by inhibitors of mitochondrial respiration.     

Differences in the tumoricidal activation of rat and mouse macrophages by endotoxins

Conventional and specific pathogen-free rat resident peritoneal macrophages were lytic to tumor cells in the presence of endotoxins even when not elicited or not stimulated in vivo or in vitro. In contrast, conventional mouse resident peritoneal macrophages were not cytolytic in the presence of endotoxins. The induction by endotoxins of rat macrophage-mediated cytolysis was only obtained after the binding of tumor cells by macrophages. Rat resident peritoneal macrophages bound faster and stronger to tumor cells than mouse resident peritoneal macrophages. These differences in binding could explain the species differences in the tumoricidal response to endotoxins.