A flow cytometric assay to quantify in vivo bacterial uptake by alveolar macrophages

Our laboratory has developed a flow cytometric assay to quantify alveolar macrophage (MФ) phagocytosis of bacteria within a live animal. MФs collected by bronchoalveolar lavage from rats infected transtracheally with Syto 9-labeled bacteria are fluorescently labeled for identification and analyzed by flow cytometry to quantify their bacterial uptake.     

CD47 promotes both phosphatidylserine-independent and phosphatidylserine-dependent phagocytosis of apoptotic murine thymocytes by non-activated macrophages

The ubiquitously expressed cell surface glycoprotein CD47 on host cells can inhibit phagocytosis of unopsonized or opsonized viable host target cells. Here we studied the role of target cell CD47 in macrophage uptake of viable or apoptotic murine thymocytes. As expected, IgG-opsonized viable CD47^−/− thymocytes were taken up more efficiently than equally opsonized Wt thymocytes. However IgG-opsonized apoptotic thymocytes from Wt and CD47^−/− mice were taken up equally. Although uptake of apoptotic thymocytes by non-activated bone marrow-derived macrophages was phosphatidylserine (PS)-independent, while uptake by non-activated resident peritoneal macrophages was PS-dependent, both macrophage populations showed a reduced uptake of non-opsonized apoptotic CD47^−/− thymocytes, as compared with the uptake of apoptotic Wt thymocytes. This difference was only seen with non-activated macrophages, and not with β-1,3-glucan-activated macrophages. CD47 promoted binding of thymocytes to macrophages, which did not require F-actin polymerization. CD47 became clustered on apoptotic thymocytes, both co-localized with or separated from, clustered PS and cholesterol-rich GM-1 domains. Thus, CD47 does not inhibit, but rather support, both PS-independent and PS-dependent uptake of apoptotic cells in the murine system. This mechanism only comes into play in non-activated macrophages.     

Externalization and recognition by macrophages of large subunit of eukaryotic translation initiation factor 3 in apoptotic cells

We previously isolated a monoclonal antibody named PH2 that inhibits phosphatidylserine-mediated phagocytosis of apoptotic cells by macrophages. We report here the identification of the cognate antigen. A protein bound by PH2 in Western blotting was identified as the 170-kDa subunit of eukaryotic translation initiation factor 3 (eIF3 p170/eIF3a). When eIF3a was expressed in a culture cell line as a protein fused to green fluorescence protein, the fusion protein was detected at the cell surface only after the induction of apoptosis. The same phenomenon was seen when the localization of endogenous eIF3a was determined using anti-eIF3a antibody, and eIF3a seemed to be partially degraded during apoptosis. Furthermore, bacterially expressed N-terminal half of eIF3a fused to glutathione S-transferase bound to the surface of macrophages and inhibited phagocytosis of apoptotic cells by macrophages when it was added to phagocytosis reactions. These results collectively suggest that eIF3a translocates to the cell surface upon apoptosis, probably after partial degradation, and bridges apoptotic cells and macrophages to enhance phagocytosis.     

Spore diffusate isolated from some strains of Aspergillus fumigatus inhibits phagocytosis by murine alveolar macrophages

Aspergillus fumigatus is a ubiquitous fungus that grows in decaying organic matter. It can cause disease in both immunodeficient and immunocompetent patients by using virulence factors to escape the host defenses. Some of these factors, such as a diffusate, released from the spores of A. fumigatus, have previously been described. This diffusate was demonstrated to inhibit oxidative burst and phagocytosis of coated red blood cells. The present study has shown that this substance can inhibit the phagocytosis of A. fumigatus spores by murine alveolar macrophages (MALU) and evaluated the action of this substance. We quantified phagocytosis by MALU cells with and without diffusate and evaluated the inhibition of phagocytosis by testing diffusates from different strains. We conclude that the spore diffusate of some strains of A. fumigatus can reversibly decrease the ability of alveolar macrophages to ingest A. fumigatus spores.     

Signaling via macrophage G2A enhances efferocytosis of dying neutrophils by augmentation of Rac activity

Phosphatidylserine (PS) and oxidized PS species have been identified as key ligands on apoptotic cells important for their recognition and removal (efferocytosis) by phagocytes, a requisite step for resolution of inflammation. We have recently demonstrated that lysophosphatidylserine (lyso-PS) generated and retained on neutrophils following short term activation of the NADPH oxidase in vitro and in vivo enhanced their clearance via signaling through the macrophage G-protein-coupled receptor G2A. Here, we investigated the signaling pathway downstream of G2A. Lyso-PS, either made endogenously in apoptosing neutrophils or supplied exogenously in liposomes along with lyso-PS(neg) apoptotic cells, signaled to macrophages in a G2A-dependent manner for their enhanced production of prostaglandin E2 (PGE2) via a calcium-dependent cytosolic phospholipase A2/cyclooxygenase-mediated mechanism. Subsequent signaling by PGE2 via EP2 receptors activated macrophage adenylyl cyclase and protein kinase A. These events, in turn, culminated in enhanced activity of Rac1, resulting in an increase in both the numbers of macrophages efferocytosing apoptotic cells and the numbers of cells ingested per macrophage. These data were surprising in light of previous reports demonstrating that signaling by PGE2 and adenylyl cyclase activation are associated with macrophage deactivation and inhibition of apoptotic cell uptake. Further investigation revealed that the impact of this pathway, either the enhancement or inhibition of efferocytosis, was exquisitely sensitive to concentration effects of these intermediaries. Together, these data support the hypothesis that lyso-PS presented on the surface of activated and dying neutrophils provides a tightly controlled, proresolution signal for high capacity clearance of neutrophils in acute inflammation.     

Phagocytic clearance of apoptotic neurons by Microglia/Brain macrophages in vitro: involvement of lectin-, integrin-, and phosphatidylserine-mediated recognition

Microglia, the tissue macrophages of the brain, play a crucial role in recognition and phagocytic removal of apoptotic neurons. The microglial receptors for recognition of apoptotic neurons are not yet characterized. Here we established a co-culture model of primary microglia and cerebellar granule neurons to examine the receptor systems involved in recognition/uptake of apoptotic neurons. Treatment with 100 microM S-nitrosocysteine induced apoptosis of cerebellar neurons as indicated by nuclear condensation and phosphatidylserine exposure to the exoplasmic leaflet of the plasma membrane. Microglial cells were added to neurons 2 h after apoptosis induction and co-cultured for 6 h in the presence of ligands that inhibit recognition by binding to respective receptors. Binding/phagocytosis was determined after combined 4', 6-diamidino-2-phenylindole/propidium iodide (for apoptotic/necrotic neurons) and lectin staining (for microglia). Uptake of apoptotic neurons was reduced by N-acetylglucosamine or galactose, suggesting that recognition involves asialoglycoprotein-like lectins. Furthermore, the inhibition of microglial binding/uptake of apoptotic neurons by RGDS peptide suggests a role of microglial vitronectin receptor. As microglia selectively bind lipid vesicles enriched in phosphatidylserine and O-phospho-L-serine interfered with the uptake of apoptotic neurons, an involvement of phosphatidylserine receptor is rather likely. Apoptotic neurons do not release soluble signals that serve to attract or activate microglia. Collectively, these results suggest that apoptotic neurons generate a complex surface signal recognized by different receptor systems on microglia.     

Programmed cell clearance: Molecular regulation of the elimination of apoptotic cell corpses and its role in the resolution of inflammation

Programmed cell clearance is a physiological process of elimination of apoptotic cell corpses. Recent studies have disclosed several ligand-receptor interactions that dictate the recognition or non-recognition of cells by macrophages and other phagocytes. The externalization of the anionic phospholipid, phosphatidylserine is effectively recognized by specific receptors on professional phagocytes and facilitates the clearance of apoptotic cells. Macrophage disposal of cells at sites of inflammation is believed to play an important role in the resolution of the inflammatory process, and recent studies have suggested a role for the NADPH oxidase in the process of macrophage elimination of activated neutrophils. The present review will focus on the molecular regulation of programmed cell clearance, and discuss the role of cell elimination in the resolution of inflammation.     

Effect of cigarette smoke and dexamethasone on Hsp72 system of alveolar epithelial cells

Smoking is the leading risk factor of chronic obstructive pulmonary disease (COPD) and lung cancer. Corticosteroids are abundantly used in these patients; however, the interaction of smoking and steroid treatment is not fully understood. Heat shock proteins (Hsps) play a central role in the maintenance of cell integrity, apoptosis and cellular steroid action. To better understand cigarette smoke-steroid interaction, we examined the effect of cigarette smoke extract (CSE) and/or dexamethasone (DEX) on changes of intracellular heat shock protein-72 (Hsp72) in lung cells. Alveolar epithelial cells (A549) were exposed to increasing doses (0; 0.1; 1; and 10 μM/μl) of DEX in the medium in the absence(C) and presence of CSE. Apoptosis, necrosis, Hsp72 messenger-ribonucleic acid (mRNA) and protein expression of cells were measured, and the role of Hsp72 on steroid effect examined. CSE reduced the number of viable cells by significantly increasing the number of apoptotic and necrotic cells. DEX dose-dependently decreased the ratio of apoptosis when CSE was administered, without change in necrosis. CSE − DEX co-treatment dose-dependently increased Hsp72 mRNA and protein expression, with the highest level measured in CSE + DEX (10) cells, while significantly lower levels were noted in all respective C groups. Pretreatment with Hsp72 silencing RNA confirmed that increased survival observed following DEX administration in CSE-treated cells was mainly mediated via the Hsp72 system. CSE significantly decreases cell survival by inducing apoptosis and necrosis. DEX significantly increases Hsp72 mRNA and protein expression only in the presence of CSE resulting in increased cellular protection and survival. DEX exerts its cell protective effects by decreasing apoptotic cell death via the Hsp72 system in CSE-treated alveolar epithelial cells.     

Adherence of Mycoplasma pneumoniae to human alveolar macrophages

Abstract The human pathogen Mycoplasma pneumoniae causes primary atypical-cold agglutinin-positive pneumonia. Since alveolar macrophages internalize mycoplasma as part of their immune defense, we studied characteristics of the human macrophage receptor for opsonized and nonopsonized M. pneumoniae . The glass-adhering subpopulation of M. pneumoniae attached more than the non-adherent subpopulation. The attachment was dose-dependent and enhanced by opsonization in the presence of human serum. It is inhibited by sulfated compounds such as dextran-sulfate and polyanetholsulfonic acid, but not by dextran or several monosaccharides, suggesting that sulfated glycolipids on the macrophage surface may act as receptors for M. pneumoniae binding. In addition, sialylated compounds, such as fetuin and α 1-acid glycoprotein, were found to be potent inhibitors of the attachment, also indicating the role of sialic acid residue in recognition and attachment of M. pneumoniae to human alveolar macrophages.     

hKv1.5 channels play a pivotal role in the functions of human alveolar macrophages

We examined the pharmacological properties, the molecular identity, and the functional roles of hKv1.5 channel in human alveolar macrophage. Some of outward K(+) current was inhibited by 4-aminopyridine and antisense oligodeoxynucleotides against hKv1.5 mRNA. Consistently, the protein and mRNA expressions of hKv1.5 channel were detected. Furthermore, the phagocytosis and migration of human alveolar macrophages were significantly suppressed when the protein expression of hKv1.5 channel was lowered by the antisense hKv1.5 oligodeoxynucleotides. These results suggest that hKv1.5 channel is expressed in human alveolar macrophages and it plays a role in phagocytosis and migration of the human alveolar macrophage.     

Macrophage phagocytosis of apoptotic neutrophils is compromised by matrix proteins modified by cigarette smoke and lipid peroxidation products

Clearance of apoptotic cells by phagocytosis plays an important role in the resolution of an inflammatory response. Macrophages interacting with extracellular matrix (ECM) proteins upregulate their phagocytic capacity. Cigarette smoke contains highly reactive carbonyls that modify proteins which directly/indirectly affects cellular function. We observed, in vitro, that human macrophages interacting with carbonyl or cigarette smoke modified ECM proteins dramatically down regulated their ability to phagocytose apoptotic neutrophils. We also show that this interaction with carbonyl-adduct modified ECM proteins led to increased macrophage adhesion in vitro. We hypothesise that changes in the ECM environment as a result of cigarette smoking affect the ability of macrophages to remove apoptotic cells. Moreover, we postulate that this decreased phagocytic activity was as a result of sequestration of receptors involved in the uptake of apoptotic cells towards that of recognition of carbonyl adducts on the modified ECM proteins leading to increased macrophage adhesion.     

Apoptosis-dependent externalization and involvement in apoptotic cell clearance of DmCaBP1, an endoplasmic reticulum protein of Drosophila

To elucidate the actions of Draper, a receptor responsible for the phagocytic clearance of apoptotic cells in Drosophila, we isolated proteins that bind to the extracellular region of Draper using affinity chromatography. One of those proteins has been identified to be an uncharacterized protein called Drosophila melanogaster calcium-binding protein 1 (DmCaBP1). This protein containing the thioredoxin-like domain resided in the endoplasmic reticulum and seemed to be expressed ubiquitously throughout the development of Drosophila. DmCaBP1 was externalized without truncation after the induction of apoptosis somewhat prior to chromatin condensation and DNA cleavage in a manner dependent on the activity of caspases. A recombinant DmCaBP1 protein bound to both apoptotic cells and a hemocyte-derived cell line expressing Draper. Forced expression of DmCaBP1 at the cell surface made non-apoptotic cells susceptible to phagocytosis. Flies deficient in DmCaBP1 expression developed normally and showed Draper-mediated pruning of larval axons, but a defect in the phagocytosis of apoptotic cells in embryos was observed. Loss of Pretaporter, a previously identified ligand for Draper, did not cause a further decrease in the level of phagocytosis in DmCaBP1-lacking embryos. These results collectively suggest that the endoplasmic reticulum protein DmCaBP1 is externalized upon the induction of apoptosis and serves as a tethering molecule to connect apoptotic cells and phagocytes for effective phagocytosis to occur.     

Sesquiterpene lactones induce distinct forms of cell death that modulate human monocyte-derived macrophage responses

Sesquiterpene lactones (SQTLs) are shown to possess anti-inflammatory as well as cytotoxic activity. No study, however, links both activities. We, therefore, hypothesized that SQTL-treated, dying cells might induce an anti-inflammatory response in cocultured THP-1 macrophages. Here we show that SQTLs bearing either an alpha,beta-unsaturated cyclopentenone or an alpha-methylene-gamma-lactone induce different forms of cell death. Whereas the cyclopentenone SQTL induced typical apoptosis, the alpha-methylene-gamma-lactone SQTLs-induced cell death lacked partly classical signs of apoptosis, such as DNA fragmentation. All SQTLs, however, activated caspases and the nuclear morphology of cell death was dependent on caspase activation. Most interestingly, alpha-methylene-gamma-lactone SQTLs induced a more pronounced phosphatidylserine (PS) exposure than the cyclopentenone SQTL. Especially, 7-hydroxycostunolide (HC), with an alpha-methylene-gamma-lactone substituted with a hydroxyl group, showed a striking fast and pronounced PS translocation. This result was in agreement with a strong activation of phagocytosis in cocultured THP-1 macrophages. Interestingly, HC-treated Jurkat cells led to an early (3.5 h) but transient increase in TNF-alpha levels in macrophage coculture. Release of TGF-beta remained unaffected after 18 h. We propose that this type of SQTL may influence local inflammation by transiently activating the immune system and help to clear cells by inducing a form of cell death that promotes phagocytosis.     

Inhibition by adiponectin of IL-8 production by human macrophages upon coculturing with late apoptotic cells

Adiponectin, an adipocyte-derived hormone, reportedly suppresses the production of TNF-alpha and IL-6 by LPS-stimulated human or porcine macrophages, and the phagocytosis of microbeads by human macrophages. In this study, we used a high molecular weight form of adiponectin purified from human plasma to examine its effects on the phagocytosis of late apoptotic cells by human macrophages and the subsequent IL-8 production. Adiponectin suppressed both the phagocytosis of apoptotic cells and the IL-8 production. In contrast, adiponectin augmented both the phagocytosis of apoptotic cells and the IL-8 production in the presence of LPS. These results suggest that adiponectin is not an anti-inflammatory hormone but rather a dual modulator of innate responses.     

Friendly fire against neutrophils: proteolytic enzymes confuse the recognition of apoptotic cells by macrophages

Physiologically the only acceptable fate for almost all damaged or unwanted cells is their apoptotic death, followed by engulfment of the corpses by healthy neighbors or professional phagocytes. Efficient clearance of cells that have succumbed to apoptosis is crucial for normal tissue homeostasis, and for the modulation of immune responses. The disposal of apoptotic cells is finely regulated by a highly redundant system of receptors, bridging molecules and 'eat me' signals. The complexity of the system is reflected by the term: 'engulfment synapse', used to describe the interaction between a phagocytic cell and its target. In healthy humans, dying neutrophils are the most abundant and important targets for such recognition and engulfment. In inflammation the scope and importance of this complicated task is further increased. Paradoxically, despite growing evidence highlighting the priority of neutrophils clearance, the recognition of these cells by phagocytes is not as well understood as the recognition of other apoptotic cell types. New findings indicate that the interaction of phosphatidylserine (PS) on apoptotic neutrophils with its receptor on macrophages is not as critical for the specific clearance of neutrophil corpses it was previously believed. In this review we focus on recent findings regarding alternative, PS-independent "eat me" signals expressed on neutrophils during cell death and activation. Based on our own research, we emphasize the clearance of dying neutrophils, especially at the focus of bacterial infection; and the associated inflammatory reaction, which occurs in a highly proteolytic milieu containing both host and bacteria-derived proteinases. In these environments, eat-me signals expressed by neutrophils are drastically modified; arguing against the phospholipid-based detection of apoptotic cells, but supporting the importance of proteinaceous ligand(s) for the recognition of neutrophils by macrophages. In this context we discuss the effect of the gingipain R (Rgp) proteinases from Porphyromonas gingivalis on neutrophils interactions with macrophages. Since the recognition of apoptotic neutrophils is an important fundamental process, serving multiple functions in the regulation of immunity and homeostasis, we hypothesize that many pathogenic bacteria may have developed similar strategies to confuse macrophage-neutrophil interaction as a common pathogenic strategy.     

Inhibition of LPS-induced activation of alveolar macrophages by high concentrations of LPS-binding protein

Lipopolysaccharide (LPS)-binding protein regulates the effects of LPS on immunocompetent cells. By catalyzing the binding of LPS to membrane CD14, LPS-binding protein (LBP) potentiates both the inflammatory response and internalization of LPS. LBP-mediated transport of LPS into high density lipoprotein particles participates in LPS clearance. Elevated serum levels of LBP have been shown to elicit protective effects in vivo. Because the expression of LBP is upregulated in lung epithelial cells upon proinflammatory stimulation, we here investigated whether LBP modulates inflammatory responses by lung specific cells. The moderate elevation of LBP concentrations enhanced both LPS-induced signaling and LPS uptake by rat alveolar macrophages, whereas strongly elevated LBP levels inhibited both. In contrast, the lung epithelial cell line A549 responded to high concentrations of LBP by an enhanced LPS uptake which did not result in cellular activation, suggesting an anti-inflammatory function of these cells by clearing LPS.     

Involvement of caspase activation through release of cytochrome c from mitochondria in apoptotic cell death of macrophages infected with Actinobacillus actinomycetemcomitans

We previously reported that infection with the periodontopathic bacterium Actinobacillus actinomycetemcomitans induced apoptosis in a mouse macrophage cell line J774.1. In the present study, we examined the involvement of cytochrome c and caspases in the induction of apoptosis in A. actinomycetemcomitans-infected J774.1 cells. Following infection, the expression levels of cytochrome c, and cleaved forms of caspase-3 and caspase-9 in the cells were examined using immunoblot analysis. Cytochrome c was released from mitochondria into the cytoplasm after A. actinomycetemcomitans-infected J774.1 cells were cultured for 6 h, and caspase-3 and caspase-9 were found to be cleaved forms in the cells. Further, caspase-9 activity was markedly increased, and phosphorylated p53 was detected in the cells 30 h following infection. These results suggest that apoptosis in A. actinomycetemcomitans-infected J774.1 cells is regulated by the release of cytochrome c from mitochondria into cytoplasm and the subsequent activation of caspases through phosphorylation of p53.     

Keratinocyte growth factor augments pulmonary innate immunity through epithelium-driven, GM-CSF-dependent paracrine activation of alveolar macrophages

Keratinocyte growth factor (KGF) is an epithelial mitogen that has been reported to protect the lungs from a variety of insults. In this study, we tested the hypothesis that KGF augments pulmonary host defense. We found that a single dose of intrapulmonary KGF enhanced the clearance of Escherichia coli or Pseudomonas aeruginosa instilled into the lungs 24 h later. KGF augmented the recruitment, phagocytic activity, and oxidant responses of alveolar macrophages, including lipopolysaccharide-stimulated nitric oxide release and zymosan-induced superoxide production. Less robust alveolar macrophage recruitment and activation was observed in mice treated with intraperitoneal KGF. KGF treatment was associated with increased levels of MIP1γ, LIX, VCAM, IGFBP-6, and GM-CSF in the bronchoalveolar lavage fluid. Of these, only GM-CSF recapitulated in vitro the macrophage activation phenotype seen in the KGF-treated animals. The KGF-stimulated increase in GM-CSF levels in lung tissue and alveolar lining fluid arose from the epithelium, peaked within 1 h, and was associated with STAT5 phosphorylation in alveolar macrophages, consistent with epithelium-driven paracrine activation of macrophage signaling through the KGF receptor/GM-CSF/GM-CSF receptor/JAK-STAT axis. Enhanced bacterial clearance did not occur in response to KGF administration in GM-CSF(-/-) mice, or in mice treated with a neutralizing antibody to GM-CSF. We conclude that KGF enhances alveolar host defense through GM-CSF-stimulated macrophage activation. KGF administration may constitute a promising therapeutic strategy to augment innate immune defenses in refractory pulmonary infections.     

THIS ARTICLE HAS BEEN RETRACTED Antifungal antibiotic hamycin increases susceptibility of Candida albicans to phagocytosis by murine macrophages

Hamycin is an antifungal antibiotic produced by Streptomyces pimprina Thirum. In the present study, the effect of hamycin on (a) the phagocytosis of Candida albicans by murine peritoneal macrophages and (b) the cell surface hydrophobicity (CSH) of C. albicans was investigated. Addition of hamycin to the culture of macrophages and Candida cells increased the susceptibility of Candida cells to the phagocytosis by macrophages. Pretreatment of Candida cells with hamycin increased their vulnerability to killing by macrophages. Examination of physico-chemical properties of Candida cell surface showed a significant decrease in the CSH. These findings suggest that the binding of hamycin to Candida cells induces biochemical/physico-chemical alterations of the surface, so that it becomes more susceptible to phagocytosis by murine macrophages.     

Antibody-independent phagocytosis of tumor cells by human monocyte-derived macrophages cultured in recombinant macrophage colony-stimulating factor

Human monocytes exposed in vitro to recombinant macrophage-colony-stimulating factor (rhMCSF) differentiate into monocyte-derived macrophages (MDM), which mediate efficient antibodydependent cytotoxicity (ADCC) against tumor cells. We and others have shown that this form of ADCC is unusual in that phagocytosis, rather than extracellular lysis, appears to play the major role in target cell killing. In this study, we asked whether the phagocytic form of cytotoxicity seen with ADCC could occur in the absence of an opsonizing antibody. We now report that, whereas cell lines derived from solid tumors are often resistant to antibody-independent cytotoxicity, malignant cells of lymphoid origin appear particularly susceptible to such antibody-independent killing. We found that all of nine lymphocytic leukemia and lymphoma cell lines tested in a total of 35 experiments, plus all four samples of fresh leukemic blasts, were consistently susceptible to antibody-independent MDM cytotoxicity. Antibody-independent cytotoxicity against these cells was efficient (40%–63% killing) at effector: target (E:T) ratios as low as 2:1. Like ADCC, antibody-independent cytotoxicity involved phagocytosis of target cells, as demonstrated by ingestion of fluorescently labeled targets and analysis by flow cytometry. At the time of phagocytosis, the majority of target cells retained membrane integrity, as indicated by the direct transfer of intracellular [⁵¹Cr]chromate from radiolabeled targets to phagocytosing MDM, without release of the label into the medium. However, in contrast to ADCC, we found that the degree of antibody-independent cytotoxicity was not a function of the E:T ratio. Instead, a constant proportion of the available target cells were killed regardless of the E:T ratio, suggesting that target cell recognition, rather than effector cell potency, might be the limiting factor in determining cytotoxicity. In additional experiments, we have also identified a second tumor cell type, nueroblastoma, as being susceptible to antibody-independent phagocytosis (all of five cell lines tested, cytotoxicity 40%–93%, E:T=3:1). Our data thus indicate that the cytotoxicity induced by rhMCSF is not confined to antibody-mediated killing, and that phagocytosis can play a significant role in target cell destruction even in the absence of opsonizing antibody.Supported in part by grants CA-33049 and CA-53624 from the National Institutes of Health, grant IRG-174b from the American Cancer Society, the Friends of Children Toys-R-Us Foundation. Inc., and the Robert Steel Foundation