The mechanism by which polymorphonuclear neutrophils kill most microorganisms includes conversion of oxygen by the cell to superoxide anion (O2-), 1 hydrogen peroxide (H202), hydroxyl radical (.OH), and, possibly, singlet oxygen (1). These toxic species kill ingested organisms, presumably through oxidation reactions and in conjunction with the contents of lysosomal granules. The mirobicidal mechanisms of macrophages are still obscure (2). However, mouse peritoneal macrophages have been shown to respond to phagocytosis or plasma membrane perturbation with the vigorous release of H202 (3) and 02-(4), and it has been suggested that the microbicidal mechanisms of macrophages may be similar to those of the neutrophil, at least for some organisms (2-5). We report here that macrophages elicited by injection of lipopolysaccharide (LPS) or obtained from animals infected with bacillus CalmetteGu~rin (BCG), shown previously to release greater amounts of 02-and H202 when stimulated, killed candida better than did resident cells. The killing of both Candida albicans and Candida parapsilosis was inhibited by scavengers of oxygen radicals, suggesting that oxygen metabolites play an important role in macrophage candidacidal activity. We also report that C. parapsilosis, which was killed more effectively than C. albicans, elicited a greater oxidative metabolic response from macrophages. Materials and Methods Macrophages. Mouse peritoneal macrophages were harvested as previously described (4).Approximately lO s washed cells in 1 ml of medium supplemented with 20% heat-inactivated fetal calf serum (FCS) (Flow Laboratories, Inc., Rockville, Md.) (4) were plated on 16-mm diameter tissue culture dishes (Costar, Data Packaging, Cambridge, Mass.) or, in phagocytosis experiments, on 13-mm diameter glass cover slips. After incubation for 120 min at 37°C in 5% CO2-95% air, plated cells were washed vigorously with medium twice, then cultured in medium with 20% FCS, penicillin, and streptomycin (4). After overnight in culture, adherent cells were washed with vigorous swirling with Hanks' balanced salt solution (HBSS; Grand Island Biological Co., Grand Island, N. Y.) before assay. Cultures of the murine cell line J774.1 and peritoneal macrophages elicited with LPS or removed from BCG-infected mice were obtained and processed as previously described. l Abbreviations used in this paper: BCG, bacille Calmette-Gu~rin; FCS, fetal calf serum; HBSS, Hanks' balanced salt solution; H202, hydrogen peroxide; LPS, lipopolysaccharide; O2-, superoxide anion; .OH, hydroxyl radical; PBS, phosphate-buffered saline; PMA, phorbol myristate acetate; SOD, superoxide dismutase.J. ExP. MED.

A B S T R A C T During phagocytosis, neutrophils take oxygen from the surrounding medium and convert it to superoxide anion (O°) and hydrogen peroxide (H202). Hydroxyl radical ( OH), a particularly potent oxidant, is believed to be produced by interaction between O2 and H202 in the presence of iron, according to the Haber-Weiss reactions. Production of OH by whole human neutrophils, by particulate fractions from human neutrophils disrupted after stimulation, and by a xanthine oxidase system was measured by conversion of a-keto-y-methiol butyric acid to ethylene. FeCl3 or ferric EDTA enhanced ethylene production in all three systems by 155-406% of base line at a concentration of 50-100 ,uM. Iron-saturated human milk lactoferrin, 100 nM, increased ethylene generation by 127-296%; and purified human neutrophil lactoferrin, 10 nM, enhanced ethylene production by 167-369%. Thus, iron bound to lactoferrin was -5,000 times more effective in producing an enhancement in ethylene generation than iron derived from FeCl3 or ferric EDTA. 02 and H202 were required for ethylene production in the presence of lactoferrin, since superoxide dismutase inhibited ethylene formation in the three systems by 76-97% and catalase inhibited by 76-98%. Ethylene production in the presence of lactoferrin was inhibited by the OH scavengers mannitol, benzoate, and thiourea by , and 76-96%, respectively. Thus, most of the ethylene production could be attributed to oxidation of a-keto-y-methiol butyric acid by OH. The ability ofneutrophil lactoferrin to provide iron efficiently to the oxygen radical-generating systems is compatible with a role for lactoferrin as regu-

Plasma membrane receptors for the Fc portion of IgG and for the major fragment of the third component of complement (C3b) t play an essential role in the function of macrophages as phagocytic cells. Fc receptors mediate both attachment and phagocytosis of particles coated with IgG antibody (1-3). Receptors for C3b on normal (resident) mouse peritoneal macrophages mediate only the attachment of particles to the cell (1, 3, 4), whereas C3b receptors on macrophages activated by infection or macrophages elicited by injection of inflammatory agents mediate both attachment and ingestion (3, 5-7).During phagocytosis, or as a consequence of perturbation of the phagocyte plasma membrane by soluble stimuli such as phorbol myristate acetate (PMA), the cell undergoes a "respiratory burst," in which it consumes oxygen and converts it to the potent microbicidal agents superoxide anion (O2-), hydrogen peroxide, hydroxyl radical, and perhaps, singlet oxygen. The role of phagocytosis through Fc or C3b receptors in stimulation of this oxidative burst has not been defined in macropbages. Studies with neutrophils in this regard suggest that the C3b receptor permits the attachment of C3b-coated particles to the cell; however, additional interaction with a second receptor (e.g., Fc receptor [8,9] or receptor for a component of fungal cell wall [9], perhaps conjugated mannose [10]) appears to be required for both phagocytosis and stimulation of the respiratory burst. In these systems, ingestion has invariably been associated with stimulation of oxidative metabolism. We report here that phagocytosis of IgG-coated sheep erythrocytes (E) by macrophages elicited a vigorous oxidative response. In contrast, phagocytosis by macrophages of E coated with IgM and complement or E treated with glutaraidehyde was not accompanied by stimulation of the respiratory burst. This dissociation could protect the host from oxidative tissue damage.

The adjuvant muramyl dipeptide (MDP) has been shown to affect a number of macrophage functions in vitro. We studied the effect of subcutaneous injection of MDP into mice. Cultured peritoneal macrophages from treated mice displayed increased spreading, total cell protein, and specific activity of beta-glucosaminidase a constituent of macrophage lysosomes, and of lactate dehydrogenase. Generation of superoxide anion (O2-) by MDP-treated macrophages stimulated by contact with phorbol myristate acetate was enhanced by over fivefold to levels achieved by macrophages from bacillus Calmette-Guérin-infected mice. The enhancement in stimulated O2- release was noted by 1 h after injection of MDP, peaked by 3 h, and remained high for at least 48 h. Priming for enhancement of O2- release by MDP was similar in athymic nude mice and in normal littermates, suggesting that mature T lymphocytes are not involved in this MDP effect. Priming for enhanced stimulated O2- release, and morphologic and enzymic changes, were not achieved by injection of the D-D stereoisomer of MDP. Phagocytosis of Candida albicans was only slightly greater by macrophages from mice give MDP, but MDP-stimulated cells killed two times more C. albicans in vitro than did cells from untreated animals. When MDP was given 18 h before, simultaneously with, or 24 h after lethal infectious challenge with C. albicans, treated mice were protected compared with controls. These results suggest that injection of MDP effectively and rapidly activates macrophages in the recipient animal. This agent should serve as an important probe of macrophage physiology and, perhaps ultimately, as a means of enhancing host defense in humans.

A B S T R A C T Alteration ofthe surface ofhuman neutrophils with the nonpenetrating, protein-inactivating agent p-diazobenzenesulfonic acid (DASA) was found to prevent activation of the respiratory burst by some stimuli, but not others. Production of superoxide anion (O°) stimulated by concanavalin A or the chemotactic peptide formyl-methionyl-leucyl-phenylalanine FMLP was inhibited by DASA pretreatment, whereas°2 production stimulated by phorbol myristate acetate (PMA), sodium fluoride, or the ionophore A23187 was not inhibited by DASA. Pretreatment with DASA inhibited oxygen uptake stimulated by FMLP, but not oxygen uptake stimulated by PMA. DASA reproducibly inhibited activities of two known surface enzymes, Mg++-ATPase and alkaline phosphatase, by 45-55% and 60-70%o, respectively. The inhibition by DASA of°2 production did not appear to be caused by interference with binding of the affected stimuli, since pretreatment with DASA did not inhibit release of the lysosomal enzymes lysozyme and myeloperoxidase induced by concanavalin A or FMLP. Membrane-rich particulate fractions from neutrophils have been shown to contain NADPH-dependent oxidative activity that is presumably responsible for the phagocytosis-associated respiratory burst ofintact cells. The PMA-activated enzyme was susceptible to inhibition if directly exposed to DASA in this particulate fraction. These findings suggest that more than one mechanism exists for activation of the respiratory burst oxidase in human neutrophils, and that the neutrophil possesses at least one oxidase that is not an ectoenzyme.

Multinucleated giant cells (MGC) ~ are found in a variety of inflammatory disease processes (1, 2). Both monocytes and macrophages appear in the lesions of these diseases before the MGC; and monocytes are thought to be the precursor to both the macrophages and multinucleated cells (1-3). The phagocytic capacity of MGC derived in vivo has been studied using cells adherent to coverslips planted under the skin of rodents (1, 3-6), and MGC derived from cultured mouse macrophages have also been shown to be phagocytic (7, 8). It has been reported that human monocytes can form variable numbers of multinucleated cells in vitro (9-12). However, functional capacities of human MGC that might relate to removal of foreign material or microorganisms have not been defined, and such functions have not been thoroughly studied with MGC of any species. We report here some functional and biochemical characteristics of MGC derived from human blood monocytes cultured in vitro. The characteristics examined are potentially important in mediating the resolution of, or in propagating, the variety of pathologic lesions in which these cells are found. Materials and Methods

Enhanced spreading of macrophages on glass or plastic surfaces has been defined as a property of activated macrophages (1, 2). Rabinovitch and DeStefano (3) demonstrated that incubation for 30 rain with trypsin, pronase, papain, or subtilisin "induced marked spreading in normal mouse peritoneal macrophages attached to glass; and G6tze et al. (4) reported that activated factor B of the properdin system (Bb), when active as a protease, stimulated pronounced macrophage spreading within 60 min. Increased activity of the hexose monophosphate shunt in resting (nonphagocytie) macrophages also was noted after exposure to proteolytic enzymes (2).Macrophages activated by infection or elicited by injection of inflammatory agents are primed so that they undergo an enhanced oxidative metabolic response when stimulated by phagocytosis or contact with surface-active agents such as phorbol myristate acetate (PMA; 5, 6). We report here that brief exposure to proteinases in vitro also primes macrophages for greater oxidative metabolism, measured as stimulated release of superoxide anion (02-) and hydrogen peroxide (H202). Materials and MethodsMacrophages. Peritoneal macrophages were harvested from mice as previously described (6).Washed cells were suspended in medium supplemented with 20% heat-inactivated fetal calf serum (FCS; 6), and 3-4 × 106 cells in a total volume of 1 ml were plated on 35-mm tissue culture dishes. After incubation for 120 min at 37°C in 5% CO2-95% air, plated cells were washed vigorously twice with Hanks' balanced salt solution (HBSS; Grand Island Biological Co., Grand Island, N. Y.), then cultured overnight in medium with 20% FCS, penicillin, and streptomycin (6). The cells adherent after overnight culture, as estimated by differential counts of stained smears (6) and by phagocytic capacity for candida (7), were >97% macrophages and <1% granulocytes. The protein content of the dishes at the time of assay was 40-90/.tg/dish. Thioglycollate-elicited cells were obtained from the peritoneum 4 d after intraperitoneal injection of thioglycollate medium (6). Louis, Mo.), chymotrypsin (Worthington Biochemical Corp.), or papain (Sigma Chemical Co.). The papain was "activated" immediately before use by incubation at 2.5 mg/ml with 2.5 mM dithiothreitol (P-L Biochemicals, Inc., Milwaukee, Wis.) for 10 rain at 37°C in 0.1 M sodium phosphate buffer, pH 7.2, with 0.01 M EDTA. The enzymes in HBSS were added to the macrophages at final concentrations of 1-250/~g/ml. Treatment of Macrophages with ProteolyticMacrophages on control dishes were incubated with HBSS alone or with HBSS containing

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