Hydrogen Peroxide Release by Rat Alveolar Macrophages: Comparison with Blood Neutrophils

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1 NFECTON AND MMUNTY, Feb. 1978, p /78/ $02.00/0 Copyright X 1978 American Society for Microbiology Vol. 19, No. 2 Printed in U.S.A. Hydrogen Peroxide Release by Rat Alveolar Macrophages: Comparison with Blood Neutrophils W. DOUGAS BGGAR,'` AND JENNFER M. STURGESS2 Department of Pediatrics and mmunology,' and Department of Pathology,2 University of Toronto, and The Hospital for Sick Children, Toronto, Ontario M5G1X8 Canada Received for publication 1 August 1977 Hydrogen peroxide release was examined using biochemical and cytochemical techniques in rat alveolar macrophages, at rest and during phagocytosis, and compared with rat blood neutrophils. Using biochemical techniques, alveolar macrophages released small amounts of hydrogen peroxide at rest, and no increase was observed after challenge with opsonized and nonopsonized zymosan particles at several particle-cell ratios (1:1 to 1:1,000). Neutrophils released similar quantities of hydrogen peroxide at rest but showed a 12-fold increase in hydrogen peroxide release following exposure to opsonized zymosan particles. Using cytochemical techniques to localize sites of hydrogen peroxide release, resting neutrophils showed little deposition of reaction product at the cell surface and occasional deposits in endocytotic vesicles. After exposure to latex particles, a dense reaction product was observed between the particle and the cell membrane, indicating significant increases in hydrogen peroxide release at the sites of particle contact with the neutrophil. The resting macrophage displayed a light, uniform precipitation of cerium over the cell surface and lining intracellular channels and endocytotic vesicles and vacuoles. Following particle exposure, there was no significant difference in the density or distribution of reaction product. These findings, together with previous studies of oxidative metabolism, suggest that alveolar macrophages do not release increased quantities of hydrogen peroxide during phagocytosis. n contrast to neutrophils, oxidative-dependent metabolic pathways may not be of primary importance for microbial killing by alveolar macrophages. Alveolar macrophages are important for lung defense, and, existing in an environment of high oxygen tension, many of their cellular functions are oxygen dependent. Although oxygen, activated products of oxygen, and oxidative phosphorylation are intimately associated with effective phagocytosis (27), their contributions to microbial killing are poorly understood and may vary considerably between macrophages from different animal species (13, 21). Rabbit alveolar macrophages produce hydrogen peroxide (14, 28), but hydrogen peroxide formation does not increase during phagocytosis, as assessed by the iodination of ingested particles (6). The lack of iodination reaction product does not reflect a deficiency in myeloperoxidase activity (7), since the reaction does not increase significantly during phagocytosis of particles coated with exogenous peroxidase (6). Similarly, there is no increase in the generation of superoxide during phagocytosis (6), based on measurement of Nitro Blue Tetrazolium dye reduction (4). These observations indicated that activated metabolites of oxygen may not be of primary importance 621 for those bactericidal mechanisms that occur in the phagocytic vacuole after particle ingestion by alveolar macrophages. Recently, attention has focused on the ability of alveolar macrophages to release bactericidal agents such as lysozyme (8) in response to extracellular particles. These agents may contribute extracellularly to bactericidal mechanisms in the peripheral lung. n the present study, the release of hydrogen peroxide at the surface of rat alveolar macrophages and rat neutrophils at rest and during phagocytosis has been compared, using biochemical and cytochemical techniques. Our findings demonstrate that alveolar macrophages release low levels of hydrogen peroxide at rest, but this is not stimulated during particle contact or ingestion. n contrast, neutrophils release little hydrogen peroxide at rest and large amounts during phagocytosis. MATERAS AND METHODS Alveolar macrophages were obtained from exsanguinated 200- to 300-g rats by bronchial lavage (25). The cell suspensions contained more than 95% alveolar

2 622 BGGAR AND STURGESS macrophages, less than 3% polymorphonuclear leukocytes, and less than 1% erythrocytes and epithelial cells. Neutrophils were obtained from human blood following dextran sedimentation and from rat blood by methods previously described (10, 16). The cells were washed twice in Hanks balanced salt solution (HBSS; Microbiological Associates, nc., Bethesda, Md.) and resuspended at a concentration of 5 x 107 cells per ml in HBSS containing 1 mg of glucose per ml. Biochemical assay for hydrogen peroxide. Hydrogen peroxide was measured fluorimetrically, using horseradish peroxidase-mediated oxidation of reduced scopoletin as described by Root et al. (26). Scopoletin (7-OH-6 methoxycoumarin; Sigma Chemical Co., St. ouis, Mo.) was prepared as a 2 mm solution in 0.5 M phosphate buffer, ph 7.0. Horseradish peroxidase (Sigma) was prepared in 0.05 M sodium phosphate buffer, ph 7.0. n some experiments, cells were preincubated with 0.1 to 50 mm 3-amino-1,2,4-triazole (Sigma) to inhibit catalase for 10 to 20 min prior to measuring hydrogen peroxide release. Extinction of the fluorescence was recorded from resting cells for 2 to 3 min in cuvettes maintained at 37 C by a constant-temperature water jacket attached to an Aminco-Bowman spectrophotofluorometer (American nstrument Co., Silver Spring, Md.). Zymosan particles (Sigma) were washed and suspended in Krebs-Ringer phosphate buffer (KRP) at ph 7.0. Zymosan particles were opsonized by incubation in 50% fresh rat serum at 37 C for 30 min and then washed three times and resuspended in KRP. Particles were added at various particle-cell ratios (1:1 to 1,000:1) to ensure maximum stimulation, and the loss of fluorescence was recorded for 5 min on a Hewlett-Packard recorder (model 7044A). The maximum rate of hydrogen peroxide release was calculated from the steepest portion of each curve. For standard curves, the extinction of fluorescence of 2,uM scopoletin was determined for hydrogen peroxide generated from glucose in the presence of glucose oxidase (26). Glucose oxidase type (Sigma), 1.6 mg/ml, generated 2 mm hydrogen peroxide from 5.5 mm glucose in KRP buffer at ph 7.4 at 37 C within 2 min. Cytochemical localization of hydrogen peroxide. Hydrogen peroxide was localized, using the cytochemical method of Briggs et al. (9) based on the precipitation of cerium salts at cell surfaces. Neutrophils and alveolar macrophages were examined both at rest and during active phagocytosis. Resting cells, not exposed to particles, were incubated in HBSS containing 1 mg of glucose per ml for 20 min at 370C. For phagocytosis, 5 x 107 cells were incubated with an excess of latex particles, 1.1-,um diameter (Dow Chemicals, Midland, Mich.), for 20 min at 370C. After incubation, the cells were washed three times in cold HBSS. n preliminary experiments with both rat and human neutrophils, no differences in cytochemical localization of reaction product or of hydrogen peroxide release were detected when alveolar macrophages were challenged in suspension or after attachment to glass surfaces. Therefore, both biochemical and cytochemical studies were carried out with cells in suspension. For electron microscopy, cells were washed in 0.1 M tris(hydroxymethyl)aminomethane-maleate buffer containing 7% sucrose, ph 7.5, fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer containing 7% sucrose, ph 7.3, at 4 C for 60 min, and then processed for electron microscopy as described in detail previously (7). Briefly, the cells were rinsed in 0.1 M sodium cacodylate buffer containing 5% sucrose at ph 6.0, postfixed in 2% osmium tetroxide in Veronal acetate buffer, ph 7.0, dehydrated in graded-ethanol solutions, and embedded in Epon epoxy resin. Thin sections were prepared and stained with 4% lead acetate and examined at 60 kv in a Philips EM 300 electron microscope. RESUTS Hydrogen peroxide release from phagocytic cells. (i) Biochemical studies. Rat neutrophils not exposed to particles released low levels of hydrogen peroxide ( nmol/2.5 x 10' cells per min). After challenge with opsonized zymosan particles, a latent period of 10 to 13 s occurred with no change in hydrogen peroxide release, followed by marked increase in hydrogen peroxide release. Release of hydrogen peroxide increased as the zymosan particle-cell ratio increased with maximum levels of hydrogen peroxide (0.640 ± nmol/2.5 x 101 cells per min) at approximately 200 particles-cell (Fig. 1). After challenge with nonopsonized zymosan particles, little change in hydrogen peroxide release was observed ( nmol/2.5 x 106 cells per min). Rat alveolar macrophages at rest showed low levels of hydrogen peroxide release ( nmol/2.5 x 106 cells per min) (Fig. 1). Following exposure to opsonized zymosan particles, no significant increase in hydrogen peroxide release occurred at any particle-cell ratio between 1:1 to 1,000:1 ( nmol/2.5 x 101 cells per min). Under these conditions, 10 z 0.8j 0 4 E o Porticle Cetl Ratio NFECT. MMUN. FG. 1. Relationship between maximum rates for H202 release from rat neutrophils (0) and rat alveolar macrophages (0) following exposure to opsonized zymosan particles at different particle-cell ratios. Mean values + standard error of the mean represent four separate experiments. 200

3 VO. 19, 1978 H202 REEASE FROM AVEOAR MACROPHAGES although the phagocytic capacity of neutrophils seen on the remaining cells. A dense reaction and macrophages at each particle-cell ratio stud - product was seen occasionally at the cell surface, ied differed, there was vigorous phagocytosis off often when the macrophage appeared to be opsonized zymosan particles as judged by optica 1 closely associated with a degenerating cell. n and electron microscopy. No increase in hydro - the absence of reduced nicotinamide adenine gen peroxide release was observed when alveola r dinucleotide (NADH), little or no reaction prodmacrophages were challenged with nonopson - uct was observed at the cell surfaces. ized zymosan particles ( nmol/2.q5 After incubation with latex particles, 25 to x 10' cells per min). No change in hydroger1 30% of the alveolar macrophages showed active peroxide activity was detected when the cell, s phagocytosis of latex particles with numerous were preincubated with aminotriazole (0.1 to 5( ) particles adherent to the cell surface, and apmm) and then challenged with zymosan parti - proximately two to six particles were enclosed cles. within phagocytic vacuoles in each of these cell (ii) Cytochemical studies. Alveolar macro - sections (Fig. 3). The latex particles were parphages isolated by lavage have been character - tially extracted during preparation for electron ized previously (7). When the cells were reacted microscopy, forming an electron-dense body surfor hydrogen peroxide release, the resting mac - rounded by an electron-lucent zone. rophage showed a light, but uniform, precipita - Macrophages containing latex particles tion of cerium on the cell surface and lining showed an even precipitation of cerium on the numerous intracellular channels, endocytoti( c plasma membrane with a density similar to that vesicles, and vacuoles (Fig. 2). Of 200 cells ex - observed in resting macrophages. Cerium preamined, this light reaction was observed in ap - cipitated around latex particles close to the cell proximately 50%, and little or no precipitate wa. s surface, in the space between the particle and ~~~~~~~~ 2 7t ~v r._sm FG. 2. Resting alveolar macrophages incubated with medium containing cerium and NADH to demonstrate hydrogen peroxide release. Uniform deposition of reaction product occurs on the cell surface and lining pinocytotic vesicles and channels (arrows) continuous with the cell surface. x15,000. nset, Detail of deposition at the cell surface, with typical needle-like crystal formation of reaction product. x24,000.

4 .s 2.;.. t 624 BGGAR AND STURGESS 3a j.--%'.4w-lq%i k4.1.'#, -.,,, -... t.;. * S,@_--ui i aez... k :Nftmir..,.t. ; s*se,, ),,4 _ 5! F,4e ';+ -A 3b : -.^....9, t. v ;.< f f ),,^z *. 4.. _.: '>, X A. vt *,. FG. 3. (a) Alveolar macrophages incubated with medium containing cerium and NADH during active phagocytosis of latex particles (). Uniform reaction product occurs over cell surface with similar density to that observed in Fig. 2. No increase in reaction product occurs at sites of ingestion ofparticles (arrows) or other regions of the cell surface (double arrows). X16,400. (b) Detail of reaction product (arrows) at the cell surface during phagocytosis of latex particles (). x43,000. NFECT. MMUN. the plasma membrane, and in some vesicles and vacuoles, which were often continuous with cerium-lined channels leading to the cell surface (Fig. 4). Other phagocytic vacuoles containing latex particles showed no cerium deposition, indicating that these were no longer in continuity with the cell surface. The intensity of the reaction product was similar for macrophages at rest and during active phagocytosis. However, the presence of cerium salts appeared to stimulate some pinocytotic activity of resting cells (Fig. 5) and, to a lesset extent, of cells phagocytosing latex particles. n the presence of cerium chloride, surface microvilli were shorter and more rounded, and elaborate networks of intracellular channels and pinocytotic vesicles were observed. When NADH was omitted from the incubation mixture, cerium precipitation was markedly reduced in both resting and phagocytosing cells (Fig. 6). This minimal reaction was observed both at the cell surface and in channels extending into the cell. Rat blood neutrophils at rest showed a relatively smooth cell surface with few microvilli. ittle deposition of cerium was observed except occasionally in indentations of the plasma membrane and in small intracellular vesicles, close to the plasma membrane (Fig. 7). n the absence of NADH, no precipitation of cerium was observed. After exposure to latex particles, more than 80% of rat neutrophils showed particles at the cell surface and within intracellular phagosomes. Of 200 cells examined, most neutrophils ingested more particles (10 to 40 per cell section) than alveolar macrophages (2 to 6 per cell section). At the cell surface, a dense cerium precipitate was seen between latex particles and the plasma membrane (Fig. 8). ittle or no reaction product was visualized on surfaces of plasma membrane which were not in contact with latex particles. Cerium precipitate-lined channels extended from the cell surface to phagocytic vacuoles containing latex particles. n some cells cerium precipitate appeared in vesicular structures associated with the phagosome, apparently representing the sites of lysosomal fusion with the phagocytic vacuole (Fig. 9). When NADH was omitted from the incubation mixture, the reaction product was significantly reduced (Fig. 10). However, occasional intracellular vacuoles containing latex particles were lined with reaction

5 4 * t,.9. -1e ' ' ' ; 4- i- re.. s *" *, -. _t~ e ^' 6 * '4q, - " t-f '1 r0 FG. 4. Alveolar macrophages incubated with medium containing cerium and NADH during active phagocytosis of latex particles (). Reaction product is deposited on the inner surface ofphagosomes, which are continuous with the cell surface via intracellular channels (arrows). Other phagosomes showing no reaction product presumably do not communicate with the extracellular environment. x36,000. FG. 5. Alveolar macrophage at rest incubated with medium containing cerium and NADH, showing the increased elaboration of plasma membrane caused by cerium ions. Reaction product lines cell surfaces (arrows). x40,ooo. 6a : 6b - <.-! = Pt... r ml T-A V. --'V 4-,-'.0.. -~ 4.#-* tq 9j4 *4 V.' ~~~~~4 ~ 4-..?t 4 V 4t,, FG. 6. Alveolar macrophage during active phagocytosis of latex particles () incubated with cerium in the absence of NADH. There is a marked reduction in reaction product. x18,000. (b) Detail of cell surface (arrows) at site of adherence of latex particles () showing lack of reaction product. X45, *4

6 626 BGGAR AND STURGESS 7 NFECT. MMUN. M.. Downloaded from FG. 7. Resting rat neutrophil reacted with medium containing cerium and NADH to demonstrate hydrogen peroxide release at the cell surface. ittle or no reaction product appears at the cell surface, but occasional deposits occur inpinocytotic vesicles (arrows). X16,000. nset, Detail of cell surface and reaction inpinocytotic vesicle. x50,000. product, and some neutrophils in close apposition to one another or to an erythrocyte showed some reaction product when NADH was omitted from the incubation mixture. DSCUSSON n blood neutrophils, oxidation-reduction reactions are initiated in the presence of foreign particles and are thought to be important for microbial killing (20, 23). The initial reaction in the formation of bactericidal compounds involves the conversion of oxygen to superoxide (5, 11, 12, 15, 19, 29), which is followed by dismutation of superoxide to hydrogen peroxide. The activated metabolites of oxygen, including superoxide (3, 30), hydroxyl radicals (2, 19, 22), singlet oxygen (1, 22, 24), and hydrogen peroxide (17, 18, 26), may all contribute to the bactericidal functions of neutrophils. Rat neutrophils release hydrogen peroxide from their surfaces in low levels, at rest, and enhanced levels during attachment and phagocytosis of particles. These changes can be detected both biochemically after the ingestion of zymosan particles and cytochemically after the ingestion of latex particles. The increases in hydrogen peroxide release following challenge with particles is very significant. ocalized concentrations of hydrogen peroxide are generated at the site of particle contact with the cell surface, whereas adjacent areas of the cell surface, not in contact with particles, show little evidence of hydrogen peroxide. t would appear, therefore, that the activation of the cell membrane depends on the contact with the extracellular particle directly, but the mechanism for this activation is not known. With zymnosan, opsonins on the particle surface may promote adherence to cells, but with latex particles the binding to cells would appear to be primarily electrostatic. on July 24, 2018 by guest

7 VO. 19, 1978 The release of hydrogen peroxide, as well as its stimulation in rat neutrophils, is similar essentially to that described previously for human neutrophils (9). The cytochemical reaction for both cells is largely NADH dependent and presumably represents NADH-oxidase activity in the plasma membrane. Rat neutrophils show somewhat lesser response to latex particles than the human neutrophils, as judged by the number of ingested particles and by the intensity of the reaction products. Alveolar macrophages release small quantities of hydrogen peroxide at rest and, judged on a biochemical assay, are similar to the amounts released from rat neutrophils. Despite vigorous phagocytosis of opsonized or nonopsonized zymosan particles or of latex particles, hydrogen peroxide release is not increased in rat alveolar macrophages as an early event in phagocytosis. The failure to detect differences between resting and phagocytosing cells may indicate that hydrogen peroxide release is not stimulated soon after particle challenge. Alternatively, failure to detect changes in hydrogen peroxide may indicate that the catabolism of hydrogen peroxide by pathways such as glutathione peroxidase and catalase may be very rapid (27). Alveolar macrophages contain glutathione peroxidase (28), but it is not known whether this is active at the 8a H202 REEASE FROM AVEOAR MACROPHAGES, K~ 627 extracellular surface. The failure of aminotriazole to influence the detection of hydrogen peroxide either biochemically or cytochemically suggests that under these experimental conditions catalase does not contribute significantly to the breakdown of hydrogen peroxide. n addition, the failure of alveolar macrophages to show increased iodination of ingested particles and enhanced Nitro Blue Tetrazolium dye reduction (6) suggests that hydrogen peroxide also does not accumulate at intracellular sites and may not participate, in a primary way, in microbial killing. t seems likely that stimulation of hydrogen peroxide production is not a feature at the cell surface either. Our findings do not rule out the possibility that other activated products of oxygen could participate in the bactericidal event. Cytochemical studies of alveolar macrophages indicate that hydrogen peroxide is released from the cell surface at rest, as judged by the uniform deposition of cerium reaction product at the cell surface. As with rat and human neutrophils, this reaction product is largely dependent on the presence of NADH, indicating that NADH-oxidase activity is present also in alveolar macrophage plasma membranes. The intensity of reaction product and distribution on the alveolar macrophage surface is greater than that ob- -'... 8b V1 FG. 8. (a) Rat neutrophil during active phagocytosis of latex particles () incubated with medium containing cerium and NADH. Reaction product is deposited between the plasma membrane and adherent particles (arrows), but little deposition occurs on cell surfaces not in contact with particles. x30,000. (b) Detail of strong reaction product (arrows) observed at the surface of some cells during phagocytosis of latex particles (). x34,000.

8 628 BGGAR AND STURGESS 10 NFECT. MMUN. " " 11-,.,;....- i..,.-..; 'wk 4:t " i* " - 9b "e 50 :. e1. f..:. AOr.4 FG. 9. Rat neutrophils during active phagocytosis of latex particles () showing localized intense reaction product during phagocytosis. (a) Release of hydrogen peroxide at cell surface during phagocytosis (arrow). x43,000. (b) Release of hydrogen peroxide in phagosome deep to the cell surface (arrow) indicative of degranulation of lysosomes into the phagocytic vacuole, presumably still in continuity with the extracellular space. X48,000. FG. 10. Rat neutrophil during active phagocytosis incubated with cerium in absence of NADH. Minimal deposition of reaction product occurs at the cell surface or in phagosomes containing latex particles (). x28,000. served with rat neutrophils at rest. These observations suggest that hydrogen peroxide detection by cytochemical techniques may be more sensitive than biochemical techniques. Some stimulation of the alveolar macrophage cell membrane may be attributed in part to the presence of cerous ions used in the cytochemical method. With cerium and other heavy metal salts, there is morphological evidence of activation or perturbation of the cell membrane (unpublished data) which is more pronounced than that observed with rat or human neutrophils under the same conditions. This finding suggests, first, that some activation of the cell surface by cerous ions may occur in the alveolar macrophage and, second, that the activation of the cell surface by particulate matter may vary for these two types of phagocytic cells. The alveolar macrophage may respond more readily to fine particulate matter, which may be important to its functioning in the lung. Another form of macrophage activation is seen after Bacille Calmette-Gu6rin immunization, which induces a population of alveolar macrophages with altered cellular functions and enhanced oxidative metabolism. n these activated cells, oxidativedependent bacterial killing may play a greater role than in normal unstimulated cells. The application of both biochemical and cytochemical studies of hydrogen peroxide release by resting and phagocytosing cells permitted a detailed comparison of their interactions with different particles. Whereas the basic metabolic response of the cell population could be judged by biochemical means, the cytochemical methodology allowed the sites of hydrogen peroxide release to be localized both at rest and during the adherence and ingestion of particles. This latter approach allows the distribution of hydrogen peroxide to be compared in individual cells, eliminating problems inherent in the biochemical assay of cell populations, such as contamination or cell degradation, which may interfere with the interpretation of experimental data.

9 VO. 19, 1978 Our findings on hydrogen peroxide production by alveolar macrophages lends support to previous studies which have indicated that oxidative-dependent bactericidal mechanisms are not of primary importance for these cells as they are for neutrophils. Alternative pathways or non-oxidative-dependent pathways appear to be more significant in the defense mechanisms of the normal unstimulated alveolar macrophage. ACKNOWEDGMENTS We are grateful for the skilled assistance of S. Omar and K. Y. ee. This work was supported by grants from the Medical Research Council of Canada (MA 5276 and MA 4858) and the Ontario Thoracic Society. W. Douglas Biggar and J. M. Sturgess are Medical Research Council Scholars. TERATURE CTED 1. Allen, R. C., S. J. Yevich, R. W. Orth, and R. H. Steele The superoxide anion and singlet molecular oxygen: their role in the microbicidal activity of the polymorphonuclear leukocyte. Biochem. Biophys. Res. Commun. 60: Babior, B. M., J. T. Curnutte, and R. S. Kipnes Biological defense mechanisms: evidence for participation of superoxide in bacterial killing by xanthine oxidase. J. ab. Clin. Med. 85: Babior, B. M., R. S. Kipnes, and J. T. Curnutte Biological defense mechanisms: the production by leukocytes of superoxide a potential bactericidal agent. J. Clin. nvest. 52: Baehner, R.., S. K. Murrmann, J. Davis, and R. B. Johnston, Jr The role of superoxide anion and hydrogen peroxide in phagocytosis-associated oxidative metabolic reactions. J. Clin. nvest. 56: Baines, B. M., R. K. Kyers, and J. J. Corman Biologic mechanisms: the production by leukocytes of superoxide, bactericidal agent. J. Clin. nvest. 52: Biggar, W. D., S. Buron, and B. Homes Bactericidal kiling by alveolar macrophages: evidence against myeloperoxidase and hydrogen peroxide bactericidal mechanisms. nfect. mmun. 14: Biggar, W. D., and J. M. Sturgess Peroxidase activity in alveolar macrophages. ab. nvest. 34: Biggar, W. D., and J. M. Sturgess Role of lysozyme in the microbicidal activity of rat alveolar macrophages. nfect. mmun. 16: Briggs, R. T., D. B. Drath, and M. J. Karnovsky ocalization of NADH oxidase on the surface of human polymorphonuclear leukocytes by a new cytochemical method. J. Cell Biol. 67: Chordirker, W. B., G. N. Bock, and J. H. Vaughan solation ofhuman polymorphonuclear leukocytes and granules: observations on early blood dilution and on heparin. J. ab. Clin. Med. 71: Curnutte, J. T., and B. M. Babior Biological defense mechanisms: the effects of bacteria and serum on superoxide production by granulocytes. J. Clin. nvest. 53: Curnutte, J. T., D. M. Whitten, and B. M. Babior Defective superoxide production by granulocytes from patients with chronic granulomatous disease. N. H202 REEASE FROM AVEOAR MACROPHAGES 629 Engl. J. Med. 290: DeChatelet,. R., D. Mullikin, and C. E. McCall The generation of superoxide anion by various types of phagocytes. J. nfect. Dis. 131: Gee, J. B.., C.. Vassallo, R. Bell, J. Kaskin, R. E. Basford, and J. B. Field Catalase-dependent peroxidative metabolism in the alveolar macrophage during phagocytosis. J. Clin. nvest. 45: Goldstein,. M., D. Roos, H. B. Kaplan, and G. Weissmann Complement and immunoglobulins stimulate superoxide production by human leukocytes independently of phagocytosis. J. Clin. nvest. 56: Hawkins, D., and S. Peeters Response of polymorphonuclear leukocytes to immune complexes in vitro. ab. nvest. 24: Homan-Muller, J. W. T., R. S. Weening, and D. Roos Production of hydrogen peroxide by phagocytizing human granulocytes. J. ab. Clin. Med. 85: yer, G. Y. N., M. F. slam, and J. H. Quastel Biochemical aspects of phagocytosis. Nature (ondon) 192: Johnston, R. B., B. Keele, H. P. Misra, J. E. ehmeyer,. S. Webb, R. Baehner, and K. V. Rajogopalan The role of superoxide anion generation in phagocytic bactericidal activity: studies with normal and chronic granulomatous disease leukocytes. J. Clin. nvest. 55: Karnovsky, M Chronic granulomatous disease-pieces of a cellular and molecular puzzle. Fed. Proc. 32: Karnovsky, M., and D. B. Drath Superoxide production by phagocytic leukocytes. J. Exp. Med. 141: Kellogg, E. W.,, and. Fridovich Superoxide. hydrogen peroxide, and singlet oxygen in lipid peroxidation by a xanthine oxidase system. J. Biol. Chem. 250: Klebanoff, S. J Antimicrobial mechanisms in neutrophilic polymorphonuclear leukocytes. Semin. Hematol. 12: Maugh, T. H., Singlet oxygen: a unique microbicidal agent in cells. Science 182: Myrvik, Q. N., E. S. eake, and B. Farriss Studies on alveolar macrophages from the normal rabbit: a technique to procure them in a high state of purity. J. mmunol. 86: Root, R. K., J. Metcalf, N. Oshino, and B. Chance H202 release from human granulocytes during phagocytosis.. Documentation, quantitation, and some regulating factors. J. Clin. nvest. 55: Rossi, F., G. Zabucchi, and D. Romeo Metabolism of phagocytosing mononuclear phagocytes, p n R. Van Furth (ed.), Mononuclear phagocytes in immunity, infection and pathology. Blackwell Scientific Publications, Oxford. 28. Vogt, M. T., C. Thomas, C.. Vassallo, R. E. Basford, and J. B.. Gee Glutathione-dependent peroxidative metabolism in the alveolar macrophage. J. Clin. nvest. 50: Weening, R. S., R. Wever, and D. Roos Quantitative aspect of the production of superoxide radicals by phagocytizing human granulocytes. J. ab. Clin. Med. 85: Yost, F. J., and. Fridovich Superoxide radicals and phagocytosis. Arch. Biochem. Biophys. 161: