Bactericidal Mechanisms in Rabbit Alveolar ... - Europe PMC

Vol. 14, No. 1 Printed in U.S.A.

INFECTION AND IMMUNITY, JUlY 1976, p. 6-10 Copyright © 1976 American Society for Microbiology

Bactericidal Mechanisms in Rabbit Alveolar Macrophages: Evidence Against Peroxidase and Hydrogen Peroxide Bactericidal Mechanisms W. DOUGLAS BIGGAR,* SUE BURON,

AND

BEULAH HOLMES

Department of Pediatrics and Immunology, University of Toronto, The Hospital for Sick Children, Toronto. Canada M5G 1X8, and Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455 Received for publication 3 February 1976

The role of peroxidase-mediated bacterial killing by rabbit alveolar macrophages was examined. During 3 h of incubation in vitro, alveolar macrophages ingested and killed greater than 88% of the Streptococcus faecalis, Proteus mirabilis, or Streptococcus pneumoniae present in the incubation mixture. Preincubation of alveolar macrophages with inhibitors of catalase, 3-amino1,2,4-triazole or sodium nitrite, did -not alter their bactericidal potential. Iodination of ingested zymosan particles, a peroxidase-dependent and hydrogen peroxide-dependent reaction, was not observed, in spite of vigorous phagocytosis by alveolar macrophages. Furthermore, iodination by alveolar macrophages was not significantly increased when peroxidase-coated zymosan particles were ingested. The results suggest that hydrogen peroxide may not be available to the phagocytic vacuole for microbial killing. Since tetrazolium dye reduction reflects the activity of an oxidase responsible for stimulated oxygen consumption by polymorphonuclear leukocytes, this reaction was also measured. Rabbit alveolar macrophages incubated with latex particles did not exhibit an increased dye reduction compared with resting cells. The absence of significant stimulation of tetrazolium dye reduction indicates that the oxidase reaction does not occur in the proximity of the phagocytic vacuole of alveolar macrophages. (8) or have very low concentrations of myeloperoxidase (25). Functionally, some peroxidase may be present in alveolar macrophages, since the 20,000 x g pellet from rabbit alveolar macrophages has been shown to be bactericidal for Escherichia coli in the presence of H202 and iodide (23). By contrast, others have not found peroxidase in rabbit alveolar macrophages (8). Catalase is associated with the 100,000 x g fraction of alveolar macrophages and is transferred to the phagocytic vacuole during phagocytosis (29). Since catalase is known to function as a peroxidase in the presence of low concentrations of H202, it might substitute for myeloperoxidase in a peroxidase-H202-halide bactericidal system (15). This report describes investigations undertaken in order to elucidate the contribution of peroxidase and hydrogen peroxide to the bactericidal capacity of alveolar macrophages.

Alveolar macrophages ingest and kill microorganisms and are regarded as a major host defense barrier against a variety of external stimuli. As phagocytic cells, alveolar macrophages and polymorphonuclear (PMN) leukocytes share certain characteristics. Phagocytosis by PMN leukocytes is accompanied by enhanced oxidative metabolism (13). These metabolic changes include increased oxygen consumption (26, 28), increased hydrogen peroxide production (11), and increased hexose monophosphate pathway activity (26). These changes are thought to contribute in a major way to the bactericidal capacity of PMN leukocytes. Hydrogen peroxide is perhaps the most important metabolic product and is believed to participate in a bactericidal reaction with myeloperoxidase and halide (13, 16, 27, 30). Increased oxidative metabolism is also associated with phagocytosis by alveolar macrophages (8), but the relationship of these metabolic changes to the bactericidal capacity of alveolar macrophages is not well understood. Alveolar macrophages kill bacteria vigorously. These cells produce hydrogen peroxide (8, 31), although by biochemical quantitation they lack

MATERIALS AND METHODS Isolation and preparation of rabbit alveolar macrophages. Rabbit alveolar macrophages were obtained by pulmonary lavage according to the method of Myrvik et al. (20). Four- to six-pound (about 1.81 to 6

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2.72 kg) New Zealand rabbits were killed by anesthesia, and the lungs were removed. The lungs were distended with 30 to 40 ml of sterile Hanks balanced salt solution (Microbiological Associates), massaged, and aspirated by gentle suction. Three to four lung washings were pooled and washed twice with Hanks balanced salt solution. More than 95% of the cells isolated were macrophages, and greater than 95% of the isolated cells excluded trypan blue. All alveolar macrophage preparations were carefully monitored for bacterial contamination, and contaminated cell preparations were discarded. The sterility of all preparations was confirmed by overnight culture on blood agar plates. Bacterial killing by alveolar macrophages. The assay for bacterial killing by alveolar macrophages has been published (5). Three test organisms, Proteus mirabilis, Streptococcus pneumoniae type 22, and Streptococcus faecalis, were used. The influence of 3-amino-1,2,4-triazole (aminotriazole) (Sigma Chemical Co.) or sodium nitrite on the bactericidal capacity of alveolar macrophages was assessed by preincubating cells with the chemical for 30 min at 37 C. Iodination by alveolar macrophages. Iodination of zymosan particles was quantitated by the method of Pincus and Klebanoff (24). Iodination by human blood leukocytes was estimated in parallel. Human blood leukocytes were isolated by dextran sedimentation (5) and contained greater than 85% PMN leukocytes. In a second series of experiments, iodination of zymosan particles coupled with horseradish peroxidase was quantitated. Horseradish peroxidase (Sigma Chemical Co., type II) was coupled to zymosan particles by using glutaraldehyde, as described by Weston and Avrameas (32). Confirmation of peroxidase activity in association with the surface of zymosan particles was obtained by light (12) and electron (9) microscopy. Tetrazolium dye reduction by alveolar macrophages. Quantitation of tetrazolium dye reduction by alveolar macrophages was estimated by using a modification (4) of the method of Baehner and Nathan (3). Extraction of neotetrazolium dye (Sigma Chemical Co.) from cells was achieved using ethyl acetate (4). Supernatant optical densities were determined in a Gilford spectrophotometer (model 240) at 515 nm, using an ethyl acetate blank. Neotetrazolium dye reduction by resting and phagocytizing alveolar macrophages was compared with dye reduction by human blood leukocytes, tested in parallel.

RESULTS Experiments in which the bactericidal capacity of rabbit alveolar macrophages were examined are summarized in Tables 1 and 2. Better than 88% of the initial bacterial inoculum (1 x 106 to 3 x 106 bacteria/5 x 106 cells) was killed by alveolar macrophages over a 3-h period of incubation. Under these experimental conditions, alveolar macrophages killed the three bacterial strains as vigorously as did human PMN leukocytes (Table 1). Effective phagocyto-

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TABLE 1. Bacterial killing by alveolar macrophages % Bacteria killed at 3 ha

Test organism

Alveolar macrophages

PMN leu-

kocytes

Streptococcus faecalis ......

93 + 3b (82-99)r n=5 Proteus mirabilis .......... 91 ± 3 (81-96) n =5 Streptococcus pneumoniae ..89 4 (73-96)

92 ± 2 (84-99) n=6 93 ± 1 (90-99) n= 6 94 ± 2 (90-99) n=5 n=5 a Determined as [(number of bacteria killed)/ (number of bacteria added)] x 100. b Mean ± standard error. c Range.

TABLE 2. Effect of aminotriazole on bacterial killing of alveolar macrophages Test Test organism organism

Aminotriazole Concn

% Bacteria killed at 3 ha

(mM)

Streptococcus faecalis (n = 3) ...........

Proteus mirabilis (n = 4) ................ a-

0 10 40

88 ± 3b (84-95)Y 89 ± 2 (85-91) 91 ± 1 (90-92)

0 40 100

88 ± 3 (81-93) 89 ± 3 (85-96) 91 ± 2 (85-99)

See Table 1.

sis by alveolar macrophages, like PMN leukocytes, required the presence of fresh or freshfrozen serum (5). The effect of aminotriazole on the bactericidal capacity of alveolar macrophages is summarized in Table 2. After preincubation with aminotriazole for 30 min at 37 C, no significant effect on the bactericidal capacity of alveolar macrophages was apparent with aminotriazole at concentrations of 10 to 100 mM. Aminotriazole did not affect the viability of alveolar macrophages as assessed by trypan blue exclusion. Furthermore, these concentrations of aminotriazole did not inhibit bacterial growth of the test organisms, P. mirabilis and S. faecalis. In other experiments (not shown), sodium nitrite (20 to 100 mM) also had no detectable effect on the bactericidal capacity of alveolar macrophages. lodination by alveolar macrophages. Table 3 summarizes the experiments that compared iodination by alveolar macrophages to iodination by human PMN leukocytes. Iodination by alveolar macrophages that had ingested zymo-

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BIGGAR, BURON, AND HOLMES

TABLE 3. Iodination by alveolar macrophages 125Iodide bounda Culture components 730 ± 106b Alveolar macrophages ..... Alveolar macrophages + con744 + 86 trol zymosan ............ Alveolar macrophages + per817 + 69 oxidase-coated zymosanc . PMN leukocytes + control zymosan ................ 151,600 ± 16,000 Control zymosan + H202 (1 222 ± 12d ,.mol/ml) ............... Peroxidase-coated zymosan + H202 (1 Amol/ml) ..... 1,970 ± 54" Alevolar macrophages + control zymosan plus H202 (1 ,umol/ml) ................ 814 ± 73 Alveolar macrophages + peroxidase-coated zymosan plus H,20 (1 ,umol/ml) .... 2,651 ± 103 a Counts per minute per 107 cells per 60 min of incubation with the exception noted below (d). b Mean + standard error for at least three experiments. c Peroxidase coated to zymosan particles by gluteraldehyde (33). d Counts per minute of incubation in the absence of phagocytes.

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ml. Addition of exogenous H202 to the complete system (including alveolar macrophages) caused a similar stimulation of iodide binding. Tetrazolium dye reduction by alveolar macrophages. Quantitation of tetrazolium dye reduction by rabbit alveolar macrophages and human PMN leukocytes is summarized in Table 4. When alveolar macrophages, stimulated by phagocytosis of latex particles, were compared with resting alveolar macrophages, the increment in tetrazolium dye reduction (0.02 optical density increment at 515 nm/20 min per 5 x 106 cells) represented a 10% increase over resting dye reduction. By contrast, tetrazolium dye reduction by PMN leukocytes, examined in parallel experiments, was increased 500% during phagocytosis. DISCUSSION Phagocytosis by PMN leukocytes is associated with a burst in oxidative metabolism, which includes increased hexose monophosphate pathway activity and increased oxygen consumption (13). A cyanide-insensitive oxidase is believed responsible for the increased rate of respiration (13). Hydrogen peroxide is produced (11, 13). Superoxide anion is also formed in the oxidase reaction (1), and this intermediate appears to mediate the reduction of nitroblue tetrazolium dye (2), also stimulated during phagocytosis by PMN leukocytes (3). Information derived from studies of leukocytes from patients with chronic granulomatous disease and patients with myeloperoxidase deficiency has emphasized the importance of H202 and myeloperoxidase in the bactericidal mechanisms of PMN leukocytes and has been recently reviewed (13, 16). Phagocytosis by alveolar macrophages is also associated with increased oxidative metabolism (8, 23), hydrogen peroxide production (8, 22), but no superoxide anion production (6, 7). Although alveolar macrophages are not known to possess a PMN leukocyte type of peroxidase (myeloperoxidase), peroxidase-like activities have been reported. Catalase is associated with the 100,000 x g pellet obtained by differential

san particles (774 + 86 counts/min) was increased very little compared with iodination by resting alveolar macrophages (730 ± 106 counts/min). Although iodination by resting PMN leukocytes and alveolar macrophages was comparable in these experiments, during phagocytosis the alveolar macrophages fixed less than 1% of the 125I bound by PMN leukocytes. This marked difference in iodination could not be explained by poor phagocytosis of zymosan, since greater than 90% of the alveolar macrophages had ingested two or more zymosan particles during the incubation period. The failure of alveolar macrophages to iodinate zymosan could be due to insufficient peroxidase and/or hydrogen peroxide within the phagocytic vacuole. Iodination of peroxidasecoated zymosan particles by alveolar macrophages was studied in order to test the possibility that peroxidase was limiting. After inges- TABLE 4. Quantitative tetrazolium dye reduction by tion of peroxidase-coated zymosan particles, no alveolar macrophages significant increase in iodination by alveolar Optical density increment macrophages was observed (Table 3). Effective Cells Mean + standard coupling of peroxidase and preservation of enRange error ero zyme activity within the alveolar macrophages 0.003-0.040 was confirmed by light (12) and electron (9) Alveolar macrophages 0.020 + 0.007 (n = 5) microscopy. Finally (Table 3), iodination by PMN 0.162 - 0.216 leukocytes (n = 0.198 ± 0.011 peroxidase-coated zymosan particles in the ab5) sence of phagocytic cells was approximately as increment at 515 nm per 20 min per 5 x 10" ninefold that of control zymosan when H202 cellsaMeasured from duplicate samples, with and without latex partiwas added at a final concentration of 1 ,umol/ cles.

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centrifugation of rabbit alveolar macrophages homogenates and is transferred to the phagolysosome during phagocytosis (29). Because catalase is known to act as a peroxidase at low concentrations of H,20, a bactericidal mechanism similar to the myeloperoxidase-H202-halide system for PMN leukocytes was suggested by Gee et al. (8). A peroxidase activity resembling that of horseradish peroxidase has been demonstrated in the 20,000 x g fraction of alveolar macrophage homogenates. This pellet, in combination with H202 and iodide, but not chloride, was shown to be bactericidal for E. coli (23). To examine the role of catalase, the bactericidal capacity of alveolar macrophages was assessed in the presence of aminotriazole or sodium nitrite, known inhibitors of catalase activity. The concentrations of aminotriazole used in these experiments were shown by Gee et al. (8) to cause a marked suppression of phagocytosis-stimulated oxidative metabolism by alveolar macrophage, indicating a role for catalase in peroxidative metabolism. However, in our studies (Table 2), the addition of aminotriazole in concentrations as high as 100 mM did not inhibit the bactericidal capacity of alveolar macrophages. These observations were not due to a toxic effect of aminotriazole on the bacteria, since these concentrations of aminotriazole did not inhibit bacterial growth. The burst of oxidative metabolism and degranulation accompanying phagocytosis by PMN leukocytes is thought to provide the agents necessary for bacterial killing with the phagocytic vacuole. Hydrogen peroxide alone has some bactericidal activity, but when hydrogen peroxide acts in concert with myeloperoxidase and a suitable oxidizable substance (iodide, chloride), this bactericidal activity is markedly enhanced (14). When the oxidizable substance is 125I, an iodination reaction occurs (24). Leukocytes that are deficient in myeloperoxidase (17, 18) or hydrogen peroxide (10, 18) kill bacteria less effectively than do normal leukocytes and fail to iodinate ingested bacteria (17). Iodination by intact PMN leukocytes can be quantitated by the conversion of 125I to a trichloroacetic acid-precipitable form (24). Since iodination of microorganisms occurs within the phagocytic vacuole and requires myeloperoxidase, hydrogen peroxide, and 1251, this reaction could provide a probe to examine some of the biochemical events taking place within the phagocytic vacuole. The observed failure of rabbit alveolar macrophages to iodinate zymosan particles (Table 3) could be the result of insufficient peroxidase content or insufficient hydrogen peroxide pro-

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duction. The observation might also be explained by an inefficient delivery of hydrogen peroxide and/or peroxidase to the phagocytic vacuole after particle ingestion. To evaluate these possibilities, the cells were allowed to ingest peroxidase-coated zymosan particles and iodination was examined after inserting peroxidase into the phagocytic vacuole. If significant iodination occurred, then the absence of stimulated iodination after ingestion of control zymosan might be explained by a deficiency of peroxidase or peroxidase-like (catalase) enzyme in the phagocytic vacuole. As shown in Table 3, no significant increase in iodination was observed after phagocytosis of peroxidase-coated particles, suggesting that hydrogen peroxide may be rate limiting for the iodination reaction. Tetrazolium dye reduction may provide a second metabolic probe for examining the phagocytic vacuole. When PMN leukocytes ingest bacteria or zymosan in the presence of tetrazolium dyes, the dye enters the phagocytic vacuole with the particle and is reduced to a blue formazan precipitate (21). Tetrazolium dye reduction appears to be due to superoxide anion production during respiration by PMN, since PMN leukocytes, depleted of superoxide anion by the introduction of superoxide dismutase, reduce 60% less dye (2). PMN leukocytes of patients with chronic granulomatous disease do not increase oxygen consumption after phagocytosis (13) and do not show increased tetrazolium dye reduction (3). Since studies of the capacity of alveolar macrophages to iodinate ingested particles suggested that hydrogen peroxide was not readily available to the phagocytic vacuole, tetrazolium dye reduction by alveolar macrophages was investigated. No increase in neotetrazolium dye reduction was measurable after phagocytosis by alveolar macrophages. These observations were confirmed by histochemical estimation of nitroblue tetrazolium dye reduction (unpublished data). These data suggest that although there is increased oxidative metabolism by alveolar macrophages after phagocytosis (22, 29), the products of the respiratory burst, hydrogen peroxide and superoxide anion, may not be readily available to the phagocytic vacuole for bacterial killing. The results further indicate that a catalase-mediated bactericidal reaction is not essential in the antibacterial mechanism(s) of the rabbit alveolar macrophage. Since alveolar macrophages ingest and kill bacteria but may do so relatively independent of peroxide-mediated reactions, alternative mechanisms of bacterial killing by alveolar macrophages should be considered.

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ACKNOWLEDGMENTS This investigation was supported by grants from the Medical Research Council of Canada (MA 5276), Public Health Service (HL06315 from the National Heart and Lung Institute), and the National Science Foundation (33923). W.D.B. is a Medical Research Council Scholar. We acknowledge the excellent technical assistance of Kim-Yin Wong.

neutrophilic polymorphonuclear leukocytes. Semin. Hematol. 12:117-142. 17. Klebanoff, S. J., and C. B. Hamon. 1972. Role of myeloproxidase-mediated antimicrobial systems in intact leukocytes. J. Reticuloendothel. Soc. 12:170-196. 18. Klebanoff, S. J., and S. H. Pincus. 1971. Hydrogen peroxide utilization in myeloperoxidase-deficient leukocytes: a possible microbicidal control mechanism. J. Clin. Invest. 50:2226-2229. 19. Lehrer, R. I. 1969. Antifungal effects of peroxidase

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