Vol. 21, No. 2
IMMUNITY, Aug. 1978, p. 669-671
0019-9567/78/0021-0669$02.00/0 Copyright i 1978 American Society for Microbiology
Printed in U.S.A.
Effect of Phorbol Myristate Acetate on Cellular Metabolism and Lysozyme Release from Alveolar Macrophages and Polymorphonuclear Leukocytes W. DOUGLAS BIGGAR Departments of Immunology and Pediatrics, University of Toronto, The Hospital for Sick Children, Toronto, Ontario, M5G 1X8 Canada Received for publication 22 February 1978
Phorbol myristate acetate stimulated oxidative metabolism in alveolar macrophages and blood neutrophils. This compound also stimulated lysozyme release from neutrophils but not from alveolar macrophages. These findings suggest that the regulation of lysozyme release from alveolar macrophages is different than for polymorphonuclear leukocytes.
Specific and azurophil granules of neutrophils contain different lysosomal enzymes. In development, the azurophil granules mature before specific granules in the neutrophil precursor cells within bone marrow (2, 3). Following particle ingestion, specific granules fuse first with phagocytic vacuoles, followed by azurophil granules (1). The lysosomal granules of alveolar macrophages are poorly defined in their composition and behavior during phagocytosis. They lack a peroxidase-containing granule similar to the azurophil granule in neutrophils (5), but contain large quantities of lysozyme. It is not known which granules contain lysozyme or what factors regulate its release from granules. Phorbol myristate acetate (PMA) stimulates many of the functional and metabolic changes that normally accompany phagocytosis (7, 13). Neutrophils exposed to PMA have increased oxidative metabolism and release the contents of specific granules, including lysozyme, extracellularly. This probe has been particularly helpful for studying the signals regulating neutrophil metabolism and lysosomal degranulation. We report here that PMA causes increased oxidative metabolism in alveolar macrophages but does not result in lysosomal enzyme release. Alveolar macrophages were ravaged from rat lungs, and neutrophils were isolated from peripheral blood as previously described (6). Greater than 95% of the cells ravaged from rat lungs were macrophages, as judged by electron microscopy. Leukocyte preparations contained 55 to 65% neutrophils, the remaining cells being monocytes, lymphocytes, and platelets. Hexose monophosphate pathway activity and oxygen consumption in challenged and unchallenged cells were determined as described previously (8). For lysozyme quantitation, cells were
washed and resuspended in Hanks balanced salt solution without serum and phenol red. The cells were incubated with and without PMA for 30 min at 37°C and then centrifuged at 250 x g. The supernatant was saved for determination of lysozyme release. The cell pellet was resuspended in 0.2 M sucrose plus 0.1% Triton X-100, homogenized with a motor-driven Teflon pestle for 5 min at 4°C, and frozen (acetone-dry ice) and thawed five times. Enzyme activity in this fraction was designated as cell associated. Enzyme activity in the supernatant, expressed as a percentage of the total enzyme activity (supernatant plus cell pellet), represented enzyme release. Lysozyme and lactic dehydrogenase were assayed as previously described (4, 6). PMA (12- O-tetradecanoyl-phorbol- 13-acetate; molecular weight, 616; Consolidated Midland Corp., Chemical Division, Katonah, N.Y.) was dissolved in dimethylsulfoxide at a concentration of 1 mg/ml and kept frozen in small aliquots at -70°C for future use. For each experiment, a fresh aliquot was thawed and diluted with 0.1 M acetic acid-sodium acetate buffer (pH 6.5) to 10 times the desired final concentration. Cells were exposed to PMA at a concentration of 0.1 ng/ml. Control cells, suspended in Hanks balanced salt solution, were exposed to the same volume of the acetate buffer alone. The effect of PMA on the hexose monophosphate pathway is reflected by the oxidation of [1-'4C]glucose by alveolar macrophages and blood neutrophils is summarized in Table 1. Glucose oxidation by unchallenged alveolar macrophages was significantly higher than glucose oxidation by neutrophils. When both cell populations were exposed to latex particles (0.8,m diameter), glucose oxidation increased. The addition of PMA (0.1 ng/ml) to resting neutro669
phils increased glucose oxidation to a greater extent than that observed following challenge with latex particles. Alveolar macrophages exposed to PMA showed a smaller increment over latex stimulation; PMA produced a significant increase in oxygen consumption in both alveolar macrophages and blood neutrophils compared to resting cells (data not shown). The release of intracellular lysozyme from alveolar macrophages and neutrophils incubated with and without PMA is summarized in Table 2. When neutrophils were incubated with PMA, greater than 25% of the intracellular lysozyme was released. This was not associated with an increased release of the cytoplasmic marker lactic dehydrogenase. Exposure of alveolar macrophages to 0.1 ng of PMA per ml did not result in an increased release of lysozyme. Similar observations were made when the concentration of PMA was increased to 1 ,ug/ml. The intracellular distribution of hydrolytic enzymes in alveolar macrophages has not been clearly defined. Monocytes, as precursor cells for alveolar macrophages, contain at least two difTABLE 1. Effect of PMA on hexose monophosphate pathway activity of alveolar macrophages and blood neutrophils Hexose monophosphate pathway activitya Cells
phages 626 ± 107 3,159 ± 1,516 Resting cells + Latex 5,962 ± 3,047 8,623 ± 6,058 + PMA (1 ng/ml) 7,209 ± 2,060 8,908 ± 4,216 'Cells were incubated for 20 min; hexose monophosphate pathway activity was calculated as the amount of '4CO2 liberated from [1-'4C]glucose and expressed as counts per minute per 20 min per 5 x 106 cells. Each value represents the mean ± standard deviation of at least three experiments. TABLE 2. Effect of PMA on lysozyme release from alveolar macrophages and blood neutrophiW' Percent lysozyme released Cells
7.4 ± 1.6 3.2 ± 3.1 Resting cells + PMA (1 ng/ml) 7.6 ± 1.4 26 ± 8.4 aCells were incubated for 30 min, and the lysozyme release was calculated. Enzyme activity in the supernatant, expressed as a percentage of the total enzyme activity (supernatant plus cell pellet), represented enzyme release. Total lysozyme activity for alveolar macrophages was 10.7 ± 1.7 pg (mean ± standard deviation) of egg white lysozyme equivalents per 106 cells and for neutrophils was 0.9 ± 0.3 ug of egg white lysozyme equivalents per 106 cells.
ferent populations of lysosomal granules (10). One granule population, containing peroxidase, diminishes in number as monocytes age (14). Thus, with aging and during the transition to macrophages, the intracellular distribution and packaging of lysosomal enzymes change. Many primary lysosomes in alveolar macrophages appear as small (60 to 80 nm in diameter) vesicles. These vesicles originate in the Golgi-endoplasmic reticulum-lysosomes (9), an area related to the Golgi complex and endoplasmic reticulum, where lysosomal enzymes are concentrated and packaged into primary lysozymes (7). Secondary lysosomes containing tubular myelin also react cytochemically for acid phosphatase and arylsulfatase (9). This finding suggests that primary lysosomes fuse and release enzymes into the phagocytic vacuoles. Further evidence of lysosomal fusion can be obtained from the finding that lysozyme is released extracellularly during phagocytosis by alveolar macrophages (6). However, the intracellular localization of lysozyme and the events regulating its release are unknown. PMA enhances cellular metabolism of alveolar macrophages, a phenomenon shared with neutrophils. Failure of PMA to induce lysozyme release may indicate that, although alveolar macrophages have large quantities of lysozyme, it is not packaged in a granule population similar to that in neutrophils. Alternatively, the signals regulating the intracellular traffic of lysosomal granules in neutrophils and alveolar macrophages may differ. Of importance, these findings document that the membrane signals for enhanced cell metabolism in alveolar macrophages can be dissociated from those regulating lysosomal fusion and degranulation. This work was supported by the Medical Research Council (MA5726) and the Ontario Thoracic Society. W.D.B. is a Medical Research Council Scholar.
LITERATURE CITED F. D. 1973. Sequential degranulation of the two 1. Bainton, types of polymorphonuclear leukocyte granules during phagocytosis of microorganisms. J. Cell Biol. 58: 249-264. 2. Bainton, D. F., and M. G. Farquhar. 1966. Origin of granules in polymorphonuclear leukocytes: two types derived from opposite faces of the Golgi complex in developing granulocytes. J. Cell Biol. 28:277-301. 3. Bainton, D. F., J. L Ullyot, and M. G. Farquhar. 1971. The development of neutrophilic polymorphonuclear leukocytes in human bone marrow: origin and content of azurophil and specific granules. J. Exp. Med. 134:907-934. 4. Bergmeyer, IHL, E. Bernt, and B. Hems. 1965. Lactic dehydrogenase, p. 736-743. In H. Bergmeyer (ed.), Methods of enzymatic analysis. Academic Press Inc.,
New York. 5. Biggar, W. D., and J. M. Sturgess. 1976. Peroxidase activity in alveolar macrophages. Lab. Invest. 34:3142. 6. Biggar, W. D., and J. M. Sturgeas. 1977. Role of lyso-
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zyme in the microbicidal activity of rat alveolar macrophages. Infect. Immun. 16:974-982. Estensen, R. D., J. G. White, and B. Holmes. 1974. Specific degranulation of human polymorphonuclear leukocytes. Nature (London) 248:347-348. Holmes, B., A. R. Page, and R. A. Good. 1967. Studies of the metabolic activity of leukocytes from patients with a genetic abnormality of phagocytic function. J. Clin. Invest. 46:1422-1432. Nichols, B. A. 1971. Normal rabbit alveolar macrophages. II. Their primary and secondary lysosomes as revealed by electron microscopy and cytochemistry. J. Exp. Med. 144:920-932. Nichols, B. A., and D. F. Bainton. 1973. Differentiation of human monocytes in bone marrow and blood. Lab. Invest. 29:27-40.
11. Nichols, B. A., D. F. Bainton, and M. G. Farquhar. 1971. Differentiation of monocytes. Origin, nature and fate of their azurophil granules. J. Cell Biol. 50:498-515. 12. Novikoff, A. B. 1973. Lysosomes: a personal account, p. 1-41. In H. G. Hers and F. Van Hoof (ed.), Lysosomes and storage diseases. Academic Press Inc., New York. 13. Repine, J. E., J. G. White, C. C. Clawson, and B. Holmes. 1974. Effects of phorbol myristate acetate on the metabolism and ultrastructure of neutrophils in
chronic granulomatous disease. J. Clin. Invest. 54:83-90. 14. Van Furth, R., J. G. Hirsch, and M. E. Fedorko. 1970. Morphology and peroxidase cytochemistry of mouse promonocytes, monocytes and macrophages. J. Exp. Med. 132:794-812.