The Effect of Phorbol Myristate Acetate on the Metabolism and Ultrastructure of Human Alveolar Macrophages John R. Holdal, MD, John E. Repine, MD, Gregory D. Beall, MD, Fred L. Rasp, Jr., MD, and James G. White, MD In the present investigation we examined the influence of the surface-active agent phorbol myristate acetate (PMA) and opsonized heat-killed bacteria (HKB) on oxygen consumption, superoxide release, and glucose oxidation of human alveolar macrophages (AM). Both PMA and HKB produced a surge in oxygen consumption, superoxide release, and oxidation of 1-14C-glucose and 6-14C-glucose by human AM. Examination of AM by electron microscopy following stimulation by these two agents demonstrated membrane ruffling, loss of microvilli, and increased vacuolization in PMA-treated cells and phagocytic vacuoles containing bacteria in HKB-treated cells. The vacuolization produced by PMA-treated AM was much less striking than the vacuolization produced in PMA-treated leukocytes. The similarity in the metabolic and some of the physical responses of AM stimulated by PMA and HKB suggest that PMA may be a useful agent for evaluating cell-membrane-related events of phagocytosis in AM. (Am J Pathol 91:469-482, 1978)

FOLLOWING ENGULFMENT OF PARTICULATES,

a

number of meta-

bolic and morphologic processes occur in phagocytic cells. The biochemical activities include uptake of oxygen, formation of superoxide anion, and oxidation of glucose via the hexose monophosphate shunt; physical events include uptake of particles, vacuole formation, and degranulation. '7 Changes that occur following phagocytosis are important because they are intimately associated with the bactericidal and digestive actions of these cells.2'8'9 Moreover, extracellular secretion of by-products released during the phagocytic reaction has been implicated in tissue damage.10,11 Recent investigations have shown that phorbol myristate acetate (PMA), the active principle of croton oil, can stimulate a series of reactions in phagocytic cells that closely resemble changes that occur following ingestion of particles. PMA stimulates neutrophils and monocytes to rapidly consume oxygen, form superoxide anion, and oxidize glucose. "2-14 From the Pulmonary section of the Department of Internal Medicine and the Hematology section of the Department of Pediatrics, University of Minnesota Medical School, Minneapolis, Minnesota. Supported in part by the Minnesota Medical Foundation, The Minnesota Lung Association, the Basil O'Connor Starter Research Fund of the National Foundation of the March of Dimes, Grants HL17132, HL11880, HL20695, CA11996, GM-AM-22167, AM15317, CA-08832, and the Leukemia Task Force. Dr. Repine is an Established Investigator of the American Heart Association. Dr. Rasp is a Research Fellow of the American Lung Association. Dr. Beall is a recipient of a Public Health Service Research Fellowship 1-T32-HL07009-01 Al from the National Institutes of Health. Accepted for publication February 14, 1978. Address reprint requests to Dr. John R. Hoidal, Department of Medicine, University of Minnesota Medical School, Box 148 Mayo Memorial Building, Minneapolis, MN 55455.

0002-9440/78/0608-0469$01.00

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PMA also produces large numbers of vacuoles in neutrophils and monocytes which are similar to vacuoles seen following ingestion of particulates.5612,16 In the present investigation we have examined the effects of PMA and the ingestion of bacteria on the metabolism and morphology of human alveolar macrophages (AM). The results indicate that, like its influence on neutrophils and monocytes, PMA causes appreciable increases in oxygen consumption, superoxide anion production, and glucose oxidation of alveolar macrophages. However, its effects on morphology differ significantly from those observed in blood cells after exposure to PMA.5'12"5-'7 Materals and Methods This investigation was approved by the Human Volunteers Committee of the University of Minnesota. Preparation of Alveolar Macrophages, Serum, Heat-Killed Bacteria, and PMA Alveolar macrophages were obtained by a standard technique from subsegmental bronchoscopic lavage of the lingula of the left lung or the middle lobe of the right lung of normal volunteers 6 or smokers 10 and nonsmokers 8 undergoing diagnostic fiberoptic bronchoscopy.18 Alveolar macrophages obtained in this fashion were greater than 90% viable as assessed by trypan blue exclusion and contained less than 5% neutrophils. Serums from 5 normal subjects were obtained by venipuncture, allowed to clot, rimmed, recovered by centrifugation, pooled, and frozen in aliquots at -70 C for not more than 1 month.1 Heatkilled bacteria (HKB) were prepared from twice-washed Staphylococcus aureus, 502A that had been grown for 17 hours at 37 C in Penassay broth (Difco) after heating at 70 C for 30 minutes. Suspensions of HKB were sonicated and diluted to yield final bacteria:macrophage ratios of 50: 1.12 As previously described, PMA (12-0-tetra-deconyl-

phorbol-13-acetate, MW616, Consolidated Midland Corp. Chemical Division, Dataral, N.Y.) was dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 2 mg/ml and stored at 4 C. Just before use, PMA was diluted with Hank's balanced salt solution (HBSS) to yield final concentrations which ranged from 0.1 to 10 jg/ml.' Measurement of Oxygen Consumption Oxygen consumption was measured using a biologic oxygen electrode monitor (Yellow Springs Instrument Company, Inc, Yellow Springs, Ohio, Model 53) attached to a 12-inch Beckrman recorder, as previously reported in detail.12 Briefly, the rate of oxygen uptake was

determined by measuring the maximal slope for 10 minutes from the recorder for 3.0-ml samples containing 4 X 10. AM/ml, 8% serum, and HBSS (unstimulated sample) or PMA or HKB (stimulated samples). Maximal rates of oxygen consumption were reached rapidly and were linear throughout the 10-minute period of evaluation. The results were averaged and expressed as the mean ul 02 consumed/20 min/5 X 10. AM. Measurement of Superoxide Anion Release

Release of superoxide anion (02) by AM was determined by modification of an established method which spectrophotometrically measured superoxide-dependent horse heart ferricytochrome C (Sigma Chemical Co., St. Louis, Mo.) reduction.' Reaction mixtures contained a 1-ml volume of buffered HBSS with 5 X 10. unstimulated AM (HBSS) or with AM stimulated by PMA or HKB. Paired mixtures were incubated for 20 minutes at 37 C or

Vol. 91, No. 3 June 1978

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kept on ice and used as blanks. Specificity of cytochrome C reduction was examined by using identical mixtures containing 50 ug/ml superoxide dismutase (SOD, 3000 units/mg, Truett Laboratories, Dallas, Tex.). At the completion of incubation, the reaction mixtures were kept at 4 C and centrifuged at 800g for 10 minutes. The spectrums for paired supernatants were measured at 550 nm using a spectrophotometer (Model 24, Beckman Instrument Inc., Fullerton, Calif.). Results of measurements of absorbance for 20 minutes were averaged and expressed as nanomoles of SOD inhibitable cytochrome C reduced (Fmo nm = 21 nm-1 cm-'). Measurement of Glucose Oxidation

Measurement of 1-14C- or 6-14C-glucose was undertaken using established techniques.5 Briefly, 14C02 produced by oxidation of I-14C- or 6-14C-glucose (New England Nuclear Corporation) in reaction mixtures containing 5 X 100 AM, 8% serum, and HBSS, PMA, or HKB was trapped during a 20-minute incubation (37 C) period on filter paper saturated with 20% sodium hydroxide. Incubation mixtures contained 1 Mmole glucose and 1 ,uCi of '4C as 1-14C- or 6-14C-glucose. Filter papers were air-dried and placed in scintillation counting vials with 10 ml counting fluid (1,4-bis-2-[5-phenyloxazolyl] benzene [POPOP] + 2,5-diphenyloxazole [PPO]) (Packard Instrument Co., Inc.) and counted. Controls were run to determine oxygen consumption, superoxide release, and glucose oxidation of HBSS, PMA, or HKB in the absence of AM and for AM treated with DMSO. Morphology Test samples of AM alone or after incubation with heat-killed bacteria, PMA, or DMSO were fixed for study in the electron microscope by methods previously reported.15,1920 Statistics

Statistical significance was determined by unpaired t test analysis.

Results Oxygen Consumption

Measurements of oxygen consumption were carried out on unstimulated or stimulated AM. Unstimulated AM consumed oxygen at a rate of 6.7 ± 0.7 M1120 min/5 X 106 cells (Table 1). The rate of oxygen consumption remained constant over a 10-minute period of observation. Addition of PMA increased oxygen uptake by AM. The maximum rate of oxygen utilization was observed when AM were treated with 0.5 ,ug PMA/ml. Oxygen consumption by AM stimulated with this concentration of PMA (11.6 ± 1.6Mu1/20 min/5 X 106 AM) was nearly double (P < 0.001) the rate observed for unstimulated AM. Heat-killed bacteria (HKB) had an influence similar to that of PMA on the uptake of oxygen by alveolar macrophages. AM stimulated by 50 HKB per cell consumed oxygen at a mean rate of 12.6 ± 1.7 M1 02/20 min/5 X 106 AM. This rate of oxygen utilization was significantly greater than the rate observed for unstimulated AM but was not different from the maximum rate produced by PMA-treated AM.

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Table 1-Oxygen (02) Consumption, Superoxide Anion (02-) Release, and Glucose Oxidation by Human Alveloar Macrophages Treated With PMA or HKB

Superoxide anion release (nmole Test conditions: Oxygen consump- cytochrome C tion (AIO0/20 AM, 8% serum, reduced/20 min/5 x 10' AM min/5 x 106 AM) and: HBSS

0.1jug PMA/ml 0.5;ig PMA/ml 1.0 g PMA/ml 10.0 gg PMA/ml 50 HKB:AM

6.7 ± 0.7 (11)* 8(1) 11.7 ± 1.6 (13)t 11.2 (2) 11.8 (2) 12.6 ± 1.7 (7)t

3.4

0.9(10) -

8.6

1.0(9)t -

6.3 i 0.7 (12)t

Glucose oxidation (cpm C 1402/20 min/5 x 106AM)

1-14C-glucose 738 ± 42(11) 1540(2) 2560 ± 68 (10)t 2636 (2) -

2579 ± 133 (7)t

6-14C-glucose 48

6 (13) -

212 i 32 (9)t -

130 i 20 (11)t

Mean ± SE t P