RES-JOURNAL OF RETICULOENDOTHELIAL SOCIETY 8, 288-300 (1970)

Effect ofOzone on Acid Hydrolases ofthe Pulmonary Alveolar Macrophage1

DANIEL J. HURST,2 DONALD E. GARDNER, AND DAVID L. COFFIN 3
Division ofHealth EffectsResearch, NationalAirPollution Cont'i·ol Administration, ConsumerProtection andEnviTonmentalHealth Service, Public Health Service, U.S. Department ofHealth, Education, and WelfaTe, Cincinnati, Ohio ·
Received November 18, 1969; accepted December 3, 1969

Ozone is a common contaminant of urban atmospheres. New Zealand rabbits were exposed to various concentrations of ozone ranging from 0.25 to 7.0 ppm for 3 hr. Alveolar macrophages

were procured by lavage and purified by centrifugation. Low levels of ozone depressed the activity of acid phosphatase, beta glucuronidase, and lysozyme in the macrophage. T\venty-four hours after a 3-hr exposure, the enzymatic activities returned to almost normal. After exposure to ozone, the macrophages exhibited an intracellular rearrangement of hydrolytic activity with the lysosomal fraction losing and the soluble cellular extract gaining activity.

The possible significance of these results to the ultimate health of animals existing in a polluted atmosphere is discussed.

INTRODUCTION The pulmonary alveolar macrophage is an actively phagocytic cell found in the pulmonary alveoli (Cappell, 1929; Low, 1953; Moore and Schoenberg, 1964). It has been considered by several investigators to contribute a vital element of the lung's defense mechanism against both inert particles and viable organisms (Robertson, 1941; Casarett and Milley, 1964; Green and Kass, 1964). It is known, for example, that the alveolar macrophage is

a labile cell that responds morphologically and biochemically in

1 The authors acknowledge the excellent technical assistance of Mr. Joseph Illing and Mrs. Betty Williford.

2 Present address: Department of Medicine, Division of Environmental Medicine, Duke University Medical Center, Durham, N.C.

sAlso associated with the Departments of Pathology and Environmental Health, University of Cincinnati, School of Medicine.

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various ways to the presence of either a "normal" bacterial or particulate challenge or to the presence of a gaseous noxious agent (Pavillard and Rowley, 1962; Green and Carolin, 1967; Green, 1968). One element of this response involves the hydrolytic enzyme system found in the macrophage. Cohn and Wiener (1963) have convincingly demonstrated that selected hydrolases from alveolar macrophages sediment together and respond with increased enzymatic activity to stimulation by BCG. Others have shown that the act of phagocytosis by the macrophage raises the cellular hydrolase activity (Sbarra and Karnovsky, 1959; Myrvik 

### et al., 1962; Saito and Suter, 1965). Not only is selected hydrolase activity enhanced by phagocytosis, but an intracellular rearrangement occurs whereby the hydrolytic enzymes are transferred from lysosomal granules to phagocytic vacuoles (Cohn and Wiener, 1963). Thus intracellular hydrolysis seems to be a vitally important mechanism in the degradation ofphagocytosed material. Ozone is a common contaminant of urban atmospheres, developing with photochemical smog as a pollutant when automobile exhaust is subjected to solar radiation. It has been shown that exposure to ozone and photochemical smog enhances mortality from pathogenic bacteria introduced into the lungs and that there is a reduction in both number and phagocytic competence of alveolar phagocytes related to such exposure (Coffin 

### et al., 1968a, b).

Because the hydrolytic enzyme system appears crucial in the response of the macrophage to ingested particulate matter, it

was felt that the effect of ozone on this particular enzyme system should be studied. This report describes the 

### in vivo effect of various atmospheric concentrations of ozone upon three representative hydrolytic enzymes of the pulmonary alveolar macrophage--acid phosphatase, lysozyme, and beta glucuronidase.

MATERIALS AND METHODS**Anin~als.** New Zealand rabbits of both sexes, weighing between 1.5-2.0kgwere used in all experiments. 

### Exposure. Ozone was generated by passing oxygen through .a
neon tube silent-arc generator. The resulting effluent was mixed with filtered room air and delivered to stainless steel animal ·exposure chambers at a rate that insured one complete chamber atmosphere change per minute. Ozone concentrations were

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measured by a standard wet method (Saltzman, 1959). The period of exposure was in all cases 3 hr. Collection of alveolar macrophages and preparat' ion of cell
extracts. Alveolar macrophages were collected by a modification of the procedure of Myrvik et al. (Myrvik et al., 1961; Coffin et al., 1968a). All glassware was siliconized where whole cells were being processed, and the cellular suspensions were maintained at 4°C. After the pulmonary lavage fluid containing variable numbers of macrophages, lymphocytes, erythrocytes, and polymorphonuclear leukocytes was spun at 500g for 20 min at 4°C, the sedimented cells were resuspended in 2 cc 0.9% saline. To obtain pure macrophages from the solution containing a mixed population of cells, this resuspension was carefully layered over 5 cc of isotonic bovine albumin, pH 7.4 (Path-o-cyte 2, Pentex Inc.), contained in a 15-cc conical centrifuge tube (Roberts et al., 1958; Leake and Myrvik, 1961).

The specific gravity of the stock albumin solution had been previouslyadjustedto 1.062byuse oftheformula:



### DaVa 

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### D =

_____ __:_ 

### Va 

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### D = specific gravity desired 

### Da = specific gravity of the stock albumin solution 

### Va = volume of albumin added Db = specific gravity of the isotonic saline Vb = volume of saline to be added (Roberts 

### et al., 1958).

It was found that the interface between the cellular suspension and the albumin should be slightly turbid to effect a better separation. Thetubes were then spun in a swingingbucket rotor (International No. 240) at 650g for 15 min. This afforded an excellent separation, with the macrophages remaining at the interface while the neutrophils, lymphocytes ,and erythrocytes sedimented to the bottom of the tube. Using a siliconized Pasteur pipette, the interface layer was removed, washed twice with 0.9% saline, and resuspended to a final volume of 3 cc. At this point white cell counts were determined using disposable blood-diluting pipettes (Unopette, Becton, Dickinson and Co.) and a hemacytometer. Smears for differential counts were fixed and stained with Giemsa's solution. By the selective flotation 1 

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### 291
technique described above, an initial lung lavage solution which contained a mixture of macrophages, neutrophils, lymphocytes, and erythrocytes could be fractionated into two parts, one of which contained, on an average, 92% macrophages. This is the macrophage fraction which was used in all enzyme determinations. The macrophage suspensions were adjusted to contain 2 X 

### 107 

### cells in a volume of 3 cc, and this suspension was subjected to sonic oscillation at 20,000 Hz for 45 sec. The tube containing the cells was held in an ice bath at 2°C at all times. Phase contrast microscopy of this sonicate revealed virtually complete cellular lysis. The sonicate was cleared of disrupted cellular membranes, unbroken cells, and nuclei by centrifugation at 1000g for 10 min, and the resulting supernatantwas used for enzymatic analysis. In the series of experiments designed to investigate the subcellular localization of acid hydrolytic activity, the macrophages

were resuspended in 0.25 M 

### sucrose and homogenized at 4

## oc in

a Teflon and glass system for three 2-min periods separated by

a 1-min rest. The quality of the homogenization procedure was checked by phase contrastmicroscopy. An aliquot ofthe original homogenate was frozen and thawed six times and reserved for enzymatic analysis. The remaining homogenate was centrifuged at 15,000g (Sorvall Rotor HB-4) for 15 min. This resulted in a 15,000g pellet and supernatant, each of which was brought to volume, frozen, and thawed six times and an aliquot used directly for enzymatic analysis. 

### EnzymaticAnalysis
Acid phosphatase was measured by the method of Hofstee (1954). The assay mixture contained 0.1 cc cellular extract, 2.0

cc 0.2 M 

### acetatebuffer (pH 5.1), 0.4 cc water, and 0.5 cc 3.65 X 

### 10-a M 

### o-carboxyphenylphosphate (Worthington Biochemical Co.)

. Appropriate reagent and tissue blanks were used. The liberation of salicyclic acid was followed at 298 mp. 

### for 20 min at 25°C. Results were expressed as net change in absorbance per minute permillion macrophages. Lysozyme was assayed by a modification ofthe method of Litwack (1955). The reaction mixture consisted of 0.5 cc cell extract and 2.5 cc .15 M 

### phosphate buffer (pH 6.2) in 0.9% saline containing 0.25 mgjcc 

### Micrococcus lysodiekticus. The clearing of the bacterial suspension was followed at 645 mp. 

### for292 HURST, GARDNER, AND COFFIN

5 min at 25°C. The lysozyme concentration was determined by comparing the change in percentage transmission of the assay mixture between 30 and 60 sec with a standard solution of egg white lysozyme. Beta glucuronidase was determined by the method of Fishman 

### et al. (1948). The assay mixture contained 0.4 cc cell extract, 1.0

cc 0.1 M acetate buffer (pH 4.5)
, and 0.1 cc 0.01 M phenolphthalein glucuronic acid (Sigma Chemical Co.). The reaction

was allowed to proceed for 30 min at 37°C and then terminated by the addition of 1.0 cc 5% trichloroacetic acid. The precipitation was cleared by centrifugation, 4.0 cc glycine buffer (pH 10.0) was added, and the solution was brought to a final volume of 8.0 cc. The liberated phenolphthalein was measured at 545 mp. in Bausch & Lombe Spectronic 20 colorimeter. Values are expressed as micrograms phenolphthalein liberated per million cells.

RESULTS Activities of acid phosphatase, lysozyme, and beta glucuronidase were determined for alveolar macrophages from normal rabbits and from rabbits which had been exposed to various concentrations of ozone. In the first series of experiments the exposure was for 3 hr followed by immediate sacrifice and enzyme assay. In the second series two groups of animals were exposed to two different levels of ozone (3.0 and 7.0 ppm) for 3 hr, removed to room atmosphere for 24 hr, and sacrificed; the enzyme assays were then performed. In all experiments each control and each exposed group contained six animals, and there

was no pooling of specimens. In Table 1 it is seen that for each of the macrophage hydrolytic enzymes studied there was a significant reduction in activity in the animals which had been exposed to ozone as compared with controls. This decrease in activity appears dose dependent. A reduction was noted as low as 0.25 ppm ozone with the maximal depression occurring at approximately 3.0 ppm ozone. In the cellular preparations as studied, lysozyme activity was reduced most, followed by acid phosphatase and beta glucuronidase. Following a 3-hr exposure at 3.0 and 7.0 ppm ozone then removal to ambient atmosphere for 24 hr, the enzymatic activities returned almost to normal. Figure 1 graphically represents the effect of ozone upon the hydrolases studied. The control group is represented as 100% 

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- GLUCURONIDASE

tA. ACID PHOSPHATASE

tA. LYSOZYME

FIG. 1. Effect of various levels of ozone on the beta glucuronidase, acid phosphatase, and lysozyme activities of the pulmonary alveolar macrophage. Each point represents mean of six animals.

activity or "normal" while any change in the experimental group is represented as a percent of control. The activity of all three enzymes is seen to fall rapidly upon exposure to quite low concentrations of ozone and then to reach maximal depression at approximately 50% of normal activity at 3 ppm ozone. Figure 2 shows the return toward normal hydrolase activity of the group that was returned to ambient atmosphere after exposure to

ozone. In all cases except acid phosphatase from macrophages exposed to 7.0 ppm ozone there was over 90o/o recovery of activity at 24 hr. The effect of ozone upon the intracellular distribution of the hydrolytic enzymes studied is seen in Fig. 3. For each level of

ozone there is shown the percentage of total enzymatic activity that resides in both the 15,000g supernatant and particles. Total enzymatic activity was taken as the activity found in the original homogenate after freezing and thawing six times. The norll!al or nonexposed group is represented at zero ppm ozone. The intracellular distribution between the 15,000g supernatant and particles in the normal animals agrees well with the results of Cohn and Wiener (1963) who used BCG-induced alveolar macrophages. The 15,000g pellet contains some unbroken cells in

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ACID PHOSPHATASE

HOURS AFTER 3-hr EXPOSURE, 7 ppm FIG. 2. Hydrolytic enz:vme activity of the pulmonary alveolar macrophage immediately and 24 hr after an acute 3-hr in vivo exposure to ozone. Each bar represents mean of six animals.

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ozone. Each bar represents mean of six animals.296 HURST, GARDNER, AND COFFIN

addition to nuclei, mitochondria, and lysosomes, but the lysosomal fraction has been shown to contain neariy all intracellular acid hydrolytic activity (Cohn and Wiener, 1963) so no further organelle separation was attempted. Recovery of enzymatic activity as compared with the original homogenate averaged 93%. Exposure of alveolar macrophages 

### in vivo to ozone caused an intracellular rearrangement of acid hydrolytic activity. With increasing concentrations of ozone the activity residing in the 15,000g (lysosomal) pellet diminishes and is found in higher concentrations in the supernatant. Lysozyme appears to be the most easily displaced, while the activity of beta glucuronidase in the 15,000g particles is lowered by only 38%. The shift in location of activity appears to be an actual rearrangement in subcellular localization rather than a simple inactivation as the activity in the supernatant fraction is rising as that in the particulate isfalling.

DISCUSSION The data presented here indicate that atmospheric ozone reduces the activity of three selected acid hydrolases found in pulmonary alveolar macrophages and that removal from an atmosphere containing ozone causes the activity to be restored. In addition ozone causes a subcellular rearrangement of hydrolytic activity with the lysosomal fraction losing activity and the supernatant gaining. It is significant, we believe, that atmospheric levels of ozone
as low as 0.25 and 0.50 ppm have an effect upon**th~** enzymes studied. These levels are equivalent to the total atmospheric oxidant concentration, which is composed primarily of ozone and found in some instances of photochemical smog. Ithas been well documented that the average oxidant (ozone) concentration for

an 8-hr period on a day with severe air pollution in Los Angeles is 0.2 ppm with maximal levels of 0.9 ppm (Miller 

### et al., 1956). The question arises as to whether this lowering of hydrolytic enzyme activity in an actively phagocytic cell is related to the increased susceptibility to infection and mortality found in animals that have been exposed to low levels of ozone then challenged with an aerosolized bacteria (Purvis 

### et al., 1961; Coffin 

### et al., 1968b).As previouslymentioned, the acid hydrolyticenzymes
arefound in high levels in phagocytic vacuoles and in lysosomes. If indeed ozone causes a .e:eneralized reduction in the activity of 

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### enzymes involved in the intracellular degradation of inhaled organic particulate matter, then this could be a significant factor in explaining the above-mentioned increases in infection and mortality. Extending this concept to human pulmonary disease and itsrelationship to air pollutants would not seem out of place; and the implications involved, we believe, are quite significant. Recently, in fact, Roque (1968) 

### has shown histochemically that alveolar macrophages from the lungs of chronic cigarette smokers contain less hydrolase as well as oxidoreductase activity whencompared with a group of nonsmokers. Purvis (1961) 

### has shown in mice that a bacterial challenge 27 

### hr after a 3-hr exposure to ozone produces mortality identical with that of a control group. With this in mind, it was of considerable interest to learn in our studies that the hydrolase activity in the macrophage returns almost to normal 24 hr after exposure to ozone. Whether this return to normal activity is a reflection of a reversible enzyme inhibition, a new synthesis of enzyme, or simply an influx of a nevv population of cells into an injured lung is not clear at this time. Preliminary studies in this laboratory, however, indicate that oxygen consumption of macrophages previously exposed to ozone as measured by the Warburg technique is considerably higher than nonexposed cells. This could represent an intracellular reparative process as a reaction to injury. If this were the case, it seems logical to

assume that protein (enzyme) synthesis would be increased. Further, Cohn and Benson (1965) 

### have indicated that hydrolytic enzymes in macrophages cultured 

### in vitro appear to be degraded and resynthesized rapidly while Heise and Myrvik (1967) 

### reported that nonphagocytosing pulmonary alveolar macrophages cultured 

### in vitro rapidly synthesize lysozyme and release it into the extracellular medium. Inhibitors of RNA and protein synthesis depress this production and release suggesting

a 

### de novo synthesis bythe macrophage. The loss of activity of acid phosphatase and lysozyme seemed to parallel each other while beta glucuronidase was apparently less susceptible to the noxious agent. This may reflect the stronger binding of beta glucuronidase by some component of the lysosome. Isenberg and Stjernholm (1968) 

### have shown that detergents will release appreciable amounts of hydrolytic activity from polymorphonuclear leukocyte lysosomes, but that heparin is needed to liberate significant amounts of beta glucuronidasef lfolzman, Gardner, and Coffin (1968) 

### have shown 

### a

### 298 liURST, GARDNER, AND COFFIN

C65% decrease in the lysozyme released from rabbit alveolar macrophages previously exposed to 10 ppm ozone and held 

### in ·" tissue culture media. Our studies show an analogous reduction in the intracellular lysozyme activity of a pure population of

macrophages. The fact that ozone apparently causes a loss of hydrolytic activity from lysosomes and an increase in activity in the nonly-
_., sosomal fraction raises interesting possibilities as to the mecha. 1

· :nism of ozone toxicity upon the macrophage. It seems unlikely \ that elemental ozone would have a direct effect upon intact f 

# •· jntracellular lysosomal membranes by the very nature of its rel- 1**~atively** unstable configuration and the fact that it would be • reduced on contact with extracellular fluid and with cellular

### ~ membrane constituents. :More plausible would be the production r of a toxic reaction product from the association of ozone with 

### f either the cell membrane or an extracellular substance, which is \( then pinocytosed or actively transported across the cell membrane. This product could then damage the lysosome and cause ( release of the contained enzymes, or could alter the intracellular · metabolism and cause a shunting of the energy pathways in

... [ such a manner thatthe integrity of the lysosomal particles could not be maintained. Whether this is identical with or related to the process of autophagy remains to be seen. 

# r If the cell membrane itself were damaged, the lowering of 

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### in vitro in the absence of phago-

. cytosis secrete lysozyme and acid phosphatase into the culture mediumbutarenotthemselves depleted ofhydrolytic activity. Our present studies show an intracellular reduction in hydrolytic activity after an 

### in vivo exposure to ozone. Further experi- ; ments are in progress designed to investigate both intra- and 

### J extracellular hydrolase activity of the alveolar macrophage after '1

an 

### in vitro exposure to ozone. This should give information concerning a possible cellular membrane effect. Evidence pointing toward a cell membrane insult by ozone are the unpublished observations of Dowell (1969) showing an increased alveolar macrophage osmotic fragility after an 

### in m:vo exposure to ozone. Since the alveolar macrophage is important not only in the lungs' defense against inhaled particulates, but also in interferon production (Acton and Myrvik, 1966) 

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of antibody synthesis (Nossal and Austin, 1966; Fishman 

### et al., 1963; Uhr and Weissmann, 1965), any alteration of its function by a noxious agent (pollutant) could prove perilous for the ultimate health of the animal.

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Acton, J., and Myrvik, Q. N. (1966). Production of interferon by alveolar

macrophages. J. Bacteriol. 91, 2300-2304. Cappell, D. F. (1929). Intravital and supravital staining. III. The nature

of the normal lining of the pulmonary alveoli and the origin of the alveolar phagocytes in the light of vital and supravital staining. J . Pathol. Bacteriol. 32, 675-707. Casarett, L. J., and Milley, P. S. (1964). Alveolar reactivity following

inhalation of particles.Health Phys. 10, 1003-1011. Coffin, D. L., Gardner, D. E., Holzman, R. S., and Wolock, F J. (1968a).

Influence of ozone on pulmonary cells. Arch. Environ. Health 16, 633-636. Coffin, D. L., Blommer, E. J., Gardner, D. E., and Holzman, R. (1968b).

Effect of air pollution on alteration of susceptibility to pulmonary infection. In "Proceedings of the 3rd Annual Conference on Atmospheric Con**ta~ination** in Confined Spaces," pp. 71-80. Aerospace Medical Research Laboratories, Wright-Patterson Air Force Base, Ohio. Cohn, Z. A., and Wiener, E. (1963). The particulate hydrolases of macrophages I. Comparative enzymology, isolation and properties. II. Biochemical and morphological response to particle ingestion. J. Exp. Med. 118, 991-1009. Cohn, Z. A., and Benson, B. (1965). The ,in vitro differentiation of mononuclear phagocytes. III. The reversibility of granule and hydrolytic enzyme formation and the turnover of granule constituents. J. Exp. Med. 122, 455-466. Dowell, A. R., Lohrbauer, L. A., Hurst, D. J., and Lee, S. D. (1969).

Unpublished observations. Fishman, M., Hammerstrom, R. A., and Bond, V. P. (1963). In vitro transfer of macrophage RNA to lymph node cells. Nature London 198, 549. Fishman, W. H., Springer, B., and Brunetti, R. (1948). Application of an

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by glutathione and cysteine. Science 162, 810-811. Heise, E. R., and Myrvik, Q. N. (1967). Secretion of lysozyme by rabbit

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ofphosphomonoesterases.Arch.Biochem. Biophys. 51, 139-145.I ' 300 HURST, GARDNER, AND COFFIN

Holzman, R. S., Gardner, D. E., and Coffin, D. L. (1968). In 'Vivo inactivation of lysozyme by ozone. J. Bacterial. 96, 1562-1566. Isenberg, J. N., and Stjernholm, R. L. (1968). Selective release of a lysoso- (

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. loendothel. Soc. 5, 556-557. Leake, E. S., and Myrvik, Q. N. (1961). Lysozyme content of**~**

epithelioid-like cells recovered from the pulmonary alveoli of rabbits 

### J undergoing a granulomatous response. Atti II Symposium Internaztion- 1 ale sul Lisozima di Fleming, Vol. 1, Sec. 4, p. 35-37. Cesano Boscone,**r~** Milan, Italy. Litwack, G. (1955). Photometric determination of lysozyme activity. PrfJc.

Soc. Exp. Biol. Med. 89, 401-403. Low, F. N. (1953). The pulmonary alveolar epithelium of laboratory mammals and man. Anat. Rec. 117, 241-263.

1111 Miller, A., Brown, C., and Frankson, K. D. (1956). Photochemical reactions 1

of nitrogen dioxide and hydrocarbons in air. Science 123, 934.**~** Moore, R. D

., and Schoenberg, M. (1964). Alveolar lining cells and pulmo- {.

nary reticuloendothelial system of the rabbit. Amer. J. Pathol. 4!l, 991-1006. Myrvik, Q. N., Leake, E. S., and Fariss, B. (1961). Studies on pulmonary t

alveolar macrophages from the normal rabbit: a technique to procure L them in a high state of purity. J. Immunol. 86, 128-132. 

# r
Myrvik, Q. N., Leake, E. S., and Oshima, S. (1962). A study of macro-r

...

_ , phages and epithelioid-like cells from granulomatous (BCG-induced) lungs of rabbits. J. Immunol

.. 89, 745-751. Nossal, G. J. V., and Austin, C. M. (1966). Mechanism of induction of

immunological tolerance, II. Aust. J. Exp. Biol. Sci. 44, 327-340. 

## r Pavillard, E. R. J., and Rowley, D. (1962). A comparison of the phagocytic J

and bactericidal ability of guinea pig alveolar and mouse peritoneal

_ r _. macrophages. Austr. J. Exp. Biol. Sci. 40, 207-214. > Purvis, M. R., Miller, S., and Ehrlich, R. (1961). Effect of atmospheric pol- 

### I

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### i

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### ~~

phagocystosis. I. Metabolic changes during the ingestion of particles by polymorphonuclear leukycytes. J. Biol. Chem. 234, 1355-1362. Uhr. J. W., and Weissmann, G. (1965). Intracellular distribution and degradation of bacteriophage in mammalian tissues. J. Immunol. 94, 544-550.**ctiva~**

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## { Announcement The Seventh National Meeting of the Reticuloendothelial Society will be held at the Augusta Town House in Augusta, Georgia, December 2-5, 1970. The keynote address will be deliveredbyDr. Richmond T. Prehn, Institutefor CancerResearch, Philadelphia. In addition to contributed papers, the meeting will include n symposium on "Current Concepts of Acquired Cellular Immunity" with discussion of the following topics: Antitissue vs. antimicrobial cellular immunity, Nature and biological activities lymphotoxins of animals and man, Lymphocyte products in delayed hypersensitivity, Mechanisms in antimicrobial cellular immunity, and the Role of lymphocytes and other factors in antimicrobial cellular immunity. A second symposium, "Immune Responses to Neoplasms," is also scheduled with emphasis on the following topics: Immunologic regulation ofinduced autochthonous neoplasms, Enhancing antibodies in human and animal neoplasms, Immunologic studies of human neoplasms, and Immune responsesto humanmelanomas. The deadline for submitting abstracts is September 14. For further information please write to the meeting chairman, Dr. Sherwood 

### M. Reichard, Medical College of Georgia, Augusta, Georgia 30902.

301