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

Effect of Ozone on Acid Hydrolases of the Pulmonary 

Alveolar Macrophage1 

DANIEL J. HURST,2 DONALD E. GARDNER, AND DAVID L. COFFIN 3 

Division of Health Effects Research, 

National Air Pollution Cont'i·ol Administration, 

Consumer Protection and EnviTonmental Health Service, 

Public Health Service, 

U.S. Department of Health, 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. 

s Also associated with the Departments of Pathology and Environmental 

Health, University of Cincinnati, School of Medicine. 

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OZONE AND MACROPHAGE HYDROLASES 289 

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 of phagocytosed 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 1.5-2.0 kg were 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 
I 

di 

290 HURST, GARDNER, AND COFFIN 

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 

previously adjusted to 1.062 by use of the formula: 

D = _____ 

DaVa + DbVb, __:_ 

Va + Vb 

where 

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. The tubes were then spun in a swinging bucket 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 

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OZONE AND MACROPHAGE HYDROLASES 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 supernatant was 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 contrast microscopy. An aliquot of the 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. 

Enzymatic Analysis 

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 acetate buffer (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 per million macrophages. 

Lysozyme was assayed by a modification of the 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. for 
292 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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TABLE 1 

EFFECT OF OZONE ON ACID HYDROLASE ACTIVITY OF THE PULMO="!ARY ALVEOLAR 

Ozone level 

(ppm) 

0 

0.25 0.50 

1.00 

3.00 

5.00 

7.00 

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(24-hr delay) 

7.00 

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Acid phosphatase 

(D.A/mh/10 6 cells) 

Meana ± SEM Range 

0.079 ± 0.008 0.049-0.105 0.070 ± 0.004 0.061-0.082 0.054 ± 0.004 0.040-0.066 

0.038 ± 0.003 0.029-0.046 0.035 ± 0.003 0.025-0.047 0.033 ± 0.001 0.029-0.036 0.031 ±0.001 0.028-0.036 

0.074 ± 0.005 0.062-0.089 0.061 ± 0.004 0.050-0.072 

a Mean of six animals. 

Lysozyme 

(pg lysozyme/10 6 cells) (p

Meana ± SEM Range Mean

0.145 ± 0.005 0.138-0.169 0 . 620 .1 09 ± 0 . 003 0.101-0.120 0.580.087 ± 0.004 0.077-0.102 0 . 54

0.051 ± 0.003 0.043-0.065 0 . 440.068 ± 0.002 0.062-0.074 0.370.058 ± 0.002 0.051-0.064 0.370.058 ± 0.002 0.050-0.064 0.370.133 ± 0.005 0.115-0.147 0 . 58

0.141 ±0.003 0.133-0.150 0 . 59
294 HURST, GARDNER, AND COFFIN 

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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 90 o/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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OZONE AND MACROPHAGE IiYDROLASES 295 

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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 is falling. 

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. It has 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 previously mentioned, the acid hydrolytic enzymes 

are found 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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OZONE AND MACROPHAGE HYDROLASES 297 

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 its relationship 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 

when compared 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 by the 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 

macro phages. 

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 that the 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 f 

<total cellular hydrolase activity found could be explained by a '' 

·'leakage of intracellular enzyme through an altered membrane. j 

As mentioned previously, Heise and Myrvik (1967) have shown 1 

that alveolar macrophages held in vitro in the absence of phago- . 

cytosis secrete lysozyme and acid phosphatase into the culture 

medium but are not themselves depleted of hydrolytic 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) and in the initiation 
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OZONE AND MACROPHAGE HYDROLASES 299 

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. 

REFERENCES 

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 Conta~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 

improved glucuronidase assay method to the study of human blood beta 

glucuronidase. J. Biol. Chern. 173, 449-456. 

Green, G. M., and Kass, E. H. (1964). The role of the alveolar macrophage 

in the clearance of bacteria from the lung. J. Exp. Med.119, 167-176. 

Green, G. M., and Carolin, D. (1967). The depressant effect of cigarette 

smoke on the in vitro antibacterial activity of alveolar macrophages. N. 

Engl. J. M ed. 276, 421-427. 

Green, G. M. (1968). Cigarette smoke: protection of alveolar macrophages 

by glutathione and cysteine. Science 162, 810-811. 

Heise, E. R., and Myrvik, Q. N. (1967). Secretion of lysozyme by rabbit 

alveolar macrophages in vitro. J. Reticuloendothel. Soc. 4, 510-523. 

Hofstee, B. H. J. (1954). Direct and continuous spectrophotometric (lSS;::ty 

of phosphomonoesterases. 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- ( 

mal enzyme: solubilization of beta glucuronidase by heparin. J. Reticu1 

•. 

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. A nat. 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 

lutants on susceptibility to respiratory infections. J. Infec. Dis. 109, i 

Ro~e~~!~ s., Watne, A., McGrath, R., McGrew, E., and Cole, W. (1958).1 

Technic and results of isolation of cancer cells from the circulating . 

blood. Arch. Sur g. 76, 334-346. 

Robertson, 0. H. (1941). Phagocytosis of foreign material in the lung. 

Physiol. Rev. 21, 112-139. 

Roque, A. L., and Pickren, J. W. (1968) Enzymatic changes in fluorescent 

alveolar macrophages of the lungs of cigarette smokers. Acta. Cytol. 12, 

420-429. 

Saito, K., and Suter, E. (1965). Lysosomal acid hydrolases in mice infected 

with BCG. J. Exp. Med. 121, 727-737. 

, 

I 

·.A 

·i 

Saltzman, B. E., and Gilbert, N. ( 1959). Iodometric microdetermination of 

organic oxidants and ozone. Anal. Chem. 31, 1914-1920. 

Sbarra, A. J., and Karnovsky, M. L. (1959). The biochemical basis of ~~ phagocystosis. I. Metabolic changes during the ingestion of particles by 

polymorphonuclear leukycytes. J. Biol. Ch em. 234, 1355-1362. 

Uhr. J. W., and Weissmann, G. (1965). Intracellular distribution and degradation of bacteriophage in mammalian tissues. J. Immunol. 94, 

544-550. 
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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 delivered by Dr. Richmond T. Prehn, Institute for Cancer Research, 

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 of induced autochthonous neoplasms, Enhancing antibodies in human and animal 

neoplasms, Immunologic studies of human neoplasms, and Immune responses to human melanomas. 

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