In Vivo Inactivation of Lysozyme by Ozone

Transcription

1 JOURNAL of BACTERIOLOGY, Nov. 1968, p Copyright 1968 American Society for Microbiology Vol. 96, No. 5 Printed in U.S.A. In Vivo Inactivation of Lysozyme by Ozone R. S. HOLZMAN, D. E. GARDNER, AND D. L. COFFIN National Center for Air Polltion Control, Brea of Disease Prevention and Environmental Control, U.S. Department of Health, Edcation, and Welfare, Cincinnati, Ohio 45237, and Department of Pathology, University of Cincinnati School of Medicine, Cincinnati, Ohio Received for pblication 22 Jly 1968 Bronchial mcs contains large qantities of the antibacterial enzyme lysozyme. Actely exposing mice or rabbits to ozone redced the amont of active lysozyme obtainable by bronchoplmonary lavage. The effect was proportional to ozone concentration, as well as to dration of exposre. Enzyme activity retrned to normal levels dring the 12 hr following exposre. Ozone (5,liters/liter; 5 parts per million) for 3 hr redced lysozyme levels approximately 3%. Stdies of the release of lysozyme by alveolar cells spport the theory that loss of activity is de to in vivo oxidation of lysozyme. Certain air polltants impair the mechanisms by which the lng inactivates and eliminates inspired bacteria. Ozone (3) and nitrogen dioxide (NO2) each prolong the presence of cltivable bacteria in the lngs of actely exposed animals (4). The alveolar macrophage has been implicated as a key factor in the "plmonary clearance" of inspired bacteria, and 3 and NO2 depress the macrophage's rate of phagocytosis (3, 5). Bacterial inactivation occrs throgh other mechanisms in addition to phagocytosis. Bronchial mcs contains specific antibodies and a grop of poorly characterized nonspecific bacterostatic and bactericidal sbstances active against both gram-positive and gram-negative organisms (8). The most widely known and stdied of these sbstances is lysozyme. Althogh the clinical importance of this enzyme is not clearly delineated, it has tberclostatic activity and is bacteriolytic for a variety of organisms (Q. Myrvik, and E. S. Leake, npblished data). The spectrm of ssceptible microorganisms is concentration-dependent (12). Lysozyme's bactericidal activity is enhanced by low ph, complement, chelating agents, and trypsin (8). At least one investigator has noted that among bronchitic patients, the activity of lysozyme was less in bronchial mcs than in other body secretions (2). Lysozyme represents an easily assayed enzyme of possible significance as a factor in the body's defense against infection. This paper describes a reprodcible method of extracting and assaying the activity of lysozyme in plmonary secretions. The techniqe was applied to mice and rabbits to determine the effect of exposre to oxidant air polltants on plmonary lysozyme. MATERIALS AND METHODS Lysozyme was assayed by sing 1 mg of Micrococcs lysodeiktics per 1 ml in ph 6.1 bffer (.6 M Na3PO4,.9% NaCI) as the sbstrate. A portion of enzyme (.3 ml) was added to 3 ml of sbstrate at 22 C. The change in optical density (OD) was measred on a recording spectrophotometer at 45 nm. The concentration of lysozyme was estimated by comparing the change in OD occrring between 1 and 3 min after mixing to that obtained with standards of 4, 2, 1, 5, and 2.5,g of hen's egg white lysozyme per ml. All specimens to be compared were assayed on the same day, and a standardization crve was inclded in each day's rn. Barrier-sstained white mice or New Zealand rabbits were exposed to either 3 or NO2 in a stainlesssteel chamber, previosly described (7). 3 was generated by passing oxygen throgh a neon tbe silentarc generator. NO2 was obtained from commercially available cylinders. Each polltant was mixed with filtered room air and delivered to the chamber at a rate to insre one complete air change per minte. Concentrations were monitored by standard wet methods at 3-min intervals (1). The effects of polltant concentration and dration of exposre were stdied independently. In one experiment, grops of mice were exposed to varios levels of 3 for 3 hr and killed immediately. In a second experiment, mice were exposed to 5 liters of 3 per liter (5 parts per million) for varios drations. After exposre, the animals were killed with pentobarbitol. Washings were obtained from mice by exposing the trachea and by cannlating it with a blnt 18-gage needle. Bffered saline soltion (1 ml), ph 7.2, warmed to 37 C, was instilled in the lngs via syringe and, after 1 min, was rapidly flshed in and ot for times. Washings from two mice were pooled 1562

2 VOL. 96, 1968 IN VIVO INACTIVATION OF LYSOZYME BY OZONE 1563 and centrifged at 4 C for 15 min at 1, rev/min. The spematant flid was frozen at -6 C and was stored ntil assay. Washings were obtained from rabbits with 3 ml of bffer and a large bore cannla. All other procedres were the same. By this method, a reprodcible measre of the lysozyme extractable from plmonary secretions was obtained. To demonstrate that the release of lysozyme by plmonary cells was not contribting significantly to the extract's activity, the rate of release of this enzyme from plmonary cells was stdied. Rabbits were exposed to 1,liters of 4 per liter (1 parts per million) or to ambient air for 3 hr. Cells were obtained by the previosly described techniqe of plmonary lavage (3). The yields from two rabbits were combined, washed three times, and resspended in tisse cltre media (no. 199 in Hanks' soltion) to a final concentration of 1 to 1 million cells per ml. Siliconized flasks containing the sspension were incbated at 37 C in a shaker bath. Samples were taken at intervals and were centrifged at 4 C for 15 min at 1, rev/ min. The rate of release of lysozyme was expressed as micrograms per million cells per hor. Total cell conts did not change dring the incbation period, and more than 99% of the cells remained viable when tested by the eosin-y techniqe (6). Normally >9% of the cells obtained by plmonary lavage were mononclear macrophages. After 3 exposre, however, approximately 4% of the cells were polymorphonclear lekocytes. Becase of this change in composition, a comparison poplation of more than 9% polymorphs was obtained from the peritoneal cavities of both normal and 3-exposed rabbits by instilling.1% glycogen for 4 hr prior to sacrifice. RESULTS Stdies on lysozyme release from plmonary and peritoneal cells from both exposed and nex- TABLE 1. Rate of release of lysozyme from the cells of rabbits Mean per cent Mean per cent Lysozyme (,g Cell sorce No. of rabbits million cells)a Macrophages Heterophiles per hor per Plmonary Unexposed A.28 1,liters of Os per liter of air per 3 hr Peritoneal Unexposed i.82 1,liters of Os per liter of - air per 3 hr a Mean pls or mins standard error. 2 - I- C z 15 DUMTION VARIED I,, AJ ppm VARIED CONCENTRATION x DURATION [ppm ozone] x [hors of exposre] FIG. 1. Effect of 3 on plmonary lysozyme extractable from mice. (Each point is a pooled sample from two mice.)

3 1564 HOLZMAN, GARDNER, AND COFFIN J. BACTERIOL. 2l ol_\ o EXPOSURE. ppm/3 hors posed animals are smmarized in Table 1. The mean rate of release from nexposed plmonary macrophages was.615,g per hr per million cells. Immediately after exposre to 1,liters of 3 per liter, the macrophage-heterophile mixtre had a mean release rate of.443 Ag per hr per million cells. The plmonary macrophages from normal rabbits and the mixtre of the two cell types obt - 5,3; a <.5 FIG. 2. Effect Of s on plmonary lysozyme extractable from rabbits. (Each point is a pooled sample from two rabbits.) tained from the lngs of exposed animals had similar release rates to that of the peritoneal heterophiles. Since the normal rabbit lng yields approximately 4 million cells, the total rate of release wold be expected to be 4 x.615 = 24.6 Mg per hr per lng. Since or washings were only in contact with the cells for 1 min before refrigeration and since the mean lysozyme content was 22.5,g/ml in the washing spernatant flid, it wold have taken almost 6 min to achieve this level by celllar release. The lysozyme concentration in the spernatant liqid after 1 min, therefore, reflects primarily the extraction of solble lysozyme from bronchial mcs and possibly from the alveolar lining. Exposre of mice to 3 linearly decreased the activity of lysozyme that was extractable (Fig. 1). After 3 hr at 5 Mliters/liter, activity had decreased approximately 3%. The magnitde of the effect is proportional to the prodct of the concentration and dration. These findings were confirmed in rabbits (Fig. 2) exposed to 1 Mliters of Ox per liter for 3 hr. Under these conditions, a 65% redction in lyoszyme was observed. Forty mice were exposed to 5.5 MAliters of OT per liter for 3 hr and sacrificed immediately and at 3-hr intervals thereafter. After 12 hr, extractable lysozyme had retrned to approximately preexposre levels (Fig. 3). Analysis of variance for 25, 1 1 V 2 15~ - l.' 1 _ 4 LId 1-I ppm OZONE WITHIN EXPOSED ERROR ANALYSIS OF VARIANCE df MS 'ERSUS EXPOSED T 8T8 CONTROL V LINEAR RE:GRESSION F a 1 U. 3 -<T.V 8.9 < : TIME, hors FIG. 3. Retrn of lyso2yme to normal levels following exposre to 3. 3

4 VOL. 96, 1968 IN VIVO INACTIVATION OF LYSOZYME BY OZONE 1565 I- 15 o 4 v~~~~8 o 1 :L z 1o z LJ 5 ANALYSIS OF VARIANCE df MS F a BETWEEN 7 W3 <1 n.s. WITHIN FIG. 4. DURATION, hors Failre of 3 gliters of NO2 per liter to inactivate plmoniary lysozyme. trend showed that the rate of recovery was linear (P <.1), with no significant deviations from linearity. Mice exposed to 3,liters of NO2 per liter for 3 hr showed no change in extractable lysozyme activity (Fig. 4). This failre of NO2 to react with lysozyme in the lngs of exposed animals can be dplicated in the test tbe. When 5 Aliters of 3 per ml was bbbled throgh a soltion of hen's *egg liter (1 parts per million) white lysozyme, the enzyme was rapidly inactivated. Concentrations as high as 1,liters of NO2 per liter (1 parts per million) failed to alter the enzyme in vitro. DISCUSSION All three of the major oxidant air polltants, peroxyacetyl nitrate (PAN), 3, and NO2, altered the activity of enzyme systems. Chemical reactions between the polltant and enzyme, as well as secondary celllar responses to polltant-indced alteration in the extracelllar milie, have been reported. For example, levels of PAN that inhibit the growth of plant cells in vivo also inactivate in vitro the enzymeb involved in the synthesis and resorption of cell walls (L. Ordin, et al., npblished data). In vitro stdies by J. B. Mdd (npblished data) sggest that reactivity of PAN to proteins is de almost exclsively to oxidation of slfhydryl grops. 3 shares this ability, bt, in addition, is highly active against doble bonds. Ozonolysis is sed by the organic chemist to fragment complex nsatrated molecles. In fact, the reaction with lysozyme observed by s in vivo has been stdied in vitro by Previero (9), who showed that when lysozyme is dissolved in formic acid and treated with high concentrations of 3, tryptophan's indole ring is split to form N'-formylkynrenine, whereas other amino acids are nchanged. Mdd has demonstrated that this reaction may also occr in aqeos soltion at netral ph. 3 depresses levels of alkaline phosphatase and 5- ncleotidase in homogenates of the lngs of exposed animals (11). Unlike PAN and 3, no direct reaction between NO2 and an enzyme has been reported. Both acte and chronic exposres to NO2 increased oxygen consmption and depressed levels of aldolase and lactic dehydrogenase observed in ginea pig lng, liver, kidney, and spleen. We believed that an indirect mechanism was responsible for these widespread enzymatic changes (1). As yet, this mechanism is ndefined. As NO2 is inspired, it cold react with the water, which satrates alveolar air to form nitros and nitric acids. Both of these acids, when present in appropriate concentrations, may react with tryptophan. The failre of 3,liters of NO2 per liter to alter the lysozyme level after 3 hr of exposre sggests that nder these conditions the concentrations of acid were too low to permit sch reactions to occr. LITERATURE CITED 1. Bckley, R. D., and. J. Balchm Enzyme alterations following nitrogen dioxide exposre. Arch. Environ. Health 14: Brgi, H., J. Regli, and T. Medici Lysozyme in sptm in chronic bronchitis. Schweiz. Med. Wochschr. 96:

5 1566 HOLZMAN, GARDNER, AND COFFIN J. BACFERIOL. 3. Coffin, D. L., D. E. Gardner, R. S. Holzman, and F. J. Wolock Inflence of ozone on plmonary cells. Arch. Environ. Health 16: Ehrlich, R Effect of nitrogen dioxide on resistance to respiratory infection. Bacteriol. Rev. 3: Green, G. M., and E. H. Kass Role of the alveolar macrophage in the clearance of bacteria from the lng, J. Exptl. Med. 119: Hanks, J. H., and J. H. Wallace Determination of cell viability. Proc. Soc. Exptl. Biol. Med. 98: Hinners, R. G., J. K. Brkart, and C. L. Pnte Animal inhalation exposre chambers, Arch. Environ. Health 16: Miller, T. E., and D. W. Watson Innate immnity. Med. Clin. N. Am. 49: Previero, A., M. A. Caletti-Previero, and P. Jolles Localization of non-essential tryptophan resides for the biological activity of lysozyme. J. Mol. Biol. 24: Saltzman, B. E. Selected methods for the measrement of air polltants. U.S. Dept. of Health, Edcation, and Welfare, PHS Pblication no. 999-AP-11, Scheel, L. D.,. J. Dobrogorski, J. T. Montain, J. L. Svirbely, and H. E. Stokinger Physiologic, biochemical, immnologic, and pathologic changes following ozone exposre. J. Appl. Physiol. 14: Work, E Factors affecting the ssceptibility of bacterial cell walls to the action of lysozyme. Proc. Royal Soc. (London) Ser. B 167: