INFECTION AND IMMUNITY, Apr. 1985, p. 57-61 Vol. 48, No. 10019-9567/85/040057-05$02.00/0Copyright C 1985, American Society for Microbiology

Modulation of Pulmonary Clearance of Bacteria by AntioxidantsEDWARD L. PESANTIl12* AND KENNETH M. NUGENT3t

School of Medicine, Veterans Administration Medical Center, Newington, Connecticut 061111; Department of Medicine,University of Connecticut Health Center, Farmington, Connecticut 060372; and Veterans Administration Medical Center

and University ofIowa Hospitals, Iowa City, Iowa 552423

Received 10 September 1984/Accepted 18 December 1984

To further delineate the mechanisms underlying murine pulmonary defenses against bacterial infection, westudied the effects of antioxidant enzymes and hydroxyl radical scavengers on pulmonary clearance processes.Intratracheal injection of catalase and superoxide dismutase resulted in prolonged intraalveolar residence ofthe enzymes, but caused no decrease in rates of clearance of either Staphylococcus aureus 502A or Pseudomonasaeruginosa PAO1. In contrast, dimethylsulfoxide and dimethylthiourea caused significant depression ofclearance of P. aeruginosa without altering clearance of S. aureus. These results provide further differentiationbetween clearance processes affecting gram-negative and gram-positive bacteria and suggest that murineclearance of gram-negative organisms may be in part mediated by reactions which generate hydroxyl anion. Invivo administration of agents which inhibit hydrogen peroxide-, superoxide-, or hydroxyl anion-mediatedreactions do not alter normal clearance of S. aureus.

Since the initial studies of pulmonary defenses in mice byKass and co-workers (10, 19), substantial evidence has beenaccumulated to indicate that clearance of small inocula ofgram-positive bacteria is mediated by intrinsic pulmonaryclearance mechanisms, whereas that of gram-negativebacteria and of larger inocula of gram-positive microbesinvolves both intrinsic mechanisms and recruited polymor-phonuclear leukocytes (PMNs) (25, 35). Since Staphylococ-cus aureus is cleared from murine lungs without significantcontribution from PMNs, phagocytosis and intracellularkilling of bacteria by resident alveolar macrophages werethought responsible for the bactericidal action (10). How-ever, recent data from several investigators have indicatedthat actual. phagocytosis of bacteria may not be necessaryfor intrapulmonary kill of gram-pbsitive microbes (4, 5, 24)And that the majority of inhaled staphylococci are killedwithout being ingested by resident alveolar macrophages(24). The assumption that phagocytosis of microbes is not anecessary precursor of intrapulmonary bactericidal activitydoes not exclude phagocytes from a major or even primaryrole in that activity. Both macrophages and PMNs areknown to be active secretory cells, releasing enzymes,nonenzymatic proteins, and reactive oxygen derivatives intothe environment (23, 36).

Reactive oxygen metabolites, including superoxide, per-oxide, and hydroxyl anion, secreted by phagocytes or othersecretory cells, could form the basis of alveolar bactericidalactivity (9, 30, 31). The possibility that such soluble oxidantsmay participate in intrapulmonary bactericidal activity hasnot been evaluated in vivo. Since previous data have shownthat the bactericidal activity of these substances can beinhibited by enzymes or scavengers (21, 22, 28, 31), weevaluated the impact of selected antioxidants on the rate ofpulmonary clearance of bacteria in mice.

* Corresponding author.t Present address: University of Texas Health Center, Tyler, TX

75710.

MATERIALS AND METHODS

Bacteria used in these studies were S. aureus 502A andPseudomonas aeruginosa PAO1, both of which we havepreviously used to study pulmonary clearance in mice (27),and a streptomycin-dependent mutant of PAO1 (PAO1strd),kindly provided by C. Cox, Department of Microbiology,The University of Iowa. The bacteria were grown overnightin tryptic soy broth and washed with sterile phosphate-buff-ered saline (PBS; pH 7.4) prior to use. PAO1strd was grownin tryptic soy broth containing 200 ,ug of streptomycin sulfateper ml. Since PAOlstrd cannot grow in the absence of strep-tomycin, it cannot grow in tissues of mice not treated withstreptomycin. It was used in limited numbers of experimentsto directly confirm the assumption that observed alterationsin bacterial clearance were secondary to depressions ofintrapulmonary killing of P. aeruginosa.Two methods of delivery of the inocula, intratracheal

injection and aerosol delivery, were utilized in these studies.Aerosolization was performed as previously described (26,27) by using a Plexiglas aerosol chamber with the aerosolgenerated by a DeVilbiss ultrasonic nebulizer. Intratrachealinjection of bacteria was performed on animals lightly anae-

sthetized with ketamine and xylazine. A midline incisionwas made to expose the trachea, and 0.05 ml of a PBSsuspension of washed bacteria suspension, followed by 0.1ml of air, was injected directly into the trachea through a

30-gauge needle. The incision was closed with wound clips.Aniimals were sacrificed at the termination of aerosol deliv-ery or 1 h after intratracheal injection, as well as 6 and 24 hlater, to determine intrapulmonary survival of bacteria.Inocula were adjusted to result in delivery of 1 x 106 to 2 x

106 viable bacteria to each animal. Female BALB/c mice (20to 25 g) (Charles River Breeding Laboratories, Inc.) were

used for all studies.A number of compounds capable of inhibiting production

or activity of toxic oxygen radicals were tested. Catalase(5,000 U; Sigma Chemical Co.) plus superoxide dismutase(SOD) (2,500 U; Sigma) were administered concurrentlywith bacteria by intratracheal injection. The persistence of

57

58 PESANTI AND NUGENT

TABLE 1. Rates of clearance of S. aureus from murine lungs'Route of Elimination

Group (no. of mice) S. aureus half-life (h)administration

Control (12) AER 3.2Control (49) IT 3.1DMSO-IP (6) AER 3.2DMSO-IP (18) IT 2.9Catalase + SOD (36) IT 2.6Indomethacin (30) IT 3.5

a Elimination half-life was determined from slopes of calculated regressionlines with the natural logarithm of the concentration of viable S. aureus cellsplotted against sampling time. The slopes of the lines were compared byanalysis of variance; none of the differences approached statistical signifi-cance. The inoculum was adjusted so that, in all experiments, 1 x 106 to 2 x106 S. aureus cells were recovered from the lungs at the first sampling period.For indomethacin treatment, data are combined from animals to which thedrug was administered for 2 weeks (n = 18) and overnight (n = 12). All miceinjected intraperitoneally with DMSO (DMSO-IP) received 165 mg of thedrug. AER, Aerosol delivery; IT, intratracheal injection.

the enzymes in the alveolar spaces was evaluated in separateexperiments by measuring the activity of the enzymes incentrifuged lavage samples at 1, 6, and 24 h after intratra-cheal injection of normal mice with the two enzymes or withPBS. Catalase activity was measured by polarographic elec-trode determination of release of 02 from H202 in PBS (pH7.4) (21). SOD activity was measured spectrophotometri-cally by determining the inhibition of xanthine-xanthineoxidase-mediated reduction of cytochrome c (22). Lavagefluid samples were diluted with PBS such that the measuredreaction was comparable to that in standards containing 0.1U of catalase or 1.0 U of SOD.The possibility that inhibition of hydroxyl radical activity

by dimethylsulfoxide (DMSO) or dimethylthiourea (DMTU)(30) would retard clearance of bacteria was also evaluated.For these experiments, DMSO (50 to 165 mg per mouse) orDMTU (0.3 mg per mouse) was administered intraperitone-ally 30 min before challenge with S. aureus by either theintratracheal or aerosol route. In other experiments, theeffects of intranasal administration of 0.05 ml of a 10%solution of DMSO were tested. Drug levels were not mneas-ured; however, the mice obviously exhaled DMSO for theduration of the experiments. Both compounds also causedthe animals to become lethargic and lessened the dose ofketamine required for anaesthesia.Because we have previously reported that prolonged

administration of glucocorticoids inhibited pulmonary clear-ance of staphylococci in mice (27), and because reactiveoxygen derivatives are released during prostaglandin synthe-sis (18, 34), we also studied clearance of S. aureus in micepretreated, either overnight or for 2 weeks, with indometh-acin administered in drinking water (50 mg/liter), resulting iningestion of ca. 8 mg/kg per day. Indomethacin (100 ,ug) wasalso administered intratracheally with the bacterial inocu-lum.

Intrapulmonary survival of inhaled or injected bacteriawas determined by quantitative cultures on brain heartinfusion agar of suitably diluted samples of lungs ground inglass tissue grinders. No efforts were made to separatelyevaluate intracellular and extracellular bacterial popula-tions; only total lung counts were determined. For S. aureusand P. aeruginosa PA0lstrd, the rate of in vivo clearancewas assessed by calculating the half-life of the decrease inthe number of viable organisms by regression analysis oflogarithmically transformed viable counts. The significance

of differences between experimental and control groups wasdetermined by analysis of variance of the slopes of thecalculated regression lines. Clearance of the parental strainof PAO1 was not monoexponential, so calculations of half-lives were not feasible; differences between control andexperimental groups were analyzed by the x2 test or thefourfold table test for small samples (6).

RESULTSAlthough aerosol delivery and direct intratracheal injec-

tion of bacteria are both used in studies of murine pulmonaryantibacterial activity, there has been no direct comparison ofthe two methods. Since it was impractical to administer theinhibitors by aerosolization, we first evaluated the compara-bility of clearance rates of S. aureus after aerosol deliveryand intratracheal injection. As is apparent in Table 1, thehalf-life of elimination was independent of the means ofadministration of the bacteria. And none of the agentsthought capable of inhibiting intraalveolar oxidative reac-tions altered rates of clearance of S. aureus from murinelungs (Table 1). Administration of S. aureus in combinationwith quantities of catalase and SOD sufficient to maintainconcentrations of the enzymes over 100 times normal levelsfor most of the time of the experiments (Fig. 1) resulted inbacterial clearance rates that were identical to those incontrol animals. In contrast to previous data concerning therapid disappearance of catalase and SOD from murineperitoneal cavities (21), both enzymes were very slowlycleared from alveolar spaces, persisting in cell-free lavagefluids with an elimination half-life of ca. 18 h. Thus, itappears unlikely that the lack of effect was due to insufficientdelivery or rapid clearance or inactivation of the inhibitors.

Similarly, treatment with DMSO (150 mg per mouse), adose which was ca. one-fourth of the acute 50% lethal doseof the compound for normal mice (data not shown), did notadversely influence the intrapulmonary elimination of viableS. aureus cells. These studies were complicated by thefrequent development of secondary infections in the animalsinjected with DMSO. Although animals injected with S.

1 0000 -

'n-

1000-

100 -

10 -

A

1 6

B

½

24 1 6

HOURS

24

FIG. 1. Means and standard errors of total alveolar space en-zyme content after intratracheal injection of catalase (5,000 U) (A)and SOD (2,500 U) (B). The mean (.--) and upper range (- - -) ofvalues frotn 24 control mice, sacrificed at 1, 6, and 24 h afterinjection, are also illustrated. Control mice for catalase determina-tions were injected with SOD, and control mice for SOD determi-nations were injected with catalase.

4

INFECT. IMMUN.

ANTITOXIDANT MODULATION OF PULMONARY CLEARANCE 59

aureus alone did not die during the course of the experi-ments, ca. 10% of the mice injected with DMSO died within24 h, and many others were ill at the time of sacrifice. In thelatter group, quantitative cultures showed that S. aureus wascleared normally, but the animals had developed overwhelm-ing infections (2107 CFU per animal) with gram-negativeorganisms (not further identified). The sterility of the dilu-ents and the DMSO was verified. In addition, 10 mice givenDMSO (165 mg) but not anaesthesized suffered no mortality;cultures of lungs of these animals, sacrificed at 24 h, con-tained <103 bacteria. Two of eight mice anaesthesized afterDMSO treatment appeared toxic at 24 h, and culture of lungsshowed >107 gram-negative rods; the lungs of the sixwell-appearing animals contained <103 CFU. Thus, it ap-peared that DMSO had no effect on clearance of S. aureusbut may have altered defenses against gram-negative bacte-ria, so that the animals manifested increased susceptibility toinfections with indigenous flora.

This possibility was directly assessed by evaluating theeffects of various doses of intraperitoneally administeredDMSO on clearance of intratracheally injected P. aerugi-nosa PAO1. DMSO caused dose-dependent inhibition ofPseudomonas clearance (Fig. 2). The majority of animalswhich received 165 mg of DMSO intraperitoneally failed toclear PAO1, instead allowing the organism to replicate(Table 2). This resulted in the deaths, with in excess of 107PAO1 cells per animal, of 25% of the animals; most of thesurviving mice appeared ill. Even when administered as a10% solution intranasally, DMSO caused a detectable in-crease in susceptibility to PAO1 infection (Table 2). Similarinhibition of clearance of strain PAO1 also resulted frompretreatment of mice with DMTU (0.3 mg per mouse intra-peritoneally), another hydroxyl radical scavenger (Table 2);as with DMSO, this compound was administered at one-fourth the 50% lethal dose for normal mice. Since pulmonarybacterial clearance is best viewed as the net result ofbacterial growth and host-mediated killing (15), we alsotested the effect of DMSO on clearance of an organismwhich cannot grow in mice, PAO1strd. Elimination ofPAO1strd

100-

75-

50-

25-

.01..1-N0-.01~ ~ ~ ~

)kN%

0 82 110 138 165DMSO mg/mouse

FIG. 2. Dose-response curve illustrating the relationship be-tween intraperitoneal dose of DMSO and efficiency of clearance ofstrain PA01 from murine lungs. The percentage of animals elimi-nating >90%o of the initial inoculum within 24 h is plotted against thedose of DMSO. Each group consisted of eight animals.

TABLE 2. Pulmonary clearance of P. aeruginosa PAO1 at 24 hafter intratracheal injectiona

No. (%) of animals with:

Group Decreased CFU (fold) Increased CFU (fold).10 <10 <10 -10

Parent PAO1Control 25 (92%) 0 1 (4%) 1 (4%)SOD + CAT 29 (91%) 3 (9o) 0 0DMSO-IP` 2 (7%) 2 (7%) 5 (19%) 18 (67%)DMSO-INc 12 (44%) 4 (15%) 7 (26%) 4 (15%)DMTUd 0 1 (10%) 1 (10%) 8 (80%)

PAOls'rdControl 12 (100%o) 0 0 0DMSO-IPe 4 (33%) 8 (67%) 0 0a The numbers of viable PAO1 cells at 24 h was divided by the numbers of

viable PA01 cells at 1 h after injection to determine the ratios for this table.For each experimental group, the numbers of animals (%) which reduced theintrapulmonary numbers of viable PAO1 cells or allowed growth of theorganisms is indicated, and the groups are further subdivided on the basis ofwhether the observed change was less than or greater than 10-fold. The lack ofeffect of SOD plus catalase (CAT) and the depression of clearance afterDMSO administration, either intraperitoneal injection of 165 mg(DMSO-IP) orintranasal instillation of 0.05 ml of a 10%o solution (DMSO-IN), are evident.Similarly, PAO1strd, which could not grow in normal mice, is eliminated lessefficiently in DMSO-treated mice.

b Significantly different from the control group by the x2 test. An additionalnine mice in this group were dead at 24 h.

c Significantly different from the DMSO-IP and control groups by the x2test.

d Significantly different from the control group by the x2 test.Significantly different from the control group (PAO01srd) by the fourfold

table test.

was also impaired by DMSO treatment of the mice (Table 2),supporting the assumption that diminished intraalveolar kill-ing of P. aeruginosa was largely responsible for the dimin-ished clearance of the organism. As determined by hemo-cytometer counts of the lavaged leucocytes at 1, 6, and 24 hafter bacterial inoculation, DMSO (165 mg intrjtperitoneally)did not alter the intraalveolar PMN recruitment after Pseu-domonas infection. Catalase and SOD did not alter clear-ance of strain PAO1 (Table 2).

DISCUSSIONSeveral separate but related issues were addressed in

these experiments. Although it has been established that thedose of organisms delivered to the airspaces determines themechanisms involved in pulmonary antibacterial activity(36) and various groups have achieved comparable results byusing different modes of delivering the bacteria, there hasbeen no previous demonstration that clearance rates areindependent of the means of delivery of the organisms. Ourdata confirm the tacit assumption that delivery of bacteria bydirect intratracheal injection resulted in rates of eliminationof viable intrapulmonary organisms that were identical tothose observed after inoculation by aerosolization; net elim-ination half-lives approximated 3 h in all control groups.

Catalase and SOD-at levels which were greater thanfivefold higher than those which completely inhibited kill ofS. aureus 502A by hydrogen peroxide or xanthine plusxanthine oxidase, potassium iodide, and lactoperoxidase inthe test tube (data not shown) (21, 23)-were without effecton intrapulmonary bactericidal activity against either gram-negative or gram-positive bacteria. The lack of effect of theinhibitory enzymes on clearance despite their persistence inthe alveoli (elimination half-life, 18 h) appears to virtuallyexclude participation of secreted peroxide or superoxide in

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60 PESANTI AND NUGENT

clearance processes. Our previous observations (26) havesuggested that murine pulmonary clearance of S. aureusmay be primarily an extracellular process, with little demon-strable contribution of intracellular bactericidal activity.Even if it is assumed that intracellular functions contributesubstantially to pulmonary clearance mechanisms, the bio-chemical mechanism of that action is also not clearly de-fined. Data have been published that cast doubt on the roleof oxidants in the microbicidal activity of alveolar macroph-ages (1-3, 20). Both catalase (17, 21, 37) and SOD (17, 37)have previously been shown to be effective in diminishingthe intracellular bactericidal activity of PMNs when used invitro at concentrations that were lower than those weachieved in the alveolar spaces. Presumably, over the 24 h ofthe current experiments, the enzymes would have hadconstant access to the phagolysosomes of alveolar phago-cytes. Thus, the available data from in vitro and in vivoexperiments do not support the assumption that hydrogenperoxide and superoxide are important antibacterial com-pounds in murine lung defenses.Although we could not demonstrate any impact of catalase

and SOD on pulmonary defenses against either S. aureus orP. aeruginosa, DMSO and DMTU treatment markedly im-paired Pseudomonas clearance. Although the biochemicaleffects of DMSO and DMTU are not as confidently inter-pretable as those of the antioxidants enzymes, they areprimarily used, both in vivo and in vitro, to evaluate the roleof hydroxyl anion in pathogenic mechanisms (11, 12, 16, 29).The results of our experiments utilizing those inhibitorssuggest that hydroxyl radicals figure prominently in pulmo-nary bactericidal activity against P. aeruginosa. Since it hasbeen demonstrated that PMNs figure prominently in pulmo-nary defenses against gram-negatvie organisms (28), it seemsreasonable to postulate that inhibition of PMN hydroxylradical generation is responsible for the actions of DMSO.However, hydroxyl radical is thought to be formed fromsuperoxide and hydrogen peroxide within the lysosomes ofphagocytes (12, 38). The necessity for direct interferencewith hydroxyl radical function and the lack of effect ofcompounds which should have interfered with generation ofthose radicals in difficult to explain. The difference mayrelate to factors which affect in vivo activity of inhibitors,such as a greater ability of DMSO to penetrate intact cells orto a combined local and systemic effect of DMSO. It is alsopossible that DMSO is acting by mechanisms other thanhydroxyl radical scavenging. Alternatively, it is possible thatthe active hydroxyl radical in these defenses is generatedthrough intermediates other than hydrogen peroxide andsuperoxide. Of relevance in this regard is the recent sugges-tion that hydroxyl radical may be generated via the lipoxyge-nase pathway of arachidonic acid metabolism (34). How-ever, our experimental observations do support the hypoth-esis that hydroxyl radical mediates much of the antibacterialactivity in pulmonary defenses in which PMNs are involved.This hypothesis also provides a possible explanation for thewell-demonstrated effects of ethanol, another scavenger ofhydroxyl radical, on pulmonary defense mechanisms (sum-marized in reference 13).Other mechanisms whereby toxic oxygen derivatives could

be generated were also considered. Alveolar macrophagesare active in synthesis and secretion of prostaglandins andleukotrienes (14, 33), and it seemed a reasonable hypothesisthat oxidants generated as byproducts of the cyclooxygen-ase pathway (18) may participate in pulmonary clearance ofstaphylococci. Inhibitors of the cyclooxygenase pathwayhave been shown to alter host defenses against other mi-

crobes, both inhibiting defenses, as is the case in pulmonaryclearance of Streptococcus pneumoniae in rats (7) or ofrhinovirus in humans (32), and potentially enhancing de-fenses, as may be seen after indomethacin administration incases of immunodeficiency syndrome (8). However, wecould not demonstrate any effect of large doses of indo-methacin on clearance of small inocula of staphylococcifrom murine lungs. This observation casts doubt on the invivo significance of cycloexygenase pathways in generalnonimmune pulmonary defenses and, at the very least,suggests that the inhibitory effects of glucocorticoids onmurine pulmonary clearance of S. aureus (25, 27) cannot beexplained on that basis.The results of these experiments also provide another

example of the difference between clearance processes af-fecting gram-negative and gram-positive bacteria, in thatDMSO, which profoundly inhibited clearance of strain PA01,was without effect on the elimination of S. aureus. Clearanceof S. aureus proceeded normally after DMSO administra-tion, even in animals succumbing to gram-negative bacterialsuperinfections. Thus, it seems reasonable to conclude that,whatever the mechanism underlying staphylococcal clear-ance in murine lungs, oxidant radical damage contributeslittle if any to the overall outcome. Similarly, althoughscavengers of hydrogen peroxide and superoxide could notbe shown to alter elimination of P. aeruginosa, the dataimply that reactions involving hydroxyl radicals may con-tribute to murine pulmonary defenses against gram-negativebacteria.

ACKNOWLEDGMENTS

This work was supported in part by the Veterans Administrationand by Public Health Service grant AI-19772 from the NationalInstitutes of Health.The technical assistance of Maureen Allison-Gay and Lisa Wen-

dell is gratefully acknowledged.

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