INFECTION AND IMMUNITY, Jan. 1985, p. 277-2810019-9567/85/010277-05$02.00/0Copyright C 1985, American Society for Microbiology

Anaerobic Phagocytosis, Killing, and Degradation of Streptococcuspneumoniae by Human Peripheral Blood Leukocytes

MAGNUS THORE,1,2t* STURE LOFGREN,2 ARNE TARNVIK,2 TOR MONSEN,2 EVA SELSTAM,3 ANDLARS G. BURMAN2

Departments of Otorhinolaryngology,l Clinical Microbiology,2 and Plant Physiology,3 University of Umea, S-901 85Umea, Sweden

Received 9 April 1984/Accepted 28 September 1984

Encapsulated Streptococcus pneumoniae of serotypes 2, 9N, 14, 21, and 23F and an unencapsulated variantof type 2 pneumococci were efficiently phagocytosed by both aerobically and anaerobically incubated humanleukocytes. In the presence of 02, the pneumococci rapidly lost their viability, whereas during anaerobiosis,killing was considerably delayed. Type 14 pneumococci radiolabeled with [14C]choline or [14C]ethanolamine forcell wail teichoic acid, [14C]uracil for nucleic acids, or [14C]arachidonic acid for unsaturated cytoplasmicmembrane lipids were used in studies of the fate of bacterial macromolecules after phagocytosis. Thedegradation of teichoic acid, RNA, and DNA during anaerobiosis approached that recorded in air at 60 minof incubation (45 to 70% and 55 to 75%, respectively). In contrast, the marked loss of [14C]arachidonic acidfrom pneumococcal membrane lipids observed in aerobic leukocytes did not occur during anaerobicincubation. Hence, lipid peroxidation could be involved in the rapid aerobic leukocyte killing of pneumococci,whereas a different leukocyte function of as yet unknown nature appears to be responsible for the killing seen

in anaerobiosis. Autolysis-resistant type 14 pneumococci were obtained by substituting ethanolamine forcholine in a defined culture medium. Differences between such bacteria and normal (autolytic) pneumococci intheir killing and degradation by leukocytes were not detected in either the presence or the absence of 02. Theaerobic and anaerobic handling of phagocytosed pneumococci by human blood leukocytes thus proceededindependently of the bacterial autolytic system.

Streptococcus pneumoniae is the organism most fre-quently isolated in acute otitis media and in acute sinusitis(13). Although purulent maxillary sinus effusion invariablyhas a P02 approaching zero (3), allowing growth of strictlyanaerobic species of bacteria (10), the interaction betweenStreptococcus pneumoniae and phagocytes has so far beenstudied under aerobic conditions only. In the presence of air,properly opsonized pneumococci are rapidly killed by hu-man peripheral blood leukocytes (PBLs) (12). During thisprocess, peroxidation of unsaturated fatty acids in bacterialmembrane lipids (27) and degradation of cell wall andintracellular pneumococcal macromolecules (30) are ob-served.Whereas phagocytosis of various particles can occur in

the absence of 02 (25), the subsequent killing of manybacterial species seems to depend on the availability of 02

and its reduction by the phagocyte to reactive intermediates(15, 20). However, some microorganisms, such as strepto-cocci of low virulence, are efficiently killed by leukocytesthrough 02-independent mechanisms also (7, 20). Whethermore virulent streptococcal species, such as Streptococcuspneumoniae, are killed and degraded by human phagocytesin the absence of 02 is a question highly relevant, e.g., withregard to the host defense in sinusitis (3) and perhaps also inotitis media. It was addressed by using an anaerobic in vitrophagocytosis model. Furthermore, the possible role of thepneumococcal autolytic system in killing and degradation ofthe bacteria was studied.

* Corresponding author.t Present address: Department of Clinical Bacteriology, Central

Hospital, S-721 89 Vasteras, Sweden.

MATERIALS AND METHODS

Bacteria, growth conditions, and preparation of bacterialsuspensions. Five clinical isolates of encapsulated Strepto-coccus pneumoniae (types 2, 9N, 14, 21, and 23F) and anunencapsulated variant of a type 2 strain (S. pneumoniaeATCC 27336) were used. Streptococcus faecalis C3 wasobtained from S. E. Holm, Department of Clinical Micro-biology, University of Umea, Umea, Sweden. Staphylococ-cus aureus phage type 52/80 I was a recent clinical isolate.

Streptococcus faecalis, Staphylococcus aureus and theunencapsulated pneumococci were subcultivated on bloodagar (Oxoid Ltd., Hants, England). To maintain their viru-lence, encapsulated pneumococci were subjected to animalpassage with a mouse peritonitis model (5) and stored at-70°C in brain heart infusion broth (BHI; Difco Laborato-ries, Detroit, Mich.) supplemented with 20% glycerol untilused.

Exponential-phase bacteria were obtained by subcultiva-tion of overnight cultures of Streptococcus pneumoniae,Streptococcus faecalis, and Staphylococcus aureus in freshBHI for 5 h at 37°C. The bacteria were then washed once inphosphate-buffered saline (PBS) (12.6 mM KH2PO4, 54 mMNa2HP04, 85 mM NaCl [pH 7.4]) and suspended at a densityof 0.5 x 108 to 2.0 x 108 CFU/ml in RPMI 1640 mediumcontaining 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (RPMI-HEPES; GIBCO Bio-cult,Glasgow, Scotland) before being mixed with leukocytes.A defined medium for Streptococcus pneumoniae (A.

Tomasz, Bacteriol. Proc., p. 29, 1964) was used for radiola-beling of cell wall teichoic acid and nucleic acids (see below)and to obtain pneumococci with modified (autolysis-resist-ant) cell walls (22, 29). Such cells were produced by growingthe pneumococci in defined medium supplemented withethanolamine (40 mg/liter) instead of choline (4 mg/liter) (29).

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The bacteria appeared mostly as diplococci when grown inthe choline-supplemented medium, whereas growth in theethanolamine-supplemented medium resulted in chains ofpneumococcal cells (6 to 12 cocci per chain). During storagefor 24 h at 20°C, a suspension of pneumococci grown incholine medium lysed completely, whereas the turbidity of asuspension of ethanolamine-grown bacteria was virtuallyunchanged.

Radiolabeling of Streptococcus pneumoniae serotype 14.[I-4C]arachidonic acid was used for labeling of bacteriallipids (27). Arachidonic acid in 10 ,ul of toluene (0.2 ,uCi, 57.6,uCi/,umol; Amersham International Ltd., Amersham, UnitedKingdom) was dried in N2, 100 ,ul of 2.5% defatted bovineserum albumin (Sigma Chemical Co., St. Louis, Mo.) wasadded (26), and the mixture was equilibrated at 8°C over-night. Two milliliters of a diluted overnight pneumococcalBHI culture (ca. 106 CFU) was added, and incubation wascontinued for 5 h at 37°C. The bacteria were harvested bylow-speed centrifugation, washed once in PBS, suspended infresh BHI supplemented with 0.1% defatted bovine serumalbumin, and allowed to grow further for 1 h at 37°C. Thelabeled pneumococci were then washed twice in Hanksbalanced salt solution (HBSS) containing 1% defatted bovineserum albumin and were finally suspended in HBSS contain-ing HEPES (20 mM) at a density of 2 x 108 CFU/ml.The fate of [14C]arachidonic acid incorporated into pneu-

mococcal lipids was checked by thin-layer chromatography(Silica Gel 60 H; E. Merck AG, Darmstadt, West Germany)of extracts of total lipids with chloroform:methanol:aceticacid:water (85:15:10:3.5 [vol/vol]) (14) or petroleum ether(boiling point, 40 to 60°C):diethyl ether:formic acid (85:15:1[vol/vol]) (23) as solvents. Reference phospholipids, diglycer-ide (1,2-destearoyl-sn-glycerin) and fatty acid (heptadeca-noic acid) were used for localization of acyl lipids beforetheir quantification by liquid scintillation counting of[14C]arachidonic acid. At least 92% of the [14C]arachidonicacid associated with the bacteria was in esterified form; 76 +3% was found in phospholipids, and 16 ± 5% was found indiglycerides. To estimate the amount of [14C]arachidonicacid incorporated into bacterial lipids as compared with thetotal amount of bacterial fatty acids, an analysis of bacterialacyl group composition was performed. In brief, lipid ex-tracts were transmethylated (80°C for 30 min in N2) with BF3(14%) in methanol (21). Fatty acid methyl esters wereseparated by gas-liquid chromatography, using a Varian3200 chromatograph (equipped with a Hewlett-Packard 3390integrator) and a 140 to 185°C program. It was found that28.3 ± 0.3% of the fatty acids in normal (BHI-grown)pneumococci were unsaturated, but no arachidonic acid wasdetected. Also, after labeling with [14C]arachidonic acid,28.9 ± 1.7% of pneumococcal fatty acids were unsaturated,and arachidonic acid constituted 9.2 ± 0.7% of the unsatur-ated fatty acids.

For labeling of bacterial cell wall teichoic acid, 1.8 ,uCi of[methyl-14C]choline chloride (58 ,uCi/,mol; Amersham) wasadded to 2 ml of choline-supplemented defined medium.Alternatively, 1.8 ,uCi of [2-14C]ethanol 2-amine hydro-chloride (60 ,uCi/,mol; Amersham) was added to the etha-nolamine-supplemented medium (30). Nucleic acids werelabeled with 0.45 ,uCi of [2-14C]uracil (58 ,uCi/,umol; Amer-sham) added to 2 ml of either of the two defined media (30).The labeling media were inoculated with pneumococci thatwere pregrown for 60 min in the respective isotope-freemedium; growth and incorporation of label was allowed for5 h at 37°C. The labeled log-phase pneumococci werecollected, washed in isotope-free medium, allowed to grow

in such medium for 1 h at 37°C, washed twice in HBSS, andfinally suspended in HBSS-HEPES at a density of 2 x 108CFU/ml.

Sera. Pooled normal human serum (PNHS) was preparedwith blood from four healthy donors. Immune human serumrequired for phagocytosis of the encapsulated type 2 Strepto-coccus pneumoniae strain was obtained from one individual4 weeks after a standard vaccination with Pneumovax (MerckSharp & Dohme, Rahway, N.J.). The sera were stored inaliquots at -70°C.Antibody titers of PNHS and immune human serum to

individual pneumococcal serotypes were determined by M.Koskela, University of Oulu, Oulu, Finland, using an en-zyme-linked immunosorbent assay technique (16). The im-munoglobulin G titer against pneumococcal serotype 2 po-lysaccharide in immune human serum was about sixfold thatin PNHS (data not shown).

Preparation of human leukocytes. PBLs were obtainedfrom heparinized venous blood by dextran sedimentation(17). Polymorphonuclear leukocytes were prepared fromheparinized venous blood by using the Hypaque-Ficoll tech-nique (8). Leukocytes were suspended at a density corre-sponding to 5 x 107 polymorphonuclear leukocytes per ml ofthe appropriate buffer (RPMI-HEPES for killing experi-.ments and HBSS-HEPES for experiments with radiolabeledbacteria). In preliminary experiments, we observed no dif-ference between PBL and polymorphonuclear leukocytepreparations with regard to phagocytic killing of pneumo-cocci or degradation of their macromolecules. For practicalreasons, PBLs were used throughout.

Experimental cell mixtures. The procedure was essentiallyas previously described (28). For aerobic experiments, 100-,lt samples of PBL suspension in capped plastic tubes (12 by75 mm; Becton Dickinson Labware, Oxnard, Calif.) weregently waggled at 37°C for 1 h before the addition of theamount of serum desired; 50 ,ul of salt solution (10.0 mMMgSO4, 31.5 mM CaCl2) was added to each sample, and thevolume was adjusted to 400 ,ul with the appropriate buffer.The experiment was started by adding 100 [lI of bacterialsuspension per tube. The cell mixtures were then incubatedat 37°C under waggling.

For anaerobic experiments, a glove box with an atmos-phere of 10% H2 and 5% CO2 in N2 was used (28). Buffers,BHI, salt solutions, and tubes were stored in the box for atleast 3 days before use. The PBL preparations were broughtinto the box after the final washing and were suspended inanaerobic buffer. Samples (100 ,il) of PBL suspension wereadded to plastic tubes, equilibrated for 2 h at 20°C, andfurther incubated anaerobically under waggling (1 h at 37°C).Experimental cell mixtures were prepared as in the aerobicexperiments (see above) and incubated in the box (37°C)under waggling.

Assay of phagocytosis and killing. Phagocytosis and killingwere measured as previously described (4, 28). Briefly, zerotime and 30-, 60-, and 90-min samples (50 Rd) of the experi-mental cell mixtures were diluted in 5 ml of PBS at 0°C andsonicated for 8 s at an amplitude of 18 ,um with a 150 W MSEsonicator with a 145- by 35-mm tip (Fisons Scientific Equip-ment, Crawley, United Kingdom). This treatment was suf-ficient to lyse the leukocytes without notably affecting thenumber of viable bacteria. Total numbers of viable bacteria,i.e., viable bacteria associated with PBLs (phagocytosedbacteria) as well as viable bacteria free in suspension, werethen assayed by viable counting of the sonic extract. Parallel50-pld samples from the experimental cell mixtures werediluted in 5 ml of PBS at 0°C and centrifuged (110 x g for 10

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min at 4°C) to remove the PBLs. Viable bacteria notassociated with PBLs (nonphagocytosed bacteria) were thenassayed by viable counting of the sonicated supernatants.Total viable bacteria and nonphagocytosed bacteria wereexpressed as the percentage of input viable bacteria.

Assay of degradation of macromolecules. Samples weredrawn from the experimental mixtures at zero time and 60min and were subjected to viable counting (as describedabove) and determinations of radioactivity in pneumococcalmacromolecules. Control mixtures containing serum andpneumococci but no PBLs were always incubated andanalyzed in parallel.For analysis of cell wall teichoic acid, 100-,ul samples were

precipitated with 4 ml of 10% trichloroacetic acid (TCA) (15h at 0WC). The precipitates were collected on glass microfiberfilters (type GF/C; Whatman, Maidstone, United Kingdom).After drying, radioactivity from [14C]choline or [14C]ethanol-amine retained on the filters was determined by liquidscintillation counting.

Nucleic acids were assayed by using duplicate 100-,usamples. One sample was immediately precipitated withTCA, yielding the total incorporation of [14C]uracil, and theother sample was diluted in 2 ml of 1 N NaOH and incubatedovernight at 37°C before TCA precipitation and counting.Since the precipitable radioactivity from [14C]uracil remain-ing after alkali treatment is in DNA (30), labeled RNA couldbe calculated by subtracting the DNA radioactivity from thetotal precipitable [14C]uracil.To estimate the degradation of arachidonic acid, the

experimental mixture was incubated for 60 min at 37°C, andthe lipids of a 400-,u sample were extracted with 8 ml ofchloroform:methanol (2:1 [vol/vol]) (9, 27), using sonicationat 30 ,um two times for 30 s. After incubation (60 min at20°C), 1.6 ml of 0.1 N KCI was added, and the extractionmixture was centrifuged (300 x g for 5 min). The chloroformphase containing the water-insoluble lipids was collectedand taken to dryness before measurement of radioactivityfrom intact [14C]arachidonic acid by liquid scintillation count-ing. As control, labeled bacteria were incubated in serum (60min at 37°C), after which 100 ,ul of PBL suspension wasadded, and a 400-,ul sample was immediately drawn andsubjected to lipid extraction.

Control of anaerobiosis. The redox potential of the an-aerobic experimental mixtures was monitored with a freshlysanded platinum electrode and, as reference, a hydrogenelectrode (electrodes P101 and K410, respectively; Radiom-eter, Copenhagen, Denmark). The redox potential was -229+ 4 mV when measured immediately before and at the endof anaerobic 60-min incubations.

Staphylococcus aureus is known to be killed less effi-ciently by anaerobically than by aerobically incubated hu-man phagocytes (20) and was therefore included in ourexperiments as a biological control of the anaerobiosis of theleukocytes. More than 80% of the staphylococci were killedwithin 60 min of incubation under aerobic conditions, butonly about 15% were killed during anaerobiosis. Streptococ-cus faecalis is reportedly killed by phagocytes equallyefficiently under aerobic as under anaerobic conditions (20)and was also included as a reference. Also, the enterococciwere efficiently killed under both conditions.

Statistical method. Student's t test was used.

RESULTSPhagocytosis and killing of Streptococcus pneumoniae by

human PBLs. Serum supplement was required for phago-cytosis of each of the pneumococcal strains by human PBLs.

For opsonization of encapsulated bacteria of serotypes 9N,14, 21, and 23F, 10% PNHS was found to be sufficient,whereas for encapsulated type 2 pneumococci, 20% immunehuman serum was required. For purposes of comparison,the latter supplement was also used in all experiments withunencapsulated type 2 pneumococci. None of the serumsupplements used was cidal to any of the bacterial strainsstudied.

Phagocytosis of Streptococcus pneumoniae by humanPBLs proceeded as efficiently in the absence as in thepresence of 02 (Table 1). During aerobiosis, phagocytosiswas immediately followed by killing of the Streptococcuspneumoniae cells irrespective of their serotype and, for type2 pneumococci, independently of the presence of capsule(Table 1). Thus, 94 to 99.8% of the pneumococci were killedduring a 60-min incubation of bacteria and PBLs in air.Under anaerobic conditions, killing of all pneumococcalstrains studied still occurred (anaerobic killing versus con-trols without PBLs; P < 0.01) but always after considerabledelay (more than 30 min). However, after 60 min of an-aerobic incubation, the pneumococcal viable counts haddecreased by 69 to 86% (Table 1), and after 90 min more than92% of encapsulated type 2 pneumococci were killed in theabsence of air (data not shown). The contribution by thepotent autolytic system present in normal Streptococcuspneumoniae to its killing by human PBLs was evaluated bycomparing pneumococci pregrown in the presence of choline(normal, autolytic) or ethanolamine (autolysis resistant; seeabove). Autolytic and autolysis-resistant pneumococci werekilled with equal efficiency by human PBLs both in thepresence of air and during anaerobiosis (data not shown).

Degradation of Streptococcus pneumoniae macromoleculesby human PBLs. Degradation of bacterial cell wall polymer(teichoic acid), DNA, RNA, and lipids after phagocytosiswas studied by using specifically radiolabeled encapsulatedpneumococci of serotype 14. The serum supplement used(15% PNHS) had no detectable degradative effect on the

TABLE 1. Phagocytosis and killing of Streptococcus pneumoniaebelonging to five different capsular serotypes and an

unencapsulated variant of one of them (2-) during aerobic andanaerobic mixed incubation with human PBLs and serum

Sero- Time of Phagocytosis (%)a Killing (%)atyero incubation(min) Anaerobic Aerobic Anaerobic Aerobic

2 30 80.7 8.7b 84.6 ± 1.1 12.0 ± 19.2 84.6 ± 4.760 96.7 ± 1.2 94.0 0.5 77.0 ± 8.1 94.5 ± 2.4

2- 30 91.5 ± 2.5 99.2 ± 0.1 29.3 ± 14.8 96.0 ± 0.360 95.9 ± 2.5 99.8 ± 0.0 69.6 ± 4.3 99.1 ± 0.2

9N 30 92.1 ± 1.3 92.4 ± 0.2 52.5 ± 4.9 86.6 ± 1.460 99.5 ± 0.1 98.1 ± 1.2 85.7 ± 3.5 96.7 ± 1.6

14 30 84.6 8.6 82.3 ± 11.8 27.0 ± 12.6 80.0 10.060 98.3 ± 1.0 97.9 1.0 68.9 5.0 96.1 ± 3.1

21 30 94.4 ± 3.8 98.1 ± 0.6 35.2 ± 11.6 93.2 ± 0.560 98.9 ± 0.8 99.9 ± 0.1 86.5 ± 5.8 99.8 ± 0.1

23F 30 70.5 ± 3.5 98.9 ± 0.2 3.8 ± 13.8 97.7 ± 1.360 99.0 ± 0.3 99.8 ± 0.1 84.0 ± 1.0 99.4 ± 0.4

a Percent reduction of input viable counts.b Arithmetical mean + standard error of the mean from two to five

experiments. Control preparations of pneumococci in buffer showed onlyslight increases in CFU per milliliter during incubation.

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LIPID TA RNA DNA TA RNA DNA

FIG. 1. Degradation of radiolabeled cell wall teichoic acid (TA),RNA, DNA, and unsaturated fatty acids in membrane lipids ofphagocytosed Streptococcus pneumoniae serotype 14 after 60 minof aerobic and anaerobic mixed incubation with human PBLs. (A)Results with normal (autolytic) pneumococci; (B) results withautolysis-resistant pneumococci (see text). Arithmetical means +

standard errors of the means from three to five experiments areindicated. Symbols: [, aerobic incubation; C, anaerobic incuba-tion. Degradation of TA, RNA, and DNA was independent of 02

and autolysis (aerobiosis versus anaerobiosis and autolytic versusautolysis-resistant pneumococci; P > 0.05). Degradation of lipidswas greater in air than under anaerobiosis (P < 0.05).

bacteria (data not shown). Degradation was studied at 60min of incubation, i.e., when a majority of the pneumococcihad been killed, under both aerobic and anaerobic condi-tions (see above).

After a 60-min aerobic incubation of pneumococci andPBLs in serum, 58% of the radioactive choline incorporatedinto pneumococcal teichoic acid ceased to be in high-molec-ular-weight form (TCA precipitable) as compared with 45%for anaerobic conditions (P > 0.05; Fig. 1). Also, 70 to 75%of the pneumococcal RNA and about half of their DNA was

degraded after 60 min under both anaerobic and aerobicconditions (P > 0.05; Fig. 1).

Teichoic and nucleic acids in autolysis-resistant pneumo-cocci were degraded by aerobic and anaerobic human PBLsto the same extent (P > 0.05) as were these macromoleculesin normal (autolytic) pneumococci (Fig. 1). Hence, theendogenous autolytic system of Streptococcus pneumoniaeapparently did not contribute either to the killing (see above)or to the aerobic or anaerobic phagocytic degradation of itscell envelope, which would render pneumococcal nucleicacids accessible to PBL nuclease(s).

[14C]arachidonic acid incorporated into Streptococcuspneumoniae lipids was used to study the degradation of thepneumococcal cytoplasmic membrane by the phagocytes.At 60 min of aerobic incubation of bacteria and PBLs, half ofthe arachidonic acid radioactivity (48 ± 3%) could no longerbe recovered in lipid extracts, whereas after anaerobicincubation, virtually all of the lipid label (92 ± 5%) remainedextractable (P < 0.05; Fig. 1A).

DISCUSSIONThe present data demonstrated that encapsulated Strepto-

coccus pneumoniae of serotypes 2, 9N, 14, 21, and 23F andunencapsulated pneumococci of serotype 2 were phago-cytosed with comparable efficiency in air and under an-

aerobic conditions by human PBLs. Analogous results havebeen reported for several other particles (20, 25).As also found for certain other bacteria (18, 20, 28), an

02-dependent killing system(s) of the leukocytes was re-

quired for maximum killing of Streptococcus pneumoniae.This was previously suggested from aerobic experiments inwhich the influence of 02-dependent bactericidal mecha-nisms was minimized by the use of phagocytes from apatient with chronic granulomatous disease and a mutantstrain of Streptococcus pneumoniae deficient in H202 pro-duction (24).

Streptococcus pneumoniae was shown to be killed byhuman PBLs under anaerobic conditions also. This findingseems highly pertinent to our understanding of the hostdefense in acute sinusitis (see above) and has not, to ourknowledge, been reported before. Measurements of redoxpotential and functional controls (see above) as well as lipidanalyses (see below) indicated that residual 02 was not thecause of the delayed anaerobic killing observed. Apparently,our data reflected the existence of 02-independent bacteri-cidal leukocyte functions not yet elucidated.Few studies of the degradation of phagocytosed Strepto-

coccus pneumoniae by human phagocytes are available, inparticular in which 02-dependent killing mechanisms areexcluded (6). As shown here and earlier by Shohet et al. (27),unsaturated pneumococcal cytoplasmic membrane lipidswere degraded by human PBLs under aerobic conditions, asindicated by considerable losses of [14C]arachidonic acid.Neither we nor Shohet et al. (27) recorded any significantdegradation of lipids under conditions in which 02-dependent bactericidal mechanisms probably were nonfunc-tional. Hence, the proposal by the latter workers thataerobic polymorphonuclear leukocytes (PMNs) attack bac-terial membrane lipids mainly by means of an 02-dependentperoxidation of unsaturated fatty acids was supported here.It is conceivable that such a process causes irreversible(lethal) membrane damage and contributes to the acceler-ated cidal activity of PBLs against pneumococci seen in thepresence of 02.

In contrast to pneumococcal lipids, the bacterial cell wallteichoic acid, RNA, and DNA were attacked by humanPBLs almost as efficiently under anaerobic as under aerobicconditions. Thus, leukocyte degradation of lipids was bettercorrelated with killing than was degradation of the otherbacterial macromolecules studied. The anaerobic break-down of pneumococcal nucleic acids must reflect severedamage to their cytoplasmic membrane, rendering bacterialRNA and DNA accessible to leukocyte nuclease. Althoughan effect on lipids seems unlikely (see above and reference27), the mechanism of the anaerobic attack by leukocytes onpneumococcal membranes is not presently known.

Elsbach and Weiss (7) have isolated and studied anantibacterial cationic granular protein from human and rab-bit PMNs. This factor is, however, active only againstgram-negative bacteria such as Escherichia coli, which arekilled in part due to triggering of their own autolytic enzymesystem by the granular protein (7). On the other hand,preparations of other cationic granular protein fractions (2)and granulocyte lactoferrin (1) are reportedly cidal to Strep-tococcus pneumoniae. Also, an acid environment lethal topneumococci (19) may develop in the phagosomes duringanaerobiosis (11). Apparently, human PMNs contain several02-independent bactericidal functions with different speci-ficities (7).The presence of capsular type 2 polysaccharide offered no

protection to the pneumococci against aerobic or anaerobickilling by human PBLs. Similarly, autolysis-resistant Strep-tococcus pneumoniae type 14 cells were killed and degradedto the same extent as were normally autolytic type 14bacteria under both aerobic and anaerobic conditions. Also,

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Tomasz and co-workers (30) found that aerobically incu-bated rabbit PMNs killed and degraded autolytic and auto-lysis-resistant pneumococci with equal efficiency. Thus, theaerobic and anaerobic decay of phagocytosed pneumococcimust be attributed solely to activities of the human PBLswithout any significant contribution by the autolytic systemof the bacteria.

ACKNOWLEDGMENTSWe thank W. Sjoberg, S. Granstrom, and Lena Ohlund for tech-

nical assistance and M. Koskela for antibody determinations.Grants were obtained from the Swedish Medical Research Coun-

cil (B84-16x-06 562-02) and the Faculty of Medicine, University ofUmea, Sweden.

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