Endotoxin In Vitro Interactions with Human Neutrophils: Depression ...

INFECTION AND IMMUNITY, Sept. 1979, P. 912-92100 19-9567/79/09-0912/ 10$02.()0/0

Vol. 25, No. 3

Endotoxin In Vitro Interactions with Human Neutrophils:Depression of Chemiluminescence, Oxygen Consumption,

Superoxide Production, and KillingRICHARD A. PROCTOR

Departments ofMedical Microbiology and Medicine, University of Wisconsin, Madison, Wisconsin 53706

Received for publication 22 June 1979

Endotoxin was shown to depress neutrophil bactericidal activity while enhanc-ing Nitro Blue Tetrazolium reduction and hexose monophosphate shunt activity.Separation of bactericidal action from oxidative metabolism suggests that thetoxic effect of endotoxin might involve the formation of reactive oxygen radicalssuch as superoxide. Chemiluminescence often accompanies metabolic activationof polymorphonuclear neutrophils (PMNs). However, human PMNs did not showchemiluminescence when challenged with endotoxin (lipopolysaccharide; LPS) or

lipid A. Superoxide formation was also unaffected by endotoxin. In contrast,preincubation of PMNs with LPS for 30 min produced significant depression ofchemiluminescence, oxygen consumption, and superoxide formation. Decreasedchemiluminescence was not the result of complement consumption. In a cell-freesystem, superoxide was not scavenged by LPS, nor did LPS stimulate superoxidedismutase. Oxidase enzymes for reduced nicotinamide adenine dinucleotide or

reduced nicotinamide adenine dinucleotide phosphate harvested from brokencells were not affected by LPS. The toxicity of LPS may reside in its ability toactivate the PMNs while simultaneously blocking bactericidal capacity.

There is general agreement that exposure ofpolymorphonuclear neutrophils (PMNs) toendotoxin (lipopolysaccharide; LPS) in vitro re-sults in enhanced hexose monophosphate shunt(HMPS) activity (10, 11, 23, 41), increased gly-colysis (10, 11, 23, 28), increased lysosomal en-zyme release (22), and accelerated Nitro BlueTetrazolium (NBT) reduction (19, 29, 33, 34, 40,41). In contrast, various authors have found thatLPS challenge increases (23, 45), decreases (28),or has no effect (11) on neutrophil oxygen con-sumption. This disparity of results might be dueto the different species used in these experiments(human, guinea pig, rabbit), differences in meth-ods of obtaining PMNs (i.e., from exudates orvenous blood), the presence or absence of serumin the system, and the relative insensitivity ofthe Warburg manometric technique (48).

Understanding the oxidative changes that oc-cur in LPS-challenged human PMNs is criticalsince oxygen-derived radicals are instrumentalin neutrophil bactericidal activity (3). This com-munication describes the effects of endotoxin onhuman neutrophil oxygen consumption, HMPSactivity, chemiluminescence (CL), superoxideproduction, reduced nicotinamide adenine di-nucleotide (phosphate) (NADPH-NADH =

[NAD(P)H]) oxidase activity, and bactericidalactivity.

MATERIALS AND METHODS

Preparation of neutrophils. Venous blood fromhealthy donors was heparinized (2 U of aqueous so-dium heparin per ml of blood; grade 1; Sigma ChemicalCo., St. Louis, Mo.) and combined with 1 to 2 volumesof 3% dextran (average molecular weight, 264,000;Sigma) in isotonic saline (pH 7.4). After 20 min oferythrocyte sedimentation at room temperature, theleukocyte-rich supernatant was separated and thencentrifuged (200 x g, 5 min). Contaminating erythro-cytes were lysed by addition of 0.155 M NH4Cl, 10 mMethylenediaminetetraacetic acid, and 0.3 mM NaHCO:3at 37°C. The preparation was washed twice with'ianks balanced salt solution without phenol red(HBSS; GIBCO, Grand Island, N.Y.) (pH 7.4). Greaterthan 95% of PMNs appeared viable as assessed by 1%trypan blue exclusion. Differential and quantitativeleukocyte counts were performed. Suspensions ofPMNs were adjusted with HBSS to final concentra-tions as noted in legends of figures.LPS was added to PMNs either simultaneously

with or 30 min before challenge with other substances.Some of the PMNs were preincubated with LPS at37°C for 30 min, and the rest of the PMNs weresimilarly incubated as controls. After 30 min of incu-bation of LPS with PMNs, greater than 91% remainedviable as determined by trypan blue exclusion.

Phagocytizable particles. Escherichia coli(ATCC 25922), E. coli 0:6 (a clinical isolate fromurine), and Staphylococcus aureus (ATCC 25923)were grown overnight in tryptic soy broth (Difco Lab-

912

ENDOTOXIN IN VITRO INTERACTIONS 913

oratories, Detroit, Mich.). (Serotyping was performedin the laboratory of Calvin Kunin, William S. Middle-ton V.A. Hospital, Madison, Wis.) Suspensions of bac-teria were washed three times in HBSS. Single batchesof lyophilized E. coli (ATCC 25922) and S. aureuswere stored at -20'C and used in oxygen consumptionstudies. Before lyophilization, bacteria were suspendedin sterile distilled water. Those bacteria used for bac-tericidal assay (E. coli 0:6 and S. aureus) were ob-tained from overnight cultures and adjusted to anoptical density of 0.6 at 620 nm. A 1:1,000 dilution gave1 x 10' to 4 x 10' bacteria per ml. Both E. coli 0:6and S. aureus were resistant to serum bactericidalaction.Zymosan (Sigma) was prepared using the method

of Rosen and Klebanoff (38). Bacteria and zymosanwere preopsonized by incubation with serum (37"C, 30min) and then washed twice. Serum was obtained fromhealthy donors and stored at -40'C for less than 6weeks before use.

Latex beads (0.81 pm, Difco) were washed twice andthen adjusted to 4 x 109 particles per ml. Endotoxin-coated latex beads were prepared by incubation ofLPS with latex beads (1 mg of LPS per ml of latexsuspension). Binding was confirmed by specific agglu-tination of triply washed latex beads with specificrabbit antisera to E. coli serotype 0111:B4 (antiseradonated by Calvin Kunin). Normal rabbit sera failedto agglutinate endotoxin-coated beads. The endotoxinantisera did not agglutinate uncoated latex beads.Endotoxin-coated latex beads were also adjusted to 4x 109 particles per ml.Endotoxin. A single lot (no. 740856) of E. coli

endotoxin 0111:B4 prepared by the Westphal methodwas obtained commercially (Difco) and used through-out. LPS caused gelation of Limulus amoebocyte ly-sate at 1 to 5 ng/ml (Sigma) (43) and demonstrated anintraperitoneal 50% lethal dose for C57BL mice(Sprague-Dawley) of 150 Mg. This LPS containedabout 1% protein by weight. Lipid A was a gift fromPaul Madson (William S. Middleton Memorial V. A.Hospital, Madison, Wis.) which had been preparedfrom Salmonella in the laboratory of 0. Westphal(Freiburg, Germany) as previously described (17).Lipid A was solubilized by adding bovine serum albu-min.Oxygen measurements. Oxygen consumption by

PMNs was measured polarographically with a Clarkoxygen electrode (Gibson Medical Electronics, Mid-dleton, Wis.). Measurements were made at 370C for 3to 5 min after challenge with phagocytizable particles.Experimental conditions are noted in Table 1, footnotea.

Bactericidal assay. PMN bactericidal activity wasmodified using the method of Quie et al. (36). Dupli-cate 1-ml reaction mixtures in HBSS containing 10%serum, 5 x 10" PMNs, and 1 x 10' to 4 X 10' bacteriawere rotated end-over-end for 120 min at 37"C. Sam-ples were taken at 30-min intervals and diluted withdistilled water. The number of bacteria per milliliterwas determined by the pour-plate method using tryp-tic soy agar (Difco).

Quantitative NBT test. The quantitative NBTdye reduction test was performed as previously de-scribed (4). A brief description of the method, includ-

ing the anaerobic modification, follows. PMNs weredivided into equal portions and held on ice either inroom air or in a Coy anaerobic glove box (Ann Arbor,Mich.) for 5 h. After the cells were allowed to warmfor 30 min at 37°C, 4 x 10` PMNs, 0.05 ml of 2C mMKCN, 0.4 ml of 0.1% NBT, 100 yg of LPS, and HBSSsufficient to make a volume of 1.1 ml were added andincubated either aerobically or anaerobically for 30min at 37°C. Reactions were terminated by adding 4.0ml of ice-cold 0.4 N HCI. Pellets were extracted with4.0 ml of pyridine, and optical density was read at 515nm.HMPS activity. HMPS activity was determined as

previously described (35). Briefly, 5 x 10" PMNs, 1.0MuCi of D-[1-'4C]glucose or D-[6-'4C]glucose, 2 mMKCN, and challenge substance (as noted in Table 3)were incubated in glucose-free HBSS (total volume,1.0 ml) at 37°C on a reciprocating shaker. The reactionwas terminated by addition of 1.0 ml of 10% trichlo-roacetic acid. Released 14C02 was trapped on filterpaper impregnated with methyl benzethonium hy-droxide and then counted in a Packard Tri-Carb liquidscintillation counter.

CL. CL was measured in a Packard Tri-Carb liquidscintillation counter by the method of Allen et al. (1).Reaction mixtures were maintained at 37°C until theywere counted for 1 min. Concanavalin A (ConA) wasobtained from Sigma.

Superoxide measurements. In the cell-free sys-tem, superoxide production was measured by usingthe method of McCord and Fridovich (31). Reactionmixtures containing 0.15 mM xanthine (Sigma), 3.6 x10 9 M xanthine oxidase (Boehringer MannheimCorp., New York, N.Y.) and 30 MM horse heart ferri-cytochrome (type III, Sigma) in 3 ml of 0.05 M potas-sium phosphate buffer (pH 7.8) were incubated at20°C. Reaction mixtures containing superoxide dis-mutase (>3,000 U/mg; Truett Labs., Dallas, Tex.)received sufficient enzyme to cause a 50% decrease incytochrome c reduction. When superoxide (02-) mea-surements were performed on PMNs, reaction mix-tures contained 75 MM horse ferricytochrome c, 5 x10" PMNs, and 10% serum in HBSS, according to themethod of Goldstein et al. (21). An extinction coeffi-cient of 2.11 x 104 M cm-' at 550 nm (reduced-oxidized) was used to calculate micromoles of ferro-cytochrome c formed. When present, LPS was at afinal concentration of 100 jg/ml.NAD(P)H oxidase activity. Neutrophil oxidase

activity was measured by the fluorometric method ofDeChatelet (14, 26) and is briefly described below.Reaction mixtures containing 5 x 10' PMNs in phos-phate-buffered saline (pH 7.4) were equilibrated for 30min at 37°C before challenge with preopsonized zym-osan particles (final concentration, 3 mg/ml). To thosetubes receiving LPS, LPS (100 Mg/ml) was addedeither at the beginning or the end of the equilibrationperiod. Three minutes after zymosan was added, thereaction was stopped by adding 2.0 ml of iced 0.68 Msucrose. The cells were homogenized (greater than95% breakage) and then centrifuged at 500 x g, andfinally the supernatant was removed and centrifugedat 27,000 x g. The protein concentration in the high-speed pellet was adjusted to 1 mg/ml, and 0.3-mlsamples were used for assay in the NAD(P)H oxidase

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914 PROCTOR

assay. Assay mixtures (1.0 ml) contained 0.3 ml ofpellet protein solution, 2.0 mM KCN, 0.1 M 2-(N-morpholino)ethanesulfonic acid (pH 6.0) buffer, andeither 0.2 mM NADPH or 0.8 mM NADH. After 30min of incubation at 370C, reactions were stopped bythe addition of 1 ml of 0.4 M HCl04, and precipitatedprotein was removed by centrifugation. A 0.1-ml su-pernatant was incubated with 0.15 ml of 10 N NaOHfor 1 h at 20'C. Then 1.6 ml of water was added, andfluorescence was read on an Aminco-Bowman spectro-fluorometer (excitation, 365 nm; emission, 448 nm).Nanomoles of NADH or NADPH formed were deter-mined by comparison to relative fluorescence of stan-dard curves, run in parallel daily. All determinationswere performed in duplicate. Direct addition of LPSto the assay mixture was found not to alter oxidaseactivity.

RESULTSEffect of endotoxin on neutrophil bacte-

ricidal activity. The effects of graded doses ofLPS on PMN function are shown in Fig. 1. Whenno LPS was present, approximately 1.5 log10bacteria were killed in 120 min. Preincubation ofLPS with PMNs (Fig. 1A and C) inhibited killingof E. coli at 10 jig of LPS per ml or greater andresulted in a dose-dependent reduction in thekilling of S. aureus. When LPS was added si-multaneously with E. coli or S. aureus (Fig. 1Band D), PMN bactericidal action was again re-duced, but to a lesser extent. Of particular inter-est were the rates of killing of E. coli during thefirst 30 min. With simultaneous addition of 10 to100 pg of LPS per ml and E. coli to PMNs (Fig.1B), the rates of killing progressed rapidly for 30min and then plateaued. After 30 min of incu-bation, the contours of the growth curves in Fig.1B were nearly identical to those in Fig. 1A.That these bacteria were resistant to killing

by 10% serum was demonstrated by their growthwhen no PMNs were present (Fig. 1). Uptake ofbacteria was not affected by the presence ofLPS, as determined by the percentage of PMNsthat had phagocytized bacteria or by the numberof bacteria phagocytized per PMN.

Effect of endotoxin on neutrophil oxygenconsumption. Neutrophils rapidly consumedoxygen when phagocytizing E. coli, S. aureus, orzymosan (Table 1). Concomitant addition ofLPS with these same particles allowed the phag-ocytically induced increase in oxygen consump-tion to proceed normally. In contrast, preincu-bation of LPS with PMNs resulted in a markedreduction of oxygen consumption during phag-ocytosis. Challenge of PMNs with LPS aloneresulted in rates of oxygen consumption nearlyidentical to that of resting PMNs, even whenreaction rates were monitored for as long as 45min. Addition of 0.1 ml of 0.01 M KCN to thereaction mixtures did not alter oxygen uptake in

two of the experiments done in duplicate. Sincethe bacteria were in lag phase (lyophilized andreconstituted for 30 to 60 min before use), theydid not show oxygen uptake when added to thereaction chamber alone.

Effect of LPS on neutrophil HMPS activ-ity. Challenge of neutrophils with E. coli or S.aureus resulted in an approximate 10-fold in-crease in [1-'4C]glucose metabolism and a 2-foldincrease in [6-'4C]glucose metabolism over rest-ing values (Table 2). Addition of endotoxin re-sulted in a twofold (10 Mg) to threefold (100 Mg)increase in [1-'4C]glucose oxidation and a 40 to75% increase in [6-'4C]glucose oxidation.Effect of LPS on NBT dye reduction. Re-

duction of NBT dye by PMNs was enhanced byaddition of either latex spheres or LPS (Table3). This reduction increased with increasing con-centrations of LPS. Addition of larger quantitiesof LPS (up to 1,000 4g/ml) resulted in no greaterreduction than occurred at 100 tg/ml.Because particle-induced NBT reduction by

neutrophils is reportedly mediated solely by su-peroxide (an oxygen-derived radical) in wholePMNs (6), we elected to measure LPS-inducedNBT reduction anaerobically (Fig. 2). A consist-ent increase in NBT reduction occurred whetherthe cells were held aerobically or anaerobicallyfor 5 h. A larger mean increase in optical densitywas seen under aerobic (0.067 ± 0.021) condi-tions as compared to anaerobic conditions (0.039± 0.009). Also, total change in optical densitywas smaller after the PMNs were held (bothaerobic and anaerobic) for 5 h at 4°C than whencells were used immediately. Cell suspensionswere warmed for 30 min at 37°C before use toallow microtubule function to be restored.

Effect of LPS on PMN CL. When PMNswere challenged with concentrations of LPSfrom 0.1 to 100 pg/ml, there was no change inCL as compared to resting cells. Table 4 sum-marizes the data for E. coli O111:B4 LPS ob-served at 25 min. Challenge with endotoxinsfrom E. coli 055:B5 (Difco) and Salmonellatyphimurium (Difco) also failed to induce CL.Also, when observations were performed at 5- to15-min intervals over a 1- to 180-min time span,LPS did not stimulate PMN CL. Furthermore,no enhancement of neutrophil CL followed lipidA challenge (Table 4). To be certain that LPSwas entering the PMNs, LPS was bound to latexparticles. Whether LPS was absent, added si-multaneously, or bound to the latex beads, noalteration in latex-induced CL was noted (Table4).

Addition of S. aureus and E. coli to PMNsresulted in a 20- to 30-fold increase in CL (Table5). This increase was not affected by the simul-

INFECT. IMMUN.


70() 50#9/0s0o<mLiji 50#g

FIG.~~~1.Efc fenooi nnutohlbceicdlatvt. 0 Ms 10#9 eu, BS n

>6.0-0

0

0 30 60 90 120 0 30 60 90 120

TIME (min)FIG. 1. Effect of endotoxin on neutrophil bactericidal activity. 5 x 106 PMNs, 10% serum, HBSS, and

bacteria (S. aureus or E. coli 0:6) were incubated with LPS (O to 100 pg/ml). (A) and (C) Data when LPS waspreincubated with PMNs; (B) and (D) effects of simultaneous additions. Each point represents the mean offive to six experiments done in duplicate. One standard deviation is shown for 120-min values.

taneous addition of LPS, whereas simultane-ously added lipid A caused a consistent, but notstatistically significant, reduction. In contrast,preincubation of neutrophils with LPS or lipidA for 30 min produced a highly significant re-duction in bacteria-induced CL. A similar reduc-tion in ConA-stimulated CL was noted in thosePMNs preincubated with LPS (Table 5).Because LPS is known to activate the alter-

native pathway of complement (18, 32), studieswere undertaken to assess the role of comple-ment in the LPS-altered CL response of PMNs.When heated serum replaced fresh serum (Fig.3), a marked reduction in light production was

obtained. Nevertheless, the PMNs preincubatedwith LPS still showed less CL in heated serumthan those PMNs that were either unexposed toLPS or added to the system simultaneously withLPS. PMNs challenged with preopsonized bac-teria in heated serum showed the same CL val-ues as those challenged with bacteria in freshserum, whereas the LPS-preincubated PMNsshowed significantly less light production.

Effect of endotoxin on neutrophil super-oxide production. To be sure that LPS wasnot decreasing CL by light quenching, by scav-enging light-producing radicals, or by enhancingsuperoxide dismutase activity, 02 formation

VOL. 25, 1979

TABLE 1. Effect of endotoxin in particle-induced neutrophil oxygen consumption

Oxygen consumption' by:Particles P

PMNs PMNs + LPS PMNs + LPS-PI

None 4.38 ± 0.5 (14) 4.87 ± 0.5 (13) 4.31 ± 0.4 (13)E. coli 48.6 ± 8.7 (6) 47.8 ± 10.8 (6) 28.0 ± 3.5 (6) <0.02S. aureus 102 ± 13 (5) 94 ± 14 (5) 75 ± 2.5 (5) <0.02Zymosan 73.9 ± 5.1 (4) 71.7 ± 5.1 (4) 54.8 + 4.8 (5) <0.02

Nanomoles of oxygen consumed per minute ± 1 standard deviation by 5 x 10' PMNs per 1.5 ml in 10%serum after challenge with 3.5 mg of washed zymosan or lyophilized bacteria at 37"C. LPS was used at 67 Ag/ml. LPS-PI, LPS preincubated with PMNs for 30 min before challenge. Parentheses indicate number of tests induplicate. Significance was determined by paired Student's t test.

TABLE 2. Effect of endotoxin on neutrophil HMPS TABLE 3. Effect of endotoxin on NBT dye reductionnotiz litv

HMPS activity"PMNs (5x106/ml) IV [1-14C]glucose glucose

Resting 18 1,503 + 148 210 ± 18+ E. coli (0.35 mg) 6 14,865 + 1,308 448 ± 39+ S. aureus (0.35 mg) 6 16,021 + 1,872 398 ± 41+ LPS (1,000jig) 4 5,081 + 639 423 ± 68+ LPS (100lug) 8 4,869 + 424 381 ± 52+ LPS (10 jig) 8 3,077 + 326 300 ± 37+ ConA (100,Lg) 4 5,232 + 717 583 ± 55

'N, Number of experiments, performed in triplicate.Mean disintegrations per minute per 5 x 10' PMNs, re-

covered as "4CO. from the oxidation of glucose after 30 min ofincubation (±1 standard deviation).

was measured. When xanthine is metabolized tohypoxanthine by xanthine oxidase, 02- is formed(15, 16). Production of 02- was found to beunaffected by the addition of LPS. When suffi-cient superoxide dismutase was added to thereaction mixture to decrease the amount of cy-tochrome c reduction by 50%, addition of LPSdid not affect superoxide dismutase activity.

Release of 02 from whole cells, as measuredby cytochrome c reduction, was greatly acceler-ated by the addition of E. coli or S. aureus (Fig.4). PMNs challenged simultaneously with LPSand bacteria release 02- in quantities nearlyidentical to PMNs challenged with bacteriaalone. In contrast, preincubation of PMNs withLPS resulted in a significant decrease in thebacteria-induced neutrophil 02 formation.

Effect of LPS on NAD(P)H oxidase activ-ity. The NADPH oxidase activity in neutrophilschallenged with zymosan showed a 60% increaseas compared to that in resting PMNs (Table 6).In contrast, the NADH oxidase activity fromthese same zymosan-challenged PMNs demon-strated a less than 9% increase. Whether LPSwas added simultaneously or preincubated withthe PMNs, it did not alter the NADPH oxidaseor NADH oxidase activities of resting or phag-ocytizing PMNs.

PMNs ODr,," P

Resting 0.101 ± 0.008 (10)+ Latex 0.369 ± 0.048 (8) <0.001+ LPS (100 pg) 0.223 ± 0.032 (10) <0.001+ LPS (10 jg) 0.198 ± 0.049 (10) <0.01+ LPS (1 Mig) 0.136 ± 0.011 (10) <0.05

" OD515, Optical density at 515 nm of 5 x 10" PMNsafter 15 min.

.15

E 13E

_ 11I-)

(nZ .090

ANAEROBICRESTIGI

* = RESTING

AEROBIC

IB

FIG. 2. Effect of anaerobic environment on endo-toxin-induced NBT reduction. The change in opticaldensity was measured after 15 min of incubation inreaction mixtures containing 5 x 106 PMNs, 100 Mgof LPS, 0.1% NBT, 20 mM KCN, and HBSS. Experi-ments were paired so that the set (resting + LPS set)of anaerobic data appearing furthest to the left in (A)corresponds to the aerobic set in (B), and so forth.

DISCUSSIONThese experiments suggest that endotoxin dis-

rupts human PMN function by disassociatingglucose metabolism from oxygen consumptionand bactericidal activity. Activation of the PMNHMPS by LPS has been established (10, 11, 23,47), and this was reconfirmed in our studies

916 PROCTOR INFECT. IMMUN.


TABLE 4. Effect ofLPS and lipid A on neutrophilCLa

Additive Concn, Ag/ml (la- CL (cpm ± 1Additivetex, ml) SD)

LPS - 3,998 + 6580.1 4,132±4311.0 4,367 ± 1,264

10.0 4,004 ± 458

Lipid A - 3,813 ± 4110.001 4,124 ± 1,9560.01 3,784 ± 1,4130.1 3,656±861

LPS + latex (ml) - (-) 3,521 ± 673- (0.2) 22,637 ± 3,826100 (free) (0.2) 24,184 ± 7,244Bound (0.2) 21,463 ± 3,789

'Reaction mixtures, containing 5 x 106 PMNs, 10%serum, E. coli O111:B4 LPS (0 to 100 jig/ml) or lipidA (0 to 100 jqg/ml), and HBSS sufficient to make 7.0ml of total volume, were incubated for 25 min at 370C.Peak CL occurred at 25 min whether LPS or lipid Awas added simultaneously or was preincubated withthe PMNs. CL (counts per minute [cpm]) was meas-ured in a Packard Tri-Carb scintillation counter inout-of-coincidence mode utilizing dark-adapted, sili-conized glass counting vials. Latex particles (0.81-ttm)were incubated with LPS (1 mg/ml) at 370C to achieveLPS binding. Unbound LPS was washed from latexparticles. Specificity was assured by group-specificantisera. LPS-free latex particles were also washedthree times. Uptake of latex particles was determinedmicroscopically. Variation from four experiments donein duplicate is given as 1 standard deviation (SD). Allvalues were taken 25 min after test substance wasadded. -, No LPS added.

using E. coli O11L:B4 LPS in human PMNs.Separation of HMPS activation from oxygen

consumption and 02 production is unusualamong activators of PMN metabolism (3, 13).Sodium fluoride is the only other agent known

to stimulate glucose metabolism (39, 42) but toinduce little (24) or no (9) PMN CL relative tothe quantity of 02 produced. In contrast toLPS, sodium fluoride does induce the release oflarge quantities of 02 from human PMNs (9,12, 24). It has been postulated that sodium flu-oride fails to produce CL because it is a nonoxi-dizable agent. However, LPS is a soluble, poten-tially oxidizable compound which did not stim-ulate CL or the release of02 in our experiments.Another soluble agent, ConA, also stimulates CL(Table 5) and 02 (22). Thus, it cannot be pos-tulated that LPS fails to induce CL or 02 pro-duction because it is a soluble, nonoxidizablesubstance.

Preincubation of PMNs with lipid A and LPSdepressed phagocytosis-induced CL and 02 re-

lease. ConA-stimulated CL was also reducedafter preincubation of PMNs with LPS. Thus,LPS interfered with fullPMN excitation by bothparticulate and surface-active agents. Interest-ingly, the PMN CL after E. coli challenge was

consistently less than that produced by S. au-reus. Whether this is due to in situ LPS remainsto be determined.

Failure of LPS to stimulate oxygen consump-tion is consistent with the lack of CL, since lightemission from PMNs is felt to arise from theoxygen-derived by-products of the respiratoryburst (2, 9, 38, 49). Previous studies had reported

TABLE 5. Effect ofpreincubation of endotoxin or lipid A on neutrophil CL"Neutrophil CL

PMNsE. coli S. aureus ConA

Resting 4,322 ± 419 4,168 ± 375 4,031 ± 383No LPS 93,471 + 10,328 121,653 ± 8,407 15,981 ± 3,428+ LPS 95,684 ± 9,872 120,281 ± 7,378 20,119 ± 3,137+ LPS-PI 54,634 ± 8,366 78,059 ± 9,462 9,846 + 1,020Significance (P) <0.01 <0.01 <0.02

Resting 4,322 ± 419 4,168 ± 375 -

No lipid A 99,113 ± 6,544 128,481 ± 9,006 -+ Lipid A 90,764 ± 1,738 107,524 ± 7,656 -+ Lipid A-PI 52,279 ± 3,787 74,710 ± 8,091 -Significance (P) <0.01 <0.01

"'Effect of preincubation of endotoxin and lipid A on ConA and bacteria-induced neutrophil CL. See Table4 for reaction conditions and measurements. Lyophilized E. coli ATCC 25922 and S. aureus ATCC 25923 wereat 0.5 mg/ml in final reaction mixture. ConA was used at 30 pg/ml. LPS or Lipid A, Endotoxin or lipid A addedat time zero along with bacteria. LPS-PI or Lipid A-PI, Endotoxin or lipid A added 30 min before bacterialchallenge. All PMNs were incubated at 370C for 30 min whether LPS was present or absent. CL was measured25 min after the addition of bacteria. Data represent six experiments done in duplicate. Significance wasdetermined by paired Student's t test; values for simultaneously added LPS or lipid A were compared withpreincubated values. -, Not done.

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2

(-)

FRESH SERUM HEATED SERUM HEATED SERUMPRE-OPSONIZED BACTERIA

FIG. 3. Effect of heated serum and endotoxin on bacteria-induced neutrophil CL. See Fig. 1 for reactionconditions. Complement was inactivated by heating at 56"C for 30 min (heated serum). A 35-mg sample oflyophilized bacteria plus 1 ml of fresh serum was incubated at 370C for 30 min. These bacteria were washedthree times. Nonpreopsonized bacteria were handled similarly, but HBSS replaced serum. Final bacterialconcentrations were 0.5 mg/ml. Experiments were performed six times in duplicate. Significance wasdetermined by the paired Student 's t test.

that LPS caused increased (23, 45), decreased(28), or unchanged (11) PMN oxygen consump-

tion. Variations in the species studied, in concen-trations of PMNs, serum, and LPS, in the du-ration of incubation, and in methods of harvest-ing cells probably all contribute to the disparateresults. The Warburg manometric techniqueemployed to determine oxygen consumption ineach of these studies may show a severalfoldintra-experimental variation unless meticulouscare is given to the rate of shaking, vapor pres-sure from released C02, etc. (48). By using higher

concentrations of PMNs (5 x 107/ml) andshorter reaction times and by measuring oxygenchanges polarographically, technological limita-tions of prior methods were greatly obviated.Another cell that responds to LPS challengesimilarly to the human PMN is the S91 mela-noma (50). Endotoxin stimulated glycolysiswithout affecting oxygen uptake.Both oxygen consumption and bactericidal

activity were reduced by preincubation of PMNswith LPS. Since oxygen is crucial to the bacte-ricidal action of PMNs (3), it is not surprising

INFECT. IMMUN.


zi

-50-z

40400

w

RESTING +EC *EC +EC RESTING USA +SA +SAPMSN *LPS +LPS-PI P1*4 +LPS +LPS-PI

FIG. 4. Effect ofendotoxin on neutrophil superoxide reduction of cytochrome c. A mixture of5 x 106 PMNs,75p ferricytochrome c, HBSS, and 10%o serum was incubated at 370C. E. coli ATCC25922, 5. aureus ATCC25923, and 1(X0 pg of LPSper ml were added as noted. SA, S. aureus; EC, E. coli; LPS-PI, LPSpreincubatedwith PMNs (370C, 30 mmn) before bacterial challenge. Values represent means ± 1 standard deviation of sixexperiments done in duplicate. Significance was determined by paired Student's t test.

TABLE 6. Effect of endotoxin on NAD(P)H oxidaseactivity'

nmol oxidized by:

Protein N NADPH ox- NADH oxi-

idase dase

Resting PMN 9 10.0 + 0.79 4.81 ± 1.06PMN + zymosan 9 16.0 ± 3.15 5.23 ± 1.01PMN + zymosan + 7 16.0 ± 5.15 6.06 ± 0.90LPS

PMN + zymosan + 9 15.6 ± 2.57 5.69 ± 0.98LPS-PI

PMN + LPS 8 10.9 ± 0.70 5.07 ± 0.72Spontaneous 9 10.6 ± 3.29 5.06 ± 0.68

a he number of nanomoles (±1 standard deviation)of either NADPH or NADH oxidized by 0.3 mg ofprotein is shown. N, Number of experiments done induplicate. LPS-PI, Endotoxin preincubated for 30 minwith PMNs before phagocytic activation.

that PMNs utilizing less oxygen kill with de-creased efficiency. Interestingly, adding bacteriaand LPS to PMNs simultaneously also resultedin a similar, but delayed, reduction in bacteri-cidal activity. The bactericidal assay was the onetest that extended for more than 30 min, thusaffording simultaneously added LPS an oppor-tunity to interact with the PMNs. Indeed, thecontours of the bactericidal curves were nearlyidentical after 30 min.

Previous work by Cohn and Morse (11)showed enhanced killing of Staphylococcus al-bus by rabbit peritoneal PMNs in the presenceof 10% serum and LPS, but reduced killing with

1% serum and 10 to 50 /ig of LPS per ml. TheLPS enhancement of the bactericidal activity ofrabbit exudative PMNs contrasts with the re-duced activity seen with human venous PMNs.Most likely, this represents another example ofthe considerable variation in response to LPSamong different species. With human PMNs,varying the concentration of serum did not alterthe response to LPS, but only reduced the extentof phagocytosis at low concentrations of serum.The diminished bactericidal activity observed

was not the result of decreased phagocytosis.This is in accord with a previous report whichactually demonstrated accelerated phagocytosiswhen LPS and 10% serum were present withbacteria and PMNs (11).

It is well established that LPS-challengedPMNs demonstrate enhanced NBT reduction(19, 29, 33, 34, 40, 41). NBT reduction is report-edly mediated solely by O2 in whole cells, butnot in sonicated PMNs (6). When PMN sonicextracts were utilized, anaerobic NBT reductionwas 67% as efficient as aerobic NBT reduction.Since LPS did not stimulate oxygen uptake,alternative NBT-reducing substances must beavailable to LPS-challenged PMNs, perhaps viaanother NBT reductase(s) (5, 25). LPS mightallow the NBT dye to penetrate the intact PMNor phagosomal membrane and thus to come incontact with non-oxygen-dependent reducingcompounds. LPS has been shown to enhanceinulin entry into PMNs (23).

Neutrophil NAD(P)H oxidase(s) activates theHMPS by producing NADP, the rate-limiting

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920 PROCTOR

compound for the HMPS (7). NeutrophilNADH oxidase or NADPH oxidase activitieswere unaltered by the addition of LPS. Thus,nonoxidative pathways must be available forproduction of NADP from NADPH. One suchpossibility might be via the glutathione reduc-tase pathway (27, 37).The multiple effects that LPS has on the

PMN are possibly mediated through membranealterations. LPS is known to bind to PMNs (8,30), perhaps via a specific membrane receptor(44). Partial removal of sialic acid from the hu-man PMN membrane causes the PMN to re-

spond much like a PMN preincubated with LPS(46). Sialic acid-deficient PMNs showed normalphagocytosis and HMPS activation by latex par-

ticles, but marked inhibition of ConA- or phag-ocytosis-stimulated 02- production (46). An-other possibility is that alterations) in the oxi-dase enzyme(s) in the intact cell might return tonormal during isolation. Since the NADPH ox-

idase is thought to be an allosteric enzyme (14),LPS might act by maintaining the enzyme in a

less active form in whole cells. Alternatively,LPS might interfere with the delivery of oxygento the oxidase enzyme. In any event, the strikingsimilarlity between LPS-challenged and neura-

minidase-treated PMNs suggests that LPS ac-

tion may well be mediated by interactions withcell membrane sialic acid groups.

ACKNOWLEDGMENTS

I thank Kunin, Craig, and Kornguth for their help andencouragement, and Patricia Prochniak for her excellent tech-nical assistance.

I was a recipient of a Bristol Fellowship in InfectiousDisease.

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