Vol. 31, No. 1INFECTION AND IMMUNITY, Jan. 1981, p. 228-2350019-9567/81/010228-08$02.00/0

Evaluation of the Opsonic Requirements for Phagocytosis ofStreptococcus pneumoniae Serotypes VII, XIV, and XIX by

Chemiluminescence AssayKATHERINE K. MATTHAY,12* WILLIAM C. MENTZER 1,2 DIANE W. WARA,1 HAIGANOUSH K.

PREISLER, 2 NATASHA B. LAMERIS,' AND ARTHUR J. AMMANN" 2

Department ofPediatrics' and Northern California Comprehensive Sickle Cell Center,2 University ofCalifornia, San Francisco, California 94143

A luminol-enhanced chemiluminescence assay was used to investigate opsonicrequirements for phagocytosis of Streptococcus pneumoniae serotypes VII, XIV,and XIX. After opsonization with whole immune sera (with antibody and totalcomplement pathway), heat-inactivated immune sera (with antibody alone), ormagnesium dichloride-ethylene glycol tetraacetic acid-chelated immune sera(with antibody and alternative complement pathway), live S. pneumoniae cellswere incubated at 37°C with normal polymorphonuclear leukocytes while serialchemiluminescence measurements were recorded. The amount of chemilumines-cence observed correlated closely with evidence of phagocytosis as observed bymicroscopy. Complement was required for efficient opsonization, since all threeserotypes showed a slower rise and less integral chemiluminescence after opso-nization with heat-inactivated serum as compared with whole serum. The alter-native pathway provided opsonic activity equal to that of the total complementpathway for type XIX, but only intermediate activity for types VII and XIV.Type-specific antibody was also required for effective opsonization of all threeserotypes since chemiluminescence was markedly reduced when bacteria wereopsonized with antibody-depleted serum (serum absorbed with type-specific S.pneumoniae cells at 4°C). Thus, chemiluminescence proved to be an effectivemeans of defining the requirement for both antibody and complement in theopsonization and phagocytosis of S. pneumoniae.

The recently introduced 14-valent pneumo-coccal polysaccharide vaccine provides protec-tion against serotypes responsible for more than80% of pneumococcal infections (7). The anti-body response and clinical effectiveness of suchpolyvalent vaccines have been demonstrated inasplenic (3, 43) and normal (33, 39, 45) individ-uals. Nonetheless, apparent vaccine failure hasrecently occurred in patients with either an in-adequate antibody response (1, 18, 32) or aninfection with a serotype not included in the 14-valent vaccine (6). The vaccine would be ex-pected to be less effective in younger patients(11) or those with an altered immune status (30,37) because they form less antibody after vacci-nation. In addition, certain serotypes, such as6A, which has been associated with three of thereported cases of vaccine failure (1, 10, 32), oftenelicit a weaker antibody response (45). Whatevertheir origins, these cases of vaccine failure high-light the need to establish the relation betweenin vitro measures of antibody response and thedegree of protection afforded by the vaccine invivo. Development of a convenient assay of theeffectiveness of vaccine-induced antibody in op-

sonization and phagocytosis of pneumococciwould be particularly useful in this regard. It isalso essential to know whether antibody alonewill suffice for opsonization or whether an intacttotal or alternative complement pathway (AP)is also necessary and which, if any, of the varioustypes of Streptococcus pneumoniae can be op-sonized efficiently via the AP alone.Many previous investigations into the opsonic

requirements of S. pneumoniae relied on rela-tively insensitive morphological techniques (46)or on measurement of complement consumption(8, 13, 40), which is not a functional assay ofphagocytosis. Bacterial killing assays (15) pro-vide only an indirect measure of phagocytosisand are laborious to perform. Giebink and co-workers recently have used a more direct phag-ocytic assay that measures uptake of radiola-beled S. pneumoniae to define opsonic require-ments for several pneumococcal serotypes (17,19) and assess vaccine response in splenecto-mized individuals (16).

Quantitative measurement of chemilumines-cence (CL), the light emitted by polymorpho-nuclear leukocytes (PMNs) and monocytes dur-

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CHEMILUMINESCENCE AND PNEUMOCOCCAL OPSONINS 229

ing phagocytosis, is another technique of poten-tial value in assessing the opsonization of S.pneumoniae. CL is thought to result from relax-ation of electronically excited carbonyl groupsgenerated by singlet molecular oxygen (2). Gran-ulocyte CL may be induced or enhanced even inthe absence of phagocytosis by numerous solu-ble factors, such as sodium fluoride (22), N-for-mylmethionyl peptides (24), concanavalin A, cy-tochalasin E, calcium ionophone A23187, andphorbol myristate acetate (23), and diminishedby defects of oxidative metabolism, such as

chronic granulomatous disease (42) or myelo-peroxidase deficiency (34). However, in the pres-ence of normal PMN function, CL has beendemonstrated to be a useful assay for phagocy-tosis of several bacterial species, including groupB streptococci, Pseudomonas aeruginosa,Escherichia coli, Salmonella typhinurium, andStaphylococcus aureus. Bacterial killing assays(5, 21), morphological assays (25), radiolabeledbacterial uptake studies (36), and correlationwith the amount of opsonins (5, 41) have allconfirmed the validity of CL as a phagocyticassay for these organisms.We describe below a quantitative assay of

phagocytosis in which the CL of normal PMNswas measured during ingestion ofS.pneumoniaetypes VII, XIV, and XIX opsonized with im-mune serum. These three types are common

pathogens (9, 20, 29, 38) which are all includedin the 14-valent pneumococcal polysaccharidevaccine and have reportedly varying opsonicrequirements (13, 17, 40). Opsonic requirementswere studied by using whole serum (with anti-body and total complement pathway), 2.5 mMmagnesium dichloride-10 mM ethyleneglycol-tetraacetic acid (MgEGTA)-chelated serum

(with antibody and AP only) and heat-inacti-vated serum (with antibody alone). Type-spe-cific antibody absorptions were also performedto further delineate opsonic requirements.

MATERIALS AND METHODSPreparation of bacteria. S. pneumoniae types

VII, XIV, and XIX were obtained from the AmericanType Culture Collection (Rockville, Md.) and main-tained by serial passage weekly in brain heart infusionbroth with 10% rabbit serum in an incubator at 370Cwith a 5% CO2 atmosphere. Purity checks were per-

formed routinely on blood agar plates. Although bac-teria were not passed through mice, each type was

replaced monthly by using a fresh lyophilized sampleof the original culture. Bacteria for opsonization ex-

periments were cultured in an incubator for 18 h inbrain heart infusion broth with 10% rabbit serum in a

5% CO2 atmosphere, centrifuged at 2,400 x g for 10min, and then washed once in calcium- and magne-

sium-free phosphate-buffered saline (PBS). The bac-teria were then resuspended in PBS at a concentration

of approximately 5 x 108 to 109 colony-forming unitsper ml, equal to an optical density of 0.9 at 620 nm asmeasured in a Gilford 4200 S spectrophotometer (Gil-ford Instrument Laboratories, Inc., Oberlin, Ohio).Antibody absorption. Whole serum (0.1 ml) was

added to a dry pellet from a 0.5-ml suspension of S.pneumoniae of the desired type and mixed at 0°C toprevent fixation of the complement. After 30 min, thebacteria were removed by centrifugation at 2,400 x gfor 5 min, and the supernatant was transferred to afresh pellet of S. pneumoniae. This absorption proce-dure was performed 3 to 5 times to remove the major-ity of the antibody. Total hemolytic complementmeasurements before and after five absorptionsshowed preservation of more than 80% of the originalcomplement activity. Quantitative antibody was meas-ured by radioimmunoassay (35) on undiluted samplesof serum.

Opsonization procedure. Serum was obtainedfrom healthy adult donors three weeks after immuni-zation with the 14-valent pneumococcal polysaccha-ride vaccine (Pneumovax; Merck Sharpe & Dohme)and stored in 1-ml samples at -70°C. Just before use,the opsonin was thawed and kept at 0°C. For completecomplement inactivation, verified by hemolytic com-plement assay, serum was heated to 56°C for 30 min.For inactivation of the classical pathway only, serumwas chelated with MgEGTA for 10 min at ambienttemperature (12, 14). Preservation ofAP activitiy afterchelation with MgEGTA was verified by the capacityof the chelated serum to maximally opsonize zymosanas measured by CL with PMNs. A mixture of 0.5 ml ofS. pneumoniae (109 colony-forming units per ml) andthe desired amount of serum was placed in a tightlycapped sterile plastic test tube (12 by 75 mm; Falcon2054, Falcon Plastics, Oxnard, Calif.). The final op-sonizing concentration (14% serum for most experi-ments) was adjusted as desired with PBS to a finalopsonizing volume of 0.725 ml. The tubes were tum-bled at 10 rpm at 37°C for 30 min. After opsonization,bacteria were washed twice with PBS and then sus-pended in 0.5 ml of PBS.

Cell preparation. Blood obtained from healthyadult donors was anticoagulated with heparin (10 U/ml) and allowed to sediment with 4.5% dextran (aver-age molecular weight, 500,000) at 37°C for 20 min inan inverted plastic syringe. The leukocyte-rich plasmawas aspirated, and the leukocytes were centrifuged at260 x g for 10 min. The remaining erythrocytes werelysed at 0°C for 10 min to prevent quenching of CLwith a solution of 0.8 NH4C1 (8.32 g/liter), NaHCO3(0.84 g/liter), and disodium ethylenediaminetetraace-tic acid (0.0432 g/liter) (4). The remaining leukocyteswere washed twice with PBS containing 0.2% glucose,suspended, and adjusted to a concentration of 106PMN/ml with PBS-glucose containing 1 yMluminol (5-amino-2,3-dihydro-1,4-phthalazine-dione;Eastman Kodak Co., Rochester, N.Y.) for amplifica-tion of the CL. The final cell composition was morethan 65% PMNs and less than 5% monocytes. Sincelymphocytes do not produce CL and monocytes giveonly 20 to 30% the CL of PMNs, the mononuclear cellsdid not interfere with the assay (27).CL assay. A 0.5-ml sample of the PMN suspension

(5 x 105 cells) was pipetted into a borosiliate tube (6

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by 50 mm) and allowed to equilibrate at 37°C for 10min. to reach baseline CL in the Picolite Luminometer(Packard Instrument Co., Inc., Rockville, Md.), a light-tight, temperature-controlled instrument designed forCL measurements. A 50-,il sample of the opsonizedbacterial suspension was then injected through a rub-ber septum and mixed with a 50-ul syringe five times.Light emission was quantitated for 30 s per sample at6-min intervals for 72 min. All samples were run induplicate (see K. Matthay, D. Wara, W. Mentzer, N.Lameris, and H. Preisler, Clin. Res. 28:109, 1980).Data analysis. Results were corrected by subtract-

ing the CL value obtained when unopsonized bacteriaand PMNs were incubated. The integral of the CLcurve during the entire period of observation yieldeda smaller standard deviation than either peak CL orthe initial slope and was therefore used for all subse-quent analyses. A total reaction time of 72 min waschosen because the maximal CL had usually beenachieved for all three serum treatments within thatperiod. More prolonged incubation of PMNs resultedin gradual diminution of ability to generate CL. Unlessotherwise indicated, the term CL will henceforth referto the corrected integral CL. The intrinsic variabilityof the assay was assessed on three separate occasionsby opsonizing six replicate tubes of S. pneumoniaetype VII with serum from the same donor at the sametime and using a single preparation of PMNs. Thecoefficient of variation was 12.1%.The CL of three different donor PMN preparations

assayed simultaneously in triplicate with a single se-rum source and type of S. pneumoniae showed signif-icant variation among donors (P < 0.001) not relatedto sex. Therefore, experiments to examine the effectof any alteration of complement or antibody werealways run with a concurrent unaltered control usingthe same PMNs.Morphological correlation of phagocytosis

with CL. Duplicate vials containing 5 x 105 PMNsand 50 pl of preopsonized S. pneumoniae cells in atotal volume of 0.55 ml were prepared for simultaneousCL assay and morphological assay for each serotypeand serum treatment. Two drops of the phagocyticmixture were withdrawn after 20 min of incubation,and slides were prepared by spinning at 1,000 rpm for5 min in a cytocentrifuge (Shandon Elliott). The slideswere fixed in methanol and then stained with Giemsastain. Percent phagocytosis was determined by count-ing 200 PMNs, scoring those with ingested bacteria,and then correcting by subtracting the percent phago-cytosis when PMNs were incubated with unopsonizedbacteria.Complement determinations. Total complement

was measured by a standard hemolytic assay (28) withsheep erythrocytes (Colorado Serum Company, Den-ver, Colo.) sensitized with immune rabbit serum for 30min at 37°C. Sufficient CaCl2 was added to test sera

containing MgEGTA to provide optimal levels of un-

chelated calcium for complement activation. One mil-liliter of each of four serum dilutions was incubatedwith 0.5 ml of a 0.2% suspension of sensitized sheeperythrocytes in Veronal buffer with 0.1% gelatin at37°C for 1 h. The absorbance at 414 nm of the super-natant after dilution with 3.5 ml of gel-Veronal buffer

with 0.1 M ethylenediaminetetraacetic acid was re-

corded, and the percent lysis was calculated.

RESULTS

Correlation of CL with phagocytosis.Morphological phagocytosis correlated closelywith CL regardless of whether complement was

present (whole immune serum), absent (heat-inactivated immune serum), or active only in thealternative pathway (MgEGTA-chelated im-mune serum) during opsonization. Table 1 givesthe results of simultaneous experiments in whichthe percentage of ingested bacteria was assessedmicroscopically on Giemsa-stained smears ob-tained from the phagocytic mixture and com-

pared with duplicate vials assayed for CL. Com-parison of the ratios of the altered serum towhole serum after correction for the unopso-nized control yielded virtually identical percent-ages for the CL and morphological methods.Dependence of CL on concentration of

luminol, PMNs, and opsonized bacteria. CLincreased linearly with increasing luminol con-

centrations, with mean peak CL with opsonizedzymosan of 7.0 x 104 cpm at 0.1 ,uM luminol andmean peak CL of 6.8 x 106 cpm at 10 ,iM luminol.CL also varied directly with PMN number (Fig.1). Changing the bacterium/PMN ratio alteredthe kinetics and peak height (Fig. 2) as well astotal integral CL. The CL peak occurred pro-gressively earlier as the bacterium/PMN ratioincreased. In experiments (n = 4) with S. pneu-moniae type VII the maximal CL appeared after72 min when the bacterium/PMN ratio was 66:1, after 48 min when the ratio was 100:1, after 44min when the ratio was 150:1, and after 33 minwhen the ratio was 200:1.Complement requirements for opsoniza-

tion and phagocytosis. The CL produced byphagocytosis of bacteria preopsonized in wholeimmune serum (total complement pathway) was

compared with the CL after opsonization inheated immune serum (no complement activity)

TABLE 1. Morphological correlation of CL withphagocytosis after 20 min of incubationa

CL (cpm) for se- % Phagocy-tosis for se-Opsonin rotype: rotype:

VII XIX VII XIX

Whole immune serum 35,763 53,340 69.5 48.0MgEGTA-chelated 34,956 69,648 69.0 61.0immune serum

Heated immune serum 9,022 17,492 10.0 17.0Unopsonized 5,264 8,362 0.0 15.0

a All values are the mean of duplicate determina-tions.

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CHEMILUMINESCENCE AND PNEUMOCOCCAL OPSONINS 231

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FIG. 1. Effectrepresents the min duplicate; 5preopsonized inthe test number o,CL is equal to th4by the mean inteWmixture containi

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rotypes VII, XIV, and XIX is shown in Fig. 4.Inactivation of complement by heating signifi-cantly (P < 0.001) decreased CL to less than30% of the whole-serum activity for types VIIand XIX and 50% of the whole-serum activityfor type XIV. Furthermore, for types VII andXIV relative CL with heated serum was partic-ularly low during the first 20 min but less abnor-mal during the remainder of the 70-min incu-bation, indicating that phagocytosis occurredmore slowly without complement. In contrast,relative CL in the MgEGTA-chelated sampleswas greater at 20 min than at 70 min for all three

, , , , , , , , serotypes, indicating that the AP was particu-2 4 6 8 10 larly active early in the reaction. In fact, early in

oS PMNs the course of incubation with type XIX, opso-nization with the AP alone resulted in more CLofPMN number on CL. Each point than was seen if the total complement pathwayean oftwo separate experiments run wassen lthe atal mplese differyx 167 S. pneumoniae type VII cells were present, although at 70 mi these differ-14% immune serum were added to ences had disappeared.PfPMNs in 1 Mluminol. Thepercent Total hemolytic complement activity was de-e mean integral CL at 70min divided termined for S. pneumoniae types VII and XIV!gral CL ofa simultaneous reference before and after opsonization with 14 and 70%ng 5 x 105 PMNs. whole or MgEGTA-chelated immune serum

(Table 2). Complete consumption of comple-ment was noted after opsonization of each sero-type, even in serum from which much of theantibody had been removed by absorption. Inthe absorption experiment, complement con-sumption was calculated on the basis of thechange from whole serum, since less than 20% of

11llthetotal hemolytic complement was lost in the

/1/ 100:1

24 48 72MINUTES

FIG. 2. Effect of bacteria/PMN ratio on kineticsand magnitude of CL. Various numbers of S. pneu-moniae type VII cells preopsonized in 14% wholeimmune serum were added to 5 x 10O PMNs in 1 stMluminol.

or MgEGTA-chelated immune serum (AP only).A representative example of CL resulting fromphagocytosis of S. pneumoniae type VII afteropsonization with whole or complement-de-pleted immune serum is shown in Fig. 3. Similarresults were noted with the other two pneumo-coccal serotypes, although the quantitative in-terrelationships between the various serumtreatments differed for each type. The effect ofcomplement on the rate and amount of integralCL after opsonization with immune sera of se-

160

MINUTESFIG. 3. Effect of complement on opsonization and

phagocytosis ofS. pneumoniae type VII. CL ofPMNsincubated with unopsonized S. pneumoniae type VIIcells or with S. pneumoniae type VII cells pre-opso-nized with whole immune serum (complement intact),heat-inactivated serum (no complement activity), or

MgEGTA-chelated serum (AP only).

WHOLE SERUM

HEATED

MEGTA

UNOPSONMED

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232 MATTHAY ET AL.

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HEATED M9EGTA HEATED MEGTA

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HEATED MbEGTA HEATED M9EGTA

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FIG. 4. Effect of complement on opsonization and phagocytosis ofS. pneumoniae types VII, XIV, and XIX.Integral CL at 20 and 70 min after opsonization with heated (no complement activity) or MgEGTA-chelated(AP only) immune serum is shown as a percentage of the CL after opsonization with intact serum (100%activity). Median and interquartile ranges are shown.

TABLE 2. Complement (CH5o) consumption duringopsonization of S. pneumoniae

ComplementCml consumption

Op8Onin ~ComPle- (%) of sero-Opsonin ment Mo eo

pathway' type:

VII XIV

Present study14% Serum TP 100 10014% Serum plus AP 51 90MgEGTA

71% Serum TP 100 10071% Serum plus AP 50 50MgEGTA

71% Serum (absorbed) TP 100 10071% Serum (absorbed) AP 100 20b

plus MgEGTA

Previous studies'100% Serum (40) TP 44 50-75

100% Serum plus AP 100 50MgEGTA (13)(40) 0 50-75

100% Serum (absorbed) AP 50 50plus MgEGTA (13)

a TP, total complement pathway intact; AP, APonly.

b This is the same amount lost in three absorptionseven without subsequent opsonization.'Numbers within parentheses indicate references.

absorption procedure. When only the AP wasactive in 14% serum, type XIV consumed about90% of the complement. Complement consump-tion during opsonization of type VII averaged50% but varied considerably on different days(range, 0 to 100%; n = 5). Both types consumed50% of the complement in MgEGTA-chelated71% serum, indicating that complement could befixed via the AP during opsonization.Antibody requirements for opsonization

and phagocytosis. The opsonic requirementfor antibody in the presence or absence of com-plement was determined for types VII, XIV, andXIX by absorption of type-specific antibodyfrom whole, heated, or MgEGTA-chelated se-rum before use in the CL assay. The residualtype-specific antibody after 3 to 5 absorptionswas less than 150 ng/ml by radioimmunoassay.This represented a 80 to 96% decrease. Thespecificity of the absorption procedure for anti-body was documented in two ways. First, afterabsorption of immune serum with type XIV, CLwas ablated for type XIV but preserved for typeVII. Similarly, immune serum absorbed withtype VII retained opsonic activity for type XIVbut not for type VII, as assessed by CL. Second,quantitation of antibody by radioimmunoassayshowed a decrease in the specific antibody forthe serotype used in the absorption, but a lesserdecrease for antibody to the two other serotypes.Furthermore, the addition ofwhole human com-plement to the absorbed sera did not restoreopsonic activity, whereas it did restore full op-sonization potential to heat-inactivated serum.

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CHEMILUMINESCENCE AND PNEUMOCOCCAL OPSONINS 233

Table 3 shows the quantitative relationshipbetween CL and the amount of antibody presentduring opsonization in sera with and withoutcomplement activity. For all three serotypes,there was almost no residual CL when opsoni-zation was carried out by using sera lackingspecific antibody, regardless of the presence or

absence of complement. A further experimentcompared CL by using whole immune serumfrom three individuals with differing antibodylevels. Peak CL was greatest in the individualwith the highest antibody level and least in theindividual with the lowest antibody level. Forindividuals with serotype XIX antibody levels of0, <50, 122, and 765 ng/ml, the peak CL valueswere 2,770, 6,339, 9,367, and 15,911 cpm, respec-tively.

DISCUSSION

The preceding results validate CL as a phag-ocytic and opsonic assay for S. pneumoniae,confirm that the antibody produced after im-munization with pneumococcal polysaccharidevaccine is opsonically active, and show the ne-

cessity for both type-specific antibody and com-

plement in opsonization of S. pneumoniae inthis assay.Complement is clearly essential for the opso-

nization of all three serotypes, since the S. pneu-moniae cells coated with antibody alone were

ingested more slowly and were associated withless than half of the total CL seen when thecomplement was intact. The AP was responsiblefor an appreciable portion ofthe opsonic activityfor types VII and XIV. In fact, with type XIX,opsonic activity equalled that seen with unche-lated (whole) serum. Despite the importance ofthe complement, it was ineffective as an opsoninby itself (without antibody) since the antibody-poor absorbed sera did not opsonize effectivelyeven with the complement present.Complement assays confirmed the participa-

tion of the complement in the opsonic process,since all of the complement was consumed byopsonization of types VII and XIV. In the caseof type VII, only half of the complement was

TABLE 3. Effect of type-specific antibody absorptionon opsonization of S. pneumoniae

Relative CL (% of unab- Type-specificsorbed value) antibody

Serotype (ng/ml)

Whole Heated MgEGTA Whole/ab-

VII 15 21 8 804/142XIV 13 0 11 685/70XIX 14 8 4 635/23

consumed via the AP, consistent with the CLresults. Type XIV consumed 90% of the availa-ble complement via the AP. However, the widevariation in AP complement consumption fromexperiment to experiment made meaningfulcomparisons with CL impossible. Furthermore,despite the fact that all of the complement wasconsumed by opsonization in antibody-poor ab-sorbed serum, opsonization and phagocytosis asmeasured by the CL assay were markedly di-minished. The limitations of using complementconsumption as an opsonic assay are also appar-ent in the divergent results of previous investi-gators (Table 2). On the other hand, functionalassays of opsonic activity by radiolabeled bac-terial uptake have given results similar to thoseof the present study for S. pneumoniae type VII(26).The antibody absorption experiments clearly

demonstrate that type-specific antibody pro-duced in response to the pneumococcal polysac-charide vaccine is functional and important evenfor opsonization via the AP for serotypes VII,XIV, and XIX.The CL assay has also been valuable in more

clearly delineating the kinetics of phagocytosisof S. pneumoniae prepared with differing opso-nins. It has permitted continuous measurementsduring phagocytosis of preopsonized particlesand by isolating the opsonic step from the phag-ocytic step has permitted evaluation of the rel-ative effectiveness of opsonization. The durationof opsonization, which may affect the relativecontributions of the classical pathway or the APto the opsonization of S. pneumoniae (44), canalso be assessed easily by use of the CL assay.From examination of either the actual CL curves(Fig. 3) or the integral CL (Fig. 4), the differencein opsonic effectiveness of different test sera canbe evaluated. We have used both areas and peakheights (approximately equivalent relation-ships) to show the differences in the amount ofCL associated with the use of complement-al-tered sera. Light emission has been shown inthis investigation to be proportional both to thenumber of ingested particles (Table 1) and tothe number of opsonized particles present in thephagocytic mixture (Fig. 2). Examination of theCL curves shows that opsonization by comple-ment-depleted serum gives a late low peak sim-ilar to that observed when relatively few bacteriaare present (Fig. 2). Such a finding suggests thatthe presence of antibody alone without comple-ment results either in opsonization of fewer bac-teria or in slower phagocytosis. If the latter weretrue, the integral of the curve for heated serumought to equal the integral for whole serum iffollowed to the base line, which it does not. On

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the other hand, the curve for MgEGTA-chelatedserum rises just as quickly or more quickly thanthat of whole serum, whereas the integral isoften less than that ofwhole serum. This impliesthat although the number of opsonized particlesmust be fewer, the opsonin may work so effec-tively that phagocytosis occurs more quicklythan when the total complement pathway isintact.The CL assay of pneumococcal opsonization

and phagocytosis described in this report maybe of value in investigations of patients withincreased susceptibility to pneumococcal infec-tions. For example, a recent report (31) de-scribed a decrease in peak CL with S. pneumo-niae type XIX opsonized with patient serumjust before an episode of type XIX pneumococ-cal sepsis. However, an isolated abnormal resultsuch as that obtained above should be inter-preted with caution, since we and other investi-gators have demonstrated a pronounced varia-bility in CL peak height with the same opsonicsource but different PMNs or even with PMNsfrom the same donor on different days.

Despite some limitations, we have shown that,with appropriate controls, PMN CL provides aneffective quantitative measurement of pneumo-coccal opsonization and phagocytosis. The func-tional opsonic requirements we have definedstress the need for both quantitative antibodyand complement determinations when evaluat-ing individuals at particular risk of pneumococ-cal sepsis.

ACKNOWLEDGMENTSWe thank Gerald Schiffman for the quantitative antibody

detenninations. We would also like to acknowledge Ann Shi-geoka, Harry Hill, Burton Anderson, and Dean Linscott forgenerous technical advice and to thank Keith Hadley for theuse of his microbiological facilities. We gratefully thank themany willing hospital and laboratory personnel who donatedtheir blood. Finally, we thank Debora Springer and May Fongfor their excellent secretarial assistance.

This work was supported in part by grants from the KoretFoundation and from the National Institutes of Health (HL20985).

LITERATURE CITED1. Ahonkhai, V. I., S. H. Landesman, S. M. Fikrig, E. A.

Schmalzer, A. K. Brown, C. E. Cherubin, and G.Schiffman. 1979. Failure of pneumococcal vaccine inchildren with sickle-cell disease. N. Engl. J. Med. 301:26-27.

2. Allen, R. C. 1977. Evaluation of serum opsonic capacityby quantitating the initial chemiluminescent responsefrom phagocytizing polymorphonuclear leukocytes. In-fect. Immun. 15:828-833.

3. Ammann, A. J., J. Addiego, D. W. Warn, B. L. Lubin,W. B. Smith, and W. C. Mentzer. 1977. Polyvalentpneumococcal-polysaccharide immunization of patientswith sickle-cell anemia and patients with splenectomy.N. Engl. J. Med. 297:897-900.

4. Andersen, B. R., and A. M. Brendzel. 1978. Use of a

unique chemiluminescence spectrometer in a study offactors influencing granulocyte light emission. J. Im-munol. Methods 19:279-287.

5. Anderson, D. C., M. S. Edwards, and C. J. Baker.1980. Luminol-enhanced chemiluminescence for evalu-ation of type III group B Streptococcal opsonins inhuman sera. J. Infect. Dis. 141:370-381.

6. Appelbaum, P. C., B. S. Shaihk, M. D. Widome, R. A.Gordon, and R. Austrian. 1979. Fatal pneumococcalbacteremia in a vaccinated, splenectomized child. N.Engl. J. Med. 300:203-204.

7. Austrian, R., R. ML Douglas, G. Schiffman, A. M.Goetzee, H. J. Koornhof, S. Hayden-Smith, and R.D. W. Reid. 1976. Prevention of pneumococcal pneu-monia by vaccination. Trans. Assoc. Am. Physicians89:184-194.

8. Bjornson, A. B., M. H. Gaston, and C. L. Zellner. 1977.Decreased opsonization for Streptococcus pneumoniaein sickle cell disease: studies on selected complementcomponents and immunoglobulins. J. Pediatr. 91,371-378.

9. Burke, J. P., J. 0. Klein, H. M. Gezon, and M. Finland.1971. Pneumococcal bacteremia-review of 111 cases,1957-1969, with special reference to cases with undeter-mined focus. Am. J. Dis. Child. 121:353-359.

10. Cadman, E., M. S. Cohen, R. K. Root, J. L. Ryan, andD. R. Minor. 1978. Impaired response to pneumococcalvaccine in Hodgkin's disease. N. Engl. J. Med. 299:1317-1318.

11. Cowan, M. J., A. J. Ammann, D. W. Wara, V. M.Howie, L. Schultz, N. Doyle, and M. Kaplan. 1978.Pneumococcal polysaccharide immunization in infantsand children. Pediatrics 62:721-727.

12. Des Prez, R. M., C. S. Bryan, J. Hawiger, and D. G.Colley. 1975. Function of the classical and alternatepathways of human complement in serum treated withethylene glycol tetraacetic acid and MgClrethyleneglycol tetraacetic acid. Infect. Immun. 11:1235-1243.

13. Fine, D. P. 1975. Pneumococcal type-associated variabil-ity in alternate complement pathway activation. Infect.Immun. 12:772-778.

14. Fine, D. P., S. R. Marney, Jr., D. G. Colley, J. S.Sergent, and R. M. Des Prez. 1972. C3 shunt activa-tion in human serum chelated with EGTA. J. Immunol.109:807-809.

15. Forsgren, A., and P. G. Quie. 1974. Influence of thealternate complement pathway on opsonization of sev-eral bacterial species. Infect. Immun. 10:402-404.

16. Giebink, G. S., J. E. Foker, Y. Kim, and G. Schiffman.1980. Serum antibody and opsonic responses to vacci-nation with pneumococcal capsular polysaccharide innormal and splenectomized children. J. Infect. Dis. 141:404-412.

17. Giebink, G. S., J. V. Grebner, Y. Kim, and P. G. Quie.1978. Serum opsonic deficiency produced by Strepto-coccus pneumoniae and by capsular polysaccharide an-tigens. Yale J. Biol. Med. 51:527-538.

18. Giebink, G. S., G. Schiffman, W. Krivit, and P. G.Quie. 1979. Vaccine-type pneumococcal pneumonia. J.Am. Med. Assoc. 241:2736-2737.

19. Giebink, G. S., J. Verhoef, P. K. Peterson, and P. G.Quie. 1977. Opsonic requirements for phagocytosis ofStreptococcuspneumoniae types VI, XVIII, XXIII, andXXV. Infect. Immun. 18:291-297.

20. Gray, B. M., G. M. Converse HI, and H. C. Dillon, Jr.1979. Serotypes of Streptococcus pneumoniae causingdisease. J. Infect. Dis. 140:979-983.

21. Grebner, J. V., E. L. Mills, B. H. Gray, and P. G. Quie.1977. Comparison of phagocytic and chemilumines-cence response of human polymorphonuclear neutro-phils. J. Lab. Clin. Med. 89:153-159.

22. Harvath, L., H. J. Amirault, and B. R. Andersen.

INFECT. IMMUN.

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CHEMILUMINESCENCE AND PNEUMOCOCCAL OPSONINS 235

1978. Chemiluminescence of human and canine poly-morphonuclear leukocytes in the absence of phagocy-tosis. J. Clin. Invest. 61:1145-1154.

23. Harvath, L, and B. R. Andersen. 1979. Defective ini-tiation of oxidative metabolism in polymorphonuclearleukocytes. N. Engl. J. Med. 300:1130-1135.

24. Hatch, G. E., D. E. Gardner, and D. B. Menzel. 1978.Chemiluminescence of phagocytic cells caused by N-formylmethionyl peptides. J. Exp. Med. 147:182-195.

25. Hemming, V. G., R. T. Hall, P. G. Rhodes, A. 0.Shigeoka, and H. R. Hill. 1976. Amesmment of groupB streptococcal opsonins in human and rabbit serum byneutrophil chemiluminescence. J. Clin. Invest. 68:1379-1387.

26. Hof, D. G., J. E. Repine, P. K. Peterson, and J. R.Hoidal. 1980. Phagocytosis by human alveolar macro-phages and neutrophils: qualitative differences in theopsonic requirements for uptake of Staphylococcus au-reus and Streptococcus pneumoniae in vitro. Am. Rev.Respir. Dis. 121:65-71.

27. Johnston, R. B., Jr., J. E. Lehmeyer, and L. A. Guth-rie. 1976. Generation of superoxide anion and chemi-luminescence by human monocytes during phagocytosisand on contact with surface-bound immunoglobulin G.J. Exp. Med. 143:1551-1566.

28. Kabat, E., and M. M. Mayer. 1961. Complement andcomplement fixation, p. 149-153. In Experimental im-munochemistry, 2nd ed. Charles C. Thomas, Publisher,Springfield, Ill.

29. Loda, F. A., A. M. Collier, W. P. Glezen, K. Strangert,W. A. Clyde, Jr., and F. W. Denny. 1975. Occurrenceof Diplococcus pneumoniae in the upper respiratorytract of children. J. Pediatr. 87:1087-1093.

30. Minor, D. R., G. Schiffman, and L S. McIntosh. 1979.Response of patients with Hodgkin's disease to pneu-mococcal vaccine. Ann. Intern. Med. 90:887-892.

31. Moore, D. H., P. G. Shackelford, A. M. Robson, andG. M. Rose. 1980. Recurrent pneumococcal sepsis anddefective opsonization after pneumococcal capsularpolysaccharide vaccine in a child with nephrotic syn-drome. J. Pediatr. 96:882-885.

32. Overturf, G. D., R. Field, and R. Edmonds. 1979. Deathfrom type 6 pneumococcal septicemia in a vaccinatedchild with sickle-cell disease. N. Engl. J. Med. 300:143.

33. Riley, L. D., P. I. Tarr, M. Andrews, M. Pfeiffer, R.Howard, P. Challands, G. Jennison, and R. M.Douglas. 1977. Lmmunisation with a polyvalent pneu-mococcal vaccine. Lancet i:1338-1341.

34. Rosen, H., and S. J. Klebanoff. 1976. Chemilumines-cence and superoxide production by myeloperoxidase-

deficient leukocytes. J. Clin. Invest. 58:50-60.35. Schiffman, G., R. M. Douglas, M. J. Bonner, M. Rob-

bins, and R. Austrian. 1980. A radioimmunoassay forimmunologic phenomena in pneumococcal disease andfor the antibody response to pneumococcal vaccines. I.Method for the radioimmunoassay of anticapsular an-tibodies and comparison with other techniques. J. Im-munol. Methods 33:133-144.

36. Shigeoka, A. O., J. I. Santos, and H. R. Hill. 1979.Functional analysis of neutrophil granulocytes fromhealthy, infected, and stressed neonates. J. Pediatr. 95:454-460.

37. Siber, G. R., S. A. Weitzman, A. C. Aisenberg, H. J.Weinstein, and G. &hiffm. 1978. Impaired anti-body response to pneumococcal vaccine after treatmentfor Hodgkin's disease. N. Engl. J. Med. 299:442-448.

38. Siegel, J. D, C. S. Poziviak, and R. H. Mlchaels. 1978.Serotypically defined pneumococcal infections in chil-dren. J. Pediatr. 93:249-250.

39. Smit, P., D. Oberholzer, S. Hayden-Smith, H. J.Koornhof, and M. R. Hilleman 1977. Protective ef-ficacy of pneumococcal polysaccharide vaccines. J. Am.Med. Assoc. 238:2613-2616.

40. Stephens, C. G., R. C. Williams, Jr., and W. P. Reed.1977. Classical and alternative complement pathwayactivation by pneumococci. Infect. Immun. 17:296-302.

41. Stevens, P., and L S. Young. 1977. Quantitative gran-ulocyte chemiluminescence in the rapid detection ofimpaired opsonization of Escherichia coli. Infect. Im-mun. 16:796-804.

42. Stjernholm, R. L, R. C. Allen, R. H. Steele, W. W.Waring, and J. A. Harris. 1973. Impaired chemilu-minescence during phagocytosis of opsonized bacteria.Infect. Immun. 7:313-314.

43. Sullivan, J. I., H. D. Ochs, G. Schiffman, M. R. Ham-merschlag, J. Miser, E. Vichinbky, and R. J. Wedg-wood. 1987. Immune response after splenectomy. Lan-cet i:178-181.

44. Tofte, R. W., P. K. Peterson, Y. Kim, and P. G. Quie.1980. Influence of serum concentration on opsonizationby the classical and alternative complement pathways.Infect. Immun. 27:693-696.

45. Weibel, R. E., P. P. Vella, A. A. McLean, A. F. Wood-hour, W. L Davidson, and M. R. Hilleman. 1977.Studies in human subjects of polyvalent pneumococcalvaccines (39894). Proc. Soc. Exp. Biol. Med. 166:144-150.

46. Winkelstein, J. A., and RH H. Drachman. 1968. Defi-ciency of pneumococcal serum opsonizing activity insickle-cell disease. N. Engl. J. Med. 279:459-466.

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