INFECTION AND IMMUNITY, Dec. 1979, p. 808-812 Vol. 26, No. 30019-9567/79/12-0808/07$02.00/0

Quantitation of the Third Component of Human ComplementAttached to the Surface of Opsonized Bacteria: Opsonin-

Deficient Sera and Phagocytosis-Resistant StrainsHENRI A. VERBRUGH,'* WILLEMIEN C. VAN DIJK,' MARIJKE E. VAN ERNE,1 ROEL PETERS,

PHILLIP K. PETERSON,2 AND JAN VERHOEF'

Laboratory ofMicrobiology, State University of Utrecht School ofMedicine, 3511 GG Utrecht, TheNetherlands,' and Department ofMedicine, University ofMinnesota School ofMedicine, Minneapolis,

Minnesota 554552

Received for publication 18 September 1979

The role of the third component of human complement (03) in the opsonizationof bacteria in nonimmune human sera was evaluated. The amount of C3 thatbecomes attached to the surface of bacteria upon incubation in serum wasmeasured in a quantitative fluorescent immunoassay using fluorescein-conjugatedmonospecific antiserum to human C3. The intensity of the fluorescence fromopsonized bacteria was found to be directly proportional to the absolute amountof C3 fixed, and this enabled the detection of as few as 300 molecules of bound C3per bacterium. In normal serum the rate of C3 fixation was closely correlatedwith an increase in opsonization of the bacteria for human PMNs. Both C3fixation and opsonization were maximal after 15 min of incubation. C3 fixationwas also observed, albeit at a significantly slower rate, in human serum with anonfunctional classical pathway but an intact alternative complement pathwayand in serum deficient in immunoglobulins. Again, the kinetics of C3 fixationcorrelated with bacterial opsonization. Using a total of 21 strains of severalbacterial species, including Staphylococcus aureus and Escherichia coli, encap-sulation of bacteria was found to interfere with the process of C3 fixation innormal human serum, rendering these organisms resistant to subsequent phago-cytosis by human polymorphonuclear leukocytes.

Recognition of invading bacterial pathogensby human phagocytes requires the participationof serum factors (opsonins) including immuno-globulins and the complement system. Deficien-cies in this phagocytosis-promoting activity ofserum have been associated with an increasedsusceptibility to bacterial infections in a varietyof diseases such as hypogammaglobulinemia(18), active systemic lupus erythematosus (14),congenital complement deficiencies (2, 15), sic-kle cell anemia (12), and cystic fibrosis (3). Inpatients with gram-negative bacteremia, a de-crease in the opsonic activity of serum has beencorrelated with an increase in mortality (1). Thephagocytosis-enhancing capacity of serum fromnonimmune individuals depends largely uponthe heat-labile complement system (15, 24, 26,30). Activation of complement (C) via both theclassical and the alternative pathways can resultin the fixation of opsonically active C3b mole-cules to the surface of bacteria (10, 16, 27).However, certain bacteria such as encapsulatedstrains of Staphylococcus aureus, Escherichiacoli, and pneumococci are able to resist phago-cytosis in the presence of normal serum, render-

ing them more virulent than unencapsulatedstrains (13, 17, 23, 25).

In the present report, the kinetics of bacterialC3 fixation in human serum were studied usinga quantitative fluorescent immunoassay employ-ing fluorescein-conjugated antiserum monospe-cific for human C3. Opsonic recognition of bac-teria by human polymorphonuclear leukocytes(PMNs) was shown to be closely related to theamount of C3 bound to their surfaces. Opsonicdefects in serum from patients could be detectedwith this method. Furthermore, it was shownthat encapsulated strains of several bacterialspecies failed to bind significant amounts of C3in nonimmune human serum and were ineffi-ciently phagocytized by human PMNs.

MATERIALS AND METHODS

Bacterial strains and cultural conditions. S.aureus Cowan I, Wood 46, H, and Ev (29, 30) and S.aureus M (encapsulated) and M variant (unencapsu-lated), kindly provided by M. A. Melly, VanderbiltUniversity School of Medicine, Nashville, Tenn. (17),were used in this study.

Five encapsulated strains of E. coli containing808

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KINETICS OF BACTERIAL C3 FIXATION 809

polysaccharide K antigens and five strains without theK antigen, reported previously (28), were also studied.Clinical isolates of Salmonella typhi (Vi antigen posi-tive), Klebsiella pneumoniae (mucoid colonies), andProteus mirabilis were likewise tested. All strainswere maintained on blood agar plates at 40C. In studiesof bacterial C3 fixation, organisms were freshly grownin 5 ml of Mueller-Hinton broth (Difco Laboratories,Detroit, Mich.) for 18 h at 370C with shaking. Afterincubation, the bacteria were washed three times withsterile isotonic phosphate-buffered saline (pH 7.4) andadjusted to a concentration of 5 x 108 colony-formingunits per ml of phosphate-buffered saline by using aspectrophotometric method confirmed by pour-platecolony counts (30).

Radioactive labeling of bacteria. In studies ofbacterial opsonization, organisms were radioactivelylabeled by adding 0.02 mCi of [methyl-3H]thymidine(specific activity, S Ci/mmol; The RadiochemicalCentre, Amersham, England) to the medium as pre-viously described (28, 30).Serum sources. Normal human sera were obtained

from 20 healthy donors and were stored at -70'Cindividually and as a serum pool. Each serum con-tained normal levels of total hemolytic complement(range, 80 to 125%) and C3 (range, 0.8 to 1.6 mg/mn).Also quantitative immunoglobulins were within nor-mal ranges. In addition, sera were obtained from eachof the following patients: (i) a 12-year-old patient witha primary X-linked immunoglobulin deficiency (Bru-ton type, with 0.05 mg of immunoglobulin G per ml ofserum and undetectable immunoglobulins M, A, E,and D; total hemolytic complement and C3 werewithin normal limits); (ii) a 6-year-old male with Med-iterranean-type kala azar of 1 year duration (this hy-perglobulinemic serum had a total hemolytic comple-ment of 74% and contained 0.4 mg of C3 per ml); (iii)a 16-year-old female with active systemic lupus ery-thematosus (total hemolytic complement, 45%, andC3, 0.3 mg/ml; quantitative immunoglobulins withinnormal limits); (iv) a 19-year-old female with systemiclupus erythematosus and an undetectable serum totalhemolytic complement (<10%) due to a complete ab-sence of the Cls subcomponent of complement factorC1 (20) (quantitative immunoglobulins and C3 werewithin normal ranges); and (v) a 21-year-old femalepatient with an inherited complete and selective ab-sence of C2 (29), kindly provided by L. Berrens, StateUniversity of Utrecht School of Medicine, Utrecht,The Netherlands (this serum also had normal levelsof immunoglobulins).

Total hemolytic complement of sera was titratedwith optimally sensitized sheep erythrocytes as waspreviously described (29). The levels of immunoglob-ulins and C3 were determined by Mancini radial im-munodiffusion (RID plates; ICL Scientific, Euclid,Calif.).

Isolation of human PMNs. Suspensions of purePMNs were prepared from fresh heparinized venous

blood, obtained from healthy volunteers, as previouslydescribed (29). Final leukocyte suspensions were ad-justed to a concentration of 5 x 106 PMNs per ml ofHanks balanced salt solution with 0.1% gelatin.

Opsonization procedure. Shortly before use, serawere thawed and diluted to the desired concentration

in Veronal-buffered saline with ionic strength of 0.147and with 0.15 mM Ca2 , 1 mM Mg2+, and 0.1% (wt/vol) gelatin (GVB2+). To study bacterial C3 fixationand opsonization via the alternative C pathway, C1was blocked by chelation of undiluted serum withethylene glycoltetraacetic acid in the presence ofMgCl2 (MgEGTA) in a final concentration of 10 mM(8) and further dilution in calcium-free buffer. Heat-inactivated serum was prepared by heating thawedsamples of serum at 56°C for 30 min.

Opsonization mixtures were constituted by adding0.2 ml of the washed bacterial suspension to 0.8 ml ofserum in polypropylene vials (Biovials; Beckman, Chi-cago, Ill.) in a 37°C water bath. At indicated intervals,incubation was stopped by adding 2.5 ml of ice-coldphosphate-buffered saline to the vials and washing thebacteria three times with phosphate-buffered salineby centrifugation (1,600 x g for 15 min at 4°C). Bac-teria treated as described here were designated "op-sonized washed bacteria."Measurement of bacterial C3 fixation. The

amount of C3 attached to opsonized washed bacteriawas quantitatively measured in a fluorescent immu-noassay modified from Gillis and Thompson (11).Briefly, opsonized washed bacteria were pelleted andsubsequently reacted with 500 id of ½o fluoresceinisothiocyanate-conjugated goat antiserum monospe-cific for human C3 (Wellcome, Beckenham, England)for 15 min at room temperature. To remove non-reacted conjugate, the bacteria were then washedthree times with phosphate-buffered saline and finallyresuspended in 2.5 ml of phosphate-buffered saline.Fluorescence was measured in a Perkin-Elmer 204Fluorescence Spectrophotometer (Perkin-Elmer, Nor-walk, Conn.) with excitation and analyzing wave-lengths of 485 and 525 nm, respectively. Results wererecorded as percentages of fluorescence, ranging fromzero (control of bacteria opsonized with GVB2+ alone)to 100% (maximal emission intensity in the seriestested).Measurement of bacterial opsonization. Bac-

terial opsonization was assessed in a quantitativephagocytosis assay employing [3H]thymidine-labeledbacteria and isolated human PMNs, as described ear-lier (30). A 0.2-ml sample of the opsonized washedbacteria, containing 107 colony-forming units, was in-cubated in polypropylene vials with 0.2 ml of theleukocyte suspension, containing 106 PMNs. The per-centage of bacteria taken up by the PMNs after 12min of incubation in a 370C shaking water bath wastaken as a measure of bacterial opsonization (29).Uptake of bacteria, opsonized with GVB2+ alone, neverexceeded 1%.

Radioiodination of human C3. In one experi-ment, human C3 (Cordis Laboratories, Miami, Fla.)was labeled with '25I1 to a specific activity of 3.2 x 104cpm/pg of C3 by the method of Bolton and Hunter(4). The C3 preparation used was functionally pureand found free of contaminating immunoglobulins byMancini radial immunodiffusion. Fifty milligrams ofC3 was reacted with 200 p1 of "uI-containing acylatingagent (Bolton-Hunter reagent; the RadiochemicalCentre) for 10 min at 4°C, chromatographed on Seph-adex G-25 (Pharmacia, The Hague, The Netherlands),concentrated by ultrafiltration, and stored at -70°C

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until use. l25I-C3 was added to normal pooled serumor to heat-inactivated pooled serum to yield a final C3specific activity of 1.2 x 106 cpm/ml of serum. '25Iradioactivity of labeled C3, of serum to which radio-active C3 was added, and of opsonized washed bacteriawas measured directly with an autogamma spectrom-eter (Packard Instrument Co., Downers Grove, Ill.).

RESULTSEffect of serum dilution on C3 fixation.

The amount of C3 that becomes attached to thesurface of S. aureus Ev during opsonization inserial dilutions of pooled normal human serumis shown in Fig. 1. This bacterial strain fixed C3maximally when incubated in undiluted serumand in serum diluted 1:2 or 1:4. At these serumconcentrations we invariably obtained over 80%of C3-specific fluorescence from the opsonizedstaphylococci. At lower serum concentrations,however, the amount of C3 fixation decreasedaccordingly, and less than 10% of the fluores-cence remained when bacteria were opsonized in1:128-diluted serum. Opsonization in heat-inac-tivated pooled serum resulted in less than 2% ofC3 fluorescence, regardless of the serum concen-tration used (not shown in the figure). Thesefindings indicate that S. aureus Ev will bind C3in normal human serum via the heat-labile com-plement system and that the bacterial surface issaturable with respect to the amount of C3 thatcan be fixed. The amount of C3 attached to thebacteria did not increase significantly at serumconcentrations over 20% and, indeed, tended tobe slightly lower at 100% than at 50% serum(Fig. 1).Sensitivity of the C3 fluorescent immu-

noassay. To determine the minimal amount ofsurface-bound C3 required to detect C3 fixationin the fluorescent immunoassay, S. aureus Evwas opsonized in serial dilutions of pooled serumto which 1251_C3 was added. Serum dilutionsranged from 1:4 to 1:64. After incubation, theamount of C3 attached to the opsonized washedstaphylococci was measured for each dilution ofserum both by direct measurement ofthe uptakeof 125I and by recording the fluorescence afterfluorescein isothiocyanate-conjugated anti-C3treatment. The intensity of the 03-specific flu-orescence was found to be directly proportionalto the absolute amount of C3 attached to thebacteria, which could be calculated from theuptake of the 1251_C3 tracer (correlation coeffi-cient, r = 0.99; least squares method) (Fig. 2).The fluorescent immunoassay could detect 10 to20 ng of bound C3/10' colony-forming units, andthe maximum amount of C3 fixation by staphy-lococci in normal serum was approximately 0.5,tg/108 colony-forming units. These data estab-lish the specificity and sensitivity of the quanti-

INFECT. IMMUN.

LUXL- 40 -

~ 0

1 2 4 8 16 32 64 128

SERUM DILUTION (reciproc.)

FIG. 1. Intensity of C3-specific fluorescence of S.aureus Ev after incubation in serial dilutions ofnor-mal pooled human serum for 60 min at 370C. Thewashed opsonized bacteria were reacted with fluo-rescein isothiocyanate-conjugated anti-C3 serum,and the fluorescence was recorded as percent C3fixed. Symbols represent the means ± standard errorof the mean of five experiments done on 5 separatedays.

100 r .0.99U.U

C~ 80

U- 60-zw

WJ 40

LDL20X1 0

L. 20- /

0.1 0.2 0.3 0.4 0.5,UgC3/10 CFU

FIG. 2. Rectilinear correlation between the re-corded intensity of C3-specific fluorescence and theabsolute amount ofC3 fixed to S. aureus Ev. Bacteriawere incubated for 60min in serial dilutions ofpooledserum with added 1251-C3. The absolute amount offixed C3, expressed as micrograms of C3 per 108colony-forming units, was calculated from the uptakeofradioactivity from the '251-C3 tracer. r, Correlationcoefficient.

tative fluorescent immunoassay, which will reli-ably detect as few as 300 molecules of C3 at-tached to the surface of a bacterium (using185,000 as the molecular weight of C3 and 6.023X 1023 as Avogadro's number).Kinetics of bacterial C3 fixation. To study

the kinetics of the process of C3 fixation and itsrelation to bacterial opsonization, 3H-labeled S.aureus Ev was incubated in 20% pooled serum

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KINETICS OF BACTERIAL C3 FIXATION 811

for 0.25, 0.5, 1.0, 5, 15, and 30 min at 370C. Afterincubation, the amount of C3 attached to theopsonized washed staphylococci was deter-mined, and opsonization of these bacteria was

measured. C3 fixation was quantitated by im-munofluorescence, and the percent of washedbacteria taken up by PMNs after 12 min ofincubation was taken as a measure of bacterialopsonization. In Fig. 3 the kinetics of the proc-

esses of bacterial C3 fixation and opsonizationare shown to be closely related. In normal serum,

S. aureus Ev rapidly accumulated C3 (over 60%C3 fixation was found after 5 min of incubation),and this was paralleled by an equally rapid in-crease in bacterial opsonization (over 85% up-

take of the bacteria by PMNs was observed after5 min of opsonization). When 20% heat-inacti-vated serum was used as the opsonic source, C3fixation was -1.5% and there was maximally 15%uptake of staphylococci by PMNs, even after 60min of opsonization (data not shown). Fromthese observations it was concluded that forefficient opsonization in nonimmune human se-rum, C3 fixation on the bacterial surface is an

absolute requirement and that the processes ofC3 fixation and opsonization are thereforeclosely related kinetically.C3 fixation and opsonization in sera from

patients. The possibility of using the fluores-cent C3 fixation assay in the detection of opsonicdefects in human sera was evaluated by studyingS. aureus Ev C3 fixation and opsonization insera from 20 healthy donors and in sera from 5patients with well-defined deficiencies of poten-tially important opsonic factors. After 5, 15, and45 min of incubation in each of the sera, diluted1:5, the amount of C3 attached to the staphylo-cocci was determined. The results, expressed as

percentages of C3 fluorescence, are given in Ta-ble 1. The mean amount of C3 bound to thebacteria after 5, 15, and 45 min of incubation insera from 20 healthy donors was 45, 74, and 82%,respectively. In contrast, a significantly slowerrate of bacterial C3 fixation was found in sera

from the five patients. After 5 and 15 min ofincubation, the amount of C3 attached to thestaphylococcal surface varied from 6 to 28% andfrom 13 to 44%, respectively. These values werewell below the ranges of C3 fixation observedwith sera from the healthy donors (Table 1).Except for the Cls deficient serum, no significantdifferences in C3 fixation remained between pa-tients' sera and normal sera after 45 min ofincubation. These results indicate that deficien-cies in opsonic serum factors can be detected bykinetic analysis of the process of bacterial C3fixation.To correlate these findings with bacterial op-

sonization, the uptake of the opsonized washed

staphylococci by human PMNs was also studied.To further evaluate the role of the classical andalternative pathways of complement activation,bacterial opsonization was likewise measured inpooled normal serum in which the classical path-way was selectively blocked by MgEGTA che-lation. The percent phagocytosis ofradioactivelylabeled staphylococci was determined after op-

sonizing the bacteria for 1, 5, 15, 30, and 60 min.

LC3 FIXATION

100 11m PHAGOCYTOSIS

Z 60

I;A40

20I

Q25 0.5 1.0 5.0 15 30

INCUBATION TIME (min)FIG. 3. Kinetics of S. aureus Ev C3 fixation and

opsonization in 20% pooled serum. [3Hithymidine-labeled staphylococci were incubated for the indi-cated times, washed, and assayed for surface-boundC3 by quantitative immunofluorescence as well as forphagocytosis by human PMNs. Phagocytosis was

quantitated by measuring the percent PMN-associ-ated radioactivity after 12 min of incubation. Barsrepresent means of two experiments done in dupli-cate.

TABLE 1. Amount of C3 attached to S. aureus Evafter opsonization in sera from healthy donors and

from patientsBacteria-associated C3b after op-

Serum source~asonization for:

5 min 15 min 45 min

Healthy donors' 45 74 82(35-75) (50-95) (62-100)

Patients with:Agammaglobulin- 9 16 84emia

Cls deficiency 6 13 58C2 deficiency 12 28 76Systemic lupus ery- 25 44 63thematosus

Kala azar 28 40 73

All sera were tested at 1:5 dilution.bExpressed as percentages of C3-specific fluorescence after

fluorescein isothiocyanate-conjugated anti-C3 staining ofwashed opsonized staphylococci as described in the text.

e N = 20; mean (total range in parentheses).

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S. aureus Ev was rapidly opsonized in pooledserum and in individual sera from healthy do-nors (Fig. 4a). Chelation of pooled serum withMgEGTA resulted in a less efficient opsonicsource. Although bacterial opsonization waseventually effected, the rate of opsonization inMgEGTA-chelated serum was significantly re-duced (Fig. 4a). The rate of bacterial opsoniza-tion in serum from each of the five patients wassimilarly decreased (Fig. 4b). The kinetics ofopsonization in sera from the healthy donorsand patients correlated well with the kinetics ofbacterial C3 fixation presented in Table 1. Wetherefore concluded that bacterial opsonizationand the opsonic capacity of sera from nonim-mune individuals can best be evaluated by ki-netic studies, either by using a functional phag-ocytosis assay or by quantitating bacterial C3fixation.C3 fixation of "phagocytosis-resistant

strains." We next tested the hypothesis thatresistance to phagocytosis of encapsulated bac-terial strains is related to a resistance to opson-ization in nonimmune serum and that this phe-nomenon would be reflected in low levels of C3fixation by these strains. Twenty-one encapsu-lated and unencapsulated strains of several bac-terial species were studied. Radioactively la-beled bacteria were incubated in normal pooledserum, diluted 1:5, for 30 min at 370C. Afterincubation, both the amount of C3 attached tothe opsonized washed bacteria and their uptakeby human PMNs were measured. A close cor-relation was found between the amount of sur-face-bound C3 and the phagocytosis of encap-sulated as well as unencapsulated strains (Fig.5). In general, encapsulated strains of S. aureus,

100 (a) (b)Ig def

xC2- def80~~~~~~~~~~~ ~~Kala-azar

S LE

60 Cls -def

C)4040

20/A NormaL serum

-N A MgEGTA serum

OPSONIZATION TIME (min)FIG. 4. The kinetics of opsonization of S. aureus

Ev (a) in pooled normal and MgEGTA-chelated se-

rum and (b) in sera from patients with indicateddiseases. The total range of values for phagocytosisof bacteria incubated individually in sera from 20healthy donors is indicated by the bars in (a). Percentphagocytosis represents the percentage of staphylo-cocci taken up by human PMNs after 12 min ofincubation.

1UU

80

060

00Sru.r 401

20

20 40 60

% C3 Fixation80 100

FIG. 5. Correlation between phagocytosis and theamount of C3 fixed to 21 encapsulated and unencap-sulated strains of several bacterial species. Bacteriawere opsonized with 20% pooled serum for 30 min. C3fixation was quantitated by immunofluorescence, andthe percentage of bacteria phagocytized by humanPMNs after 12 min of incubation was determined.Symbols: S. aureus (0, encapsulated; 0, unencapsu-lated), E. coli (A, encapsulated; A, unencapsu-lated), K. pneumoniae (+), S. typhi (U), P. mirabilis(x).

E. coli, K. pneumoniae, and S. typhi showed lowlevels of C3 fluorescence and were poorly phag-ocytized by PMNs, and unencapsulated strainsof E. coli and S. aureus readily fixed C3 andwere efficiently phagocytized. Intermediate lev-els of C3 fixation and phagocytosis were ob-served with three strains of P. mirabilis. Al-though only a relatively small number of strainswere investigated, these findings support theconcept that encapsulation of bacteria interfereswith the process of opsonization by C3 moleculesin nonimmune human serum.

DISCUSSION

Considerable evidence points to a form ofbound C3 as an agent that elicits recognition byphagocytic cells. Particles coated sequentiallywith antibody and classical C components (C1,C4, and C2) become opsonized only when C3 isadded (22, 27). In fresh normal human serum C3can become firmly attached to bacteria by acti-vation of either the classical or the alternative Cpathway (26). The main purpose of the presentinvestigation was to study the nature of therelationship between bacterial C3 fixation andphagocytosis by human PMNs. To evaluate thisrelationship, sera with specific opsonic defects incomplement or immunoglobulin were used, andbacteria that resist phagocytosis were studied.The amount of C3 bound to opsonized bacte-

ria was measured in a quantitative fluorescentimmunoassay employing fluorescein-conjugatedantiserum specific for human C3. By comparing

no

x xA

* -+A

+ B, II

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KINETICS OF BACTERIAL C3 FIXATION 813

the absolute amount of C3 taken up by thebacteria with the intensity of fluorescence afterstaining with the fluorescent conjugate, the im-munofluorescence assay was shown to accu-rately measure the amount of C3 attached toopsonized bacteria, detecting as few as 300 mol-ecules of C3 per bacterium. Thus, this methodappears to be a reliable alternative to morecumbersome assays using radioiodinated C3 forquantitating particle-bound C3 (6, 21, 27).The amount of C3 that will become attached

to the surface of bacteria upon incubation inserum from nonimmune individuals is clearlyshown in this report to be dependent upon: (i)the concentration of serum chosen, (ii) the du-ration of the incubation, (iii) the presence orabsence of a functional classical pathway, alter-native complement pathway, or both, and (iv)on the particular bacterial strain studied. Innormal pooled serum, the amount of C3 boundto an unencapsulated strain of S. aureus after60 min of incubation was found to be maximalwhen serum concentrations of 25% or higherwere used, indicating that bacterial surfaces aresaturable with respect to the amount of acti-vated C3 that can be accepted. At higher dilu-tions of serum, the amount of C3 that becameattached to the bacteria decreased accordingly.Fifty percent C3 fixation, i.e., bacteria half-sat-urated with C3, was observed at 5 to 10% serumconcentration, and less than 10% C3 fixationremained when serum was diluted to 1:128.When kinetic studies were performed, the

process of C3 fixation to the surface of bacteriaproved to be closely related to the process ofbacterial opsonization. When normal human se-rum was used, a rapid increase in C3 fixation ofbacteria was correlated with a rapid rate ofopsonization. Both properties were maximalwithin 15 min of incubation in normal serum.Such a correlation between C3 fixation and op-sonization has been reported for lipopolysaccha-ride-coated paraffin oil particles upon incubationin fresh human serum containing 125I1C3 (27).The key role of C3 fixation in opsonization of

bacteria is further evidenced by our studies onthe kinetics of C3 fixation and opsonization insera from patients with well-defined deficienciesof complement (Cls, C2, or C3) and immuno-globulins. The amount of bacterial C3 fixationin patients' sera after incubation times of 5 and15 min was well below the total ranges of C3fixation found in sera from 20 healthy donors.This diminished rate of C3 fixation was associ-ated with a significantly reduced rate ofbacterialopsonization in patients' sera as compared tonormal donor sera. The Cls- and C2-deficientpatients provided sera with a nonfunctionalclassical pathway but intact alternative comple-

ment pathway, and the immunoglobulin-defi-cient serum contained less than 5 mg of immu-noglobulin G per 100 ml, with undetectable im-munoglobulin M, forming Cl-activating antigen-antibody complexes on the bacterial surface.Upon more prolonged incubation in these defi-cient sera, however, over 50% C3 fixation wasobserved, indicating that activation of C3 couldbe mediated via the alternative complementpathway and in the absence ofimmunoglobulins,albeit at a significantly slower rate. A significantreduction in the rate of bacterial opsonization innormal serum was also observed when C1 acti-vation was blocked by MgEGTA chelation.The impairment in opsonization in comple-

ment-deficient human serum found in this studyis consistent with previous observations. Defec-tive opsonization has been reported in humansera deficient in Cls (29), C2 (15, 16, 29, 30), andC4 (5), and in MgEGTA-chelated serum (7, 9,28-30). In addition, a delay in the generation ofchemotactic complement factors, primarily C3aand C5a, has been observed when Clr- and C2-deficient serum were used (8). These data col-lectively support the concept that optimal op-sonization of bacteria in the nonimmune hostrequires an intact classical complement pathwayand that activation of the classical and alterna-tive complement pathways can be distinguishedby kinetic analysis. Kinetic analysis may there-fore be necessary to detect opsonic defects insera from patients. In this study we have shownthat opsonic defects can be detected by measur-ing the rate of C3 fixation to the surface ofbacteria.The presence of a capsule is a convincing

virulence factor in strains of several differentbacterial species, and this phenomenon corre-lates well with the ability of encapsulated strainsto resist phagocytosis (13, 17, 23, 25). By sepa-rating the process of opsonization and phagocy-tosis, it was recently shown that heavily encap-sulated strains of S. aureus and E. coli areineffectively opsonized by the classical and thealternative complement pathways in normal hu-man serum (19, 25, 28). It was concluded in thesestudies that encapsulation interferes with theprocess of complement-dependent opsonizationdue to masking by the capsule of other, comple-ment-activating, cell wall components. In thepresent study we demonstrated that the phago-cytosis of encapsulated and unencapsulated bac-teria varies directly with the amount of C3 thatbecomes fixed to their surfaces upon incubationin serum. Unencapsulated strains of several bac-terial species, including S. aureus and E. coli,readily fixed C3 in nonimmune serum and were

efficiently phagocytized by human PMNs,whereas encapsulated strains showed lower lev-

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els of C3 fixation and were poorly taken up bythe phagocytes. It is possible that, in addition tothe quantitative relationship between bacterialC3 fixation and opsonization described in thisstudy, other factors, such as the localization ofbound C3 and certain physicochemical proper-ties of the bacterial capsule itself, are also ofimportance in determining the resistance tophagocytosis of some encapsulated bacteria. Infuture studies several of these characteristicswill be investigated, and the influence of anti-capsular antibodies on the processes of C3 fixa-tion and opsonization will be evaluated.

LITERATURE CITED

1. Alexander, J. W., M. A. McClellan, C. K. Ogle, and J.D. Ogle. 1978. Consumptive opsinopathy: possiblepathogenesis in lethal and opportunistic infections.Ann. Surg. 184:672-678.

2. Alper, C. A., N. Abramson, R. B. Johnston, Jr., J. H.Jandl, and F. S. Rosen. 1970. Increased susceptibilityto infection and the third component of complement(C3). N. Engl. J. Med. 282:349-354.

3. Biggar, W. D., B. Holmes, and R. A. Good. 1971.Opsonic defect in patients with cystic fibrosis of thepancreas. Proc. Natl. Acad. Sci. U.S.A. 68:1716-1719.

4. Bolton, A. E., and W. N. Hunter. 1973. The labeling ofproteins to high specific radioactivity by conjugation toa '251-containing acylating agent. Application to theradioimmuno-assay. Biochem. J. 133:529-539.

5. Clark, R. A., and S. J. Klebanoff. 1978. Role of theclassical and alternative complement pathways inchemotaxis and opsonization: studies of human serumdeficient in C4. J. Immunol. 120:1102-1108.

6. Dalmasso, A. P., and H. J. Muller-Eberhard. 1967.Physicochemical characteristics of the third and fourthcomponent of complement after dissociation from com-plement-cell complexes. Immunology 13:293-305.

7. Forsgren, A., and P. G. Quie. 1974. Influence of thealternative complement pathway on opsonization ofseveral bacterial species. Infect. Immun. 10:402404.

8. Gallin, J. I. 1975. Abnormal chemotaxis: cellular andhumoral components. In J. A. Bellanti and D. H. Dalton(ed.), The phagocytic cell in host resistance, p. 227-248.Raven Press, New York.

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