INFECTION AND IMMUNrrY, Oct. 1977, p. 14-22Copyright i 1977 American Society for Microbiology

Vol. 18, No. 1Printed in U.S.A.

Complement Levels in Pneumococcal PneumoniaJ. DONALD COONROD* AND BARBARA RYLKO-BAUER

Veterans Administration Hospital* and the Division of Infectious Diseases, Department ofMedicine,University ofKentucky School ofMedicine, Lexington, Kentucky 40506

Received for publication 2 May 1977

Levels of complement proteins and functional activity of the alternate comple-ment pathway were assessed in 39 patients with pneumococcal pneumonia. Meanlevels of C3 and properdin and the functional activity of the alternate pathwayin acute sera were significantly (P < 0.05) below normal, whereas levels ofcomponents of the early classical pathway were normal. Although levels of factorB were in the normal range, they correlated significantly with C3 levels; therewas no significant relation between C3 levels and C4 or Clq levels. The 19patients with pneumococcal pneumonia and bacteremia had significantly lowermean values of properdin and factor B than the 20 patients without bacteremia,suggesting a more severe depression of the alternate complement pathway withbacteremia. During convalescence, complement levels were normal or elevatedin most of the patients, but mean levels of properdin remained significantlybelow normal in bacteremic patients. Functional activity of the alternate pathwayalso remained below normal. These results indicate that there is a selectivedepression of the alternate pathway in patients with pneumococcal pneumonia,and they are consistent with the concept that the alternate pathway has animportant role in host defenses in pneumococcal infection.

Both the classical and alternate (properdin)complement pathways have been shown to par-ticipate in opsonization of pneumococci (9, 26).The alternate pathway may be the more impor-tant determinant of natural immunity to pneu-mococci in humans, but comparative studies ofthe two pathways in naturally occurring pneu-mococcal infection have been few. Reed et al.(16) demonstrated recently that levels of factorB, a component of the alternate pathway, werebelow normal in patients with pneumococcalinfections, whereas levels of components of theearly classical pathway (Clq and C4) werewithin normal limits. These findings suggested aselective alteration of the alternate complementpathway in patients with pneumococcal infec-tion. In the present studies, complement com-ponents of the classical and alternate pathwaysand functional activity of the alternate pathwaywere measured in patients with pneumococcalpneumonia. The results showed a selective de-pression of the alternate pathway during theacute phase of the infection, with the depressionbeing more marked in patients with pneumoniaaccompanied by pneumococcal bacteremia.

MATERIALS AND METHODSPatients. Thirty-nine patients who were admitted

to the hospital with fever, purulent sputum, and aclinical history suggestive of acute bacterial pneu-monia were studied. All had roentgenographic evi-

dence of pulmonary consolidation. Cultures of bloodand sputum were obtained before antibiotic therapy,and Streptococcus pneumoniae was isolated from cul-tures of the blood of 19 cases, in pure culture fromtranstracheal aspirates of 14 cases, and as the pre-dominant organism from expectorated sputum of 6cases. The latter six patients showed clinical improve-ment within 72 h on low doses of penicillin G. Themean age of the subjects was 56.8 years (range, 25 to80 years). Thirty-one of the patients were males; therewere 32 whites and 7 blacks. Thirteen of the patientswere considered by their physicians to be alcoholics,and 11 subjects had clinical and laboratory evidenceof chronic obstructive airways disease. None of thepatients had documented collagen-vascular disease,neoplastic disease, cirrhosis, or sickle cell disease.Acute sera were obtained within 48 h after admissionto the hospital. Convalescent sera were obtained 7 to21 days after admission (mean, 13.5 days), exceptfrom three patients who died and from three individ-uals who were lost to follow-up. Blood samples wereallowed to clot at room temperature for 30 to 60 min,and the serum was stored in multiple small samples at-700C.Complement proteins. Levels of complement pro-

teins were measured by a modification of the radialimmunodiffusion method of Mancini et al. (12). Cali-bration curves were prepared with dilutions of refer-ence standards for all determinations, and internalcontrols were included where appropriate. The diam-eters of precipitin rings were measured to the nearest0.1 mm with a magnifying (x3.5) ocular and an illu-minated viewer. Properdin levels were determinedwith an optimal dilution (1:50) of anti-properdin serum

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incorporated into agarose (0.6%) in 0.01 M phosphate-buffered saline (pH 7.0) with 0.01 M ethylenediamine-tetraacetate (EDTA) and 0.1% sodium azide. Wells of3-mm diameter were filled with 20 pl of serum. Precip-itin reactions were maximal after incubation for 48 hat 370C in a moist box. The antiproperdin serum(kindly provided by Ann B. Bjornson, University ofCincinnati College of Medicine) was produced withhuman properdin purified by the method of Penskyet al. (14). The antiserum gave a single precipitin linein immunodiffusion with human serum and gave areaction of identity against properdin and an antipro-perdin serum provided originally by Clark West (Uni-versity of Cincinnati). Clq and C5 levels were deter-mined with an optimal dilution (1:40 and 1:15, respec-tively) of antiserum incorporated into 1% agarose inVeronal buffer (pH 8.6) and 0.04 M EDTA. Wells of3-mm diameter were used, and precipitin reactionswere developed for 24 h at room temperature. Antiserato Clq and C5 were obtained from Behring Diagnostics(Somerville, N.J.) and gave a single precipitin linewith human serum. Levels of properdin, Clq, and C5were expressed as percentages of standard referencesera. C3, C4, and factor B levels were measured withcommercially available immunoplates and referencestandards, obtained, respectively, from Hyland Labo-ratories (Costa Mesa, Calif.), Meloy (Springfield, Va.),and Behring Diagnostics. Precipitin reactions for thesecomponents were developed for 24 h at room temper-ature. In some cases, serum C3 levels were measuredby radial immunodiffusion with antiserum, which wasspecific for the B-antigenic determinant of the nativeC3 molecule. This antiserum (kindly provided by AnnBjornson) was prepared according to the method ofWest et al. (23); it gave a single precipitin line againstfresh normal serum but no reaction with aged humanserum.Normal levels of complement proteins were estab-

lished with sera from 30 adults (mean age, 51.3 years,and range, 19 to 65 years; there were 26 whites and 4blacks).

Immunoelectrophoresis. Electrophoresis of se-rum C3 and factor B was performed with 1.5% Nobleagar (Difco, Detroit, Mich.) in 0.02 M EDTA andVeronal buffer (pH 8.2; ionic strength, 0.05). Electro-phoresis was conducted at 200 V for 90 min, andprecipitin reactions were obtained with antiserum toC3 (Hyland Laboratories) or factor B (Behring Diag-nostics). Controls consisted of fresh and aged normalhuman serum.Hemolytic complement. Serum hemolytic com-

plement levels were measured as 50% hemolytic units(CH5w) by a spectrophotometric method, with a reac-tion volume of 4.0 ml (13). Normal levels were estab-lished with sera from 51 subjects (mean age, 48.3years, and range, 19 to 65 years; 43 whites and 8 blacks).Zymosan test. The functional activity of the alter-

native complement pathway was measured by theconsumption of total hemolytic complement afterzymosan activation. A modification of the method ofFine et al. (4) was used. Zymosan (Nutritional Bio-chemicals Corp., Cleveland, Ohio) in normal salinewas boiled for 2 h, washed three times in normalsaline, and brought to a final concentration of 2 mg/ml. A stock solution of 0.1 M ethyleneglycol-bis(/B-

aminoethyl ether)-N,N'-tetraacetic acid (EGTA)(Sigma Chemical Co., St. Louis, Mo.) and 0.02 MMgCl2 was prepared in normal saline, with adjustmentof the pH to 7.45 before dilution to final volume withsaline. In the routine test, 1 ml of fresh serum wasmixed with 0.1 ml of this buffer, and the mixture wasincubated at room temperature for 5 min. The serumwas divided into two (0.5-ml) aliquots; one aliquot wasmixed with 0.5 ml of zymosan and the other aliquot,which served as the control, was mixed with 0.5 ml ofnormal saline. The samples were mixed and incubatedat 37°C for 45 min. They were remixed once by inver-sion at the middle of the incubation period. Zymosanwas removed by centrifugation at 2,500 x g for 10 minat 40C. To recalcify, 0.05 ml of CaCl2 (0.1 M) wasadded to each aliquot. The CH5o titer of the sampleswas determined, and the number of CH5o units con-sumed by zymosan was taken as the difference inCHM content of the control and the zymosan-treatedsamples. This test for measurement of alternate path-way activity was validated by the following studies.The amount of spontaneous activation (or deteriora-tion) of complement in saline control samples wasdetermined by comparing the CHso content of thesesamples with the CHso content of the original sera.With 25 normal sera, the mean (± standard deviation[SD]) reduction in CHso content due to spontaneousactivation was 16.5 ± 5.9%. The efficacy of EGTA-chelated serum in blocking activation by the classicalpathway was tested by using optimally sensitizedsheep erythrocytes (2 x 109/ml) in the test in thesame manner that zymosan was usually used. Withfive normal sera, the mean (±SD) reduction in CH50content by sensitized sheep erythrocytes was 19.3 ±6.5%. In contrast, with 51 normal sera, the mean (±SD)reduction in CHw content by using zymosan in thetest was 82.1 ± 18.9%.

Since Mg levels are an important determinant ofzymosan-induced consumption of complement inEGTA-chelated serum, the effect of varying Mg levelsin the test was determined with Mg-EGTA buffers ofvarying Mg content. Maximum activation by zymosanwas achieved in Mg-EGTA buffers with a Mg contentof 0.01 to 0.03 M. The Mg content (0.02 M) of theMg-EGTA buffer routinely used in the test thus pro-vided a twofold excess ofMg over the minimal amountneeded for maximal complement activation. In addi-tional studies, serum Mg levels were measured in 25acute sera and 21 convalescent sera from patients withpneumococcal pneumonia. The assays were performedby atomic absorption by Joseph Boone at the Univer-sity of Kentucky laboratories. Mg levels for all thesera fell within the normal range (0.0006 to 0.0013M), and the mean levels of Mg in the acute sera(0.00094 M) and the convalescent sera (0.00095 M)were not significantly different from the normal meanvalue (0.00093 M).The results obtained by zymosan activation in che-

lated serum were highly reproducible; six replicatesamples tested on the same day for each of threeindividuals varied by less than 0.5% from the meanvalue, and results with sera tested on three differentoccasions during a 6-month period for five individualsshowed less than 7% variation from the mean. Underthe conditions of the assay, the amount of available

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hemolytic complement that was consumed by zymo-san in 51 normal sera correlated with the total com-plement content ofthe sera (r = 0.76;P< 0.001). There-fore, results obtained with patients' sera in the assaywere compared with the results of normal sera con-taining the same level (±5%) of hemolytic comple-ment. Where there were several normal sera of thesame complement level, the results were averaged foruse in the comparison.Antigens. Soluble pneumococcal antigens were

measured by counterimmunoelectrophoresis, withmethods that have been described elsewhere (1).

Statistics. Probability values for differences in fre-quency were determined by Fisher's exact test. Linearregression coefficients were calculated with a Monroemodel 1860 calculator. Complement levels were com-pared with the t statistic, with correction for differ-ences in variances of groups (7). A probability value(P) of c0.05 was considered significant.

RESULTSComplement levels. Hemolytic comple-

ment activity and levels of several proteins ofthe classical and alternate complement path-ways were measured in acute sera from 39 pa-tients with pneumococcal pneumonia. The re-sults are shown in Table 1. Mean levels of serumC3 and properdin, an alternate pathway com-ponent, were significantly lower in patients thanin normal controls. Hemolytic complement lev-els in the patients' acute sera were lower thannormal, but not significantly so (P < 0.1). Inconvalescent sera from 33 of the 39 patients,mean levels of Clq, C3, and C5, as well as factorB, an altemate pathway component, were sig-nificantly higher than normal. Mean levels ofhemolytic complement and all components ex-

cept C4 and C5 were significantly higher inconvalescent than in acute sera for the 33 pa-tients with both values. C4 levels varied littleduring the course of the infection, and mean C5levels were above normal in both acute andconvalescent sera.

C3 levels were measured in 31 acute sera and27 convalescent sera with antiserum that wasspecific for the B-antigenic determinant of thenative C3 molecule. The results with antiseraspecific for B antigen were virtually identicalwith those obtained in the routine test for C3,and linear regression analysis showed a coeffi-cient of correlation (r) between the two of 0.95.This finding suggested that C3 levels, as mea-

sured in the routine studies, were indicative ofthe levels of functionally active C3.

Immunoelectrophoresis of C3 and of factor Bin 20 acute and 15 convalescent sera was carriedout with the antisera that had been used forroutine measurement of C3 and of factor B.Fresh and aged normal human sera served as

controls. The precipitin reactions obtained withC3 and factor B from each of the patients' sera

were identical with the results obtained withfresh normal human serum. None of the pa-

tients' sera contained detectable quantities ofthe characteristic determinants of C3 and factorB which were observed with aged human se-

rum. These findings suggested that the C3 andfactor B proteins measured in patients' serawere the native molecules and not inactive de-rivatives.Alternate pathway activity. Functional

activity of the alternate complement pathwaywas evaluated by measuring zymosan-inducedconsumption of hemolytic complement (CH,o)in sera treated with EGTA to block the classi-cal complement pathway. Since the quantity ofcomplement consumed by zymosan varied withthe complement content of the sera, the resultswith patients' sera were compared with normalsera of the same total content of hemolytic com-plement. The functional activity of the alternatepathway was markedly depressed in the acutephase of pneumococcal pneumonia. Mean con-

sumption of complement in acute sera from the

TABLE 1. Serum complement levels in pneumococcal pneumonia

Complement component (mean ± SD)

Group Clq (% of ref- C4 (mg/100 Properdin (% Factor B C3 (mg/1i0 CS (% of ref- Hemolyticerence stan- n;/10 of reference (mg/ B l) Ml) erence stan- complement

dard) standard) m) dard) (units)Normals ................. 98.4 + 9.2 36.5 ± 6.6 98.1 ± 12.6 20.9 ± 3.2 132.9 ± 20.6 98.4 ± 15.4 92.8 ± 17.6Patients with pneumococcal

pneumoniaAcute (n = 39) ... 97.2 ± 27.1 39.2 ± 13.2 62.9 ± 22.0 19.7 ± 6.7 116.6 ± 34.4 107.4 ± 30.1 83.8 ± 17.8Convalescent (n - 33) 107.5 ± 21.2 37.9 ± 9.2 90.9 ± 17.9 27.7 ± 7.8 161.9 ± 44.6 111.7 ± 22.0 99.4 ± 16.8

P valueAcute vs normala NSb NS <0.001 NS <0.02 NS NSConvalescent vs normal" <0.05 NS NS <0.001 <0.005 <0.05 NSAcute vs convalescent, <0.01 NS <0.001 <0.001 <0.001 NS <0.005a ttest for independent variables.'NS, Not significant (P > 0.05).c Paired (dependent) t test for patients with both values.

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COMPLEMENT IN PNEUMONIA 17

39 patients with pneumonia was 63.0% of theconsumption in matched normal human sera (P< 0.001). In convalescent sera from 24 of the 39patients, functional activity of the alternatepathway was 84.9% of the normal value-a sig-nificant increase from the acute sera (P < 0.001)though still significantly below the normal level(P < 0.05).Complement levels and bacteremia. To

define the complement profile in pneumococcalpneumonia further, complement levels in pa-

tients with bacteremic pneumococcal pneu-

monia were determined and compared with thelevels of cases of abacteremic pneumococcalpneumonia. Of the 39 patients in the study, 19had pneumococcal bacteremia and 20 had sterileblood cultures. Levels of components of theclassical pathway and of hemolytic complementof these two groups of patients are tabulated inTable 2. Mean levels of C3 and hemolytic com-

plement in acute sera from bacteremic patientswere significantly below normal, whereas thecorresponding mean values for abacteremic pa-

tients did not differ significantly from normal.Levels of C5 and hemolytic complement for bac-teremic patients were significantly below thecorresponding values for abacteremic patients.Clq levels in bacteremic patients were lowerthan normal and lower than Clq for abacteremicpatients, but these differences were not signifi-cant (P< 0.20). With convalescent sera, elevatedlevels of several of the classical pathway com-

ponents and hemolytic complement were ob-served in both groups of patients, and therewere no significant differences between the twogroups.

Values for properdin levels are shown in Fig.

1. With acute sera, 16 of 19 bacteremic patientswere more than 2 SDs below the normal mean,

and 10 of 20 abacteremic patients also had sub-normal values. Mean properdin levels of bothgroups were significantly below normal (P <0.001). However, the mean properdin level(±SD) for bacteremic patients (52.8 ± 17.8%)was significantly below the mean properdin level

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ACUTE CONVALESCENTFIG. 1. Serum levels ofproperdin in patients who

had pneumococcal pneumonia with bacteremia (0)or without bacteremia (0). The shaded area repre-

sents the normal mean ± 2 SDs.

TABLE 2. Components of the classical complement pathway and hemolytic complement in bacteremic versusabacteremic pneumococcal pneumonia

Complement component (mean ± SD)a

Group HemolyticClq C4 C3 C5 complement

Normals ......... 98.4 ± 9.2 36.5 ± 6.6 132.9 ± 20.6 98.4 ± 15.4 92.8 ± 17.6Pneumonia, acute serum

Bacteremic (n = 19) 88.2 ± 27.1 33.0 ± 14.4 106.4 ± 28.6 97.7 ± 27.6 78.0 ± 19.2Abacteremic (n = 20) ............ 97.2 ± 22.6 39.3 ± 11.5 126.2 ± 37.2 117.0 ± 30.1 89.6 ± 15.2

P valuebBacteremic vs normal .......... NSc NS <0.005 NS <0.02Abacteremic vs normal ......... NS NS NS <0.02 NSBacteremic vs abacteremic .... NS NS NS <0.05 <0.05

Pneumonia, convalescent serumBacteremic (n = 16) ............. 106.0 ± 25.5 37.8 ± 10.7 161.4 ± 32.7 109.9 ± 22.7 103.9 ± 17.2Abacteremic (n = 17) ............ 108.4 ± 16.6 38.0 ± 7.9 162.4 ± 54.0 113.5 ± 22.0 95.4 + 16.3

P valuebBacteremic vs normal .......... NS NS <0.005 NS <0.05Abacteremic vs normal ......... <0.05 NS <0.05 <0.02 NSBacteremic vs abacteremic ..... NS NS NS NS NS

aUnitS of measurement are as indicated in Table 1.b ttest for independent variables.c NS, Not significant (P > 0.05).

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18 COONROD AND RYLKO-BAUER

for abacteremic patients (73.0 ± 21.5%) (P <0.005). With convalescent sera (Fig. 1), proper- _

-

din levels remained more than 2 SDs below 220normal in 4 of 16 bacteremic patients, whereas 0all abacteremic patients had levels in the normalrange. The mean properdin level (±SD) in con- 180valescent sera from bacteremic patients was 83.2 00± 17.8%. This level was significantly below nor-mal (P < 0.005) and also lower than the mean 0 0/ 0

level of convalescent sera from abacteremic pa- ° 140 o 0tients (98.5 ± 14.8%) (P < 0.02). °oFactor B levels are given in Fig. 2. The major- , 7

ity of patients in both groups had normal levels /of factor B in acute sera. However, the mean rr 100 -

level of factor B in acute sera from bacteremic 0o opatients (17.4 ± 6.3 mg/100 ml) was significantly 0 0

below nornal (P < 0.05) and significantly lowerthan the mean level for abacteremic patients 60 -

(22.0 ± 6.5 mg/100 ml) (P < 0.02). During con-valescence, mean factor B levels were elevatedin both the bacteremic group (27.1 ± 6.2 mg/100 ml) and the abacteremic group (28.2 ± 7.5 20 -

mg/100 ml) (P < 0.001 for both groups).Linear regression analysis showed that factor 0 5 15 25 35

B levels in acute sera correlated significantlywith C3 levels, and the values for bacteremic FACTOR B, mg/100 mlpatients clustered in the lower range for both FIG. 3. Correlation of serum C3 and factor B 1ev-proteins (Fig. 3). Similar analyses of Clq and 04 els in acute sera of 19 patients who had pneumococ-with C3 levels failed to show any significant re- cal pneumonia with bacteremia (0) and 20 patientslationship (r = 0.19 for C3 and Clq; r = 0.25 for who had pneumococcal pneumonia without bactere-C3 and C4). mia (0). The coefficient of correlation (r) was 0. 79, as

Results of studies of the functional activity of determined by linear regression analysis (P < 0.001).the alternate pathway for bacteremic and abac-

teremic patients are shown in Fig. 4. Bothgroups of patients had depressed alternate path-way activity in acute sera as compared with

45 - matched normal subjects (P < 0.001). The de-o° pression was more pronounced in bacteremic

0 patients; these individuals averaged 57.9% of the0 mean complement consumption achieved by

o Go normal subjects, whereas the comparable figureo 0 _ g for abacteremic patients was 68.2% of normal._oA.. .. t. , However, the difference between the two groupsE5 of patients was not statistically significant. Dur-25 ing convalescence, alternate pathway activity

was within the normal range for most patients* in both groups. For bacteremic subjects, com-

plement consumption by zymosan in the conva-

; 15t: SWlescent sera averaged 81.0% of the mean valuefor normals, and abacteremic patients averaged88.1% of the normal value. Neither group of pa-

5 -tients differed significantly from normal.

Clinical features. Clinical characteristics ofl______________ l_____________ patients with bacteremic or abacteremic pneu-

ACUTE CONVALESCENT mococcal pneumonia were compared in an ef-FIG. 2. Serum levels of factor B in patients who fort to gain insight into the reasons for differ-

had pneumococcal pneumonia with bacteremia (0) ences in their complement levels (Table 3). Theor without bacteremia (0). The shaded area repre- two groups did not differ significantly in age,sents the normal mean ± 2 SDs. race, or sex, but bacteremic patients did appear

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COMPLEMENT IN PNEUMONIA 19

FIG. 4.Cosmto0fhmltccmlmn

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ptetwhhapneumococcal pneumonia withbceei 0 or

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thmas 2Ss ornrra sbjcs hoetoa

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painswohdpneumococcal pneumonia with or~~~~without bacteremia(0.Tesaearsrpeen

Blood culture

Patients Posi- Nega- P value

tive tiveTotal (no.) 19 20Mean age (yrs) 55.2 58.4 NSaMales (no.) 18 13 NSCaucasians (no.) 14 18 NSAdmission granulocyte count:Mean x 10'/mm3 9.2 16.8 < 0.005Count < 4,000/mm3 (no.) 6 0 0.05

Pulmonary findings:Multilobar pneumonia (no.) 9 7 NSPleural fluid (no.) .... 6 0 0.05

Hospital course:Mean days of fever, >37.8oC. 5.0 2.5 < 0.05Mean days in the hospitalb 17.1 8.8 < 0.005Deaths (no.) ...3 0 NS

Associated diseases (no.)Chronic alcoholism 10 3 0.05Chronic obstructive airways dis-

ease ... 4 7 NSDiabetes millitus 2 2 NSOthers, 3 4 NSa NS, Not significant (P > 0.05).h Deceased patients excluded.c Includes peptic ulcer disease, atherosclerotic heart dis-

ease, and essential hypertension.

to have a more severe illness. There were signif-icantly more pleural effusions in bacteremic pa-tients, as judged by roentgenograms obtained inthe lateral decubitus position, and three pa-tients in the bacteremic group had documentedpleural empyema. Fever persisted longer in bac-teremic patients, and they had a significantlylonger stay in the hospital. The only three

deaths in the study occurred in patients withbacteremia.Both groups of patients had similar underly-

ing illnesses, with the exception that signifi-cantly more bacteremic patients were consid-ered to be alcoholics by their physicians. How-ever, as indicated in Table 4, mean complementlevels for 10 alcoholic patients with pneumococ-cal bacteremia did not differ significantly frommean complement levels of 9 bacteremic pa-tients who were not alcoholics.Pneumococcal serotypes. The possibility

of a relationship between the serotype of theinfecting pneumococcus and complement levelswas considered. Seven of the 39 patients in thestudy were infected with type 3 pneumococci;four cases were due to type 4, and three caseseach were caused by types 6, 19, and 23. Therewere 14 different pneumococcal types associ-ated with the remaining 19 cases. The numberof patients infected with any given serotype wastoo small to warrant statistical analysis of theresults. However, it was apparent that alteredcomplement levels were associated with pneu-monia caused by any one of a variety of pneu-mococcal serotypes.Capsular polysaccharide antigenemia.

Complement levels were evaluated in relation tocapsular polysaccharide antigenemia. Capsularpolysaccharide was detected by counterimmu-noelectrophoresis in acute sera from 11 of the 39patients. Since each of these 11 patients wasalso bacteremic, complement levels could not beevaluated independently of bacteremia. The an-tigenemic patients were therefore comparedwith the eight bacteremic patients who were notantigenemic. Hemolytic complement and meanlevels of Clq, C3, C4, C5, and factor B were notsignificantly different in the two groups. The

TABLE 4. Complement parameters for acute sera ofalcoholic or nonalcoholic subjects with bacteremic

pneumococcal pneumoniaMean + SDa

Complementparameter Alcoholic Not alcoholic

(n =10) (n = 9)

Clq .......... 93.0 + 24.6 82.3 + 30.5C4 27.6 ± 8.3 39.1 ± 17.6Properdin ... 55.1 + 16.6 50.2 ± 21.0Factor B .... 16.3 ± 5.7 18.6 + 7.1C3 .. ... 1105.2 + 26.2 107.8 ± 32.6C5 .I..... 93.9 ± 26.1 102.5 ± 30.5Hemolytic com- 76.5 ± 16.5 79.7 ± 22.6plement

CHso consumed 56.4± 19.2 60.1±14.2by zymosan 56. 4I 19j2 6

a Units of measurement are as indicated in Table 1.Alcoholic and nonalcoholic subjects did not differ sig-nificantly in any complement parameter (P > 0.05).

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20 COONROD AND RYLKO-BAUER

mean properdin level of antigenemic patientswas 45.4% of normal, whereas the correspondingvalue of non-antigenemic patients was 62.0% ofnornal; mean consumption of hemolytic com-plement by zymosan was lower in the sera ofantigenemic patients (48.7% of normal) than ofnon-antigenemic individuals (76.6% of normal).However, neither of these differences was signif-icant (P < 0.10).

DISCUSSION

Our studies indicate that there are abnormali-ties of several components of complement in pa-tients with pneumococcal pneumonia. Acutesera from 39 patients showed a significant de-pression of mean levels of C3, which is a pivotalcomplement component for activation of theterminal complement sequence by either theclassical or alternate complement pathway. Instudies of the alternate pathway, mean levels ofproperdin were significantly decreased, andfunctional activity of the alternate complementpathway, as measured by zymosan stimulation,was also markedly depressed. Although levelsof factor B were in the normal range, they cor-

related significantly with C3 levels, suggestinga common determinant for levels of C3 andfactor B. In contrast, studies ofthe early classicalpathway showed normal mean levels of Clq andC4, and levels of these proteins did not correlatesignificantly with C3 levels. Total hemolyticcomplement in acute sera was at the lower limitof normal, a finding which could have reflectedlowered levels of C3 or changes in levels of otherterminal components of the classical pathway.Taken together, these findings strongly suggestthat there is a selective depression of the alter-nate complement pathway in patients withpneumococcal pneumonia. These observationsare consistent with the early report of Hinz (8),who noted diminished properdin activity incases of pneumococcal pneumonia, and with therecent observations of Reed et al. (16), who ob-served decreased C3 and factor B levels, withnormal Clq and C4 levels, in patients with acutepneumococcal infections.

In extending these observations, we comparedcomplement levels of the patients with pneu-monia who had an accompanying pneumococcalbacteremia with those who were abacteremic.Properdin levels and functional activity of thealternate pathway were below normal in bothgroups of patients, but properdin levels were

significantly lower in the bacteremic patients. Inaddition, bacteremic patients had lower levels offactor B, C5, and hemolytic complement thanthe abacteremic subjects. Clq and C4 levels didnot differ significantly in the two groups.

There was, therefore, evidence of selective de-pression of the alternate complement pathwayin both bacteremic and abacteremic patients,but the changes in complement were greater indegree in the bacteremic individuals.The reasons for depression of the alternate

pathway in pneumococcal pneumonia and forthe observed differences in complement levelsof bacteremic and abacteremic patients are notclear. One possible explanation for the findingsmay be a selective activation and consumptionof alternate pathway proteins in pneumococcalpneumonia. In varying degrees, most serotypesof pneumococci are able to activate the alternatepathway in vitro (24), and it is by this processthat opsonically active C3b is thought to befixed to pneumococci. Conceivably, the extentof consumption of complement by pneumococciin patients with pneumonia could depend onthe concentration of organisms in various tissuesand on whether organisms were present in thecirculation. In vitro, more than 105 pneumococciper ml are needed to consume measurableamounts of complement by the alternate path-way (3). This level of organisms may be attainedreadily in the lung, but there are seldom morethan a few hundred pneumococci per milliliterin the circulation in pneumococcal pneumonia(5, 21). The effects of a sustained bacteremia atthis level, as may occur in pneumococcal pneu-monia, are not known. Robertson et al. (17) instudies of pneumococcal pneumonia in dogs con-cluded that there was a loss of bactericidal ac-tivity for pneumococci in the blood after thedevelopment of pneumococcal bacteremia; ces-sation of bacteremia was accompanied by anincrease in the bactericidal activity of the blood.Unfortunately, in these early studies the specificserum factors responsible for the bactericidalcapacity of the serum were not defined, and wecan only surmise that complement was a factor.In the present studies, patients with bacteremiahad evidence of a more severe infection thandid abacteremic patients, as has been noted bya number of investigators in both the pre- andpostantibiotic eras (21, 22). If increased utiliza-tion of complement were a major determinantof complement levels in pneumococcal pneu-monia, it seems possible that the extent of theinfection could have influenced the amount ofcomplement consumed. However, it is not cer-tain that the reduction in complement levels weobserved was due to increased consumption ofcomplement. Low complement levels in severaldisease states have been shown to be due toincreased complement consumption, but frac-tional catabolic rates must be determined toprove hypercatabolism.

Patients who develop pneumococcal pneu-

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VOL. 18, 1977

monia could have some impairment of the alter-nate pathway antedating the infection. Thereare precedents for this possibility. It is estab-lished, for example, that individuals with sicklecell disease fail to fix the essential C3 opsonin topneumococci to a normal degree (25). This fail-ure is due to reduced functional activity of thealternate pathway (10), which may be caused bya deficiency of C3 proactivator convertase (al-ternate pathway factor lp) (11). Individuals withsickle cell disease are especially susceptible tosevere infections, and particularly to pneumo-coccal meningitis (18, 20). If there were preex-isting abnormalities of the alternate pathway inthe patients we studied, these abnormalitiesmight have predisposed to the development ofpneumococcal pneumonia or of pneumococcalbacteremia. This possibility cannot be excludedby the present results. Studies of complementlevels in convalescent sera did establish thatmost of the patients were able to increase com-plement levels in the nornal range as the infec-tion subsided. Mean levels of factor B, proper-din, and C3, and the functional activity of thealternate pathway all increased significantly be-tween the acute and convalescent phases of in-fection, and factor B and C3 were significantlyabove the normal range during convalescence.These latter changes could reflect the functionof factor B and C3 as so-called acute-phasereactants (19) and do not necessarily indicatethat the patients had normal levels of the com-ponents at the time the infection was acquired.Mean properdin levels returned to the normalrange for the pneumonia patients as a wholeduring convalescence, but the mean level forbacteremic patients remained significantly be-low normal during the period in which the pa-tients were observed. In addition, mean func-tional activity of the altemate pathway wasslightly below normal during convalescence. Onthe basis of these findings we cannot exclude apreexisting abnormality of complement in atleast some of the patients.We attempted to exclude from the study pa-

tients who had diseases known to be associatedwith decreased complement levels. There were,however, 13 patients who were considered bytheir physicians to be alcoholics. Alcoholism,when complicated by hepatic cirrhosis, has beenassociated with depressed levels of C3, factor B,C4, C5, and hemolytic complement (2, 6, 15).None of the patients included in the presentstudy had clinical or laboratory evidence of cir-rhosis, but an attempt was made to evaluate thepossible effects of alcoholism on complementlevels. Alcoholic and nonalcoholic subjects withbacteremic pneumococcal bacteremia werecompared. No significant differences between

COMPLEMENT IN PNEUMONIA

these two groups were found, suggesting thatalcoholism per se was not a major determinantof the low complement levels seen in bacteremicindividuals.

Activation of complement by pneumococciseems to be initiated principally by antigens inthe cell wall, but certain type-specific capsularpolysaccharides can also activate the alternatepathway. Capsular polysaccharide is occasion-ally present in the circulation in pneumococcalpneumonia, but the concentration of circulatingantigen is usually much lower than the quantityof antigen required for activation of comple-ment in vitro (24). In the present study, comple-ment levels in patients with antigenemia did notdiffer significantly from the complement levelsof non-antigenemic patients. Properdin levelsand functional activity of the alternate pathwaywere lower in antigenemic than in non-antige-nemic individuals, but these differences werenot statistically significant (P < 0.10).

ACKNOWLEDGME?NTISThis work was supported by the Medical Research Service

of the Veterans Administration.We are indebted to David Drennan for assistance in col-

lecting the clinical samples and to Peter Walzer and WardBullock for reviewing the manuscript. We also thank Ann B.Bjomson for helpful discussions of the work and Grant W.Somes for help with the statistics.

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12. Mancini, G., A. 0. Carbonara, and J. F. Heremans.1965. Immunochemical quantitation of antigens by sin-gle radial immunodiffusion. Int. J. Immunochem.2:235-254.

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14. Pensky, J., C. F. Hinz, Jr., E. W. Todd, R. J. Wedg-wood, J. T. Boyer, and I. H. Lepow. 1968. Propertiesof highly purified human properdin. J. Immunol.100:142-158.

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24. Winklestein, J. A., J. A. Bocchini, Jr., and G. Schiff-man. 1976. The role of the capsular polysaccharide inthe activation of the alternative pathway by the pneu-mococcus. J. Immunol. 116:367-370.

25. Winklestein, J. A., and R. H. Drachmann. 1968. Defi-ciency of pneumococcal serum opsonizing activity insickle cell disease. N. Engl. J. Med. 279:459-466.

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