INncTION
AND
IMMUNITY, Aug. 1977, p. 296-302
Copyright 0 1977 American Society for Microbiology
Vol. 17, No. 2 Printed in U.S.A.
Classical and Alternative Complement Pathway Activation by Pneumococci CHRISTOPHER G. STEPHENS, RALPH C. WILLIAMS, JR., AND WILLIAM P. REED*
Department ofMedicine of the Veterans Administration Hospital* and Bernalillo County Medical Center, The University of New Mexico School of Medicine, Albuquerque, New Mexico 87108 Received for publication 14 January 1977
Sixty-two strains of Streptococcus pneumoniae were studied for their abilities consume selected components of classical and alternative complement pathways in human sera. The classical pathway was blocked by chelating calcium with ethyleneglycol-bis(,8-aminoethyl ether)-N,N-tetraacetic acid and by removing C4. The alternative pathway was blocked by removing factor B. Each strain's activation of the two pathways was compared with its nonimmune reactivity with the Fc region of immunoglobulin G (IgG). Activation of the classical complement pathway appeared to be independent of such Fc reactivity. Highly Fc-reactive strains, however, were shown to activate the alternative pathway more effectively than did less Fc-reactive strains. Since pneumococcal activation of the alternative pathway requires non-immunospecific IgG, these findings suggest that nonimmune binding of IgG on the pneumococcal surface endows it with complement-activating properties. to
Alternative complement pathway activation by several Streptococcus pneumoniae serovars has recently been demonstrated (5). Such activation requires an immunoglobulin which need not show specific antibody reactivity for the pneumococcal polysaccharide (30). The immunoglobulin's role in this interaction between pneumococci and the alternative system has not been well characterized. The Fc region of immunoglobulin G (IgG) reacts directly with the capsular type-specific polysaccharide of some pneumococcal serovars (27). Such Fc reactivity might cause approximation of non-immunospecific IgG molecules on the pneumococcal surface to such a degree that the complement-activating properties of aggregated immunoglobulins would be acquired (25). This study demonstrates a correlation between the Fc reactivity of a pneumococcal serovar and its ability to activate the alternative pathway, suggesting that accumulation of IgG on the bacterial surface is involved in alternative pathway activation. (This work was presented at the Western Section, American Federation for Clinical Research, Carmel, Calif., February 1976 [Clin. Res. 24:115A, 1976].)
described agammaglobulinemic serum with no measurable IgA or IgM and less than 50 mg of IgG per ml by radial immunodiffusion (19) was also used. Classical and alternative complement pathways were inactivated by several procedures. Ethylenediaminetetraacetic acid (EDTA; as the disodium and tetrasodium salts; Sigma, St. Louis, Mo.) at a concentration of 0.01 M in undiluted sera inactivated both pathways. The classical pathway was selectively blocked with 0.01 M ethylene glycol-bis(,8-aminoethyl ether)-N,N'-tetraacetic acid (EGTA; Sigma) and 0.01 M MgCl2(5). Samples of the normal serum were depleted of C4 or factor B by incubation with Sepharose-bound unfractionated anti-human C4 (Meloy, Springfield, Va.) or anti-,12 glycoprotein II (Behring, Woodbury, N.Y., lot 1548P) at 4°C in the presence of EDTA, which was later removed by dialysis (19). Levels of type-specific antipneumococcal antibodies were determined by hemagglutination of erythrocytes coated with pneumococcal polysaccharides (1). Several serovar polysaccharides were kindly provided by Robert Austrian (University of Pennsylvania, Philadelphia, Pa.). and others were prepared by standard techniques (17). Polysaccharide concentration were determined by the phenosulfuric acid procedure (4). Bacteria. S. pneumoniae isolated in routine diagnostic cultures were typed, preserved by lyopilization, and grown in the presence of 5% C02 in trypMATERIALS AND METHODS tose-phosphate broth (Difco, Detroit, Mich.) at 370C. Sera. Normal serum was collected in two closely A type 25 pneumococcus was obtained from the spaced bleedings from a healthy adult donor. Serum American Type Culture Collection (Rockville, Md.). was stored in samples at -70°C and thawed once The Fc reactivities of previously untested strains immediately before each experiment. A previously were determined (27). Escherichia coli 014, a serum296
VOL. 17, 1977
COMPLEMENT ACTIVATION BY PNEUMOCOCCI
sensitive laboratory strain, was grown overnight in tryptose-phosphate broth at 37°C. All bacteria were washed by centrifugation in normal saline and suspended to an optical density of 0.20 at 640 nm in a Coleman Jr. II spectrophotometer (approximately 4 x 109 organisms/ml). Pneumococci prepared as above were used in most complement depletion tests. In some experiments bacteria were precoated with immunoglobulin fractions by incubation for 30 min at 37°C with an equal volume of the individual immunoglobulin fraction, each in approximately equimolar concentrations [IgG = 20 mg/100 ml, Fc = 6 mg/100 ml, Fab = 6 mg/ 100 ml, F(ab')2 = 10 mg/100 ml]. IgG was derived from Cohn fraction II prepared by diethylaminoethyl-cellulose chromatography (26). Fc, Fab, and F(ab')2 were prepared from IgG as previously described (27). Bacterial suspensions were then washed in saline and resuspended to their previous concentrations. Fc-reactive pneumococcal strains (27) were thereby coated with IgG or some of its fragments. Some pneumococcal strains were also coated with specific typing antisera (Statens Seruminstitut, Copenhagen, Denmark), using 0.02 ml of antiserum per 4 x 109 organisms, to demonstrate classical complement activation. Immunochemical complement assays. In sera treated with EDTA or EGTA, 0.5 ml of 100 mm CaCl2 per ml of serum was added to saturate the chelators before measurement of complement components. Levels of C4, C3, and factor B were measured by radial immunodiffusion (13, 14) with specific antiC4, anti-C3 (Meloy), and anti-,82 glycoprotein II antisera. The standards used were commercial C4 and C3 (Meloy) and factor B, provided by Otto Goitze (Scripps Research Institute, La Jolla, Calif.). Functional complement assays. Hemolytic complement was measured in 50% hemolytic complement (CH50) units (11). A small-volume method used 5 x 106 sensitized erythrocytes and varying dilutions of serum in 0.7 ml of saline solution containing optimal Ca2+ and Mg2+. A functional assay for the overall acitivity of the alternative complement pathway used serum-sensitive (21) E. coli 014. Washed organisms (4 x 109/ml) were incubated for 30 min at 370C with an equal volume of IgG (75 mg/100 ml) prepared from pooled normal sera by diethylaminoethyl-ion-exchange chromatography followed by Sephadex G200 gel filtration, after which they were washed, resuspended to the previous concentration, and then further diluted 1:50 in saline. In a manner analogous to the assay for hemolytic complement, 0.1 ml of diluted test serum (1:10, 1:50, 1:100) was incubated with 0.1 ml of sensitized E. coli, 0.1 M EGTA and MgCl2, and 0.7 ml of saline for 30 min at 370C. The classical pathway was thus blocked, and killing could occur only by the alternative pathway. A 0.001-ml sample of the reaction mixture was removed with a calibrated loop and diluted in 1.0 ml of saline, and then 0.2 ml was plated in warm Penassay agar (Difco). Bacterial counts were determined at 48 h with a Biotron automatic colony counter. The per-
297
cent bacterial killing by each serum dilution was calculated, using the bacterial count of a sample in which saline replaced diluted serum to indicate the number of viable E. coli in the original samples. The percent killing by each serum dilution replaced percent hemolysis in the standard hemolytic complement assay calculations. The term CBs, representing the serum dilution required for killing 50% of 8 x 108 IgG-coated E. coli 014, was used to emphasize the conceptual similarity between bactericidal and hemolytic complement assays. Complement depletion test. Serum samples (0.2 ml) treated with EGTA, EDTA, or saline were incubated with 0.4 mg of zymosan, 107 sheep erythrocytes (Colorado Serum Co., Denver, Colo.) sensitized with anti-sheep hemolysin (5), or 8.0 x 108 pneumococci, each in 0.2 ml of saline, for 1 h at 37°C. Organisms, zymosan, or erythrocytes were removed by centrifugation, and the remaining sera were tested for levels of various complement components. C3, C4, factor B, CH50, and CB,0 levels were expressed as the percent reduction from simultaneous values obtained with untreated serum from the same sample. It was recognized that after such treatment immunochemical quantitation of complement components such as C3 would depend on relative amounts of antibody to C3b, C3d, and C3c in the available antisera. For this reason, relative changes in components such as C3 were closely correlated in all instances with the functional CH. and CB,0 assays. EDTA-treated sera were used as negative controls throughout these experiments. Significant consumption of all complement components by pneumococci, zymosan, and sensitized erythrocytes in normal and agammaglobulinemic sera was prevented by EDTA.
RESULTS Levels of complement components in normal serum controls were found to be near the expected values (22). The means and standard deviations were: C3 = 1.32 + 0.11 mg/ml, C4 = 0.21 ± 0.05 mg/ml, B = 145 + 16 ,g/ml, and CHw = 44 + 8 units. Treatment of sera with EGTA or EDTA and subsequent saturation of the chelator with CaCl2 did not alter its CH50. Factor B-depleted serum had unmeasurable factor B levels and 35% normal CH50, but less than 12% normal CB50. The C4-depleted serum had no measurable C4 and a 98% loss of hemolytic complement, and yet retained 87% of the normal CB,. The agammaglobulinemic serum demonstrated normal levels of hemolytic (51 CH50 units) and bactericidal (41 CB5o units) complement. Complement levels were tabulated as percent consumption of normal values. In Table 1, for instance, after normal serum without EGTA was incubated with zymosan, a C3 level of 0.92 mg/ml was measured. Since 0.92 mg/ml repre-
298
INFECT. IMMUN.
STEPHENS, WILLIAMS, AND REED
TABLE 1.
Consumption of complement components by zymosan, sensitized erythrocytes, and selected pneumococcal strains %
Serum
C3 With No EGTA EGTA
C4 No With EGTA EGTA
Consumption
Factor B No With EGTA EGTA
CH50 No With EGTA EGTA
CB50 No With EGTA EGTA
Normal with: 30 30 33 82 66 88 74 4 22 22 Zymosan 35 6 33 23 4 16 97 19 86 12 Sensitized erythrocytes 62 62 51 60 30 38 26 24 12 44 Type 5 pneumococcia 45 44 33 28 7 28 82 62 92 68 Type 19 pneumococci 17 12 4 44 3 23 32 21 29 26 Type 7 pneumococci 9 45 7 7 7 79 18 42 25 22 Type 9 pneumococci C4 depleted with: +b 58 61 + 27 Zymosan 12 + 23 + 4 Sensitized erythrocytes + + 43 38 50 Type 19 pneumococci 12 + + 9 15 Type 7 pneumococci Factor B depleted with: + 23 12 18 + Zymosan + 14 16 32 + Type 19 pneumococci + 28 34 + 18 Type 17 pneumococci a Representative strains shown here are arranged in decreasing order with respect to their Fc reactivity
(27). b
+, Not measurable due to absence of complement component.
sented 70% of the untreated serum C3 level measured concurrently, 30% of C3 had been consumed. Thus, the less remaining complement component, the greater the percent consumption. Zymosan, a known activator of the alternative system, consumed sizable fractions of most complement components in both untreated and EGTA-treated sera (Table 1). Zymosan consumed significant portions of C3 and factor B in C4-depleted sera, but not in factor B-depleted serum. Pneumococci activating the alternative system should display a similar pattern. Consumption of complement components by hemolysin-sensitized erythrocytes, as expected with pneumococci unable to activate the alternative system, was sharply reduced by EGTA and C4 depletion, demonstrating their dependence on the classical system. In Table 1, pneumococcal serovars are arranged in order of decreasing Fc reactivity, types 5 and 19 being highly reactive and types 7 and 9 much less so (27). Specific antibodies for each pneumococcal type in Table 1 were present, so activation of the classical pathway in the absence of EGTA would be expected. The highly Fc-reactive types 5 and 19, like zymosan, consumed relatively large portions of all complement components, even when the classical pathway was blocked by EGTA or C4 depletion. In factor B-depleted serum, however, the consumption of C3 and CH,, by type 19 was pre-
vented. Thus, the highly Fc-reactive strains resembled zymosan in their activation of the alternative pathway. The less Fc-reactive types 7 and 9 paralleled results with sensitized erythrocytes in that complement consumption was prevented by C4 depletion and EGTA. Low levels of C4 depletion found in the presence of EGTA need not indicate Cl classical pathway activity (16, 21). A summary of the data on complement depletion by all pneumococcal serovars is presented in Table 2. Serovars are arranged in order of decreasing Fc reactivity. Percent consumption represents average findings for all strains of a given serovar. In most, but not all, cases, consumption was uniform among the strains of a given serovar. Table 2 was divided into upper and lower halves for statistical purposes; the upper half contained 31 strains from 14 of the more Fc-reactive serovars and the lower half contained 31 strains from 14 serovars with less Fc reactivity. Some serovars in both upper and lower halves of Table 2, the high- and low-Fc-reactive serovars, respectively, consumed complement components in normal serum without EGTA. Ranges of serovar-specific antibody titers and prominent C4 depletion were dispersed throughout the high- and low-Fc-reactive serovars, suggesting that a portion of the observed complement depletion with no EGTA occurred through the classical pathway. Using the chi-
COMPLEMENT ACTIVATION BY PNEUMOCOCCI
VOL. 17, 1977
299
TABLE 2. Consumption of complement components in normal serum by pneumococci % Consumptiona
Serotype°
Strateis
33 5 19 29 20 10
2 2 3 2 3 3
16
1
14 17 1
1 3 1
0
25 3 24
1 5 3
0 1:8 0
11
1
+
15 6 31 8 18 39 7
1 3
0
0
+
++
0 0
0 0
0 0
++
++
+
+
++
0
+
0 0
+
+
+
0 0
0 0
0 0
+
++
+
+ 0 +
0 0
0 0
0 0
++
+
++
0 0 +
4 21 12 9
13 42 23
1:2 1:8 1:16
1:4
1 4 3 2 3 4 1 1 4 1 1 2
1:4
1:16
No EGTA C3 + ++ ++ + ++ ++
Factor B + + ++ + + ++ + 4
P
With EGTA
CH.
CBS,,
C3
++ +++ ++ +++ +++ ++
+ ++ +++ ++ ++
C4 Factor B
CH.,, CB5
0.6 >0.3 >0.1
AND
IMMUNITY, Aug. 1977, p. 296-302
Copyright 0 1977 American Society for Microbiology
Vol. 17, No. 2 Printed in U.S.A.
Classical and Alternative Complement Pathway Activation by Pneumococci CHRISTOPHER G. STEPHENS, RALPH C. WILLIAMS, JR., AND WILLIAM P. REED*
Department ofMedicine of the Veterans Administration Hospital* and Bernalillo County Medical Center, The University of New Mexico School of Medicine, Albuquerque, New Mexico 87108 Received for publication 14 January 1977
Sixty-two strains of Streptococcus pneumoniae were studied for their abilities consume selected components of classical and alternative complement pathways in human sera. The classical pathway was blocked by chelating calcium with ethyleneglycol-bis(,8-aminoethyl ether)-N,N-tetraacetic acid and by removing C4. The alternative pathway was blocked by removing factor B. Each strain's activation of the two pathways was compared with its nonimmune reactivity with the Fc region of immunoglobulin G (IgG). Activation of the classical complement pathway appeared to be independent of such Fc reactivity. Highly Fc-reactive strains, however, were shown to activate the alternative pathway more effectively than did less Fc-reactive strains. Since pneumococcal activation of the alternative pathway requires non-immunospecific IgG, these findings suggest that nonimmune binding of IgG on the pneumococcal surface endows it with complement-activating properties. to
Alternative complement pathway activation by several Streptococcus pneumoniae serovars has recently been demonstrated (5). Such activation requires an immunoglobulin which need not show specific antibody reactivity for the pneumococcal polysaccharide (30). The immunoglobulin's role in this interaction between pneumococci and the alternative system has not been well characterized. The Fc region of immunoglobulin G (IgG) reacts directly with the capsular type-specific polysaccharide of some pneumococcal serovars (27). Such Fc reactivity might cause approximation of non-immunospecific IgG molecules on the pneumococcal surface to such a degree that the complement-activating properties of aggregated immunoglobulins would be acquired (25). This study demonstrates a correlation between the Fc reactivity of a pneumococcal serovar and its ability to activate the alternative pathway, suggesting that accumulation of IgG on the bacterial surface is involved in alternative pathway activation. (This work was presented at the Western Section, American Federation for Clinical Research, Carmel, Calif., February 1976 [Clin. Res. 24:115A, 1976].)
described agammaglobulinemic serum with no measurable IgA or IgM and less than 50 mg of IgG per ml by radial immunodiffusion (19) was also used. Classical and alternative complement pathways were inactivated by several procedures. Ethylenediaminetetraacetic acid (EDTA; as the disodium and tetrasodium salts; Sigma, St. Louis, Mo.) at a concentration of 0.01 M in undiluted sera inactivated both pathways. The classical pathway was selectively blocked with 0.01 M ethylene glycol-bis(,8-aminoethyl ether)-N,N'-tetraacetic acid (EGTA; Sigma) and 0.01 M MgCl2(5). Samples of the normal serum were depleted of C4 or factor B by incubation with Sepharose-bound unfractionated anti-human C4 (Meloy, Springfield, Va.) or anti-,12 glycoprotein II (Behring, Woodbury, N.Y., lot 1548P) at 4°C in the presence of EDTA, which was later removed by dialysis (19). Levels of type-specific antipneumococcal antibodies were determined by hemagglutination of erythrocytes coated with pneumococcal polysaccharides (1). Several serovar polysaccharides were kindly provided by Robert Austrian (University of Pennsylvania, Philadelphia, Pa.). and others were prepared by standard techniques (17). Polysaccharide concentration were determined by the phenosulfuric acid procedure (4). Bacteria. S. pneumoniae isolated in routine diagnostic cultures were typed, preserved by lyopilization, and grown in the presence of 5% C02 in trypMATERIALS AND METHODS tose-phosphate broth (Difco, Detroit, Mich.) at 370C. Sera. Normal serum was collected in two closely A type 25 pneumococcus was obtained from the spaced bleedings from a healthy adult donor. Serum American Type Culture Collection (Rockville, Md.). was stored in samples at -70°C and thawed once The Fc reactivities of previously untested strains immediately before each experiment. A previously were determined (27). Escherichia coli 014, a serum296
VOL. 17, 1977
COMPLEMENT ACTIVATION BY PNEUMOCOCCI
sensitive laboratory strain, was grown overnight in tryptose-phosphate broth at 37°C. All bacteria were washed by centrifugation in normal saline and suspended to an optical density of 0.20 at 640 nm in a Coleman Jr. II spectrophotometer (approximately 4 x 109 organisms/ml). Pneumococci prepared as above were used in most complement depletion tests. In some experiments bacteria were precoated with immunoglobulin fractions by incubation for 30 min at 37°C with an equal volume of the individual immunoglobulin fraction, each in approximately equimolar concentrations [IgG = 20 mg/100 ml, Fc = 6 mg/100 ml, Fab = 6 mg/ 100 ml, F(ab')2 = 10 mg/100 ml]. IgG was derived from Cohn fraction II prepared by diethylaminoethyl-cellulose chromatography (26). Fc, Fab, and F(ab')2 were prepared from IgG as previously described (27). Bacterial suspensions were then washed in saline and resuspended to their previous concentrations. Fc-reactive pneumococcal strains (27) were thereby coated with IgG or some of its fragments. Some pneumococcal strains were also coated with specific typing antisera (Statens Seruminstitut, Copenhagen, Denmark), using 0.02 ml of antiserum per 4 x 109 organisms, to demonstrate classical complement activation. Immunochemical complement assays. In sera treated with EDTA or EGTA, 0.5 ml of 100 mm CaCl2 per ml of serum was added to saturate the chelators before measurement of complement components. Levels of C4, C3, and factor B were measured by radial immunodiffusion (13, 14) with specific antiC4, anti-C3 (Meloy), and anti-,82 glycoprotein II antisera. The standards used were commercial C4 and C3 (Meloy) and factor B, provided by Otto Goitze (Scripps Research Institute, La Jolla, Calif.). Functional complement assays. Hemolytic complement was measured in 50% hemolytic complement (CH50) units (11). A small-volume method used 5 x 106 sensitized erythrocytes and varying dilutions of serum in 0.7 ml of saline solution containing optimal Ca2+ and Mg2+. A functional assay for the overall acitivity of the alternative complement pathway used serum-sensitive (21) E. coli 014. Washed organisms (4 x 109/ml) were incubated for 30 min at 370C with an equal volume of IgG (75 mg/100 ml) prepared from pooled normal sera by diethylaminoethyl-ion-exchange chromatography followed by Sephadex G200 gel filtration, after which they were washed, resuspended to the previous concentration, and then further diluted 1:50 in saline. In a manner analogous to the assay for hemolytic complement, 0.1 ml of diluted test serum (1:10, 1:50, 1:100) was incubated with 0.1 ml of sensitized E. coli, 0.1 M EGTA and MgCl2, and 0.7 ml of saline for 30 min at 370C. The classical pathway was thus blocked, and killing could occur only by the alternative pathway. A 0.001-ml sample of the reaction mixture was removed with a calibrated loop and diluted in 1.0 ml of saline, and then 0.2 ml was plated in warm Penassay agar (Difco). Bacterial counts were determined at 48 h with a Biotron automatic colony counter. The per-
297
cent bacterial killing by each serum dilution was calculated, using the bacterial count of a sample in which saline replaced diluted serum to indicate the number of viable E. coli in the original samples. The percent killing by each serum dilution replaced percent hemolysis in the standard hemolytic complement assay calculations. The term CBs, representing the serum dilution required for killing 50% of 8 x 108 IgG-coated E. coli 014, was used to emphasize the conceptual similarity between bactericidal and hemolytic complement assays. Complement depletion test. Serum samples (0.2 ml) treated with EGTA, EDTA, or saline were incubated with 0.4 mg of zymosan, 107 sheep erythrocytes (Colorado Serum Co., Denver, Colo.) sensitized with anti-sheep hemolysin (5), or 8.0 x 108 pneumococci, each in 0.2 ml of saline, for 1 h at 37°C. Organisms, zymosan, or erythrocytes were removed by centrifugation, and the remaining sera were tested for levels of various complement components. C3, C4, factor B, CH50, and CB,0 levels were expressed as the percent reduction from simultaneous values obtained with untreated serum from the same sample. It was recognized that after such treatment immunochemical quantitation of complement components such as C3 would depend on relative amounts of antibody to C3b, C3d, and C3c in the available antisera. For this reason, relative changes in components such as C3 were closely correlated in all instances with the functional CH. and CB,0 assays. EDTA-treated sera were used as negative controls throughout these experiments. Significant consumption of all complement components by pneumococci, zymosan, and sensitized erythrocytes in normal and agammaglobulinemic sera was prevented by EDTA.
RESULTS Levels of complement components in normal serum controls were found to be near the expected values (22). The means and standard deviations were: C3 = 1.32 + 0.11 mg/ml, C4 = 0.21 ± 0.05 mg/ml, B = 145 + 16 ,g/ml, and CHw = 44 + 8 units. Treatment of sera with EGTA or EDTA and subsequent saturation of the chelator with CaCl2 did not alter its CH50. Factor B-depleted serum had unmeasurable factor B levels and 35% normal CH50, but less than 12% normal CB50. The C4-depleted serum had no measurable C4 and a 98% loss of hemolytic complement, and yet retained 87% of the normal CB,. The agammaglobulinemic serum demonstrated normal levels of hemolytic (51 CH50 units) and bactericidal (41 CB5o units) complement. Complement levels were tabulated as percent consumption of normal values. In Table 1, for instance, after normal serum without EGTA was incubated with zymosan, a C3 level of 0.92 mg/ml was measured. Since 0.92 mg/ml repre-
298
INFECT. IMMUN.
STEPHENS, WILLIAMS, AND REED
TABLE 1.
Consumption of complement components by zymosan, sensitized erythrocytes, and selected pneumococcal strains %
Serum
C3 With No EGTA EGTA
C4 No With EGTA EGTA
Consumption
Factor B No With EGTA EGTA
CH50 No With EGTA EGTA
CB50 No With EGTA EGTA
Normal with: 30 30 33 82 66 88 74 4 22 22 Zymosan 35 6 33 23 4 16 97 19 86 12 Sensitized erythrocytes 62 62 51 60 30 38 26 24 12 44 Type 5 pneumococcia 45 44 33 28 7 28 82 62 92 68 Type 19 pneumococci 17 12 4 44 3 23 32 21 29 26 Type 7 pneumococci 9 45 7 7 7 79 18 42 25 22 Type 9 pneumococci C4 depleted with: +b 58 61 + 27 Zymosan 12 + 23 + 4 Sensitized erythrocytes + + 43 38 50 Type 19 pneumococci 12 + + 9 15 Type 7 pneumococci Factor B depleted with: + 23 12 18 + Zymosan + 14 16 32 + Type 19 pneumococci + 28 34 + 18 Type 17 pneumococci a Representative strains shown here are arranged in decreasing order with respect to their Fc reactivity
(27). b
+, Not measurable due to absence of complement component.
sented 70% of the untreated serum C3 level measured concurrently, 30% of C3 had been consumed. Thus, the less remaining complement component, the greater the percent consumption. Zymosan, a known activator of the alternative system, consumed sizable fractions of most complement components in both untreated and EGTA-treated sera (Table 1). Zymosan consumed significant portions of C3 and factor B in C4-depleted sera, but not in factor B-depleted serum. Pneumococci activating the alternative system should display a similar pattern. Consumption of complement components by hemolysin-sensitized erythrocytes, as expected with pneumococci unable to activate the alternative system, was sharply reduced by EGTA and C4 depletion, demonstrating their dependence on the classical system. In Table 1, pneumococcal serovars are arranged in order of decreasing Fc reactivity, types 5 and 19 being highly reactive and types 7 and 9 much less so (27). Specific antibodies for each pneumococcal type in Table 1 were present, so activation of the classical pathway in the absence of EGTA would be expected. The highly Fc-reactive types 5 and 19, like zymosan, consumed relatively large portions of all complement components, even when the classical pathway was blocked by EGTA or C4 depletion. In factor B-depleted serum, however, the consumption of C3 and CH,, by type 19 was pre-
vented. Thus, the highly Fc-reactive strains resembled zymosan in their activation of the alternative pathway. The less Fc-reactive types 7 and 9 paralleled results with sensitized erythrocytes in that complement consumption was prevented by C4 depletion and EGTA. Low levels of C4 depletion found in the presence of EGTA need not indicate Cl classical pathway activity (16, 21). A summary of the data on complement depletion by all pneumococcal serovars is presented in Table 2. Serovars are arranged in order of decreasing Fc reactivity. Percent consumption represents average findings for all strains of a given serovar. In most, but not all, cases, consumption was uniform among the strains of a given serovar. Table 2 was divided into upper and lower halves for statistical purposes; the upper half contained 31 strains from 14 of the more Fc-reactive serovars and the lower half contained 31 strains from 14 serovars with less Fc reactivity. Some serovars in both upper and lower halves of Table 2, the high- and low-Fc-reactive serovars, respectively, consumed complement components in normal serum without EGTA. Ranges of serovar-specific antibody titers and prominent C4 depletion were dispersed throughout the high- and low-Fc-reactive serovars, suggesting that a portion of the observed complement depletion with no EGTA occurred through the classical pathway. Using the chi-
COMPLEMENT ACTIVATION BY PNEUMOCOCCI
VOL. 17, 1977
299
TABLE 2. Consumption of complement components in normal serum by pneumococci % Consumptiona
Serotype°
Strateis
33 5 19 29 20 10
2 2 3 2 3 3
16
1
14 17 1
1 3 1
0
25 3 24
1 5 3
0 1:8 0
11
1
+
15 6 31 8 18 39 7
1 3
0
0
+
++
0 0
0 0
0 0
++
++
+
+
++
0
+
0 0
+
+
+
0 0
0 0
0 0
+
++
+
+ 0 +
0 0
0 0
0 0
++
+
++
0 0 +
4 21 12 9
13 42 23
1:2 1:8 1:16
1:4
1 4 3 2 3 4 1 1 4 1 1 2
1:4
1:16
No EGTA C3 + ++ ++ + ++ ++
Factor B + + ++ + + ++ + 4
P
With EGTA
CH.
CBS,,
C3
++ +++ ++ +++ +++ ++
+ ++ +++ ++ ++
C4 Factor B
CH.,, CB5
0.6 >0.3 >0.1
