Antibody to the type 3 capsule facilitates immune adherence of pneumococci to erythrocytes and augments their transfer to macrophages

Abstract

Streptococcus pneumoniae has been shown to bind to erythrocytes via a process called immune adherence. This adherence and the subsequent transfer of pneumococci from erythrocytes to macrophages are both dependent on complement C3 deposition onto the pneumococcal surface. The observation that anti-capsule antibody increases C3 deposition on the pneumococcal capsule indicated that anti-capsule antibody may also facilitate the clearance of pneumococci through immune adherence. Using pneumococcal strain WU2 (capsule type 3) and its nonencapsulated mutant JD908, we found that monoclonal antibody (MAb) to type 3 capsule increases complement C3, C1q, and C4 deposition on WU2 and enhanced the immune adherence of WU2 to erythrocytes. The MAb to type 3 capsule also enhanced the transfer of WU2 from erythrocytes to macrophages. Moreover, the transfer reaction was inhibited by preincubating macrophages with anti-CR3 or anti-Fc gammaRIII/II MAb, indicating that CR3 and Fc gammaRIII/II on macrophages mediate this process. The transfer reactions of JD908 (opsonized with complement) and WU2 (opsonized with complement plus MAb to type 3 capsule) were similarly inhibited by anti-CR3 MAb, but only the latter was inhibited by anti-Fc gammaRIII/II MAb. This finding indicates that although complement and the macrophage receptor CR3 are essential for the transfer reaction, if antibody is present it can further enhance the transfer reaction through a process dependent on Fc gammaRIII/II. Using pre- and postvaccination sera of people immunized with the 23-valent pneumococcal polysaccharide vaccine, we confirmed that human anti-capsule antibodies are also able to increase the immune adherence of pneumococci and their transfer to macrophages.

FIG. 1.

Effects of heat-inactivated MAb to type 3 capsule (16.3, ascites fluid) on C3 (A), C1q (B), and C4 (C) deposition on WU2 (Cps3⁺) and its nonencapsulated mutant JD908 (Cps3⁻). Pneumococci were opsonized in NHS supplemented with different concentrations of MAb to type 3 capsule at 37°C for 30 min. FITC-conjugated goat-anti-human C3, C1q, or C4 antibody was added. After incubation on ice for 30 min, the bacteria were washed and fixed for FACS analysis. Complement deposition was measured by MF of the gated bacteria. Error bars indicate the standard errors of triplicate samples. The amount of MAb to capsule is presented as the percentage of MAb-containing ascites fluid in the final mixture of pneumococci, serum (complement source), and ascites fluid. The amounts of pneumococci and complement remained constant.

FIG. 2.

Effects of MAb to type 3 capsule on the adherence of WU2 (Cps3⁺) and JD908 (Cps3⁻) to erythrocytes. (A) Erythrocyte adherence of WU2 and JD908 in the presence of NHS alone or supplemented with 4% MAb to type 3 capsule. (B) Adherence of WU2 and JD908 to erythrocytes in the presence of NHS in relation to the concentration of MAb to type 3 capsule. The adherence of pneumococci to erythrocytes was measured by MF of erythrocytes. The asterisk indicates that the adherence of WU2 to erythrocytes was significantly higher than that of JD908 opsonized in NHS plus 4% heat-inactivated MAb to type 3 capsule (16.3, ascites fluid) (P = 0.0001, Student's two-tailed t test). Error bars indicate the standard errors of triplicate samples.

FIG. 3.

Adherence of type 3 pneumococci to erythrocytes in the presence of mouse serum. Sera from a wild-type mouse (C57BL/6), a C3-deficient mouse (C3^−/−), a C1q-deficient mouse (C1q^−/−), and a heat-inactivated C1q-deficient mouse (HI-C1q^−/−) were used to opsonize type 3 pneumococcal strains WU2 and A66.1. The adherence of WU2 (A) and A66.1 (B) to erythrocytes in various mouse sera with or without supplementation with heat-inactivated MAb to type 3 capsule (16.3, ascites fluid) was measured by MF of erythrocytes. The portions of adherence of pneumococci to erythrocytes mediated by different complement components are indicated in panel B.

FIG. 4.

Influence of MAb to type 3 capsule on the transfer reaction of WU2 (Cps⁺) and JD908 (Cps3⁻). FITC-labeled pneumococci were opsonized in NHS supplemented with different concentrations of MAb to type 3 capsule. Erythrocytes were added and incubated at 37°C for 30 min. After washing, J774A.1 macrophages were added and the incubation was repeated. Erythrocytes in the samples were then lysed and washed off. The macrophages were fixed for FACS analysis. The MF of macrophages was used to measure the transfer reaction of pneumococci. The natural fluorescence of macrophages was subtracted from each sample. The subtracted values were never more than 3% of the total MF of macrophages observed after the transfer reaction.

FIG. 5.

Inhibition of macrophage transfer reaction of pneumococci by anti-CR3 MAb (A) and anti-FcγRIII/II MAb (B). The macrophages were preincubated with different concentrations of anti-CR3 MAb or anti-FcγRIII/II MAb before the transfer reaction was conducted. Error bars indicate the standard errors of triplicate samples.

FIG. 6.

Adherence of pneumococci to erythrocytes opsonized with pre- and postvaccination sera of people immunized with the 23-valent pneumococcal polysaccharide vaccine. Type 4 (A), 6B (B), and 23F (C) pneumococci were examined. Error bars indicate the standard errors of triplicate samples.

FIG. 7.

Macrophage transfer reaction of pneumococci obtained with pre- and postvaccination sera. Type 4, 6B, and 23F pneumococci were opsonized with pre- and postvaccination sera of a person (donor 1, as in Fig. 6) immunized with the 23-valent pneumococcal polysaccharide vaccine. Error bars indicate the standard errors of triplicate samples.