Complement receptor 1 expression on mouse erythrocytes mediates clearance of Streptococcus pneumoniae by immune adherence

Abstract

Complement-containing immune complexes can be presented to phagocytes by human erythrocytes bearing complement receptor 1 (CR1). Although this has long been assumed to be a mechanism by which humans are able to protect themselves from "extracellular" bacteria such as pneumococci, there is little direct evidence. In these studies we have investigated this question by comparing results for erythrocytes from transgenic mice expressing human CR1 on their erythrocytes to the results for wild-type mouse erythrocytes that do not express CR1. We demonstrate that human CR1 expression on murine erythrocytes allows immune adherence to beads opsonized with either mouse or human serum as a source of complement. The role of CR1 in immune adherence was supported by studies showing that it was blocked by the addition of antibody to human CR1. Furthermore, human CR1 expression enhances the immune adherence of opsonized pneumococci to erythrocytes in vitro, and the pneumococci attached to erythrocytes via CR1 can be transferred in vitro to live macrophages. Even more importantly, we observed that if complement-opsonized pneumococci are injected intravenously with CR1(+) mouse erythrocytes into wild-type mice (after a short in vitro incubation), they are cleared faster than opsonized pneumococci similarly injected with wild-type mouse erythrocytes. Finally, we have shown that the intravenous (i.v.) injection of pneumococci into CR1(+) mice also results in more rapid blood clearance than in wild-type mice. These data support that immune adherence via CR1 on erythrocytes likely plays an important role in the clearance of opsonized bacteria from human blood.

FIG. 1.

Mouse erythrocytes expressing human CR1 bind mouse complement-opsonized particles and human complement-opsonized particles. Mouse (A and C) or human (B and D) complement-opsonized beads were incubated with WT mouse erythrocytes (E) (A and B) or CR1⁺ mouse erythrocytes (C and D) for 15 min at 37°C. Cells were washed twice and analyzed by flow cytometry. Shifts in the CR1⁺ erythrocyte population in the presence of Ca²⁺ and Mg²⁺ were observed, in comparison to the absence of Ca²⁺ and Mg²⁺ to block complement activation. Some CR1⁺ erythrocytes were pretreated with anti-human CR1 monoclonal antibody 3D9, which led to an inhibition of the shift.

FIG. 2.

Immune adherence of pneumococci to erythrocytes obtained from WT mouse, CR1⁺ mouse, or human erythrocytes. The bacteria were opsonized with WT mouse serum (A) or C3-deficient mouse serum (B), followed by incubation with WT mouse, CR1⁺ mouse, or human erythrocytes (E). (A) The immune adherence of pneumococci to either CR1⁺ mouse or human erythrocytes, assessed as the mean fluorescence (MF) of erythrocytes, was significantly higher than that to WT mouse erythrocytes (*, P < 0.01 by a Student's two-tailed t test). (B) The increased immune adherence exhibited with CR1⁺ mouse and human erythrocytes was C3 dependent. Error bars indicate the standard deviations (SD) of data from triplicate samples.

FIG. 3.

Adherence of pneumococci to CR1⁺ erythrocytes is inhibited in the presence of anti-CR1 antibody. FITC-labeled Streptococcus pneumoniae BG7322 was opsonized in mouse serum and then incubated with WT mouse erythrocytes (E) or CR1⁺ mouse erythrocytes that had been pretreated with either anti-CR1 antibody 3D9 or control IgG for 1 h at room temperature. While a shift in the CR1⁺ population was present in comparison to the WT population, the shift was not observed for CR1⁺ erythrocytes pretreated with monoclonal antibody 3D9 to human CR1.

FIG. 4.

Macrophage transfer reaction of pneumococci from WT mouse, CR1⁺ mouse, or human erythrocytes. The transfer reaction was conducted by incubating J774A.1 cells with pneumococci adherent to erythrocytes for 30 min prior to fixation and subsequent analysis by flow cytometry. The amount of bacteria transferred to macrophages was measured as the mean fluorescence (MF) acquired by the macrophages. The asterisk indicates that significantly more pneumococci were transferred from erythrocytes (E) of CR1⁺ mouse and human than from WT mouse (P < 0.01 by a Student's two-tailed t test). Error bars indicate the SD of data from triplicate samples.

FIG. 5.

Blood clearance of BG7322 (A) and TIGR4 (B) combined with erythrocytes. Pneumococci were opsonized with WT mouse serum, followed by incubation with WT or CR1⁺ murine erythrocytes. A mixture containing 2 × 10⁵ bacteria and 5 × 10⁶ erythrocytes (E) was injected i.v. into WT mice (n = 5). Blood was drawn at different time intervals to evaluate the bacterial clearance. The asterisk indicates that BG7322 or TIGR4 combined with CR1⁺ mouse erythrocytes was cleared from blood significantly faster than when combined with WT mouse erythrocytes (P = 0.006 [A] and P = 0.02 [B] by a Student's two-tailed t test). Error bars indicate the standard errors of the means (SEM).

FIG. 6.

Bacterial clearance and survival of WT and CR1⁺ transgenic mice infected with Streptococcus pneumoniae BG7322. (A) Blood clearance and/or growth of pneumococci in WT and CR1⁺ transgenic mice. WT mice and transgenic mice were infected i.v. with 2 × 10⁵ CFU of pneumococci. Bacteremia was determined at different time points. Data are presented as the mean CFU/ml of blood ± SEM for eight mice in each group. The asterisks indicate that bacteremia in deficient mice is significantly different (P < 0.001 by a Student's two-tailed t test). (B) Number of mice not yet moribund at each day following their infection as described above. It took a day longer for 50% of the CR1⁺ mice to become moribund compared to the C57BL/6 mice. This difference, however, was not statistically significant (P = 0.2 by two-tailed Mann-Whitney U test).