These studies provide the first in vivo
evidence that CR1 expression on erythrocytes has an actual, and not just a theoretical (24
), effect on the clearance of pneumococci from the blood. We have demonstrated that human CR1 expression on mouse erythrocytes enhances the immune adherence of pneumococci to erythrocytes, facilitates the transfer of pneumococci to phagocytes, and improves the clearance of pneumococci from the blood of infected mice.
Prior studies demonstrated the activity of human soluble CR1 (sCR1) in mouse models of disease and showed that sCR1 and its fragments are active against the mouse alternative pathway of complement activation, which suggests at least some interaction between sCR1 and mouse C3b (25
). However, the behavior of sCR1 may differ from that of membrane-bound CR1. Our data establish both in vitro
and in vivo
that mouse complement can interact with human CR1 expressed on murine erythrocytes in the transgenic model. This was demonstrated by comparisons of the results obtained with wild-type erythrocytes to those obtained with CR1+
transgenic erythrocytes. It was also demonstrated by showing that the CR1-dependent enhancement of the binding of complement-coated pneumococci or beads to erythrocytes could be blocked by the use of a monoclonal antibody to CR1.
Moreover, the CR1 transgenic mouse was also shown to clear bacteriophage
X174 in the presence of a bispecific monoclonal antibody heteropolymer that can bind both CR1 and bacteriophage
). This CR1-dependent clearance of bacteriophage
X174 in the mouse was similar to what was observed previously for baboons, which have erythrocyte-based immune adherence mediated by CR1-like molecules that are glycosylphosphatidylinositol (GPI) anchored (5
). These combined findings suggest that the CR1 transgenic mouse is a useful surrogate animal model for studying the clearance of immune complexes through IA.
Our in vitro
data using blocking antibody against human CR1 demonstrate that transgenic CR1 RBCs bind to the pneumococcus in a manner that is dependent on CR1 and C3b. Nearly all of the pneumococcal association with CR1+
RBCs was inhibited by 3D9, an antibody that inhibits the binding of CR1 to C3b/C4b (19
). Also, data from the use of serum from C3-deficient mice as a complement source suggest that mouse C1q, C4b, and MBL play a minimal role of the attachment to CR1 in IA in our model since the absence of C3 by itself largely abrogated IA. The serum used as a complement source for the opsonization of bacteria was obtained from C57BL/6 mice that lacked significant antibody against pneumococci. Therefore, the deposition of C1q, C4, and MBL on the pneumococcal strains in our experimental system is expected to be low. However, C3 deposition can still be achieved because even in the presence of normal serum lacking detectable antibody to pneumococci, there is a measurable but low-level activation of C3 through the classical pathway, which is greatly amplified by the alternative pathway (9
), thus making the total amount of C3 products available to bind CR1 much greater than that of C1q or C4. Previous studies using human complement indicated that the interaction between CR1 and the immune complexes is made largely through C3b (13
). Although it is possible that C3b fragments generated through the activation of mouse complement C3 might play a critical role in the CR1-dependent IA that we have observed, our studies have not experimentally examined this issue.
We observed a significantly greater virulence of our BG7322 infections shown in Fig. than those shown in Fig. . This was reflected by the over 1,000-fold-more CFU at 24 h in C57BL/6 wild-type mice. This difference could be due to inadvertent differences in the phases of growth of the bacterial stock between the experiments, which were performed at two different sites. Although the difference makes interpretations of the data more complex, it also provides additional strength to the findings, since the presence of CR1 was associated with greater protection against bacteremia in the experiments using mice regardless of the experimental site.
In the studies shown in Fig. , BG7322 pneumococci injected with CR1+ erythrocytes were cleared significantly faster than in mice where there were no CR1+ cells. Moreover, four of the five mice given BG7322 and WT cells died within 48 h, and all five mice given bacteria and CR1+ cells lived. In the studies shown in Fig. , where the BG7322 infections rapidly progressed in wild-type mice, nearly 10-fold-lower levels of CFU were present in CR1+ mice than in wild-type mice at early time points, but no difference in the rates of survival of the mice was observed.
Another interesting observation was that although the 10-fold effect of CR1 expression on CFU levels in transgenic mice (Fig. ) occurred within 4 to 6 h of infection, the difference in CFU levels showed no further increase at 24 h postinfection. This finding was in contrast to the data shown in Fig. , where the greatest differences in circulating pneumococci in the absence and presence of CR1 occurred at 22 to 30 h postinfection. This difference in the kinetics of clearance may be a reflection of the 10- to 1,000-fold-higher bacterial burden observed for C57BL/6 mice (Fig. ) from the 6-h time point on. Previous studies demonstrated that when the blood levels of pneumococci in mice exceed 106
, as occurred in the studies where CR1+
and WT mice were injected with pneumococci, a very protective inflammatory response was elicited (3
). It is possible that once this inflammatory response developed, CR1 expression no longer provided an advantage for the clearance of pneumococci from blood.
Our finding that pneumococcal clearance can be enhanced by CR1 is consistent with the work of others who have shown that nonbacterial immune complexes bound to heterologous CR1-expressing erythrocytes can be removed by phagocytes in the liver (16
). Even so, the ability of our studies and previous studies to see CR1-dependent clearance in the mouse seemed remarkable considering the fact that since the mouse host lacks CR1, it thus has lacked evolutionary pressure to maintain or develop a CR1-dependent clearance mechanism.
The findings presented here support the possibility that pathogens, in addition to pneumococci, that have been opsonized with complement may be cleared from the blood of humans more rapidly because of the presence of CR1 on human erythrocytes. We are actively investigating this possibility with bacterial, fungal, and viral pathogens. Interestingly, we have observed an increase in the binding of adenovirus with transgenic CR1 erythrocytes in vivo
, indicating that CR1 affects viral pathogenesis by altering the delivery of the virus to different organs (10
). Future studies with the CR1 transgenic mouse will allow a greater understanding of the role of IA in vivo