Targeted complement inhibition by C3d recognition ameliorates tissue injury without apparent increase in susceptibility to infection

Abstract

Previous studies indicate a pivotal role for complement in mediating both local and remote injury following ischemia and reperfusion of the intestine. Here, we report on the use of a mouse model of intestinal ischemia/reperfusion injury to investigate the strategy of targeting complement inhibition to sites of complement activation by linking an iC3b/C3dg-binding fragment of mouse complement receptor 2 (CR2) to a mouse complement-inhibitory protein, Crry. We show that the novel CR2-Crry fusion protein targets sites of local and remote (lung) complement activation following intestinal ischemia and reperfusion injury and that CR2-Crry requires a 10-fold lower dose than its systemic counterpart, Crry-Ig, to provide equivalent protection from both local and remote injury. CR2-Crry has a significantly shorter serum half-life than Crry-Ig and, unlike Crry-Ig, had no significant effect on serum complement activity at minimum effective therapeutic doses. Furthermore, the minimum effective dose of Crry-Ig significantly enhanced susceptibility to infection in a mouse model of acute septic peritonitis, whereas the effect of CR2-Crry on susceptibility to infection was indistinguishable from that of PBS control. Thus, compared with systemic inhibition, CR2-mediated targeting of a complement inhibitor of activation improved bioavailability, significantly enhanced efficacy, and maintained host resistance to infection.

Figure 1

In vitro characterization of recombinant CR2-Crry and Crry-Ig. (A) Flow cytometric analysis of binding of recombinant fusion proteins to C3-opsonized CHO cells; Crry-Ig (light gray line), CR2-Crry (dark gray line) and PBS control (black line). Representative of 3 separate experiments. (B) Inhibition of complement-mediated lysis by recombinant fusion proteins. Antibody-sensitized CHO cells were incubated with 15% rat serum (which resulted in approximately 90% lysis in the absence of inhibitor), and lysis was determined after 1 hour at 37°C. Background lysis determined by incubating cells in heat-inactivated serum was less than 5% and was subtracted. Mean ± SD; n = 4.

Figure 2

Quantitative evaluation of intestinal IRI with different doses of CR2-Crry and Crry-Ig. H&E-stained sections of intestine were assessed for mucosal injury score using the criteria described by Fleming et al. (46) (A) as well as for villi height (B). All measurements were obtained at ×200 magnification. Mean ± SD; n = 4–7 mice per group.

Figure 3

Representative sections of H&E-stained intestinal sections from BALB/c mice treated with different doses of CR2-Crry and Crry-Ig. Sham-operated and PBS controls are included for comparison. Photomicrograph magnification, ×200; high-power inserts, ×400. Each panel is representative of 4–7 mice per group.

Figure 4

Evaluation of remote organ injury of the lung following intestinal IRI. (A) H&E-stained sections of lungs from CR2-Crry, Crry-Ig, sham-operated, and PBS groups. (B) Quantitative evaluation of the degree of alveolar wall expansion in the lungs measured as described in Methods. Mean ± SD; n = 4–7 mice per group.

Figure 5

IRI-induced C3 deposition demonstrated by immunofluorescent staining. Intestinal sections were stained for C3 expression and visualized by confocal microscopy. C3 deposition is denoted by green fluorescence on apical epithelial cells (arrows). Each image is representative of 4–7 mice per group.

Figure 6

The dose-dependent effect of CR2-Crry and Crry-Ig on neutrophil infiltration into the intestine and lung. Neutrophil infiltration was assessed by immunohistochemical staining with mAb GR1. The number of neutrophils per high-power field (hpf) was counted in 3 fields per slide. Mean ± SD; n = 4–7 mice per group.

Figure 7

Mean serum circulatory levels of CR2-Crry. The serum levels of CR2-Crry were measured by ELISA in serum prepared from blood collected at indicated times following injection (0.1 mg). A t_1/2 of 8.7 hours was calculated. Mean ± SD; n = 4–7 mice per group.

Figure 8

Biodistribution and localization of CR2-Crry and Crry-Ig following intestinal IRI. (A) ¹²⁵I-labeled CR2-Crry and ¹²⁵I-labeled Crry-Ig were injected 30 minutes after reperfusion; tissue distribution was assessed in all major organs 2 hours after reperfusion (mean ± SD; n = 3). (B–E) Representative anti-CR2 immunohistochemistry photomicrographs. (B) Intestine. Arrows indicate endothelial and epithelial CR2 immunostaining. (C) Intestine. Arrow indicates specific localization of CR2 immunostaining to endothelium of vessel beneath crypts of epithelial cells. (D and E) Lung. Arrows indicate endothelial and parenchymal wall CR2 immunostaining. There was no immunoreactivity with isotype control (data not shown). Magnification, ×200 (B and D); ×400 (C and E).

Figure 9

Effect of administration of CR2-Crry and Crry-Ig at different doses on systemic complement inhibition. Serum was isolated from animals following intestinal IRI and complement activity assessed using zymosan particles and flow cytometry to detect surface-bound C3. Mean ± SD; n = 3 mice per group.

Figure 10

Survival analysis in CLP model of sepsis. Mice were administered a single dose of PBS (n = 18), Crry-Ig (1.0 mg; n = 8), or CR2-Crry (0.1 mg; n = 10) or multiple doses of CR2-Crry (0.1 mg; n = 10) and monitored. Inj., injection.