Complement activation in factor D-deficient mice

Abstract

To assess the contribution of the alternative pathway in complement activation and host defense and its possible role in the regulation of systemic energy balance in vivo, factor D-deficient mice were generated by gene targeting. The mutant mice have no apparent abnormality in development and their body weights are similar to those of factor D-sufficient littermates. Complement activation could not be initiated in the serum of deficient mice by the alternative pathway activators rabbit erythrocytes and zymosan. Surprisingly, injection of cobra venom factor (CVF) caused a profound and reproducible reduction in serum C3 levels, whereas, as expected, there was no C3 reduction in factor B-deficient mice treated similarly. Studies of C3 and factor B activation in vitro by CVF demonstrated that in factor D-deficient serum the alpha chain of C3 was cleaved gradually over a period of 60 min without detectable cleavage of factor B. CVF-dependent C3 cleavage in the deficient serum required the presence of Mg(2+), whereas in normal mouse serum the presence of divalent cations was not required. These results suggest that in mouse proteolytic cleavage of factor B by factor D is not an absolute requirement for the zymogen to active enzyme conformational transition of CVF-bound factor B. Kinetics of opsonization of Streptococcus pneumoniae by C3 fragments was much slower in factor D-deficient serum, suggesting a significant contribution of the alternative pathway to antibacterial host defense early after infection.

Figure 1

Targeted disruption of the murine Df. (A) Schematic presentation of the Df allele, the targeting vector, and the targeted Df allele after recombination. Open boxes denote exons and thick lines intron sequences. The 1.1-kb neo^r gene cassette and the TK gene from herpes simplex virus 1 (HSV-1) are shown. Restriction fragments and probes used for Southern blotting are indicated. Restriction enzyme cleavage sites: H, HindIII; E, EcoRI; Sm, SmaI. (B) Southern blot analysis of HindIII-restricted tail DNA from offspring of F₁ Df^+/− intercross. (C) Western blot analysis of serum factor D of the same litter. Markers are on the right.

Figure 2

Flow cytometric analysis of C3 deposition on zymosan. Zymosan particles (1 × 10⁶) were incubated with 10% wild-type (black) or factor D-deficient (gray) serum in Mg²⁺-EGTA or EDTA buffer. C3 deposition was allowed to occur at 37°C for 15 min and detected by FITC-conjugated F(ab)′₂ fragments of goat anti-mouse C3 IgG. In the reconstitution assay, purified human factor D was supplemented at 0.5 μg/ml of deficient serum.

Figure 3

Consumption of serum C3 by CVF. Factor D-sufficient or -deficient and factor B-deficient mice were treated with single doses (30 μg) of CVF. Mouse sera were collected at the indicated time intervals for 9 days. Concentration of serum C3 was measured by ELISA.

Figure 4

Kinetics of CVF-mediated activation of the alternative pathway. Pooled mouse serum from wild-type (Left) or factor D-deficient (Right) mice was incubated with an equal volume of CVF (75 μg/ml) in Ca²⁺-Mg²⁺, Mg²⁺-EGTA (*) or EDTA (**) buffer at 37°C for the indicated time periods. Duplicate aliquots of the mixtures were subjected to 10% SDS/PAGE under reducing conditions. C3 (A) and factor B (B) and their activation fragments were detected by goat anti-C3 and anti-factor B IgG, respectively. Markers are on the left.

Figure 5

C3 opsonization of S. pneumoniae. Heat-killed S. pneumoniae R36A (1 × 10⁷) were incubated with 10% wild-type (black) or factor D-deficient (gray) serum at 37°C for 15 min (A) or for indicated time intervals (B). Surface-bound IgM (A Upper) and C3 (A Lower and B) were stained by FITC-conjugated goat IgG anti-mouse μ-chain and C3, respectively, and analyzed by flow cytometry.