Analysis of C4 and the C4 binding protein in the MRL/lpr mouse

Abstract

Systemic lupus erythematosus is a complement-mediated autoimmune disease. While genetic deficiencies of classical pathway components lead to an increased risk of developing systemic lupus erythematosus, end organ damage is associated with complement activation and immune complex deposition. The role of classical pathway regulators in systemic lupus erythematosus is unknown. C4 binding protein (C4bp) is a major negative regulator of the classical pathway. In order to study the role of C4bp deficiency in an established murine model of lupus nephritis, mice with a targeted deletion in the gene encoding C4bp were backcrossed into the MRL/lpr genetic background. Compared with control MRL/lpr mice, C4bp knockout MLR/lpr mice had similar mortality and similar degrees of lymphoproliferation. There were no differences in the extent of proteinuria or renal inflammation. Staining for complement proteins and immunoglobulins in the kidneys of diseased mice revealed no significant strain differences. Moreover, there was no difference in autoantibody production or in levels of circulating immune complexes. In comparison with C57BL/6 mice, MRL/lpr mice had depressed C4 levels as early as 3 weeks of age. The absence of C4bp did not impact serum C4 levels or alter classical pathway hemolytic activity. Given that immune complex renal injury in the MRL/lpr mouse is independent of Fc receptors as well as the major negative regulator of the classical pathway, new mechanisms for immune-complex-mediated renal injury need to be considered.

Figure 1

No difference in survival between knockout MRL mice and control MRL mice. C4bp^-/-MRL/lpr (KO MRL) mice (solid line, n = 38) and littermate control (CTRL MRL) mice (dashed line, n = 34) from the F6 backcross were followed for up to 34 weeks. Mortality was quantified using Kaplan–Meier analysis. P = 0.15, KO MRL mice versus CTRL MRL mice (log-rank).

Figure 2

Renal histopathology in knockout MRL mice and control MRL mice. Sections showing the renal histopathology of C4bp^-/-MRL/lpr(KO MRL) mice and littermate control (CTRL MRL) mice. (a) Representative formalin-fixed sections from the kidney stained with periodic acid Schiff (0.75NA, 400× magnification). Glomeruli with crescentic changes are shown. (b) Sections stained with periodic acid Schiff showing perivascular inflammation around branching arteries (white arrows) (0.15NA, 50× magnification).

Figure 3

Similar serum autoantibody titers and circulating immune complexes in knockout MRL and control MRL mice. (a) Sera from C4bp^-/-MRL/lpr (KO MRL) mice (▲, solid line, n = 15) and littermate control (CTRL MRL) mice (◆, dashed line, n = 8) were tested for binding to double-stranded DNA by ELISA using serial serum dilution (x axis). Pooled normal mouse serum from nonautoimmune mice (■, NMS) was used as a negative control. P > 0.05, KO MRL mice versus CTRL MRL mice. OD450, optical density at 450 nm. (b) Sera from 20-week-old KO MRL mice (▲, n = 15) and littermate control mice (◆, CTRL MRL, n = 8) were tested for binding to human C1q by ELISA. Data for each individual mouse are shown and mean increases in immune complex levels are displayed as solid lines. P > 0.05, KO MRL mice versus CTRL MRL mice. AU, arbitrary units.

Figure 4

Serum C3 and C4 levels in knockout MRL mice, control MRL mice, and nonautoimmune mice. (a) Serum C3 levels from 20-week-old C4bp^-/-MRL/lpr (KO MRL) mice (n = 6) and littermate control (CTRL MRL) mice (n = 5) were measured by ELISA, and means values for 1:4,000 dilution are shown. P > 0.05 at all dilutions tested. OD450, optical density at 450 nm. (b) Serum C4 levels from 20-week-old KO MRL mice (n = 13) and CTRL MRL mice (n = 9) were compared with levels from mice at different ages as well as 20-week-old KO C57BL/6 (B6) mice, CTRL B6 mice, and C4-deficient B6 mice (n = 3 for each). Serum levels were measured by serial dilutions using sandwich ELISA, and mean values for 1:200 dilution are shown. *P < 0.001.