Role of complement receptor 1 (CR1; CD35) on epithelial cells: A model for understanding complement-mediated damage in the kidney

Abstract

The regulators of complement activation gene cluster encodes a group of proteins that have evolved to control the amplification of complement at the critical step of C3 activation. Complement receptor 1 (CR1) is the most versatile of these inhibitors with both receptor and regulatory functions. While expressed on most peripheral blood cells, the only epithelial site of expression in the kidney is by the podocyte. Its expression by this cell population has aroused considerable speculation as to its biologic function in view of many complement-mediated renal diseases. The goal of this investigation was to assess the role of CR1 on epithelial cells. To this end, we utilized a Chinese hamster ovary cell model system. Among our findings, CR1 reduced C3b deposition by ∼ 80% during classical pathway activation; however, it was an even more potent regulator (>95% reduction in C3b deposition) of the alternative pathway. This inhibition was primarily mediated by decay accelerating activity. The deposited C4b and C3b were progressively cleaved with a t½ of ∼ 30 min to C4d and C3d, respectively, by CR1-dependent cofactor activity. CR1 functioned intrinsically (i.e, worked only on the cell on which it was expressed). Moreover, CR1 efficiently and stably bound but didn't internalize C4b/C3b opsonized immune complexes. Our studies underscore the potential importance of CR1 on an epithelial cell population as both an intrinsic complement regulator and an immune adherence receptor. These results provide a framework for understanding how loss of CR1 expression on podocytes may contribute to complement-mediated damage in the kidney.

Keywords: Cofactor activity; Complement activation; Complement receptor 1; Decay accelerating activity; Epithelial cells; Immune complexes.

Fig. 1

Structure of CR1. Diagrammatic representation of the most common size allelic form of CR1 containing 30 complement control repeats (CCPs). There are three C4b binding sites (CCPs 1–3; 8–10 and 15–17) and two C3b binding sites (CCPs 8–10 and 15–17). CCPs 22–28 bind C1q, ficolins and mannose binding lectin (MBL). TM, transmembrane domain; IC, intracytoplasmic domain; the four long homologous repeats (LHR) of this protein are demarcated: 1–7, 8–14, 15–21, 22–28. The functional sites in 8–10 and 15–17 are nearly identical. Repeats in yellow are required for C4b binding and decay accelerating activity while those in purple are required for C3b and C4b binding and cofactor activity. (Modified from: Park et al., 2014).

Fig. 2

CR1 expression by transfected CHO cells. (A) Flow cytometry to compare expression of CR1 on transfected CHO cells. RCHO served as a negative control. Rabbit polyclonal anti-CR1 Ab was used as the primary Ab and FITC-labeled donkey anti-rabbit IgG as the secondary Ab. Red line, RCHO; green line, CR1–10 k; blue line, CR1–200k; orange line, CR1–2 m. Representative of four independent experiments (also see Table 1). (B) Western blot of CR1 in lysates of transfected CHO cells. The solubilized cell preparations were analyzed on 6% gel under non-reducing conditions. Following transfer, the blot was developed with the polyclonal anti-CR1 Ab (same Ab as in A). These cell lysates were also analyzed under reducing conditions and the relationship between full length CR1 and Pro-CR1 was the same and additional bands were not detected. Lane 1, RCHO; lanes 2, 3 and 4, sCR1 (15 ng, 20 ng, 25 ng); lanes 5–7, CR1–200 k (1x, 2x, 5x); lanes 8 and 9, CR1–2 m (1x, 2x). 1x equals 20,000 cell equivalents for CR1–200 k and 10,000 cell equivalents for CR1–2 m. Representative experiment of four. (C) Western blot showing a longer exposure of lanes 5–7.

Fig. 3

Kinetic analysis of C4b cleavage by CR1 following classical pathway activation. (A) Sensitized CR1–200 k cells were exposed to C7-deficient human serum for 5–90 min and surface C4b (via its C4c epitope) and C4d fragments were detected by flow cytometry. Monoclonal Ab to C4c detects uncleaved C4b [C4b contains the C4c fragment (see Fig. 1 supplement)] while monoclonal anti-C4d Ab detects C4b and the C4d fragment (5 min, anti-C4d MFI = 726). FITC-labeled goat anti-mouse served as a secondary Ab. The solid light line represents unsensitized cells exposed to secondary Ab only. Representative of three independent experiments. (B) Sensitized RCHO cells were exposed to C7-deficient human serum for 5–90 min and C4 fragments were detected by flow cytometry (5 min, anti-C4d MFI = 726). Representative of three independent experiments. (C) Time course of C4d generation. The kinetics of C4b cleavage, based on the decrease in the anti-C4c signal, closely fit an exponential decay curve with T ½ of ~ 30 min. SEMs were too small to be depicted on the plot. Data points are averages of values obtained from three independent experiments as shown in the representative panel A.

Fig. 4

Kinetic analysis of C3b cleavage by CR1 following classical pathway activation. (A) Sensitized CR1–200k cells were exposed to C7-deficient human serum for 5–90 min and surface C3b and its fragments detected by flow cytometry. Monoclonal anti-C3c detects uncleaved C3b and iC3b [C3c is contained in C3b and iC3b (see Fig. 2 supplement)] while monoclonal anti-C3d Ab detects C3b, iC3b and C3d (5 min, anti-C3d MFI = 726). FITC-labeled goat anti-mouse served as the secondary Ab. The solid light line represents unsensitized cells exposed to secondary Ab only. Representative of three independent experiments. (B) Sensitized RCHO cells were exposed to C7- deficient human serum for 5–90 min and surface C3 fragments were detected by flow cytometry (5 min, anti-C3d MFI = 2224). Representative of three independent experiments. (C) Time course of C3d generation. The steady anti-C3d signal indicates that the number of C3 fragments bound to the cell surface remained constant over the course of the experiment. The kinetics of iC3b cleavage based on the reduction in the anti-C3c signal, closely fit an exponential decay curve with T ½ ~ 24 min. SEMs ranged between 0.004 and 0.007 and therefore were too small to be depicted on the plot. Data points are averages of three independent experiments (representative experiment in panel A).

Fig. 5

CR1 efficiently inhibits alternative pathway activation. Flow cytometry analysis depicting C3b deposition on sensitized cells exposed to C7-deficient human serum (10%) diluted in AP buffer for 5 min. Murine monoclonal Abs to human C3c and C3d were used as the primary Abs and FITC-labeled goat anti-mouse was the secondary Ab. (A) On RCHO, C3b deposition was substantial but there was no degradation. (B) The C3b deposition on CR1–200 k cells was decreased compared to RCHO. C3b on CR1–200 k is cleaved to C3c (released) and C3dg (remains covalently bound to surface). The solid light line represents unsensitized cells exposed to secondary Ab. Representative of three independent experiments. Similar results were obtained when RCHO and CR1–200 k cells were exposed to C7- deficient human serum for 5–90 min.

Fig. 6

CR1 protects cells by an intrinsic mechanism. Flow cytometry analysis demonstrating C3b deposition and degradation after complement activation on CR1–200 k and RCHO cells in a 1:1 ratio. The sensitized cell mixtures were incubated with 10% C7-deficient human serum in GVB or in Mg2+−EGTA buffer for 60 min. Monoclonal Abs to C3c and C3d were used as primary Abs; FITClabeled goat anti-mouse was the secondary Ab. [A] (i) Monoclonal Ab to C3d detected two peaks in the cell mix, the first represents CR1–200 k and the second represents RCHO. [A] (ii) and (iii) For comparison, individual populations of RCHO and CR1–200 k are also shown in the middle and right hand panels, respectively. [B] (i) Monoclonal Ab to C3c also detected two peaks in the 1:1 cell mix, the first represents CR1–200 k and the second represents RCHO. [B] (ii) and (iii) For comparison, RCHO and CR1–200 k alone are shown in the middle and right hand panels, respectively. (C) CR1–200 k and RCHO mixed in 4:1 ratio. Monoclonal Ab to C3c used as primary Ab; FITC-labeled goat anti-mouse was the secondary Ab. Light red line represents unsensitized cells exposed to secondary Ab only. Results shown are representative of three independent experiments.

Fig. 7

CR1 binds opsonized immune complexes. CR1–200 k cells were incubated with C3b/C4b opsonized or non-opsonized control IC (rabbit peroxidase anti-peroxidase) for 30 min at 37 °C. FITC-labeled anti-rabbit IgG was used to detect IC. To assess specificity of this interaction, cells were pretreated with mAb 3D9, which blocks CR1 interactions with C3b and C4b. Solid line (light), cells plus non-opsonized IC; solid line (dark), cells plus C3b/C4b opsonized IC; dotted line, cells preincubated with 3D9 plus C3b/C4b opsonized IC. Representative of four independent experiments.

Fig. 8

Comparison of immune complex binding on the surface of red blood cells to CR1-expressing CHO cells. (A) Red blood cells and CR1-expressing CHO cells were incubated with serum opsonized IC (¹²⁵I-labeled BSA/anti-BSA) for 0–30 min. The percent of IC binding to the cells was calculated by counting radioactivity in the cell pellets and supernatants (see Section 2). Grey line, RBC; black line, CR1–2 m (B) CR1–200 k and CR1–2 m cell lines were harvested in amounts that would give equivalent total CR1 numbers. 1 × 10⁷ cells of CR1–200 k (2 × 10⁵ CR1/cell) and 1 × 10⁶ cells of CR1–2m (2 × 10⁶ CR1/cell) were incubated with serum-opsonized IC for 30 min and percent binding calculated.