C5a receptor mediates neutrophil activation and ANCA-induced glomerulonephritis

Abstract

Anti-neutrophil cytoplasmic autoantibody (ANCA)-induced necrotizing crescentic glomerulonephritis (NCGN) requires complement participation in its pathogenesis. We tested the hypothesis that the anaphylatoxin C5a is pivotal to disease induction via the neutrophil C5a receptor (C5aR). Supernatants from ANCA-activated neutrophils activated the complement cascade in normal serum, producing C5a. This conditioned serum primed neutrophils for ANCA-induced respiratory burst; neutrophil C5aR blockade abrogated this priming, but C3aR blockade did not. Furthermore, recombinant C5a but not C3a dosage-dependently primed neutrophils for ANCA-induced respiratory burst. To test the role of C5aR in a model of NCGN, we immunized myeloperoxidase-deficient mice with myeloperoxidase, irradiated them, and transplanted bone marrow from wild-type mice or C5aR-deficient mice into them. All mice that received wild-type marrow (six of six) but only one of eight mice that received C5aR-deficient marrow developed NCGN (P < 0.05). Albuminuria and neutrophil influx into glomeruli were also significantly attenuated in the mice that received C5aR-deficient marrow (P < 0.05). In summary, C5a and the neutrophil C5aR may compose an amplification loop for ANCA-mediated neutrophil activation. The C5aR may provide a new therapeutic target for ANCA-induced necrotizing crescentic glomerulonephritis.

Figure 1.

Normal TNF-α–primed neutrophils were stimulated for 45 min with buffer control (buffer), 125 μg/ml normal healthy IgG (Ctrl IgG), or 125 μg/ml ANCA IgG (five independent experiments using MPO-ANCA in two experiments and PR3-ANCA in three experiments). Supernatants from the cell suspensions were incubated for 15 min with normal serum, and C5a generation was measured by a C5a-specific ELISA. Data are means ± SEM (given as ratio to buffer-incubated normal serum [−], which was set at baseline to 100%). **P < 0.01.

Figure 2.

Normal TNF-α–primed neutrophils were stimulated for 45 min with buffer control (buffer), 125 μg/ml normal IgG (Ctrl IgG), or 125 μg/ml ANCA IgG (four independent experiments using MPO-ANCA in two experiments and PR3-ANCA in two experiments). Supernatants of these were incubated for 15 min with normal serum. This conditioned serum was used in different neutrophils for priming, and these cells were analyzed for PR3 membrane expression by FACS. Data are means ± SEM (given as ratio to buffer-incubated normal serum [−], which was set at baseline to 100%). **P < 0.01.

Figure 3.

Normal TNF-α–primed neutrophils were stimulated as described already. Supernatants from these cells were incubated for 15 min with normal serum. This conditioned serum was used in different neutrophils for priming (45 min at 37°C), after which cells were activated with a mAb to PR3. ROS production was measured after 45 min of stimulation with αPR3, and data are means ± SEM (given as ratio to buffer-incubated normal serum [−], which was set at baseline to 100%). **P < 0.01.

Figure 4.

(A and B) Normal neutrophils were tested for C3aR and C5aR expression by FACS. A representative example is shown. The gray line depicts the matching isotype control. (C) TNF-α–primed neutrophils were stimulated with a mAb to PR3 for 45 min. Supernatants of these were incubated for 15 min with normal serum (NS), C3-depleted serum, or C5-depleted serum. These different conditioned sera were used in different neutrophils for priming (45 min at 37°C), after which cells were activated with a mAb to PR3. ROS production was measured after 45 min of stimulation with αPR3, and data are means ± SEM (given as MFI; five independent experiments performed in duplicate). (D) Neutrophils were preincubated with a blocking antibody to C5aR or a C3aR blocker, afterward primed with conditioned serum for 45 min and subsequently activated with a mAb to PR3. ROS production was measured after 45 min of stimulation with αPR3, and data are means ± SEM (given as MFI; data from three independent experiments performed in duplicate). **P < 0.01.

Figure 5.

Normal TNF-α–primed neutrophils were stimulated as described already. Supernatants from these cells were incubated with normal serum. Before co-incubation, PMSF or SOD was added to the supernatants, or supernatants were heat inactivated. This conditioned serum was used in different neutrophils for priming, before analysis of cells for membrane-PR3 expression by FACS. Data are means ± SEM (given as ratio to buffer-incubated normal serum [−], which was set at baseline to 100%). **P < 0.05.

Figure 6.

(A) Normal neutrophils were primed with buffer (cells), 2 ng/ml TNF-α, or C5a in different concentrations for 25 min at 37°C. Afterward, neutrophils were stimulated with an activating mAb to MPO (αMPO Ab) or a matching isotype control (isotype mAb). ROS production was measured by the ferricytochrome assay, and data are the 45-min time point (data from four independent experiments performed in duplicate). (B) Neutrophils were primed as described already and subsequently activated with 125 μg/ml normal IgG (Ctrl IgG), PR3-ANCA IgG (PR3 IgG), or MPO-ANCA IgG (MPO IgG); activation was measured by the ferricytochrome assay, and data are the 45-min time point. □, Unprimed cells; ▪, C5a-primed cells; , TNF-α–primed cells (data from three independent experiments each using two different PR3-ANCA IgG, two different MPO-ANCA IgG, and two different normal IgG, done in duplicate).

Figure 7.

Mice were killed 8 wk after BM transplantation, and renal tissue was examined. (A) The extent of glomerular crescents and necrosis was expressed as the mean percentage of glomeruli with crescents and necrosis. (B) Glomerulus from a C5aR^−/− mouse with no lesion.(C through E) WT mice developed glomerular crescents and necrosis. (F and G) Examination for glomerular neutrophil infiltration showed that C5aR^−/− mice had fewer glomerular neutrophils (F) than did WT mice (G). **P < 0.01.