C5a receptor (CD88) blockade protects against MPO-ANCA GN

Abstract

Necrotizing and crescentic GN (NCGN) with a paucity of glomerular immunoglobulin deposits is associated with ANCA. The most common ANCA target antigens are myeloperoxidase (MPO) and proteinase 3. In a manner that requires activation of the alternative complement pathway, passive transfer of antibodies to mouse MPO (anti-MPO) induces a mouse model of ANCA NCGN that closely mimics human disease. Here, we confirm the importance of C5aR/CD88 in the mediation of anti-MPO-induced NCGN and report that C6 is not required. We further demonstrate that deficiency of C5a-like receptor (C5L2) has the reverse effect of C5aR/CD88 deficiency and results in more severe disease, indicating that C5aR/CD88 engagement enhances inflammation and C5L2 engagement suppresses inflammation. Oral administration of CCX168, a small molecule antagonist of human C5aR/CD88, ameliorated anti-MPO-induced NCGN in mice expressing human C5aR/CD88. These observations suggest that blockade of C5aR/CD88 might have therapeutic benefit in patients with ANCA-associated vasculitis and GN.

Figure 1.

C5aR (CD88) deficiency ameliorates, C5L2 deficiency enhances and C6 deficiency has no effect on anti-MPO–induced GN. WT B6, C5L2^−/− B6, C5aR/CD88^−/− B6, WT C3H/HeJ, and C6^−/− C3H/HeJ were injected intravenously with 50 µg/g body weight mouse antimouse MPO IgG and euthanized on day 6. Glomerular crescents were calculated as percentage of glomeruli with crescents by counting all glomeruli in cross-sections of both kidneys, which averaged approximately 80 per cross-section. (A) In contrast to histologically unremarkable glomeruli (upper left, C5aR^−/− mouse), glomeruli with crescents had increased extracapillary cells often with adjacent segmental fibrinoid necrosis (upper right, C5L2^−/− mouse; lower left, C6^−/− mouse; lower right, hC5aR mouse). (B) All WT B6 mice developed glomerular crescents with an average of 8.1% of glomeruli with crescents, B6 mice with knocked-out C5aR/CD88 were protected from induction of GN by anti-MPO IgG, with only 1 of 6 mice developing rare crescents, and B6 mice with knockout of C5L2 had more severe disease, with an average of 18% crescents per mouse (P=0.0035). (C) All WT C3H/HeJ mice and all five C6 knock out C3H/HeJ mice developed NCGN, with an average of 5.2% of glomeruli with crescents in WT mice and 4.4% in C6^−/− mice (P=0.63). Thus, the absence of C6 did not influence pathogenicity of anti-MPO. Magnification, ×400, periodic acid-Schiff stain.

Figure 2.

C5aR/CD88 small molecule antagonist CCX168 ameliorates anti-MPO–induced GN. Daily administration of 30 mg/kg CCX168, an orally active small molecule antagonist of human C5aR, markedly reduced the severity of anti-MPO–induced NCGN in hC5aR knock-in mice (7 days after anti-MPO injection). (A) Glomerular crescent formation induced by anti-MPO was reduced from 30.4% with vehicle alone to 3.3% with CCX168 (P<0.0001). (B) Urine hematuria, proteinuria, and leukocyturia were reduced in mice receiving CCX168 (dipstick scale, 0–4+). (C) Glomerular crescent and necrosis formation was reduced in animals receiving CCX168. Data were analyzed by Mann-Whitney t test; **P<0.01 and ***P<0.001. Data are presented as the median with SEM. BID, twice daily; QD, daily.

Figure 3.

Leukocytes in mice with human C5aR knock-in and mouse C5aR knock-out express human C5aR and respond to human C5a, and CCX168 inhibits activation via human C5aR. (A) Mouse and human C5aR expression in isolated leukocytes from hC5aR knock-in mice. Flow cytometric leukocyte staining with antibodies specific for mouse or human C5aR is shown in blue with isotype controls (green line) shown for comparison. (B) Chemotaxis of hC5aR knock-in cells in response to a dose range of human C5a in the absence (square) or presence (circle) of CCX168 (100 nM) showing inhibition of chemotaxis by CCX168. Migration signal is a measure of cell numbers migrating between ChemoTX chambers based on intensity of fluorescence of a DNA-binding fluorescent marker. (C) Effects of oral pretreatment with vehicle or a single dose of CCX168 on cell count in the peritoneal lavage 24 hours after intraperitoneal thioglycollate injection. (D) Schematic of the C5a-induced leukopenia study in hC5aR knock-in mice. One hour after oral administration of CCX168, blood was drawn 1 minute before and 1 minute after intravenous (IV) administration of C5a (20 μg/kg); leukocyte concentrations were determined in these blood samples. (E) Following the study outline shown in panel D (n=4 mice/group), CCX168 inhibited the transient depletion of blood leukocytes caused by intravenous administration of C5a. Percentage change from baseline is shown (0, no change; 100, no leukocytes in blood). CCX168 was administered as an oral 30 mg/kg dose in these studies. **P<0.01 t test.