A Pro-Inflammatory Role of C5L2 in C5a-Primed Neutrophils for ANCA-Induced Activation

Background: The complement system is crucial for the development of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). In particular, C5a and its receptor on neutrophils, CD88, play a central role. The functional role of the second receptor of C5a, C5L2, remains unclear. In the current study, we investigated the role of C5L2 in C5a-primed neutrophils for ANCA-induced activation. Methods: The effect of blocking C5L2 by anti-human C5L2 blocking antibody were tested on respiratory burst and degranulation of C5a-primed neutrophils activated with ANCA, as well as on membrane-bound proteinase 3 (mPR3) and concentration of myeloperoxidase (MPO) in supernatant of C5a-primed neutrophils. An antagonist for CD88 was also employed. Results: Blocking C5L2 resulted in a significantly decreased MPO concentration in the supernatant of C5a-primed neutrophils. mPR3 expression increased from 209.0 +/- 43.0 in untreated cells to 444.3 +/- 60.8 after C5a treatment (P<0.001), and decreased to 375.8 +/- 65.44, 342.2 +/- 54.3 and 313.7 +/- 43.6 by pre-incubating blocking C5L2 antibody at 2.5 mg/ml, 5 mg/ml or 10 mg/ml (compared with C5a-priming group, P<0.001, P<0.001, and P<0.001), respectively. In C5a-primed neutrophils, subsequently activating with MPO-ANCA-positive IgG, the MFI value was 425.8 +/- 160.6, which decreased to 292.8 +/- 141.2, 289.7 +/- 130.0 and 280.3 +/- 136.4 upon pre-incubation with mouse anti-human C5L2 blocking antibody at 2.5 mg/ml, 5 mg/ml or 10 mg/ml (compared with C5a-primed neutrophils, for MPO-ANCA-positive IgG-induced activation, P<0.05, P<0.05, and P<0.05), respectively. Blocking C5L2 also resulted in significantly decreased C5a-primed neutrophils for PR3-ANCA-positive IgG-induced activation. Moreover, the lactoferrin concentration in the supernant significantly decreased in pre-incubation with anti-human C5L2 blocking antibody, compared with C5a-primed neutrophils induced by PR3- or MPO-ANCA-positive IgG. Conclusions: C5L2 may be implicated in the pro-inflammatory role in C5a-primed neutrophils for ANCA-induced activation.