Discussion
All three complement activation pathways converge at the generation of C3 convertase that cleaves C3 into anaphylatoxin C3a and opsonin C3b [18]. The association of C3b with the C3 convertase results in the formation of a C5 convertase cleaving C5 into anaphylatoxins C5a and C5b. This cleavage triggers the terminal complement cascade leading to the assembly of MAC (C5b-9). In our and previous studies, the terminal pathway components levels, that is C5a and MAC, were elevated in patients with active AAV compared to healthy controls. This finding indicates that complement activation occurs in the course of systemic vasculitis. In our patients, effective remission induction treatment was associated with a decrease in C5a levels, whereas MAC levels did not change. Nevertheless, the meta-analysis showed that both C5a and MAC levels in patients with remission of AAV were lower compared to patients with active disease. Remission induction therapy also resulted in a decrease in C3a levels. Activation of complement in AAV occurred irrespective of ANCA-serotype, severity of disease or previous immunosuppression, since we detected no difference between C5a and MAC levels in the groups of patients with MPO-ANCA or PR3-ANCA vasculitis, active MPA or GPA, severe or non-severe vasculitis, newly diagnosed or relapsing disease, predominant granulomatous or vasculitic or mixed disease, immunosuppressive-naïve or previously immunosuppressed patients. C5a and MAC levels did not correlate with BVAS, proteinuria, serum creatinine or laboratory markers of inflammation and were increased only in a proportion of patients with active AAV. Of note, C5a concentrations exceeded the upper reference level only in a quarter of patients who showed clinical signs of active AAV. Therefore, analysis of both complement components alone does not allow for distinction between active disease and remission.
We also measured factor B and properdin levels that are components of the alternative complement pathways. Factor B can be cleaved by factor D into Ba and Bb and is necessary for the formation of the alternative pathway C3 convertase (C3bBb), whereas properdin increases the half-life of the convertase activity and promotes constant cleavage of C3 into C3a and C3b. Median factor B levels were elevated in patients with active AAV compared to healthy controls and decreased after remission. However, we detected no difference between median properdin levels in AAV patents and healthy volunteers. These data were confirmed by our meta-analysis.
Deposition of Bb in glomeruli of patients with AAV correlated with the proportion of crescents, the extent of interstitial infiltrates, interstitial fibrosis and tubular atrophy, and inversely with the proportion of normal glomeruli [19]. Moreover, plasma Bb concentrations correlated with common pathway components levels (C3a, C5a, and MAC), clinical and laboratory signs of vasculitis activity (BVAS, erythrocyte sedimentation rate (ESR)), and the proportion of total and cellular crescents in kidney biopsies [8]. These findings indicate that circulating Bb levels may reflect both systemic and renal disease activity of AAV. In another study that analyzed 187 renal biopsy samples from patients with AAV, properdin staining was associated with the proportion of cellular crescents, and the presence of properdin correlated with the level of proteinuria [5]. In our study, factor B or properdin levels did not correlate with proteinuria, serum creatinine, BVAS or laboratory markers of inflammation (ESR, C-reactive protein). In a small group of patients with kidney biopsies, factor B levels correlated with C3 deposition in the glomeruli, whereas all other correlations between complement components and histological parameters were not significant.
A crucial role of complement activation in AAV pathogenesis makes targeting complement components an attractive therapeutic strategy. Currently, two C5a inhibitors are in clinical development for AAV: avacopan, an oral C5a receptor (C5aR) inhibitor, and IFX-1, a monoclonal antibody to C5a [20]. Efficacy, safety and steroid-sparing effects of avacopan in patients with GPA/MPA were shown in two phase II trials, CLEAR and CLASSIC [20, 21], whereas IFX-1 has entered phase II development. The positive results of the phase III trial of avacopan, ADVOCATE, in AAV patients were recently announced, though not published yet. C5a also interacts with C5L2 receptors that compete with C5aR for binding of anaphylotoxins and, therefore, may have anti-inflammatory effects. Knockout of C5L2 in mice resulted in a more severe MPO-ANCA associated glomerulonephritis [4]. On the contrary, C5L2 was upregulated in glomeruli in patients with ANCA-associated glomerulonephritis [7]. These contradictory experimental and clinical data suggest that C5L2 may have different roles and functions in various systems, cells, tissues, organs, and/or species [22].
A major limitation of our study is the relatively small sample of patients, particularly studied sequentially both prior to and after achievement of remission. However, our findings are in accordance with previous studies and were reinforced by the meta-analysis of all available data on activation of the complement system in AAV.
In summary, our findings and the results of the meta-analysis provided additional evidence for activation of the complement system via the alternative pathway in AAV. Signs of complement activation were found only in a proportion of patients with active AAV, whereas achievement of clinical remission following immunosuppressive therapy did not always lead to normalization of various complement component levels. Therefore, we cannot conclude that any plasma complement component alone may be useful as a biomarker of the disease activity or a guide for treatment decisions. However, studies of the complement system in AAV and other autoimmune diseases pave the way to new drugs development and can turn the dream of steroid-free regimens into a reality. In addition, investigations of key signaling pathways and molecules (i.e., macrophage migration inhibitory factor, sphingosine-1-phosphate, high mobility group box 1) of C5a-mediated neutrophil priming and activation by ANCA may provide new insights into the complex AAV pathogenesis.