Discussion
Our study showed that anti-properdin IgG were present in 22.5 % of patients with LN and correlated with some clinical parameters. These antibodies were specific and enhanced the deposit of C3 activation fragments on apoptotic cells.
Properdin stabilizes the alternative pathway C3 and C5 convertases and hence it is tempting to speculate that autoantibodies binding to it may enhance the stabilization capacity, increasing the half-life of these otherwise labile enzymes. Such enhanced stabilization could result in complement overactivaition and pathological consequences similar to the C3Nef, found in C3 glomerulopathies [27]. LN is a hallmark of a disease with complement overactivaiton in the kidney and indeed we discovered that more than 20% of the patients in our cohort were positive for anti-properdin IgG. Anti-properdin IgG titres showed a trend to negative correlation with eGFR, suggesting a possible association with renal damage. Moreover, higher levels of anti-properdin IgG were related to high levels of anti-dsDNA and ANA and to low concentrations of C3 and C4. The correlation with C3 complement consumption could be either related to the overall autoimmunity status, where the anti-properdin IgG are just an epiphenomenon or could indicate functional relevance.
To understand whether anti-properdin IgG affect the functions of properdin as complement regulator, their functional consequences were characterized. The anti-properdin IgG formed stable complexes with its target. Except to stabilize the C3bBb, it is reported that properdin binds to C3b, promoting it subsequent association with Factor B [28]. We found that in some patients anti-properdin positive IgG weakly increase the binding of properdin to C3b and to pro-convertase (C3bB) and did not affect the alternative complement pathway C3 convertase unlike C3NeF which react with C3 convertase and stabilized it [19]. In a case report anti-properdin positive IgG activated complement in serum [18]. We did not detect fluid phase complement activation by anti-properdin IgG, contrary to autoantibodies against other alternative pathway components, such as anti-Factor B, anti-C3b or anti-FH [29-32]. A possible explanation for the weak or absent effect of anti-properdin IgG could be the usage of low pH elution buffer for IgG purification, which may have a dramatic effect on the biological activity of IgG and their antigen-binding behavior [33]. Nevertheless, purified IgG showed strong and dose-dependent interaction with properdin by SPR, suggesting preserved binding capacity. Another possibility is that the epitopes of anti-properdin IgG are outside of the C3-convertase binding region of properdin. Indeed, predicted epitopes showed higher density of antigenic determinants outside the C3bBb-binding area, suggesting that the effect of these antibodies may be indirect, and likely affecting other functions.
It has been reported that properdin binds specifically to late apoptotic cell, but not to early ones and this occurs independently of C3b [8]. We performed an analysis with late apoptotic cells in order to understand weather anti-properdin affect the C3b and properdin deposition. Anti-properdin IgG did not contribute to properdin deposition on late apoptotic cells in all studied patients. Nevertheless, we found that anti-properdin increased the C3b deposition in 2/4 tested patients to similar levels as anti-C3b IgG from LN patients, which have overt functional consequences [30]. These two patients were the same in whom anti-properdin slightly increased binding of properdin to C3b and pro-convertase, but not to the convertase. The C3 levels in both patients were in the reference range. This suggests that in those patients there was not excessive consumption of C3, following by increased C3b deposition. Taken together, these results suggest that anti-properdin IgG could contribute to the complement overactivaiton in a subgroup of patients, but that this is not a general phenomenon and the functional consequences of these autoantibodies are rather weak.
Further we explored whether the anti-properdin positivity could serve as a biomarker in combination with other characteristics of LN patients to predict flares and severity. Anti-C1q are the more often parameters correlated with the renal flares in LN. It is known that they are associated with LN activity and severity with renal histological lesion [34-40]. Anti-C1q are positively associated with BLAG renal score [41] as well as with SLEDAI score [42]. The combination of anti-C1q and anti-dsDNA was reported as a stronger marker for renal involvement and increased specificity for the identification of LN activity. Julkunen et al., 2012 found that anti-C1q and complement C3 and C4 are better markers for lupus nephritis activity than anti-dsDNA, and that anti-dsDNA and complement C3 and C4 were better than anti-C1q to evaluate the overall and nonrenal activity of SLE [43]. In our study anti-C1q alone and in combination with anti-dsDNA and in combination with anti-dsDNA and serum levels of C3 and C4 could significantly increase the specificity but decreased the sensitivity for identification of patients in category A according to BILAG Renal score. These findings confirmed established trends in the study of Chi et al., 2015 who evaluated the role of anti-C1q alone and in combination with other serological markers to identified patients with active LN [35]. Anti-properdin alone could not be determinant for high category of LN according to the BILAG Renal score. But in combination with anti-dsDNA anti-properdin could significantly increase sensitivity and NPV in the identification of patients in A BILAG category. The high NPV of anti-properdin and anti-dsDNA combination suggested that patients will not have severe nephritis in the absence of anti-properdin and anti-dsDNA. Although anti-properdin did not associate with more active and severe LN, they were significantly associated with renal flares. We found that pathological high levels of anti-properdin were associated with some renal histologic lesions, such as “Wire loop” deposits, fibrous and cellular crescents.
In conclusion, we found the presence of anti-properdin autoantibodies in the patients’ sera with LN. Their presence correlate with clinical parameters and affect properdin function in a subgroup of patients. Although likely not a driver of the disease, these autoantibodies may be a contributing factor with pathological relevance for LN.