Introduction
Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are primary systemic vasculitides affecting small and medium-sized vessels, and are associated with ANCA against proteinase 3 (PR3) and myeloperoxidase. AAV include granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), Churg-Strauss syndrome, and isolated pauci-immune necrotizing crescentic glomerulonephritis also designated as renal limited vasculitis (RLV) [
1,
2]. Disease relapses are common in AAV and occur in up to 60% of patients, especially in patients with GPA and PR3 ANCA [
3‐
7]. Risk factors for relapses in AAV include the persistence of PR3 ANCA after induction of remission, upper and lower airway involvement, cardiovascular involvement, and chronic nasal carriage of
Staphylococcus aureus, particularly strains that express the toxic shock syndrome toxin-1 superantigen gene [
3,
5,
6,
8]. A recent meta-analysis showed that the rise in ANCA titers or their persistence during remission is only modestly associated with an increased risk of relapses in AAV patients [
9]. There is thus an unmet need for biomarkers predicting which AAV patient is prone to relapse.
High-mobility group box-1 (HMGB1) is a nuclear protein that binds DNA and modulates chromosomal architecture. Once released into the extracellular space, after cell death or upon activation, HMGB1 acts as a danger-associated molecular pattern or as an alarmin and stimulates inflammatory and immunological activities that include cytokine production, chemotaxis, cell proliferation, angiogenesis and cell differentiation. HMGB1 has to bind to the receptor for advanced glycation end-products (RAGE) and toll-like receptor (TLR)-2, TLR-4 and TLR-9 in order to exert its actions [
10,
11].
In systemic lupus erythematosus (SLE), serum HMGB1 has been shown to be a biomarker of disease activity, especially in patients with lupus nephritis. Moreover, patients with active lupus nephritis present higher HMGB1 levels in urine compared with SLE patients without active nephritis and with controls [
12‐
14]. Furthermore, levels of antibodies to HMGB1 are higher in patients with active SLE than in patients with quiescent disease and in controls [
13]. In AAV, a cross-sectional study showed increased serum levels of HMGB1 in patients with active GPA [
15]. In addition, one study found an association with granulomatous manifestations and another with biopsy-proven renal involvement [
16,
17].
Until now, HMGB1 levels have not been evaluated longitudinally as a biomarker of disease activity or as a predictor of ensuing relapses in patients with AAV. The aims of this study were to evaluate whether serial levels of HMGB1 reflect changes in disease activity and/or predict the occurrence of relapses, and to analyze whether AAV patients have antibodies to HMGB1.
Discussion
In this study, we evaluated serum HMGB1 levels as a biomarker of disease activity in AAV and investigated anti-HMGB1 antibodies in AAV patients with active disease. We observed that even though a significant correlation was found between HMGB1 and CRP levels at presentation, only AAV patients without active renal disease had significantly higher serum HMGB1 levels than HC. Serum HMGB1 levels decreased significantly in a median of 3 months after presentation but then returned to levels similar to those found at baseline and no significant fluctuation was seen over time in AAV patients, not prior to or during disease relapses. Only a minority of AAV patients with active disease develop anti-HMGB1 antibodies.
Anti-HMGB1 antibodies have been described in sera of patients with septic shock, polymyositis, dermatomyositis and in active SLE [
13,
20]. In critically ill patients with septic shock, anti-HMGB1 antibodies were associated with a better prognosis and increased survival [
21]. In patients with SLE, anti-HMGB1 antibodies were positively correlated with disease activity and anti-double-stranded DNA titers and were negatively correlated with serum complement levels [
13,
20]. In this study, only a small minority of AAV patients were positive for anti-HMGB1 antibodies. Moreover, no significant association between anti-HMGB1 antibodies and BVAS score could be observed. A previous study also failed to demonstrate anti-HMGB1 antibodies in 22 patients with AAV and active renal involvement [
22].
The technique used to measure serum HMGB1 levels is a relevant issue because serum HMGB1 levels are usually five times higher when a western blot technique is used in comparison with ELISA, although both assays correlate well [
23]. The binding of HMGB1 to different serum/plasma molecules, especially IgG
1, interferes with its detection by ELISA systems, which is considered the main reason for this discrepancy [
24]. Moreover, the depletion of IgG from the sera of SLE patients lowered serum HMGB1 levels detected by ELISA, indicating that this interference might in part be due to anti-HMGB1 antibodies in SLE [
13]. We therefore firstly tested anti-HMGB1 antibodies in AAV patients before choosing the technique to measure serum HMGB1. Since only a few AAV patients presented anti-HMGB1 antibodies, the ELISA technique was used to measure serum HMGB1 in the present study. Nevertheless, current ELISA methods seem to have limitations in assessing HMGB1 levels as a surrogate marker of active disease in different scenarios due to potential interference in its detection by serum factors, including anti-HMGB1 antibodies. Perhaps only free HMGB1 could be detected by current ELISA systems instead of total HMGB1 [
24,
25]. Furthermore, it is now known that functionality of HMGB1 is affected by the redox state of its three cysteine residues (C23, C45 and C106) and future methods to detect HMGB1 should take this functional nuance into account. The all-thiol form of HMGB1 has only chemotactic activity while disulfide-bonded HMGB1 (between C23 and C45) induces cytokine release through binding of TLR-4. No cytokine-stimulating or chemotactic activity is found in the fully oxidized HMGB1 [
26,
27].
We observed that AAV patients with renal involvement presented similar HMGB1 levels in comparison with HC while AAV patients without active nephritis had significantly higher HMGB1 levels than HC. We therefore speculate that the reason for finding serum HMGB1 levels in AAV patients at presentation similar to HC, in contrast to other studies evaluating HMGB1 in AAV [
15,
17], could be the high number of patients with active renal involvement evaluated in the present study (75.0%). Although Bruchfeld and colleagues have previously described higher serum HMGB1 levels in AAV patients with biopsy-proven active nephritis in comparison with patients without active renal inflammation, no systematic comparison was made with AAV patients presenting active disease in other organs or systems [
16]. In line with our results, Henes and colleagues described lower serum HMGB1 levels in GPA patients with predominantly vasculitic manifestations when compared with those with predominantly granulomatous manifestations. Active nephritis was the most frequent feature observed among GPA patients with vasculitic manifestations. Granulomatous inflammation may have contributed to higher HMGB1 levels in GPA patients with granulomatous manifestations [
17]. In our study, patients with GPA and localized disease presented higher levels of HMGB1 at baseline than patients with generalized disease. Also, AAV patients with pulmonary nodules and/or lung infiltrates had higher levels of HMGB1 than those with alveolar hemorrhage. Differences, however, were not significant in both situations. Nonetheless, the presence of granulomatous manifestations in AAV patients did not seem to influence HMGB1 levels in AAV patients with simultaneously active nephritis, indicating that HMGB1 levels were mostly influenced by renal involvement. The increased expression of HMGB1 in renal tissue [
16] and the necrotizing nature of glomerulonephritis in AAV indicate that both active release by activated cells and passive release by dying necrotic cells could be the source of extracellular HMGB1 in renal involvement of AAV besides systemic inflammation. However, whether leakage of HMGB1 into urine due to renal damage contributes to lower serum HMGB1 levels in parallel with increased urinary levels of HMGB1 in active glomerulonephritis in AAV is still unknown.
Serum HMGB1 levels have been correlated with disease activity in AAV in cross-sectional studies. Nevertheless, longitudinal evaluation of serum HMGB1 levels demonstrated only a significant decrease in HMGB1 levels approximately 3 months after presentation. Thereafter, with ongoing remission, serum HMGB1 returned to levels similar to those found at presentation. Serum HMGB1 levels prior to relapses and during relapses were somewhat higher than baseline levels but this difference was not significant. Achievement of disease remission in response to sustained immunosuppressive therapy may be the reason for lower HMGB1 levels within 3 months after disease presentation. Although approximately one-half of AAV patients at presentation were already under immunosuppressive therapy for a median 3 weeks and no difference in serum HMGB1 levels could be found between patients with and without therapy, both treated patients and untreated patients had a similar median BVAS at baseline evaluation. After achieving remission most AAV patients were still on oral prednisolone and cyclophosphamide, whereas during ongoing remission immunosuppressive therapy was tapered and cyclophosphamide was changed to azathioprine. Prior to relapses in the majority of AAV patients, immunosuppressive therapy was withdrawn (data not shown). This reduction in immunosuppressive therapy may therefore account for the significant increase in serum HMGB1 levels from early remission to ongoing remission.
In this study, no difference regarding serum HMGB1 levels could be found during follow-up between relapsing and nonrelapsing AAV patients and fluctuations of serum HMGB1 levels during the remission period or prior to a relapse were not associated with an increased risk of relapses in AAV. Hence, fluctuations of serum HMGB1 levels cannot be used as a surrogate marker for disease activity in AAV.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
AWSdS contributed to the study design, collected data from patients' medical records, performed laboratory tests, conducted the statistical analysis and drafted the manuscript. JW contributed to the study design, performed laboratory tests and revised the manuscript. JB contributed to the study design, performed laboratory tests, helped the interpretation of results and revised the manuscript. PCL contributed to the study design, helped the interpretation of data and revised the manuscript. CAS participated in the acquisition of data, interpreted results and revised the manuscript. MB conceived the study, contributed to the study design, interpretation of data and revised the manuscript. CGMK conceived the study, contributed to the study design and interpretation of data and revised the manuscript.