Increased circulating levels of proteinase 3 in patients with anti-neutrophilic cytoplasmic autoantibodies-associated systemic vasculitis in remission

Abstract

SUMMARYIn systemic small vessel vasculitides, patients form autoantibodies against neutrophil granular proteins, anti-neutrophilic cytoplasmic autoantibodies (ANCA). Some correlation is seen between ANCA titre and disease activity, but whether this is cause or effect is still unknown. It has been reported that levels of proteinase 3 (PR3), one of the main ANCA antigens, are increased in patients with active disease. An increased level of circulating antigen could mean a predisposition to autoimmunity. In order to explore this we measured PR3 levels in patients with stable disease. In addition we measured neutrophil gelatinase-associated lipocalin (NGAL) as a specific marker of neutrophil degranulation, cystatin C as a marker of renal function as well as C-reactive protein (CRP), IL-6 and sTNFr1 as markers of inflammation. PR3, NGAL, IL-6 and sTNFr1 were measured in plasma by the ELISA technique. In the PR3 ELISA, we used anti-PR3 monoclonal antibodies as capture-antibodies and affinity-purified rabbitanti-PR3 antibodies for detection. PR3-ANCA, myeloperoxidase (MPO)-ANCA, CRP and cystatin C were measured by routine methods. PR3 was significantly raised ( P < 0·0001) in vasculitis patients (median 560 m g/l, range 110-3940, n = 59) compared with healthy blood donors (350 m g/l, 110-580, n = 30) as well as disease controls (360, 110-580, n = 46). No correlation was seen with disease activity, inflammation or renal function. The raised NGAL levels correlated strongly with decreased renal function ( r = 0·8, P < 0·001). After correcting for this, slightly increased levels (110, 42-340, n = 59) were observed compared with healthy blood donors (81, 38-130, n = 25), but not compared with the disease controls (120, 57-260, n = 48). In the disease controls, there was a significant correlation between NGAL and proteinase 3 ( r = 0·3, p < 0·05), but this was not the case in the vasculitis patients. Whether patients had PR3-ANCA or MPO-ANCA was of no significance. In our measurements, we found significantly raised levels of PR3 in plasma from patients with small vessel vasculitis, regardless of ANCA specificity. This was not due to decreased renal function, ongoing inflammation or neutrophil activation. Plausible mechanisms for this include defects in the reticuloendothelial system, genetic factors and selective neutrophil degranulation or leakage.

History, Classification and Pathophysiology of Small Vessel Vasculitis

“…Experiments performed by our group, have shown that the plasma levels of mature PR3 as well as pro-PR3 are elevated in AASV [114,115,116]. It was also observed that mPR3 + neutrophils are more abundant in AASV compared to healthy donors, which agrees with previous studies suggesting that a high percentage of mPR3 + cells may be a risk factor for vasculitis [78,115].…”

Neutrophil Gelatinase-Associated Lipocalin and Acute Kidney Injury after Cardiac Surgery

“…However, these reports may not be applicable to an adult cardiac surgery population. NGAL is expressed by atherosclerotic vascular tissue (21,22), which may itself be up-regulated in the early postoperative period, and chronic renal disease is a marker for generalized atherosclerosis, potentially enhancing the contribution of extrarenal NGAL in such patients. Furthermore, although animal models indicate systemic NGAL filtered at the renal glomerulus is rapidly and efficiently reabsorbed by the proximal renal tubule (20), this may not be the case in the setting of chronic renal insufficiency.…”

The Contribution of Innate Immunity to the Pathogenesis of ANCA-associated Vasculitis

“…In addition to the histological findings of NETs in biopsies, elevated levels of NETs in the circulations have also been identified in patients (Table I) (Kessenbrock et al 2009, Surmiak et al 2015, Surmiak et al 2016). Similar results as for NETs have been obtained for NET-associated components such as HMGB1, calprotectin, PR3, MPO, and NE as well as mtDNA when measured separately in the circulation (Table 1) (Henshaw et al 1994, Haubitz et al 1997, Ohlsson et al 2003, Wibisono et al 2010, Bruchfeld et al 2011, Pepper et al 2013, Surmiak et al 2016). …”

“…Our finding suggests a dual role for ANCA of which some also are involved in clearance of NET remnants from the circulation, possibly through opsonization. We have previously also reported on an inverse relationship between ANCA and PR3/α1-antitrypsin complexes in the circulation (Ohlsson et al 2003).…”