Background Osteopontin is a pleiotropic cytokine involved in the recruitment and retention of neutrophils to sites of inflammation, which are the primary targets cells in antineutrophil cytoplasmic autoantibody-associated vasculitis (AAV). Osteopontin may play a role in the pathogenesis of AAV.
Methods 24 patients with systemic AAV and six patients with limited granulomatous disease were included. 19 patients were followed up at 6 and 12 months after the initiation of immunosuppressive therapy. 21 matched healthy volunteers and 20 body mass index and glomerular filtration rate-matched patients with IgA nephropathy were included as controls. Plasma levels of osteopontin were measured by ELISA. Disease activity was gauged by the Birmingham vasculitis activity score (BVAS) and C-reactive protein (CRP).
Results Osteopontin levels are elevated compared with controls (healthy p<0.001; IgA p<0.001).Osteopontin levels decrease significantly during follow-up (p=0.02). Osteopontin levels correlate with disease activity (BVAS r=0.93; CRP r=0.73; all p<0.001) as well as erythrocyturia (r=0.7, p<0.001) and proteinuria (r=0.54, p=0.007).
Conclusions Active AAV is characterised by increased plasma levels of osteopontin, which decrease dramatically with successful therapy. Osteopontin may mediate the inflammatory process in AAV through the recruitment of neutrophils.
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Osteopontin is a pleiotropic cytokine that is broadly expressed and upregulated during inflammation, cancer and various other conditions.1,–,3 Osteopontin has an arginine–glycine–aspartic acid cell binding sequence. Via this sequence, osteopontin interacts with a variety of cell surface receptors.4,–,6 It is involved in the recruitment and retention of macrophages and leucocytes to sites of inflammation.4 5 7
Gene profiling experiments found osteopontin messenger RNA to be highly increased in experimental crescentic glomerulonephritis.8 9 Similarly, a dramatically enhanced glomerular expression of osteopontin was found in human crescentic glomerulonephritis.10 Inducible expression of osteopontin in the tubular epithelium seems to be associated with interstitial monocyte infiltration and subsequent tubulointerstitial changes in human myeloperoxidase–antineutrophil cytoplasmic autoantibody (ANCA)-associated glomerulonephritis.11
Although osteopontin is involved in the recruitment and retention of neutrophils to sites of inflammation and despite the increased expression found in myeloperoxidase–ANCA-associated glomerulonephritis, circulating levels of osteopontin have not been studied in systemic ANCA-associated vasculitis (AAV).
We thus hypothesise that osteopontin is elevated in active systemic AAV and during follow-up, correlating with markers of disease activity including the Birmingham vasculitis activity score (BVAS) and C-reactive protein (CRP).
All patients were recruited from the Department of Nephrology at Hannover Medical School between 2007 and 2008. The study was carried out in accordance with the Declaration of Helsinki and approved by the institutional review board. Informed consent was obtained. The diagnosis of AAV was established in accordance with the Chapel Hill classification.12 Disease activity was assessed using the BVAS.13 The diagnostic criteria for granulomatous AAV/Wegener's granulomatosis (WG) were typical presentation with involvement of the upper respiratory tract (as described in the American College of Rheumatology criteria for WG) and/or granulomatous inflammation on histology. The diagnostic criteria for microscopic polyangiitis were necrotising pauci-immune vasculitis or glomerulonephritis of the small vessels without granuloma. Plasma from a total of 30 patients with active AAV was analysed at initial presentation, plasma of 19 patients was analysed during follow-up at 6 and 12 months after the initiation of immunosuppressive therapy. Twenty-four patients presented with systemic AAV, six patients with limited granulomatous disease (LGD).
Twenty-one age and body mass index (BMI)-matched healthy volunteers as well as 20 matched patients with regard to BMI and glomerular filtration rate (GFR) with IgA nephropathy served as controls. In all but two patients with systemic AAV, oral prednisolone (1 mg/kg body weight) was started on day 4 after 500 mg prednisolone was given on days 1–3 intravenously. By day 15, tapering of the steroid regimen was initiated, with a reduction of 10 mg/week. Cyclophosphamide pulses were administered intravenously for 3–6 months. The characteristics of patient groups and disease controls are shown in table 1.
Peripheral blood samples at initial presentation were obtained before or within 24 h of the initiation of treatment. All samples were centrifuged and plasma was stored at −80°C. Leucocyte counts, CRP and serum creatinine were measured by a standard technique. GFR was estimated using the modification of diet in renal disease (MDRD) formula. Proteinuria was quantified by 12-h urine collections. Erythrocyturia was quantified by AUTION MAX AX-4280 automated urine chemistry analyser (Iris Diagnostics, Chatsworth, California, USA).
ELISA for the detection of osteopontin plasma levels
Osteopontin plasma levels were measured using a commercially available ELISA (R&D Systems, Minneapolis, Minnesota, USA). The detection limit was 0.011 ng/ml and the intra and interassay coefficients of variance were 2.6% and 5.4%, respectively.
Differences between patients and controls were evaluated using the Mann–Whitney U test (two-sided). Correlations between osteopontin and parameters of disease activity were calculated using Spearman's test and linear regression analysis. Sensitivity, specificity and predictive values were calculated using receiver–operator characteristics curves. Statistical significance was accepted at 5% probability levels. Data are displayed as mean±SD. Data analysis was performed using SPSS and GraphPad Prism.
Plasma concentrations of osteopontin are increased in active AAV
Plasma osteopontin concentrations are elevated eightfold in patients with systemic active AAV (248.5±156.2 ng/ml) compared with healthy controls (32.3±7.7 ng/ml, p<0.001) and 2.2-fold compared with GFR-matched patients with IgA nephropathy (115.7±74.9 ng/ml, p<0.001) (figure 1A). Osteopontin levels in patients with LGD are elevated 1.8-fold compared with healthy controls (56.8±28.9 ng/ml, p=0.001). Osteopontin levels in systemic AAV are significantly higher compared with patients with LGD (248.5±156.2 ng/ml vs 56.8±28.9 ng/ml, p=0.006) (figure 1B).
Plasma concentrations of osteopontin decline during therapy
Osteopontin concentrations decline significantly during 12 months of immunosuppressive treatment (p=0.02) (figure 1C). After 12 months of therapy osteopontin levels had decreased substantially (65.9±67.2 ng/ml), but were still significantly higher than in healthy controls (p=0.02). The consistent decline in osteopontin levels was observed in all 17 patients who entered complete remission (BVAS 0 after 12 months).
Circulating osteopontin levels correlate with disease activity and erythrocyturia/proteinuria
Circulating osteopontin showed a strong positive correlation with BVAS and CRP levels that was confirmed by linear regression analysis (BVAS r=0.93; CRP r=0.73; n=30, all p<0.001) (figure 2A,B). In 24 patients with systemic AAV and biopsy-confirmed necrotising pauci-immune glomerulonephritis, we quantified proteinuria and erythrocyturia to address vascular inflammation within the renal vasculature. Osteopontin was associated with erythrocyturia (r=0.7, p<0.001) and proteinuria (r=0.54, p=0.007).
Application of different osteopontin cut-off concentrations identifies active AAV
We calculated receiver–operator characteristics curves to address the diagnostic value of osteopontin as a novel disease marker for active systemic AAV. When active AAV was compared with healthy controls, an osteopontin value greater than 39.96 ng/ml resulted in a specificity of 90.48% and a sensitivity of 93.3% in diagnosing active AAV (area under the curve (AUC) 0.93±0.04, 95% CI 0.84 to 1.016, p<0.001). When comparing active AAV with patients in remission an osteopontin value greater than 71.9 ng/ml resulted in a specificity of 100% and a sensitivity of 81.8% (AUC 0.9±0.07, 95% CI 0.75 to 1.07, p=0.001). Comparing active AAV with patients with IgA nephropathy, an osteopontin value greater than 103.4 ng/ml reveals the diagnosis of active AAV with a specificity of 60% and a sensitivity of 70% (AUC 0.68±0.07, 95% CI 0.53 to 0.83, p=0.034).
In the present study, we investigated circulating plasma levels of osteopontin in patients with active systemic AAV and LGD at baseline as well as during follow-up at 6 and 12 months. Age and BMI-matched healthy individuals and BMI and GFR-matched patients with IgA nephropathy served as controls. The results are as follows: (1) mean osteopontin concentrations are elevated eightfold in active AAV with renal involvement compared with healthy controls and 2.2-fold compared with patients with IgA nephropathy. (2) Osteopontin levels in systemic AAV are significantly higher compared with patients with LGD. (3) Osteopontin levels decline dramatically after the initiation of successful immunosuppressive therapy and (4) clinical markers of disease activity including BVAS and CRP are closely associated with individual osteopontin concentrations. (5) Erythrocyturia and proteinuria, indicating involvement of the renal vasculature, were closely linked to osteopontin concentrations. (6) Different osteopontin cut-off values detected active AAV with good specificity and sensitivity, distinguishing between AAV and patients in remission and disease controls (IgA nephropathy).
Our findings suggest that osteopontin might be linked to neutrophil chemotaxis in AAV. Subsequently, neutrophils are primed and activated, adhere to endothelial cells and release toxic oxygen species and proteases. The importance of osteopontin in neutrophil chemotaxis was exemplified by the impaired recruitment of osteopontin −/− neutrophils in experimental colitis, which was restored after the administration of exogenous osteopontin.14 The increased level of osteopontin would support a role for osteopontin in an autocrine feedback loop, which could potentiate the disease process by promoting the further accumulation of monocytes and macrophages to the site of infammation. Osteopontin might thus be central to the perpetuation of disease activity by influencing the availability of the primary target cells in AAV.
The striking correlation of osteopontin with erythrocyturia/proteinuria, identifiying renal tissue as an important localisation of osteopontin action in AAV, is well in line with previous findings involving human crescentic glomerulonephritis. Monocytes present in the human glomerular crescent were shown to express osteopontin protein and mRNA at high levels.10 This expression supports a role for osteopontin in the formation and progression of the crescentic lesion via its chemotactic and signalling properties. In patients with stable renal function (no changes in GFR over time) osteopontin levels showed a dramatic decline in the same fashion as in patients with impaired renal function on admission. GFR-matched patients with IgA nephropathy showed significantly lower levels of osteopontin.
Elevated osteopontin levels in AAV can thus only be attributed to renal function to a small extent.
Our results may offer the possibility of using osteopontin inhibitors as a novel therapeutic target of neutrophil chemotaxis, because monoclonal osteopontin antibodies have been used with great success in collagen-induced arthritis.15
Competing interests None.
Patient consent Obtained.
Ethics approval This study was conducted with the approval of the Hanover Medical School, Ethics Committee.
Provenance and peer review Not commissioned; externally peer reviewed.
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