Calprotectin has a pathogenic pro-inflammatory role in anti-neutrophil cytoplasmic antibody associated vasculitis and glomerulonephritis

Abstract

BackgroundCalprotectin, an endogenous toll-like receptor 4 (TLR 4) agonist that is expressed in neutrophils, monocytes, and infiltrating macrophages, promotes endothelial activation and transcription of pro-inflammatory cytokines. We investigated calprotectin in renal biopsy samples and serum of patients with anti-neutrophil cytoplasmic antibody associated vasculitis (AAV) and in mice deficient in calprotectin (cal−/−), and we assessed the interaction of calprotectin with macrophages and endothelial cells in vitro. MethodsWe examined renal biopsy samples with immunohistochemistry. Serum calprotectin levels were measured with ELISA, and cell surface expression with flow cytometry. Accelerated nephrotoxic nephritis experiments were performed on both wild-type (WT) and cal−/− mice. Macrophages were isolated from WT, TLR4−/−, and cal−/− mice, and kidney endothelial cells from WT mice, and stimulated with calprotectin and supernatants harvested. Phagocytosis with opsonised beads was compared between WT and cal−/− macrophages. FindingsPatients with active AAV glomerular lesions demonstrated the most calprotectin positivity in renal biopsy samples, sclerotic lesions the least (p<0·05), linking calprotectin with disease activity. Serum levels in patients were significantly higher than in controls. In limited systemic disease, calprotectin levels assessed at 1 and 6 months after treatment predicted relapse (sensitivity 78·6%, specificity 92·3%). Patients had persistently higher monocyte and neutrophil cell surface calprotectin expression than did healthy controls suggesting a persistently activated state. Cal−/− mice were protected from renal disease with less macrophage and T-cell infiltration, less thrombosis, and preserved renal function. Calprotectin stimulation of WT macrophages and endothelial cells increased production of tumour necrosis factor α, interleukin 6, and interleukin 8 (p<0·05), an effect abrogated in TLR4−/− and cal−/− macrophages. Additionally, cal−/− macrophages had decreased phagocytosis ability compared with WT (p<0·005). Together these data demonstrate a positive amplification of inflammation mediated by calprotectin. InterpretationSerum calprotectin is a potential biomarker in AAV, and may predict relapse. Calprotectin contributes to pathogenesis by promoting leukocyte and endothelial cell activation in a positive feedback loop. FundingUK Medical Research Council.