Background Systemic onset Juvenile Idiopathic Artritis (sJIA) is considered an autoinflammatory disease, with increased circulating neutrophil counts and extremely high serum levels of S100 proteins and interleukin (IL)-18. We have previously shown that upon stimulation with S100 proteins and ATP, peripheral blood mononuclear cells (PBMCs) of healthy donors produce high levels of IL-1β and IL-18; however this cytokine production is decreased in PBMCs of sJIA patients with active disease and partly restored when patients are in remission.1 Given the observed hyporesponsiveness of PBMCs from sJIA patients, we hypothesized that neutrophils might play an important role in the inflammatory cascade of sJIA.
Objectives Our objective was to investigate the role of neutrophils in sJIA, with a focus on the relation between neutrophils, S100 proteins and IL-18.
Methods We determined ex vivo cell frequencies of patients at disease onset, patients with inactive disease and healthy controls by flow cytometric analysis, using fluorescent beads for absolute counts. Neutrophils from healthy donors were sorted and subsequently primed with S100 proteins in a time series up to 20 hours with or without Formyl-Methionyl-Leucyl-Phenylalanine (fMLP) for the last 10 minutes. Elastase and cytokine levels in supernatant were measured by Luminex, neutrophil activation markers were analysed by flow cytometry.
Results Patients with new onset sJIA had significantly elevated neutrophil counts compared to healthy controls and patients with inactive disease. Immature neutrophils (CD16dimCD62L+) and normally matured neutrophils (CD16+CD62L+) were significantly higher in patients with new onset sJIA compared to healthy controls or patients with inactive disease, while hypersegmented neutrophils (CD16+CD62Ldim) were comparable between patients and controls.
Given the high serum levels of S100 proteins in active sJIA, we investigated if neutrophils could be activated by these proteins. Priming with S100A9 before stimulation with fMLP increased activation and degranulation of specific granules and secretory vesicles compared to unprimed cells. This was reflected by an increased expression of CD11b, CD66b and CD35. Upon stimulation with S100A9, we observed an increase in elastase secretion in the culture supernatant, as well as a minor increase in IL-6, IL-18 and TNF-α.
Conclusions Patients with new onset sJIA have increased neutrophil counts. S100 proteins prime neutrophils and enhance degranulation upon a second stimulus such as fMLP. We are now exploring whether IL-18 or other pro-inflammatory mediators elevated in sJIA plasma can also prime neutrophils. Further, we will investigate whether ex vivo neutrophils from sJIA patients are more activated or respond differently to stimulation compared to patients with inactive disease or healthy controls.
N. Ter Haar, D. Holzinger, W. de Jager, R. Scholman, S. de Roock, B. Vastert. IL-18 production upon s100 stimulation is reduced in active sJIA patients compared to sJIA patients in remission and healthy controls. Pediatric Rheumatology 2014, 12(Suppl 1):O24
Acknowledgements We would like to acknowledge D. Holzinger, T. Vogl, D. Foell and J. Roth for providing the S100 proteins.
Disclosure of Interest None declared