Background and Objectives Neutrophils play a pivotal role in inflammation and contribute to systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) pathogenesis. Interferon (IFN)-α is involved in lupus development and might contribute locally to RA development. Activated plasmacytoid dendritic cells (pDC) are important producers of IFN-α but represent a minor cell population. On the other hand, neutrophils represent 50% of total blood leukocytes. Although neutrophils are not considered as IFN-α-producing cells, we have investigated whether they may produce this cytokine and the stimuli involved.
Materials and Methods PBMC and neutrophils were isolated from healthy individuals, SLE and RA patients. Mouse neutrophils were purified from the bone marrow. Cells were activated with different stimuli and IFN-α production/secretion was estimated by flow cytometry, ELISA and a bioassay. Neutrophil activation was verified by flow cytometry and ELISA. Gene expression was analysed by qRT-PCR. Neutrophil extracellular trap (NET) induction was estimated by confocal microscopy. Chromatin, a major autoantigen in SLE, was purified from calf thymus.
Results Isolated neutrophils produce IFN-α upon stimulation with Toll-like receptor (TLR) 9 and TLR7/8 agonists. IFN-α secretion by neutrophils was observed with neutrophils from both healthy donors and SLE and RA patients. Similar results were obtained with mouse neutrophils. IFN-α production by neutrophils was associated with IL-8, IL-6 and TNF-α secretion, CD66b up-regulation, ROS production and increased gene expression levels of IFN-α, IFN-β and IL-6. In low responders, PBMC sustain IFN-α secretion by neutrophils in co-cultures. Neutrophil priming is not required but GM-CSF acts synergistically with TLR9 agonists. Particularly, neutrophils respond to all types (A, B and C) of CpG-oligonucleotides. pDC are more efficient than neutrophils in producing IFN-α at the single cell level but this was largely compensated by the 200-fold excess of neutrophils in whole blood. Importantly, neutrophil-derived IFN-α was detected in response to free chromatin, a lupus autoantigen, and was associated with neutrophil extracellular trap (NET) formation (NETosis).
Conclusions Neutrophils represent an important source of IFN-α. IFN-α was detected at the mRNA and protein levels and in an active and secreted form. Both normal as well as SLE and RA neutrophils produce IFN-α in response to specific stimuli. Therefore, neutrophils represent also important targets for future therapies aiming at influencing IFN-α levels.
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