Fig. 7

OPN inhibits histone-induced NETs formation. a The release of NETs by neutrophils after exposure to histones in the presence or absence of OPN was assessed by fluorescence microscopy. Freshly isolated human neutrophils were preincubated with CTH (50 μg/mL) in the presence (1:1) or absence of OPN for 3 h at 37 °C. NETs release was detected by fluorescence microscopy using antibodies to neutrophil elastase (red) and staining of DNA by DAPI (blue) under ×40 magnification. PMA-stimulated neutrophils releasing NETs were used as a positive control. b The formation of NETs was quantified and is presented as the percentage of cells forming NETs. The effect of preincubation of CTH with OPN is also shown. n = 6; *P = 0.0022. c The effect of OPN on histone-induced MPO release from neutrophils was measured and is expressed as relative fluorescence units (RFU). d The release of NETs-associated DNA from activated neutrophils was quantified in the cell supernatant by PicoGreen analysis. Preincubation of OPN with CTH significantly reduced the amounts of DNA released into the supernatants (n = 5; **P = 0.0079)