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. 2019 Jun 14;10:1346. doi: 10.3389/fimmu.2019.01346

Figure 3.

Figure 3

Mono-nucleosome-induced PMN activation leads to secretion of soluble CEACAM8. (A) Chromatin was further purified by ultracentrifugation on 5–29% sucrose gradients to get mono-nucleosomes. Purified nucleosomes were analyzed by 1.5% agarose gel electrophoresis (left) and 18% SDS-PAGE (right). Molecular weight markers are indicated. bp, base pairs. The doublet at ~35 kDa represents histone H1. (B) Freshly isolated human PMN were cultured for 30 min or 14 h with different stimuli and the release of soluble CEACAM8 was estimated in the supernatants by ELISA. PMA, phorbol myristate acetate; Gradient, empty sucrose gradient loaded with nuclei lysis buffer instead of chromatin; Nuc, purified mono-nucleosomes (2.5, 5, 10, 20 indicate concentrations in μg/ml). As a control, nucleosomes were incubated without (w/o) PMN. (C,D) PMN were activated as in (B) for 14 h and cell activation was estimated by flow cytometry (C) or ELISA (D). Plasma membrane CD66b (CEACAM8) and CD11b expression and IL-8 secretion were determined. MFI, mean fluorescence intensity. In (D), PMN were cultured with or without polymyxin B (PB, a LPS antagonist). Shown is one representative experiment of three independent experiments using different donors and different purifications of nucleosomes. Mean and SD of triplicates are shown. (E–G) Chromatin-induced PMN activation leads to increased phagocytic activity and oxidative burst. Freshly isolated human PMN were cultured for 2 h in medium supplemented with the chromatin purification buffer (E) or stimulated with 20 μg/ml purified mono-nucleosomes (F) or 5 ng/ml LPS (G), in the presence of dichlorofluorescin diacetate (which is oxidized in dichlorofluorescein (DCF), x axis) and phycoerythrin-conjugated microspheres (y axis) to measure oxidative burst and phagocytosis, respectively, by flow cytometry. Shown is one representative experiment of three independent experiments using different donors. Mean fluorescence intensities for both axes are depicted below each corresponding dot-plot. LPS, lipopolysaccharides. *p < 0.05; **p < 0.01; ***p < 0.001 for PMN cultured with nucleosomes vs. the purification buffer.