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. 2016 Dec 12;2:16084. doi: 10.1038/cddiscovery.2016.84

Figure 1.

Figure 1

GrA enhances the LPS-induced proinflammatory cytokine release from monocytes. (a) Human monocytes were incubated with increasing concentrations of GrA(-SA) with or without LPS (5 ng/ml) for 6 h. Tumor necrosis factor-α (TNFα) levels in the culture supernatants were determined. Data are expressed as mean±S.D. and are representative of at least three independent experiments with normal donors (*P<0.05; **P<0.01; ***P<0.001, compared with LPS control). TNFα release was not statistically different between GrA plus LPS and GrA-SA plus LPS (P=0.113). (b) Human monocytes were treated with GrA(-SA) (400 nM) with or without LPS (2.5 ng/ml) for 4, 6 or 8 h. TNFα was detected in the culture supernatants. Data are expressed as mean±S.D. and are representative of at least three independent experiments with normal donors (*P<0.05; **P<0.01, as compared with LPS control for the same time point). (c) GrA (7.5 μM) was treated with DCI (150 μM) for 30 min, dialyzed overnight, and residual GrA activity (2 μM) was kinetically monitored by chromogenic substrate Z-Phe-Arg-pNA (1 mM) hydrolysis at OD405. (d) Human monocytes were treated with GrA(-DCI) (500 nM) with or without LPS (1 ng/ml) for 6 h. TNFα was detected in the culture supernatants. Data are expressed as mean±range and are representative of three independent experiments in duplo with normal donors. (e, f) Human monocytes were treated with GrA(-SA) (400 nM) with or without LPS (2.5 ng/ml) for 4, 6 or 8 h. Cytokines interleukin-6 (IL-6) (e) and IL-8 (f) were detected in the culture supernatants. Data are expressed as mean±S.D. and are representative of at least three independent experiments with normal donors (*P<0.05; **P<0.01, as compared with LPS control for the same time point). (g) The magnitude of the GrA synergistic effect is similar to that of GrK. Human monocytes were incubated with GrA(-SA) or GrK (400 nM) with or without LPS (2.5 ng/ml) for 6 h, after which TNFα levels in the culture supernatants were measured. Data are expressed as mean±S.D. and are representative of six independent experiments with normal donors (*P<0.05; **P<0.01; ***P<0.001, compared with LPS control). (h) GrA is not cytotoxic to human monocytes. Human monocytes were incubated with GrA(-SA) (500 nM) for 0–6 h. Relative cell viability was determined by WST-1 assay. Data (n=3 per treatment) are depicted as mean±S.D. (% of medium control) and are representative of at least two independent experiments with normal donors.