Figure 6.

Inflammasome-active neutrophils maintain phagocytosis, degranulation, and migration activities, but do not trigger efferocytic anti-inflammatory macrophage polarization. (A) Mouse BMNs or BMDMs were untreated (Unt) or treated with LPS (0.25 μg/mL, 2.5 h) alone, LPS followed by ATP (2.5 mM, 1 h), or cytochalasin D (Cyto D, 10 μM, 30 min) as determined using flow cytometric analysis after incubating with zymosan-FITC (5 particles/cell, 30 min) (n = 3). (B) Quantification of MMP-9 in the culture supernatants of mouse BMNs treated with LPS (0.25 μg/mL, 2.5 h) followed by ATP (2.5 mM, 1 h), washed and treated with PMA (1 μM, 2 h) (n = 3). (C, D) Quantification of track length (C) and track straightness (D) of BMNs treated with LPS (0.25 μg/mL, 2.5 h) alone or followed by ATP (2.5 mM), as determined using in vitro migration assays. (E) Representative migration tracks of BMNs treated as in (C, D). (F) Experimental scheme for determining the potential effect of inflammasome-active neutrophils on efferocytosis. (G–I) Mouse BMNs were untreated or treated with LPS (0.25 μg/mL, 2.5 h) followed by ATP (2.5 mM, 2 h) or staurosporine (2 μg/mL, 5 h). Mouse peritoneal macrophages were treated with the injured BMNs (1:2.5 ratio, macrophages:neutrophils) for 1 h, washed and treated with LPS (0.1 μg/mL) for 18 (H) Quantification of IL-10 (G), TNF-α (H), or Il-6 (I) mRNA levels in mouse peritoneal macrophages treated as above (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, n.s. not significant.