Volume 123, Issue 5, Pages 1543-1553 (November 2002) The role of dietary microparticles and calcium in apoptosis and interleukin-1β release of intestinal macrophages  Stephen M. Evans, Paul Ashwood, Alice Warley, Fatmire Berisha, Richard P.H. Thompson, Jonathan J. Powell  Gastroenterology  Volume 123, Issue 5, Pages 1543-1553 (November 2002) DOI: 10.1053/gast.2002.36554 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 IL-1β release (pg/106 cells) from normal intestinal cell isolates over time after stimulation with 1 ng/mL of LPS (A), 5 μg/mL of TiO2 (B), 4 mmol/L of Ca2+ (C), or the TiO2-Ca2+-LPS conjugate (D). Data are mean ± SEM (n = 4–24 per time point) and are baseline subtracted (i.e., IL-1β levels from stimulated cells minus IL-1β levels from paired unstimulated cells). Gastroenterology 2002 123, 1543-1553DOI: (10.1053/gast.2002.36554) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 IL-1β (pg/106 cells) secretion over time from intestinal cells isolated from patients with (▴) or without (●) IBD. Cells were stimulated with 4 mmol/L of calcium (A) or the TiO2-Ca2+-LPS conjugate (B). Data are baseline subtracted (mean ± SEM; n = 9 for 15- and 24-hour cultures and n = 2–9 for other time points). Gastroenterology 2002 123, 1543-1553DOI: (10.1053/gast.2002.36554) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 IL-1β release (pg/106 cells) of cultured (24-hour) intestinal mononuclear cells, stimulated with calcium (open symbols) or the TiO2-Ca2+-LPS conjugate (closed symbols) against baseline (i.e., unstimulated) IL-1β levels from paired cultures. Triangles represent IBD patients (n = 9); circles represent non-IBD patients (n = 9) (r = 0.71; P < 0.0001). Gastroenterology 2002 123, 1543-1553DOI: (10.1053/gast.2002.36554) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 The inhibition of IL-1β–converting enzyme (ICE) activity in Ca2+-stimulated PBMNC cultures with the ICE inhibitor Z-Val-Ala-Asp(OMe)-CH\-(2)F. PBMNC (106 cells) cultures were pretreated with increasing doses (0–60 μmol/L) of ICE inhibitor before 24 hours of incubation with 160 μg/mL of Ca2+. Data are mean ± SEM; n = 8. Gastroenterology 2002 123, 1543-1553DOI: (10.1053/gast.2002.36554) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 IL-1ra (pg/106 cells) secretion (A) and the IL-1ra/IL-1β ratio (B) in intestinal cells isolated from patients with (n = 11) and without (n = 12) IBD. Cells were incubated for 24 hours and either unstimulated (□) or stimulated with calcium (▩) or with the TiO2-Ca2+-LPS conjugate (■) (mean ± SEM; *P = 0.02; **P = 0.001 vs. unstimulated). Gastroenterology 2002 123, 1543-1553DOI: (10.1053/gast.2002.36554) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Percentage cell death in the macrophage gate of isolated intestinal cells, cultured for 24 hours, as measured by propidium iodide incorporation, from patients with (IBD; n = 9) and without (Normal; n = 8) IBD. Cells were incubated for 24 hours and either unstimulated (□) or stimulated (▩) with calcium or with the TiO2-Ca2+-LPS conjugate (■) (mean ± SEM; **P < 0.001 vs. unstimulated). Gastroenterology 2002 123, 1543-1553DOI: (10.1053/gast.2002.36554) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 IL-1β production of intestinal cells in the presence and absence of calcium and increasing concentrations of citrate. Control cells had no added calcium or citrate (0,0 is control; 4,0 is 4 mmol/L of calcium without citrate; 4,4 is 4 mmol/L of calcium and citrate; and 4,8 is 4 mmol/L of calcium and 8 mmol/L of citrate). Citrate alone had no effect (data not shown) (mean ± SEM; P < 0.01 for 4,0 vs. other combinations). Gastroenterology 2002 123, 1543-1553DOI: (10.1053/gast.2002.36554) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 Electron micrograph showing subcellular detail of a macrophage in lamina propria cell culture. The dark intralysosomal material is calcium phosphate, confirmed by x-ray microanalysis, the formation of which was abolished by adding citrate to the culture medium. Gastroenterology 2002 123, 1543-1553DOI: (10.1053/gast.2002.36554) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 9 The effect of cytochalasin D pretreatment on the stimulation of monocytes. PBMNC (106 cells) cultures were pretreated with increasing doses (0–8 μg/mL) of cytochalasin D for 30 minutes before stimulation with 160 μg/mL of Ca2+ for 24 hours. The monocyte population was assessed for phagocytosis (▴) and propidium iodide incorporation (○) by FACS analysis of 10,000 events within the phagocyte gate. Culture supernatants were analyzed by specific ELISA for IL-1β (pg/mL) (■). Data are expressed as the percentage change in response compared with no cytochalasin D pretreatment (mean ± SEM; n = 8). Gastroenterology 2002 123, 1543-1553DOI: (10.1053/gast.2002.36554) Copyright © 2002 American Gastroenterological Association Terms and Conditions