1. Cytokines and Peroxisome Proliferator-Activated Receptor γ Ligand Regulate Phagocytosis by Pancreatic Stellate Cells 
Kyoko Shimizu, Makio Kobayashi, Junko Tahara, Keiko Shiratori 
Gastroenterology 
Volume 128, Issue 7, Pages (June 2005)
DOI: /j.gastro
Copyright © 2005 American Gastroenterological Association Terms and Conditions

2. Figure 1
Morphological analysis of phagocytic cells in the pancreas of 4-month-old male WBN/Kob rats (n = 5). H&E-stained sections show focal inflammation that includes inflammatory cell infiltration, hemorrhage, fibrosis, and parenchymal destruction (A). Stellate cells that have ingested brown pigment (*) are seen in the periacinar region of the pancreas at high magnification (B). The immunohistochemical study showed GFAP-positive cells (*) in the periacinar region (C). Phagocytic cells whose cytoplasm is full of ingested pigment are seen in the periacinar region of the pancreas, where there was mild to moderate inflammation (D). Some GFAP-positive cells (E) were identical to the cells that had internalized pigment in the negative control (F). (G) and (H) are higher magnifications by digital zoom of the boxed areas marked with the solid line and dotted line in (E) and (F), respectively. Some cells positive for α-SMA whose cytoplasm contains ingested brown pigment are seen in the areas of moderate to severe inflammation in the pancreas (I). Cells that have internalized pigment are seen in the adjacent section, which was used as a negative control (J)
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
Immunoelectron-microscopic appearance of the pancreas of male WBN/Kob rats (n = 5). Pancreata of male WBN/Kob rats were fixed and examined by immunoelectron microscopy after incubation with anti-GFAP antibody (A and C) or anti-α-SMA antibody (B and D), followed by incubation with colloidal gold-conjugated secondary antibody and then double-staining with uranyl acetate and lead citrate. Phagocytized electron-dense bodies were observed in the cytoplasm of the cells (A and B) (original magnification, 28,500×). Higher magnifications showed that gold particles had accumulated on the fibrils of these cells (C and D) (original magnification, 57,000×). These results indicate that GFAP- and α-SMA-positive cells have a phagocytic capacity.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
Phagocytosis of apoptotic cells by PSCs in an animal model of acute pancreatitis (n = 5). Acute pancreatitis was induced by ligation of the pancreatic duct at its entrance to the duodenum, and the bile was returned to the duodenum. Apoptotic cells and α-SMA were labeled with FITC (green) and rhodamine (red), respectively. α-SMA-positive cells had ingested apoptotic cells into their cytoplasm, thus suggesting that PSCs had internalized apoptotic cells (original magnification, 400×).
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
Annexin V/FITC staining for PMNs. Freshly isolated PMNs and aged PMNs that had been incubated for 24 hours after isolation were stained with annexin V/FITC and PI and examined with a Lazar scanning microscope. The plasma membranes of aged PMNs were positive for annexin V/FITC, and this suggests that they were undergoing apoptosis. Some aged PMNs positive for both annexin V/FITC and PI were in the late stage of apoptosis or were already dead. By contrast, no annexin V-/FITC or PI was detected in freshly isolated PMNs.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
PSCs were cultured in a Lab-Tek Chamber Slide and then allowed to interact with aged PMNs. Uningested aged PMNs were washed away with cold saline, and the PSCs that had interacted with aged PMNs were identified by staining for myeloperoxidase. Aged PMNs had attached to the surface of the PSCs (A) and had been ingested into their cytoplasm (B).
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Interaction assay with aged PMNs. PSCs were cultured for 48 hours in the presence or absence of FCS or troglitazone (TRO) in concentrations of 5, 10, or 20 μmol/L. The number of ingested PMNs was increased by the addition of FCS compared with serum-free medium. Troglitazone significantly and dose-dependently increased the number of PMNs ingested. *P < .001, FCS(−) vs FCS(+) or TRO. Each experiment was performed in triplicate and repeated at least 5 times.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
Bead phagocytosis assay. PSCs were cultured in the absence and presence of FCS or 20 μmol/L troglitazone (TRO) for 48 hours. TNF-α (10 ng/mL), IL-1β (10 ng/mL), or TGF-β (10 ng/mL) was added 12 hours before the addition of 20 μmol/L troglitazone. Cells were incubated with fluorescence-labeled latex beads, and internalization was analyzed by flow cytometry. When troglitazone was added, the size of the peaks with higher levels of fluorescence intensity and mean fluorescence intensity (MFI) was increased. By contrast, TGF-β, TNF-α, or IL-1β markedly inhibited TRO-induced bead uptake. Each experiment was performed in duplicate and repeated at least 3 times.
Gastroenterology  , DOI: ( /j.gastro )
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9. Figure 8
Cells were cultured in the presence or absence of 20 μmol/L troglitazone (TRO) and then incubated with FITC-conjugated anti-mouse monoclonal antibody against CD36. FITC-conjugated mouse IgMκ was used as a negative control. Cell fluorescence was measured by FACScan and CellQuest software. CD36 expression was significantly increased when cells had been treated with TRO. Each experiment was performed in duplicate and repeated 3 times.
Gastroenterology  , DOI: ( /j.gastro )
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10. Figure 9
A rabbit polyclonal CD36 antibody was added 12 hours before the addition of 20 μmol/L troglitazone (TRO), and bead internalization was assayed by flow cytometry. Blockade with CD36 antibody decreased TRO-induced bead internalization to the same level as in the absence of TRO. Each experiment was performed in duplicate and repeated 3 times. MFI, mean fluorescence intensity.
Gastroenterology  , DOI: ( /j.gastro )
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11. Figure 10
Western blot analysis was performed after culture of PSCs in the absence and presence of 20 μmol/L troglitazone (TRO). CD36 was up-regulated by TRO, whereas α-SMA expression was down-regulated. Each experiment was repeated 3 times.
Gastroenterology  , DOI: ( /j.gastro )
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12. Figure 11
PSCs at 80%–90% confluence were cotransfected with PPAR-γ siRNA by using a reporter plasmid and LipofectAMINE 2000 reagent. After incubation for 24 hours, the cells were treated with 20 μmol/L troglitazone (TRO) for 12 hours and harvested for reporter assay. The relative light units of firefly luciferase activity were measured with a luminometer, and all values were normalized for transfection efficiency against the relative light units from Renilla luciferase. Each transfection was performed in triplicate and repeated at least 3 times. Approximately 15-fold greater luciferase activity generated by the reporter gene was induced in the presence of TRO. PPAR-γ siRNA completely blocked TRO-stimulated reporter activity. *P < .01, PPAR-γ siRNA(−) vs PPAR-γ siRNA(+). Each transfection was performed in duplicate and repeated 3 times.
Gastroenterology  , DOI: ( /j.gastro )
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13. Figure 12
PPAR-γ, CD36, and α-SMA protein expression in the presence of 20 μmol/L troglitazone (TRO) was measured by Western blot analysis after transfection with PPAR-γ siRNA or with green fluorescent protein (GFP) siRNA as a negative control. Transfection with PPAR-γ siRNA caused a significant reduction in PPAR-γ and CD36 expression compared with GFP siRNA transfection. In contrast, the expression of α-SMA was slightly increased by the disruption of endogenous PPAR-γ expression. Each experiment was repeated 3 times.
Gastroenterology  , DOI: ( /j.gastro )
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14. Figure 13
Inhibitory effect of PPAR-γ siRNA on phagocytosis. At 24 hours after siRNA transfection, cells were exposed to 20 μmol/L troglitazone (TRO) for 48 hours and then cultured with aged PMNs or fluorescence-labeled latex beads. Green fluorescent protein (GFP) siRNA was used as a negative control. Phagocytic activity was measured by PMN phagocytosis assay (A) and bead phagocytosis assay (B). TRO significantly increased the number of ingested PMNs in cells transfected with GFP siRNA, but not in cells transfected with PPAR-γ siRNA. Bead internalization was not markedly blocked by silencing PPAR-γ expression (solid line), but it was slightly less than when expression of an unrelated gene was silenced (dotted line). *P < .05, TRO vs FCS. Each experiment was performed in duplicate and repeated at least 3 times.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

15. Figure 14
Effect of phagocytosis on activation of PSCs. Cells were serum-starved for 48 hours. The medium was then replaced with FCS-containing medium, and the cells were incubated with aged PMNs for 48 hours in the presence or absence of 20 μmol/L troglitazone (TRO). (A) The rate of cell proliferation was measured by a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Exposure to troglitazone or aged PMNs significantly inhibited cell proliferation. When cells were incubated with aged PMNs in the presence of TRO, cell proliferation was inhibited additively. *P < .05 vs control; #P < .05 vs TRO or PMN. (B) α-SMA expression was measured by Western blot analysis. Ingestion of aged PMNs did not affect α-SMA expression by PSCs. Each experiment was performed in triplicate and repeated at least 5 times.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions