1. Activation of Human Neutrophil by Cytokine-Activated Endothelial Cells
by Tatsuji Takahashi, Fumihiko Hato, Takahisa Yamane, Hiroshi Fukumasu, Kenichi Suzuki, Sachio Ogita, Yoshiki Nishizawa, and Seiichi Kitagawa
Circulation Research
Volume 88(4):
March 2, 2001
Copyright © American Heart Association, Inc. All rights reserved.

2. Cytokine-activated ECs induce O2− release from neutrophils.
Cytokine-activated ECs induce O2− release from neutrophils. ECs (1.5×105 cells) were untreated or pretreated with IL-1β (1.25 ng/mL) or TNF-α (39.22 ng/mL) for 4 hours, washed, and then cocultured with neutrophils (6×105 cells) for 3 hours. Human neutrophils spontaneously released O2− in the presence of IL-1β–activated or TNF-α–activated ECs (A). CTL (control) indicates untreated ECs. Furthermore, the amount of O2− release from neutrophils on the cocultures with ECs pretreated with the indicated dose of IL-1β or TNF-α at 4 hours was examined (B). Results represent ≥5 separate experiments, each done in duplicate. *P<0.05 and **P<0.01 compared with CTL.
Tatsuji Takahashi et al. Circ Res. 2001;88:
Copyright © American Heart Association, Inc. All rights reserved.

3. Effect of supernatants from activated ECs and coculture between activated ECs and neutrophils on neutrophil activation.
Effect of supernatants from activated ECs and coculture between activated ECs and neutrophils on neutrophil activation. ECs were untreated or pretreated with IL-1β (1.25 ng/mL) or TNF-α (39.22 ng/mL) for 4 hours, washed, and then incubated with or without neutrophils (6×105 cells) for 3 hours at 37°C. The conditioned medium was aspirated, and the supernatants were centrifuged to remove cells. After 3 hours of incubation with supernatants (50%), the O2− release by neutrophils (3×105cells) was measured as described in Materials and Methods. Data are representative of 5 independent experiments, each done in duplicate. *P<0.01 compared with CTL.
Tatsuji Takahashi et al. Circ Res. 2001;88:
Copyright © American Heart Association, Inc. All rights reserved.

4. Activation of MAPK 3 subtypes in response to TNF and IL-1.
Activation of MAPK 3 subtypes in response to TNF and IL-1. HUVECs were exposed for 10 minutes to TNF (39.22 ng/mL) or IL-1 (1.25 ng/mL). Phosphorylation of ERK1/2, p38 MAPK, and JNK1/2 was analyzed by immunoblotting using Ab against phosphorylated form of each protein (A through C). The cell lysates equivalent to 2×106 cells were loaded onto each lane. Immunoblots of total ERK1/2, p38 MAPK, and JNK1/2 were performed to confirm equal protein loading. Data are representative of 3 separate experiments. IB indicates immunoblotting.
Tatsuji Takahashi et al. Circ Res. 2001;88:
Copyright © American Heart Association, Inc. All rights reserved.

5. Neutrophils adherent to activated ECs and ICAM-1 expression on IL-1–activated or TNF-activated ECs. We counted the number of neutrophils adherent to activated ECs with a phase-contrast microscopy after 5 minutes of coculture (A).
Neutrophils adherent to activated ECs and ICAM-1 expression on IL-1–activated or TNF-activated ECs. We counted the number of neutrophils adherent to activated ECs with a phase-contrast microscopy after 5 minutes of coculture (A). ICAM-1 expression on TNF (39.22 ng/mL)-activated ECs for indicated time was analyzed by FACS (B). After ECs were treated with the indicated concentration of the cytokine for 4 hours, ICAM-1 expression was analyzed (C). M.F.I. indicates mean fluorescence intensity. Results represent 4 separate experiments. *P<0.05 compared with CTL.
Tatsuji Takahashi et al. Circ Res. 2001;88:
Copyright © American Heart Association, Inc. All rights reserved.

6. Fixed EC-neutrophil interaction assay.
Fixed EC-neutrophil interaction assay. TNF-treated or IL-1–treated EC monolayers (1.5×105 cells) were fixed with 1% paraformaldehyde for 30 minutes and washed 3 times thoroughly. After neutrophils (6×105 cells) were incubated with nontreated or cytokine-treated fixed ECs in the presence of supernatants from activated ECs (A) and rh GM-CSF (B) for 3 hours, the amount of O2− release was measured. CTL-fixed, IL-1–fixed, and TNF-fixed ECs indicate nontreated, IL-1β (1.25 ng/mL)–treated, and TNF-α (39.22 ng/mL)–treated fixed ECs, respectively. Results represent 4 separate experiments, each done in duplicate. CTL-EC sup, IL-1-EC sup, and TNF-EC sup indicate supernatant from nonactivated, IL-1–activated, and TNF-activated ECs, respectively. *P<0.05, #P<0.05, and §P<0.05 compared with CTL-EC sup, IL-1-EC sup on CTL-fixed ECs, and TNF-EC sup on CTL-fixed ECs, respectively. †P<0.05 compared with the absence of rh GM-CSF. ¶P<0.05 compared with CTL-fixed ECs.
Tatsuji Takahashi et al. Circ Res. 2001;88:
Copyright © American Heart Association, Inc. All rights reserved.

7. Effects of various inhibitors on EC-neutrophil interaction and supernatant-induced O2− release assays.
Effects of various inhibitors on EC-neutrophil interaction and supernatant-induced O2− release assays. The magnitudes of O2− release from neutrophils on coculture between cytokine-treated ECs (1.5×105 cells) and neutrophils (6×105 cells) in the absence or presence of anti–GM-CSF Ab (55 μg/mL), YM264 (2.5×10−5 mol/L), PD98059 (1×10−5 and 5×10−5 mol/L), SB (1×10−6 and 1×10−7 mol/L), ADA (1.18 μg/mL), and DMPX (2.5×10−5 mol/L) for 3 hours were measured (A). After neutrophils (3×105 cells) were incubated with activated EC supernatant in the absence or presence of various inhibitors, the O2− release was measured (B). PD indicates PD98059; SB, SB Results represent ≥3 independent experiments, each done in duplicate. *P<0.05 compared with the absence of inhibitors.
Tatsuji Takahashi et al. Circ Res. 2001;88:
Copyright © American Heart Association, Inc. All rights reserved.

8. GM-CSF production of activated ECs
GM-CSF production of activated ECs. PD98059 and SB were added to EC monolayers 15 minutes before treatment, treated with IL-1 or TNF for 4 hours, washed, and then incubated with HBSS for 3 hours.
GM-CSF production of activated ECs. PD98059 and SB were added to EC monolayers 15 minutes before treatment, treated with IL-1 or TNF for 4 hours, washed, and then incubated with HBSS for 3 hours. The supernatants were centrifuged to remove cells, and GM-CSF concentrations in the conditioned medium were determined by ELISA (R&D Systems). Results represent 5 separate experiments, each done in duplicate.
Tatsuji Takahashi et al. Circ Res. 2001;88:
Copyright © American Heart Association, Inc. All rights reserved.

9. Effect of exogenous PAF on GM-CSF–induced O2− release from neutrophils.
Effect of exogenous PAF on GM-CSF–induced O2− release from neutrophils. The amounts of nonstimulated or rh GM-CSF (50 pg/mL)–induced O2− release from neutrophils in the absence or presence of varying concentrations of exogenous PAF were measured (A). Neutrophils were incubated with or without varying concentrations of exogenous rh GM-CSF in the absence or presence of PAF (2×10−8 mol/L) for 3 hours (B). Results represent ≥3 separate experiments, each done in duplicate. *P<0.05 and #P<0.05 compared with control and 0, respectively.
Tatsuji Takahashi et al. Circ Res. 2001;88:
Copyright © American Heart Association, Inc. All rights reserved.