Figure 1.
TWEAK and Fn14 are upregulated in LPS-induced CV.
BALB/c mice were treated with LPS or equal volume of PBS s.c. into the left ear and challenge with LPS 150 µg or PBS i.p. respectively 24 h later. 6 h after challenge, sera and tissue samples were collected. (ten mice per group). (A) Serum TWEAK levels were determined by ELISA. Values are expressed as Mean ± SD. P-values are based on the Mann–Whitney U-test. (B) The expression of TWEAK and its receptor Fn14 on endothelial cells in the skin were detected by immunohistochemistry. Scale bars, 50 µm.
Figure 2.
TWEAK priming induces a local CV.
Mice were treated as described (six mice per group). (A) 6 h after LPS challenge, the hemorrhagic lesions in the ears were observed in TWEAK priming group. Tissue samples were stained for hematoxylin–eosin (B) and Masson's trichrome (C). Representative images were shown from three experiments. Scale bars, 50 µm. (D) The number and size of hemorrhagic lesions were analyzed according to the semiquantitative score as described 21 at different time points. (E) Increased FITC-BSA extravasation was found in the left ear from TWEAK priming group and LPS priming group. Values are expressed as Mean ± SD; Mann–Whitney U-test *P<0.05, **P<0.01, compared with PBS priming group; ##P<0.01, compared with TWEAK priming group.
Figure 3.
TWEAK neutralization blocks LPS induced CV.
Mice were treated as described (six mice per group). (A) 6 h after systemic LPS challenge, the representative macroscopic appearance of the hemorrhagic lesions in the ears was shown. Tissue samples were collected and stained for hematoxylin–eosin (B) and Masson's trichrome (C). Scale bars, 50 µm. (D) The hemorrhagic lesions in different group were determined by a semiquantitative score as described 21 at the indicated time points. (E) Extravasation of FITC-BSA was assessed spectrophotometrically in the ear from CV mice treated with anti-TWEAK mAb or IC mAb or PBS-treated mice (control group). Values are expressed as Mean ± SD; Mann–Whitney U-test *P<0.05, **P<0.01, compared with IC mAb-treated group; ##P<0.01, compared with control group.
Figure 6.
TWEAK does not induce cell apoptosis in HMEC-1 cells.
Cells were treated with TWEAK 500 ng/ml for 12 h. After treatment, cells were suspended in binding buffer and double-stained with FITC-labled Annexin V and propidium iodide. Apoptotic rate of HDMECs were determined by flow cytometry.
Figure 4.
TWEAK priming upregulates but TWEAK blockade suppresses adhesion molecules expression and MPO activity.
Mice were treated as described above, tissue sample were collected 6 h after systemic LPS challenge (six mice per group). Skin sections were stained with antibodies against E-selectin (A) and ICAM-1 (B). These observations were representative of multiple experiments and microscopic fields. Scale bars, 10 µm. (C) MPO activity was assessed for the quantification of PMNs accumulation in tissue samples by a colorimetric assay. Values are expressed as Mean ± SD; Mann–Whitney U-test **P<0.01.
Figure 5.
Cells were stained for Fn14 expression using mouse anti-Fn14 mAb (dark grey histogram) or IC mAb (light grey histogram) and PE-labeled secondary antibody. After that, Cells were resuspended in PBS and analyzed by flow cytometry for fluorescence.
Figure 7.
TWEAK enhances adhesion molecule expression in HDMECs.
Cells were treated with TWEAK (1–100 ng/ml), TNF-a 10 ng/ml, TWEAK 100 ng/ml plus anti-human Fn14 mAb or IC mAb for 6 h. After treatment, cells were stained with FITC-labeled antibodies against E-selectin and ICAM-1. The expression of E-selectin (A and C) and ICAM-1 (B and D) were quantitatively analyzed by flow cytometry and depicted in the dark grey histograms. Cells stained with FITC-labeled IC mAb were depicted in the light grey histograms. Values are expressed as Mean ± SD; one-way analysis of variance n = 4.*P<0.05, **P<0.01, compared with untreated group; ##P<0.01, compared with TWEAK 100 ng/ml-treated group.
Figure 8.
TWEAK induces the adhesion of human PMNs to HDMECs.
Cells were treated with TWEAK (1–100 ng/ml), TNF-a 10 ng/ml or TWEAK 100 ng/ml plus anti-human Fn14 mAb or IC mAb for 6 h, and then co-incubated with fluorescent-labeled PMNs for 0.5 h. After treatment, PMNs bound to HDMECs were lysed. Fluorescent intensities of cell lysate were counted on a spectrofluorometer. Values are expressed as Mean ± SD; one-way analysis of variance n = 4. *P<0.05, **P<0.01, compared with untreated group; ##P<0.01, compared with TWEAK 100 ng/ml-treated group.
Figure 9.
TWEAK activates MAPKs family proteins in HDMECs.
(A) Cells were treated with TWEAK 50 ng/ml for various intervals. After treatment, cells were lysed, and 30 µg of protein samples were used for western blots analysis of the indicated proteins. Representative blots were shown from three experiments. (B and C) Cells were pre-treated with various MAPKs inhibitors (SB203580, SP600125 and PD98059) for 0.5 h followed by incubation with TWEAK 100 ng/ml for a further 4 h. The mRNA expression levels of E-selectin (B) and ICAM-1 (C) were determined by real-time quantitative PCR. Values are expressed as Mean ± SD; one-way analysis of variance n = 4. **P<0.01, compared with untreated group; ##P<0.01, compared with TWEAK -treated group.
Figure 10.
SB203580 and SP600125 but not PD98059 block TWEAK-induced adhesion molecules protein expression.
Cells were pre-treated with various MAPKs inhibitors (SB203580, SP600125 and PD98059) for 0.5 h followed by incubation with TWEAK 100 ng/ml for a further 6 h. After treatment, cells were stained with FITC-labeled antibodies against E-selectin and ICAM-1. The expression of E-selectin (A and B) and ICAM-1 (A and C) were quantitatively analyzed by flow cytometry and depicted in the dark grey histograms. Cells stained with FITC-labeled IC mAb were depicted in the light grey histograms. Values are expressed as Mean ± SD; one-way analysis of variance n = 4. **P<0.01, compared with untreated group; ##P<0.01, compared with TWEAK 100 ng/ml-treated group.