Figure 1.
Ang-1 inhibited LPS-induced cytokines production in P815 mast cells.
Quantitative RT-PCR (A and B) and ELISA (C and D) were employed for detection of mRNA and the secretion of cytokines production in duplicates, respectively. A and C: LPS dramatically increased TNF-α mRNA production and protein secretion in P815 mast cells (column 2 versus column 1). Addition of Ang-1 abrogated the induction of LPS on mast cells (column 3 versus column 2). Soluble form of Tie2 (sTie-2) and RGD reversed the inhibition of Ang-1 on LPS-induced TNF-α production (column 4, 5 versus column 3). *P<0.05. B and D: IL-6 mRNA and secretion were both significantly increased in mast cells upon LPS treatment. Ang-1 could decrease the induction while sTie-2 and RGD exerts opposite effects. *P<0.05.
Figure 2.
Ang-1 inhibited LPS-induced IκB phosphorylation and NF-κB nuclear translocation in P815 mast cells.
A: Immunofluorescence showed Tie-2 receptor expression. Primary antibody in negative control was monoclonal IgG. B: Western blotting was performed to analyze phosphorylation levels of IκB in mast cells in response to different stimuli. C and D: Densitometric analysis was used to calculate the relative ratio of p-IκB/β-actin (C) and IκB/β-actin (D). Ratio of the control group was arbitrarily presented as 1. *P<0.05. E: NF-κB translocation was detected by confocal microscopy. In quiescent P815 mast cells, NF-κB exerts primarily in the cytoplasm (first panel). With LPS treatment for 2 h, NF-κB translocates into the nucleus (second panel). Ang-1 nearly abolished LPS induced translocation of NF-κB (third panel) which was reversed by soluble form of Tie2 (sTie-2) (forth panel) and RGD (fifth panel). F: NF-κB translocation was also detected by Western blot. Fibrillarin was used as internal control. G: Densitometric analysis was used to calculate the relative ratio of NF-κB/Fibrillarin. Ratio of the control group was arbitrarily presented as 1. Data are mean ± SD of the ratios from 3 independent experiments. *P<0.05.
Figure 3.
Ang-1 suppressed compound 48/80 induced mast cells degranulation.
Degranulation was determined by staining with dyes and measuring the release of histamine and tryptase. A: Mast cells degranulation was observed by microscope 20 min after compound 48/80 10 µg/mL treatment. Cells were stained with alcian blue (a–e) and toluidine blue (f–j) (100×). (a, f) control group; (b, g) compound 48/80-treated cells; (c, h) Ang-1 100 ng/ml-treated cells; (d, i) Soluble form of Tie2 (sTie-2)-treated cells and (e, j) RGD-treated cells. B and C: Quantification of P815 mast cells degranulation by compound 48/80. It was performed in a blinded fashion. D: Degranulation stimulated by compound 48/80 was determined by measuring the release of tryptaseβ2 (mMCP-6) through commercial ELISA kit in duplicates. The data shown are mean±SD of 3 separate experiments. E: Degranulation stimulated by compound 48/80 was determined by measuring the release of histamine through OPT-fluorometric assay as previously reported in duplicates. F: Mast cells degranulation were incubated with 250 ng/ml before DNP-BSA 10 µg/ml treatment for 20 min. Degranulation was determined by measuring the release of tryptase-β2 (mMCP-6) through commercial ELISA kit in duplicates. The data shown are mean ±SD of 3 separate experiments. G: Mast cells degranulation were incubated with 250 ng/ml before DNP-BSA 10 µg/ml treatment for 20 min. Degranulation was determined by measuring the release of histamine through OPT-fluorometric assay as previously reported in duplicates. *P<0.05.
Figure 4.
Ang-1 suppressed compound 48/80 stimulated and FcεRI-mediated Ca2+ mobilization.
A and B: Ca2+ response to compound 48/80 (A) or IgE-DNP/DNP-BSA (B) activation in control, Ang-1-treated, sTie2-treated and RGD treated cells. C and D: Statistical analysis of the amplitude of the compound 48/80 stimulated (C) and FcεRI-induced (D) Ca2+ increase in both groups. The amplitude of the Ca2+ response was represented as the highest observed level of ΔF/F0. Data indicate mean ± SD from four independent experiments. *P<0.05,**P<0.001.
Figure 5.
Ectopic expression of Ang-1 in vivo protected against IgE-dependent PCA and anaphylaxis shock.
A: The measurements of serum Ang-1 levels by ELISA in 7, 14, 21 and 28 days after intravenous injection of 1×106 TU LV-Ang-1. Each data point indicates mean±SD of 5 mice. B and C: The anti-allergic effect was firstly assessed using IgE-DNP/DNP-BSA induced PCA mouse model. PCA mice showed ear Evans blue exudation (B), and the absorbance value was detected (C). D: Serum histamine of systemic anaphylaxis shock mice was determined through OPT-fluorometric assay as previously reported. Each data point indicates mean ±SD of 6 mice. E: ELISA kit detected serum tryptase-β2 of systemic anaphylaxis shock mices. Each data point indicates mean±SD of 6 mice. F and G: Peritoneal TNF-α and IL-6 levels in systemic anaphylaxis shock mices were detected by ELISA. Data indicate mean±SD from four independent experiments. H: In compound 48/80 induced systemic anaphylaxis shock model,mesentery mast cells degranulation was detected by toluidine blue staining. Quantification of mesentery mast cells degranulation by compound 48/80 was performed in a blinded fashion. I: Lung injury was detected by hematoxylin and eosin staining in systemic anaphylaxis shock mices. J: Statistical analysis of the lung injury score in systemic anaphylaxis shock mices by assessment infiltration of numerous polymorphonuclear leukocytes, interstitial spaces and hemorrhage, as described in materials and methods. It was performed in a blinded fashion. Each data point indicates mean±SD of 5 mice. *P<0.05.
Table 1.
Effects of Ang-1 on compound 48/80-induced systemic anaphylactic shock.